Study of the urban-impact on microbial communities and their virulence factors and antibiotic resistance genomes in the Nandu River, Haikou / 中华流行病学杂志

Objective: To explore the changes in bacterial community structure, antibiotic resistance genome, and pathogen virulence genome in river water before and after the river flowing through Haikou City and their transmission and dispersal patterns and to reveal anthropogenic disturbance's effects on microorganisms and resistance genes in the aquatic environment. Methods: The Nandu River was divided into three study areas: the front, middle and rear sections from the upstream before it flowed through Haikou City to the estuary. Three sampling sites were selected in each area, and six copies of the sample were collected in parallel at each site and mixed for 3 L per sample. Microbial community structure, antibiotic resistance, virulence factors, and mobile genetic elements were analyzed through bioinformatic data obtained by metagenomic sequencing and full-length sequencing of 16S rRNA genes. Variations in the distribution of bacterial communities between samples and correlation of transmission patterns were analyzed by principal co-ordinates analysis, procrustes analysis, and Mantel test. Results: As the river flowed through Haikou City, microbes' alpha diversity gradually decreased. Among them, Proteobacteria dominates in the bacterial community in the front, middle, and rear sections, and the relative abundance of Proteobacteria in the middle and rear sections was higher than that in the front segment. The diversity and abundance of antibiotic resistance genes, virulence factors, and mobile genetic elements were all at low levels in the front section and all increased significantly after flow through Haikou City. At the same time, horizontal transmission mediated by mobile genetic elements played a more significant role in the spread of antibiotic-resistance genes and virulence factors. Conclusions: Urbanization significantly impacts river bacteria and the resistance genes, virulence factors, and mobile genetic elements they carry. The Nandu River in Haikou flows through the city, receiving antibiotic-resistant and pathogen-associated bacteria excreted by the population. In contrast, antibiotic-resistant genes and virulence factors are enriched in bacteria, which indicates a threat to environmental health and public health. Comparison of river microbiomes and antibiotic resistance genomes before and after flow through cities is a valuable early warning indicator for monitoring the spread of antibiotic resistance.

Portación nasal de Staphylococcus aureus en el personal de salud de áreas críticas de un Hospital Pediátrico durante julio-setiembre 2018 / Nasal carriage of Staphylococcus aureus in health personnel in critical areas of a Pediatric Hospital during July-September 2018 / Transporte nasal de Staphylococcus aureus em profissionais de saúde em áreas críticas de um Hospital Pediátrico durante julho-setembro de 2018

Las infecciones intrahospitalarias (IIH) son causa de elevada morbimortalidad y representan un problema sanitario importante. El personal de salud es reservorio y potencial transmisor de los agentes etiológicos de las mismas. S. aureus es uno de los microorganismos implicados, por lo tanto es importante conocer la frecuencia de portación en el personal de salud y establecer el perfil de susceptibilidad antimicrobiana para contribuir con la elaboración de medidas de prevención incluyendo actividades educativas. Objetivo: Conocer la frecuencia de portación de S. aureus, distribución y antibiotipos de las cepas presentes en el personal sanitario del Hospital Pediátrico de Referencia (HPR). Materiales y métodos: Se realizó un estudio descriptivo durante el periodo julio-setiembre del año 2018. Se incluyeron muestras de hisopados nasales de trabajadores de la salud de distintas áreas de internación que consintieron participar en el estudio. Se excluyeron aquellos que recibieron antibióticos dentro de los 3 meses previos al estudio. Las muestras fueron sembradas en agar sangre ovina al 5% (ASO) y se incubaron a 35-37ºC en aerobiosis por 24-48 horas. La identificación de las colonias sospechosas de Staphylococcus aureus por métodos convencionales y MALDI-TOF. El patrón de resistencia antimicrobiana de S. aureus se detectó por disco-difusión. En los cultivos resistentes a meticilina (SAMR) se determinó la presencia del gen mecA y se realizó la tipificación del SCCmec por pruebas de reacción en cadena de polimerasa. Resultados: Se obtuvieron 225 hisopados a partir de 225 trabajadores, presentaron desarrollo 212. En 49 se recuperaron cultivos de S. aureus. Correspondieron a SAMR 11 de las 49 cepas, todas portaban el gen mecA. Hubo predominio en el personal de enfermería (7/11), en los servicios de hemato-oncología (3/11) y cuidados intensivos neonatales (4/11). Asociaron resistencia a macrólidos y clindamicina 8 de 11 aislamientos SAMR, a gentamicina 2 y a mupirocina uno. El SCCmec más frecuentemente identificado fue el tipo IV (7/11). Conclusiones: Los resultados muestran la presencia de cepas SAMR entre el personal de salud del CHPR y aportan información complementaria para efectuar prevención y control de las IIH, actuando sobre todo en el personal de salud encargado de la atención de pacientes susceptibles.

Hospital-acquired infections (IIH) are a cause of high morbidity and mortality and represent a major health problem. Health personnel are reservoirs and potential transmitters of their etiological agents. S. aureus is one of the microorganisms involved, therefore it is important to know the frequency of carriage in health personnel and establish the antimicrobial susceptibility profile to contribute to the development of prevention measures, including educational activities. Objective: To know the frequency of carriage of S. aureus, distribution and antibiotypes of the strains present in the health personnel of the Reference Pediatric Hospital (HPR). Materials and methods: A descriptive study was carried out during the period July-September 2018. Nasal swab samples from health workers from different hospitalization areas who agreed to participate in the study were included. Those who received antibiotics within 3 months prior to the study were excluded. The samples were seeded in 5% sheep blood agar (ASO) and incubated at 35-37ºC in aerobiosis for 24-48 hours. Identification of suspicious Staphylococcus aureus colonies by conventional methods and MALDI-TOF. The antimicrobial resistance pattern of S. aureus was detected by disc diffusion. In methicillin-resistant cultures (MRSA), the presence of the mecA gene was determined and SCCmec was typified by polymerase chain reaction tests. Results: 225 swabs were obtained from 225 workers, 212 showed development. S. aureus cultures were recovered from 49. 11 of the 49 strains corresponded to MRSA, all of them carried the mecA gene. There was a predominance in the nursing staff (7/11), in the hematology-oncology services (3/11) and neonatal intensive care (4/11). They associated resistance to macrolides and clindamycin in 8 of 11 MRSA isolates, 2 to gentamicin, and 1 to mupirocin. The most frequently identified SCCmec was type IV (7/11). Conclusions: The results show the presence of MRSA strains among the health personnel of the CHPR and provide complementary information to carry out prevention and control of IIH, acting especially on the health personnel in charge of the care of susceptible patients.

As infecções hospitalares (HII) são causa de alta morbidade e mortalidade e representam um importante problema de saúde. Os profissionais de saúde são reservatórios e potenciais transmissores de seus agentes etiológicos. O S. aureus é um dos micro-organismos envolvidos, por isso é importante conhecer a frequência de portadores em profissionais de saúde e estabelecer o perfil de suscetibilidade antimicrobiana para contribuir no desenvolvimento de medidas de prevenção incluindo atividades educativas. Objetivo: Conhecer a frequência de portadores de S. aureus, distribuição e antibiótipos das cepas presentes no pessoal de saúde do Hospital Pediátrico de Referência (HPR). Materiais e métodos: Foi realizado um estudo descritivo durante o período de julho a setembro de 2018. Foram incluídas amostras de swab nasal de profissionais de saúde de diferentes áreas de internação que concordaram em participar do estudo. Aqueles que receberam antibióticos nos 3 meses anteriores ao estudo foram excluídos. As amostras foram semeadas em 5% de ágar sangue de carneiro (ASO) e incubadas a 35-37ºC em aerobiose por 24-48 horas. Identificação de colônias suspeitas de Staphylococcus aureus por métodos convencionais e MALDI-TOF. O padrão de resistência antimicrobiana de S. aureus foi detectado por difusão em disco. Em culturas resistentes à meticilina (MRSA), a presença do gene mecA foi determinada e SCCmec foi tipificado por testes de reação em cadeia da polimerase. Resultados: 225 swabs foram obtidos de 225 trabalhadores, 212 apresentaram desenvolvimento. Culturas de S. aureus foram recuperadas de 49. 11 das 49 cepas correspondiam a MRSA, todas carregavam o gene mecA. Houve predominância na equipe de enfermagem (7/11), nos serviços de hematologia-oncologia (3/11) e de terapia intensiva neonatal (4/11). Eles associaram resistência a macrolídeos e clindamicina em 8 de 11 isolados de MRSA, 2 à gentamicina e 1 à mupirocina. O SCCmec mais frequentemente identificado foi o tipo IV (7/11). Conclusões: Os resultados mostram a presença de cepas de MRSA entre os profissionais de saúde do CHPR e fornecem informações complementares para realizar a prevenção e controle da HII, atuando principalmente sobre os profissionais de saúde responsáveis ​​pelo atendimento de pacientes suscetíveis.

Environmental risks of antibiotics in soil and the related bioremediation technologies / 生物工程学报

Antibiotics are widely used and prevalently distributed in the environment. The issue of antibiotic resistance genes has posed a huge threat to the global public health. Soil is an important sink of antibiotics in the environment. Antibiotic exposure may introduce adverse effects on soil organisms, and bring indirect but potential risks to human health. Therefore, it is urgent to take actions to remediate antibiotics-contaminated soil. This review summarized effects of antibiotics on phenotype growth of plants, physiological characteristics and community structure of animals, composition and structure of microbial communities, and transmission of antibiotic resistance genes among organisms in soil. Additionally, the potential and prospects of employing antibiotic-resistant soil plants, animals, microorganisms, and their combinations to treat antibiotics-contaminated soil were illustrated. Last but not least, the unaddressed issues in this area were proposed, which may provide insights into relevant research directions in the future.

Application of CRISPR in evolution analysis, detecting and typing, virulence and antibiotic resistance regulation in food-borne pathogens / 生物工程学报

Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein gene system can limit the horizontal gene transfer, thereby effectively preventing the invasion of foreign gene elements such as bacteriophages. CRISPR arrays of different bacteria are diverse. Based on the differences in the CRISPR system, this review summarizes the application of CRISPR in food-borne pathogen evolution analysis, detection and typing, virulence and antibiotic resistance in recent years. We also address bacterial detection typing method developed based on the characteristics of CRISPR arrays and the association of CRISPR with virulence and drug resistance of food-borne pathogens. The shortcomings of CRISPR in evolution, detection and typing, virulence and resistance applications are analyzed. In addition, we suggest standardizing CRISPR typing methods, improving and expanding the CRISPR database of pathogenic bacteria, and further exploring the co-evolution relationship between phages and bacteria, to provide references for further exploration of CRISPR functions.

Escherichia coli diarrogénicos, identificación de patotipos y fenotipos de resistencia antimicrobiana en aislados cubanos / Diarrheagenic Escherichia coli: identification of pathotypes and antimicrobial resistance phenotypes in Cuban isolates

Introducción: En los países en vías de desarrollo las enfermedades diarreicas agudas son causa frecuente de morbilidad y mortalidad. Entre las primeras causas se encuentra Escherichia coli diarrogénicos, que afecta a pacientes en edades extremas de la vida y con inmunodeficiencias. Objetivo: Identificar los patotipos de Escherichia coli diarrogénicos que más inciden y los fenotipos de resistencia antimicrobiana expresados por el patotipo predominante. Métodos: Se estudiaron 184 aislamientos procedentes de 15 centros provinciales de Higiene, Epidemiología y Microbiología de Cuba e Isla de la Juventud. La investigación se realizó desde julio de 2012 hasta febrero de 2014. La identificación de género, especie y patotipos fue realizada por métodos de diagnóstico convencional y molecular. La determinación de la susceptibilidad antimicrobiana se realizó por el método de Bauer y Kirby, y las normas del Clinical and Laboratory Institute Standards de 2013. Resultados: Se identificaron 108 (58 por ciento) Escherichia coli diarrogénicos. Los patotipos confirmados fueron: en la PCR múltiple 1, 5 (6 por ciento) de Escherichia coli enteropatogénico, 4 (4 por ciento) de enterotoxigénico y 0 (0 por ciento) de enterohemorrágico. La PCR múltiple 2 reveló 72 (82 por ciento) Escherichia coli enteroagregativo, que resultó el predominante en el estudio. La PCR 3 (simple) detectó 7 (8 por ciento) de enteroinvasivo. El 100 por ciento del patotipo predominante mostró resistencia, al menos a un antimicrobiano de los probados, un solo patron de resistencia a dos antimicrobianos, y nueve patrones de multirresistencia. Conclusiones: El estudio demuestra la importancia del uso de pruebas moleculares rápidas para la confirmación de los patotipos de E. coli diarrogénicos, los que provocan deshidratación ligera, complicaciones graves y la muerte. Se logra identificar los cuatro patotipos más frecuentes y E. coli enteroagregativo, el de mayor incidencia en la población estudiada. El patotipo predominante mostró altos porcentaje de resistencia antimicrobiana a betalactámicos y buena sensibilidad antimicrobiana a los aminoglucósidos y cefalosporinas de tercera generación. La investigación aporta conocimientos, no revelados en estudios anteriores con aislados cubanos, lo que es considerado de alto valor para los clínicos, pediatras y epidemiólogos del país(AU)

Introduction: Acute diarrheal disease is a frequent cause of morbidity and mortality in developing countries. One of the leading causes is diarrheagenic Escherichia coli, which affects patients at extreme ages and with immunodeficiencies. Objective: Identify the most active pathotypes of diarrheagenic Escherichia coli and the antimicrobial resistance phenotypes expressed by the prevailing pathotype. Methods: A study was conducted from July 2012 to February 2014 of 184 isolates obtained from 15 provincial Hygiene, Epidemiology and Microbiology Centers in Cuba and the Isle of Youth. Identification of the genus, species and pathotypes was based on conventional and molecular diagnostic methods. Determination of antimicrobial susceptibility was performed by the Bauer-Kirby method in compliance with guidelines from the Clinical and Laboratory Standards Institute of 2013. Results: A total 108 (58 percent) diarrheagenic Escherichia coli were identified. The following pathotypes were confirmed: Multiplex PCR 1 revealed 5 (6 percent) enteropathogenic, 4 (4 percent) enterotoxigenic and 0 (0 percent ) enterohemorrhagic Escherichia coli. Multiplex PCR 2 found 72 (82 percent) enteroaggregative Escherichia coli, which was the prevailing type in the study. PCR 3 (simple) detected 7 (8 percent) enteroinvasive Escherichia coli. 100 percent of the prevailing pathotype displayed resistance to at least one of the antimicrobials tested, a single resistance pattern to two antimicrobials, and nine multiresistance patterns. Conclusions: The study showed the importance of the use of rapid molecular tests to confirm diarrheagenic E. coli pathotypes, which cause mild dehydration, serious complications and death. Identification could be done of the four most common pathotypes and enteroaggregative E. coli, the one with the highest incidence in the study population. The prevailing pathotype displayed high percentages of antimicrobial resistance to beta-lactams and good antimicrobial sensitivity to third-generation cephalosporins and aminoglycosides. The study contributed knowledge not revealed by previous research about Cuban isolates. Such information is considered to be highly valuable for clinicians, pediatricians and epidemiologists in the country(AU)

Characteristics of virulence, resistance and genetic diversity of strains of Salmonella Infantis isolated from broiler chicken in Brazil / Características de virulência, resistência e diversidade genética de estirpes de Salmonella Infantis isoladas de frangos de corte no Brasil

Salmonella Infantis is frequently associated with human infections worldwide and is transmitted by consumption of contaminated foods, particularly those of animal origin, especially the chicken meat. We aimed to evaluate virulence characteristics, antimicrobial resistance and the genetic similarity of 51 strains of S. Infantis isolated from samples of poultry origin. The strains were isolated from 2009 to 2010 in a company with full cycle of broiler's production in the state of São Paulo, Brazil. The antimicrobial susceptibility test was performed and, by PCR, we evaluated the presence of the genes lpfA (hem-adhesion), agfA (hem-biofilm) and sefA (hem-adhesion) and resistance genes to beta-lactams (blaTEM, blaSHV, bla CTX-M and blaAmpC ). The phylogenetic relationship was determined by RAPD-PCR method. Among the drugs tested, the highest percentages of resistance were to amoxicillin (35.3%) and to sulfonamide (15.7%). Eleven antimicrobial resistance patterns were identified (A1 to A11), none of them presented a multiresistance profile (> 3 antimicrobials classes). There was 100% of positivity for the agfA gene, 92.2% for the lpfA gene, and no strain presented the sefA gene. Most of the isolates showed similarities in virulence potential, since they were simultaneously positive for two studied genes, agfA and lpfA (92.2%, 47/51). Of the 18 (35.3%) strains resistant to antimicrobials of the β-lactam class, 10 (55.5%) were positive to blaAmpC gene, five (27.8%) for blaCTX-M , two (11.1%) to blaSHV and no strain presented the blaTEM gene. The phylogenetic evaluation has shown the presence of five clusters (A, B, C, D and E) with similarity greatSalmonella Infantis is frequently associated with human infections worldwide and is transmitted by consumption of contaminated foods, particularly those of animal origin, especially the chicken meat. We aimed to evaluate virulence characteristics, antimicrobial resistance and the genetic similarity of 51 strains of S. Infantis isolated from samples of poultry origin. The strains were isolated from 2009 to 2010 in a company with full cycle of broiler's production in the state of São Paulo, Brazil. The antimicrobial susceptibility test was performed and, by PCR, we evaluated the presence of the genes lpfA (hem-adhesion), agfA (hem-biofilm) and sefA (hem-adhesion) and resistance genes to beta-lactams (blaTEM, blaSHV, bla CTX-M and blaAmpC ). The phylogenetic relationship was determined by RAPD-PCR method. Among the drugs tested, the highest percentages of resistance were to amoxicillin (35.3%) and to sulfonamide (15.7%). Eleven antimicrobial resistance patterns were identified (A1 to A11), none of them presented a multiresistance profile (> 3 antimicrobials classes). There was 100% of positivity for the agfA gene, 92.2% for the lpfA gene, and no strain presented the sefA gene. Most of the isolates showed similarities in virulence potential, since they were simultaneously positive for two studied genes, agfA and lpfA (92.2%, 47/51). Of the 18 (35.3%) strains resistant to antimicrobials of the ß-lactam class, 10 (55.5%) were positive to blaAmpC gene, five (27.8%) for blaCTX-M , two (11.1%) to blaSHV and no strain presented the blaTEM gene. The phylogenetic evaluation has shown the presence of five clusters (A, B, C, D and E) with similarity greater than 80%, and three distinct strains which were not grouped in any cluster. Cluster B grouped 33 strains, all positive for lpfA and agfA genes, from both, the broiler farming facility and the slaughterhouse, persistent throughout all the study period. This cluster also grouped 18 strains clones with genetic similarity greater than 99%, all isolated in the slaughterhouse. The presence of virulence genes associated with persistent strains clones for a long period, warns to the possibility of S. Infantis to form biofilm, and should be constantly monitored in broilers' production chain, in order to know the profile of the strains that may contaminate the final product and evaluate the hazards that represents to public health.er than 80%, and three distinct strains which were not grouped in any cluster. Cluster B grouped 33 strains, all positive for lpfA and agfA genes, from both, the broiler farming facility and the slaughterhouse, persistent throughout all the study period. This cluster also grouped 18 strains clones with genetic similarity greater than 99%, all isolated in the slaughterhouse. The presence of virulence genes associated with persistent strains clones for a long period, warns to the possibility of S. Infantis to form biofilm, and should be constantly monitored in broilers' production chain, in order to know the profile of the strains that may contaminate the final product and evaluate the hazards that represents to public health.(AU)

Salmonella Infantis é frequentemente associada a infecções humanas no mundo todo sendo transmitida pelo consumo de alimentos contaminados, principalmente aqueles de origem animal, com destaque para a carne de frango. Objetivou-se avaliar características de virulência, resistência antimicrobiana e a similaridade genética de 51 estirpes de S. Infantis isoladas em amostras de origem avícola. As estirpes foram isoladas no período de 2009 a 2010 em uma empresa com ciclo completo de produção de frango de corte, localizada no estado de São Paulo, Brasil. Foi realizado o teste de susceptibilidade antimicrobiana e pela técnica de PCR, foi avaliada a presença dos genes lpfA (fímbria-adesão), agfA (fímbria-biofilme) e sefA (fímbria-adesão) e os genes de resistência aos beta-lactâmicos (bla TEM, blaSHV, blaCTX-M e blaAmpC ). A relação filogenética foi determinada pelo método de RAPD-PCR. Dentre as drogas testadas, os maiores percentuais de resistência foram para amoxacilina com 35,3% e sulfonamida com 15,7%. Onze perfis de resistência aos antimicrobianos foram identificados (A1 a A11), sendo que nenhum deles apresentou perfil de multirresistência (>3 classes de antimicrobianos). Houve 100% de positividade para o gene agfA, 92,2% para o gene lpfA e nenhuma estirpe apresentou o gene sefA. A maioria dos isolados apresentaram semelhanças no potencial de virulência, pois foram positivos simultaneamente para dois genes estudados, agfA e lpfA (92,2% - 47/51). Das 18 (35,3%) estirpes resistentes aos antimicrobianos da classe dos ß-lactâmicos, 10 (55,5%) foram positivas para o gene blaAmpC , cinco (27,8%) para blaCTX-M , duas (11,1%) para blaSHV e nenhuma estirpe apresentou o gene bla TEM . A avaliação filogenética demonstrou a presença de cinco clusters (A, B, C, D e E) com similaridade superior a 80%, e três estirpes distintas que não foram agrupadas em nenhum dos clusters. O cluster B agrupou 33 estirpes, todas positivas para os genes lpfA e agfA, provenientes tanto do aviário quanto do matadouro frigorífico, persistentes durante todo o período do estudo. Este cluster ainda agrupou 18 estirpes clones com similaridade genética superior a 99%, todas isoladas no matadouro frigorífico. A presença dos genes de virulência, associada à persistência das estirpes clones durante um longo período do estudo, alertam para a possibilidade de S. Infantis em formar biofilme, devendo ser constantemente monitorada na cadeia de produção avícola, especialmente no ambiente de abate, de forma a conhecer o perfil das estirpes que podem contaminar o produto final e assim avaliar os perigos que representam para a saúde pública.(AU)

Overview of antibiotic resistance genes database / 生物工程学报

The discovery of antibiotics is a big revolution in human history, and its clinical application has saved countless lives. However, with the widespread and abuse of antibiotics, many pathogens have developed resistance, and even "Super Bacteria" resistance to multiple drugs have evolved. In the arms race between humans and pathogens, humans are about to face a situation where no medicine is available. Research on microbial antibiotic resistance genes, resistance mechanisms, and the spread of resistance has attracted the attention of many scientific researchers, and various antibiotic resistance gene databases and analysis tools have emerged. In this review, we collect the current databases that focus on antibiotics resistance genes, and discuss these databases in terms of database types, data characteristics, antibiotics resistance gene prediction models and the types of analyzable sequences. In addition, a few gene databases of anti-metal ions and anti-biocides are also involved. It is believed that this summary will provide a reference for how to select and use antibiotic resistance gene databases.

Relationships between phagocytosis, mucoid phenotype, and genetic characteristics of Klebsiella pneumoniae clinical isolates

Abstract INTRODUCTION The relationships between phagocytosis, and mucoid phenotype, plasmid profile and virulence, and resistance genetic characteristics of Klebsiella pneumoniae clinical isolates were evaluated. METHODS Thirty isolates were used to determine the mucoid aspect. Four were selected for analysis of phagocytosis by alveolar macrophages. RESULTS Thirty percent of the samples presented the mucoid phenotype. The phagocytosis rate ranged from 21.5% to 43.43%. Phagocytosis was not correlated with the plasmid profile, but was apparently correlated with mucoid phenotype and antibiotic susceptibility. CONCLUSIONS: Several virulence factors act in parallel in K. pneumoniae to impair host defense.

Genomic basis of antibiotic resistance in Vibrio parahaemolyticus strain JPA1

A multi-resistant strain of Vibrio parahaemolyticus was isolated from a tropical estuary in Rio de Janeiro, Brazil. Genome sequencing was conducted to establish the molecular basis of antibiotic resistance in this organism. The genetic content of this strain revealed it to be a non-virulent lineage that nevertheless possesses several antibiotic resistance determinants.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Detecção e caracterização de elementos conjugativos integrativos em bactérias isoladas de amostras ambientais / Detection and characterization of integrative conjugative elements in bacteria isolated from environmental samples

O reconhecimento da resistência antimicrobiana como um fenômeno emergente em saúde pública, tem constituído um problema em nível mundial. O abuso na utilização de antibióticos na medicina humana e veterinária, e na agricultura, tem originado incremento na diversidade de micro-organismos resistentes, refletindo em falha terapêutica. Os mecanismos de resistência a antibióticos em micro-organismos são mediados principalmente por genes adquiridos de DNA exógeno. A dinâmica da transferência horizontal é realizada por meio de elementos genéticos móveis que carregam genes de resistência. A ampla distribuição deste tipo de estruturas, como o elemento SXT, isolado inicialmente em V. cholerae, tem contribuído para a disseminação de complexos específicos clonais em determinadas áreas geográficas. Este estudo pioneiro no Brasil pesquisou a presença de elementos SXT, em espécies bacterianas do grupo das gama proteobactérias em espécies ambientais, determinou suas características estruturais e funcionais, incluindo genes de resistência a antibióticos, bem como a sensibilidade aos antibióticos dentre os isolados bacterianos que os abrigam. O resultado foi a classificação de 43 elementos SXT obtidos no Brasil, através da comparação com aqueles descritos na literatura. Dentre os elementos SXT obtidos, quatro são albergados por Morganella morganii, fato inédito na literatura. O conhecimento da evolução bacteriana constitui importante ferramenta para estabelecer estratégias eficazes de controle e tratamento de infecções, sem aumentar a pressão seletiva sobre os micro-organismos, bem como instrumento preciso e de grande importância para subsidiar estudos epidemiológicos.

Recognition of antimicrobial resistance as an emerging phenomenon in public health has been a problem worldwide. The abuse in the use of antibiotics in human and veterinary medicine, and agriculture, has caused an increase in the diversity of resistant microorganisms, reflecting in treatment failure. The mechanisms of antibiotic resistance in microorganisms are primarily mediated by genes acquired from exogenous DNA. The dynamics of the horizontal transfer is performed by mobile genetic elements which carry resistance genes. The wide distribution of these structures, such as the SXT element originally isolated from V. cholerae, has contributed to the spread of specific clonal complexes in certain geographical areas. This pioneering study in Brazil researched the presence of SXT elements in the group of bacterial species in environmental gamma-proteobacteria species, determined their structural and functional characteristics, including genes for resistance to antibiotics and the antibiotic susceptibility among bacterial isolates that harbor them. The result was the classification of 43 SXT elements found in Brazil, by comparison with those found in the literature. Among the SXT elements found, four are sheltered by Morganella morganii, unprecedented in the literature. Knowledge of bacterial evolution is an important to establish effective strategies to control and treat infections without increasing the selective pressure on microorganisms, as well as a precise instrument and very important tool to support epidemiological studies.

Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems / Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems.

The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.

Genetic site determination of antibiotic resistance genes in escherichiacoli isolated from different sources of human infection

This study includes isolation of Escherichia coli from different sources of human infections [urine, stool, burns, wounds, and cerebrospinal fluid]. In addition to these, a few samples were taken from sewage water. Eighty-three isolates of E. coli were obtained from 264 samples. According to the resistance to antibiotics, isolates were classified into 41 groups. The isolates varied in their resistance to tested antimicrobials. Isolate E48 was resistant to all antimicrobials under study, while isolate E37 was resistant only to three antimicrobials. All isolates showed resistance of 97.59% to Chm and less sensitivity to Amk [2.40%]. The transformation was conducted successfully by plasmid DNA of isolate E48 and failed by plasmid isolate E38. The results cleared that the genes responsible for resistance to Amk, Chm, Cln, Dox, Kan, Lin, Pac, Tet, Tob, and Tri were located on the plasmid DNA, while the genes responsible for resistance to Cef, Cph, Cip, Gen, Gul, Nal, Nit, Pip, and Rif were located on the chromosome. It appeared from the electrophoresis run DNA samples on gel that E. coli K12JM83 strain obtained two plasmids through the transformation process

Emergence and dissemination of antibiotic resistance: A global problem.

Antibiotic resistance is a major problem in clinical health settings. Interestingly the origin of many of antibiotic resistance mechanisms can be traced back to non-pathogenic environmental organisms. Important factors leading to the emergence and spread of antibiotic resistance include absence of regulation in the use of antibiotics, improper waste disposal and associated transmission of antibiotic resistance genes in the community through commensals. In this review, we discussed the impact of globalisation on the transmission of antibiotic resistance genes in bacteria through immigration and export/import of foodstuff. The significance of surveillance to define appropriate use of antibiotics in the clinic has been included as an important preventive measure.

Caracterização fenogenotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite bovina / Phenogenotypical characterization of antimicrobial resistance in Staphylococcus spp. isolated from bovine mastitis

A utilização de antibióticos no controle das infecções intramamárias e na eliminação de prováveis fontes de infecção nas fazendas leiteiras se constitui em importante medida de controle. No entanto, o uso inadequado de antibióticos no tratamento da doença pode selecionar cepas resistentes e comprometer a eficiência do tratamento. Bactérias do gênero Staphylococcus spp. estão entre os principais agentes etiológicos da mastite bovina e são freqüentemente resistentes aos antimicrobianos, em especial aos beta-lactâmicos, principalmente por dois mecanismos distintos: a produção da enzima extracelular beta-lactamase, codificada pelo gene blaZ, e a produção de PBP2a ou PBP2´, uma proteína ligante de penicilina de baixa afinidade, codificada pelo gene mecA. A expressão do gene mecA é constitutiva ou induzida por antibióticos betalactâmicos, como a oxacilina e cefoxitina. O gene mecA está inserido no cromossomo através de um elemento genético móvel, denominado cassete estafilocócico cromossômico mec (SCCmec). O presente estudo avaliou o perfil fenogenotípico de resistência aos beta-lactâmicos em 250 isolados de Staphylococcus spp., utilizando os marcadores oxacilina e cefoxitina, de modo a produzir dados que possam contribuir para o conhecimento da resistência antimicrobiana em algumas propriedades leiteiras das regiões Sul-Fluminense e Metropolitana do Estado do Rio de Janeiro com o objetivo de subsidiar a implementação de medidas de controle dessa enfermidade. A avaliação da resistência foi feita a partir de 8 diferentes testes fenotípicos, sendo obtidos 54 perfis. Os testes de difusão em disco simples e ágar screen com oxacilina foram utilizados como "padrão ouro" para os cálculos dos valores de sensibilidade, especificidade e predição por serem preconizados pelo CLSI veterinário. O teste de difusão em disco simples com cefoxitina foi o de melhor desempenho na predição da resistência a oxacilina. Na avaliação genotípica, não foi detectado qualquer isolado positivo para o gene mecA, já os genes mecI e mecRI foram detectados igualmente em 11,6% (29/250) dos Staphylococcus spp. avaliados. Foram detectados os quatro tipos de cassete mec analisados (I, II, III e IV), sendo o tipo I o que teve mais ampla distribuição entre as regiões estudadas. Gene blaZ foi detectado em 5,2% (13/250) dos isolados, sendo que nestes, todo o sistema blaZ-blaI- blaR1 foi detectado em 23,1% (3/13) dos isolados.

The use of antibiotic in the control of intramammary infections and in the elimination of its possible sources in dairy farms is an important control measure. However, the inappropriate use of antibiotics can result in the appearance of resistant strains and compromise the efficiency of the treatment. Besides Staphylococcus spp. are among the main pathogens of bovine mastitis, they are often resistant to antibiotics, especially beta-lactamics, mainly by two distinct mechanisms: the production of extracellular enzyme beta-lactamase, encoded by the blaZ gene, and production of PBP2a or PBP2' a penicillin-binding protein with low affinity, encoded by the mecA gene. The expression of mecA gene is constitutive or induced by beta-lactamic antibiotics, such as oxacillin and cefoxitin. The mecA gene is inserted into the chromosome through a staphylococcal mobile genetic element, called staphylococcal cassette chromosome mec (SCCmec). The present study evaluated the phenogenotypical resistance profile to beta-lactam antibiotics of 250 Staphylococcus spp isolates, using oxacillin and cefoxitin as markers in order to produce data to the knowledge of resistance in dairy farms located in the South-Fluminense and the Metropolitan regions of the State of Rio de Janeiro to support the implementation of measures to control this disease. The assessment of resistance was made through 8 different phenotypic tests and yielded 54 profiles. Disk diffusion and agar screen with oxacillin were used as "gold standard" for the calculation of sensitivity, specificity and prediction once they are recommended by the CLSI veterinarian as standardized tests. Disk diffusion with cefoxitin achieved the best performance in the prediction of oxacillin resistance. Genotypic detection of mecA do not provided any positive isolate, otherwise mecI and mecRI genes were also detected in 11.6% (29/250) of the studied Staphylococcus spp. Four cassette mec types were detected (I, II, III and IV), being type I the most disseminated one. Gene blaZ was detected in 5.2% (13/250) isolates. From these 13 blaZ positive isolates, the whole system comprising blaR1-blaI-blaZ was detected in 23.1% (3/13) isolates.

In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China

Streptococcus suis is a swine pathogen and also a zoonotic agent. The formation of biofilms allows S. suis to become persistent colonizers and resist clearance by the host immune system and antibiotics. In this study, biofilm forming potentials of various S. suis strains were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and tissue culture plates stained with crystal violet. In addition, the effects of five antimicrobial agents on biofilm formation were assayed in this study. S. suis produced biofilms on smooth and rough surface. The nutritional contents including glucose and NaCl in the growth medium modulated biofilm formation. There was a significant difference in their biofilm-forming ability among all 46 S. suis strains. The biofilm-forming potential of S. suis serotype 9 was stronger than type 2 and all other types. However, biofilm formation was inhibited by five commonly used antimicrobial agents, penicillin, erythromycin, azithromycin, ciprofloxacin, and ofloxacin at subinhibitory concentrations, among which inhibition of ciprofloxacin and ofloxacin was stronger than that of other three antimicrobial agents.Our study provides a detailed analysis of biofilm formation potential in S. suis, which is a step towards understanding its role in pathogenesis, and eventually lead to a better understanding of how to eradicate S. suis growing as biofilms with antibiotic therapy.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis / Resistência antimicrobiana e detecção dos genes mec e blaZ em Staphylococcus coagulase-negativos isolados de mastite bovina

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

O presente estudo avaliou o perfil fenogenotípico de resistência aos antimicrobianos em espécies de Staphylococcus coagulase-negativo (ECN) isoladas do leite de vacas com mastite, em especial considerando a oxacilina. Dos 100 isolados de ECN, S.xylosus foi a espécie predominante, seguida por S. cohnii, S. hominis, S. capitis and S. haemolyticus Apenas 6% dos isolados foram fenotipicamente suscetíveis aos agentes antimicrobianos testados no ensaio de difusão em disco. O percentual de resistência à penicilina e ampicilina foi significativamente maior que aos outros antimicrobianos. Quatro isolados foram positivos para o gene mecA (4%), sendo todos representados pela espécie S.xylosus. O gene blaZ foi detectado em 16% dos isolados (16/100), sendo todos mecA positivos e a presença de ambos os genes foi correlacionada com a resistência fenotípica aos beta-lactâmicos. Foi possível concluir que as espécies de ECN provenientes de leite bovino apresentaram distintos perfis de resistência antimicrobiana, avaliados por testes fenotípicos e genotípicos, podendo dificultar a adoção de medidas de tratamento e manejo dos animais.

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil / Resistência antimicrobiana de Staphylococcus spp. de mastite de pequenos ruminantes no Brasil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.

O presente trabalho teve como objetivo determinar os padrões de resistência a agentes antimicrobianos e identificar marcadores moleculares de resistência em Staphylococcus spp. (n=210) isolados de mastite de pequenos ruminantes no Brasil. Os padrões de resistência a agentes antimicrobianos foram avaliados pelo teste de difusão em disco e pela detecção da presença dos genes mecA, blaZ, ermA, ermB, ermC e msrA via PCR. O teste da bomba de efluxo foi realizado utilizando brometo de etídio e a produção de biofime foi determinada pelo teste do vermelho congo em paralelo com o PCR para detecção do gene icaD. Os isolados foram mais resistentes a amoxicilina (50,0%), estreptomicina (42,8%), tetraciclina (40,4%), lincomicina (39,0%) e eritromicina (33,8%). Setenta e um (33,8%) isolados foram sensíveis a todas as drogas testadas e 41 foram positivos para a bomba de efluxo. Embora a resistência fenotípica a oxacilina tenha sido observada observada em 12,8% dos isolados, nenhum possuiu o gene mecA. Entretanto, 45,7% dos isolados continham a gene blaZ, indicando que a produção de beta-lactamases foi o principal mecanismo associado com a resistência dos Staphylococcus aos beta-lactâmicos. Os outros determinantes de resistência a agentes antimicrobianos ermA, ermb, ermC e msrA foram observados em 1,4%, 10,4%, 16,2% e 0,9% dos isolados respectivamente. Além disso, o gene icaD foi detectado em 32,9% dos isolados. Setenta e três isolados (54 de cabras e 19 de ovelhas) foram negativos para todos os genes de resistência testados e 69 isolados apresentaram dois ou mais genes de resistência. A associação entre blaZ, ermA, ermB, ermC e bomba de efluxo foi observada em 17 isolados dos quais 14 eram oriundos de cabras e três de ovelhas. Os dados obtidos no presente estudo indicam a resistência dos isolados aos beta-lactâmicos, o que pode estar associado ao uso sem controle veterinário destas drogas nos animais.

Antibiotic resistance genes & susceptibility patterns in staphylococci.

Background & objectives: This study was carried out to evaluate the association between the antibiotic susceptibility patterns and the antibiotic resistance genes in staphylococcal isolates obtained from various clinical samples of patients attending a teaching hospital in Hatay, Turkey. Methods: A total of 298 staphylococci clinical isolates were subjected to antimicrobial susceptibility testing. The genes implicated in resistance to oxacillin (mecA), gentamicin (aac(6’)/aph(2”), aph(3’-IIIa, ant(4’)-Ia), erythromycin (ermA, ermB, ermC, and msrA), tetracyclin (tetK, tetM), and penicillin (blaZ) were amplified using multiplex PCR method. Results: Methicillin resistance rate among 139 Staphlococcus aureus isolates was 16.5 and 25.9 per cent of S. aureus carried mecA gene. Of the 159 CoNS isolates, methicillin resistance rate was 18.9 and 29.6 per cent carried mecA gene. Ninety four isolates identified as gentamicin resistant phenotypically, contained at least one of the gentamicin resistance genes [aac(6’)/aph(2”), aph(3’)-IIIa, ant(4’)-Ia], 17 gentamicin-susceptible isolates were found as positive in terms of one or more resistance genes [aac(6’)/aph(2”), aph(3’)-IIIa, ant(4’)-Ia] by multiplex PCR. A total of 165 isolates were resistant to erythromycin, and contained at least one of the erythromycin resistance genes (ermA, ermB, ermC and msrA). Phenotypically, 106 staphylococcal isolates were resistant to tetracycline, 121 isolates carried either tetK or tetM or both resistance genes. The majority of staphylococci tested possessed the blaZ gene (89.9%). Interpretation & conclusions: The present results showed that the phenotypic antibiotic susceptibility patterns were not similar to those obtained by genotyping done by multiplex PCR. Rapid and reliable methods for antibiotic susceptibility are important to determine the appropriate therapy decisions. Multiplex PCR can be used for confirmation of the results obtained by conventional phenotypic methods, when needed.

Avaliação da resistência a antibióticos beta-lactâmicos em isolados de Staphylococcus spp / Evaluation of beta-lactam antibiotic resistance in isolates of Staphylococcus spp

O uso indiscriminado de antibiótico favorece o surgimento de cepas de Staphylococcus spp. resistentes à oxacilina. O mecanismo dessa resistência é devido à produção da proteína PBP2a, codificada pelo gene mecA. Alguns isolados clínicos apresentam o gene mecA, porém são sensíveis a concentrações de oxacilina e cefoxitina, ou seja, nesses isolados, denominados pré-MRS ou OSMRS, a presença do mecA não está associada à sua expressão. Acredita-se que, ao sofrerem pressão seletiva pelo uso indiscriminado de antibióticos, isolados pré-MRS podem tornar-se resistentes. O objetivo desse trabalho foi determinar se o surgimento de cepas de Staphylococcus resistentes à oxacilina e cefoxitina ocorre devido ao estímulo da expressão da PBP2a (indução) ou por seleção antibiótica de linhagens resistentes. Para isso, foram analisadas 42 isolados de Staphylococcus spp., vinte e cinco Staphylococcus aureus e 17 Staphylococcus Coagulase Negativa (SCN) de um Hospital Universitário de Recife-PE. Antes da exposição ao antibiótico a Concentração Inibitória Mínima (MIC) dos isolados foi determinada, o reconhecimento da PBP2a foi avaliado pelos testes de Western blot e Imunofluorescência Indireta (IFI) e realizadas provas de indução de resistência e de seleção por uso de antibiótico. O Western blot e a IFI mostraram que em algumas amostras havia expressão da PBP2a mesmo na ausência do antibiótico. No teste de indução com oxacilina houve uma estabilização do MIC e os isolados não alcançaram o MIC de resistência sugerindo um mecanismo de seleção. No teste para confirmação do processo de seleção, foi observada variabilidade entre as colônias de um mesmo isolado caracterizando um perfil de heteroresistência, enquanto alguns isolados se mostraram homogêneos, com alto nível de resistência. Esses achados reforçam a importância da detecção do gene mecA e da proteína PBP2a para conduta de prescrição de antibióticos na prática clínica.