To combat the ever-growing clinical burden imposed by antibiotic-resistant pathogens, multiple-antibiotic treatments are increasingly being considered as promising treatment options. The impact of multiple-antibiotic treatments on the evolution of resistance is not well understood however, and debate is ongoing about the effectiveness of various multiple-antibiotic treatments. In this thesis, I investigate how aspects of multiple-antibiotic treatments impact the rate of evolution of antibiotic resistance in the opportunistic human pathogen Pseudomonas aeruginosa. In particular, I look at the impact of interactions between antibiotics in combination on the evolution of resistance, and how creating heterogeneity in the antibiotic environment by rotating the antibiotics used may change the rate of evolution of resistance. I characterise the interactions present in 120 combinations of antibiotics and find that the type of interaction can be predicted by the mechanism of action of the antibiotics involved. I investigate the effect of a subset of these combinations on the evolution of antibiotic resistance. My results refute the influential but poorly-evidenced hypothesis that synergistic combinations accelerate the evolution of resistance, even when synergistic combinations have the same inhibitory effect on sensitive bacteria as additive or antagonistic antibiotic combinations. I focus on a combination of the antibiotics ceftriaxone and sulfamethoxazole and test whether it is more effective in preventing the evolution of resistance than predicted by the inhibitory effect of the combination on sensitive bacteria. I do not find the combination to be more effective than predicted. Finally, I create heterogeneous antibiotic environments by rotating the antibiotic present at different rates. For the first time in a laboratory setting, I test how varying the rate of fluctuation in the antibiotics present in a heterogeneous antibiotic environment impacts the rate of evolution of resistance. Unexpectedly, I find the rate of evolution of resistance increases with increasing levels of antibiotic heterogeneity.

In the first part of my thesis I focus on mapping problems associated with antibiotic therapy and subsequent development of antibiotic resistance. Tracking resistance is based primarily on data collection and evaluation of the results set sensitivity from around the world. Antibiotic resistance is a natural phenomenon that can be observed in the evolution of microbes as one of the mechanisms of adaptation to new conditions in the environment. For this work I have chosen the following research questions. Do the incidence of ESBL strains in the České Budějovice Hospital a.s. increase over time? Are these values comparable to those achieved in another region, namely in Moravian hospitals the University Hospital of Olomouc, Ostrava University Hospital and Regional University Hospital of T. Bata in Zlin? The data collection I made in collaboration with the laboratory technicians and doctors at Hospital?s Bacteriology Laboratory in České Budějovice. Bacteries tested for the detection of ESBL production originated from biological materials, witch came from patients of hospital in České Budějovice. The first objective was to compare the results achieved in the České Budějovice Hospital in the period of 2007 to 2012. If we look at the total number of ESBL strains that have been isolated since 2007, values have upward trend. While in 2007 there were only 64 strains a year later, the number more than doubled. In 2010, the value soared to 281 tribes and in the year 2012, the number was 321 tribes. The incidence of ESBL strains in 2007 increased about five times. In the long term we can say the numbers have increasing tendency and the range of each species in the production of ESBL has significantly changed. In 2007, it was K. pneumoniae strains that dominated the statistics, but over time the strains of E. coli came forefront. Values of 2012 suggest that the presence of ESBL strains of K. pneumoniae is again almost equal to the number of E. coli strains. The second objective was to compare the results of the 2012 with study of the Prevalence of ESBL-positive Enterobacteriaceae in large Moravian hospitals. In the general overview of ESBL producers values in Hospital České Budějovice (5.23%) are comparable to those in Ostrava (4.9%) and in Zlín (4.3%). Number of strains in the Hospital in Olomouc (11.8%) is about twice as high as the numbers in České Budějovice. In this comparison the České Budějovice Hospital is one of the hospitals with a lower incidence of ESBL producers. The České Budějovice Hospital is below the national average, which originate from an elaborate system of care for patients with colonization or infection with ESBL strains, and from therapy control system using antibiotic center. These results may serve to the Hospital in České Budějovice for statistical purposes, and also for proposals for improving patient care. In the discussion, I pointed out the danger of the spread of resistant strains of bacteria in the community and also the associated risks that mentioned bacteria mean for patients injured in mass accidents or disasters. In these cases, number of infections including ESBL producers can penetrate through open wounds into the affected body. Unlike conventional sensitive bacteria those strains are resistant to commonly used antibiotics and thereby endanger the lives of people affected by the accident.

Terapia empírica com antimicrobiano de amplo espectro faz parte do manejo inicial padrão de pacientes com neutropenia febril (NF). Evidências suficientes de quais esquemas antibióticos devem ser inicialmente prescritos já existem; embora, nenhum estudo randomizado tenha avaliado se a aderência a programas de controle de antimicrobianos (PCAs) resulta em diminuição das taxas de mortalidade por esta síndrome. No presente estudo de coorte prospectivo, realizado em um hospital terciário no período de outubro de 2009 a agosto de 2011, avaliou-se o impacto da aderência ao PCA, aferida através da prescrição antimicrobiana inicial, na mortalidade em 295 episódios de NF (em 145 indivíduos adultos) que necessitaram de tratamento endovenoso hospitalar. Após análise multivariada através de regressão de Cox, incluindo outros preditores de mortalidade, a aderência ao PCA mostrou-se fator de proteção independente para morte 28 dias após início do episódio de NF (razão de hazard ajustada[HR], 0.29; intervalo de confiança de 95% [IC 95%], 0.11 a 0.72). Os fatores de risco encontrados para a não-aderência ao PCA foram presença de hipotensão (risco relativo ajustado[RR], 1.90; IC 95%, 1.37 a 2.63), diarreia (RR, 2.13; IC 95%, 1.66 a 2.73), dor perianal (RR, 2.08; IC 95%, 1.54 a 2.82), suspeita de foco infeccioso em cavidade oral (RR, 2.45; IC 95%, 1.75 a 3.43) e manifestações cutâneas de infecção (RR, 2.34; IC 95%, 1.81 a 3.04). A escolha antimicrobiana inicial é particularmente importante no manejo inicial do paciente com febre em vigência de neutropenia; a aderência ao PCA, que preconiza o uso racional de antibióticos, mostrou ser efetiva na redução de mortalidade durante o curso da doença. A presença de fatores modificadores da terapia inicial representa risco para não-adesão ao programa de controle de antimicrobianos. / Empirical therapy with broad-spectrum antimicrobial is part of the initial management of patients with febrile neutropenia (FN). Enough evidence on which antibiotics schemes should be initially prescribed already exists; however, no randomized study has evaluated whether adherence to antimicrobial stewardship programs (ASPs) results in lower rates of mortality from this syndrome. In the present prospective cohort study performed in a tertiary hospital, from October 2009 to August 2011, we evaluated the impact of adherence to ASP, measured by initial antimicrobial prescribing, in mortality of 295 episodes of FN (in 145 adults) that required intravenous inpatient treatment. After multivariate analysis through Cox regression, including other predictors of mortality, adherence to ASP proved to be an independent protective factor for death 28 days after the beginning of the episode of FN (adjusted hazard ratio [HR], 0.29; 95% confidence interval [95% CI], 0.11 to 0.72). The risk factors found to noncompliance to ASP were presence of hypotension (adjusted relative risk [RR], 1.90; 95% CI, 1.37 to 2.63), diarrhea (RR, 2.13; 95% CI, 1.66 to 2.73), perianal pain (RR, 2.08; 95% CI, 1.54 to 2.82), suspected source of infection in oral cavity (RR, 2.45; 95% CI 1.75 to 3.43) and cutaneous manifestations of infection (RR, 2.34; 95% CI, 1.81 to 3.04). The choice of antimicrobial is particularly important in the initial management of patients with fever in the presence of neutropenia; the adherence to ASP, which calls for rational use of antibiotics, was effective in reducing mortality during the course of the disease. The presence of signs or symptoms that demand changes in the initial therapy poses risks to nonadherence to the antimicrobial management program.

Terapia empírica com antimicrobiano de amplo espectro faz parte do manejo inicial padrão de pacientes com neutropenia febril (NF). Evidências suficientes de quais esquemas antibióticos devem ser inicialmente prescritos já existem; embora, nenhum estudo randomizado tenha avaliado se a aderência a programas de controle de antimicrobianos (PCAs) resulta em diminuição das taxas de mortalidade por esta síndrome. No presente estudo de coorte prospectivo, realizado em um hospital terciário no período de outubro de 2009 a agosto de 2011, avaliou-se o impacto da aderência ao PCA, aferida através da prescrição antimicrobiana inicial, na mortalidade em 295 episódios de NF (em 145 indivíduos adultos) que necessitaram de tratamento endovenoso hospitalar. Após análise multivariada através de regressão de Cox, incluindo outros preditores de mortalidade, a aderência ao PCA mostrou-se fator de proteção independente para morte 28 dias após início do episódio de NF (razão de hazard ajustada[HR], 0.29; intervalo de confiança de 95% [IC 95%], 0.11 a 0.72). Os fatores de risco encontrados para a não-aderência ao PCA foram presença de hipotensão (risco relativo ajustado[RR], 1.90; IC 95%, 1.37 a 2.63), diarreia (RR, 2.13; IC 95%, 1.66 a 2.73), dor perianal (RR, 2.08; IC 95%, 1.54 a 2.82), suspeita de foco infeccioso em cavidade oral (RR, 2.45; IC 95%, 1.75 a 3.43) e manifestações cutâneas de infecção (RR, 2.34; IC 95%, 1.81 a 3.04). A escolha antimicrobiana inicial é particularmente importante no manejo inicial do paciente com febre em vigência de neutropenia; a aderência ao PCA, que preconiza o uso racional de antibióticos, mostrou ser efetiva na redução de mortalidade durante o curso da doença. A presença de fatores modificadores da terapia inicial representa risco para não-adesão ao programa de controle de antimicrobianos. / Empirical therapy with broad-spectrum antimicrobial is part of the initial management of patients with febrile neutropenia (FN). Enough evidence on which antibiotics schemes should be initially prescribed already exists; however, no randomized study has evaluated whether adherence to antimicrobial stewardship programs (ASPs) results in lower rates of mortality from this syndrome. In the present prospective cohort study performed in a tertiary hospital, from October 2009 to August 2011, we evaluated the impact of adherence to ASP, measured by initial antimicrobial prescribing, in mortality of 295 episodes of FN (in 145 adults) that required intravenous inpatient treatment. After multivariate analysis through Cox regression, including other predictors of mortality, adherence to ASP proved to be an independent protective factor for death 28 days after the beginning of the episode of FN (adjusted hazard ratio [HR], 0.29; 95% confidence interval [95% CI], 0.11 to 0.72). The risk factors found to noncompliance to ASP were presence of hypotension (adjusted relative risk [RR], 1.90; 95% CI, 1.37 to 2.63), diarrhea (RR, 2.13; 95% CI, 1.66 to 2.73), perianal pain (RR, 2.08; 95% CI, 1.54 to 2.82), suspected source of infection in oral cavity (RR, 2.45; 95% CI 1.75 to 3.43) and cutaneous manifestations of infection (RR, 2.34; 95% CI, 1.81 to 3.04). The choice of antimicrobial is particularly important in the initial management of patients with fever in the presence of neutropenia; the adherence to ASP, which calls for rational use of antibiotics, was effective in reducing mortality during the course of the disease. The presence of signs or symptoms that demand changes in the initial therapy poses risks to nonadherence to the antimicrobial management program.

Francisella tularensis est une bactérie à Gram négatif intracellulaire facultative, agent causal de la tularémie. Cette zoonose est induite principalement par deux sous espèces : F. tularensis subsp. tularensis (type A) et F. tularensis subsp. holarctica (type B) retrouvées respectivement en Amérique du Nord et dans tout l’hémisphère Nord. Cette seconde sous espèce, moins virulente que la première induit majoritairement des formes cliniques de sévérité moyenne à modérée dites ganglionnaires. Leur traitement est basé sur l’utilisation des antibiotiques de la classe des fluoroquinolones ou des tetracyclines, l’utilisation des aminosides étant réservée aux formes graves. Les adénopathies évoluent cependant souvent vers la suppuration et la chronicité (20 à 40% des cas), malgré l’administration d’un traitement antibiotique adapté.Les travaux réalisés visent à étudier l’hypothèse de l’émergence de la résistance bactérienne chez Francisella, expliquant ces échecs thérapeutiques. Ils sont basés sur le développement et l’étude d’un modèle d’évolution in vitro de la bactérie en présence de ciprofloxacine, une fluoroquinolone. Nos travaux ont confirmé la capacité de la bactérie à évoluer vers un haut niveau de résistance à ces antibiotiques, corrélée à l’accumulation de mutations dans les gènes codant pour les topoïsomérases de type II. De plus, nous avons observé la présence sur l’ensemble des souches de F. tularensis subsp. holarctica d’un niveau de résistance cliniquement significatif induit par des mutations modifiant la sous-unité GyrA de l’ADN gyrase sur les acides aminés en position 83 et 87. La recherche de ce marqueur dans des prélèvements de patient en échec thérapeutique suite à divers traitements antibiotiques s’est avérée infructueuse.Après avoir vérifié l’action de l’antibiotique sur les bactéries dans le compartiment intracellulaire (fibroblates), nous avons recherché les autres mutations induites lors de l’évolution de Francisella en présence de fluoroquinolones. Cette étude a permis l’implication de plusieurs systèmes de transports transmembranaires dans la résistance antibiotique. Nous avons également révélé l’existence d’une seconde cible majeure impliquée dans le métabolisme du fer de la bactérie. L’altération de cette cible (FupA/B) en plus d’être associée à une augmentation de la résistance aux fluoroquinolones est corrélée à une forte diminution de la capacité de la bactérie à se multiplier dans les cellules phagocytaires. / Francisella tularensis is a gram-negative facultative intracellular bacterium, causing tularemia. This zoonosis is mainly related to two subspecies: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) in North America and throughout the Northern Hemisphere, respectively. Infections with this second subspecies, less virulent than the first one, predominantly induce glandular clinical forms of mild to moderate severity. Their treatment is based on antibiotherapy using a fluoroquinolone or a tetracycline. The use of aminoglycosides is reserved for severe clinical forms. The lymph nodes infection, however, often become chronic (20 to 40% of cases), despite administration of an appropriate antibiotic treatment.The aim of this study was to verify the hypothesis of the emergence of bacterial resistance in Francisella, which could explain treatment failures. It is based on the development and study of an in vitro evolutionary experiment of the bacterium in the presence of ciprofloxacin, a fluoroquinolone. Our work confirmed the bacterium's ability to evolve towards a high-level of resistance to fluoroquinolones, this evolution being correlated with the accumulation of mutations in the genes encoding for type II topoisomerases. In addition, we observed in all strains of F. tularensis subsp. holarctica resistant to fluoroquinolones at a clinically significant level, the presence of mutations altering the GyrA subunit of DNA gyrase at amino acids positions 83 and 87. The research of this marker in clinical samples from patients with treatment failure following appropriate antibiotic treatment was however unsuccessful.After checking the action of antibiotics on bacteria internalized in the intracellular compartment in fibroblast cells, we looked for other mutations induced during the evolution of Francisella to resistance to fluoroquinolones. This study unveiled the involvement of several transmembrane transport systems in antibiotic resistance. We also revealed the existence of a second major target involved in Francisella iron metabolism. The alteration of this target (FupA/B), in addition to being associated with an increase in fluoroquinolone resistance, is correlated with a sharp decrease in the ability of the bacteria to multiply in phagocytic cells.

Uropathogenic E. coli (UPEC) is the most frequent cause of urinary tract infection (UTI), being responsible for up to 85% of community acquired and 40% of nosocomial cases. UPEC strains harbour various virulence factors that contribute to their ability to cause disease. The high prevalence across the globe of multidrug resistant UPEC is a significant threat to therapy. Virulent and resistant UPEC strains have been recognised as belonging to major lineages and we have only recently begun to understand the factors contributing to their successful global dissemination. Work in this thesis was carried out to identify the population structure of E. coli isolates recovered from urosepsis and biliary sepsis, to reveal any differences in genetic background. A total of 100 isolates from the blood and urine of 50 patients presenting with urosepsis and 27 isolates from cases of biliary sepsis were subjected to genotypic and phenotypic analysis, including MLST, virulence gene detection and antibiogram and metabolic profiling. Urosepsis paired isolates showed identical genotypes and antimicrobial resistance profiles. However, several pairs of isolates showed discrepant metabolic activity profiles suggesting niche specific regulation of metabolism. Members of the ST131 clone were significantly associated with antibiotic resistance and ST38 isolates were associated with the highest level of metabolic activity. An in vivo infection model was used to investigate the virulence potential of isolates from the major UPEC lineages. Galleria mellonella larvae inoculated with ST69 and ST127 isolates showed significantly higher mortality rates than those infected with other strains. However, one isolate of ST127 (strain EC18) was avirulent and comparative genomic analyses with a single virulent ST127 strain revealed an IS1 mediated deletion in the O-antigen cluster in strain EC18, which is likely to explain the lack of virulence in the larvae and demonstrates the importance of this cell surface molecule in the model system. Finally, a total of 202 UPEC isolates were recovered from community and hospital urine samples from a tertiary care hospital in Riyadh, Saudi Arabia. Molecular epidemiological investigation of the strains was carried out to examine the overall UPEC population structure, for the first time in any part of Saudi Arabia. The most common lineages were ST131 (17.3%), ST73 (11.4%), ST38 (7.4%), ST69 (7.4%) and ST10 (6.4%). The findings highlight the successful spread of multidrug resistant, CTX-M positive ST38, ST131 and ST405 UPEC in Saudi Arabia. The high proportion (35%) of ESBL producing E. coli isolates is a particular concern and is driving frequent prescription of carbapenem antibiotics. A total of four isolates of ST38 were positive for aggR, which is a virulence marker of enteroaggregative E. coli (EAEC); ST38 strains that cause UTI but have an EAEC genetic background are becoming recognised as novel UPEC and this clonal group warrants further study.

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>

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.

The prevalence of antibiotics on farms for therapeutic and prophylactic use in animals can cause negative effects on biomethane production during anaerobic digestion. Previous literature has found decreased biomethane production rates from a variety of antibiotics, but biogas inhibition differs between studies of continuous and batch reactors and the type of antibiotic studied. Cephalosporin drugs are the most common antibiotic class used to treat mastitis in dairy cows and can retain most of their bioactivity after excretion. Ceftiofur is a commonly used cephalosporin drug but no previous study investigating the effect of Ceftiofur on biomethane during continuous anaerobic digestion has been performed. The aim of this study was to examine the effect on biomethane production when manure from cows treated with Ceftiofur was anaerobically digested. Laboratory sized anaerobic digesters (AD) were run at thermophilic (55°C) temperatures and a 10 day hydraulic retention time. Manure from cows treated with Ceftiofur were fed to the antibiotic treatment reactors for 50 days. The reactor performance was measured by i) biomethane production, ii) waste stabilization in terms of solids and chemical oxygen demand, iii) change in mass of Ceftiofur and iv) change in concentration of antibiotic resistant genes, specifically cfx(A), mef(A), and tet(Q). There was statistically significant decrease in cumulative gas production due to the addition of Ceftiofur into the reactors, but no significant difference between treatments in waste stabilization in terms of percent volatile solids (VS) and total chemical oxygen demand (TCOD) reduction. Anaerobic digestion decreased the amount of Ceftiofur in manure, and the amount of Ceftiofur in the reactors reduced over the time of the experiment. Change in antibiotic resistant genes (ARGs) were gene dependent over time. Concentrations of tet(Q) reduced significantly between feed and effluent of both treatments, and cfx(A) reduced significantly for the control treatment but not the Ceftiofur treatment. Concentrations of mef(A) increased over time in both treatments. Overall, the addition of Ceftiofur in continuously operated anaerobic digesters negatively affected biomethane production, a value-added product responsible for on-farm renewable energy. However, anaerobic digestion does decrease the mass of Ceftiofur within manure, thereby reducing the environmental loading from run-off from farms. / Master of Science / Anaerobic digestion is a biological treatment technology used on farms to treat manure. It can be used to reduce potential environmental damage from contaminants and manure, homogenize manure for fertilizer, and produce methane gas for renewable energy. An emerging challenge in manure management is the presence of antibiotics such as ceftiofur used in animal production to prevent and treat illnesses. When antibiotics are used on livestock, they are excreted from the animal in manure. When the manure is added to the digester, the antibiotic molecules within the manure can kill the bacteria responsible for manure homogenization and gas production. Ceftiofur is a type of cephalosporin antibiotic used to treat dairy cows for mastitis, a bacterial infection of the udder. When the cows are treated with Ceftiofur, it can remain in the excreted manure and enter the digester. The use of antibiotics on farms is also leading to a global phenomenon known as antibiotic resistance. The bacteria that are exposed to antibiotics can develop mutations to become immune to the antibiotic, and can spread the mutations through antibiotic resistant genes (ARGs). ARGs can spread to bacteria which have never been exposed to antibiotics, making them resistant. This causes a significant concern in regards to disease treatment across the world as the efficacy of antibiotics is threatened. Understanding how ARGs move and how they can be eliminated is crucial to preventing global antibiotic resistance. The purpose of this study was to assess the effect of anaerobic digestion on Ceftiofur and ARGs. Four continuous lab-scale anaerobic digesters, two using control manure and two using manure from cows treated with Ceftiofur, were run at 55˚C for a period of 50 days. Over time, the reactor with manure from cows treated with the Ceftiofur antibiotic produced less gas as compared to the control digesters. The amount of Ceftiofur within the digesters decreased over time, demonstrating anaerobic digestion’s ability to degrade the antibiotic molecule. The effect of anaerobic digestion on the ARG concentration was gene specific. The concentration of the tet(Q) gene, a gene responsible for resistance against the very common antibiotic tetracycline, was reduced by anaerobic digestion. These results demonstrate that anaerobic digestion is a technology which can reduce the environmental impact of manure from Ceftiofur-treated cows. This shows that manure treatment can be a first step in combating antibiotic resistance across the globe.