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.

Comparative gut microbiota and resistome profiling of intensive care patients receiving selective digestive tract decontamination and healthy subjects.

BACKGROUND: The gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed "selective decontamination of the digestive tract" (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes ("the resistome") of ICU patients during SDD and of healthy subjects. RESULTS: From ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6')-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects. CONCLUSIONS: The gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.

The Two-Component System ChtRS Contributes to Chlorhexidine Tolerance in Enterococcus faecium.

Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrug-resistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNA-binding response regulator (ChtR), which contributed to CHX tolerance in E. faecium Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX- and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR Transmission electron microscopy showed that upon challenge with CHX, the ΔchtR and ΔchtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans Morphological abnormalities were also observed upon exposure of the ΔchtR and ΔchtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials.

Genetic Interaction of Aspergillus nidulans galR, xlnR and araR in Regulating D-Galactose and L-Arabinose Release and Catabolism Gene Expression.

In Aspergillus nidulans, the xylanolytic regulator XlnR and the arabinanolytic regulator AraR co-regulate pentose catabolism. In nature, the pentose sugars D-xylose and L-arabinose are both main building blocks of the polysaccharide arabinoxylan. In pectin and arabinogalactan, these two monosaccharides are found in combination with D-galactose. GalR, the regulator that responds to the presence of D-galactose, regulates the D-galactose catabolic pathway. In this study we investigated the possible interaction between XlnR, AraR and GalR in pentose and/or D-galactose catabolism in A. nidulans. Growth phenotypes and metabolic gene expression profiles were studied in single, double and triple disruptant A. nidulans strains of the genes encoding these paralogous transcription factors. Our results demonstrate that AraR and XlnR not only control pentose catabolic pathway genes, but also genes of the oxido-reductive D-galactose catabolic pathway. This suggests an interaction between three transcriptional regulators in D-galactose catabolism. Conversely, GalR is not involved in regulation of pentose catabolism, but controls only genes of the oxido-reductive D-galactose catabolic pathway.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

Comparison of an ST80 MRSA strain from the USA with European ST80 strains.

OBJECTIVES: A number of community-acquired MRSA (CA-MRSA) clonal lineages dominate worldwide. ST80 was dominant in Europe and has increasingly been described from the Middle East. Here we report the whole genome sequence of the first ST80 CA-MRSA from the USA. METHODS: CA-MRSA isolate S0924 was obtained from a patient admitted to Cook County Hospital (Chicago, IL, USA) who came from Syria; the isolate belonged to spa type t044 and ST80. The whole genome sequence of S0924 was determined and compared with three previously published whole genome sequences of ST80 CA-MRSA from Europe and a newly sequenced ST80 CA-MRSA from the Netherlands (S1475). RESULTS: Based on spa typing, SCCmec type and virulence gene profile, this US ST80 isolate is indistinguishable from the European CA-MRSA ST80 clone. SNP analysis within the conserved core genome showed clear differences between the strains with up to 144 SNPs differing between S0924 and strain S1800, an ST80 MRSA from Greece. The gene content showed 21 regions of difference between the US and European isolates, although these were largely restricted to mobile genetic elements. Phylogenetic reconstruction indicated that the European strains were more closely related to each other than to the US strain. The SNP data suggest that a common ancestor existed around two decades ago, indicating that the US and European ST80 strains are clonally linked. CONCLUSIONS: These data combined with the country of origin of the patient suggest that ST80 S0924 was probably relatively recently introduced into the USA from Syria.

Shared reservoir of ccrB gene sequences between coagulase-negative staphylococci and methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci (CoNS) is caused by expression of the low-affinity penicillin-binding protein (PBP) 2a encoded by the mecA gene. This gene is carried on the staphylococcal cassette chromosome mec (SCCmec) of which several types and subtypes have been described. CoNS and S. aureus share SCCmec types and it has been suggested that CoNS are a potential reservoir of mecA for S. aureus. Evidence for this is mainly based on PCR typing of SCCmec or on sequence-based methods including only a limited number of strains. In this study, we determined the genetic relatedness of ccrB sequences contained in SCCmec elements of a spatio-temporally diverse and comprehensive collection of methicillin-resistant CoNS and S. aureus. METHODS: Part of the ccrB genes of 367 methicillin-resistant CoNS and 94 methicillin-resistant S. aureus (MRSA) were sequenced and compared. RESULTS: The data revealed that 92 of 94 (98%) MRSA isolates carried ccrB genes, involving different ccrB alleles, which were indistinguishable from ccrB genes of methicillin-resistant CoNS. In total, 273 of 367 (74%) CoNS shared ccrB gene sequences with MRSA. CONCLUSIONS: The high rate of identical ccrB sequences in a geographically, temporally and genotypically diverse set of S. aureus and CoNS isolates indicates frequent horizontal transfer of SCCmec between CoNS and S. aureus, which may have contributed to the emergence of MRSA.

Aspergillus cibarius sp. nov., from traditional meju in Korea.

Aspergillus cibarius sp. nov. isolated from meju, a brick of dried fermented soybeans in Korea, is described. The species was also found from black bean, bread and salami in the Netherlands. It is characterized by abundant yellow to reddish brown ascomata and small lenticular ascospores (4.5-5.5 µm) with a wide furrow, low equatorial crests and tuberculate or reticulate convex surface. The species was resolved as phylogenetically distinct from the other reported Aspergillus species with an Eurotium teleomorph based on multilocus sequence typing using partial fragments of the ß-tubulin, calmodulin, ITS and RNA polymerase II genes.

The recombinase IntA is required for excision of esp-containing ICEEfm1 in Enterococcus faecium.

Comparative genome analysis of Enterococcus faecium recently revealed that a genomic island containing the esp gene, referred to as the esp-containing pathogenicity island (esp PAI), can be transferred by conjugation and contains a partial Tn916-like element and an integrase gene, intA. Here, we characterize the role of intA in the excision of the esp PAI. An intA insertion-deletion mutant in E. faecium E1162 (E1162ΔintA) was constructed and in trans complemented with wild-type intA (E1162ΔintA::pEF30). Circular intermediates (CI) of excised esp PAI were determined using inverse PCR analysis on purified chromosomal DNA from strains E1162, E1162Δesp, E1162ΔintA, and E1162ΔintA::pEF30. In E1162 and E1162Δesp, CI of the esp PAI were detected. No CI were detected in E1162ΔintA, while in the complemented strain E1162ΔintA::pEF30 CI formation was restored, indicating that intA is essential for excision and subsequent mobilization of the esp-containing genomic island in E. faecium. Based on the fact that this island can be mobilized and is self-transmissible, we propose to change the name of the esp PAI to ICEEfm1.