Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey

ABSTRACT: Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

RESUMO: Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey / Caracterização genômica perfis de resistência antimicrobiana, produção de enterotoxinas e biofilme Staphylococcus aureus resistente à meticilina isolado de origem clínica e de produtos de origem animal no leste da Turquia

Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks

Background: Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran. Methods and results: Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates. Conclusion: In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.(AU)

Antimicrobial susceptibility and molecular characterization of Salmonella serovar Ndolo isolated from outbreaks in cattle and horses / Susceptibilidade antimicrobiana e caracterização molecula de isolados de Salmonella sorovar Ndolo de bovinos e equinos

The present study aimed to describe and characterize, for the first time, two outbreaks of salmonellosis caused by Salmonella Ndolo in foals and calves in Brazil and compare the isolated strains with S. Ndolo previously identified in asymptomatic reptiles. The affected calves and foals presented fever, lethargy, and profuse diarrhea. Isolated strains were subjected to antimicrobial susceptibility testing, characterized according to virulence genes, and fingerprinted by ERIC-PCR. Salmonella Ndolo was identified in fecal samples from two foals and four calves. One isolate from a calf was resistant to amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, and florfenicol. Strains from two other calves were resistant to oxytetracycline. All virulence genes tested were present in the isolates, and two major clusters of closely related strains were identified by ERIC-PCR, each per outbreak. This is the first report of Salmonella Ndolo infection in domestic and symptomatic animals. Previously, this serovar had been identified only in human infections. The presence of relevant virulence genes in all Salmonella Ndolo isolates and the detection of antimicrobial multi-resistant strains highlighted the importance of monitoring serovars associated with salmonellosis in domestic animals.(AU)

O objetivo do presente estudo foi descrever e caracterizar, pela primeira vez, dois surtos de salmonelose causados por Salmonella Ndolo em potros e bezerros do Brasil e comparar esses isolados com Salmonella Ndolo previamente identificada em répteis assintomáticos. Os animais infectados apresentaram febre, letargia e diarreia profusa. Os isolados foram submetidos a testes de susceptibilidade a antimicrobianos e foram caracterizados conforme a presença de genes de virulência e diversidade genética, utilizando-se o ERIC-PCR. Salmonella Ndolo foi identificado em amostras fecais de dois potros e quatro bezerros. Um isolado de bezerro foi resistente a amoxicilina/ácido clavulanico, trimethoprima/sulfametoxazol e florfenicol. Estirpes de dois outros bezerros foram resistentes a oxitetraciclina. Todos os genes de virulência testados foram identificados nos isolados e dois grandes grupos de estirpes geneticamente relacionadas foram identificados pelo ERIC-PCR, um para cada surto. Esse é o primeiro relato de Salmonella Ndolo em animais domésticos e sintomáticos. Previamente, esse sorovar foi identificado apenas em infecções humanas. A presença de fatores de virulência relevantes em todos os isolados e a detecção de estirpes multirresistentes a antimicrobianos destaca a importância do monitoramento de sorovares associados a salmonelose em animais domésticos.(AU)

Molecular epidemiology of Listeria monocytogenes isolated from different sources in Brazil / Epidemiologia molecular de Listeria monocytogenes isoladas de diferentes fontes no Brasil

Listeria monocytogenes is an important foodborne pathogen that primarily affects pregnant women, neonates, the elderly and immune-compromised individuals, and it may cause abortion, septicemia, and meningitis. From the 13 capsular groups described, serotypes 4b, 1/2b and 1/2a are most closely related to human infection. For this reason, serotyping has limited value as an epidemiological tool; thus, improved discriminatory typing methods are required to enhance knowledge of L. monocytogenes contamination and infection. The aim of this study was to characterize the genetic diversity of L. monocytogenes isolates in the pork processing industry in Sao Paulo, Brazil and human infection isolates by ERICPCR and single enzyme AFLP. Serotypes 1/2c and 4b were frequent among isolates from pork and slaughterhouse/market environments, whereas serotypes 4b and 1/2a were observed among human isolates. ERIC-PCR and AFLP revealed 34 and 31 distinct profiles, respectively, which had tendencies of separation according to serogroup and isolate origin. The genetic profiles from slaughterhouse and market environments suggest the possibility of different sources of Listeria contamination in the environment, although in certain cases, continuous contamination caused by the persistence of clonal strains is also a possibility.(AU)

Listeria monocytogenes é um importante patógeno de origem alimentar que afeta principalmente grávidas, neonatos, idosos e indivíduos imunocomprometidos, e pode causar abortamento, septicemia e meningite. Dos 13 grupos capsulares descritos, os sorotipos 4b, 1/2b e 1/2a são os mais relacionados à infecção humana. Por esta razão, a sorotipagem possui valor limitado como ferramenta epidemiológica e, dessa forma, métodos mais discriminatórios são necessários para melhorar o conhecimento sobre a contaminação e a infecção por L. monocytogenes. O objetivo deste estudo foi caracterizar a diversidade genética de isolados de L. monocytogenes da indústria de processamento de carne suína noEstado de São Paulo, Brasil, e compará-los a isolados de casos de infecção humana através do ERIC-PCR e AFLP com uma única enzima. Os sorotipos 1/2c e 4b foram frequentes em carne suína e ambientes de abatedouros e mercados, enquanto os sorotipos 4b e 1/2a foram observados nos isolados de humanos. ERIC-PCR e AFLP resultaram em 34 e 31 perfis distintos, respectivamente, com uma tendência a separar de acordo com o sorogrupo e a origem do isolado. Os perfis genéticos de ambiente dos abatedouros e mercados sugerem a possibilidade de diferentes origens de contaminação por Listeria nos ambientes estudados, porém, em alguns casos, é possível que ocorra a persistência de cepas clonais causando contaminação contínua.(AU)

Patógenos Respiratórios dos Suínos: Diversidade genética de 15 Mycoplasma hyopneumoniae em Minas Gerais e dinâmica de 16 colonização e resposta imune por Haemophilus parasuis

A doença de Glasser, causada pela bactéria Haemophilus parasuis, e a pneumonia enzoótica, causada pelo Mycoplasma hyopneumoniae, são duas das principais enfermidades sistêmicas e respiratórias de suínos na creche, recria e terminação. Os fatores que desencadeiam a doença não foram completamente esclarecidos. Várias estratégias já foram utilizadas para controlar estas doenças, mas nenhuma delas é completamente eficaz. Em relação ao M. hyopneumoniae, o primeiro capítulo teve como objetivo obter um panorama sobre a diversidade genética desse agente relacionado a lesões pneumônicas em suínos de rebanhos das diferentes mesorregiões produtoras do estado de Minas Gerais. Isso foi possível através da genotipagem pela análise de MLVA, uma técnica altamente discriminatória para a espécie. Os resultados mostraram que há alta prevalência de granjas positivas para o agente e um alto polimorfismo das cepas circulantes no estado de Minas Gerais, sem um padrão genotípico específico de determinada região geográfica. Em relação ao H. parasuis, o segundo capítulo objetivou esclarecer, através de um estudo longitudinal em uma granja comercial de ciclo completo, o efeito da associação de diferentes antimicrobianos aplicados no manejo em diferentes fases de criação dos suínos sobre a dinâmica de colonização por H. parasuis e resposta imune humoral contra essa bactéria. Os resultados mostraram que o uso intenso de antimicrobianos na creche pode reduzir a pressão de infecção na granja, mas interfe na colonização pela bactéria e desloca o início da resposta imune ativa para semanas posteriores. Nas condições de campo encontradas nesse estudo, não existiram diferenças significativas no tratamento com enrofloxacina sobre a dinâmica de colonização em leitões clinicamente saudáveis. Em relação à cinética de colonização global dos leitões, nem todos os leitões são expostos ao agente e recebem imunidade materna em uma idade precoce a níveis detectáveis, e a presença de anticorpos maternos parece reduzir a colonização até mesmo por cepas possivelmente comensais

Glassers disease, caused by the bacterium Haemophilus parasuis, e enzootic pneumonia, caused by Mycoplasma hyopneumoniae, are two of the main causes of systemic and respiratory diseases in swine after weaning, respectively. The factors involved in the occurrence of disease are not completely clarified. Several strategies used to control these diseases are not completely efficient. In relation to Mycoplasma hyopneumoniae, the first chapter focused on obtaining an overview about the distribution and genetic diversity of this agent related to pneumonic lesions of swine reared in herds from different production areas of Minas Gerais state. This was accomplished by the use of MLVA genotyping analysis, which has proved worldwide to be a highy discriminatory technique for this bacterium. The results showed that there is a high prevalence of positive farms for the agent and a high polymorphism among circulating strains of the state of Minas Gerais, without a specific genotypic pattern of a geographic region. Regarding H. parasuis, the second chapter aimed to clarify, though a longitudinal study in a commercial farm, the effect of the association with different antimicrobials used in different stages of pig breeding on colonization dynamics by H. parasuis and on humoral response against this bacterium. Results showed that the intensive use of antibiotics in nursery can reduce the infection pressure in the farm, but interfers with the colonization by bacteria and shifting the onset of active immune response to subsequent weeks. In the field conditions of this study, there was no significant difference on the treatment with enrofloxacin on the colonization dynamics in clinically healthy piglets. Regarding the global colonization kinetics, not all piglets are exposed to the agent and receive maternal immunity in an early age, and the presence of maternal antibodies seems to reduce colonization even by putative comensal strains.

Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections

Pathogenic strains of Escherichia coli are the most common bacteria associated with urinary tract infections in both humans and companion animals. Standard biochemical tests may be useful in demonstrating detailed phenotypical characteristics of these strains. Thirteen strains of E. coli isolated from dogs with UTIs were submitted to biochemical tests, serotyping for O and H antigens and antimicrobial resistance testing. Furthermore, the presence of papC, sfa, and afa genes was evaluated by PCR, and genetic relationships were established using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The antimicrobial that showed the highest resistance rate among the isolates was nalidixic acid (76.9%), followed by cephalotin (69.2%), sulfamethoxazole + trimethoprim (61.5%), tetracycline (61.5%), streptomycin (53.8%), ciprofloxacin (53.8%), ampicillin (46.2%), gentamicin (30.8%) and chloramphenicol (23.1%). No isolate was resistant either to meropenem or nitrofurantoin. Among the five clusters that were identified using ERIC-PCR, one cluster (A) had only one strain, which belonged to a serotype with zoonotic potential (O6:H31) and showed the genes papC+, sfa+, afa-. Strains with the genes papC-, sfa+, afa- were found in two other clusters (C and D), whereas all strains in clusters B and E possessed papC-, sfa-, afa- genes. Sucrose and raffinose phenotypic tests showed some ability in discriminating clusters A, B and C from clusters D and E.

ERIC-PCR genotypic characterization of Haemophilus parasuis isolated from Brazilian swine

Haemophilus parasuis infection, known as Glässer's disease, is characterized by fibrinous polyserositis, arthritis and meningitis in piglets. Although traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, the molecular-based methods are alternatives for species-specific tests and epidemiologic study. The aim of this study was to characterize H. parasuis strains isolated from different states of Brazil by serotyping, PCR and ERIC-PCR. Serotyping revealed serovar 4 as the most prevalent (24 %), followed by serovars 14 (14 %), 5 (12 %), 13 (8 %) and 2 (2 %), whereas 40 % of the strains were considered as non-typeable. From 50 strains tested 43 (86%) were positive to Group 1 vtaA gene that have been related to virulent strains of H.parasuis. ERIC-PCR was able to type isolates tested among 23 different patterns, including non-typeable strains. ERIC-PCR patterns were very heterogeneous and presented high similarity between strains of the same animal or farm origin. The results indicated ERIC-PCR as a valuable tool for typing H. parasuis isolates collected in Brazil.

Polyclonal endemicity of Pseudomonas aeruginosa in a teaching hospital from Brazil: molecular typing of decade-old strains

Pseudomonas aeruginosa infections cause significant mortality and morbidity in health care settings. Strategies to prevent and control the emergence and spread of P. aeruginosa within hospitals involve implementation of barrier methods and antimicrobial stewardship programs. However, there is still much debate over which of these measures holds the utmost importance. Molecular strain typing may help elucidate this issue. In our study, 71 nosocomial isolates from 41 patients and 23 community-acquired isolates from 21 patients were genotyped. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was performed. Band patterns were compared using similarity coefficients of Dice, Jaccard and simple matching. Strain similarity for nosocomial strains varied from 0.14 to 1.00 (Dice); 0.08 to 1.00 (Jaccard) and 0.58 to 1.00 (simple matching). Forty patterns were identified. In most units, several clones coexisted. However, there was evidence of clonal dissemination in the high risk nursery, neurology and two surgical units. Each and every community-acquired strain produced a unique distinct pattern. Results suggest that cross transmission of P. aeruginosa was an uncommon event in our hospital. This points out to a minor role for barrier methods in the control of P. aeruginosa spread.(AU)