Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Future potential of metagenomics in clinical laboratories

INTRODUCTION: Rapid and sensitive diagnostic strategies are necessary for patient care and public health. Most of the current conventional microbiological assays detect only a restricted panel of pathogens at a time or require a microbe to be successfully cultured from a sample. Clinical metagenomics next-generation sequencing (mNGS) has the potential to unbiasedly detect all pathogens in a sample, increasing the sensitivity for detection and enabling the discovery of unknown infectious agents. AREAS COVERED: High expectations have been built around mNGS; however, this technique is far from widely available. This review highlights the advances and currently available options in terms of costs, turnaround time, sensitivity, specificity, validation, and reproducibility of mNGS as a diagnostic tool in clinical microbiology laboratories. EXPERT OPINION: The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionise clinical microbiology. However, its role as a diagnostic tool has yet to be widely established, which is crucial for successfully implementing the technique. A clear definition of diagnostic algorithms that include mNGS is vital to show clinical utility. Similarly to real-time PCR, mNGS will one day become a vital tool in any testing algorithm

Enterococcus faecium:from microbiological insights to practical recommendations for infection control and diagnostics

Early in its evolution, Enterococcus faecium acquired traits that allowed it to become a successful nosocomial pathogen. E. faecium inherent tenacity to build resistance to antibiotics and environmental stressors that allows the species to thrive in hospital environments. The continual wide use of antibiotics in medicine has been an important driver in the evolution of E. faecium becoming a highly proficient hospital pathogen.For successful prevention and reduction of nosocomial infections with vancomycin resistant E. faecium (VREfm), it is essential to focus on reducing VREfm carriage and spread. The aim of this review is to incorporate microbiological insights of E. faecium into practical infection control recommendations, to reduce the spread of hospital-acquired VREfm (carriage and infections). The spread of VREfm can be controlled by intensified cleaning procedures, antibiotic stewardship, rapid screening of VREfm carriage focused on high-risk populations, and identification of transmission routes through accurate detection and typing methods in outbreak situations. Further, for successful management of E. faecium, continual innovation in the fields of diagnostics, treatment, and eradication is necessary

New Topoisomerase Inhibitors:Evaluating the Potency of Gepotidacin and Zoliflodacin in Fluoroquinolone-Resistant Escherichia coli upon tolC Inactivation and Differentiating Their Efflux Pump Substrate Nature

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux-bypassing drugs and provide AcrB targets with critical relevance for this purpose

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

Within-patient plasmid dynamics in Klebsiella pneumoniae during an outbreak of a carbapenemase-producing Klebsiella pneumoniae

INTRODUCTION: Knowledge of within-patient dynamics of resistance plasmids during outbreaks is important for understanding the persistence and transmission of plasmid-mediated antimicrobial resistance. During an outbreak of a Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae, the plasmid and chromosomal dynamics of K. pneumoniae within-patients were investigated. METHODS: During the outbreak, all K. pneumoniae isolates of colonized or infected patients were collected, regardless of their susceptibility pattern. A selection of isolates was short-read and long-read sequenced. A hybrid assembly of the short-and long-read sequence data was performed. Plasmid contigs were extracted from the hybrid assembly, annotated, and within patient plasmid comparisons were performed. RESULTS: Fifteen K. pneumoniae isolates of six patients were short-read whole-genome sequenced. Whole-genome multi-locus sequence typing revealed a maximum of 4 allele differences between the sequenced isolates. Within patients 1 and 2 the resistance gene- and plasmid replicon-content did differ between the isolates sequenced. Long-read sequencing and hybrid assembly of 4 isolates revealed loss of the entire KPC-gene containing plasmid in the isolates of patient 2 and a recombination event between the plasmids in the isolates of patient 1. This resulted in two different KPC-gene containing plasmids being simultaneously present during the outbreak. CONCLUSION: During a hospital outbreak of a KPC-producing K. pneumoniae isolate, plasmid loss of the KPC-gene carrying plasmid and plasmid recombination was detected within the isolates from two patients. When investigating outbreaks, one should be aware that plasmid transmission can occur and the possibility of within- and between-patient plasmid variation needs to be considered

Limited Multidrug Resistance Efflux Pump Overexpression among Multidrug-Resistant Escherichia coli Strains of ST131

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years E. coli ST131 has emerged as a major cause of extraintestinal infection frequently exhibiting an MDR phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones however, is not well studied. We characterized strains from an international collection of clinical MDR-E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by Principal Component Analysis (PCA). PCA revealed a group of 17/34 MDR-E. coli exhibiting increased susceptibility to treatment with NMP suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed "enhanced efflux phenotype" [EEP]). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino-acid substitutions occurring in AcrR, MarR and SoxR. qRT-PCR of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC-system in EEP strains, whereas in the remaining strains we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC-efflux-pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP