Antibiotics are substances that inhibit the growth of microorganisms and are widely used in modern medicine. High consumption of antibiotics correlates with their elevated occurence in sewage systems, from where they are further released into the environment. The threat of their occurrence in the environment is in triggering the formation and spread of antibiotic resistance. This thesis focuses on the removal of selected antibiotics and partly on the antibiotic resistance from wastewater by advanced oxidation processes (AOPs). AOPs are based on the creation of highly reactive hydroxyl radicals, which are able to oxidize even highly persistent substances. The thesis focuses on the purification of effluents from municipal wastewater treatment plants (WWTP), which are identified as the main source of pharmaceutical pollution in the environment, by AOPs mainly based on ozonation. These processes were tested under laboratory conditions on model water as well as under real conditions as the tertiary stage of the treatment at Brno-Modřice WWTP under various operating states. As the results of the performed experiments show, it could be concluded that the studied AOPs are capable of effective degradation of studied antibiotics from the treated medium. Under real conditions, the antibiotics sulfamethoxazole, trimethoprim, azithromycin and clarithromycin were monitored. Output concentrations in each operational state were achieved to be below the limit of detection for all four antibiotics. The application of AOPs based on ozonation as the tertiary step of the treatment also had a positive effect on the reduction of microbial contamination and antibiotic resistance. Within the pilot plant experiments, a reduction of up to 4 orders of magnitude of E.coli, coliform microorganisms and a reduction of their resistant strains in the range of 1.4 - 4.0 logs were observed, compared to the effluent from the WWTP.

<p>  </p> <p>Infections of dermal wounds is a growing burden for the healthcare industry, with a 2017 market exceeding $17.5 USD. As the number of patients with severe infections continues to increase year after year, there is an alarming downward trend in efficacy for traditional antibiotic treatments. In large part, this is due to the increasing development of antibiotic resistance within common bacteria strains. As microbes evolve to protect themselves from previously effective drugs, there is a growing need for new antimicrobial therapies. While alternatives exist in the market, they are largely impaired by non-selective toxicity which can cause further damage to the cells in the wound bed, as is the case with silver and other strong antiseptics, or the need for high energy, specialized equipment, as with cold atmospheric surface treatments. Gaseous ozone is a promising alternative therapy for treating these wound infections. Because ozone is a strong natural oxidant, it exhibits significant antimicrobial properties, and has also been shown to help stimulate natural wound healing in many cases. Herein is presented the design of a portable system for the topical delivery of gaseous ozone as an antimicrobial treatment for infected dermal wounds. This includes the design and characterization of the portable system and a custom ozone application dressing, the characterization of the safety and efficacy of the system using <em>in vitro</em> and <em>in vivo</em> models, and a disposable system for wound infection monitoring. The system utilizes a portable corona discharge generator to produce gaseous ozone from the ambient environment. The ozone gas is delivered through a dressing engineered to have a hydrophobic interface at the wound bed and disperse the ozone gas across the patch surface for more uniform application. The antimicrobial strength and biocompatibility of the system was optimized at varying ozone output levels. Additionally, an adjunct therapy of topical antibiotics was shown to significantly increase the strength of the treatment without leading to greater cytotoxicity. This synergistic effect between ozone and antibiotics was shown to circumvent natural bacterial resistances to antibiotics, which will have a major impact on the wound care industry. This adjunct treatment was then validated on a porcine animal model for safety and pilot results for efficacy testing. Finally, the pH sensor which can be incorporated with use of the ozone therapy enables objective monitoring of wound condition and is able to signal when appropriate infection therapy should begin. As it stands, this portable ozone wound treatment system shows great promise as an alternative therapy to improve the quality of live for millions of patients.</p>

Antibiotic resistance is critical to global health. This thesis examines the relationship between antibiotic resistance and genomic variations in E. coli from wastewater. E. coli is of interest as it causes urinary tract and other infections. Wastewater is a good source because it is a melting pot for E. coli from diverse origins. The research delves into two key aspects: including or excluding antibiotic resistance data and the level of granularity in representing genomic variations. The former is important because there is more genomic data than antibiotic resistance data. Consequently, relying solely on genomic data, this thesis studies positive selection in E. coli to identify mutations and genes favored by evolution. This study demonstrates the preferential selection of known antibiotic resistance genes and mutations, particularly mutations located on functionally important locations of outer membrane porins, and may hence have a direct effect on structure and function. Encouraged by these results, the study was expanded to include antibiotic resistance data and to examine genomic variations at three resolution levels: single mutations, unitigs (genome words) that may contain multiple mutations, and whole coding genome using machine learning classifier models that capture dependencies among multiple mutations and other genomic variations. Representation of single mutations detects well-known resistance mutations as well as potentially novel mechanisms related to biofilm formation and translation. By exploring larger genomic units such as genome words, the analysis confirms the findings from single mutations and additionally uncovers joint mutations in both known and novel genes. Finally, machine learning models, including AI language models, were trained to predict antibiotic resistance based on the whole coding genome. This achieved an accuracy of over 90% in predicting antibiotic resistance when sufficient data were available. Overall, this thesis unveils new antibiotic resistance mechanisms, conducts one of the largest studies of positive selection in E. coli, and stands out as one of the pioneering studies that utilizes AI language models for antibiotic resistance prediction.

Antibiotic resistance in multidrug resistant bacteria cause high mortality rates worldwide, where there has been over 1,000,000 deaths reported as of the year 2019. Antibiotics were thought to be the cure for fighting infectious diseases and preventing further spreading of infection. This became a major problem due to bacteria evolving and developing mechanisms for resistance. The purpose of this review was to see if there are differences in the resistance mechanism of gram negative and gram positive bacteria, focusing mainly on the six most common multidrug resistant pathogenic bacteria; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae. The results show that there is a difference in the resistance mechanism between gram positive and gram negative multidrug resistant bacteria. The difference in resistant mechanisms is due to the cell wall compositions of gram negative and gram positive bacteria. The main difference as to why the gram negative bacteria have more resistance is due to the outer membrane. Antibiotics have a hard time to diffuse through and into the cell, that is they can easily decrease their outer membrane permeability. Gram positive bacteria lack an outer membrane which makes them become more susceptible to antibiotics. The most common antibiotic resistance mechanisms in gram negative bacteria are outer membrane mechanisms such as lipid A and lipopolysaccharide modification as well as mutations in porin channels. On the other hand, the most common resistance mechanisms for gram positive bacteria are point mutations especially in penicillin binding proteins as well mutations in the rpoB gene. One important gram positive bacteria is Methicillin resistant Staphylococcus aureus, which developed a new mechanism against antibiotics, a missense mutation and mutation on the promoter region in penicillin binding protein 4. Recently new research has come forward showing that N-chlorotaurine (NCT) inhibits resistance in both gram positive and gram negative multidrug resistant bacteria. The research on NCT is still fairly new and only time will tell if this method of inhibiting resistance will be used in the future. This review highlights the importance and concern of multidrug resistance bacteria, especially due to bacteria being able to rapidly evolve when antibiotics are used incorrectly. It is important to understand the differences in resistance between gram negative and gram positive bacteria and how resistance spreads. This knowledge can be used to develop antibiotics that treat infections. It is however still a challenge to overcome resistance in multidrug resistant bacteria due to evolutionary adaptation especially through horizontal gene transfer, where resistant bacteria can adapt to changing conditions. / Antibiotikaresistens hos multiresistenta gramnegativa och grampositiva bakterier orsakar hög dödlighet över hela världen, där det har rapporterats över 1,000,000 dödsfall för år 2019. Antibiotika ansågs vara botemedlet för att bekämpa infektionssjukdomar och förhindra ytterligare spridning av infektioner. Detta blev ett stort problem på grund av att bakterier utvecklades mekanismer för resistens, vilket gör att de kan överleva när de behandlas med antibiotika. Syftet med denna studien är att se om det finns skillnader i resistensmekanismener för gramnegativa och grampositiva bakterier, med fokus på de sex vanligaste multiresistenta bakterierna; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae och Streptococcus pneumoniae. Resultaten visar att det finns en skillnad i resistensmekanismen mellan grampositiva och gramnegativa multiresistenta bakterier. Skillnaden i resistenta mekanismer beror på cellväggssammansättningen av gramnegativa och grampositiva bakterier. Den största skillnaden till varför de gramnegativa bakterierna har mer resistens beror på det yttre membranet. Antibiotika har svårt att penetrera genom och in i cellen genom att minska deras yttre membranpermeabilitet. Grampositiva bakterier saknar ett yttre membran som gör att de blir mer mottagliga för antibiotika. De vanligaste antibiotikaresistensmekanismerna hos gramnegativa bakterier är yttre membranmekanismer som lipid A och lipopolysackaridmodifiering samt mutationer i porinkanaler. De vanligaste resistensmekanismerna för grampositiva bakterier är punktmutationer, särskilt i penicillinbindande proteiner samt mutationer i rpoB genen. En viktig grampositiv bakterie är Meticillin-resistent Staphylococcus aureus, som utvecklade en ny mekanism mot antibiotika, en missense-mutation och mutation på promotorregionen i penicillinbindande protein 4. Nyligen har ny forskning kommit fram som visar att N-klorotaurin (NCT) hämmar resistens i både grampositiva och gramnegativa multiresistenta bakterier. Forskningen om NCT är fortfarande ny och bara tiden kommer att utvisa om denna metod för att hämma resistens kommer att användas i framtiden. Den här studien belyser vikten och oron för multidresistena bakterier, särskilt på grund av att bakterier snabbt kan utveckla antibiotikaresistens när antibiotika används på fel sätt. Det är viktigt att förstå skillnaderna i resistens mellan gramnegativa och grampositiva bakterier och hur resistens sprids inom resistenta bakterier. Denna kunskap kan användas för att utveckla antibiotika som behandlar infektioner orsakade av både gramnegativa och grampositiva bakterier. Det är fortfarande en utmaning att övervinna resistens hos multiresistenta bakterier på grund av evolutionär anpassning särskilt genom horisontell genöverföring, där resistenta bakterier kan anpassa sig till förändrande förhållanden.

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.

No description available.

No description available.

<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>