Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

<p>Abstract</p> <p>Background</p> <p>Since bacteria embedded in biofilms are far more difficult to eradicate than planktonic infections, it would be useful to know whether certain <it>Staphylococcus aureus </it>lineages are especially involved in strong biofilm formation. For this reason, <it>in vitro </it>biofilm formation of 228 clinical <it>S. aureus </it>isolates of distinct clonal lineages was investigated.</p> <p>Results</p> <p>At 0.1% glucose, more than 60% of the <it>S. aureus </it>strains associated with multilocus sequence typing (MLST) clonal complex (CC)8 produced large amounts of biomass, compared to 0-7% for various other clonal lineages. Additionally, <it>S. aureus </it>bloodstream isolates associated with MLST CC8 and CC7 had similar biofilm forming capacities as their commensal counterparts. Furthermore, strong biofilm formation could not be attributed to a specific accessory gene regulator (<it>agr</it>) genotype, as suggested previously. The <it>agr </it>genotypes were strictly associated with the clonal lineages. Moreover, strong biofilm formation was not related to slime formation. Congo red agar (CRA) screening is therefore not useful as a qualitative screening method for biofilm formation.</p> <p>Conclusion</p> <p>The adherence to polystyrene surfaces under physiologic glucose concentration (0.1%) was dependent on the clonal lineage. Strains associated with MLST CC8 were markedly more often classified as strong biofilm former at glucose concentrations of 0%, 0.1% and 0.25%.</p> <p>The present study reveals that the MLST CC8 associated genetic background was a predisposing factor for strong biofilm formation <it>in vitro</it>, under all tested glucose concentrations.</p

MRSA Prevalence and Associated Risk Factors among Health-Care Workers in Non-outbreak Situations in the Dutch-German EUREGIO

Preventing the spread of methicillin-resistant Staphylococcus aureus (MRSA) in healthcare facilities is a major infection control target. However, only a few studies have assessed the potential role of healthcare workers (HCWs) for MRSA dissemination. To investigate the MRSA prevalence and the risk factors for MRSA colonization among HCWs, nasopharyngeal swabs were taken between June 2010 and January 2011 from 726 employees from nine acute care hospitals with different care levels within the German part of a Dutch-German border region (EUREGIO). The isolated MRSA strains were investigated using spa typing. The overall MRSA prevalence among HCWs in a non-outbreak situation was 4.6% (33 of 726), and was higher in nurses (5.6 %, 29 of 514) than in physicians (1.2%, 1 of 83). Possible risk factors associated with MRSA colonization were a known history of MRSA carriage and the presence of acne. Intensive contact with patients may facilitate MRSA transmission between patients and HCWs. Furthermore, an accumulation of risk factors was accompanied by an increased MRSA prevalence in HCW

Molecular characterization of methicillin-resistant Staphylococcus aureus bloodstream isolates from

Objectives: The objectives of this study were (i) to investigate the genetic background of methicillinresistant Staphylococcus aureus (MRSA) bloodstream isolates from Croatia and (ii) to monitor the prevalence of Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) among these isolates. Methods: Eighty-two hospital-acquired MRSA bloodstream isolates, collected in 2001 and 2002 in Croatia, were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). The presence of genes encoding PVL and TSST-1 was investigated by realtime PCR. Results: All strains were multiresistant and were distributed among 16 different similarity groups as determined by PFGE. Two of the groups, groups H and K, harboured the majority of the MRSA strains with 52 and 12%, respectively. The predominant SCCmec type found among the isolates was type I (89%). Eleven per cent of the strains harboured a modified SCCmec type III, which contained, in contrast to the regular type III, an additional dcs region. One strain harboured a novel SCCmec type, containing the ccrC gene in combination with the mecI gene, the dcs region, the locus between pI258 and Tn554 (locus E) and the locus between Tn554 and orfX (locus F). MLST showed the presence of ST111-MRSA-I and ST247-MRSA-I among Croatian MRSA isolates. All isolates were negative for both PVL and TSST-1. Conclusions: These results indicate the emergence of ST111-MRSA-I and ST247-MRSA-I in Croatia among MRSA bloodstream isolates. The virulence factors PVL and TSST-1 were not present among these isolates

Characterization of a CTX-M-15 Producing Klebsiella Pneumoniae Outbreak Strain Assigned to a Novel Sequence Type (1427)

Extended-spectrum ß-lactamase producing Klebsiella pneumoniae have emerged as one of the major nosocomial pathogens. Between July and September 2012, a CTX-M-15 producing K. pneumoniae caused an outbreak in a university hospital in the Netherlands. The outbreak isolates were characterized and assigned to a novel sequence type (ST1427). An epidemiological link between affected patients was supported by patient contact tracing and whole-genome phylogenetic analysis. Genetic diversity was detected among multiple isolates obtained from different body sites of the index patient, which may relate to antibiotic treatment and/or host adaptation. Environmental contamination caused by the outbreak clone was found in the patient rooms even on medical equipment. The novel clone was not closely related to any known endemic/epidemic clone, but carried a set of a plasmid-borne resistance genes (blaCTX-M-15, blaTEM-1, blaOXA-1, aac(6')-Ib-cr, qnrB1, tetA(A), aac(3)-II). Analysis of its virulence factors revealed a previously uncharacterized capsular biosynthesis region and two uncharacterized fimbriae gene clusters, and suggested that the new clone was not hypervirulent. To our knowledge, this is the first outbreak report of K. pneumoniae ST1427, and our study could be of help to understand the features of this newly emerging clone

Cross-Border Dissemination of Methicillin-Resistant Staphylococcus aureus, Euregio Meuse-Rhin Region

MRSA clones were associated with hospital-associated clonal complexes and with Panton-Valentine leukocidin–positive community-associated MRSA

Evaluation of an Accelerated Workflow for Surveillance of ESBL (CTX-M)-Producing Escherichia coli Using Amplicon-Based Next-Generation Sequencing and Automated Analysis

Outbreak management of extended spectrum β-lactamase (ESBL)-producing pathogens requires rapid and accurate diagnosis. However, conventional screening is slow and labor-intensive. The vast majority of the screened samples are negative and detection of non-outbreak-related resistant micro-organisms often complicates outbreak management. In a CTX-M-15-producing Escherichia coli outbreak, 149 fecal samples and rectal eSwabs were collected by a cross-sectional survey in a Dutch nursing home. Samples were processed by routine diagnostic methods. Retrospectively, ESBL-producing bacteria and resistance genes were detected directly from eSwab medium by an accelerated workflow without prior enrichment cultures by an amplicon-based next-generation sequencing (NGS) method, and culture. A total of 27 (18.1%) samples were positive in either test. Sensitivity for CTX-M detection was 96.3% for the phenotypic method and 85.2% for the NGS method, and the specificity was 100% for both methods, as confirmed by micro-array. This resulted in a positive predictive value (PPV) of 100% for both methods, and a negative predictive value (NPV) of 99.2% and 96.8% for the phenotypic method and the NGS method, respectively. Time to result was four days and 14 h for the phenotypic method and the NGS method, respectively. In conclusion, the sensitivity without enrichment shows promising results for further use of amplicon-based NGS for screening during outbreaks

Use of whole-genome sequencing to trace, control and characterize the regional expansion of extended-spectrum beta-lactamase producing ST15 Klebsiella pneumoniae

The study describes the transmission of a CTX-M-15-producing ST15 Klebsiella pneumoniae between patients treated in a single center and the subsequent inter-institutional spread by patient referral occurring between May 2012 and September 2013. A suspected epidemiological link between clinical K. pneumoniae isolates was supported by patient contact tracing and genomic phylogenetic analysis from May to November 2012. By May 2013, a patient treated in three institutions in two cities was involved in an expanding cluster caused by this high-risk clone (HiRiC) (local expansion, CTX-M-15 producing, and containing hypervirulence factors). A clone-specific multiplex PCR was developed for patient screening by which another patient was identified in September 2013. Genomic phylogenetic analysis including published ST15 genomes revealed a close homology with isolates previously found in the USA. Environmental contamination and lack of consistent patient screening were identified as being responsible for the clone dissemination. The investigation addresses the advantages of wholegenome sequencing in the early detection of HiRiC with a high propensity of nosocomial transmission and prolonged circulation in the regional patient population. Our study suggests the necessity for interinstitutional/regional collaboration for infection/outbreak management of K. pneumoniae HiRiCs

Reprint of "Application of next generation sequencing in clinical microbiology and infection prevention"

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM (TM) (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements

The molecular evolution of hospital- and community-associated methicillin-resistant Staphylococcus aureus

Staphylococcus aureus can cause a wide variety of infections, ranging from minor skin infections to post-operative wound infections. Its adaptive power to antibiotics has resulted in the emergence of methicillin-resistant S. aureus (MRSA) in the beginning of the 1960s. Resistance to methicillin and all other beta-lactam antibiotics is caused by the mecA gene, which is situated on a mobile genomic island, the Staphylococcal Cassette Chromosome mec (SCCmec). Seven main SCCmec types, I to VII, have been distinguished. The most important methods used to study the molecular epidemiology of MRSA are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing and SCCmec typing. These methods have been used to investigate the evolution of the MRSA clones that have emerged since the 1960s, and to study their worldwide dissemination. Early MRSA clones were hospital-associated (HA-MRSA). However, from the late 1990s, community-associated MRSA (CA-MRSA) has emerged. CA-MRSA harbors SCCmec type IV, V or VII, has a genetic background that is often distinct from HA-MRSA, and is often associated with the toxin Panton-Valentine leukocidin (PVL). However, the distinction between HA-MRSA and CA-MRSA is beginning to blur, and CA-MRSA is endemic in many US hospitals nowadays. This review describes the latest developments concerning the structure of SCCmec, the methods used to investigate the molecular epidemiology of MRSA, the molecular evolution of MRSA as well as the major challenges that are awaiting researchers in the near future

The evolution of Staphylococcus aureus

A broad variety of infections, ranging from minor infections of the skin to post-operative wound infections can be caused by Staphylococcus aureus. The adaptive power of S. aureus to antibiotics leaded, in the early 1960s, to the emergence of methicillin-resistant S. aureus (MRSA). The cause of resistance to methicillin and all other beta-lactam antibiotics is the mecA gene, which is situated on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). Seven major variants of SCCmec, type I to VII, are distinguished. The most important techniques used to investigate the molecular epidemiology of S. aureus are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), S. aureus protein A (spa) typing and SCCmec typing (only for MRSA). These techniques have been used to study the evolution of the MRSA clones that have emerged since the early 1960s, and to study their subsequent worldwide dissemination. The early MRSA clones were hospital-associated (HA-MRSA). However, from the late 1990s, community-associated MRSA (CA-MRSA) clones emerged worldwide. CA-MRSA harbors SCCmec type IV, V or VII, the majority belong to other S. aureus lineages compared to HA-MRSA, and CA-MRSA is often associated with the presence of the toxin Panton-Valentine leukocidin (PVL). However, during recent years, the distinction between HA-MRSA and CA-MRSA has started to disappear, and CA-MRSA is now endemic in many US hospitals. MRSA probably originated trough the transfer of SCCmec into a limited number of methicillin-sensitive S. aureus (MSSA) lineages. This review describes the latest observations about the structure of SCCmec, the techniques used to study the molecular epidemiology and evolution of S. aureus as well as some challenges that researchers face in the future