Primer probes for targeted identification and detection of Bifidobacterium animalis and their application / 中国药科大学学报

@#In this study, in order to overcome the shortcomings of the current methods used to identify Bifidobacterium animalis, such as long time, complicated operation and low adaptability of experimental environment, specific primer probes were designed based on ERIC-PCR technology to identify and detect B.animalis.Based on the genomic DNA of B.animalis HP-B1124, the ERIC-PCR reaction conditions of B.animalis HP-B1124 were optimized, and the ERIC-PCR fragments were obtained one by one and sequenced.Two pairs of specific primer probes were designed.The accuracy, specificity, limitation and universality of the two pairs of primer probes were evaluated, and the two pairs of specific primer probes were used for testing the products containing B.animalis in the commercially published formula.The two pairs of specific primer probes designed in this study could be used for identified strains of B.animalis more simply, quickly and targeted.This method has optimized the current relatively traditional methods of pure culture and plate counting identification of B.animalis, and has solved the high requirements of SNP genotyping technology and real-time fluorescence quantitative PCR method for experimental equipment and reagents in the identification of B.animalis to a certain extent.It has the characteristics of low cost, high specificity and earn a broad market development prospect.

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.

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.

Molecular characteristics of Acinetobacter baumannii and the changes of efflux pump expression under tigecycline pressure / 医学研究生学报

Objective　Acinetobacter baumannii (A. baumannii) is a commonly infective bacterium in the hospital. This study aims to analyze its molecular epidemiological characteristics, detect the carrying rate of efflux pump and regulatory protein genes, and investigate the effects of tigecycline on the efflux pump and expression of regulatory protein genes.　Methods　A total of 183 A. baumannii strains were collected from inpatients of the affiliated hospital of Jiangsu University from May 2017 to March 2019. They were divided into an antimicrobial-resistant group (one or more antimicrobial-resistant strains, 139 strains) and a sensitive group (the drugs in the drug sensitivity test were all non-resistant strains, 44 strains). Repeated sequence PCR was used for homology analysis of the strains, and pulse-field gel electrophoresis (PFGE) was used as the gold standard for homology analysis to verify and compare some strains. PCR was used to detect the occurrence of drug resistance-related genes. Based on homology analysis, efflux pump carrying rate detection and antibiotics sensitivity test results, 6 clinical strains carrying all efflux pump genes but different resistance phenotypes were selected as experimental strains, including sensitive strains (SAB), the multidrug resistance strain (MDRAB) and the extensively drug-resistant strain (XDRAB). All strains were induced in vitro with the minimum inhibitory concentration (MIC) of tigecycline. The induced strains were categorized as induction group, and the same strains cultured in LB agar without tigecycline was used as a control group. MIC was used to analyze the tigecycline susceptibility, and RT-qPCR was used to detect the gene expression of efflux pumps, such as TetB, AbaQ and regulatory proteins (AdeS and BaeS), in drug-resistant strains.　Results　Homology analysis showed that there were 45 clonal groups in the detected clinical isolates, with no obvious outbreak of epidemic clonal groups. Efflux pumps and regulatory proteins were widely distributed in the clinical isolates, and the expression of AdeB, TetB, AbeS, AdeS in MDRAB and XDRAB is significantly higher than that insensitive group SAB. Continuous in vitro induction with tigecycline could increase the antimicrobial resistance of some clinical strains and even significantly increase the expression levels of efflux pumps and regulatory proteins.　Conclusion　A. baumannii is widely distributed in the clinic, and efflux pumps and regulatory proteins might play an important role in drug resistance process. The unreasonable use of tigecycline could enhance the tolerance of A. baumannii by up-regulating the expression of some bacterial efflux pumps.

Genomic and proteomic approach as a tool to discrimination of Escherichia coli strains of biotechnological interest

The Methods based on genomic and proteomic approaches are described as effective tools for the identificationof microorganisms. The development of methodologies capable of differentiating, interspecifically, pathogenic,wild and genetically modified Escherichia coli strains is desirable for the fields of healthcare and Biotechnology.The purpose of this study was to verify the viability of ERIC-PCR and MALDI TOF methods in differentiatinglineages of Escherichia strains. For this purpose, laboratory Escherichia coli ATCC8739, Escherichia coliW3110, Escherichia coli BL21DE3+, Escherichia coli JM109, Escherichia coli MC 1061 and Escherichiacoli DH5α were subjected to ERIC-PCR and MALDI TOF mass spectrometry analyzes. Genomic (ERIC-PCR)and proteomic (MALDI-TOF) methods were able to discriminate between different lineages of Escherichiacoli strains including lineages of Escherichia coli K-12. However, the MALDI TOF proteomic approachrevealed being able to differentiate interspecifically lineages of Escherichia coli strains. The determination ofthe most frequent masses found in the studied Escherichia coli strains in addition to future experiments ofpeptide sequencing profile and SDS-PAGE can be used as a guideline for validating a method for proteomicidentification of these strains.

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

Abstract 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.

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

ABSTRACT: 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.

RESUMO: 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.

Molecular characterization of Mycobacterium tuberculosis and Mycobacterium bovis isolates by Enterobacterial Repetitive Intergenic Consensus-PCR

Tuberculosis (TB) is an infectious disease in which the molecular typing methods allow to have important information about the dynamics of transmission and to assist properly in disease control. Although the ERIC-PCR (Enterobacterial repetitive intergenic consensus-PCR) assay is fast and easy to perform, scarce studies have reported its use in epidemiological studies in TB outbreaks. In this study, we aimed to genotype Mycobacterium tuberculosis and M. bovis isolates by ERIC-PCR and compare its discriminatory power with two other classically used methods: 12 loci-MIRU (Mycobacterial Interspersed Repetitive Units) and Spoligotyping. The M. tuberculosis isolates studied were from northwestern and southwestern and M. bovis from northwestern Parana, Brazil. ERIC-PCR rendered banding patterns with great diversity (1 to 12 bands) of molecular sizes, ranging from 100 to 1600 bp. ERIC-PCR showed to be fast, simple and affordable to differentiate isolates. ERIC-PCR would be an important tool in the epidemiology of TB as screening in case of outbreak, which demands rapid intervention. However if any doubt persist, as it may occur with the application of only one genotypic method, other genotyping methods should be applied and carefully interpreted, always with additional epidemiological information.

Gut microbiota at different stages of HBV infection / 实用医学杂志

Objective To investigate the correlation between gut microbiota and hepatitis B virus(HBV) infection at different stage. Methods 84 patients aged 25 ~ 40 were enrolled in the study,the ALT,AST and HBV antigens and antibodies qualitatively detected. Then they were divided into four groups based on HBV related antigens and antibodies qualitative detection according to guideline for chronic HBV infection:48 in the protective antibody positive group where vaccine injection was performed for producing protective antibodies ,14 in the HBV recovery group 9 in chronic HBV infection group And 13 in antibody negative group where vaccine injection was done. Their fecal samples were collected and total DNA were extracted and subjected to enterobacterial repetitive intergenic consensus sequence polymerase chain reaction(ERIC-PCR). After ERIC-PCR,gel electrophoresis and genetool software were used to analyse the differences in the amount of bands and the position of main bands between groups. Results The amounts of bands in the antibody negative and chronic HBV infected groups were significantly smaller than the protective antibody group and HBV recovery group. The position of main band in the protective antibody group and HBV recovery group were wildly spread within groups ,and most main bands in the HBV recovery group were above 282 bp. The main bands in the antibody negative and chronic HBV infected groups were centralized in 60 ~ 70 bp. Conclusions Compared with the protective antibody group and HBV recovery group ,the richness of gut microbiota in the antibody negative and chronic HBV infected groups are significantly decreased and lack of microbiota above 200 bp in ERIC-PCR.

Carbapenemase genes and homology of Acinetobacter baumannii in two hospitals of Qingdao / 中国感染控制杂志

Objective To investigate antimicrobial resistance,distribution,and carriage of carbapenemase genes of Acinetobacterbaumannii(AB)from two hospitals in Qingdao.Methods 145 AB isolates collected from two hospi-tals (78 from hospital A,67 from hospital B)were performed antimicrobial susceptibility testing,carbapenemase genes were amplified by polymerase chain reaction (PCR);homology analysis were conducted with enterobacterial repetitive intergenic consensus (ERIC)-PCR.Results AB from hospital A were generally resistant to 16 commonly used antimicrobial agents,with the lowest resistant rate of 3.85% to cefoperazone/sulbactam,followed by resist-ance rate of 16.67% to minocycline,resistant rates to the other antimicrobial agents were all>73% . AB from hos-pital B were generally resistant to 23 commonly used antimicrobial agents,but the resistance rates to minocycline and tigecycline were both 0,resistance rates to amikacin and levofloxacin were 23.88% and 38.81% respectively, resistant rates to the other antimicrobial agents were all >64% . All strains carried OXA-5 1 gene,the carriage rates of OXA-23 gene in carbapenem-resistant group were 86.76% (59/68)and 56.67% (34/60)in hospital A and B re-spectively,the difference was significant(χ2= 14.53,P<0.001);OXA-58 gene was detected in 3 isolates in hospi-tal A but not detected from hospital B. 145 AB strains were classified into 8 types,the major prevalence types were type A (n= 71)and E(n= 37);the major prevalence types in hospital A were type A (46.15% )and E(41.03% ), hospital B were type A (52.24% )and C (17.91% ).Conclusion Antimicrobial resistance of clinically isolated AB is serious and prevailed in two hospitals. OXA-23 and OXA-51 genes play an important role in AB resistance to car-bapenems.

Genomic Diversity of Cholera Outbreak Strains in East Malaysia

Thirty one Vibrio cholera isolates recovered from cholera outbreak in Bintulu, Sarawak (Malaysia) were detected with the presence of ctx gene by using specific PCR. These isolates were further characterized and differentiated by using the Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) and BOX-PCR to determine their genomic fingerprints. The specific PCR result confirmed the identities of 27 isolates out of 31 as pathogenic V. cholerae. The ERIC-PCR generated several genetic profiles consisting of 4-6 bands with sizes in the range of 100 to 600 bp, while the BOX-PCR produced profiles numbering 2-7 bands in the sizes between 200 to 1000 bp. Based on the dendrogram generated from the DNA fingerprinting profiles (ERIC-PCR and BOX-PCR), all of the isolates can be divided into 2 main clusters that is further divided into 2 sub-clusters. The low genetic diversity of the isolates indicated the outbreak of V. cholerae in the study area was due to the contamination from a single or few sources of V. cholerae.

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.

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.

Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil / Genes de multirresistência, incluindo blaKPC e blaCTX-M-2, em Klebsiella pneumoniae isoladas em Recife, Brasil

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care. METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (blaTEM, blaSHV,blaCTX-MblaKPC, blaVIM, blaIMP, and blaSPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented blaSHV, 62.5% presented blaCTX-M-2, 29% blaTEM, and 41.7% blaKPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried blaSHVblaTEM, blaCTX-M-2 and blaKPC simultaneously. CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.

INTRODUÇÃO: A prevalência de cepas de Klebsiella pneumoniae resistentes a cefalosporinas e carbapenêmicos está aumentando no Brasil, com sérias consequências em termos de desfechos dos pacientes e cuidados gerais. MÉTODOS: Este estudo caracterizou 24 isolados clínicos de K. pneumoniae provenientes de dois hospitais de Recife, Brasil, através do perfil de susceptibilidade a antimicrobianos, análise de genes de β-lactamase (blaTEM,blaSHV,blaCTX-MblaKPC,blaVIM, blaIMP,and blaSPM), perfil plasmidial e ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTADOS: A análise da ERIC-PCR e do perfil plasmidial agrupou os isolados em 17 e 19 perfis, respectivamente. Seis isolados de um hospital apresentaram o mesmo padrão de ERIC-PCR, indicando disseminação clonal. Todos os isolados apresentaram blaSHV, 62,5% apresentaram blaCTX-M-2, 29% blaTEM e 41,7% blaKPC. Genes de metalo-β-lactamase blaVIM, blaIMP e blaSPM não foram detectados. Onze isolados foram identificados carreando, pelo menos, três dos genes de β-lactamase estudados, dentre estes, dois isolados continham blaSHV,blaTEM, blaCTX-M-2 e blaKPC simultaneamente. CONCLUSÕES: O acúmulo de genes de resistência em algumas cepas, observado nesse estudo, impõem limitações nas opções terapêuticas disponíveis para o tratamento de infecções causadas por K. pneumoniae em Recife, Brasil. Estes resultados devem alertar as autoridades médicas brasileiras para estabelecer rigorosos métodos para controlar eficientemente a disseminação de genes de resistência a antimicrobianos no ambiente hospitalar.

Aislamiento e identificación de cepas del género Bifidobacterium presentes en productos lácteos fermentados tipo yogur / Isolation and identification of strains belonging to the Bifidobacterium genus found in fermented yoghurt type milk products

La identificación de microorganismos probióticos del género Bifidobacterium es de gran importancia por su uso como suplemento que favorece la salud del consumidor. En Venezuela son pocos los estudios sobre caracterización microbiológica de estas bacterias y no existen métodos oficiales para su estudio en alimentos. Esta investigación reporta la estandarización de técnicas microbiológicas y moleculares para el aislamiento e identificación de bifidobacterias aisladas de dos productos tipo yogur, I con probiótico y II sin probiótico. Se analizaron 10 muestras de cada yogur, una por semana, aislando 3 colonias por muestra. Los resultados mostraron que de los 60 aislados analizados, 27 colonias del Yogur I y 11 del Yogur II concordaron con las características de bifidobacterias. Se comparó el crecimiento bacteriano en dos medios de cultivo (MRS-m, RCA), sembrando por profundidad en placas y en tubos Miller-Pricket, obteniéndose mejores resultados con el medio MRS-m y las siembras por profundidad en tubos. De las extracciones de ADN se obtuvieron los patrones de ERIC-PCR y REP-PCR, determinándose que 34 aislados eran clones indistinguibles, mostrando el patrón de B. lactis utilizado como control positivo. Esta metodología puede ser utilizada por la industria y los entes encargados del control de la calidad de los productos probióticos.

The identification of probiotic microorganisms belonging to the Bifidobacterium genus is very important due to their use as supplements favorable for consumer’s health. In Venezuela there have been few studies of the microbiological characterization of these bacteria and there are no official methods for their study in food. This investigation reports the standardization of microbiological and molecular techniques for the isolation and identification of bifidobacteria isolated from two yoghurt type products: I with probiotic and II without probiotic. Ten samples from each yoghurt type product were analyzed, one per week, and 3 colonies were isolated per sample. Results showed that of the 60 isolates analyzed, 27 colonies of Yoghurt I and 11 of Yoghurt II coincided with the characteristics of bifidobacteria. Bacterial growth was compared in two culture media (MRS-m, RCA), inoculating in-depth in plates and Miller-Pricket tubes; the best results were obtained with MRS-m medium and in-depth inoculations in tubes. By DNA extraction we obtained ERIC-PCR and REP-PCR patterns, determining that 34 isolates were indistinguishable clones, showing the same pattern of the B. lactis used as positive control. This methodology can be used by the industry and the institutions in charge of quality control of probiotic products.

Serotyping & molecular characterization for study of genetic diversity among seafood associated nontyphoidal Salmonella serovars.

Background & objectives: Infections due to seafood associated Salmonella serovars are great risk to public health. Different phenotypic characteristics have been used previously for epidemiological investigation of Salmonella. Beyond the phenotypic characterization, a reliable genetic level discriminatory method is required. Therefore, this study was attempted to use different phenotypic and molecular fingerprinting methods for investigation of genetic diversity among seafood associated nontyphoidal Salmonella serovars. Methods: Fifty eight seafood associated Salmonella isolates were included in this study. All isolates were serotyped and epidemiological investigation was carried out using molecular fingerprinting methods, random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR) along with whole cell protein profiling using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in our study. Results: Among the 58 Salmonella isolates, S. Weltevreden was observed to be the most predominant serovar. Typing of Salmonella serovars using RAPD and ERIC-PCR suggested the existence of a genetic diversity. Though both PCR based techniques were found to have a good discriminatory index, a better discriminatory ability was observed when the results obtained by the two techniques were combined and taken for composite analysis. Protein profiling of whole cells using SDS-PAGE demonstrated the presence of several bands with two bands of sizes 38 kDa and 46 kDa common among all 58 isolates. Interpretation & conclusions: Our study shows that use of protein profiling in combination with established typing methods such as RAPD and ERIC-PCR may provide useful information in typing of non-typhoidal Salmonella isolates associated with seafood and to develop strategies to protect public from Salmonella infections.

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 Glãsser'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 percent), followed by serovars 14 (14 percent), 5 (12 percent), 13 (8 percent) and 2 (2 percent), whereas 40 percent of the strains were considered as non-typeable. From 50 strains tested 43 (86 percent) 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.

Molecular characterization of clinical multiresistant isolates of Acinetobacter sp. from hospitals in Porto Alegre, State of Rio Grande do Sul, Brazil / Caracterização molecular de isolados clínicos Acinetobacter sp. multirresistentes em hospitais de Porto Alegre, Estado do Rio Grande do Sul, Brasil

INTRODUCTION: Hospitals around the world have presented multiresistant Acinetobacter sp. outbreaks. The spread of these isolates that harbor an increasing variety of resistance genes makes the treatment of these infections and their control within the hospital environment more difficult. This study aimed to evaluate the occurrence and dissemination of Acinetobacter sp. multiresistant isolates and to identify acquired resistance genes. METHODS: We analyzed 274 clinical isolates of Acinetobacter sp. from five hospitals in Porto Alegre, RS, Brazil. We evaluated the susceptibility to antimicrobial, acquired resistance genes from Ambler's classes B and D, and performed molecular typing of the isolates using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) technique. RESULTS: A high (68 percent) percentage of multiresistant isolates of Acinetobacter sp. was observed, and 69 percent were resistant to carbapenems. We identified 84 percent of isolates belonging to species A. baumannii because they presented the gene blaOXA-51. The gene blaOXA-23 was detected in 62 percent of the isolates, and among these, 98 percent were resistant to carbapenems. Using the ERIC-PCR technique, we identified clones of Acinetobacter sp. spread among the four hospitals analyzed during the sampling period. CONCLUSIONS: The data indicate the dissemination of Acinetobacter sp. isolates among hospitals and their permanence in the hospital after one year.

INTRODUÇÃO: Hospitais no mundo todo têm apresentado surtos de Acinetobacter sp. multirresistentes. A disseminação destes isolados com uma variedade cada vez maior de genes de resistência torna difícil o tratamento destas infecções e seu controle dentro do ambiente hospitalar. Este trabalho teve como objetivo avaliar a ocorrência e disseminação de isolados de Acinetobacter sp. multirresistentes e identificar genes de resistência adquirida. MÉTODOS: Foram avaliados 274 isolados clínicos de Acinetobacter sp. obtidos de cinco hospitais da Cidade de Porto Alegre, RS, Brasil. Avaliamos o perfil de suscetibilidade a antimicrobianos, genes de resistência adquirida das classes B e D de Ambler e realizamos a tipificação molecular dos isolados utilizando a técnica de enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). RESULTADOS: Encontramos uma alta (68 por cento) porcentagem de isolados de Acinetobacter sp. multirresistentes e 69 por cento dos isolados apresentaram resistência aos carbapenêmicos. Foram identificados 84 por cento de isolados pertencentes a espécie A. baumannii, pois apresentaram o gene blaOXA-51. Em 62 por cento dos isolados, foi detectado o gene blaOXA-23, sendo que 98 por cento destes isolados foram resistentes aos carbapenêmicos. Através da tipificação molecular pela técnica de ERIC-PCR identificamos clones de Acinetobacter sp. disseminados entre quatro dos hospitais analisados e nos anos de 2006 e 2007. CONCLUSÕES: Os dados obtidos indicam a disseminação de isolados de Acinetobacter sp. entre hospitais assim como sua permanência no ambiente hospitalar após um ano.

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.

Genotipificación de Acinetobacter baumannii multi-resistente aislado en el hospital Dr. Domingo Luciani de caracas / Genotypification of multiresistant Acinetobacter baumannii isolated in the Dr. Domingo Luciani Hospital of caracas

Las técnicas de genotipificación tienen un rol fundamental en el estudio de las infecciones nosocomiales. Las infecciones nosocomiales son producidas principalmente por microorganismos que son resistentes a los antimicrobianos, que por lo general han sido seleccionados por el uso inadecuado de la terapia antimicrobiana. Entre las especies que causan frecuentemente este tipo de infecciones se encuentra A. baumannii multi-resistente. En esta investigación se planteó genotipificar mediante las técnicas ERIC-PCR y REP-PCR 19 cepas de A. baumannii multi-resistente aisladas en el hospital Dr. Domingo Luciani de Caracas. La confirmación molecular de la especie A. baumannii se realizó mediante la detección de la oxacilinasa OXA 51 por PCR, el 100% de los aislados incluidos en el estudio resultaron positivos para la detección del gen blaOXA-51-Like. La susceptibilidad antimicrobiana y la detección fenotípica de mecanismos de resistencia se efectuaron de acuerdo a las normas de la CLSI 2009. Se determinó policlonalidad en los 19 aislados de A. baumannii, con el predominio de cuatro clones en la Unidad de Terapia Intensiva de Adultos y el área de Hospitalización del Hospital Dr. Domingo Luciani de Caracas. La correlación de los datos epidemiológicos con las características de la resistencia y la información molecular de cada una de las muestras permitió identificar dos patrones de infección: infecciones de origen endógeno, las cuales se caracterizaron por la diversidad genética de los aislamientos, e infecciones cruzadas, debido al hallazgo de cepas estrechamente relacionadas en espacios cercano o distantes del centro de salud. Se demostró que ERIC-PCR y REP-PCR bajo las condiciones estandarizadas en este estudio son técnicas confiables desde el punto de vista de la estabilidad de los marcadores moleculares y la reproducibilidad para caracterizar brotes ocasionados por A. baumannii, considerándose la técnica REP-PCR más adecuada para estudios de genotipificación...

The genotypification techniques have a fundamental role in the study of nosocomial infections. These infections are produced principally by microorganisms that are antimicrobial resistant, that have benn selected by the inadequate use of antimicrobial therapy. Between the species that frequently cause these type of infections is the A baumannii multi-resistant. In this investigation we established to genotypificate by ERIC-PCR y REP-PCR techniques 19 strains of A baumannii multi-resitant isolated in the Dr. Domingo Luciani Hospital of Caracas. The molecular confirmation of the species was realized by the detection of the oxacilianse OXA 51 by PCR. 100% of the isolates included in the study resulted positive for the gen bla OXA-51-Like. The antimocrobial susceptibility and the phenotypic detection of resistance mechanism were done according the CLSI 2009 normative. We determined policlonality on the 19 isolates of A. baumannii, with the predominance of 4 clones in the Intensive Therapy unit and the hospitalization area of the hospital. The correlation of the epidemiological data with the resistance characteristics and the molecular information of each sample allowed us to identificate two patterns of infections: endogen origin infection, which was charaterized by the genetic diversity of the isolates and cross infections, due to the finding of strains closely related in spaces near o distant ffrom the health center. We demostrated that ERIC-PCR and REP-PCR under standarized conditions in this study are good techiques fron the point of view of the stability of the molecular markers and the reproducibility to characterize outbreaks occasioned by A. baumannii, consideratin the REP-PCR technique, the most adequate for genotypification of this strain.