Editorial on the Research Topic. Antimicrobial and Anticancer Peptides.

Avian pathogenic Escherichia coli and Clostridium perfringens cause economic and welfare concerns to the broiler industry. The recent shift to no antibiotics ever (NAE) production has increased disease incidence. The objectives of this study were to determine the influence of season, age of flock, and sample type on E. coli prevalence and virulence and to identify C. perfringens prevalence and toxinotypes in NAE farms. Results indicated high prevalence of virulent E. coli; prevalence of virulent E. coli decreased from Spring to Summer. Virulent E. coli showed high resistance to antimicrobials. Serogroups O8 and O78 were most prevalent in virulent E. coli. C. perfringens prevalence was very low and all recovered isolates were toxinotype A with variation in netB, cpb2, and tpeL presence. In conclusion, NAE farms should have measures to control E. coli infections, especially in spring season. Further studies are required to confirm the lower prevalence of C. perfringens.

Bacillus anthracis is a soil dwelling microbe with pronounced pathogenic potential. Historically, anthrax has infected livestock and man. In the modern-age, anthrax is a bioterrorism concern with major incidents every decade. While the threat of large scale attacks is currently viewed as unlikely, the threat is consistent and constant. Current methods to defend against such an attack focus on antibiotics and containment of public panic. Antibiotic resistance, while not currently an issue for anthrax, could easily become so with genetically engineered weaponized strains created by rogue states or independent actors. This project evolved from collaborations between the Grose lab and the Robison lab, both housed in the Microbiology and Molecular Biology Department at Brigham Young University in Provo, Utah. Two undergraduates in the Grose lab isolated 23 genetically distinct phage that infect the non-pathogenic Bacillus anthracis Sterne strain. Results from spot testing on a diverse library of 11 fully virulent strains that represent the extant genetic diversity of pathogenic B. anthracis in BYU’s BSL-3 facility give credence to the idea that phage could be useful in containing this pathogen. Phage were isolated from environmental samples using enrichment culture, high titer lysates of isolated phage were created, and differential assays were performed. Experiments to show phage differences included electron microscopy, restriction digests, and spot testing using different isolates of B. anthracis. These data identified several novel phage that could infect a wide variety of virulent B. anthracis isolates. Preliminary results also showed most of these phage to be different both morphologically and genetically.We propose that phage therapy deserves further research, public awareness, and increased understanding for governmental regulatory awareness.

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<p>The  Pakistan  poultry  industry  has  developed  into  the  11thlargest  poultry  industry  in  the world  and  poultry  products  provide  high-quality  and  affordable  protein  sources  to  communities throughout the country. However, <em>Salmonella </em>Gallinarum, the etiological agent for fowl typhoid, is  endemic  in  Pakistan  with  infections  leading  to  high  mortality  and  substantial  economic  loss. Currently, <em>Salmonella </em>Gallinarum  infectionsin  Pakistan  poultry  are  controlled  with  antibiotics. The continued emergence of antibiotic resistance, however, has led to global initiatives to reduce the  use  of  antibiotics  in  both  human  and  veterinary  medicine.  Concurrently,  the  Pakistan government  recently  introduced  new  national  policies  that  limit  the  use  of  antibiotics  for performance  in  livestock  and  poultry  production.  As  such,  controlling  bacterial  infections  in poultry  without  increasing  the  likelihood  of  antibiotic use could  ensure  the  sustainability  of Pakistan  poultry  production  without  posing  risks  to  public  health.  Toward  this  end,  we hypothesized that <em>Salmonella</em> Gallinarum infections inchickens could be prevented or otherwise controlled through the use of phages. To test this hypothesis, wastewater samples were collected from Lahore, Pakistan and different cities of Indiana, US and processed to isolate bacteriophages. The  phages  were  characterized  in  terms  of  morphology,  host  spectra,  lytic  capacity,  genomic sequencing,  and  survivability  in  different  environments. Transmission  electron  microscopy showed these phages belonged to myoviridae (n = 5) and podoviridae (n = 1) families. Spectrum analysis  revealed  that  each  phage  lysed  at  least  8  out  of  10  different  strains  of <em>Salmonella </em>Gallinarum and significantly reduced (P < 0.05) <em>Salmonella </em>Gallinarum when co-cultured in liquid medium with the bacterium. Stability of the phages was tested insimulated gastric fluid (SGF; pH= 2.5) andsimulated intestinal fluid (SIF; pH~6.8). Results showed that phage concentrationswere reduced to undetectable levels when exposed to SGF for more than 5 minutes. However, exposure to SIF did not result in appreciable reductions in phage concentrations. To mitigate potential effects of  gastric  environments,  phages  were  encapsulated  using  a  sodium  alginate-based  method.  In contrast  to  unprotected  phages,  encapsulated  phages  remained  viable  (~100%)  after  30  minutes exposure to SGF. Additionally, encapsulation efficiencies ranged between 90-99%. Encapsulated phages were sequentially incubated in SGF (30 minutes) and SIF(120 minutes) to determine the rate  of  release  of  the  phages  from  capsules. All  phages  were  released from  capsules after  60 minutes  of  exposureto  SIF. To  determine  if  the  phages  effectively  controlled <em>Salmonella </em>Gallinarum infections in chickens, 100, day-old Jumbo Cornish Rock Cross birds were randomly assigned  to  one  of  four  treatments:  1)  Control 1  (bacterial  challenge,  no  phage  treatment);  2) Control 2 (no phage or bacterial challenge); 3) challenged with SalmonellaGallinarum and treated with  unprotected  phages;  and  4)  challenged  with <em>Salmonella</em> Gallinarum  and  treated  with encapsulated phages. At7 d of age, chicks receiving the bacterial challenge were administered 5 X106CFU (500 μL) of <em>Salmonella</em> Gallinarum. For birds in phage treatment groups, the phages were administered (500 uL; 5 X108 PFU/mL or g) at 0, 12, and 24 hours post-challenge. Six birds from each group were euthanized at 1, 2, and 4 days post-challenge (dpc) and cecal SalmonellaGallinarum  concentrations  were  quantified.  At 1  dpc, birds  treated  with  unprotected  and encapsulated  phages  had significantly lower (P  <  0.05) SalmonellaGallinarum concentrations(4.36 ± 0.20and 5.05 ± 0.22 logCFU/g, respectively) than those found in untreated birds (5.71 ± 0.13). Likewise,  at4  dpc, <em>Salmonella </em>Gallinarum concentrationsin  ceca  of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 0.05; 3.26 ± 0.62 and 4.02 ± 0.15 log  CFU/g,  respectively)  than  those  found  in untreated  birds(4.65  ±  0.08log  CFU/g). A second trial was conducted with higher challenge doses (1 mL at 1× 109CFU) and an additional treatment including a mixture (1:1) of unprotected and encapsulated phages. At1dpc, <em>Salmonella</em> Gallinarum concentrations  in the ceca  of  birds  treated  with unprotected  phages,  encapsulated phages, and a mixture of unprotected  and encapsulated phages  were significantly lower(4.28 ± 0.11, 3.72 ± 0.40, and 3.81 ± 0.36log CFU/g, respectively) than found in those of untreated birds (5.26 ± 0.19log CFU/g). At 2 dpc, concentrations of<em> Salmonella </em>Gallinarumin the ceca of birds treated  with  unprotected,  encapsulated,  and  a mixture  of  unprotected  and  encapsulated  phages were significantly  lower  (P  <  0.05; 4.31  ±0.53, 3.96  ±0.61,  and 4.38  ±  0.44logCFU/g, respectively) than those found in the ceca of untreated birds (5.72 ± 0.27logCFU/g).However, no significant differences were found in concentrations of <em>Salmonella</em> Gallinarum in the ceca of birds treated with encapsulated phages versus those treated with unprotected phagesor a mixture of   encapsulated   and   unprotected   phages.   Similarly,   at   4   dpc, <em>Salmonella </em>Gallinarum concentrations in the ceca  of  birds  treated  with unprotected  phages, encapsulated  phages,  and  a mixture of unprotected and encapsulated phages were significantly lower (3.17 ± 0.45, 3.56 ± 0.51, and 3.81 ± 0.54log CFU/g, respectively) than found in those of untreated birds (5.79 ± 0.08log CFU/g). At  7  d  post-challenge,  concentrations of <em>Salmonella</em> Gallinarum in  the  ceca  of  birds treated  with mixture  of  unprotected  and  encapsulated phages(2.40  ±  0.55log  CFU/g)  were significantly lower (P  <  0.05) than  those  found  in the ceca  of  untreated  birds(7.08  ±  0.19log CFU/g). Similarly,  concentrations of<em> Salmonella</em> Gallinarum  in the  ceca of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 0.05; 4.29 ± 0.39and 4.60 ± 0.37 log  CFU/g,  respectively)  than  those  found  in  untreated  birds.  Taken  together,  these  data indicate that <em>Salmonella </em>Gallinarum infections could be controlled with phage-based treatments. Additionally, the use of a mixture of unprotected and encapsulated phages may be more effective, presumably  by  allowing  unprotected  phages  to  act  immediately  in  the  proximal  gastrointestinal tract  (GIT;  e.g.,  crop)  with  encapsulated  phages  having  greater  activity  once  released  from capsules in the distal small intestine. While no deleterious effects of the phages were observed on the chickens themselves, continuing studies should more comprehensively assess host-response to phage treatment including potential impact on microbial communities throughout the chicken GIT.</p>

ACKNOWLEDGEMENTS I would like to take this moment to extend my utmost appreciation for all the support provided by my supervisor, Dr. Russell de Souza. He assisted me along the way and ensured that I was always on the right path to achieve all my goals and checkpoints in every circumstance. I would also like to thank my committee for providing me with fantastic support: Ms. Laura Banfield for always being there to help solve any problem in this process, and Dr. Zain Chagla for providing a plethora of knowledge from the technical perspective of infectious disease and being so patient. Special thanks to Dr. Alexandra Mayhew for her support in our prevalence meta-analysis. Finally, I would like to thank my family, my wife, Dr. Sanjida Rowshan Anannya, for whom I am here today, and my parents, siblings, and in-laws; you are always there for me in every walk of life. You are why I have gotten to where I am today and are my daily inspiration. / Background: Bacteria is a major cause of many infectious diseases, and the treatment for these diseases is antibiotics designed to kill or subdue the growth of the bacteria. However, bacteria evolve, and if an antibiotic prescription is not the right antibiotic for the right patient at the right time with the correct dose and the right route, Antimicrobial Resistance (AMR) may result. During this pandemic, the use of antibiotics to treat hospitalized COVID-19 patients without any bacterial coinfection threatens the effectiveness of antibiotic treatment for current and future bacterial infections. Methods: A systematic search was conducted of the Embase, Medline, Web of Science, and Cochrane Library databases by generating search terms using the concepts of “COVID-19,” “Bacterial Coinfection,” “Secondary bacterial infection,” and “Antimicrobial resistance” to identify studies that reported the prevalence of antibiotic prescription for the treatment of COVID-19 in hospitalized patients with and without bacterial coinfection. The pooled estimate of the percentage of the total and confirmed appropriate antibiotic prescriptions provided to hospitalized COVID-19 patients was generated using a random effect meta-analysis with inverse variance weighting. Result: Of 157,623 participants from 29 studies included in our review, 67% (CI 64% to 71%, P<0.00001) were prescribed antibiotics, among which 80% (CI 76% to 83%, P<0.00001) prescriptions were given for the COVID-19 patients without any bacterial coinfections. The use of antibiotics varied during the pre-immunosuppressive period (before 16 June 2020) and post-immunosuppressive period of the pandemic and between the High-Income Countries and Upper and Lower Middle-Income Countries. Conclusion: This Systematic Review and Meta-analysis finds greater than expected use of antibiotics to treat hospitalized COVID-19 patients without bacterial coinfections, which can worsen AMR globally. Clear and concrete guidelines for the use of antibiotic prescriptions to treat COVID-19 patients, strict monitoring, and compliance with Antimicrobial Stewardship are needed to prevent over-prescription. / Thesis / Master in Advanced Studies (MAS) / Bacteria is a major cause of many infectious diseases. Before the discovery of Antibiotics in 1928, hundreds of thousands of people used to die due to infectious diseases caused by bacteria. While Antibiotics are essential to treat bacterial infectious diseases, overuse or misuse can accelerate Antibiotic Resistance, a phenomenon when bacteria change and/or develop the ability to escape the drugs designed to kill them. Self-medication, availability of antibiotics without a prescription, and inappropriate dosing of antibiotics can worsen the situation. During the COVID-19 pandemic, antibiotics were commonly prescribed as part of the treatment regime for COVID-19, even when a clear bacterial infection was not identified. In our Systematic Review and Meta-analysis, we aimed to see the frequency of antibiotic prescriptions to treat hospitalized COVID-19 patients without any bacterial coinfections.

Biocides and antibiotics are commonly used in Irish agriculture. This could lead to accumulation at sublethal levels in water and resistance development. The risk of this has earlier been assessed as non-existent. However, resistant strains have been found in Irish farm waste water. Due to possible horizontal gene transfer between bacterial populations the risk may be higher. Antibiotic resistance mechanisms have worked against certain biocides and antibiotic resistant strains have in certain cases showed reduced biocidal susceptibility. The aim of this project was to characterise bacterial populations from Irish aquatic samples and investigate their susceptibility to agriculturally common biocides and relevant antibiotics. Isolates from Dublin Bay water samples (n=15) were characterised using basic techniques. Minimum inhibitory concentration (MIC) tests with a broth microdilution method were performed with eight biocides and complimentary minimum bactericidal concentration (MBC) tests. Antibiotic disc diffusions were performed with eight antibiotics. The samples contained gram-negative isolates (n=3), Staphylococcus aureus (n=1) and Bacillus spp. (n=8) isolates. All isolates were on average resistant towards methylated spirits and iodine at the 2% v/v starting concentration. MIC values for Tri Scrub and the generic biocide were high. All MBC values were on average higher than the corresponding MIC values. A significant amount of the Bacillus spp. isolates were resistant towards β-lactams. As there is statistical uncertainty around the results, further investigations are needed. In conclusion, a trend of both high MIC and MBC values while showing resistance towards the largest number of antibiotics could be seen in Bacillus spp. isolates.

Bacterial resistance to antibiotics has become a worldwide health problem, resulting in untreatable infections and escalating healthcare costs. Wastewater treatment plants are a critical point of control between anthropogenic sources of pathogens, antibiotic resistant bacteria (ARBs), antibiotic resistance genes (ARGs), and the environment through discharge of treated effluent and land application of biosolids. Recent studies observing an apparent resuscitation of pathogens and pathogen indicators and the widening realization of the importance of addressing environmental reservoirs of ARGs all lead toward the need for improved understanding of ARG fate and pathogen inactivation kinetics and mechanisms in sludge stabilization technologies. This research has investigated the fate of two pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, and various ARGs under pasteurization, anaerobic digestion, biosolids storage, and land application conditions. Pathogen die-off occurs at a rate specific to each pathogen and matrix in ambient and mesophilic temperature environments. Viable but nonculturable (VBNC) states are initiated by thermal treatments, such as thermophilic digestion and possibly pasteurization, and allow the persistence of pathogen cells and any ARGs contained therein through treatment and into the receiving environment where resuscitation or transformation could occur. Raw sludge ARG content does affect digester effluent quality, although the predominant mechanisms of ARG persistence may be different in mesophilic versus thermophilic digestion. In both thermophilic and mesophilic digestion, a correlation was observed between raw sludge and digester ARGs associated with Class 1 integrons, possibly as a result of horizontal gene transfer. ARB survival was shown to contribute to ARG content in mesophilic digestion, but not thermophilic digestion. Thermophilic digestion may achieve a higher ARG reduction because of reduced microbial diversity compared to mesophilic digestion. However, it is evident that horizontal gene transfer still does occur, particularly with highly mobile integrons, so that complete reduction of all ARGs would not be possible with thermophilic digestion alone. Surprisingly, the experiments that introduced various concentrations of antibiotic sulfamethoxazole and antimicrobial nanosilver did not induce enhanced rates of horizontal gene transfer. Finally, ARG concentrations in biosolids increased during cold temperature storage suggesting that there is a stress induction of horizontal gene transfer of integron-associated ARGs. / Ph. D.

Food-borne illness outbreaks are occasionally associated with fresh-vegetable consumption, in part due to lack of a microbial inactivation step before consumption. Raw manure or improperly composted manure applied as soil amendments is an established source of pathogenic bacterial contamination. However, less is known about whether such soil amendments could serve as a source of transmission of antibiotic-resistant bacteria (ARB) or antibiotic-resistance genes (ARGs) via fresh produce. As such knowledge is developing, it is useful to identify strategies for mitigating ARGs and ARB on vegetable surfaces, especially those that are synergistic with known benefits in terms of general pathogen reduction on fresh produce. Sanitizers play an important role in post-harvest processing of vegetables, especially in terms of disinfecting the wash water and preventing cross-contamination. Further, temperature and time of storage of vegetables are critical to prevent the growth of microorganisms. To provide a background inoculum representing potential pre-harvest carryover of ARB and ARGs, carrots or romaine lettuce leaves were dipped in a slurry derived from composted manure from dairy cows previously dosed with antibiotics and further inoculated with multi-drug resistant E. coli O157:H7, a human pathogen, and a spoilage-associated and opportunistic pathogenic strain of Pseudomonas aeruginosa. Inoculated carrots (n=3, 25 g) were washed with water containing different sanitizers (sodium hypochlorite or peroxyacetic acid) or unwashed (control), packaged and stored at 10ºC for 7d or 2ºC for up to 60 d. Inoculated lettuce leaves (n=3, 100 g) were washed with sodium hypochlorite, packaged in modified atmosphere conditions (98% nitrogen), irradiated (1.0 kGy) and subsequently stored at 4ºC for 14 d. The effect of post-harvest treatment were compared at various times by enumeration on selective media. In addition, cultureindependent techniques were also performed to determine changes to the surficial carrot and lettuce microbiota by sequencing bacterial 16S rRNA gene amplicons. The effect of post-harvest treatments on the types and relative abundance of ARGs, also known as the “resistome,” were profiled by shotgun metagenomic sequencing and qPCR. Addition of a sanitizer during wash, storage temperature, and duration of storage affected the bacterial community structures on carrots, represented by the weighted Unifrac distance matrices (ANOSIM, R=0.465). Storage of sanitizer-washed carrots at 10ºC was associated with an increase in relative abundance of Pseudomonadaceae compared to 2ºC storage for 7 d (Wilcoxon, p<0.05). Increase in storage temperature from 2ºC (optimum) to 10ºC (temperature abuse) of sanitizer-washed carrots resulted in enrichment of ARGs conferring resistance to the following antibiotic classes: multidrug, peptide, polymyxin, quinolone, triclosan, aminoglycoside, bacitracin, β-lactam, and fosfomycin. Irradiation resulted in significant reductions (~3.5 log CFU/g) of inoculated antibiotic-resistant E. coli O157:H7 and Pseudomonas sp. on lettuce surfaces (ANOVA, p<0.05). The lettuce resistome, represented by the Bray-Curtis similarity of ARG occurrence, was affected by irradiation (ANOSIM, R=0.406). Irradiation of lettuce followed by 14 d of storage at 4ºC resulted in 2-4-fold reductions in relative abundance of ARGs encoding resistance to the following antibiotic classes: triclosan, quinolones, multidrug, polymyxin and β-lactam (Wilcoxon, p<0.05). No additional increase or reduction of the tet(A) gene present on inoculated P. aeruginosa was evident after 14d storage at 4ºC on irradiated samples. Results of this study suggest that inclusion of a sanitizer in wash water, irradiation, and storage at optimum refrigerated temperatures may offer effective strategies to combat proliferation of antibiotic resistant bacteria and antibiotic resistance genes on fresh produce. Further research is needed develop interventions that can mitigate tet(A) and other ARGs on produce that were not significantly reduced by irradiation. This study will guide future research on microbiome and metagenome of processed produce and assessment of critical control points to reduce the risk of antibiotic resistance from farm-to-fork. / PHD / Post-harvest interventions; such as washing, irradiation and cold storage, are employed to provide safe and wholesome fresh vegetables to consumers. Washing of vegetables in water that includes a sanitizing agent, such as chlorine or peroxyacetic acid (POAA), removes soil from the surface, reduces the bacteria in wash water and prevent cross-contamination between vegetables. It has an additional benefit to reduce microorganisms on produce surfaces that may cause the vegetables to spoil or result in illness in humans. Low temperature storage of produce, usually 0-5ºC, decreases the respiration rate of vegetables and reduces growth of microorganisms during storage. Some of the spoilage and/or pathogenic bacteria may also be antibiotic-resistant, which are commonly termed as antibiotic-resistant bacteria (ARB). Antibiotic resistance is a significant public health concern that leads to ineffective medical treatments, prolonged duration of illnesses and increased hospitalization costs. Antibiotic resistance is encoded by genes that confer resistance to wide range of antibiotic classes, including antibiotics used to treat human illnesses. These genes are termed as antibiotic resistance genes (ARGs). In this study we examined the effect of three common post-harvest interventions, washing with sanitizers, gamma irradiation, and cold storage to reduce antibiotic-resistant bacterial pathogens and antibiotic-resistant spoilage bacteria on carrots and lettuce. Storage temperature, inclusion of sanitizer in wash water, and length of chilled storage significantly influenced the diversity of bacteria found on carrot surface. Inclusion of either sanitizer in the wash water significantly reduced the populations of antibiotic-resistant E. coli O157:H7 (a pathogenic bacterium that causes a dangerous form of gastrointestinal illness) and Pseudomonas sp. (a bacterial species that commonly causes food spoilage). Storage at recommended temperature (2ºC) did not allow these bacteria to regrow and also reduced total ARGs on carrot surfaces. Washing of lettuce with sodium hypochlorite followed by irradiation (1.0 kGy) and storage at recommended temperature (4ºC) were effective in reducing the populations of antibiotic-resistant E. coli O157:H7 and Pseudomonas sp., and additionally reduced the number of some ARGs conferring resistance to select classes of antibiotics, including triclosan, quinolones, multidrug, polymyxin and β-lactam antibiotics on the lettuce surface. A novelty of this research is that it employed new, cutting-edge “metagenomic” DNA sequencing technique to identify and track antibiotic resistance through the various post-harvest interventions. Overall results of this research suggest that inclusion of sanitizer in wash water for fresh produce, followed by storage at refrigerated temperatures below 4ºC may reduce the risk posed by antibiotic resistant bacteria and antibiotic resistance genes on produce.