Limited influence of hospital wastewater on the microbiome and resistome of wastewater in a community sewerage system.

Effluents from wastewater treatment plants (WWTPs) have been proposed to act as point sources of antibiotic-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in the environment. Hospital sewage may contribute to the spread of ARB and ARGs as it contains the feces and urine of hospitalized patients, who are more frequently colonized with multi-drug resistant bacteria than the general population. However, whether hospital sewage noticeably contributes to the quantity and diversity of ARGs in the general sewerage system has not yet been determined.Here, we employed culture-independent techniques, namely 16S rRNA gene sequencing and nanolitre-scale quantitative PCRs, to assess the role of hospital effluent as a point source of ARGs in the sewerage system, through comparing microbiota composition and levels of ARGs in hospital sewage with WWTP influent with and without hospital sewage.Compared to other sites, hospital sewage was richest in human-associated bacteria and contained the highest relative levels of ARGs. Yet, the relative abundance of ARGs was comparable in the influent of WWTPs with and without hospital sewage, suggesting that hospitals do not contribute importantly to the quantity and diversity of ARGs in the investigated sewerage system.

New methods to analyse microarray data that partially lack a reference signal.

BACKGROUND: Microarray-based Comparative Genomic Hybridisation (CGH) has been used to assess genetic variability between bacterial strains. Crucial for interpretation of microarray data is the availability of a reference to compare signal intensities to reliably determine presence or divergence each DNA fragment. However, the production of a good reference becomes unfeasible when microarrays are based on pan-genomes.When only a single strain is used as a reference for a multistrain array, the accessory gene pool will be partially represented by reference DNA, although these genes represent the genomic repertoire that can explain differences in virulence, pathogenicity or transmissibility between strains. The lack of a reference makes interpretation of the data for these genes difficult and, if the test signal is low, they are often deleted from the analysis. We aimed to develop novel methods to determine the presence or divergence of genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach for which reference DNA was not available for some probes. RESULTS: In this study we have developed 6 new methods to predict divergence and presence of all genes spotted on a multistrain Staphylococcus aureus DNA microarray, published previously, including those gene spots that lack reference signals. When considering specificity and PPV (i.e. the false-positive rate) as the most important criteria for evaluating these methods, the method that defined gene presence based on a signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics. For this method specificity was 100% and 82% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively, and PPV were 100% and 76% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively. CONCLUSION: A definition of gene presence based on signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics, allowing the analysis of 6-17% more of the genes not present in the reference strain. This method is recommended to analyse microarray data that partially lack a reference signal.

Transition of Enterococcus faecium from commensal organism to nosocomial pathogen.

The Gram-positive species Enterococcus faecium has long been thought of as a harmless commensal of the mammalian GI tract. In the last two decades, however, E. faecium has become an important cause of nosocomial bacteremias. These infections are often difficult to treat owing to the resistance of E. faecium to a large number of antibiotics. In this article, we review the recent transition of E. faecium from commensal to nosocomial pathogen. We focus on population biology-based studies, which suggest that several clonal populations of E. faecium are mostly responsible for causing infections. We also discuss the role of the accessory genome of E. faecium in contributing to the infectious phenotype and examine the role that surface proteins of E. faecium may have in colonization and infection.

[High-risk clonal complexes CC2 and CC9 are widely distributed among Enterococcus faecalis hospital isolates recovered in Spain]. / Los complejos clonales de alto riesgo CC2 y CC9 están ampliamente representados en cepas hospitalarias de Enterococcus faecalis aisladas en España.

INTRODUCTION: Our previously described multilocus sequence typing (MLST) scheme for Enterococcus faecalis has provided insight into the population structure and global epidemiology of this organism. Two high-risk complexes, CC2 and CC9, especially adapted to the hospital environment and widely distributed in Europe and America, were identified. The purpose of this study was to define the presence of CC2 and CC9 among E. faecalis strains isolated in Spain. METHODS: A total of 81 E. faecalis isolates recovered from several sources and geographic areas of Spain were characterized using MLST. Because of their clinical and epidemiological interest, strains were included from each of the vancomycin-resistant E. faecalis hospital outbreaks described in Spain. RESULTS: Among the isolates, CC2 and CC9 were detected in the hospital setting. Included in these CC were the vancomycin-resistant E. faecalis isolates causing hospital outbreaks in La Coruña, Palma de Mallorca and Valencia, as well as vancomycin-susceptible hospital isolates. The Index of Association (Ia), which measures linkage disequilibrium between alleles, revealed an epidemic population structure on a background of high recombination rates. CONCLUSIONS: High-risk complexes (CC2 and CC9) particularly adapted to the hospital environment were detected in Spain. Evolution of these CC in different areas depended on the local gene pool. Future infection control policies should be orientated to detect high-risk CC with the aim of predicting potential trends toward acquisition of specific resistance, such as to vancomycin.

Los complejos clonales de alto riesgo CC2 y CC9 están ampliamente representados en cepas hospitalarias de Enterococcus faecalis aisladas en España / High-risk clonal complexes CC2 and CC9 are widely distributed among Enterococcus faecalis hospital isolates recovered in Spain

Introducción. El desarrollo de un esquema de Multilocus sequence typing (MLST) para Enterococcus faecalis ha permitido conocer los primeros datos de su estructura poblacional y epidemiología global. Se ha detectado la dispersión en Europa y América de dos complejos clonales (CC) de alto riesgo denominados CC2 y CC9 que están especialmente adaptados al medio hospitalario. El objetivo de este trabajo ha sido definir la presencia de ambos CC en cepas de E. faecalis aisladas en España. Métodos. Se han caracterizado por MLST 81 cepas de E. faecalis aisladas de diversos orígenes y correspondientes a diferentes años y regiones españolas. Debido a su importancia clínica y epidemiológica se han incluido cepas representantes de cada uno de los brotes hospitalarios por E. faecalis resistente a vancomicina descritos en España. Resultados. En el medio hospitalario se detectó la dispersión de CC2 y CC9. En estos CC se han agrupado las cepas de E. faecalis resistentes a vancomicina causantes de los brotes hospitalarios en La Coruña, Palma de Mallorca y Valencia, así como cepas de origen hospitalario sensibles a la vancomicina. La utilización del índice de asociación (Ia), que estima el equilibrio de ligamiento en la población estudiada, reveló una estructura poblacional epidémica cuya variabilidad genética es debida a procesos de recombinación. Conclusión. En España se han detectado los CC de alto riesgo CC2 y CC9, que han evolucionado localmente de forma diferente en función de la carga genética del entorno. Las medidas de control de la infección deberían ir encaminadas a la detección de estos CC de alto riesgo, ya que su presencia en los hospitales podrían predecir tendencias futuras en cuanto a la adquisición de determinadas resistencias como es el caso de la vancomicina (AU)

Introduction. Our previously described multilocus sequence typing (MLST) scheme for Enterococcus faecalis has provided insight into the population structure and global epidemiology of this organism. Two high-risk complexes, CC2 and CC9, especially adapted to the hospital environment and widely distributed in Europe and America, were identified. The purpose of this study was to define the presence of CC2 and CC9 among E. faecalis strains isolated in Spain. Methods. A total of 81 E. faecalis isolates recovered from several sources and geographic areas of Spain were characterized using MLST. Because of their clinical and epidemiological interest, strains were included from each of the vancomycin-resistant E. faecalis hospital outbreaks described in Spain. Results. Among the isolates, CC2 and CC9 were detected in the hospital setting. Included in these CC were the vancomycin-resistant E. faecalis isolates causing hospital outbreaks in La Coruña, Palma de Mallorca and Valencia, as well as vancomycin-susceptible hospital isolates. The Index of Association (Ia), which measures linkage disequilibrium between alleles, revealed an epidemic population structure on a background of high recombination rates. Conclusions. High-risk complexes (CC2 and CC9) particularly adapted to the hospital environment were detected in Spain. Evolution of these CC in different areas depended on the local gene pool. Future infection control policies should be orientated to detect high-risk CC with the aim of predicting potential trends toward acquisition of specific resistance, such as to vancomycin (AU)