Bacterial Conjugation- Definition, Principle, Process ...

Two problems not commonly discussed prior to the novel Coronavirus outbreak are the emergence of infectious disease and the related increasing prevalence of antimicrobial resistance. Here, I will explain the science behind these problems and some solutions that can be driven by legislation. My background is more squarely rooted in the science, so I apologize if I lean too heavily in this area as opposed to the economics and policy focus of this subreddit. I frequent this sub and enjoy the discourse here, and in my area this is one topic that overlaps with public health policy that I am passionate about.
To understand emerging disease and antimicrobial resistance, it’s important to understand evolution
The novel coronavirus, SARS-CoV2, is an example of an emerging infectious disease. SARS-CoV2 is a disease that, prior to 2019, had not to the best of our knowledge infected a human being. The genetic makeup of the virus indicates that the virus is natural, originating likely as a bat or pangolin Coronavirus that acquired the ability to infect humans, and that it is not man-made (1). Why do new diseases come into existence? Why haven’t humans encountered all the diseases capable of infecting us? Furthermore, why do diseases that we had previously thought conquered have the newfound ability to harm us again, in spite of our advancements in antibiotic development?
The answer to these questions is partially answered by evolution. Several novel viruses, like SARS-CoV1, MERS, and SARS-CoV2, began as zoonosis: infection by a pathogen with an animal source. Viruses, though generally considered non-living, contain nucleic acid genomes (either RNA or DNA) similar to every other organism in the tree of life. This genome is subject to selective pressures, just as with every other nucleic-acid containing being, and mutates non-specifically (that is, an organism develops a mutation, then selective pressures have a positive, negative, or neutral effect on retaining or discarding the mutation). An animal coronavirus that recognizes surface molecules on animal cells that have some similarity to human cell surface molecules may only be a few small genome changes away from being capable of infecting humans. It is likely that SARS-CoV2 emerged in one of two ways: as either an animal virus that mutated within an animal that gained the ability to infect humans, or as an animal virus that jumped to humans, and within the human host was selected for the ability to infect humans (1). The advent of novel viruses is also facilitated by the horizontal transfer of genetic material between distinct viral lineages. In Influenza viruses, this can take the form of segments of genome being transferred wholesale between viruses. Influenza viruses contain a genome composed entirely of RNA in multiple segments of sequence. Segments “re-assort” when flu viruses of distinct lineage infect the same cell, and viral genomes are mixed during the process of producing new viruses. Alternatively, as would be the case in coronaviruses, recombination occurs through a mechanism not fully understood, where whole portions of genome are exchanged between viruses (2).
The problem of antimicrobial resistance is also best understood through evolution. To explain this phenomenon, I will describe mainly how resistance manifests in bacteria, but similar processes drive resistance to anti-virals, anti-fungals, and anti-parasitics. Antibiotics are largely derived from natural sources: as microbes compete for resources, there is a drive to reduce competitors numbers by killing them or inhibiting their growth. Antibiotics are typically small molecules that target essential processes for bacterial growth; commonly cell wall biosynthesis (preventing growth and division of the cell, an example being penicillin), protein synthesis (blocks the process of translation, an example being erythromycin), production of RNA (blocks the process of transcription, an example being rifampin) or production of DNA (blocks the process of replication, an example being fluoroquinolone). These antibiotics arose through selective pressures, and in response bacteria have developed systems to circumvent the deleterious effects of antibiotics. These include: rapidly excreting the antibiotic before it is capable of inhibiting growth (efflux pumps, a notable offender being Pseudomonas aeruginosa, a common pathogen in patients with cystic fibrosis), degrading the antibiotic (beta-lactamases are a class of enzyme that degrade beta-lactam family antibiotics, such as penicillin), modifying the antibiotic (the most common mechanism for aminoglycoside resistance is to chemically modify the antibiotic so it doesn’t work), or simply modifying the target (Streptococcus pneumoniae is a microbe that causes multiple diseases that is naturally resistant to beta-lactams by modification of the drug target, the aptly-named Penicilin-binding protein) (3). As humans, it has been beneficial to identify these natural compounds and use them medically to treat infection.
Bacteria have incredible genome plasticity, engaging in a process known as horizontal gene transfer (HGT; sometimes referred to as lateral gene transfer) that increases the prevalence of resistant microbes. Not all bacteria are capable of this set of processes, but importantly several medically important pathogens, such as E. coli, Salmonella, Yersinia pestis, Acinetobacter baumannii engage in processes that facilitate the transfer of genetic material between bacteria. There are several molecular mechanisms for HGT: bacteria-infecting viruses can transmit pieces of genetic material between similar bacteria (transduction), bacteria can form a bridge that transfers plasmids (conjugation; plasmids are typically circular pieces of DNA, and are typically maintained independently of the bacterial chromosome and commonly encode antibiotic resistance genes), or bacteria can simply pick up naked DNA in the environment and integrate that DNA into their chromosomes (natural transformation) (3). The effect of these processes is that, when a gene that imparts resistance to a particular antibiotic is introduced into a population, it may spread between members of the population, not just within the progeny of the cells that encode the resistance gene. This is especially true when a gene that imparts resistance is on a plasmid or is otherwise mobilizable (transposons, or jumping genes, are also common perpetrators of transmission in that they move somewhat readily and often encode drug resistance). The key point to understand here is that while genes are present in bacteria, either on a chromosome or on a mobilizable element, these genes are capable of moving to many other members of the same population.
To understand this in more practical terms, many people have undergone a course of antibiotics and experienced gastrointestinal distress or stomach pains. This can be attributed to disturbing your normal intestinal microbiome, as you kill off non-resistant bacteria. Now assume you have an infection of some sort, it could be anywhere in your body accessible to an orally administered antibiotic, and your doctor prescribes you an antibiotic. It is possible, and possibly probable, that within your gut are bacteria that harbor resistance genes. In the absence of the antibiotic, these are likely to have a neutral or possibly deleterious effect; think of this like a welder that is unable to remove his welding mask: it certainly helps when he is welding, but is cumbersome at other times of the day. Taking the antibiotic results in high selection for resistant microbes to grow and prosper. This allows the resistant bugs to soon outnumber the non-resistant bugs. Ultimately, this increases the concentration of the resistance genes in the population of microbes in your gut. Subsequent to this, you may encounter an infection of a gastrointestinal pathogen that, in infecting your gut, acquires the resistance genes that you selected for. In disseminating this pathogen, you are also disseminating this resistance gene. Additionally, and perhaps more importantly, in taking antibiotics you select for drug resistance in the opportunistic pathogens of your body, notably Clostridium dificile and Staphylococcus epidermidis. These microbes are capable of causing disease, but reside in you or on you and cause infection when conditions are optimal for their growth.
The problem of antimicrobial resistance is convergent with emerging pathogens, as many pathogens “re-emerge” as they develop resistance to antimicrobials. While TB cannot be said to be an emerging pathogen as the world has been experiencing a TB pandemic since at least the early 1800’s, TB is re-emerging in the since that increased drug resistance has led to strains of TB that are not treatable via the traditional course of antibiotics (4). Similarly, common pathogens such as E. coli, Klebsiella, and Clostridium dificile are bugs that have become increasingly resistant to the antibitoics used to treat them (5). Acinetobacter baumanii, a soil microbe with resistance to a spectrum of antibiotics, became a common Gulf and Iraq War wound infection. Many of these pathogens find a home in hospitals, where the use of antibiotics is prevalent and potential hosts are abundant. Furthermore, the recently emerged pathogen HIV, the causal agent of AIDS, is intersectional with that of antibiotic resistance, as infection with HIV increases susceptibility to bacterial infections due to reduced immune cell numbers; increased infection rates of Both issues, antibiotic resistance and emerging pathogens, pose a threat to human health the world over, and I will attempt to address both of these issues in this post.
The problem of emerging disease and antibiotic resistance is exacerbated by humans
To what extent do emerging diseases and antibiotic resistance affect humans? SARS-CoV2 has had an extensive impact on human health and living, and the response to shut down to stop the spread of the virus has had a large economic impact. It is impossible to accurately predict the threat posed by non-discovered viruses, so the next threat could be relatively benign, or truly horrific. This is not to fearmonger, there is no reason to suspect that such a virus is bound to steamroll us soon, but to say that the next plague may be brewing inside a pig in a Chinese farm or outside our homes in the bodies of ticks, and we would not know it. The US Center for Disease Control and Prevention (CDC) has published two Antibiotic Resistance Threat reports on the subject, in 2013 and 2019. In the 2013 edition, it was reported that 2 million people in the United States will acquire an antibiotic resistant infection, and that 23,000 will die as a direct result of that infection (5). While by 2019 this was realized to be an underestimation of the drug-resistant cases, new approaches had determined that the true value had lowered from 2013 to 2019, with an updated estimate of 2.8 million cases and 35,000 fatalities in 2019 (6). An excellent illustration of the problem can be found on page 28 of the 2013 report, which reports the introduction date (left) and the date at which resistance was observed on the right for crucial antibiotic groups. Commonly, within a decade of the introduction of an antibiotic, resistance emerges. This problem cannot be expected to go away on its own, and more than likely pathogens commonly thought vanquished will re-emerge with drug-resistant characteristics.
There are human processes that contribute to the emergence of disease and spread of antibiotic resistance. In China, Wet Markets bring together livestock from all over the country, creating an environment that is diverse in the microbial life that live commensally and parasitically in and on these animals. The proximity of these animals allows for the exchange of these microbes; these microbes are then capable of exchanging genetic material. As I described for Flu and Coronaviruses, viruses that come into contact within cells are capable of genetic recombination, a process that can result in viruses that are capable of infecting humans. This is not to say this is a common phenomenon, just that 1) the process is accelerated by live animal markets and 2) this practice and resulting genetic recombination of zoonotic viruses is thought to have contributed to both the original and novel SARS-CoV outbreaks.
In the United States, a textbook example of an emerging disease is Lyme Disease (7). Named for the town of Lyme, Connecticut, Lyme Disease is caused by the peculiar bacterium known as Borellia burgdorferi. Borellia is a corkscrew-shaped bacteria that is interesting for its ability to grow without iron (a key component of the immune response is the sequestration of iron away from pathogens). Lyme Disease is spread through ticks, and the number of infectious cases is exacerbated by reforestation and settlement close to wooded areas in suburban environments. As building projects move closer to forested areas, exposure to arthropod-borne illnesses will be expected to rise.
Beyond settlement and the wet market practice, the emergence of new infectious disease is complicated by global warming and healthcare practices. Global warming is hypothesized to drive heat resistance in fungi, potentially improving their capacity to grow within the human body (8). The pathogenic potential of fungi is hypothesized to be limited by the heat of the human body, and there is some speculation that global warming is a contributing factor to the emergence of the notorious fungal pathogen Candida auris (8). These claims should be taken with a grain of salt and evaluated critically, but it is possible that human-caused climate change will disturb the ecology of our planet with as of yet unforeseen consequences, among them the generation novel and resurgent diseases.
In healthcare, over-prescription of and a lack of regulation on antibiotics has caused the problem to worsen (5,6). When a patient receives an antibiotic, the drug has an effect on all microbes where the drug is bioavailable. This includes the intestines, which contain a resident population of microbes, and the skin, which contains Staphylococci resident species that prevent colonization by pathogenic strains of similar bacteria. These residents are then selected for their ability to resist the drug, causing an increase in resistance among the healthy microbiota. These resistance genes, as I have described, can then move between dissimilar bacteria in the same environment. If a harmful strain of E. coli is introduced into such an environment, for example, it has a higher likelihood of encountering and assimilating the genetic potential to resist antibiotics than in an environment that is naïve to the antibiotic. Patients are commonly prescribed antibiotics for infections that are more likely to be caused by a virus, or in instances where an infection is likely to run course without medical intervention. The increased exposure to antibiotics causes the microbiota to increase the concentration of resistance genes. Additionally, in places like India, the regulations on antibiotics are much more laxed than even the United States, where one is able to purchase over-the-counter antibiotics. This allows anyone to give themselves an incomplete course of antibiotics for any condition, even if the symptoms are not caused by an infection of any kind. Additionally, prescription antibiotics that have deteriorated with time, or are manufactured with subpar quality control resulting in lower concentrations, that remain in circulation exacerbate the problem by establishing sub-inhibitory concentrations of the antibiotic in the body and resulting in selection for resistance. Furthermore, environmental pollution of antibiotics into natural water sources and sewage results in increased environmental concentrations of resistance genes. These genes can spill into humans by exposure to microbes in these environments (9).
Agriculture provides another increase in the concentration of resistance genes (10). Livestock are fed antibiotics, which increase the weight of animals in an as-of-yet not understood mechanism. A deleterious consequence of this increase in yield with antibiotic usage is the increase in resistance in response to this widespread antibiotic usage. These resistance genes then find their way into humans, whether through ingestion of food contaminated with resistant microbes.
Science and technology can solve the problem, but face institutional and biological challenges
There are both institutional and scientific challenges to combating emerging disease and antibiotic resistance. Some of these problems are easily apparent as I have described above: countries with laxed restrictions on who can obtain antibiotics, countries where the drugs are used often over-prescribed, suburbanization, and global warming all contribute to the problem.
Scientifically, there are challenges in that novel diseases are difficult to combat. The novel Coronavirus had the precedent of other coronaviruses (i.e. SARS and MERS) that had been studied and their virology dissected, but that won’t necessarily be the case everytime a novel pathogen infects a human. A technological benefit to this problem is the use of meta-genomics, which allows for DNA/RNA sequencing without prior knowledge of the nucleic acid sequence of the genome. Within weeks of the first identification of the virus, its sequence was available to researchers. This was not the case during the outbreak of SARS-CoV1, when meta-genomics approaches such as Illumina Sequencing, NanoPore Sequencing, and Pacific Biosciences Sequencing were not available. In the event of a novel disease emergence, this information would be vital to combating the pathogen.
Despite not knowing necessarily what the next threat will be, expanding the human knowledge base on microbes is an essential component of any plan to fight emerging diseases. Any emerging disease is likely to be similar to other microbes that we have encountered, and knowledge of the physiology of these organisms helps to understand weaknesses, transmission, and potential therapeutic targets. The study of all microorganisms therefore benefits the effort to combat the next pandemic, as any one piece of information could be critical.
Surveillance is perhaps the most important tool to fight emerging infectious disease; knowing the problem exists is a crucial step to curbing spread. A recent example of successful surveillance can be seen in a recent PNAS publication regarding the presence of potential pandemic influenza in hogs, and the presence of antibodies against this particular class of flu viruses in swine workers (11). While at present it does not appear that the virus has acquired the ability to cause a pandemic, this knowledge allows for immunologists to potentially include viral antigens specific to this particular viral class in seasonal vaccines. Surveillance is critical in controlling both emerging diseases and antibiotic resistance: knowledge of what potential pathogens emerge where, and what microbes are exhibiting resistance to what drugs, can drive containment and treatment efforts.
To combat antibiotic resistance, new drugs must be developed, but there are hurdles in identification, validation, and production of new antibiotics. First, potential new antibiotics have to be either identified or designed. This often involves looking through filtered environmental samples to determine the presence of small molecules that inhibit bacterial growth, or chemically altering known drugs to circumvent drug resistance. This is not necessarily difficult, as there are microbes in the soil and water that produce potential therapeutics, but this does require both time and money, as well as the consideration that it is likely that resistance to that novel therapeutic exists in the environment from which it was pulled. New drugs must be safe, but due to the abundance of antibiotics presently in use and their historic efficacy, the standard for antibiotics to pass safety regulations is extremely high. As drug resistance becomes more common, it will become apparent that more and more side effects may have to be tolerated to prevent death due to bacterial infection. Finally, and the most important challenge to developing antibiotics is that the profit margin on antibiotics is low for drug companies in the present market, disincentivizing research and production of novel drugs.
In addition to stand-alone antibiotics, new inhibitors of resistance must be developed as well. Clavulanic acid is one such inhibitor, and is administered with the beta-lactam drug amoxicillin to improve its ability to kill bacteria. Bacteria that are resistant beta-lactams often encode enzymes called beta-lactamases. Beta-lactamases break open the active portion of the beta-lactam molecule, rendering it ineffective in attacking its target. Clavulanic acid is a beta-lactam itself, and is a target for the beta-lactamase enzyme; however, when the enzyme begins to degrade clavulanic acid, the enzyme becomes stuck at an intermediate step in the reaction, rendering the beta-lactamase enzyme useless. These drugs must also be explored and screened for in environmental samples, as well as developed. It is possible to take a rational approach to drug design, with increasing knowledge of how resistance mechanisms work. This means that scientists specifically look at, say, a beta-lactamase enzyme at the molecular level, and design a small molecule that will fit into the enzyme and block its function. Chemists then design the molecule to test its efficacy.
Ultimately, scientists either know how to solve the problem, or know how to get the tools they need to solve the problem. It is the institutional challenges that make the problem more difficult to solve.
How legislation can improve the ability of scientists to combat emerging disease and drug resistance
In discussing emerging diseases and antibiotic resistance, I try to draw parallels to the problem of global warming: a global problem with global solutions. I don’t have a novel solution to climate change to discuss here, other than to parrot this subreddit’s typical ideas, so I will omit that discussion here. That is to say, global warming is a driver for emerging infectious disease, and fighting global warming is important to combat the potential rise of fungal pathogens. I will, however, discuss some ideas for combating emerging disease and drug resistance. These ideas are mostly derived from scientists familiar with the problem,
Funding for research, basic and applied, is crucial. No bit of knowledge hurts in the fight against human disease. Learning how Alphaviruses replicate, determining the structure of E. coli outer membrane proteins, and examining the life cycle of the non-pathogen Caulobacter crescentus all contribute to the fight against the next disease. The more we know, the more powerful our vision is in understanding the inner machinations of disease. Every immune response, every molecular mechanism, and every aspect of microbial physiology is potentially a drug or vaccine target, a clue into pathogenesis, or an indication of how a bug is likely to spread. The Trump administration has not been kind to science funding (12). Science that does not appear to have benefit at first glance often does in the long run, and for this reason I will stress the importance of funding research of this sort, as well as funding applied research.
Knowing is half the battle. In combating emerging diseases, it is important to know they exist. As I have mentioned the example of recent viral surveillance with regard to the novel reassortment influenza viruses, I would like to stress the importance of funding surveillance programs in fighting emerging disease and drug resistance. There are currently US governmental surveillance programs that provide valuable information about the spread of drug resistance, such as NARMS in the United States (13).
In the United States, there is a need for greater accountability in using antibiotics. Resistance is unlikely to completely go away, even when the use of an antibiotic is discontinued, but the levels of resistant bacteria dwindle when the selective pressure is reduced. For this reason, several medical practitioners have proposed a rotating schedule of prescription antibiotics, that includes the retention of some new antibiotics from use. The reasoning for this is that, in the years following the halted use of a particular antibiotic, it is expected that the concentration of resistant bacteria will decrease. As I discussed with the example of always wearing a welding helmet, carrying resistance genes often imparts some form of growth defect on the resistant bacteria (for example, altering an essential gene targeted by an antibiotic may render the bacteria resistant, but there is a reason such a gene is essential, in that it’s required for growth; changing the gene in a substantive way may negatively impact its performance and by extension make these resistant bacteria less fit). A rotating cycle of what antibiotics are allowed to be prescribed, informed by surveillance data, would buy time for the development of new antibiotics as well. Additionally, higher standards should be required for the prescription of antibiotics, to increase accountability of physicians; these standards could involve clinically verifying the presence of susceptible bacteria prior to administering a drug in situations where the disease in not life-threatening.
There is a need to reduce the environmental pollution of drugs into sewage and natural bodies of water as well. This will require research into cost-effective methods for reducing the population of resistant bugs and drugs in these environments. In the case of natural bodies of water, a source of contamination is often factories where drugs are produced. Often, waters near these factories have high levels of antibiotics that select for resistance to develop and spread. This may require legislation to improve environmental outcomes, as well as surveillance of drug resistance gene levels and the levels of antibiotics in these waters to ensure compliance.
There is also a need to halt the use of antibiotics in treating livestock (14). Halting the use of antibiotics typically results in reductions of antibiotic resistant bug populations within a year or two (10). I don’t know of studies that estimate the economic cost of halting use of antibiotics in American meat, but in the case of Denmark, livestock production does not appear to have been significantly impacted.
I think that the most challenging problem will be for drug companies to develop new antibiotics when there is not presently a financial incentive to do so. Because antibiotics are still largely effective, and the financial benefit to adding an antibiotic to the market does not outweigh the cost to put a drug to market, there is not currently a large incentive to produce new drugs (15). To address this negative externality, it is necessary to generate financial incentives of some form for the production of new antibiotics. This may take the form of subsidizing antibiotic discovery efforts and drug safety trials; additionally, applied research with the goal of specifically finding new antibiotics should see increased funding.
To combat the problem overseas, it is obvious that obtaining an antibiotic course must occur through a doctor. This eliminates false self-diagnoses of bacterial infections. The problem of wet markets may be partially resolved by preventing animals that do not regularly contact each other from being traded and stored in the same vicinity as animals that are not typically encountered. This may involve limiting a particular wet market to the trade of animals that come from a particular geographic area, preventing geographically diverse microbes from encountering each other.
It's on all of us to stop the next pandemic:
If you made it this far, thank you reading this post and I hope that I have convinced you of the importance of this issue! There are simple steps that we can all take as consumers to reduce antimicrobial resistance: don’t take antibiotics unless prescribed by a doctor and buy meat that was produced without antibiotics. I welcome any and all criticism, and would love to hear people's ideas! Please let me know of any errors as well, or any missed concepts that I glossed over. I've been excited to give my two cents to this sub, and I don't want to mislead in any way.
Sources:
1: Andersen, KG, et al. 2020. The Proximal Origin of Sars-CoV-2. Nature Medicine 26: 450-452.
2: Su, Shou, et al. 2016. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Cell Trends in Microbiology 24(6): 490-502. https://doi.org/10.1016/j.tim.2016.03.003
3: Munita, JM; Arias, CA. 2016. Mechanisms of Antibiotic Resistance. Microbiology Spectrum VMBF-0016-2015. doi:10.1128 /microbiolspec.VMBF-0016-2015.
4: Shah, NS; et al. 2007. Worldwide Emergence of Extensively Drug-resistant Tuberculosis. Emerging Infectious Diseases 13(3): 380-387. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725916/
5: CDC Antibiotic Threats Report, 2013. https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf
6: CDC Antibiotic Threats Report, 2019. https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf
7: Barbour, AG; Fish, D. 1993. The Biological and Social Phenomenon of Lyme Disease. Science 260(5114):1610-1616. https://pubmed.ncbi.nlm.nih.gov/8503006/
8: Casadevall, A; Kontoyiannis, DP; Robert, V. 2019. On the Emergence of Candida auris: Climate Change, Azoles, Swamps, and Birds. mBio 10.1128/mBio.01397-19. https://mbio.asm.org/content/10/4/e01397-19
9: Kraemer, SA; Ramachandran, A; Perron, GG. 2019. Antibiotic Pollution in the Environment: From Microbial Ecology to Public Policy. Microorgansims 7(6): 180. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616856/
10: Levy, S. 2014. Reduced Antibiotic Use in Livestock: How Denmark Tackled Resistance. Environmental Health Perspectives 122(6): A160-A165. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050507/
11: Sun, H, et al. 2020. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection. Proceedings of the National Academy of Science https://doi.org/10.1073/pnas.1921186117.
12: Kaiser, J. 2020. National Institutes of Health would see 7% cut in 2021 under White House plan. Science Magazine. https://www.sciencemag.org/news/2020/02/national-institutes-health-would-see-7-cut-2021-under-white-house-plan
13: About NARMS: National Antimicrobial Resistance Monitoring System for Enteric Bacteria. https://www.cdc.gov/narms/about/index.html
14: Khachatourians, GG. 1998. Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. CMAJ 159(9):1129-1136 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1229782/
15: Jacobs, Andrew. 2019. Crisis Looms in Antibiotics as Drug Makers Go Bankrupt. The New York Times. https://nyti.ms/366f7it

Why viruses aren't alive: A reductio ad absurdum

AP Bio Guide (Units 8 in comments)

Machine-learning invented antibiotic

Preliminary Characterization of a Nisin Z Bacteriocin with Activity Against the Fish Pathogen Streptococcus iniae- Juniper Publishers

Abstract

This is a preliminary characterisation of a bacteriocin, BacL49 produced by Lactococcus lactis ssp. lactis. This bacteriocin is significant due to its activity against Streptococcus iniae, a bacterial pathogen causing severe economic losses in the global aquaculture of various fish. Spot-on-lawn and microtitre plate assays were used to test antagonistic activity of the bacteriocin. BacL49 is heat and pH stable (100 °C for 60min, pH 2.5-9.5), and sensitive to proteinase K, a-chymotrypsin, trypsin and papain. BacL49 has a bactericidal mode of action and is produced during late log phase growth. BacL49 exhibits a broad activity spectrum against S. iniae, antagonising 93.75% (45/48) of S. iniae isolates collected from a variety of hosts and environments. The apparent molecular masses of the active protein components determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 5 and 54kDa. Molecular analyses were performed to locate the genetic determinants of BacL49. PCR of chromosomal DNA successfully amplified the structural gene encoding the precursor of nisin. Subsequent analysis of nucleotide sequences of the PCR products revealed it to be identical to the nis Z structural gene of nisin Z. There is a paucity of reports examining the inhibition of S. iniae by a lactococcal bacteriocin or even L. lactis as an aquacultural probiotic. This is one of the first studies to identify nisin genes in a strain of L. lactis exhibiting activity against S. iniae. BacL49 is a candidate biocontrol agent for mitigation of this important fish pathogen.
Keywords: Bacteriocin; Lactococcus lactis; Probiotic; Streptococcus iniae; Nisin Z
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Introduction

Streptococcus iniae is one of the most economically important bacterial pathogens causing streptococcosis in fish. Globally, economic damages have been in excess of US$100 million per year [1] with as much as 70% loss in annual production in certain fish cultures [2]. S. iniae causes disease and mortality in at least 30 species of fish in marine, brackish and freshwater environments [3,4]. Outbreaks of S. iniae typically occur in warm-water, cultured situations throughout the world including Australia, Bahrain, Israel, Japan, Korea, Spain, Italy, and the United States [4,5-10]. Epizootic incidents have also been reported in wild populations [11,12], the most notable being the 1999 and 2008 Caribbean fish kills [13,14]. Zoonotic infections of S. iniae, normally caused by percutaneous injuries sustained during raw seafood preparations, have been reported in Canada, China, Hong Kong, Singapore, Taiwan, and the United States [15]. Prevention and treatment of S. iniae in aquaculture remains difficult, particularly with the industry seeking safer alternatives to antibiotics and vaccines. Antibiotics, including erythromycin have previously been successful in treating streptococcus is in fish [16]. Nonetheless, the use of antibiotics in aquaculture is gradually being eschewed due to widespread development of antibiotic resistance in the environment and in cultured fish, which causes consumer concern [16-18]. In some cases, antibiotics are also believed to merely suppress clinical symptoms without eliminating the infection, thereby promoting the development of "carrier fish" [1].
Research devoted to creating effective vaccines for prevention of S. iniae suffered a major setback when a novel serotype of S. iniae caused severe outbreaks in Israel amongst vaccinated rainbow trout (Oncorhynchus mykiss) [19,20]. Since then, commercial vaccines against S. iniae have been marketed, but are very limited geographically [21,22]. Alternative vaccines have yielded successful results but are still undergoing testing [2325]. In spite of this progress, the task of effectively vaccinati Oceanogr Fish Open Access J 3(2): OFOAJ.MS.ID.555610 (2017) each individual can be tedious, expensive, and even stressful for the fish [26]. Vaccinations can be futile when applied to juvenile fish that may not be fully immunocompetent [27]. Additionally, S. iniae is capable of surviving in the aquatic environment without a host [11,28,29] and this was speculated to have been a factor in the evolution of the new serotype by allowing the bacterium to evade the immune response of the vaccinated fish [19]. Furthermore, the outbreak in Israel is evidence of serotype diversity that could enable S. iniae to eventually overcome yet another vaccine. Thus, the quest continues for a long-term solution that can be universally and easily applied throughout the aquaculture industry [25]. Chemical-free "green solutions" appear to be the next era of therapeutics for preventing bacterial epizootics in fish [17]. Probiotics, either in the form of whole cells or cell components such as bacteriocins, are anticipated as being effective replacements for antibiotics and chemotherapeutics to fight infectious diseases [30]. Bacteriocins are a group of proteinaceous molecules that are biologically active against bacteria that are closely related to the bacteriocin producer, while the producer is immune [31-33]. Ubiquitous across all bacterial genera, these ribosomally synthesized peptides confer a selective advantage to their producers [31,32,34]. Bacteriocins are recognized as potentially useful agents in the control of bacterialinfections due to their effectiveness, non-toxicity and relatively cheap production [32,35,36]. Only two published studies on bacteriocins have tested S. iniae as an indicator species [37,38].
An inhibitory substance, identified as bacteriocin BacL49, was found during experimentation with the library of S. iniae and other aquatic bacteria from James Cook University (JCU). The bacteriocin was produced by a strain of Lactococcus lactis ssp. lactis and was observed to inhibit a large spectrum of S. iniae isolates. L. lactis, along with the rest of the lactic acid bacteria (LAB) are considered the most prolific of all the Gram-positive bacteriocinogens [32,39]. A versatile species, variousstrains of L. lactis can produce an assortment of bacteriocins that are predominantly encoded on plasmids [40-43]. Some L. lactis strains produce the bacteriocin nisin, a small (<5kDa), membrane-active member of the Lantibiotic class of LAB bacteriocins capable of antagonistic activity towards a wide range of Gram-positive bacteria [44-46]. Nisin is heat-tolerant (115-121 °C) at low pH levels, making it an ideal preservative in pasteurised and acidic food products [45,46]. Being "generally regarded as safe," nisin has achieved worldwide recognition as a non-toxic food additive in over 50 countries [31,32,45]. As produced by various strains of L. lactis, nisin occurs as natural variants with designations A, Z, Q, and F [47-49]. 106 Unlike most plasmid-encoded bacteriocins produced by L. lactis, nisin is encoded on a conjugative transposon [50-52], a chromosomally- associated segment of DNA with the capacity to repeatedly insert into and mobilise plasmids and genomes [52,53]. Thus, one objective of this study was to identify the genetic determinants of the bacteriocin BacL49 through assessing the prevalence of plasmids in L. lactis subsp. lactis L49 and examining the bacterial chromosomal DNA for nisin genes. This study also provides a brief characterisation of bacteriocin BacL49 and highlights the potential of this substance as a green solution for S. iniae infections in fish.
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Materials And Methods

Source and identification of BacL49 producer. A lawn of S. iniae AS-04-1524#1 (JCU isolate S42; Table 1) was observed as a mixed culture, with one distinct colony type antagonizing the growth of the other. Both isolates were identified using a PCR assay for the lactate oxidase (lctO) gene of S. iniae using the primer combination LOX-1/LOX-2 [54], and a 16S rDNA PCR assay using universal primers 27F/1492R. PCR products were cleaned and sequenced by Macrogen Inc. (Korea). The 16S PCR assay returned a sequence 99% identical to Lactococcus lactis ssp. lactis in BLAST. According to sequencing, the antagonizing bacterium was identified as L. lactis L49, originally isolated from a moribund sleepy cod (Oxyeleotris lineolatus) at JCU. The inhibitory substance produced by L. lactis L49 was designated as BacL49.
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*Altered TAAHL accession number. Changes were made for unknown reasons by previous researchers; it is likely these isolates are from the correct animal accession but the final number has been altered (ie. -9 and -11). **Invalid TAAHL accession number.
Bacterial growth and isolation of BacL49. Unless otherwise stated, bacteria were propagated aerobically at 28 °C in heart infusion broth (HIB) or agar (HIA) produced by the addition of 1.5% technical agar to HIB. Bacterial lawns were made by seeding HIA plates with 1ml overnight growth of bacteria, removing excess culture and allowing lawns to dry at room temperature. To produce cell-free supernatant (cfs) containing BacL49, L. lactis L49 was grown aerobically at 28 °C for 10-12h, then centrifuged at 4300g for 5min and filtered through 0.45|im. Aliquots of cfs were stored at 4 °C.
135 Assays for antagonistic activity. The activity of L. lactis L49 against 48 isolates of S. iniae (Table 1) was determined by an altered deferred antagonism method [55]. Briefly, an overnight L. lactis L49 culture was streaked in a single line on an HIA plate with a sterile loop. Bacterial isolates and a control of uninoculated broth were then streaked with sterile loops in parallel at right angles to L49. Zones of inhibition were measured after incubation for 16h. For interest, streak plates were made to determine the activity of L. lactis L49 against B antibiotic resistant human pathogens including E. coli B597, E. coli 53e, community acquired methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae. For these human isolates, one plate was incubated at 37 °C with CO2 and the other was incubated aerobically at 37 °C to ensure sufficient growth of the pathogens. Zones of inhibition were measured after 24h.
146 Antagonistic activity of BacL49 was detected qualitatively on solid media using a spot-on-lawn assay [56]. Antagonistic activity was quantified in liquid media using a modified microtitre plate assay [57]. The indicator culture was grown to an optical density of approximately 0.2 measured at 540nm, and added to two-fold dilutions of the growth medium or cfs (treated or untreated) in duplicate 96-well round bottom plates. Plates were incubated aerobically for 3h at 28 °C and the optical density measured. Antagonistic activity was defined as the reciprocal of the dilution causing 50% growth inhibition (determined by optical density) relative to the control culture without cfs (AU = arbitrary units). S. iniae S23 was used as the indicator strain for all spot-on-lawn and microtitre plate assays due to its high level of sensitivity to BacL49.
Effect of heat, pH and enzymes on BacL49 activity. To determine the heat stability of BacL49 activity, cfs samples (pH 5) were heated at 100 °C for 10, 20, 30 and 60min. Samples were cooled to room temperature before spot-on-lawn and microtitre plate assays. To determine the effect of pH on BacL49 activity, cfs samples were adjusted to pH levels between 1.5 and 9.5 using 1N NaOH or HCl. Samples were incubated with agitation for 2h, then readjusted to pH 5 (the pH level of untreated cfs following incubation) before spot-on-lawn and microtitre plate assays. To determine the effect of different enzymes on BacL49 activity, cfs samples were adjusted to pH 7.0 and treated to a final concentration of 2mg ml-1 with the following enzymes: proteinase K (40 units mg-1), a-chymotrypsin (59.3 units mg- 1), trypsin (2.6 units mg-1), pepsin, papain (19 units mg-1), and catalase (1340 units mg-1). Samples were incubated at 37 °C with agitation for 2h, then at 100 °C for 5min to deactivate enzymes. Remaining antagonistic activity was measured using the spot- on-lawn assay. An untreated cfs sample was used as a control in all assays.
170 Kinetics of production and activity. The kinetics of BacL49 production by L. lactis L49 were investigated by measuring the growth of the bacterium and activity of BacL49 produced over the same period. Overnight growth of L. lactis L49 was added to HIB (3.75% volume) and the culture was incubated with agitation. The optical density of the culture was measured at 600nm every hour until 12h, then periodically to 28h. At each reading, 1ml culture was removed to produce cfs, which was stored at 4 °C. Antagonistic activity was measured using a spot- on-lawn assay following the collection of all cfs samples.
The activity of BacL49 was determined as either bacteriostatic or bactericidal by measuring the growth of indicator strain S. iniae S23 after the addition of BacL49. Early log-phase growth of the indicator strain was distributed into triplicate 10ml aliquots. One ml of cfs was added to two indicator cultures and HIB was added to the remaining control culture. Optical density was measured at 600nm over 24h. To measure cell viability in a treated culture, mid log-phase growth of the indicator strain was distributed into duplicate 10ml aliquots. One ml cfs was added to one aliquot and HIB was added to the control. Optical density measurements at 600nm were taken over time and 10-fold serial dilutions were grown on HIA to measure cell forming units (cfu) of the indicator strain in both cultures at 48h.
Protein purification. L. lactis L49 was grown overnight in HIB previously filtered through Millipore type HA filters (to remove unwanted proteins from the media). A portion of the cfs produced from this culture was treated with ammonium sulphate in two stages. For the primary precipitation, saturated ammonium sulphate solution was slowly added to 100ml cfs while stirring to 60% saturation at room temperature. The solution was agitated 12h at 4 °C, then centrifuged at 4500g for 30min at 4 °C. The precipitate was resuspended in 10ml sterile distilled water. A small portion was removed and stored at 4 °C for the activity assay. Saturated ammonium sulphate solution was added to the remaining primary solution while stirring to 80% saturation at RT. This solution was agitated 12h at 4 °C, then centrifuged at 4500g for 30min at 4 °C. The secondary precipitate was resuspended in 1ml (1% original volume) sterile distilled water and stored at 4 °C. The activity of the ammonium sulphate precipitated (primary and secondary) proteins was tested with a microtitre plate assay.
Approximate molecular size of BacL49 was determined by tris-tricine sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Aliquots of the partially purified protein samples and untreated cfs were prepared with TruSep tricine SDS sample buffer and run on 16% tris-tricine gels with a low MW prestained protein ladder (Fermentas, Australia) at a constant 150V. Gels were silver stained using a Silver Stain Plus staining kit (BioRad, Australia) according to the manufacturer's protocol 208.
Proteins separated by SDS PAGE were eluted from the gel using a modified protocol from Busarcevic et al. [58]. Following agitation at room temperature overnight, the eluted samples were centrifuged at 10,000g for 5min and the supernatants concentrated by vacuum at 33 °C for 20min. Two controls were used: one elution made with a slice of gel from an unused well lane (containing no protein) and one with elution buffer only. Eluted samples were assayed for antimicrobial activity with a spot-on-lawn assay.
DNA purification. Isolation of plasmid DNA was carried out using the Wizard® Plus SV Minipreps DNA Purification System (Promega, Australia). Low-copy number plasmids were assumed for all strains. Genomic DNA isolations were carried out using the High Pure PCR Prep Kit (Roche, Australia) for the detection of nisin structural genes. DNA isolation procedures were performed according manufacturer specifications with the addition of a lysozyme step for Gram-positive bacteria. Negative control preparations contained no bacterial culture.
DNA products were resolved by electrophoresis on agarose gels stained with GelRedTM and then visualised under UV light. Since L. lactis strains often harbour a wide size-range (2kb to about 100kb) of plasmids (Teuber and Geis, 2006), plasmid DNA products were visualised using two different DNA ladders. These included the 1kb GeneRulerTM (Fermentas, Australia) used with 1% (w/v) agarose gels at 90V and the Lambda Mix Marker 19 (Fermentas, Australia) used with 0.5% (w/v) agarose gels at 40V.
PCR analysis of the nisin gene. Oligonucleotide primers (Macrogen, Korea) were designed using the NCBI-ORF Finder and OLIGO 7 primer analysis software (Table 2) to target the nis A structural gene. Genomic DNA samples were prepared for PCR using GoTaq® Green master mix (Promega, Australia). As L. lactis L53 was found to contain a plasmid (results not shown), this isolate was included in the procedure. For negative controls, a tube of reaction mix containing indicator strain S. iniae S23 and one without sample DNA were included for the nisin structural gene protocol, and for the PCR protocol, respectively. DNA was amplified in a thermocycler (Eppendorf, Australia) set for denaturation at 94 °C, annealing at 55 °C and extension at 72 °C. PCR products were visualised on 2% (w/v) agarose gels at 120V, along with a 50bp GeneRulerTM (Fermentas, Australia). Sequence information (Macrogen, Korea) of the PCR products was analysed using Sequencher® 5.0 software. The resulting consensus sequences were compared to DNA and protein sequences contained in the National Center for Biotechnology Information (NCBI) database via BLAST search of "highly similar" sequences.
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Results

L. lactis L49 produced antagonistic activity against 93.75% (45/48) of S. iniae isolates in the JCU library (Figure 1). L. lactis L49 failed to inhibit the growth of isolates S32, S39, S47 and the human pathogens tested. Antagonistic activity of BacL49 remained following 60min at 100 °C and over a broad pH range (pH 2.5-9.5), though activity was weaker at pH levels higher than 5.5 (Table 3). The cfs also showed antagonistic activity against the indicator after exposure to pepsin and catalase, but lost activity when exposed to proteinase K, a-chymotrypsin, trypsin and papain. L. lactis L49 began producing BacL49 at the end of the log growth phase (Figure 2). BacL49 production reached a maximum at early stationary phase, but began to drop following600min growth and continued to decrease to the end of 24h.
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Activity measured as present (+) or absent (-) for enzyme tests. Absence antagonistic activity (-) correlates to senstivity (s) to the enzyme tested, and antagonism of the indicator (+) correlates to resistance (r) to the enzyme tested; Cfs: Cell Free Supernatant.
A Growth of the indicator strain was not only inhibited following the addition of BacL49, but the optical density of the culture continued to drop steadily over time without recovery, while the control culture grew normally to a high optical density. The cell viability of the treated indicator culture dropped 10fold in the first 30min following the addition of BacL49 and continued to drop over 70min while the control culture cfu increased 10-fold.
Ammonium sulphate precipitation successfully concentrated the bacteriocin (Table 3). The primary precipitate showed 8 times the activity (in AU) of the untreated cfs and the secondary precipitate showed at least 64 times the activity of the untreated cfs (1/512 was the highest dilution made of the substances tested in the microtitre assay).
Tris-tricine SDS PAGE allowed separation of the low molecular weight proteins, and multiple protein bands were clarified after silver staining of the gel (Figure 3). Following elution of proteins from the tris-tricine gel slices, only minor antagonistic activity was produced on the indicator strain by the small molecular weight band (5kDa) but strong antagonism was produced by the large molecular weight (54kDa) band.
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Following plasmid extraction, DNA samples of L49 showed faint bands that were too unremarkable to be denoted as plasmids (Figure 4). Based on this data, further plasmid experimentation was not pursued. PCR for the nisin structural gene resulted in products just over 100bp for isolates L49 and L53 (Figure 5). No bands were detected for S23 or the control. Sequence analysis of the L49 PCR product yielded a consensus sequence of the nisin structural gene (Figure 6) that was 99% homologous to the nis A structural gene and 100% homologous to the nisZ structural gene (Table 4). The protein BLAST search showed 100% homology with the Nisin Z precursor, followed by that of nisins F and A, and then nisin Q (Table 4). A deduced amino acid sequence of BacL49 was also obtained from the BLAST search, which showed that BacL49 was identical to nisin Z.
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Discussion

The inhibitory substance BacL49 produced by L. lactis L49 has been identified as a bacteriocin due to meeting the criteria of being a biologically active protein moiety with a bactericidal mode of action [33]. BacL49 can be produced in anaerobic conditions and is capable of diffusing through solid media (results not shown), which rules out hydrogen peroxide and phage activity, respectively, as the cause of antagonism. The protein nature of BacL49 was confirmed by its sensitivity to a number of proteases and the mode of action was confirmed as bactericidal by optical density and cell viability experiments.
BacL49 is heat and pH stable, though antagonistic activity was weaker when the bacteriocin was incubated in an alkaline environment. An acidic pH can be necessary for the retention of the cationic properties of bacteriocin peptides, which appear to be crucial for their antagonistic activity [59]. Most L. lactis bacteriocins, including nisin, are heat stable and tolerant to acidic conditions [21,60-62] but many show different enzyme sensitivities. A review of publications describing Nisin Z production by L. lactis strains isolated from various sources found that nisin Z is consistently sensitive to proteinase-K, but varies in sensitivity to trypsin, a-chymotrypsin, and papain. A few published descriptions of nisin exactly matched the protease sensitivity profile of BacL49 [63-65]. Interestingly, the only other described nisin isolated from fish differs from BacL49 in sensitivity to trypsin and a-chymotrypsin [37].
A bactericidal mode of action was confirmed by the reduction over time in optical density and Viable cell count of the BacL49 treated indicator culture. It is known that many antibiotics kill bacteria by targeting lipid II, thus blocking cell wall synthesis and leading to cell lysis by pore formation [44,66,67], and it is possible that BacL49 performs in a similar manner. The bactericidal mode of action of BacL49 not only confirms this substance is a bacteriocin, it indicates that BacL49 could reduce bacterial loads in fish or the environment by destroying S.iniae cells.
Production of antagonistic activity by the two differently sized peptide bands eluted from SDS PAGE gels (54kDa, and 5kDa) suggests that two different inhibitory substances are being concurrently produced by L. lactis L49. The two component lantibiotic lacticin 3147 consists of two 3-4kDa peptides, but both are required for antagonistic activity [68]. This required cooperation is apparently not the case with the two BacL49 peptides, as evidenced by their independent production of antagonism. It is also possible that cleaving of BacL49 is occurring; the large molecular weight peptide may be an unseparated quaternary structure of the small peptide.
The results of the plasmid extraction do not support the hypothesis that L49 contained plasmids. The faint bands observed after electrophoresis were not indicative of a typical plasmid profile. Chromosomal DNA extraction results suggest that L49 possesses the structural gene encoding the nisin precursor. Interestingly, equally strong bands for the structural gene were evident for isolate L53. Whilst this result was not expected due to isolate L53 only inhibiting 30% of S. iniae isolates in the JCU library (results not shown), it was also not surprising since various strains of L. lactis have been observed to produce nisin.
Based on the NCBI BLAST search results, BacL49 is likely nisin Z. The DNA sequence was shown to differ from that of nisin A exactly as stated in Mulders et al. [48], varying at position by a C to A transversion. Comparison of the deduced amino acid sequence with those of the other nisin variants, coupled with the protein BLAST results, substantiates the nucleotide results. Nisin Z is a natural nisin variant that has only been produced by strains of L. lactis, however these strains have been isolated from a variety of sources from different environments.
Producers of nisin Z have previously been derived from dairy [51,69] and vegetables products [70,71]. Recently, it has been shown that they are also associated with mangroves [72], marine fish [37], and now with freshwater fish. The source of the producing strain could have an impact on how and in what context it can be used most effectively.
Based on the results of this study, it is likely that BacL49 is encoded on a conjugative transposon. Like plasmids, some conjugative transposons can possess a very broad host range [73], allowing for the dissemination of various traits (e.g. antibiotic resistance) across different species [53,74,75]. Further research following this study should focus on isolating this mobile genetic element from L49, characterising it, and assessing its potential novelty and uses by genetic manipulation.
Other bacteriocin-producing L. lactis strains have been isolated from freshwater fish in the past [47,60] however these studies tested bacteriocin activity against S. aureus, L. monocytogenes, and other pathogens important in food spoilage and human infection. This study reports similar findings to that in Heo et al. [37], in which a strain of L. lactis subsp. lactis was isolated from the intestine of a marine olive flounder (Paralichthys olivaceus) and was found to inhibit S.iniae during in vitro experiments. Heo et al. [37] examined the in vitro effects of nisin Z against S. iniae by combining it with varying concentrations of NaCl. The authors concluded that the ability of nisin Z to inhibit the growth of S. iniae was synergistically improved when applied in conjunction with NaCl. This is significant for marine- based aquaculture. The producer of BacL49 was isolated from a freshwater fish species, thus it would be interesting to look for any significant differences in activity between BacL49 and nisin Z originating from a marine source BacL49 could have important applications in the aquaculture industry with regard to S. iniae.
The ability to retain its activity through heating processes would allow this bacteriocin to be readily incorporated into commercial fish food. The fact that BacL49 remains active over a broad pH range is also advantageous because S. iniae can establish in a variety of organs and tissues in infected fish. BacL49 could also be added directly into the culture water as a non-toxic means of biological control of S. iniae in the culture environment and be a supplement to vaccination procedures. This environmental treatment could address the issue of S. iniae cells surviving freely in the water and evading the immune responses of vaccinated fish. Differences in activity, such as increased inhibitory specificity, distinguish bacteriocins from classical antibiotics and are another advantage to their use in a cultured environment [76]. This specificity can reduce the risk of non-target bacteria (particularly beneficial ones) being antagonized and minimize the threat of resistance development. However, the broad activity spectrum of BacL49 on different isolates of S. iniae would prove to be an advantage due to the large variation that exists between strains of this pathogen [76,77].
The pathological effects of bacteriocins must be considered before they are used in an in vivo situation, and it would be beneficial to determine whether the bacteriocin is strongly antigenic [33]. However, most bacteriocins are not toxic to animals at effective antimicrobial concentration due to their specificity [78]. Despite the results of this study showing that high concentrations of BacL49 are easy to achieve, bacteriocin delivery or retention in fish tissues could prove difficult in vivo. If this were the case, L. lactis ssp. lactis L49 could be trialed as a probiotic. Great interest has been shown towards LAB as potential probiotics, as they are well-recognised for their bacteriocinogenic capabilities and presence within the normal microbiota of fish (typically the intestine) [30,79]. Not only can they withstand acidic stomach conditions, but they can grow and colonise the intestine of fish [80]. The piscine origins of L49 may contribute to its survivability and efficacy as a potential probiotic, though there are few studies examining L. lactis as a probiotic specifically against fish pathogens. Though L. lactis has not been documented as a fish pathogen and is regularly present in the aquatic environment and the intestinal tract of both freshwater and marine fish [71,81-84], it would be necessary to confirm isolate L49 as non-pathogenic to the species of fish undergoing treatment. The elucidation of the L. lactis genome and the fact that products from the bacteria are generally regarded as safe make the bacterium a unique candidate for genetically engineered live vaccines as well [85].
BacL49 is significant because it displays a broad activity spectrum for S. iniae isolates, implicating it as a new therapeutic or preventative agent for infections caused by this economically important fish pathogen. Nisin has had a long history as a safe food additive in the manufacturing of various foods for human consumption, thus BacL49 may also be integrated with fish food without serious concern over chemical residues. Purified BacL49 should be tested in vivo to determine antigenicity of the substance in fish, and the bactericidal action of the bacteriocin should be studied in depth to identify problems that may arise with bacterial resistance.
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How to Manage Lactose Intolerance

Lactose intolerance is a condition in which people have symptoms due to the decreased ability to digest lactose, a sugar found in dairy products. Those affected vary in the amount of lactose they can tolerate before symptoms develop. Symptoms may include abdominal pain, bloating, diarrhea, gas, and nausea. These symptoms typically start thirty minutes to two hours after eating or drinking milk-based food. Severity typically depends on the amount a person eats or drinks. Lactose intolerance does not cause damage to the gastrointestinal tract.
How to Manage Lactose Intolerance – Causes, Symptoms and Treatment
Lactose intolerance is a prevalent and distressing condition that affects a surprisingly high percentage of adults. The U.S. Department of Health & Human Services approximates that about 65 percent of the human population has a reduced ability to digest lactose after infancy.
Lactose intolerance is not the same as a milk allergy and is more of a discomfort than a real over-reaction by the immune system, according to the FDA. Many people with lactose intolerance can even have small amounts of the offending foods/drinks without having symptoms.
What sort of signs of lactose intolerance may indicate that you have this common problem? Lactose intolerance symptoms typically include bloating, gas, diarrhea and other GI issues. Fortunately, by following a lactose intolerance diet and treatment plan, it’s possible to reduce (and in some cases even eliminate) the symptoms of lactose intolerance.

What Is Lactose Intolerance?

The definition of lactose intolerance, according to The National Institute of Diabetes and Digestive and Kidney Diseases, is “a condition in which you have digestive symptoms — such as bloating, diarrhea and gas — after you consume foods or drinks that contain lactose.”
Lactose is a sugar that is found in milk and dairy products. In order to digest this sugar properly, the small intestine must produce adequate amounts of the enzyme called lactase.
Lactose is found in:

Milk
Lactose
Whey
Curds
Milk by-products
Dry milk solids
Non-fat dry milk powder

Lactase is responsible for breaking down the lactose into glucose and galactose, so the body can absorb it. When the body’s ability to make lactase diminishes, the result is lactose intolerance.
It is important to note that not all dairy products cause these unpleasant symptoms of lactose intolerance. In fact, yogurt or kefir with live active cultures typically do not produce these symptoms, as the active cultures help to break down lactose prior to consumption. Also, the longer the food is fermented, the less the lactose content will be, as the healthy probiotics survive by eating the lactose sugar.

Lactose Intolerance Causes

What triggers lactose intolerance? As described above, lactose intolerance is caused by the body’s inability to effectively digest lactose due to malabsorption or low levels of lactase produced in the digestive tract. This seems to occur for several main reasons:

1. Genetics/Family History

While it has been documented only rarely, the inability to produce lactase can sometimes be congenital. Researchers believe there are genetic links to lactose intolerance causing symptoms to appear during the teenage years. However, just because you made it through your teen years without affliction doesn’t mean you are immune for life. Lactose intolerance is not very common in children under two years of age, although it’s still possible.
In addition, lactose intolerance seems to run in families, and certain ethnic groups have greater occurrences of lactose intolerance than others. Native Americans, Hispanics, Asians and those from African descent often experience intolerance more often than those of European descent.

2. Aging

As we age lactase production decreases, leading to intolerance in individuals who otherwise never had overt signs of lactose intolerance.

3. Illness and Stress

In some cases, lactose intolerance can also result from surgery, injury, illness and even certain treatments. Common conditions that can contribute include gastroenteritis, IBS, Crohn’s Disease, ulcerative colitis, celiac disease and other conditions of the digestive tract, including candida overgrowth and leaky gut. Even cases of the flu can cause intolerance (however, often the symptoms will fade over time). Additionally, taking medications including some antibiotics for extended periods of time may disrupt gut health and contribute to lactose intolerance.

Diagnosis

How do doctors test for lactose intolerance? To test for lactose intolerance in patients who are experiencing symptoms like bloating and diarrhea, doctors rely on a number of different tests, including:

A hydrogen breath test, since undigested lactose causes you to have high levels of hydrogen in your breath
Reactions to an elimination diet, in which you stop eating and drinking milk and milk products to test the effects.
A test using a stethoscope to listen to sounds within your abdomen
Discussion of symptoms, family history, medical history and eating habits
A physical exam to check for any underlying health problems that may be the real cause of symptoms

Keep in mind that a number of other conditions aside from lactose intolerance can cause similar symptoms. These include: irritable bowel syndrome, celiac disease, inflammatory bowel disease or SIBO (small bowel bacterial overgrowth). This is why doctors must rule out these causes before confirming a diagnosis of lactose intolerance.

Symptoms of Lactose Intolerance

What are symptoms of being lactose intolerant? The most common symptoms of lactose intolerance include:

Diarrhea
Gas
Stomach bloating/swelling in the abdomen
Stomach pain/cramping
Nausea, vomiting
Headaches or migraines
Acne

When do lactose intolerance symptoms start? These warning signs of lactose intolerance can arise anywhere from 30 minutes to two hours after the consumption of dairy products and can range from mild to severe. Most immediate reactions are caused by the body not having the enzymes to digest the lactose sugar, which causes the intestines to contract.
If you have had an ongoing intolerance, you might also experience issues besides digestive upset, such as more extreme headaches, migraines or bloating that can occur over the course of up to two days from these undigested particles entering your body, especially if you have leaky gut syndrome.
Can you become lactose intolerant all of a sudden? This is more common among older adults, but usually lactose intolerance is obvious from an earlier age.
How long do lactose intolerance symptoms last? The severity of lactose intolerance symptoms depends upon personal tolerations and the amount consumed. If you’re intolerant and continue consuming lactose without making any other changes, your symptoms will likely persist.

Lactose Intolerance Treatment & Diet

There is currently no permanent cure for lactose intolerance because no treatment can increase the amount of lactase your small intestine makes. However, there are steps to take to manage symptoms and avoid complications. One major concern for people who have lactose intolerance is they may not get enough of the essential nutrients found in milk products, including calcium, magnesium, vitamin D and vitamin K, for example.
While it’s an option to take dietary supplements called lactase products that help digest lactose, this will not solve the underlying problem and may not be a good long-term solution.
What foods should you avoid if you are lactose intolerant? Do you necessarily need to give up all dairy?
Depending on the severity of your intolerance, it may be necessary to take a break from dairy while you heal; however, by following a healthy lactose intolerance diet, it may not have to be a permanent sacrifice.
Some people with severe lactose intolerance will need to avoid having most or all dairy products. Others can tolerate certain kinds without experiencing a flare-up of lactose intolerance symptoms. For example, some research suggests that many people with lactose intolerance can have up to 12 grams of lactose, the amount in about 1 cup of milk, without triggering any strong symptoms. Some experts also believe that one key to consuming dairy products while eating a lactose intolerance diet is to choose raw and unpasteurized products made from raw cow, goat and sheep milk.
A study published in the Journal of the Dietetic Association indicates that consuming kefir improves lactose digestion and tolerance. Participants in the study perceived a reduction in the severity of gas by 54 to 71 percent. While kefir is a dairy product, the fermentation process breaks down the naturally occurring lactose, making it easier for the body to digest and absorb it. The result is that the majority of individuals with lactose intolerance can still enjoy some types of dairy, while reaping the health benefits.
If you need to avoid all lactose, keep a careful eye out for dairy derivatives that hide in common foods — including bread, pastries, crackers, cereals, soups, processed meats, protein bars and candy. Look at ingredient labels and avoid these foods as much as necessary to control your symptoms:

Milk
Cream
Butter
Evaporated milk
Condensed milk
Dried milk
Powdered milk
Milk solids
Margarine
Cheese
Whey
Curds

There is no FDA definition for the terms “lactose-free” or “lactose-reduced.” Even products advertised as “non-dairy” could contain trace amounts of dairy products that can lead to the disrupting symptoms of lactose intolerance. Additionally, healthy natural foods that you have eaten for years may be at the root of your lactose intolerance. When transitioning to a lactose intolerance diet, it is important to carefully read the labels of all processed foods to ensure dairy products aren’t lurking.
Ideally, the best dairy products to consume if you have lactose intolerance are the types made from raw cow or goat’s milk that have been fermented for a minimum of 24 hours.

Raw milk myths continue to cause controversy; however, many of the claims of illness are greatly exaggerated. It is estimated that raw milk is responsible for less than 50 cases of food-borne illnesses each year, while nearly 10 million Americans regularly consume raw milk.
Raw milk benefits include immune system support, healthy skin, hair and nails, increased bone density, weight loss, muscle development and neurological support.
Raw milk is beneficial because the pasteurization process dramatically reduces essential nutrients, including vitamins A, C, E and B as well as minerals such as iron, zinc and, of course, calcium. The natural enzymes that help our bodies digest dairy products are destroyed while the protein and immunoglobulin’s are damaged.

Below are additional steps to take to help manage lactose intolerance:

1. Use Organic Fermented Dairy

Fermented dairy improves the digestibility of the lactose, fats and protein in dairy, but also helps to spur healthy digestion of other foods. While the idea of drinking fermented dairy may be off-putting to some, high-quality, organic kefir is slightly tangy, creamy and ultimately satisfying.
It is similar to yogurt, just thinner and drinkable. Probiotic foods are rich in vitamins, minerals and essential amino acids. Kefir contains high levels of thiamin, B12, folate and the secret bone-builder, vitamin K.
Vitamin K2 specifically helps calcium to metabolize, creating stronger bones, which is essential to people on a lactose intolerance diet. Organic fermented dairy also helps to increase magnesium levels. Magnesium deficiency is common in people with digestive tract disorders, including celiac and Crohn’s disease and IBS … as well as lactose intolerance.
You may choose to eliminate all dairy products for a time to help reduce symptoms and help your body heal, but ideally you can begin to swap out regular dairy for fermented dairy, which can help to restore the health of the digestive tract and has enzymes that will actually aid in digestion.

2. Try Goat Milk

For many people, goat milk may be easier on the digestive system than cow milk. Goat milk is high in fatty acids, and it is more easily absorbed and assimilated in the body. The actual fat particles in goat milk are smaller and contain lower concentrations of lactose.
It takes significantly shorter time to digest goat milk products than it does cow milk products. And yet, goat milk is richer in calcium, phosphorus, iodine, potassium, biotin and pantothenic acid. In addition, its casein levels are reduced, making it friendly to those with casein sensitivity.

3. Take Digestive Enzymes That Contain Lactase

Lactase is the enzyme that is lacking in the digestive tract for individuals suffering from lactose intolerance. According to a study published in the Alternative Medicine Review, digestive enzyme supplementation can aid in the breakdown of fats, carbs and proteins, assisting in efficient digestive function
Taking specially formulated digestive supplements provide a safe treatment for digestive malabsorption disorders, including lactose intolerance.
Take a digestive enzyme at the beginning of each meal, to ensure that foods are fully digested. This also helps to decrease the probability that partially digested foods including proteins, fats and carbohydrates will sit in the gut.

4. Supplement with Probiotics

This is an essential part of a lactose intolerance diet. The live or active cultures in yogurt, kefir, fermented vegetables and supplements help to maintain a healthy digestive tract. Increasing healthy bacteria in your gut may help to spur greater lactase production, or at the very least, aid in digestion.
By adding probiotic supplements and probiotic-rich foods to your diet, you can change the balance in the gut, leading to greater nutrient absorption. Managing lactose intolerance with yogurt and probiotics is possible, according to a study published in the Journal of Applied Microbiology.
However, probiotic supplements can do significantly more for overall health and wellness than just gut health. In fact, according to a study published in Science Daily lead by Dr. Collin Hill from the University of College Cork in Ireland, probiotics may be used in the future to help control disease, without relying on antibiotics.
It is important to look for a supplement that contain probiotics plus prebiotics derived from heat resistant soil-based organisms.

5. Incorporate Calcium-Rich Foods

While calcium is often considered a powerful mineral in the fight against osteoporosis, it is much more vital to our health than just our bones. In fact, calcium-rich foods help promote heart health and manage body weight. Calcium rich foods, which everyone should incorporate in their lactose intolerance diet include raw milk, yogurt, kefir, dark greens like cooked kale, raw cheese, sardines and broccoli.

6. Add Foods Rich in Vitamin K

As mentioned above, vitamin K plays a major role in calcium absorption and bone health, but its benefits do not end there. It also helps promote brain functioning and improve insulin sensitivity. This fat-soluble vitamin is stored in the liver, and proper levels can be disrupted by antibiotic use, certain prescription cholesterol medications and IBS and leaky gut. Many people who are lactose intolerant are also vitamin K deficient, so it is important to make sure you are getting enough in your daily food routine.
Foods rich in vitamin K to add to your lactose intolerance diet include green leafy vegetables, scallions, Brussels sprouts, cabbage, broccoli, cucumbers and dried basil. In addition, fermented, organic dairy is also rich with this essential vitamin.

7. Add Bone Broth to Your Diet

Central to helping restore the gut is bone broth. This simple and tasty broth helps the body overcome food intolerances, sensitivities and even allergies, while improving joint health, boosting the immune system and reducing cellulite.
Long simmering of grass-fed beef bones or organic free-range chicken transforms the calcium, magnesium, phosphorus, sulfur and other minerals, making them easier to absorb. In addition, the natural collagen and gelatin found in the bones help to support the GI tract. Aim to consume 8 ounces to 12 ounces each day.

8. Jumpstart Your Gut Health with the GAPS Diet

The GAPS diet plan was designed by Dr. Campbell to help reduce inflammation, treat autoimmune diseases, support healthy neurological function and minimize digestive disorders. If you have experienced the symptoms of lactose intolerance for months, or years, you can jumpstart your transition by following this eating plan.
The foods consumed include many of those mentioned above, like raw fermented dairy, fruits and vegetables rich in vitamins and minerals, healthy nuts and beans, wild fish, grass-fed beef and free-range chicken.

9. Add Non-Dairy, Probiotic-Rich Foods to Your Diet

Probiotic-rich foods increase the overall health of the digestive system and can help ease common digestive upset symptoms (including poor nutrient absorption), strengthen the immune system, support weight loss and increase energy due to more vitamin B12 in the body.
Sauerkraut and kimchi are both made from fermented cabbage and other vegetables that are nutrient rich, and rich with enzymes that help digest foods. Probiotic drinks, including kvass and kombucha, are rich with healthy bacteria, which help with liver detoxification, along with coconut kefir.
Coconut kefir is easy to make at home with the same types of kefir grains used in dairy kefirs and is rich with the healthy bacteria found in organic fermented dairy products.

10. Use Coconut Oil for Cooking

Coconut oil is one of the most amazing foods on the planet, and is easily converted to energy in the body. In addition, it helps to improve digestion, burn fat, kill bad bacteria and fungus and regulate candida in the body. Coconut oil can be used for high-heat cooking, it can replace dairy in coffee and tea and it is easy to bake with. It helps to fight inflammation throughout the body, boost the immune system and even prevent bone loss. For individuals that are limiting their traditional dairy intake, coconut oil should be included in their diet.

11. Substitute Ghee for Butter

Ghee has been used for thousands of years to improve digestion function, reduce inflammation, support weight loss, strengthen bones and so much more. But the most important factor for individuals with lactose intolerance — ghee contains only trace amounts of lactose that most aren’t likely to react to. The long simmering process and skimming of the butter removes most lactose and casein, so individuals with sensitivity or allergies to dairy products should try ghee. In addition, when created from milk from grass-fed cows, levels of conjugated linoleic acid or CLA, are double or triple that of traditional grain-fed cows.
Ghee is versatile and can be used for everything from high-heat cooking to “buttering” toast. Like coconut oil, ghee is part of my healing foods diet.

Final Thoughts

Lactose intolerance is a condition in which you have digestive symptoms — such as bloating, diarrhea and gas — after you consume foods or drinks that contain lactose, including milk and dairy products.
Symptoms of lactose intolerance include bloating, gas, stomach pains, diarrhea and sometimes other issues like headaches. They usually start within about 30 minutes to two hours after consuming lactose.
Lactose intolerance is caused by factors including genetics, aging, eating a diet that contributes to leaky gut, illnesses and stress
Lactose intolerance treatment typically involves following an elimination diet to avoid dairy and addressing underlying causes. It’s important to read labels carefully and look out for all types of dairy milk, lactose, whey, curds, milk by-products, dry milk solids and non-fat dry milk powders.

The Best Milk Alternatives, According to a Dietitian
Plant-based alternatives to cow’s milk have been a thing for a while now, and the benefit of this trend is that there’s something for everyone. Whether you’re lactose-free, vegan, or allergic to tree nuts, soy, or coconut, grocery stores in 2019 definitely have something to suit your needs. But how are you supposed to navigate the growing non-dairy aisle? And are these milk alternatives any healthier than cow’s milk?
When it comes to nutrients, commercially available versions of nut, seed, and legume-based milks contain varying amounts of protein and fiber (usually 1-4g each per 1-cup serving) since they’re made by blending the predominant ingredient with water. They can contain around 100 calories or less depending on the ratio.
Consuming fewer calories can be a good thing if you’re guzzling iced coffee with unsweetened almond milk all day, but may be less desirable if you’re looking for a nutritious addition to your morning bowl of steel-cut oats. Many of these alternative milks also cost more than dairy milk, but they may be worth the spend depending on how often and where you’re using them.
As a registered dietitian, my main piece of advice is this: Don’t be swayed by trendy marketing claims on packaging. Depending on the brand and type, these milk substitutes can be sneaky sources of added sugar, so it’s crucial to check labels before you swap out a dairy-based option for a plant-based one. Here’s everything else you should be scanning the label for.

What to Look for in Alternative Milks

• At least 7-8g protein per serving
• As few ingredients as possible
• The word “unsweetened” and “0g added sugar”
• Limited saturated fat (especially in ones made with coconut or added protein)
• Less than 140mg of sodium per cup
• Fortification with calcium and vitamin D
• Nutrients you’re personally concerned about (like the omega-3’s)
The best milk alternatives are typically unsweetened soy or pea-based blends (yep, you read that right!) that are fortified with calcium and vitamin D. These two nutrients are better absorbed when consumed together and are beneficial for strong bones, hormone regulation, and general immunity — especially for your little ones. If you’re adhering to a vegan diet, you’ll want to look for blends that have vitamin B12, vitamin A, and DHA/EPA omega-3’s, as well. Be sure to skip “barista blend” milk alternatives. They’re better for frothing but often come with loads of added sugar. Instead, spice up a hot beverage with vanilla, clove, or a cinnamon stick.
Keep reading to get the low-down on the best plant-based milks you can buy (and order):

1. Soy

Arguably the OG milk substitute (and the most nutritionally similar to dairy milk), unsweetened soy packs about 80 calories per cup with 8g plant-based protein from soybeans. Soy milk is made by soaking and blending these little beans and straining out the leftover pulp before consuming. Filled with antioxidants and fiber, soy alternatives are often super nutritious and provide key polyunsaturated fats.

2. Pea Milk

A new kid on the block in the world of plant-based milks, pea milk is made from pea protein isolate, water, and other emulsifiers like algal oil, sunflower oil, and guar and gellan gums. It’s as creamy as soy with a slightly less nutty taste for 70 calories per cup. The use of algal oil provides DHA, a key omega-3 fatty acid that’s linked to immunity, heart health, and cognition. The unsweetened versions pack up to 8g protein from a nutrient-dense source.

3. Coconut Milk

Coconut milk is made from water and coconut cream (VitaCoco’s uses coconut water, too), so it has a tropical taste compared to other plant-based milks. Nutritionally, coconut milk is higher in fat and lower in carbohydrates than nut- or grain-based milks. Most of the calories come from saturated fat — just one cup has up to 4g, which is 20% of your daily value. That said, the creamy consistency and fat content help boost satiety, so you’re likely to use less of it — especially if you’re adding it to coffee and tea.

4. Oat Milk

The trendiest blend of the bunch, oat milk is a creamy, lightly flavored addition to coffee, tea, cereal or a homemade smoothie. It contains added fiber, which may make it more filling than other alternative milks, according to early research. That said, oat milk is lower in protein than non-fat cow’s milk or soy-based versions (2-4g versus 8g per cup). It also has slightly more calories than unsweetened almond milk, which can add up if you swig it frequently.

5 Almond Milk

Most commercial almond milks range between 35-90 calories per cup and there are loads of blends and unsweetened versions to choose from. They’re mostly made from almonds and water, plus other emulsifiers and fortifying nutrients. The lower-cal versions give you about 1g each of protein and fiber per serving (though Elmhurst’s blend is about 5g protein). The low protein content is something to keep in mind if you’re using almond milk as a dairy swap in homemade smoothies — you may want to pump up the protein by adding nut butter or chopped nuts.

6. Cashew Milk

Cashew milk is particularly tasty in tea or homemade tea lattes. Try it with matcha for a little midday boost of L-theanine, a compound found in matcha which is linked to cognition and focus. Cashew milk is made the same way as almond (soaking, blending with water, and straining) and is similar in nutritional composition, ranging from around 40-50 calories per cup. Cashews themselves provide zinc, copper and magnesium, which help support your immune system. The real difference between cashew and almond milk? The flavor! Go with whichever you prefer, so long as you’re choosing an unsweetened version.

7. Peanut Milk

Peanuts are like the crown jewel of foods, since they’re good for both you and the planet. They have a similar taste and nutrient profile as tree nuts (almonds, walnuts, cashews, and hazelnuts), but these legumes actually grow underground and use way less water. The best thing about peanut-based products is they’re often more cost-effective than other dairy alternatives and are higher in protein (plus, they have a creamy taste and texture).

8. Flax Milk

At 70 calories per cup, flax milk contains a little more than meets the eye. Most store-bought versions are made from a combination of water, flaxseed oil, and pea protein, which makes it similar in nutrient composition to pea milk. The alpha-linolenic acid found in flax also helps support immunity and has been linked to reducing the risk of heart disease.

9. Hemp Milk

Out of all of the hemp products currently on the market, hemp milk is a solid choice from a nutritional POV. It’s made by blending hulled hemp seeds with water, and packs magnesium, calcium, and vitamin D, depending on fortification. You’ll also get omega-3 and omega-6 fatty acids — essential nutrients for your immune system and cognition — plus about 3g of protein at 60 calories per cup. However, there’s very little fiber in hemp milk compared to ones made from other seeds. The main objection to hemp milk is the taste: Its ultra-nutty flavor can be bitter to some, especially if you’re used to the sweeter notes of other plant-based alternatives.

10. Rice Milk

Rice milk is made by blending rice with water. It’s often lower in calories than other milk alternatives in its unsweetened form, but since the flavor is very mild, most versions contain added sugar. You may be better off with an alternative grain blend unless you’re avoiding nuts, seeds, or legumes because of an allergy.

11. Walnut Milk

Walnut milk is a top pick if you’re looking to boost your intake of plant-based omega-3’s. It tastes a little more earthy than other types of milks and packs 3g of plant-based protein for 120 calories. Use it in tea or coffee to shake up your morning routine, or in smoothies to balance out sweet-tasting fruits.
Source: http://www.hiwamag.com/health/7-symptoms-lactose-intolerance-plus-11-tips-manage/

[OC] Corridors - Chapter 23: Schism (Part 2)

This is Part 2! You can find Part 1 here
“Alright, Wrixea, but I hope you have a good plan!” As if in response, clouds of Kredith Worker ships started streaming out of the remaining Hiveseeds in solar orbit, rotating their small chassis to orient their double-helical points in the direction of the incoming Forsaken vessels. Pools of light gathered behind them as they powered up their interstellar engines and hurled themselves past the Forsaken fleet. “Where are they going?” Alan asked incredulously. He scattered a dozen probes into the Ekres star below, unleashing a furious maelstrom of ravenous flames that consumed almost a fifth of the Forsaken ships in orbit over Ekres IV.
“What the fuck are those?” he asked aloud as his tactical overlay fluttered rapidly with thousands and thousands of small detonations, occuring just behind the Forsaken fleet around the star. Alan frowned as icons denoting navigational hazards filled the screen, carpeting the space behind the Forsaken forces. Another swarm of Kredith worker ships raced past his Blinkship, trailing streams of light in their wakes as followed their brethren into superspace. Detonations appeared on his overlay again, quickly replaced by increasing numbers of hazard icons. “Wrixea, what are those worker ships doing?”
Several purple explosions suddenly bloomed in the distance as Forsaken Dreadnoughts dropped into normal space in several large pieces which immediately ignited in quick succession. Voidblades spun into existence with severed wings and large gashes carved in their hulls, instantly exploding upon rematerialization. Alan widened his eyes in shock as he realized what was happening, “Wrixea, your worker ships are self-destructing in superspace!?”
“Correct, Pilot Radisson. Their overloaded interstellar engines will create temporary ruptures in superspace, preventing the Forsaken from utilizing their intended trajectories. The enemy will be destroyed as they fly through the ruptures, and we will easily reassert our claim over the Ekres Star as they mindlessly destroy themselves.” Colonykeeper Wrixea gnashed her mandibles triumphantly, waving her antennae in excitement, “They will need to re-calculate their approach vectors to compensate, but we will constantly saturate their new courses with more superspace ruptures!”
Alan watched as another group of Dreadnoughts dropped into normal space, spinning chaotically into each other and exploding. Purple fire threw itself across the tainted space, continuously flaring up as already-destroyed Forsaken vessels arrived and summarily shattered. “Are you sure this is the right thing to do, Wrixea? Are the Mindweavers sure about this?”
“To control the enemy’s movements is to control their fate! The Mindweavers learned this from you, Pilot Radisson.” Colonykeeper Wrixea proclaimed with pride. She flaired her wings emphatically, “And because of your tutelage, we shall be victorious today!”
Alan couldn’t argue with the efficacy of their new battleplan, but the way the Kredith cavalierly spent the lives of their workers gnawed at him. “But your workers… You’ve already sent over fifty thousand Kredith to their deaths, just to close off one approach vector.”
“Do not worry about the Worker drones. Their purpose is to serve the Kredith Dominion and ensure the survival of the Hiveseeds. And in this way, they have succeeded.” Colonykeeper Wrixea dismissed with a wave of her upper left insectoid limb, “New worker bodies will be rebuilt, and we will infuse them with the minds lost in these maneuvers. Nothing of value is lost, save for a small amount of biomass.”
The Hiveseeds in solar orbit fired their rivers of ion bursts with renewed vigor, pummeling what remained of the Forsaken fleet into charred and burning twisted metal. Shadowspikes ignited, vainly announcing their demise with weak flares as the Hiveseeds buried them with endless torrential ion fire. Without being harried by the Shadowspikes, the Blinkships closed in on Ekres again, littering the star’s surface with Pathfinder Probes to transport its rage to their chosen targets. Sunbursts ignited over the skies of both Ekres IV and V, annihilating the Forsaken vessels in orbit with dancing flames. Onathin Nestships poured an onslaught of photon lances into the dark fleets, slicing white-hot incisions into their black hulls and spilling their insides into the vacuum of space. Purple flares peppered the battlespace, dwarfed by sudden brilliant orbs of light as Pathfinder Probes endlessly ejected coronal mass into the Forsaken hordes.
Flockleader Wiksen chirped into the open communications channel, “Half of my forces have been destroyed, but it appears that the Forsaken numbers are thinning out. Perhaps they have reconsidered their invasion plans, in light of your recent adaptations.”
“Good, because we’re running out of Pathfinder Probes.” Alan warned through gritted teeth as he narrowly dodged a whiplash of curling flame from the star’s surface, “If they don’t let up soon –” His tactical overlay suddenly blared at him as the red signatures seemed to rotate in place, before streaming out of the Ekres Star System. “They’re retreating!”
“A victory well-earned and fought!” Colonykeeper Wrixea flapped her wings and screeched.
“I thought they had a lot more ships they could throw at us?” Alan asked, “But I’m glad they decided to retreat. It gives us time to get resupplied, and maybe our capital ship can be here for the next battle!”
Flockleader Wiksen’s feathered face suddenly appeared on the viewscreen. Singed feathers stuck out awkwardly all over his wings, and lines of green throbbed throughout his feathers from adrenaline. Both pairs of eyes drooped in stress and fatigue, and held a heavy despair within them, “You are correct, Alan Radisson. The Forsaken do have more ships, and they are sending them from their Voidbase directly to the Orkina System.”
“What?!” Alan shouted as he pulled up the long range telemetry.
“This invasion was a farce, meant to pin our forces in Ekres while they begin the assault unhindered in Orkina. We suspected they had the range necessary to reach Orkina, but with insufficient data, and the fact that the defense of Ekres was paramount…” Wiksen covered his face with a wing and shuddered, “I’ve doomed my people to defend others!”
“We shall mobilize all of our forces immediately to defend Orkina System!” Colonykeeper Wrixea exclaimed.
“Doing so would leave your Mindweavers vulnerable!” Flockleader Wiksen jabbed his talons at a screen, “Even after the success of this battle, the Forsaken have enough ships to triple the entire allied fleet! The ships left behind at their Voidbase doubles the fleet sent to attack Orkina! If we leave Orkina, they will come here and destroy the Mindweavers. There is nothing we can do to help Orkina.”
“Then we shall leave Ekres! We will not be able to reach Orkina in time to mount a successful defense, but perhaps if we rally at the Henfir System, like General Davis has suggested, we can resist further Forsaken incursion into the Onathin Sovereignty!” Colonykeeper Wrixea proposed.
“Abandon Ekres? But your Mindweavers may die from being uprooted yet again.” Flockleader Wiksen flapped his wings, “And we cannot accommodate them in Henfir.”
“They will adapt!” Wrixea insisted, “We cannot allow Onathins to suffer at the hands of the Forsaken like the Kredith already have. The Mindweavers will not tolerate injustices inflicted upon a people that have defended us to such a large extent! Abandoning Ekres is trivial. The Kredith Dominion is not made of worlds. As long as we have the Mindweavers, everything can be rebuilt and replaced! In any case, the defense of Ekres is untenable in the longer term if we are severed from the Onathin Sovereignty!”
“Well said, Colonykeeper,” Alan agreed. “I’m ordering my Blinkships to launch probes to Henfir now. I just hope we get there in time.”
The brightly lit, pristine corridors and hallways of the North American Branch of Earth Council contrasted sharply with Ambassador Evans’s mood as he made his way past them. His usual hopeful outlook on life was marred by the current state of affairs. Although Ekres had repelled the Forsaken incursion, the situation in the Onathin Sovereignty was still spiralling wildly out of control. If he were to be of any help, he’d have to start forging stronger links with the outlying Onathin systems. Months and months of Pathfinder Probe-accelerated trade amongst these systems, and with Earth, gave the Onathins in those systems an immense appreciation for humans. Perhaps there is a way I could secure these economic ties further, and form some sort of mutual defense pact? That might take some of the pressure off of Prelate Iwardion’s back. Rubbing his eyes in fatigue, Tyler retrieved his tablet from his pocket and ordered a taxi-drone to deliver him to the Vancouver Space Elevator.
His eyebrow twitched in confusion as threads of colour wafted idly throughout the hallway. Tyler curiously approached the source of the Drikenyl song and realized it was coming from Tara Yang’s infirmary. He stepped through the threshold and waved at the pair of Drikenyl who lazily floated in the water-filled observation port located on the wall directly opposite of the entrance. They twirled their whiskers in response and continued to sing, casting shimmering waves of blue and green into the room.
“Hello Tyler,” Tara Yang greeted as she noticed him wander into her infirmary, “Are you injured?” She walked over and automatically began examining him with her practiced eyes.
“No, I’m fine.” He recognized both of the Drikenyl floating beyond the glass wall. One of them had much more luxuriously reflective scales, and was the Drikenyl that they had picked up from Sechalla Station. It was the first Drikenyl ever to arrive on Earth, a fact that it liked to show off by flashing its nourished scales ostentatiously. The other Drikenyl had been singing to him when he had woken up in Tara’s infirmary a couple of weeks ago. “What about them? Are they infected with that bacteria that’s been spreading amongst the Pilgrim Drikenyl?”
“No, they’re OK. They’ve got a very effective quarantine system set up above the Salish Sea, isolating infected Drikenyl into those levitating spheres of water.” Tara took out her tablet, reading a notification that had just popped up, “I’m not sure why they’re still here, but I’m actually very close to perfecting an antibiotic that’ll be effective against it.”
“That’s good to hear, Tara.” Ambassador Evans said as his eyes wandered throughout the room. Hues of blue and green soaked through the glass window-wall and wrapped themselves around the infirmary beds, surgical armatures, Onathin laboratory equipment, before entwining around the people within the room. At the far end of the infirmary, Derek was inspecting a piece of Drikenyl hide. Rainbows flashed across his face as he fiddled with the iridescent scales. He reached up with a free hand and rapidly scratched the side of his head.
“Derek, don’t scratch like that! You’re going to inflame your scalp.” Tara chided from her lab bench, “Anyway, the bacteria infecting the Drikenyl is partly of Earth origin. That bacterial conjugation image that you keep getting from the Drikenyl is probably what happened between two bacteria, one of Drikenyl origin, and one of Earth origin, that were both benign. But together, they produced a pathogenic progeny.”
“Wait, so two usually good bacteria mated and produced bad bacteria?” Tyler clarified with raised eyebrows, “Does that happen a lot? And what are the chances that Drikenyl bacteria could mate with Earth bacteria?”
“I guess I was a little imprecise. The ‘Drikenyl bacteria’ was also originally from Earth, but changed and evolved inside the Drikenyl gastrointestinal tract due to their unique biochemical environment. Then, it randomly conjugated with another Earthborne bacteria found in the ocean. It’s a random process, and almost impossible to predict.” Tara placed the tablet down on a nearby desk and stretched while yawning, “I’m actually quite surprised at my progress! I expected this to take a lot longer, but ideas just kept popping into my head.” Tyler eyed the wisps of colour that misted around her head and smirked, “I guess your muse was working overtime.” He waved at Cerion in a corner of the lab, who waved a wing in response.
“Ambassador Evans, it is good to see you again. Have you heard any news regarding the Tymin System and my parents?” Cerion asked as she scratched at the polymer interface of the Onathin machine in front of her. A small whir seeped into the room as a spherical, glass component of the machine began to spin, casting ripples through the blue-green aura that filled the room.
“I’m sorry, Cerion. All communication with that part of the Sovereignty has been severed since Vyndres and Trennor seceded.” Ambassador Evans replied sadly, “We’re working on getting corridors established with those outlying systems, so we’ll have more news soon.”
Cerion’s crest feathers deflated, but she nodded and blinked her thanks, “Please inform me when you have any news. My parents are all I have left.”
“Absolutely.” Tyler waved at the Onathin lab equipment, eager to change the subject, “and how has the cure for the neural parasite been proceeding?”
“We’ve actually put that on hold for now.” Tara answered, “We’re still working on the parasite, but we’ve decided to focus our efforts on disrupting the neural network that the parasite forms in the Stalwart Claw hosts’ brains. If we can disrupt the network, or suppress the parasite’s ability to form these networks, I believe we can destroy its mind control abilities.”
“So you’re starting over from scratch?” Tyler asked.
“Actually, we are much, much closer to a network disruptant than an outright cure!” Tara said with excitement flashing in her eyes, “On Gorandis, when were performing high-throughput drug screening on the neural parasite, we couldn’t find any drugs that could kill the parasite in a dose that’s tolerable to Onathins. However, we found many drugs that caused the parasite to retract its pseudopodia!” “False feet?” Tyler quirked an eyebrow.
“Exactly!” Tara continued, “The parasites extend appendage structures from the rest of their body called pseudopodia, which they then use to bind with other parasites and form plaques in the brain. They also use these appendages to anchor themselves into the Onathin brain and influence neural patterns. I think that the Stalwart Claw strain is able to extend their pseudopodia to much longer lengths than the other strains, which allows them to build this neural network and gain complete neural control of the host!”
“Because of our efforts on Gorandis, we have plenty of candidates to test for their network disruption capabilities,” Cerion echoed, “And while a cure is still preferable, this may be the fastest and most time-efficient way to restore stability to my civilization. If the Stalwart Claws were to suddenly awaken from parasitic control, they would see the error of their ways and strive to unite the Sovereignty once more!”
“I hope you’re right.” Ambassador Evans rubbed his eyes again, “The situation is getting a lot worse. The Finsen Star System just voted to secede from the Sovereignty, and there’s been numerous protests and demonstrations on Onathi itself. Prelate Iwardion has invoked the Sovereignty Security Provision, bypassing some universal rights and instituting a Sovereignty-wide detainment of any Stalwart Claw affiliated Onathin,” He sighed, “Several analysts are predicting that a coup attempt on the Onathin Homeworld is likely in the next few weeks.”
Tara pursed her lips in worry, “I’m not sure if we will have something before then.”
“Is there something I can get you from the Onathin Sovereignty? Something that I could ask Steward Gredion to ship over from Sechalla Station that might speed up the project?”
Tara and Cerion exchanged glances, “Well, we need test subjects.”
“Test subjects?”
“At least samples of the parasite spore, and they have to be that specific strain of parasite that’s infecting the Stalwart Claws. We need the strain that’s forming the neural network and controlling their minds, so we can test our drugs for their neural network disruption efficacy.”
Ambassador Evans shook his head, “I’m not sure I can provide that. Maybe Steward Gredion arrested a saboteur aboard Sechalla Station that might carry the right type of parasite that you need. Diplomat Pellon might be better suited to acquiring Stalwart Claw prisoners, but…” he looked down at his tablet in worry, “I haven’t been able to contact him for the past couple of weeks. He…was in the Brildin Nexus Relay when the Stalwart Claws attacked.”
“I’m sure he’s fine, Tyler.” Tara said quickly, “He is a very resourceful fellow. He must have found a way to survive.”
“We’ll see.” Tyler glanced at the tablet and noticed that the taxi-drone he ordered was approaching the building, “If I see him, I’ll let him know what you need.”
“Where are you going?” Tara asked, eyeing Tyler’s tablet, “Not into Sovereignty Space?! It’s pretty dangerous for any human, ambassador or not!”
“I’m heading off to all of the outlying systems that we’ve been trading with. Don’t worry, none of them are in any danger of secession, and they all have very positive attitudes towards humans. I’ll be fine.”
“Still, be careful.” Worry lines etched across her face as Tara looked at Tyler, then at Derek, “I don’t want to lose another person I care about.”
Derek, oblivious to Tara’s worried stare, brandished his omni-tool and jabbed at the Drikenyl hide. With a soft plink sound, the nanite tool glanced off the scales and embedded itself into the table underneath. He frowned, and stood up straight as if to get a better view of the situation from above. A ping sounded from Tara’s tablet, sending ripples through the blue-green strands that stretched throughout the room. Tyler stared at the tinted filaments around Derek's head, wondering just what exactly the Drikenyl were doing, when Tara suddenly gasped.
“Cerion! Tyler!” She said excitedly, “The aerosol sample contains the strain of the parasite that we need!”
“What aerosol sample?” Tyler asked confusedly.
“Prelate Iwardion sent over a sample of an aerosol that the Stalwart Claws were disseminating on several core worlds, as well as a dozen outlying systems. It arrived a few days ago, and my colleagues at the European Branch of Earth Council have identified it as a compound that contains the specific parasite strain that infects the Stalwart Claws.” Tara scrolled through the data on her tablet, “It also contains several compounds that I’m not familiar with. The Europeans suspect that they’re bonding agents, but they’re not sure. Cerion, what do you think?”
The blue-feathered Onathin waddled over to Tara and peered her four eyes at the tablet. Two of them narrowed, “These compounds are derivatives of molecules commonly used to allow drugs to penetrate the primary airflow sieves that line the initial airway tract in Onathins. We use these compounds when we want patients to breathe in drugs that would immediately accumulate in the cranial vasculature.”
“What does that mean?”
“It means that the Stalwart Claws are trying to get other Onathins to breathe their specific parasite strain directly into their brains.” Tara answered grimly. “They’re poisoning their own people. Or rather, they’re trying to make more of themselves.”
“But I thought all Onathins were already infected with the neural parasite? How does this work?” Tyler asked as he stared at Tara’s tablet. Graphs and charts scrolled endlessly across the screen, unintelligible to him.
Tara nodded, “You’re right: all Onathins are already infected. But even so, the Stalwart Claw strain is undoubtedly more belligerent, and would eventually outcompete the parasites that are already present in the host.” She tapped a nearby monitor, showing the schematic of the neural mesh that the parasites form, “The neural network that the Stalwart Claw parasites build allow them to be hardier, enhance their rate of nutrient uptake, and to reproduce faster. Imagine a town. It’s easier for families to survive in their individual houses when there is a network of roads and streets to allow them to gather resources and remove wastes.” She jabbed at her tablet with a furious finger, “They’re trying to accelerate the spread of their strain with these aerosols. It’s all a part of their plan to take over the Sovereignty.”
“But herein lies our solution, Tara Yang!” Cerion chittered, “We now have a sample of Stalwart Claw parasites that they have supplemented to grow networks more rapidly. We can use these to test our formulations, and successfully devise a neural disruptant!”
Tara beamed at the both of them, “My thoughts exactly! Cerion, please spool up the zwitterionic stabilizer. We can start manufacturing Onathin drugs while we wait for the parasite sample to ship in from Europe.”
Tyler caught a faint rustle from the corner of his eye. A tear in the fabric of colour around them bounced around the room, distorting the otherwise smooth blue-green air. “What’s wrong?” he asked aloud as the Drikenyl started to flick their scales back and forth in agitation.
The enemy have advanced their forces. the First Drikenyl resonated.
Tyler’s tablet vibrated as it repeatedly sent pings crashing into the cerulean waves that were slowly receding from the room. He took it out under both Cerion and Tara’s stare, and deliberately flicked it on. A newscaster was shakily reading out the report in front of her, “Forsaken forces…have bypassed the Ekres blockade and have entered the neighbouring Orkina System. Onathin ships are scrambling to fend off the Forsaken, but there’s…there’s just so many of them.”
The newscaster popped up on one of the screens that stretched across one of the laboratory walls, “Civilian ships that are already in space are quickly evacuating, but there are still hundreds of millions of Onathins on the surface of the planet!” Tyler winced as light flickered harshly from the screen. The feed from an orbital satellite above Orkina II replaced the newscaster’s stricken face, displaying hundreds of small, sleek Onathin ships streaking away from the planet. Rivers of light bloomed from behind their slender silver bodies as they spooled up their interstellar engines and raced out of the system. Dark crescents loomed into view from the edge of the feed, reaching out with maroon lasers, hoping to slice through the evacuating transports. One of the purple beams carved across the left side of the screen, and suddenly the feed went blank. The newscaster quickly rematerialized, looking slightly more collected than before, “Reports are still coming in, but it seems that the Forsaken have amassed a fleet of at least a thousand ships to assault the Orkina System. Half of the spaceports along the western continent are still under Stalwart Claw control, and they have not lifted their launch restrictions, and are greatly limiting the rate of evacuation! We’ll have more for you after a few moments.” The newsfeed ended abruptly.
Bright green lines traced along Cerion’s feathered face as she huffed and puffed rapidly. “It…it can’t be.” She squawked involuntarily as she stumbled into a nearby chair, “I knew…I knew the Forsaken were close, and that their invasion into Sovereignty Space was a very likely possibility. I knew that it would happen. But seeing the incursion happen…I am unsure if it was ever possible to sufficiently prepare for this eventuality.” Her wings flapped subconsciously, scattering feathers across the lab equipment, and even propelling down onto the beds across the room. “What will become of my people?” she asked in quiet desolation.
Tara walked over and knelt before Cerion, studying her pulsating blood vessels. She reached up and placed a hand on her wing, “Cerion, I’m sorry that the Forsaken have invaded the Sovereignty. But we still have a lot of work to do. Now, more than ever, we need to destroy the neural parasite and re-unify your people. We still have time, but not much.” Cerion nodded feebly as Tara continued, “We must not lose hope, and we can’t give into despair. Together, we can still save lives.”
Tyler looked down at his tablet and noticed that it was still streaming a feed from the battle over Orkina II. Apparently, another orbital satellite had taken over, showing an Onathin fleet firing photons frantically at a Forsaken armada that was twice its size. The Nestships and Predator cruisers had been scattered around the planet, providing orbital support for the Sovereignty security forces battling the rebel Stalwart Claws on the surface. He raised an eyebrow, realizing that the feed was streaming specifically to his tablet, and was not being broadcasted across a newsnet. Flashing text near the bottom confirmed his suspicion.

AMBASSADORIAL ACCESS KEYS AUTHENTICATED! STREAMING…

He watched as the orbital defense satellites spat out a phalanx of light, incinerating dozens of Voidblades and Dreadnoughts. The scattered Onathin ships had finally grouped themselves into a formation between the Forsaken fleet and the planet, defiantly throwing spears of light into the oncoming horde. Dreadnoughts exploded, replaced by even more Dreadnoughts as the Forsaken armada pressed in. Dark red plasma, underscored by purple beams of light, pummeled the Onathin ships and the orbital defense network, showering the planet below with spiralling silver wreckage.
Tyler clenched his fist and shoved the tablet back into his pocket. I guess now would be the perfect time to fulfill General Davis’s request. He strode over to the Drikenyl observation port, dimly aware of Tara’s continued condolences and attempts to coerce Cerion back to work. The pair of Drikenyl had long ceased their song and stared mournfully back at Ambassador Evans as he approached. “I want to talk to the Hierarch in my office.”
The First Drikenyl twirled its whiskers and flashed blue, As you wish, Ambassador. It flaired its wingfins, flashing brilliance once more into the room, and whirled away.
Nodding at the other Drikenyl, Ambassador Evans turned on his heel and stepped purposefully towards the exit. He passed Tara and tapped her on the shoulder, “Let me know when you have something.”
She nodded as well, and turned back to Cerion. The blue Onathin was visibly shaking, but seemed to be taking measures to calm herself. Once again, the bright hallways that playfully threw his footsteps back and forth contrasted with his dreary mood. Tyler took a deep breath as he approached the elevators and strode through the threshold. His tablet cried for his attention, insisting that he see the battle of Orkina II unfold. He ignored it, instead weaving words and sentences together in his mind, preparing for his meeting with the Hierarch. It would be the first time that he would meet the leader of the Drikenyl Warship fleet, and he was unsure of how cooperative the Hierarch would be.
His office doors whispered as he approached them, revealing a decently-sized chamber with monitors that stretched across two opposing walls. The far wall, directly opposite to the office doors, had been rebuilt with wide, thick glass panes reinforced with a thin metal net, and then connected to the Drikenyl access port that snaked upwards throughout the building. Water sat almost invisibly beyond the glass wall, surrounding a trio of Drikenyl. Two of them were hurriedly applying some sort of paste to the Drikenyl between them, tending to the many gashes, burns, skin tears, and broken scales that were strewn up and down its hide. The center Drikenyl focused all three of its eyes at Ambassador Evans, seemingly analyzing him as he walked towards the glass wall.
Ambassador Evans. I have heard of your many great deeds, and of your impressive capacity for kindness. It is an honour to finally meet the Saviour of my people. Several beveled metal rectangles, traced with pulsing blue lines, were affixed to the Drikenyl Hierarch around his eyes. Tyler surmised that they fed some sort of heads-up display to the Hierarch in times of battle, since he hadn’t ever seen any Drikenyl wear any sort of ornamentation for purely asthetic purposes. Small clear tubes, filled with orange liquid interdigitated with the Hierarch’s scales throughout its body, occasionally swelling with liquid or collapsing as it emptied its contents. The comparatively fragile wingfins were covered with an iridescent membrane, flexing with the wingfins as the Hierarch maneuvered closer to the wall. It waved its whiskers at the Drikenyl Healers, Your efforts are appreciated, and will suffice for now. Please leave us.
Very well, Hierarch. One of the Healers resonated as they fluttered back, stretched their wingfins, and whirled away.
Tyler watched the Healers disappear somewhere below the window-wall before meeting the Hierarch’s three eyes with his own, “I am humbled by the presence of a talented strategic thinker and leader such as yourself, Hierarch. No doubt, the journey to Earth was perilous with the Forsaken stalking the Pilgrim Fleet in the void.”
And it would have all been for naught, if you had not so prepared a sanctuary for my people. For that, you have my eternal gratitude. The Drikenyl flashed forest green while emitting a wave of hope that bubbled within Tyler’s chest, My apologies for our late meeting. Coordinating the disembarkation of the Pilgrims, re-energizing the fleet, and acclimating to the waters of Earth have all prevented me from seeking an audience sooner.
“I understand completely, Hierarch.” Ambassador Evans placed a hand on the glass, “And I do not want to take up too much of your time, but the situation grows desperate. I believe you are aware that the Forsaken have begun their incursion into the Onathin Sovereignty?”
Correct. It is sickening that the enemy has been able to continue their rampage towards the center of the galaxy, especially after the sacrifice of our Republic. The Hierarch rotated its top eye and inspected Ambassador Evans’s hand. After a brief pause, it mirrored the gesture by placing a forelimb against his hand through the glass. If the Enemy utilizes the same movements and strategies as they did in our war, you can expect many attacks to occur behind fortified frontier systems. They will draw your forces to the contested battleground systems, and send another fleet to attack the system behind it.
Ambassador Evans nodded, “That’s what they’re doing now. They have a Voidbase behind the Ekres stronghold system, and are currently attacking the lightly defended Orkina System.” He looked down at his tablet and sighed heavily, “Millions of Onathins are dying right now, because we were forced to fend off an attack on Ekres just hours before their attack on Orkina. There was no way we could have been able to defend both systems in such a short time span.”
This will continue to be their strategy for much of the war. They use their superior numbers and uncanny mobility to their great advantage. The Hierarch informed, The war between the Republic and the Forsaken stagnated when we adapted to their tactics. In battle, our fleets could sweep away tens of thousands of Forsaken vessels with ease. We developed technology to combat the massive numerical advantage that the Enemy enjoys, and deployed sizeable defensive fleets across many of our worlds instead of focusing solely on the frontier systems.
“The difference here is that we have neither the numbers nor the technology to defend ourselves effectively against the Forsaken. We have had success with our Pathfinder Probe weaponry, but there’s just not enough of it to hold back such a large invasion force.” Ambassador Evans thumbed on his tablet, dismissing the feed from the Orkina System battle and expanding a local starmap instead. He zoomed in the starmap on the Onathi-Henfir-Brildin-Orkina-Ekres starlane. An angry red boil seethed in the void between the Ekres and Orkina systems, indicating the Voidbase and its massive escort fleet, “The Forsaken have amassed thousands of ships here, enough to dwarf the entire allied fleet three times over. Our military analysts project that after they destroy Orkina, they are going to test the Ekres defenses again, while simultaneously attacking the Brildin System. They have enough ships to even launch an additional strike on the Finsen Star System, which is on a separate Onathin star lane. Bringing a large force to defend Finsen in such a short time frame would be infeasible, even if we had the ships to spare. And even if we were able to deal with all three Forsaken fleets, the Voidbase would still be in a position to launch further attacks.”
The situation is desperate, indeed. An inquisitive shade of yellow rippled down the Hierarch’s scales, I sense you have a request you wish to make, or you would not have demanded an urgent meeting.
Ambassador Evans pursed his lips, “You are correct, Hierarch. Believe me when I say that this is not an easy request for me to make.” He straightened himself to his full height, “As Hierarch, you have command of the Drikenyl Warships that are orbiting Earth, correct?”
For now. The structure of leadership is undergoing some discussion. In times past, it was necessary to separate the Civilian Senate from the Military Hierarchy, to maximize the efficiency of our war efforts while managing a large population base spread out amongst hundreds of stars. With neither a large nor widespread population, coupled with a diminished military force, there may be governmental reformation in the coming months.
“I see.” Ambassador Evans paused, before taking a deep breath, “But before that happens, I’d like to make a formal request for military support from the Drikenyl Military Hierarchy. We need your warships to repel the Forsaken invasion.” A heavy sigh escaped him, but he continued, “I’m reluctant to ask for your soldiers to go off and fight the Forsaken so soon after arriving on Earth, but there are so many innocent Onathins that are dying right now, and many more lives will be lost if we don’t do more. I’ve witnessed the power of your warships in several memory caches, and they would certainly even the odds in our encounters with the Forsaken.”
I sense the battle between your compassion and pragmatism, and I appreciate the fierce struggle against the Forsaken. Subtle shades of blue and yellow danced through the Hierarch’s scales. But we are so few. We need to rebuild our forces while the Onathins fight the Forsaken, just as the Onathins had amassed their forces while we resisted the Forsaken. The Hierarch curled its long body towards the glass, But we will contribute to the war by sharing our defensive technologies. I have been informed that our technicians are already working closely with your scientists, aiding their understanding of Shield Fluidics.
“With limited success.” Ambassador Evans remarked grimly, “Unfortunately, none of the scientists and engineers who are working on the shield projects can converse with the same depth and detail as you and I. The lead physicist has informed me that it would take at least another few years before we can even begin to think about building our own shield cores. We need this technology now.”
Then we shall disconnect the shield cores from our warships. We will install them all across your Homeworld and create an impenetrable planetary barrier.
Ambassador Evans paused again as he chose his next words carefully. He thumbed on his tablet again and watched the casualty numbers from the ongoing battle at Orkina swell onto the screen. “Doing that will definitely protect both of our species. But it would be the wrong course of action.” He flicked a finger across his tablet and threw the scrolling casualty report onto the window-wall between them, “While we hide behind the energy shield, our friends are being slaughtered by the Forsaken. We have to do more. We should always do all that we can to help.”
I have offered the shield cores of my warship fleet. We cannot engage a Forsaken Voidbase without our defenses, nor can our fourty-seven warships fend off an armada of ten thousand vessels. The Hierarch twitched its scales restlessly, What specific task would you ask of us? What is the extent of support that you require?
“You wouldn’t be engaging the Voidbase.” Ambassador Evans stared into the Hierarch’s eyes once more while words and phrases assembled and disassembled themselves in his mind. He flicked his fingers along his tablet again, this time sending a massive schematic onto the window-wall. Tyler pointed at the blueprints of the nearly-completed capital ship, “This is what we are building just outside of the Forge. It is a massive capital ship, one that we think might even be able to destroy a Forsaken Voidbase, provided that it is equipped with certain technologies.”
You wish to install our shield cores onto your new vessel. The Drikenyl wriggled closer to the schematics, studying them curiously while absently picking at a broken scale. Whiskers traced the large, sweeping rings of the human capital ship, while a wingfin brushed over the central spherical core that was protectively surrounded by a pair of hemispherical shells. Your design is…adequate. This can be done, with some modifications. But you cannot expect my warships to challenge the Enemy without their shield cores.
“I’m not.” Ambassador Evans smiled, “I’ve seen the conglomerate ship that carried your civilians to Earth. Every passenger transport within that conglomerate ship carries its own shield core. We can install those onto our capital ship instead.”
But that would leave our people unprotected. A timid shade of orange cascaded throughout the Hierarch’s scales. If there were to be an attack, we would require those shield cores to protect my people.
“Who would attack us?” Ambassador Evans asked, disguising his incredulous tone into the inflection of a genuine question, “The Kredith are bottled up in their last star system, and the Onathins are facing a civil war and the Forsaken incursion simultaneously. Neither humans nor Drikenyl would do anything to attack our common homeworld. There is nothing left to fear except the threat that the Forsaken pose, a threat that will surely eat its way across the Onathin Sovereignty if we don’t do more to oppose it.”
Subtle shades of blue shimmered along the Hierarch’s scales, slowly replacing the orange. It narrowed its bottom eyes and studied Ambassador Evans closely, as if staring directly into his mind, What drives you to help others so fervently?

what are the steps of bacterial conjugation video

Steps of bacterial conjugation: Step I: Pilus formation. Donor cell (F + cell) produces the sex pilus, which is a structure that projects out of the cell and begins contact with an F – (recipient) cell. Step II: physical contact between donor cell and recipient cell The process of bacterial conjugation is based on the principle that the plasmid or any other genetic material is transferred from the donor cell to the recipient cell through close physical contact. Of all the conjugative plasmids, the F (fertility) plasmid of E. coli was the first discovered and is one of the best-studied. Bacterial conjugation: a two-step mechanism for DNA transport the recipient cytoplasm. This is the ﬁrst step in con-jugation. The second step is the active pumping of the DNA to the recipient, using the already available T4SS transport conduit. It is proposed that this second step is catalysed by the coupling proteins. Bacterial conjugation is now realized to be one of the principal conduits for horizontal gene transfer (HGT) among microorganisms. The process is extremely widespread and can occur intra- and intergenerically as well as between kingdoms (bacteria to yeast or to plants). DNA sequence analysis has revealed that conjugation, and in some cases transformation, two of the main conduits for HGT, are ... Bacterial conjugation is a process, where a donor cell (having fertility factor) comes in close contact with the recipient cell by forming a protuberance called conjugation tube that passes on the genetic material from one cell to other. Bacterial Conjugation Definition; Bacterial Conjugation Steps. Step 1; Step 2; Step 3; Step 4; DNA Transfer; Quiz The steps of the conjugation lab will overlap with the pGLO lab. Background. Bacterial conjugation is the transfer of a copy of a plasmid from one bacterial cell to another. In this experiment you'll allow conjugation to occur, then verify that it occured both by checking for the transfer of antibiotic resistance from one cell to another and by directly examining the cells' DNA. Bacterial conjugation is a sexual mode of genetic transfer in the sense that chromosomal material from two sexually distinct cell types is brought together in a defined and programmed process. However, in bacterial conjugation, the process involves only a portion (usually small) of the genome of one of the cells (the donor) and the complete genome of its sexual partner (the recipient), as opposed to sexual union in most higher organisms, which involves an interaction between the entire set ... Steps of Bacterial Conjugation. STUDY. Flashcards. Learn. Write. Spell. Test. PLAY. Match. Gravity. Created by. FacelesAngel. Terms in this set (6) Step 1. Donor cell (the cell with the antibiotic resistance) creates a pilus to connect with a recipient cell (the cell without the antibiotic resistance) Step 2 . The plasmid (double stranded DNA) is cut by a Relaxosome at a place called the ... The process of bacterial conjugation is based on the principle that the plasmid or any other genetic material is transferred from the donor cell to the recipient cell through close physical contact. Of all the conjugative plasmids, the F (fertility) plasmid of E. coli was the first discovered and is one of the best-studied.

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Bacterial Conjugation Lab: Step 4--Streaking the Mix ...

Bacterial conjunction lecture - This lecture explains about the different types of Bacterial conjunction mechanism including the following plasmid. It expla... Bacterial DNA can pass from one cell to another through the processes of conjugation and transduction.© 2012 Encyclopædia Britannica, Inc.http://www.britanni... None-- Created using PowToon -- Free sign up at http://www.powtoon.com/ . Make your own animated videos and animated presentations for free. PowToon is a fre... Conjugation of Dutch verbs starts with determining the stem. For all but 6 irregular verbs from the Dutch language, you can find stem in three steps, keeping... This channel is dedicated to students of biology, medicine, pharmacy, agriculture and other branches where biology science is studied. Enjoy the videos and music you love, upload original content, and share it all with friends, family, and the world on YouTube. This is the third in a multi-part series of videos about how to prep & use the Introductory Bacterial Conjugation Kit from Carolina (found here): https://www... This is the first in a multi-part series of videos about how to prep & use the Introductory Bacterial Conjugation Kit from Carolina (found here): https://www...

Bacterial Conjugation- Definition, Principle, Process ...

what are the steps of bacterial conjugation

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The Next Pandemic: Confronting Emerging Disease and Antibiotic Resistance

Why viruses aren't alive: A reductio ad absurdum

AP Bio Guide (Units 8 in comments)

1) Chemistry of Life

Content

Calculations

Labs

2) Cell Structure & Function

Content

Calculations

Labs

Relevant Experiments

3) Cellular Energetics

Content

Calculations

Labs

Relevant Experiments

4) Cell Communication & Cell Cycle

Content

Calculations

Relevant Experiments

5) Heredity

Content

Calculations

Relevant Experiments

6) Gene Expression and Regulation

Content

Calculations

Labs

Relevant Experiments

7) Natural Selection

Calculations

Labs

Relevant Experiments

Machine-learning invented antibiotic

Preliminary Characterization of a Nisin Z Bacteriocin with Activity Against the Fish Pathogen Streptococcus iniae- Juniper Publishers

Abstract

Introduction

Materials And Methods

Results

Discussion

How to Manage Lactose Intolerance

What Is Lactose Intolerance?

Lactose Intolerance Causes

1. Genetics/Family History

2. Aging

3. Illness and Stress

Diagnosis

Symptoms of Lactose Intolerance

Lactose Intolerance Treatment & Diet

Below are additional steps to take to help manage lactose intolerance:

1. Use Organic Fermented Dairy

2. Try Goat Milk

3. Take Digestive Enzymes That Contain Lactase

4. Supplement with Probiotics

5. Incorporate Calcium-Rich Foods

6. Add Foods Rich in Vitamin K

7. Add Bone Broth to Your Diet

8. Jumpstart Your Gut Health with the GAPS Diet

9. Add Non-Dairy, Probiotic-Rich Foods to Your Diet

10. Use Coconut Oil for Cooking

11. Substitute Ghee for Butter

Final Thoughts

What to Look for in Alternative Milks

1. Soy

2. Pea Milk

3. Coconut Milk

4. Oat Milk

5 Almond Milk

6. Cashew Milk

7. Peanut Milk

8. Flax Milk

9. Hemp Milk

10. Rice Milk

11. Walnut Milk

[OC] Corridors - Chapter 23: Schism (Part 2)

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