Escherichia coli and Klebsiella pneumoniae isolates were obtained from the Canadian Ward Surveillance Study (CANWARD) and underwent in vitro susceptibility testing to determine prevalence and antimicrobial resistance patterns. The prevalence was found to be relatively stable over the years although there was an increase in prevalence among the K. pneumoniae isolates; 1.1% to 1.3% to 2.5% to 2.6% in 2007, 2008, 2009, and 2010, respectively. Genotypic characterization was conducted on ESBL, AmpC, carbapenemase genes, and outer membrane porins. The highest proportion of isolates were found to produce CTX-M-15 β-lactamase. Only 1 of each KPC-producing E. coli and K. pneumoniae was found. Porin alteration was found to be a factor leading to carbapenem reduced susceptibility among isolates. Genetic relatedness of CRS/CIR E. coli and K. pneumoniae was determined using pulsed-field gel electrophoresis. The spread of these organisms was mainly due to polyclonal spread rather than one specific clone.

Escherichia coli and Klebsiella pneumoniae isolates were obtained from the Canadian Ward Surveillance Study (CANWARD) and underwent in vitro susceptibility testing to determine prevalence and antimicrobial resistance patterns. The prevalence was found to be relatively stable over the years although there was an increase in prevalence among the K. pneumoniae isolates; 1.1% to 1.3% to 2.5% to 2.6% in 2007, 2008, 2009, and 2010, respectively. Genotypic characterization was conducted on ESBL, AmpC, carbapenemase genes, and outer membrane porins. The highest proportion of isolates were found to produce CTX-M-15 β-lactamase. Only 1 of each KPC-producing E. coli and K. pneumoniae was found. Porin alteration was found to be a factor leading to carbapenem reduced susceptibility among isolates. Genetic relatedness of CRS/CIR E. coli and K. pneumoniae was determined using pulsed-field gel electrophoresis. The spread of these organisms was mainly due to polyclonal spread rather than one specific clone.

As antibiotic resistant bacterial strains are becoming more prevalent in healthcare settings, it is necessary to find alternative methods of detecting and treating these infections. One of the antibiotic resistant strains of interest is the carbapenem-resistant Enterobacteriaceae (CRE). CREs have the ability to evade some of the most potent antibiotics currently in use and employ carbapenemases to negate the effect of antibiotics. The three most common carbapenemase genes, found in carbapenem-resistant Enterobacteriaceae along with a gene found only in Escherichia coli were chosen to create a qPCR assay for rapid detection of resistant infections. The carbapenemase genes are KPC, VIM and NDM and the E. coli gene is uidA, a β-glucuronidase gene. Consensus sequences were obtained from each of the genes to account for the many variants of each gene. We were able to triplex the assay and test it against a library for twenty isolates varying by which gene they contain. Additional research has been conducted on the library of carbapenem-resistant Enterobacteriaceae using bacteriophages or phage. The Phage Hunters class isolated and identified twenty phage that infect K. pneumoniae. Out of the twenty phage, seven phage were able to effectively infect carbapenem-resistant K. pneumoniae.

Background: Gram-negative resistance is a growing problem worldwide. It is generally believed that rates of resistant bacteria within a hospital are a function of antibiotic use, resistant organisms brought into the hospital, infection control efforts, and underlying severity of patient illness. The relative contribution of each to a particular resistance phenotype is unclear. P. aeruginosa is responsible for many hospital acquired infections and it may become resistant to carbapenems. In addition, newer threats to the future utility of the carbapenems are carbapenemase-producing K. pneumoniae Purpose: To determine if there is an association between the volume and composition of antibiotic use, geography, severity of illness and rates of carbapenem-resistant P. aeruginosa and K. pneumoniae. Methods: This is a retrospective ecological longitudinal investigation within the University HealthSystem Consortium affiliated academic medical centers. Antibiotic use data between January 1, 2006 and December 31, 2009 were obtained from billing records and reported as days of therapy per 1000 patient days (DOT/1000 PD), in addition to hospital characteristics (e.g. geographical location, bed size, case mix index). “Whole house” antibiograms were obtained to determine rates and proportions of carbapenem-resistant P. aeruginosa (CR-PA) and carbapenem resistant K. pneumoniae (CR-KP). Also, CR-KP isolation was generated as a binary outcome. Generalized estimating equations (GEE) were used to model CR-KP and CR-PA. Results: CR-KP rates (1000PDs) increased from 0.07 in 2006 to 0.15 in 2009 (P= 0.0118) and CR-KP proportions increased from 1.3% in 2006 to 3.1% in 2009 (0.0003) within 40 hospitals over 2006-2009. However, CR-PA rates and proportions were stable over the same period. Geographical location, carbapenems use, and antipseudomonal penicillins use were significantly associated with CR-KP isolation. Thus, for every ten DOT/1000 PDs increase in carbapenem use, the odds of CR-KP isolation increased by 42% (P=0.0149). In contrast, for every ten DOT/1000 PDs increase in antipseudomonal penicillin use, the odds of CR-KP isolation decreased by 14%. However, there was no significant model to explain CR-PA rates and proportions. Conclusion: Carbapenems, antipseudomonal penicillins, and geographical location were identified as risk factors associated with CR-KP isolation. These findings emphasize the challenges associated with the treatment of multidrug- gram-negative bacteria.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

Background: Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) such as Escherichia coli and Klebsiella pneumoniae are among the most urgent threats of the infectious disease realm. The incidence of these infections has been increasing over the years and due to very limited treatment options, mortality is estimated at about 50%. By 2050, mortality from antimicrobial resistant infections is expected to surpass cancer at 10 million deaths annually. Methods: We evaluated 18 contemporary antimicrobials against 122 carbapenem-resistant Enterobacteriaceae using a variety of antimicrobial susceptibility testing methods according to Clinical Laboratory Standards Institute guidelines. Time-kill studies were performed on clinical isolates with variable resistance to meropenem, amikacin, and polymyxin B. Phenotypic expression assays were performed on all isolates and whole genome sequencing was performed on 8 isolates to characterize molecular resistance mechanisms. Pharmacodynamic modeling of meropenem and polymyxin B was also conducted. Results: CRE were primarily K. pneumoniae, and Enterobacter spp. 60% expressed Klebsiella pneumoniae carbapenemase (KPC) only, 16% expressed Verona Integron-encoded Metallo-beta-lactamase (VIM) only, 5% expressed KPC and VIM, and 20% expressed other mechanisms of resistance. Antimicrobial susceptibility testing indicated the most active antimicrobials against CRE were ceftazidime/avibactam, imipenem/relebactam, amikacin, tigecycline, and the polymyxins. Etest® strips did not reliably measure polymyxin B resistance. The automated testing system, BD Phoenix™, consistently reported lower MICs than the gold standard broth microdilution. Time-kill studies showed regrowth at clinically achievable concentrations of meropenem alone (4, 16, and 64 mg/L), polymyxin B alone (0.25 and 1 mg/L), or amikacin alone (8 and 16 mg/L), but combinations of meropenem with either polymyxin B or amikacin were bactericidal and synergistic. Meropenem administered simultaneously or prior to polymyxin B exhibited superior activity to polymyxin B administered first. Conclusions: Novel carbapenemase-inhibitor combinations (ceftazidime/avibactam and imipenem/relebactam) exhibit the best activity against KPC-producing CRE. The polymyxins, amikacin, and tigecycline exhibit the best activity against VIM-producing CRE. Meropenem in combination with polymyxin B is bactericidal and synergistic when the meropenem MIC is ≤32 mg/L, and meropenem should never be administered after polymyxin B. Meropenem and amikacin is bactericidal and synergistic when the amikacin MIC is ≤16 mg/L. Etest® strips should not be used for characterizing polymyxin B or colistin activity. Clinicians should be aware that automated testing systems may produce biased susceptibility results relative to the gold standard method, broth microdilution, which may influence interpretation of in vitro results.

Orientador: Carlos Magno Castelo Branco Fortaleza / Resumo: Atualmente, estamos diante de uma notável presença de isolados de enterobactérias resistentes aos carbapenêmicos em um hospital público do município de Bauru-SP desde outubro de 2012. No entanto, não estão disponíveis estudos relacionando a epidemiologia e os fatores associados à aquisição de tais isolados. Este estudo teve como objetivo identificar fatores de risco para aquisição de Enterobactérias Resistentes aos Carbapenêmicos (CRE) em pacientes internados no Hospital Estadual Bauru, os fatores associados ao desenvolvimento de quadro infeccioso em uma coorte de pacientes colonizados por CRE e os fatores preditores de óbito. Foram incluídos pacientes do local de estudo que apresentaram colonização do trato digestório por CRE, de outubro de 2012 a dezembro de 2016, dos quais foram levantados dados clínicos e demográficos. Os isolados foram identificados por métodos fenotípicos e foram testadas as suscetibilidades por concentração inibitória mínima (MIC). Realizamos um estudo de caso-controle que incluiu 427 casos e igual número de controles. Os fatores de risco observados foram queimadura (HR 3,91; IC95% 2,36-6,46; p=<0,001), índice de Charlson (HR 1,12; IC95% 1,05-1,20; p=<0,001), uso prévio de esteróides (HR 2,79; IC95% 1,94-4,02; p=<0,001) e antimicrobianos como as penicilinas/inibidores de beta-lactamases (HR 2,01; IC95% 1,43-2,82; p=<0,001), cefalosporinas de 3ª. e 4ª. gerações (HR 2,45; IC95% 1,75-3,44; p=<0,001), quinolonas (HR 1,70; IC95% 1,75-2,45; p=0,003) e anaero... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently, we are facing a remarkable presence of isolates of carbapenem-resistant enterobacteriacea in Bauru city public hospital, São Paulo state, Brazil, since october 2012. However no studies are available relating the epidemiology and the factors associated with acquisition of such isolates. The purpose this study was to identify risk factors for acquisition of Carbapenem Resistant Enterobacteriaceae in patients hospitalized at the Bauru State Hospital, factors associated with the development of infectious disease in a cohort of patients colonized by CLP and factors predicting death. We included patients from the study site who presented colonization of the digestive tract by CRE, from October 2012 to December 2016, from which clinical and demographic data were collected. Isolates were identified by phenotypic methods and susceptibilities were tested by minimum inhibitory concentration (MIC). We performed a case-control study that included 427 cases and an equal number of controls. The risk factors observed were burn (HR 3.91, 95% CI 2.36-6.46, p = 0.001), Charlson index (HR 1.12, 95% CI 1.05-1.20, p = <0.001), previous use of steroids (HR 2.79, 95% CI 1.94-4.02, p = <0.001) and antimicrobials such as penicillins / beta-lactamase inhibitors (HR 2.01, 95% CI, 43-2.82, p = <0.001), cephalosporins of 3rd. and 4ª. (HR 2.45, IC 95% 1.75-3.44, p = 0.001), quinolones (HR 1.70, IC 95% 1.75-2.45, p = 0.003) and anaerobicides (HR 1, 63, 95% CI 1.04-2.56, p = 0.03). The cohort stud... (Complete abstract click electronic access below) / Doutor

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

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