Background. Carbapenem-resistant Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, are newly emerging pathogens of public health importance. Currently no nationally representative or mandatory surveillance or reporting system exists to examine trends of these important pathogens. Objective. The purpose of the current study was to estimate trends in overall microbial burden and carbapenem resistance in E. coli and K. pneumoniae and to understand the extent to which hospitals which report to voluntary surveillance systems represent all hospitals in the United States. Design. We conducted a descriptive study to compare the hospitals participating in voluntary reporting systems of the University HealthSystem Consortium and the National Healthcare Safety Network with the Healthcare Utilization Project’s Nationwide Inpatient Sample, a nationally representative sample of hospital discharges. Methods. Descriptive analyses examined hospital characteristics (region, bed size, hospital control, teaching status, case mix index) and patient characteristics (age, sex, race/ethnicity, admission source, admission type, discharge status, primary payer) of participant hospitals versus all US hospitals. ICD-9-CM codes identified discharges coded for E. coli and K. pneumoniae diagnoses; linear regression was used to evaluate trends in overall microbial burden of E. coli and K. pneumoniae in all US Hospitals and US Academic Centers. Trends in E. coli and K. pneumoniae resistance to carbapenem were also evaluated in hospitals participating in voluntary surveillance systems (n=13). Results. Between 2002 and 2007, slight increasing trends in burden of both E. coli and K. pneumoniae were observed (E. coli: slope = 0.0537; K. pneumoniae slope = 0.0168). Hospitals participating in voluntary surveillance systems are larger and care for fewer elderly patients than all US hospitals. Conclusions. These results suggest that hospitals that participate in voluntary surveillance systems like the National Healthcare Safety Network and the University HealthSystem Consortium may underrepresent trends in smaller hospitals, as well as those that treat elderly patients. Increasing overall burden of infection due to these isolates only reinforces the importance carbapenem resistance in E. coli and K. pneumoniae. This important public health threat may warrant the creation of a national, mandatory reporting system for these and other antimicrobial resistant organisms.

Master of Science / Department of Biomedical Science / H. Morgan Scott / The modern phenomenon of increasing prevalence of antibiotic resistance in clinically relevant bacteria threatens humanity’s ability to use antibiotics to treat infection in both humans and animals. Despite the marked complexity of bacterial evolution, there is tremendous importance in unfolding the process by which antibiotic resistance genes emerge, disperse, and persist in the natural world. This thesis investigates certain aspects of this process in two experimental studies that differ primarily by scale but also by methodology. The first study examined the long-term annual prevalence of ceftiofur and tetracycline resistance in Canadian beef cattle from 2002 to 2011 at both phenotypic and genotypic levels. Ceftiofur was present at a very low prevalence (<4%) that did not statistically increase over the decade (p<0.05). Relative proportions of tetracycline genes tet(A), tet(B), and tet(C) also did not significantly change over the observation period. However, it was surprising that almost 20% of isolates recovered from nonselective agar harbored tet(C) given that current literature generally indicates that tet(C) is significantly less prevalent than tet(A) or tet(B). The usage of historical samples in addition to parallel selective plating using agar supplemented with antibiotics provided insight into systemic bias present in common microbial approaches. Long-term sample freezing significantly diminished the recoverability of E. coli over time. Additionally the usage of selective MacConkey agar containing tetracycline biased the proportions of tetracycline genes to over-represent the tet(B) gene in commensal E. coli compared to nonselective MacConkey agar. The second study attempted to explain the short-term selection effects of antibiotic treatment on the overall ecological fitness of commensal E. coli using bacterial growth parameters estimated from spectrophotometric growth curves as a simple surrogate of general fitness. Treating cattle with either tetracycline or ceftiofur was found to not only select in favor of tetracycline resistant bacteria, but also increased the overall fitness among the tetracycline resistant population. However, growth curves were unable able to explain why transiently selected resistant bacteria were eventually replaced by susceptible bacteria once the selection pressure was removed.

Made available in DSpace on 2016-08-10T10:53:36Z (GMT). No. of bitstreams: 1 MARCIANO DE SOUSA NOBREGA.pdf: 616658 bytes, checksum: 0673ba9db2600506e22ee2169eef0af7 (MD5) Previous issue date: 2012-05-14 / The increase in bacterial resistance occurs in all regions of the world, especially in critically ill patients hospitalized in intensive care units (ICUs) and that make use of several antimicrobials. Bacterial resistance complicates therapy prolongs the ICU stay and increased morbidity and mortality. Among the microorganisms that cause infections in ICUs, we highlight P. aeruginosa and A. baumannii, gram-negative high incidence of nosocomial infections and have shown a tendency to increased antimicrobial resistance. OBJECTIVE: To evaluate microbial and bacterial resistance in adult intensive care agents P. aeruginosa and A. baumannii. MATERIALS AND METHODS: Retrospective study from January 2007 to December 2010 on the sensitivity and P. aeruginosa and A. baumannii in adult ICU patients (medical and surgical) of HC / UFG, with a diagnosis of hospital infection. We analyzed the evolution of resistance, antimicrobial consumption, mortality, topography of infections and length of stay in the units. 121 cases have been cataloged. RESULTS: The mean age of patients was 51.2 ± 17.9 years, male 45.45% and 55.55% female, mean ICU stay was 26.99 days, compared with 6.22 days of the general population unit. The mortality rate was 37.19% compared to a rate of 27.17% of the population. The primary site of infection was the respiratory tract with 41.3% followed by infection of the bloodstream with 27.27%. The initial average of bacterial resistance related of P. aeruginosa was close to 50% and A. baumannii was greater than 80%, with no significant modifications to the P. aeruginosa in this period. There was a significant increase in resistance to A. baumannii to amikacin. Consumption of antimicrobial agents showed an increase of antimicrobials amikacin, imipenem and piperacillin-tazobactam in cases of infection by P. aeruginosa infections and imipenem in A. baumannii. No correlation was found between bacterial resistance and antimicrobial consumption. CONCLUSION: Mortality and length of stay were higher in the study group. The bloodstream and respiratory tract were the main sites of infection by A. baumannii and P. aeruginosa multiresistant. The susceptibility profile revealed that P. aeruginosa and A. baumannii are highly resistant to antimicrobials. There were no significant changes in the evolution of antimicrobial resistance to the antibiotics tested, except for amikacin used in infections caused by A. baumannii. The consumption of antimicrobials showed increased consumption of amikacin, imipenem and piperacillin-tazobactam in patients infected with P. aeruginosa and to A. baumannii increased consumption was only to imipenem. The relationship between consumption of antimicrobials and increased bacterial resistance was not identified. / O aumento da resistência bacteriana ocorre em todas as regiões do mundo, principalmente em pacientes graves, internados em unidades de terapia intensiva (UTIs) e que fazem uso de vários antimicrobianos. A resistência bacteriana dificulta a terapia, prolonga a permanência nas UTIs e aumenta a morbimortalidade. Dentre os microrganismos causadores de infecções em UTIs, destacam-se P. aeruginosa e A. baumannii, gram-negativos de alta incidência em infecções hospitalares e que vêm apresentando aumento da resistência aos antimicrobianos. OBJETIVO: Avaliar os aspectos microbiológicos e a resistência bacteriana em UTIs dos agentes P. aeruginosa e A. baumannii. MATERIAL E MÉTODOS: Estudo retrospectivo, de janeiro de 2007 a dezembro de 2010, sobre o perfil de sensibilidade e P. aeruginosa e de A. baumannii em pacientes de UTIs adulto (clínica e cirúrgica) do HC/UFG, com diagnóstico de infecção hospitalar. Analisou-se a evolução da resistência, consumo de antimicrobianos, mortalidade, topografia das infecções e tempo de permanência nas unidades. Foram catalogados 121 casos. RESULTADOS: A média de idade dos pacientes foi 51,2±17,9 anos, sendo 45,45% do sexo masculino e 55,55% do sexo feminino; a média de permanência na UTI foi de 27 dias, comparado com 6,22 dias da população geral da unidade. A taxa de mortalidade foi de 37,19% ante uma taxa de 27,17% da população geral. O principal local de infecção foi a via respiratória com 41,3% seguido pela infecção de corrente sanguínea com 27,27%. A taxa média inicial de resistência bacteriana da P. aeruginosa foi próxima de 50% e A. baumannii foi superior a 80%, não se observando modificações para a P. aeruginosa nesse período. Houve um aumento significativo na resistência para o A. baumannii para amicacina. O consumo de antimicrobianos apresentou aumento dos antimicrobianos amicacina, imipenem e piperacilina-tazobactam nos casos de infecção por P. aeruginosa e imipenem nas infecções por A. baumannii. Não foi encontrada correlação entre a resistência bacteriana e o consumo de antimicrobianos. CONCLUSÃO: A mortalidade e o tempo de permanência foram maiores no grupo em estudo. A corrente sanguínea e o trato respiratório foram os principais sítios de infecção por A. baumannii e P. aeruginosa multirresistentes. O perfil de susceptibilidade revelou que P. aeruginosa e A. baumannii são altamente resistentes aos antimicrobianos testados. Não houve mudanças significativas na evolução da resistência aos antimicrobianos para os antibióticos testados, exceto para a amicacina usada nas infecções por A. baumannii. O consumo de antimicrobianos aumentou para amicacina, imipenem e piperacilina-tazobactam, nos pacientes com infecção por P. aeruginosa e, para A. baumannii o aumento do consumo foi apenas para o imipenem. A relação entre o consumo de antimicrobianos e o aumento da resistência bacteriana não foi identificada.

Antimicrobial resistance (AMR) is a worldwide public health concern. While it is evident that the use of antibiotics creates selection pressure for the evolution of antibiotic resistance genes, there are still considerable knowledge gaps relating to the status quo of antibiotic use, emergence of resistant pathogens in different livestock production systems and spread within human and animal communities. This thesis includes a survey of antibiotic use in the dairy sector within a specific area of Zambia and analysis of AMR and virulence factors in E. coli isolated from dairy cattle and diarrhoea human patients with the following objectives. 1. To investigate the usage of antibiotics in the dairy sector and the drivers for use. 2. To determine the prevalence and patterns of antimicrobial resistance in E. coli isolated from faecal samples of dairy cattle. 3. To use whole genome sequencing (WGS) to investigate the molecular epidemiology of resistance determinants in E. coli strains isolated from both dairy cattle and humans. 4. To assess the zoonotic potential of isolated E. coli focusing on Shiga toxin-producing E. coli (STEC) and relationship to STEC associated with clinical disease in the UK. In view of these objectives, the first part of the work was carried out in Zambia and involved a questionnaire, a field survey, isolation of E. coli from dairy cattle faecal samples and phenotypic testing for AMR. In addition, E. coli isolates were obtained from another study that was focused on human patients presenting with diarrhoea at the University Teaching Hospital in Lusaka. The second part involved whole genome sequencing and molecular analyses of E. coli for resistance and virulence genotypes at the Roslin Institute (UK). For the field study, a stratified random sample of 104 farms was studied, representing approximately 20% of all dairy farms in the region. On each farm, faecal samples were collected from a random sample of animals and a standardised questionnaire on the usage of antibiotics was completed. An E. coli isolate was obtained from 98.67% (371/376) of the sampled animals and tested for resistance against the six types of antibiotics (tetracycline, ampicillin, sulfamethoxazole/trimethoprim, cefpodoxime, gentamicin and ciprofloxacin). These E. coli were then analysed together with those from humans for genotypes in the laboratory and from Illumina short read whole genome sequences using bioinformatics tools. Tetracylines and penicillin were the commonly used antibiotics in dairy herds. This finding was in line with the resistance phenotypes detected in E. coli isolated from the dairy cattle. The most prevalent AMR was to tetracycline (10.61; 95%CI: 7.40-13.82), followed by ampicillin (6.02; 95%CI: 3.31-8.73), sulfamethoxazole/ trimethoprim (4.49; 95%CI: 2.42-6.56), cefpodoxime (1.91; 95%CI: 0.46-3.36), gentamicin (0.89; 95%CI: 0.06-1.84) and ciprofloxacin (0%). The risk analysis indicated that AMR was associated with livestock diseases (lumpy skin disease and foot rot), exotic breeds (Jersey and Friesian), location, farm size and certain management practices. Analysis of whole genome sequences showed that isolates from humans had both higher levels and a greater diversity of resistance alleles than the cattle isolates. Common genotypes in both populations were: tetA (16%), tetB (10%), tetC (2%) for cattle isolates with tetA (32%), tetB (22%) and tetD (1%) in human isolates. Other common genotypes were blaTEM (56%), sul1 (29%), sul2 (66%), strA4 (57%) and strB1 (64%) in isolates of human origin while blaTEM (15%), sul1 (3%), sul2 (17%), strA4 (13%) and strB1 (19%) were in the cattle isolates. Whilst the E. coli isolates from cattle encoded resistance to common antibiotics of limited significance to human clinical medicine, isolates from humans had additional extended spectrum beta-lactamases (blaOXA, blaCMY, blaNDM, and blaDHA, blaOKP and blaCTX-M) that encode for resistance to essential antibiotics such as third generation cephalosporins and carbapenems. This was an evidence that AMR is an ongoing public health subject in Zambia but the exclusivity of certain resistances in the human population points to limited or no exchange of genotypes between E. coli of human origin and those from cattle. AMR in humans was probably independently selected by the use of antibiotics of clinical importance such as cephalosporin and fluoroquinolones. The virulence analysis focused on STEC, 11% (41/371) of E. coli isolates from cattle contained Shiga toxin genes (stx) while none (0/73) of the human isolates were positive. Phylogenetic analysis showed a random distribution of bovine STEC, with no indication of clonal spread. Although 89% (16/18) of the STEC tested had a cytotoxic effect on Vero cells, indicative of Shiga toxin production, only three (O45, O111, O157) belonged to one of the seven serogroups (O26, O157, O111, O103, O121, O145 and O45) associated with life-threatening enterohaemorrhagic E. coli (EHEC) infections in humans. In line with this, only the O157 serotype encoded a type 3 secretion system. This shows that, while Stx-encoding strains are common in these dairy herds of Zambia, they are not strain backgrounds known to pose an immediate threat to human health as they lack colonisation factors that are found in typical human EHEC. However, we must remain vigilant as emergence of EHEC strains in these animals remains an ever-present threat.

A doença decorrente da infecção por Salmonella é um dos maiores problemas de saúde no mundo em termos de morbidade e mortalidade. Entre as sorovariedades de Salmonella, a sorovariedade Enteritidis é a de maior ocorrência mundial e compreende linhagens que tem seu nicho biológico relacionado a frangos e ovos. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas a fim de se delinear a epidemiologia das infecções por S. Enteritidis. Entretanto a tipagem fenotípica usualmente falha em discriminar linhagens relacionadas das nãorelacionadas epidemiologicamente e apresenta problemas de reprodutibilidade que foram minimizados com a utilização de métodos genotípicos. No Brasil, poucos estudos que utilizaram técnicas moleculares na tipagem de linhagens dessa sorovariedade foram realizados. Os objetivos desse estudo foram investigar o potencial patogênico, a resistência a antimicrobianos e realizar a tipagem molecular de linhagens de Salmonella Enteritidis isoladas de humanos, de alimentos e de frangos no Brasil. Para isso foram estudadas 188 linhagens de Salmonella Enteritidis isoladas de surtos e de casos esporádicos, de humanos (67) de alimentos (61) e de frangos (60), durante o período de 1986 a 2010, de vários locais do Brasil. A susceptibilidade frente a 14 antimicrobianos foi analisada através da técnica de disco difusão e a presença de 13 genes de virulência das ilhas de patogenicidade de Salmonella I e II e do plasmídio pSEV foram pesquisados por PCR. Os mecanismos de resistência a quinolonas foram verificados através da pesquisa de genes de resistência plasmidiais e cromossomais e também através da verificação de mutações no gene gyrA por High resolution melting analysis (HRMA) seguida de sequenciamento de algumas linhagens. As linhagens também foram tipadas molecularmente pelas metodologias Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) com a enzima XbaI, Multilocus variable-number tandem repeat analysis (MLVA) e por Multilocus sequence typing (MLST). Das 188 linhagens estudadas, 42,5% foi resistente ao ácido nalidíxico e somente 0,5% foi resistente a sulfametoxazol-trimetoprima e estreptomicina. A resistência a quinolonas foi relacionada principalmente a mutações no gene gyrA. A maioria das linhagens estudadas (98,4%) apresentou todos os genes de virulência pesquisados, sendo uma linhagem negativa para o gene sipA e duas linhagens negativas para o gene prot6E. ERIC-PCR dividiu as 128 linhagens isoladas de humanos e alimentos em 55 perfis diferentes com similaridade >79,7%. PFGE dividiu essas mesmas linhagens em 68 perfis diferentes com uma similaridade >73,1%. Para as linhagens isoladas de frango, o dendrograma concatenado de ERIC-PCR e PFGE dividiu as 60 linhagens em dois grandes grupos com 73,3% de similaridade. O grupo A consistiu de linhagens isoladas tanto de material clínico de frangos (23) quanto do ambiente da granja (5) com 81,2% de similaridade. O grupo B também consistiu de linhagens isoladas tanto de casos clínicos de frangos (21) quanto do ambiente da granja (11) com 81,1% de similaridade. MLVA dividiu as 188 linhagens isoladas no Brasil e outras 100 linhagens isoladas na América do Norte em dois grandes grupos. O grupo MLVA-A apresentou 71 linhagens isoladas na América do Norte e somente três linhagens isoladas no Brasil. Essas linhagens do ii Brasil incluíram as isoladas antes do início da pandemia de S. Enteritidis se iniciar no país. Em contraste, o grupo MLVA-B agrupou 185 linhagens isoladas no Brasil e 29 linhagens isoladas na América do Norte. As linhagens presentes no grupo A, foram divididas em 34 tipos genéticos diferentes com similaridade maior do que 46%, enquanto no grupo B as linhagens se diferenciaram em 15 tipos genéticos diferentes com mais de 66% de similaridade. MLST caracterizou 44 das 46 linhagens estudadas como pertencentes ao ST 11. As outras duas linhagens apresentaram alelos que não existiam no banco de dados e caracterizaram dois novos STs, o 1632 e o 1633. Os resultados de tipagem molecular obtidos por ERICPCR, PFGE e MLVA no presente estudo, demonstraram uma alta similaridade genotípica entre linhagens de S. Enteritidis isoladas no Brasil, o que sugere que as linhagens estudadas descendem de um precursor comum que pouco se diferenciou genotipicamente ao longo de 24 anos no país. Ademais, os resultados de MLVA sugerem que um novo e prevalente subtipo foi introduzido no Brasil após 1993 e tem contaminado alimentos e infectado humanos e animais. O grande número de genes de virulência encontrados reforça o potencial das mesmas causarem doenças em humanos e animais, bem como, os riscos de sua presença em alimentos. Ademais, a grande porcentagem de linhagens resistentes ao ácido nalidíxico observadas a partir de 1996 sugere o uso de quinolonas no tratamento de infecções em animais causadas por S. Enteritidis no Brasil. / The disease caused of the infection by Salmonella is one of the major health problem worldwide in terms of morbid and mortality. Among the Salmonella serovars, the Enteritidis is the most frequent isolated one and comprises strains that have their biological niche related to chickens and eggs. Several phenotypic and genotypic methodologies were developed to trace epidemiologically the infections by S. Enteritidis. However, the phenotypic typing usually fail to discriminate related from unrelated epidemiologicaly strains and presents problems of reproducibility that were minimized with the introduction of genotypic methods. In Brazil, few studies that used molecular typing techniques to type strains of this serovar were conducted. The aims of this study were to investigate the pathogenic potential, the antimicrobial resistance and to molecularly type of Salmonella Enteritidis strains isolated from humans, food and chickens in Brazil. For this, it was studied 188 strains of Salmonella Enteritidis isolated from outbreaks and sporadic cases, from humans (67), food (61) and chickens (60), during the period of 1986 to 2010, from various places of Brazil. The susceptibility to 14 antimicrobials were analyzed by the disc diffusion technique and the presence of 13 virulence genes of the Salmonella pathogenicity islands I and II and from the pSEV plasmid were searched by PCR. The mechanisms of resistance to quinolones were verified by the search of plasmidial and cromossomal resistance genes and also by the verification of mutations in the gyrA gene by High resolution melting analysis (HRMA) followed by sequencing of some strains. The strains were also molecularly typed by the methodologies Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) using the enzyme XbaI, Multilocus variable-number tandem repeat analysis (MLVA) and by Multilocus sequence typing (MLST). From the 188 strains studied, 42.5% were resistant to nalidixic acid and only 0.5% were resistant to sulfamethoxazoletrimethoprim and streptomycin. Resistance to quinolones was related mainly to mutations in the gyrA gene. The majority of the strains studied (98.4%) harbored all the virulence genes searched, being only one strain negative for the sipA gene and two strains negative for the prot6E gene. ERIC-PCR divided the 128 strains isolated from humans and food in 55 different profiles with >79.7% of similarity. PFGE divided the same strains in 68 different profiles with a similarity of >73.1%. Regarding the strains isolated from chickens, the concatenated dendrogram of ERIC-PCR and PFGE divided the 60 strains in two major groups with a similarity of 73.3%. Group A consisted of strains isolated either from chicken\'s clinical samples (23) or from the farm environment (5) with a similarity of 81.2%. Group B also consisted of strains isolated either from chicken\'s clinical samples (21) or from the environment (11) with a similarity of 81.1%. MLVA divided the 188 strains isolated in Brazil and other 100 strains isolated from North America in two major groups. MLVA-A group consisted of 71 strains isolated in North America and only three strains isolated in Brazil. These strains from Brazil included the ones isolated before the beginning of the pandemic of S. Enteritidis in this country. In contrast, MLVA-B group clustered 185 strains isolated in Brazil and 29 strains isolated in North America. The strains in the MLVA-A group were divided in 34 different genotypic types with a similarity of 46%, while strains in iv the group B were divided in 15 different genotypic types with a similarity of 66%. MLST characterized 44 of the 46 strains studied as belonging to ST 11. The other two strains presented new alleles that characterized two new STs, the 1632 and the 1633. The results of molecular typing obtained by ERIC-PCR, PFGE and MLVA in this study showed a high genotypic similarity among S. Enteritidis strains isolated in Brazil, which suggests that the strains studied descend from a common ancestor that differed little genotypically during 24 years in the country. Moreover, the results of MLVA suggest that a new and prevalent subtype was introduced in Brazil after 1993 and has been contaminating food and infecting humans and animals. The high prevalence of virulence genes found in the strains studied reinforce their potential to cause disease in humans and animals, as well as the risks of their presence in food. Moreover, the high percentage of strains resistant to nalidixic acid observed after 1996 suggests the use of quinolones in the treatment of animal infections by S. Enteritidis in Brazil.

Listeria monocytogenes é um importante patógeno de origem alimentar que causa listeriose, infecção severa que acomete, principalmente, gestantes, idosos, crianças e imunocomprometidos, e que apresenta elevada taxa de mortalidade. A bactéria está amplamente distribuída no ambiente e é comumente encontrada em produtos cárneos. O presente estudo teve como objetivos caracterizar 439 isolados de L. monocytogenes obtidos de salsicha bovina e produtos cárneos crus (carne moída, linguiça suína e coxa de frango) refrigerados, adquiridos no comércio do município de São Paulo, e previamente submetidos à sorotipagem molecular. Os isolados foram caracterizados quanto ao perfil de susceptibilidade antimicrobiana; presença dos genes de virulência actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB e mpl; perfil genético por eletroforese em campo pulsado (PFGE) e sequenciamento parcial dos genes actA e lmo0737. Baixa frequência de resistência antimicrobiana (0,5%) foi observada entre os 416 isolados avaliados. Um isolado pertencente ao sorogrupo 1 apresentou resistência à penicilina e à clindamicina e outro identificado como 4a ou 4c apresentou resistência à tetraciclina. Todos os isolados foram positivos para os genes de virulência testados. O sequenciamento parcial do gene actA mostrou a ocorrência de 14 sequências de nucleotídeos distintas nos 97 isolados avaliados. Além disso, verificou-se a ocorrência de uma deleção de 35 aminoácidos no gene actA em 36 isolados, além de substituições de nucleotídeos que resultaram em mutações nas sequências de aminoácidos da grande maioria dos isolados. A análise filogenética do gene actA possibilitou o agrupamento dos isolados em duas linhagens distintas (I e II). Os resultados do PFGE indicaram grande variabilidade nos perfis genéticos dos isolados analisados, principalmente naqueles pertencentes aos grupos 2 (1/2c e 3c), 3 (1/2b e 3b) e 4 (4b, 4d e 4e). Os resultados deste estudo mostram que os isolados de L. monocytogenes provenientes de salsicha bovina e produtos cárneos crus comercializados no município de São Paulo, apresentam grande diversidade genética, importante potencial de virulência e baixa frequência de resistência antimicrobiana. A diversidade observada deve-se, provavelmente, à característica ubíqua deste micro-organismo, tornando-o mais susceptível a grande pressão seletiva do ambiente. / Listeria monocytogenes is an important foodborne pathogen that causes listeriosis, a severe infection that affects primarily pregnant women, elderly, children and imunocompromised individuals, and has a high mortality rate. The bacteria is widely distributed in the environment and commonly found in meat products. The present study aimed to characterize 439 isolates of L. monocytogenes obtained from pork sausage and raw chilled meat products (ground beef, beef sausage, and chicken thigh) purchased in supermarkets in the city of São Paulo, and previously submitted to molecular serotyping. The isolates were characterized for antimicrobial susceptibility profile; presence of virulence genes actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB and mpl; genetic profile by pulsed field gel electrophoresis (PFGE) and partial sequencing of genes actA and lmo0737. A low frequency of antimicrobial resistance (0.5%) was observed among the 416 evaluated isolates. One isolate belonging to serogroup 1 presented resistance to clindamycin and penicillin and another one identified as 4a or 4c was resistant to tetracycline. All isolates were positive for the tested virulence genes. The partial sequencing of the gene actA indicated the occurrence of 14 distinct nucleotide sequences in the 97 isolates tested. Furthermore, a deletion of 35 amino acids in the actA gene was detected in 36 isolates, and nucleotide substitutions that resulted in amino acid changes in the sequences of most isolates. Phylogenetic analysis of the actA gene clustered the isolates in two distinct lineages (I and II). Results of PFGE indicated a great genetic variability among isolates, especially among those belonging to groups 2 (1/2c and 3c), 3 (1/2b and 3b) and 4 (4b, 4d and 4e). The results of this study show that isolates of L. monocytogenes from pork sausage and raw meat products marketed in the city of São Paulo present a great genetic diversity, significant virulence potential and low frequency of antimicrobial resistance. The detected diversity is probably due the ubiquitous nature of these microorganisms, making them more susceptible to selective pressure of the environment.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-10-03T16:25:38Z No. of bitstreams: 1 2016 - Viviane Figueira Marques.pdf: 1883187 bytes, checksum: 9996d5ea41ecd4f93424c808c4d82ba6 (MD5) / Made available in DSpace on 2017-10-03T16:25:38Z (GMT). No. of bitstreams: 1 2016 - Viviane Figueira Marques.pdf: 1883187 bytes, checksum: 9996d5ea41ecd4f93424c808c4d82ba6 (MD5) Previous issue date: 2016-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Staphylococcus spp. plays an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant specie due to the production of virulence factors such as ?slime?, which is required for biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolates from bovine mastitis, to detect and quantify the expression of genes involved in its production and regulation, as well as to detect the phenogenotypic resistance to beta-lactam in order to evaluate the possible relation between biofilm production and antimicrobial resistance. The isolates were characterized by MALDI-TOF and phenogenotypic identification assays. Also they were submitted to the phenotypic tests to evaluate biofilm production and the susceptibility to beta-lactams. Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration in the Biofilm (MICB) were determined to three isolates presenting distinct biofilm production. Futherly, a PCR for the detection of ?slime? production genes (icaA and icaD), Bap protein (bap), beta-lactamase (blaZ) and protein altered penicillin-binding (mecA). Also, the Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Scanning Electron Microscopy (SEM) was performed to determine the most suitable time interval for biofilm analysis. Real-time PCR (qPCR) was performed at the chosen times to quantify the expression of icaA, icaD and hld genes in the three studied isolates. All 20 isolates were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II presented a prevalence of 70%. The SEM showed gradual changes in bacterial arrangement during the biofilm formation along the phases of the growth curve. The peak was reached at the stationary phase. Transcriptional analysis revealed increased expression of ica genes at 8 h of growth and of hld at 24 h. However, for the N-365 strain the ica expression was of low yield. For this study, the penicillin resistance was related to the production of beta-lactamase otherwise the high MBC detected for cefoxitin may be associated to biofilm protection, evidentiated by the fact that the isolates have MICB values higher than MICs tested for planktonic cells / Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus ? considerada a esp?cie mais relevante devido ? produ??o de fatores de virul?ncia, tais como ?slime?, o que favorece a forma??o do biofilme. O presente trabalho teve por objetivo detectar a express?o fenot?pica da forma??o de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a express?o dos genes envolvidos na sua produ??o e regula??o, al?m de detectar a resist?ncia fenogenot?pica aos beta-lact?micos para avalia??o da poss?vel rela??o da produ??o de biofilme com a resist?ncia antimicrobiana. Os isolados foram caracterizados atrav?s de testes fenogenot?picos e MALDI-TOF, submetidos ?s provas fenot?picas de detec??o da forma??o de biofilme e avalia??o da suscetibilidade aos beta-lact?micos. A Concentra??o Inibit?ria M?nima (CIM), Concentra??o Bactericida M?nima (CBM) e a Concentra??o Inibit?ria M?nima no Biofilme (CIMB) foram determinadas para tr?s isolados selecionados com base na varia??o da intensidade da produ??o de biofilme. Posteriormente, todos os isolados foram submetidos ? t?cnica de PCR para detec??o dos genes de produ??o de ?slime? (icaA e icaD), prote?na Bap (bap), beta-lactamase (blaZ) e prote?na ligante de penicilina alterada (mecA). Al?m de detec??o do sistema regulador Agr (agr RNAIII) e da tipifica??o do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletr?nica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a an?lise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a express?o dos genes icaA, icaD e hld em tr?s isolados com produ??o variada de biofilme. Todos os isolados foram produtores de biofilme e a maioria apresentou os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr tipo II mostrou preval?ncia de 70%. A MEV mostrou mudan?as graduais no arranjo bacteriano durante a forma??o de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de forma??o na fase estacion?ria. A an?lise transcricional evidenciou maior express?o dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produ??o dos genes ica. Para este estudo, a resist?ncia ? penicilina foi relacionada com a produ??o de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada ? prote??o que o biofilme oferece, epis?dio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as c?lulas planct?nicas.

A doença decorrente da infecção por Salmonella é um dos maiores problemas de saúde no mundo em termos de morbidade e mortalidade. Entre as sorovariedades de Salmonella, a sorovariedade Enteritidis é a de maior ocorrência mundial e compreende linhagens que tem seu nicho biológico relacionado a frangos e ovos. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas a fim de se delinear a epidemiologia das infecções por S. Enteritidis. Entretanto a tipagem fenotípica usualmente falha em discriminar linhagens relacionadas das nãorelacionadas epidemiologicamente e apresenta problemas de reprodutibilidade que foram minimizados com a utilização de métodos genotípicos. No Brasil, poucos estudos que utilizaram técnicas moleculares na tipagem de linhagens dessa sorovariedade foram realizados. Os objetivos desse estudo foram investigar o potencial patogênico, a resistência a antimicrobianos e realizar a tipagem molecular de linhagens de Salmonella Enteritidis isoladas de humanos, de alimentos e de frangos no Brasil. Para isso foram estudadas 188 linhagens de Salmonella Enteritidis isoladas de surtos e de casos esporádicos, de humanos (67) de alimentos (61) e de frangos (60), durante o período de 1986 a 2010, de vários locais do Brasil. A susceptibilidade frente a 14 antimicrobianos foi analisada através da técnica de disco difusão e a presença de 13 genes de virulência das ilhas de patogenicidade de Salmonella I e II e do plasmídio pSEV foram pesquisados por PCR. Os mecanismos de resistência a quinolonas foram verificados através da pesquisa de genes de resistência plasmidiais e cromossomais e também através da verificação de mutações no gene gyrA por High resolution melting analysis (HRMA) seguida de sequenciamento de algumas linhagens. As linhagens também foram tipadas molecularmente pelas metodologias Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) com a enzima XbaI, Multilocus variable-number tandem repeat analysis (MLVA) e por Multilocus sequence typing (MLST). Das 188 linhagens estudadas, 42,5% foi resistente ao ácido nalidíxico e somente 0,5% foi resistente a sulfametoxazol-trimetoprima e estreptomicina. A resistência a quinolonas foi relacionada principalmente a mutações no gene gyrA. A maioria das linhagens estudadas (98,4%) apresentou todos os genes de virulência pesquisados, sendo uma linhagem negativa para o gene sipA e duas linhagens negativas para o gene prot6E. ERIC-PCR dividiu as 128 linhagens isoladas de humanos e alimentos em 55 perfis diferentes com similaridade >79,7%. PFGE dividiu essas mesmas linhagens em 68 perfis diferentes com uma similaridade >73,1%. Para as linhagens isoladas de frango, o dendrograma concatenado de ERIC-PCR e PFGE dividiu as 60 linhagens em dois grandes grupos com 73,3% de similaridade. O grupo A consistiu de linhagens isoladas tanto de material clínico de frangos (23) quanto do ambiente da granja (5) com 81,2% de similaridade. O grupo B também consistiu de linhagens isoladas tanto de casos clínicos de frangos (21) quanto do ambiente da granja (11) com 81,1% de similaridade. MLVA dividiu as 188 linhagens isoladas no Brasil e outras 100 linhagens isoladas na América do Norte em dois grandes grupos. O grupo MLVA-A apresentou 71 linhagens isoladas na América do Norte e somente três linhagens isoladas no Brasil. Essas linhagens do ii Brasil incluíram as isoladas antes do início da pandemia de S. Enteritidis se iniciar no país. Em contraste, o grupo MLVA-B agrupou 185 linhagens isoladas no Brasil e 29 linhagens isoladas na América do Norte. As linhagens presentes no grupo A, foram divididas em 34 tipos genéticos diferentes com similaridade maior do que 46%, enquanto no grupo B as linhagens se diferenciaram em 15 tipos genéticos diferentes com mais de 66% de similaridade. MLST caracterizou 44 das 46 linhagens estudadas como pertencentes ao ST 11. As outras duas linhagens apresentaram alelos que não existiam no banco de dados e caracterizaram dois novos STs, o 1632 e o 1633. Os resultados de tipagem molecular obtidos por ERICPCR, PFGE e MLVA no presente estudo, demonstraram uma alta similaridade genotípica entre linhagens de S. Enteritidis isoladas no Brasil, o que sugere que as linhagens estudadas descendem de um precursor comum que pouco se diferenciou genotipicamente ao longo de 24 anos no país. Ademais, os resultados de MLVA sugerem que um novo e prevalente subtipo foi introduzido no Brasil após 1993 e tem contaminado alimentos e infectado humanos e animais. O grande número de genes de virulência encontrados reforça o potencial das mesmas causarem doenças em humanos e animais, bem como, os riscos de sua presença em alimentos. Ademais, a grande porcentagem de linhagens resistentes ao ácido nalidíxico observadas a partir de 1996 sugere o uso de quinolonas no tratamento de infecções em animais causadas por S. Enteritidis no Brasil. / The disease caused of the infection by Salmonella is one of the major health problem worldwide in terms of morbid and mortality. Among the Salmonella serovars, the Enteritidis is the most frequent isolated one and comprises strains that have their biological niche related to chickens and eggs. Several phenotypic and genotypic methodologies were developed to trace epidemiologically the infections by S. Enteritidis. However, the phenotypic typing usually fail to discriminate related from unrelated epidemiologicaly strains and presents problems of reproducibility that were minimized with the introduction of genotypic methods. In Brazil, few studies that used molecular typing techniques to type strains of this serovar were conducted. The aims of this study were to investigate the pathogenic potential, the antimicrobial resistance and to molecularly type of Salmonella Enteritidis strains isolated from humans, food and chickens in Brazil. For this, it was studied 188 strains of Salmonella Enteritidis isolated from outbreaks and sporadic cases, from humans (67), food (61) and chickens (60), during the period of 1986 to 2010, from various places of Brazil. The susceptibility to 14 antimicrobials were analyzed by the disc diffusion technique and the presence of 13 virulence genes of the Salmonella pathogenicity islands I and II and from the pSEV plasmid were searched by PCR. The mechanisms of resistance to quinolones were verified by the search of plasmidial and cromossomal resistance genes and also by the verification of mutations in the gyrA gene by High resolution melting analysis (HRMA) followed by sequencing of some strains. The strains were also molecularly typed by the methodologies Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-field gel electrophoresis (PFGE) using the enzyme XbaI, Multilocus variable-number tandem repeat analysis (MLVA) and by Multilocus sequence typing (MLST). From the 188 strains studied, 42.5% were resistant to nalidixic acid and only 0.5% were resistant to sulfamethoxazoletrimethoprim and streptomycin. Resistance to quinolones was related mainly to mutations in the gyrA gene. The majority of the strains studied (98.4%) harbored all the virulence genes searched, being only one strain negative for the sipA gene and two strains negative for the prot6E gene. ERIC-PCR divided the 128 strains isolated from humans and food in 55 different profiles with >79.7% of similarity. PFGE divided the same strains in 68 different profiles with a similarity of >73.1%. Regarding the strains isolated from chickens, the concatenated dendrogram of ERIC-PCR and PFGE divided the 60 strains in two major groups with a similarity of 73.3%. Group A consisted of strains isolated either from chicken\'s clinical samples (23) or from the farm environment (5) with a similarity of 81.2%. Group B also consisted of strains isolated either from chicken\'s clinical samples (21) or from the environment (11) with a similarity of 81.1%. MLVA divided the 188 strains isolated in Brazil and other 100 strains isolated from North America in two major groups. MLVA-A group consisted of 71 strains isolated in North America and only three strains isolated in Brazil. These strains from Brazil included the ones isolated before the beginning of the pandemic of S. Enteritidis in this country. In contrast, MLVA-B group clustered 185 strains isolated in Brazil and 29 strains isolated in North America. The strains in the MLVA-A group were divided in 34 different genotypic types with a similarity of 46%, while strains in iv the group B were divided in 15 different genotypic types with a similarity of 66%. MLST characterized 44 of the 46 strains studied as belonging to ST 11. The other two strains presented new alleles that characterized two new STs, the 1632 and the 1633. The results of molecular typing obtained by ERIC-PCR, PFGE and MLVA in this study showed a high genotypic similarity among S. Enteritidis strains isolated in Brazil, which suggests that the strains studied descend from a common ancestor that differed little genotypically during 24 years in the country. Moreover, the results of MLVA suggest that a new and prevalent subtype was introduced in Brazil after 1993 and has been contaminating food and infecting humans and animals. The high prevalence of virulence genes found in the strains studied reinforce their potential to cause disease in humans and animals, as well as the risks of their presence in food. Moreover, the high percentage of strains resistant to nalidixic acid observed after 1996 suggests the use of quinolones in the treatment of animal infections by S. Enteritidis in Brazil.

Listeria monocytogenes é um importante patógeno de origem alimentar que causa listeriose, infecção severa que acomete, principalmente, gestantes, idosos, crianças e imunocomprometidos, e que apresenta elevada taxa de mortalidade. A bactéria está amplamente distribuída no ambiente e é comumente encontrada em produtos cárneos. O presente estudo teve como objetivos caracterizar 439 isolados de L. monocytogenes obtidos de salsicha bovina e produtos cárneos crus (carne moída, linguiça suína e coxa de frango) refrigerados, adquiridos no comércio do município de São Paulo, e previamente submetidos à sorotipagem molecular. Os isolados foram caracterizados quanto ao perfil de susceptibilidade antimicrobiana; presença dos genes de virulência actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB e mpl; perfil genético por eletroforese em campo pulsado (PFGE) e sequenciamento parcial dos genes actA e lmo0737. Baixa frequência de resistência antimicrobiana (0,5%) foi observada entre os 416 isolados avaliados. Um isolado pertencente ao sorogrupo 1 apresentou resistência à penicilina e à clindamicina e outro identificado como 4a ou 4c apresentou resistência à tetraciclina. Todos os isolados foram positivos para os genes de virulência testados. O sequenciamento parcial do gene actA mostrou a ocorrência de 14 sequências de nucleotídeos distintas nos 97 isolados avaliados. Além disso, verificou-se a ocorrência de uma deleção de 35 aminoácidos no gene actA em 36 isolados, além de substituições de nucleotídeos que resultaram em mutações nas sequências de aminoácidos da grande maioria dos isolados. A análise filogenética do gene actA possibilitou o agrupamento dos isolados em duas linhagens distintas (I e II). Os resultados do PFGE indicaram grande variabilidade nos perfis genéticos dos isolados analisados, principalmente naqueles pertencentes aos grupos 2 (1/2c e 3c), 3 (1/2b e 3b) e 4 (4b, 4d e 4e). Os resultados deste estudo mostram que os isolados de L. monocytogenes provenientes de salsicha bovina e produtos cárneos crus comercializados no município de São Paulo, apresentam grande diversidade genética, importante potencial de virulência e baixa frequência de resistência antimicrobiana. A diversidade observada deve-se, provavelmente, à característica ubíqua deste micro-organismo, tornando-o mais susceptível a grande pressão seletiva do ambiente. / Listeria monocytogenes is an important foodborne pathogen that causes listeriosis, a severe infection that affects primarily pregnant women, elderly, children and imunocompromised individuals, and has a high mortality rate. The bacteria is widely distributed in the environment and commonly found in meat products. The present study aimed to characterize 439 isolates of L. monocytogenes obtained from pork sausage and raw chilled meat products (ground beef, beef sausage, and chicken thigh) purchased in supermarkets in the city of São Paulo, and previously submitted to molecular serotyping. The isolates were characterized for antimicrobial susceptibility profile; presence of virulence genes actA, inlA, inlC, inlJ, prfA, iap, hly, plcA, plcB and mpl; genetic profile by pulsed field gel electrophoresis (PFGE) and partial sequencing of genes actA and lmo0737. A low frequency of antimicrobial resistance (0.5%) was observed among the 416 evaluated isolates. One isolate belonging to serogroup 1 presented resistance to clindamycin and penicillin and another one identified as 4a or 4c was resistant to tetracycline. All isolates were positive for the tested virulence genes. The partial sequencing of the gene actA indicated the occurrence of 14 distinct nucleotide sequences in the 97 isolates tested. Furthermore, a deletion of 35 amino acids in the actA gene was detected in 36 isolates, and nucleotide substitutions that resulted in amino acid changes in the sequences of most isolates. Phylogenetic analysis of the actA gene clustered the isolates in two distinct lineages (I and II). Results of PFGE indicated a great genetic variability among isolates, especially among those belonging to groups 2 (1/2c and 3c), 3 (1/2b and 3b) and 4 (4b, 4d and 4e). The results of this study show that isolates of L. monocytogenes from pork sausage and raw meat products marketed in the city of São Paulo present a great genetic diversity, significant virulence potential and low frequency of antimicrobial resistance. The detected diversity is probably due the ubiquitous nature of these microorganisms, making them more susceptible to selective pressure of the environment.

This work will look at two factors that add to the diversity of carbapenem resistant bacteria. First, it focuses on the diversity of carbapenemase resistance plasmids. 446 plasmids were characterized by size, gene content and replicon groups. We identified that on average, over 30% of the encoded proteins on each plasmid have an unknown function. Plasmid sizes ranged from 1.6kb to 500kb, with an average of around 100kb and median of 80kb. Additionally, six replicon groups account for 80% of all the carbapenemase resistance plasmids. We also highlight the lack of data available for carbapenemase carrying plasmids from bacterial genera other than Escherichia and Klebsiella, and plasmids that carry the New Delhi metallo-β- lactamase or the Verona-integron encoded metallo-β-lactamase. Second, we characterized the β-lactamase diversity of a single carbapenemase resistant Klebsiella pneumoniae. This isolate encodes six distinct β-lactamases, all of which are functional, and three of which are redundant. Additionally, we determined that the CTX-M-15 cephalosporinase imparts a greater fitness when grown in aztreonam (a monobactam) than ceftazidime (a cephalosporin). Finally, we show that individually, these β-lactamases do not account for the elevated levels of resistance seen in the parent strain, indicating that the passive resistance mechanisms (i.e. efflux pumps, altered membrane porins) may play a larger role than originally thought.