Molecular Characterization and Prevalence of Antimicrobial-Resistant Escherichia coli Isolates Derived from Clinical Specimens and Environmental Habitats.

Antibiotic-resistant bacteria (ARB) are present in wastewaters as their elimination during treatment in wastewater treatment plants (WWTPs) is often impossible. Water plays an important role in the spread of these microorganisms among humans, animals and the environment. This study aimed to assess the antimicrobial resistance patterns, resistance genes and molecular genotypes by means of phylogenetic groups of E. coli isolates in aquatic habitats, including sewage and receiving water bodies, as well as clinical settings in the Boeotia regional district of Greece. The highest resistance rates among both environmental and clinical isolates were observed to be for penicillins, ampicillin and piperacillin. Resistance patterns related to extended spectrum ß-lactamases (ESBL) production and ESBL genes were also detected in both environmental and clinical isolates. Phylogenetic group B2 was predominant in clinical settings and the second most frequent among wastewaters, whereas group A was dominant in all environmental isolates. In conclusion, the studied river water and wastewaters may serve as reservoirs of resistant E. coli isolates that pose potential threats to both human and animal health.

Evaluation of the performance of Acuitas® Resistome Test and the Acuitas Lighthouse® software for the detection of ß-lactamase-producing microorganisms.

OBJECTIVES: Given the international spread of multidrug-resistant Gram-negative bacteria the need for prompt and precise characterization of underlying resistance traits has evolved into the cornerstone of infection control strategies. Novel commercial molecular tests enable rapid simultaneous testing for multiple resistance genes. We aimed to evaluate the performance of OpGen's Acuitas® Resistome Test and the Acuitas Lighthouse® software. METHODS: The test is tailored towards detecting 46 ß-lactamase genes (SHV and TEM variants associated with wild-type penicillin resistance, extended-spectrum ß-lactamases [ESBLs], acquired AmpCs and carbapenemases) via a microfluidic polymerase chain reaction (PCR) array. In total 118 isolates part of the collection of the Bacteriology Laboratory of the Hellenic Pasteur Institute, specifically 96 enterobacterial isolates and 21 Acinetobacter baumannii, of divergent origins, with previously characterized ß-lactamase content, were tested. RESULTS: In the enterobacterial group all 69 carbapenemase genes of the KPC, VIM, NDM and OXA-48 types were correctly identified (sensitivity, specificity, positive predictive value [PPV] and negative predictive value [NPV] of 100%). Non-ESBL SHV enzymes, ESBLs (CTX-M, GES, VEB types) and acquired AmpC enzymes were also correctly characterized. Of the 35 SHV-ESBLs harboured, correct identification was possible in 32/35 isolates, with overall sensitivity, specificity, PPV and NPV for the Klebsiella pneumoniae group of 89.29%, 100%, 100% and 91.18%, respectively. For the A. baumannii group the test exhibited an overall sensitivity for carbapenemase detection of 96.55% and 100% PPV. CONCLUSIONS: The OpGen Acuitas Resistome Test is an efficient molecular tool that can identify resistance threats in health care institutions with high diagnostic accuracy and be integrated into targeted surveillance protocols.

Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data.

BACKGROUND: Confidence in any diagnostic and antimicrobial susceptibility testing data is provided by appropriate and regular quality assurance (QA) procedures. In Europe, the European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP) has been monitoring the antimicrobial susceptibility in Neisseria gonorrhoeae since 2004. Euro-GASP includes an external quality assessment (EQA) scheme as an essential component for a quality-assured laboratory-based surveillance programme. Participation in the EQA scheme enables any problems with the performed antimicrobial susceptibility testing to be identified and addressed, feeds into the curricula of laboratory training organised by the Euro-GASP network, and assesses the capacity of individual laboratories to detect emerging new, rare and increasing antimicrobial resistance phenotypes. Participant performance in the Euro-GASP EQA scheme over a 10 year period (2007 to 2016, no EQA in 2013) was evaluated. METHODS: Antimicrobial susceptibility category and MIC results from the first 5 years (2007-2011) of the Euro-GASP EQA were compared with the latter 5 years (2012-2016). These time periods were selected to assess the impact of the 2012 European Union case definitions for the reporting of antimicrobial susceptibility. RESULTS: Antimicrobial susceptibility category agreement in each year was ≥91%. Discrepancies in susceptibility categories were generally because the MICs for EQA panel isolates were on or very close to the susceptibility or resistance breakpoints. A high proportion of isolates tested over the 10 years were within one (≥90%) or two (≥97%) MIC log2 dilutions of the modal MIC, respectively. The most common method used was Etest on GC agar base. There was a shift to using breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in the latter 5 years, however overall impact on the validity of results was limited, as the percentage categorical agreement and MIC concordance changed very little between the two five-year periods. CONCLUSIONS: The high level of comparability of results in this EQA scheme indicates that high quality data are produced by the Euro-GASP participants and gives confidence in susceptibility and resistance data generated by laboratories performing decentralised testing.

Laboratory evaluation of Brilliance™ CRE Agar for screening carbapenem-resistant Enterobacteriaceae: Performance on a collection of characterised clinical isolates from Greece.

The performance of Oxoid Brilliance™ CRE Agar (BCRE), a new chromogenic medium designed for screening of carbapenem-resistant Enterobacteriaceae, was evaluated on a collection of clinical isolates of enterobacteria (n=175) and non-fermenters (n=55) with known ß-lactam resistance mechanisms and levels of susceptibility to carbapenems. BCRE supported the growth of 100 of 108 enterobacterial isolates that were non-susceptible to at least one carbapenem, whilst excluding 57 of the 67 carbapenem-susceptible isolates. The eight non-susceptible isolates that did not grow on BCRE were carbapenemase-producers with low carbapenem minimum inhibitory concentrations, mostly exhibiting non-susceptibility only to one carbapenem. In total, of 107 carbapenemase-producing enterobacteria that were included in the study, 16 did not grow, with most of them being either susceptible (n=8) or intermediate-susceptible (n=5) to carbapenems. Regarding the 10 carbapenem-susceptible enterobacteria that were not excluded by BCRE, 1 produced a carbapenemase and the rest possessed strong backgrounds of various other ß-lactam resistance mechanisms. The medium allowed growth of almost all carbapenem-resistant non-fermenting isolates; nevertheless, non-fermenters were clearly differentiated from Enterobacteriaceae by colony colour and morphology.

Analysis of emergence of quinolone-resistant gonococci in Greece by combined use of Neisseria gonorrhoeae multiantigen sequence typing and multilocus sequence typing.

The prevalence of quinolone-resistant Neisseria gonorrhoeae (QRNG) in Greece remained low from 1997 to 2003 but increased dramatically from 11% to 56% between 2004 and 2007. N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were used to investigate trends in quinolone resistance from 1997 to 2007 and explore the origins of the recent increase in QRNG. We characterized 295 QRNG isolates from the study period and 233 quinolone-susceptible (QS) gonococci from 2004 and 2005, when the rapid increase in QRNG occurred. From 1997 to 1999, an outbreak of QRNG was due to the dissemination of isolates of serovar Arst that belonged to two closely related genotypes. Few QRNG isolates, of diverse genotypes, were present between 2001 and 2003, whereas the sharp increase in QRNG from 2004 onwards was due to the appearance of serovar Bropyst isolates of several major NG-MAST sequence type (STs) that previously had not been identified in Greece. These isolates were shown by MLST to be variants of a single multiply antibiotic-resistant QRNG strain (ST1901) that appeared in Greece and rapidly diversified into 31 NG-MAST STs. There were no isolates of MLST ST1901 or any of the 31 NG-MAST STs among QS isolates from 2004 and 2005 or among 8 representatives of multiresistant but quinolone-susceptible serovar Bropyst isolates circulating in Greece during the 1990 s, supporting the view that the recent increase in QRNG was due to importation of a QRNG strain(s) of MLST ST1901 into Greece. Recently, multiresistant QRNG isolates of ST1901 with reduced susceptibility to the newer cephalosporins have appeared in Greece.

Changing figures of antimicrobial susceptibility and serovar distribution in Neisseria gonorrhoeae isolated in Greece.

OBJECTIVES: To update surveillance data on antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Greece with information for the years 2005 to 2008, and analyze changes occurred from the previous 4-year period. METHODS: Annual antimicrobial susceptibility rates, susceptibility patterns, and serovars of 635 gonococci isolated in 2005 to 2008 were determined and compared to respective data concerning the gonococcal sample of 2001 to 2004. Genetic similarity of the isolates in phenotypic clusters was investigated by pulsed field gel electrophoresis. Epidemiologic information was also considered. RESULTS: Despite a reduction in the isolation frequency of penicillinase-producing strains (3.9% vs. 11.6% in the previous period), the rates of resistance and intermediate susceptibility increased for penicillin, as well as for tetracycline, erythromycin, and chloramphenicol, leaving very small proportions of isolates sensitive to these agents (4.3%, 12.8%, 10.2%, and 3.6%, respectively). Resistance to fluoroquinolones increased from 11.3% in 2004 up to 63% in 2008, and strongly correlated with multidrug-resistant isolates of Bropyst serovar, accounting for 72.6% of the quinolone-resistant strains isolated during the last 4 years. All isolates were susceptible to spectinomycin and only 2 exceeded susceptibility breakpoints set for cefotaxime, exhibiting MICs 0.75 to 1 microg/mL. These latter isolates, however, belonged to a cluster of strains with decreased susceptibility to cephalosporins (CDS, cefotaxime MICs >or=0.25 microg/mL) that emerged in late 2006 and increased in frequency up to 20.7% through 2008. Notably, CDS isolates were also quinolone-resistant and multiresistant, further contributing to the increasing rates of quinolone and multidrug resistance in the Greek gonococcal sample. CONCLUSIONS: Antimicrobial susceptibility figures of Neisseria gonorrhoeae in Greece are worsening due to changes in the synthesis of gonococcal population, resulting from high endemicity rates of multidrug-resistant strains.