Eleni Ntokou

Intensive care unit dissemination of multiple clones of linezolid-resistant Enterococcus faecalis and Enterococcus faecium

OBJECTIVES Outbreaks caused by linezolid-resistant (LR) enterococci remain rare. We report the epidemiological and molecular characteristics of the multiclonal dissemination of LR enterococci in the intensive care unit (ICU) of a Greek hospital. METHODS All LR enterococcal isolates recovered from patients hospitalized in the ICU of the University Hospital of Larissa, Greece, between January 2007 and October 2008 were included. Isolates were tested by PFGE and PCR followed by sequence analysis of the entire 23S rRNA gene. Patient records were retrieved to access patterns of acquisition and outcome. RESULTS Sixteen separate patients were infected and/or colonized by 22 LR enterococcal isolates (17 Enterococcus faecium and 5 Enterococcus faecalis). Linezolid MICs varied from 8 to 16 mg/L; 12 isolates showed cross-resistance to vancomycin. Genotyping revealed as many as seven and three PFGE types among E. faecium and E. faecalis isolates, respectively, indicating multiclonal spread of LR enterococci. Nine patients had received linezolid prior to the recovery of LR enterococci, while the remaining seven patients were not exposed to the drug. All isolates carried the mutation G2576T; the mutated position was heterogeneous in 12 isolates and homogeneous in 10. CONCLUSIONS The multiclonal composition of LR enterococci indicates that linezolid resistance possibly occurred on several independent occasions. Its acquisition was often not related to linezolid administration; patients might have acquired their LR isolate from another patient that had received linezolid or, alternatively, resistance may have arisen by mutation that occurred independently.

Hidden VIM-1 Metallo-β-Lactamase Phenotypes among Acinetobacter baumannii Clinical Isolates

ABSTRACT A total of 87 Acinetobacter baumannii nonrepetitive consecutive clinical isolates were tested for the presence of metallo-β-lactamases (MBLs). Results of phenotypic assays (MBL Etest, imipenem/imipenem-EDTA combined-disk test, and imipenem/EDTA double-disk synergy test) were negative in all cases, but molecular testing revealed the presence of two blaVIM-1-carrying isolates. One isolate had blaVIM-1 preceded by a weak P1 promoter, and both had inactivated P2 promoters and reduced blaVIM-1 expression, partially justifying the results revealing hidden MBL phenotypes.

Linezolid Dependence in Staphylococcus epidermidis Bloodstream Isolates

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.

Activity of Oxacillin versus That of Vancomycin against Oxacillin-Susceptible mecA-Positive Staphylococcus aureus Clinical Isolates Evaluated by Population Analyses, Time-Kill Assays, and a Murine Thigh Infection Model

ABSTRACT We compared the activity of dicloxacillin with that of vancomycin against 15 oxacillin-susceptible, methicillin-resistant Staphylococcus aureus (OS-MRSA) clinical isolates. By population analyses, we found that 6 OS-MRSA isolates were able to grow in the presence of up to 8 μg/ml dicloxacillin and 9 isolates were able to grow in 12 to >32 μg/ml dicloxacillin; all isolates grew in up to 2 μg/ml vancomycin. Both drugs exhibited similar bactericidal activities. In experimental infections, the therapeutic efficacy of dicloxacillin was significant (P < 0.05 versus untreated controls) in 10 OS-MRSA isolates and vancomycin was effective (P < 0.05) against 12 isolates; dicloxacillin had an efficacy that was comparable to that of vancomycin (P > 0.05) in 8 isolates. The favorable response to dicloxacillin treatment might suggest that antistaphylococcal penicillins could be used against OS-MRSA infections.

Genetic response of Salmonella enterica serotype Enteritidis to thioridazine rendering the organism resistant to the agent.

Thioridazine (TZ)-induced accumulation of the universal efflux pump substrate ethidium bromide and its subsequent efflux by Salmonella strains with various degrees of overexpressed efflux pumps takes place automatically at pH 7.4, is independent of a metabolic source, is not affected by a proton ionophore and is precluded by palmitic acid. Salmonella enterica serotype Enteritidis cultured in medium containing increasing concentrations of TZ does not grow during the first 6-8h, after which time its growth is similar to unexposed controls. At the end of a 16-h exposure period, the organism is resistant to >250mg/L TZ. Parallel assessment by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the activity of genes that regulate and code for the AcrB transporter of the main efflux pump (AcrAB) of the organism at periodic intervals suggests a sequence of activation beginning with the stress gene soxS, followed by the global regulator ramA, then by the local regulator marA and then by the transporter acrB. These activations take place during the period of no growth. By the end of a 16-h culture period, only the acrB transporter gene is still highly overexpressed. Assessment of the activity of genes of the two-component regulon PmrA/B indicates that TZ also activates this regulon. Because activation of pmrA/B also activates acrB, development of high resistance to TZ during a 16-h culture period is in part due to activation of the two-component regulon.

Emergence of the Plasmid-Mediated Quinolone Resistance Gene qnrS1 in Escherichia coli Isolates in Greece

Three major groups of the plasmid-mediated quinolone resistance (Qnr) determinants have been identified so far in Enterobacteriaceae : the QnrA group, which includes 6 variants, the QnrB group, which includes 19 variants, and the QnrS group, which includes 3 variants ([5][1]). Although Qnr proteins

Identification of the plasmid-encoded qacA efflux pump gene in meticillin-resistant Staphylococcus aureus (MRSA) strain HPV107, a representative of the MRSA Iberian clone

Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial bacterium for which prevention and control measures consist mainly of the application of biocides with antiseptic and disinfectant activity. In this study, we demonstrated the presence of the plasmid-located efflux pump gene qacA in MRSA strain HPV107, a clinical isolate representative of the MRSA Iberian clone. The existence of efflux activity in strain HPV107 due to the QacA pump was also established and this QacA efflux activity was linked with a phenotype of reduced susceptibility towards several biocide compounds. No association could be made with antibiotic resistance. This work emphasises the potential of QacA pump activity in the maintenance and dissemination of important MRSA strains in the hospital setting and, increasingly, in the community.

Wide dissemination of linezolid-resistant Staphylococcus epidermidis in Greece is associated with a linezolid-dependent ST22 clone

OBJECTIVES Dependence on linezolid was recently described as significant growth acceleration of linezolid-resistant Staphylococcus epidermidis (LRSE) isolates upon linezolid exposure. We investigated the possible contribution of linezolid dependence to LRSE dissemination in Greece. METHODS Linezolid resistance rates were estimated in six tertiary hospitals located throughout Greece between 2011 and 2013. Sixty-three randomly selected LRSE recovered in these hospitals during this period were studied. Growth curve analysis was conducted with and without linezolid. Clonality of the isolates was investigated by PFGE and MLST. RESULTS During the study period, the LRSE rate in the participating hospitals rose significantly from 6.9% to 9% (P = 0.006); the increase was more prominent in ICUs (from 15.1% to 20.9%; P = 0.005). Forty-seven (74.6%) of the 63 LRSE, derived from all study hospitals, clearly exhibited linezolid dependence, growing significantly faster in the presence of 16 and 32 mg/L linezolid. Of note, 61 (96.8%) LRSE exhibited a single macrorestriction pattern and belonged to ST22, which included all linezolid-dependent LRSE. The remaining two LRSE belonged to unique STs. Five of six linezolid-dependent isolates tested also exhibited linezolid dependence upon exposure to 8 mg/L linezolid. Interestingly, five of six ST22 linezolid-non-dependent isolates tested developed linezolid dependence when linezolid exposure preceded growth analysis. CONCLUSIONS The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.

Macrolide resistance determinants among Streptococcus pneumoniae isolates from carriers in Central Greece

BackgroundWe sought to characterize the temporal trends in nasopharyngeal carriage of macrolide-resistant pneumococci during a period with increased heptavalent pneumococcal conjugate vaccine (PCV7) coverage in Central Greece.MethodsStreptococcus pneumoniae isolates were recovered from 2649 nasopharyngeal samples obtained from day-care center attendees in Central Greece during 2005–2009. A phenotypic and genotypic analysis of the isolates was performed, including the identification of macrolide resistance genes mef(A), subclasses mef(A) and mef(E), as well as erm(B).ResultsOf the 1105 typeable S. pneumoniae isolates, 265 (24%) were macrolide-resistant; 22% in 2005, 33.3% in 2006, 23.7% in 2007, and 20.5% in 2009 (P=0.398). Among these macrolide-resistant pneumococci, 28.5% possessed erm(B), 24.3% erm(B)+mef(E), 41.8% mef(E), and 5.3% mef(A). A mef gene as the sole resistance determinant was carried by 31% of macrolide-resistant isolates belonging to PCV7 serotypes and 75.8% of the non-PCV7 serotypes. Across the 4 annual surveillances, pneumococci carrying mef(A) gradually disappeared, whereas serotype 19F isolates carrying both erm(B) and mef(E) persisted without significant yearly fluctuations. Among isolates belonging to non-PCV7 serotypes, macrolide-resistance was observed in those of serotypes 6A, 19A, 10A, 15A, 15B/C, 35F, 35A, and 24F. In 2009, ie 5 years after the introduction of PCV7 in our country, 59% of macrolide-resistant pneumococci belonged to non-PCV7 serotypes.ConclusionsAcross the study period, the annual frequency of macrolide-resistant isolates did not change significantly, but in 2009 a marked shift to non-PCV7 serotypes occurred. Overall, more than half of the macrolide-resistant isolates possessed erm(B) either alone or in combination with mef(E). erm(B) dominated among isolates belonging to PCV7 serotypes, but not among those of non-PCV7 serotypes.