Highlighting extended-spectrum beta-lactamase-producing Klebsiella pneumoniae outbreak by routine genomic typing.

Whole genome sequencing has become the gold standard for any microbiological investigations. Taking the opportunity to doing it prospectively and routinely allowed to detect undeclared outbreaks. Thanks to that, we investigated and ended a rare epidemic extended-spectrum beta-lactamase-producing Klebsiella pneumoniae ST584 strain on two intensive care units over a 4-month period.

Emergence of optrA-mediated linezolid resistance in enterococci from France, 2006-16.

OBJECTIVES: To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates. METHODS: The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments. RESULTS: Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases. CONCLUSIONS: There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not).

The tigecycline evaluation and surveillance trial; assessment of the activity of tigecycline and other selected antibiotics against gram-positive and gram-negative pathogens from France collected between 2004 and 2016.

Background: A high level of antibiotic consumption in France means antimicrobial resistance requires rigorous monitoring. The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) is a global surveillance study that monitors the in vitro activities of tigecycline and a panel of marketed antimicrobials against clinically important Gram-positive and Gram-negative isolates. Methods: Annually clinically relevant strains were prospectively included in the survey through a national network of hospital-based laboratories. MICs were determined locally by broth microdilution using CLSI guidelines. Antimicrobial susceptibility was assessed using European Committee on Antimicrobial Susceptibility Testing breakpoints. Results: Thirty-three centres in France collected 26,486 isolates between 2004 and 2016. Enterococcus species were highly susceptible (≥94.4%) to linezolid, tigecycline and vancomycin. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), were susceptible (≥99.9%) to tigecycline, vancomycin and linezolid. Between 2004 and 2016, 27.7% of S. aureus isolates were MRSA, decreasing from 28.0% in 2013 to 23.5% in 2016. Susceptibility of Streptococcus pneumoniae isolates was 100% to vancomycin, and > 99.0% to levofloxacin, linezolid and meropenem; 3.0% were penicillin-resistant S. pneumoniae (100% susceptibility to vancomycin and linezolid). Escherichia coli isolates were highly susceptible (> 98.0%) to meropenem, tigecycline and amikacin. The rate of extended-spectrum ß-lactamase (ESBL) positive E. coli increased from 2004 (3.0%), but was stable from 2012 (23.1%) to 2016 (19.8%). Susceptibility of Klebsiella pneumoniae isolates was 99.4% to meropenem and 96.5% to amikacin. The proportion of ESBL-positive K. pneumoniae isolates increased from 2004 (7.5%) to 2012 (33.3%) and was highest in 2016 (43.6%). A. baumannii was susceptible to meropenem (81.0%) and amikacin (74.9%); none of the 6.2% of isolates identified as multidrug-resistant (MDR) was susceptible to any agents with breakpoints. P. aeruginosa isolates were most susceptible to amikacin (88.5%), and MDR rates were 13.6% in 2013 to 4.0% in 2016; susceptibility of MDR isolates was no higher than 31.4% to amikacin. Conclusions: Rates of MRSA decreased slowly, while rates of ESBL-positive E. coli and K. pneumoniae increased from 2004 to 2016. Susceptibility of Gram-positive isolates to vancomycin, tigecycline, meropenem and linezolid was well conserved, as was susceptibility of Gram-negative isolates to tigecycline and meropenem. The spread of MDR non-fermentative isolates must be carefully monitored.

Genetic characterization of a VanG-type vancomycin-resistant Enterococcus faecium clinical isolate.

Objectives: To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon. Methods: The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin. Results: E. faecium 16-346 was resistant to rifampicin (MIC >4 mg/L), erythromycin (MIC >4 mg/L), tetracycline (MIC >16 mg/L) and vancomycin (MIC 8 mg/L), but susceptible to teicoplanin (MIC 0.5 mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5 kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin. Conclusions: This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium.

Nationwide molecular epidemiology of methicillin-resistant Staphylococcus aureus responsible for horse infections in France.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in horse infections is not well documented, especially in France. The aim of the study was to evaluate the prevalence of MRSA isolates in horse infections from 2007 to 2013 in France and to characterize phenotypically and genotypically this collection. RESULTS: Out of 1393 S. aureus horse isolates, 85 (6.1%) were confirmed to be MRSA. Interestingly, the prevalence of MRSA significantly increased from 2007-2009 to 2010-2013 (0.7 vs. 9.5%, P <0.0001). Resistance to methicillin was due to the presence of the mecA gene in 84 strains (98.8%) while one strain (1.2%) possessed the mecC gene. The vast majority of the strains (83/85, 97.6%) was resistant to at least three different classes of antibiotics. Multi-locus sequence typing (MLST) showed that MRSA strains belonged mainly since not all belong to two sequence types (STs): ST398 (53/85, 62.4%) and ST8 (28/85, 32.9%). It is worth to note that all ST398 MRSA isolates were detected in the period 2010-2013. Other molecular typing methods were also used, such SCC mec analysis, spa typing and rep-PCR (Diversilab, bioMérieux). All these four techniques were in good agreement, with spa typing and rep-PCR being more discriminative than MLST and SCC mec typing. CONCLUSIONS: This study is the first epidemiological study in France with extensive characterization of MRSA isolates associated with horse infections in stud farms. It shows that there is a significant increase of MRSA prevalence between 2007 and 2013, which mainly results from the spread of ST398 clones. It also highlights the importance of horses as a potential reservoir of important antimicrobial resistance genes.

Decrease of Staphylococcus aureus Virulence by Helcococcus kunzii in a Caenorhabditis elegans Model.

Social bacterial interactions are considered essential in numerous infectious diseases, particularly in wounds. Foot ulcers are a common complication in diabetic patients and these ulcers become frequently infected. This infection is usually polymicrobial promoting cell-to-cell communications. Staphylococcus aureus is the most prevalent pathogen isolated. Its association with Helcococcus kunzii, commensal Gram-positive cocci, is frequently described. The aim of this study was to assess the impact of co-infection on virulence of both H. kunzii and S. aureus strains in a Caenorhabditis elegans model. To study the host response, qRT-PCRs targeting host defense genes were performed. We observed that H. kunzii strains harbored a very low (LT50: 5.7 days ± 0.4) or an absence of virulence (LT50: 6.9 days ± 0.5). In contrast, S. aureus strains (LT50: 2.9 days ± 0.4) were significantly more virulent than all H. kunzii (P < 0.001). When H. kunzii and S. aureus strains were associated, H. kunzii significantly reduced the virulence of the S. aureus strain in nematodes (LT50 between 4.4 and 5.2 days; P < 0.001). To evaluate the impact of these strains on host response, transcriptomic analysis showed that the ingestion of S. aureus led to a strong induction of defense genes (lys-5, sodh-1, and cyp-37B1) while H. kunzii did not. No statistical difference of host response genes expression was observed when C. elegans were infected with either S. aureus alone or with S. aureus + H. kunzii. Moreover, two well-characterized virulence factors (hla and agr) present in S. aureus were down-regulated when S. aureus were co-infected with H. kunzii. This study showed that H. kunzii decreased the virulence of S. aureus without modifying directly the host defense response. Factor(s) produced by this bacterium modulating the staphylococci virulence must be investigated.

Novel chromosome-encoded erm(47) determinant responsible for constitutive MLSB resistance in Helcococcus kunzii.

OBJECTIVES: The aim of the study was to identify the determinant responsible for erythromycin resistance in Helcococcus kunzii clinical isolate UCN99 and to characterize the genetic support and environment of this novel gene. METHODS: MICs were determined using the broth microdilution method according to EUCAST guidelines. The entire genome sequence of H. kunzii UCN99 was determined using a 454/Roche GS Junior sequencer. The fragment encompassing the new resistance gene and its own promoter was cloned into the pAT29 shuttle vector and the recombinant plasmid pAT29Ωerm(47) was expressed in both Staphylococcus aureus and Streptococcus agalactiae. The transcription start site (TSS) was experimentally determined by 5' RACE-PCR. RESULTS: UCN99 exhibited a constitutive macrolide/lincosamide/streptogramin B (MLSB) resistance phenotype, suggesting the presence of an Erm protein. WGS allowed the identification of a novel gene, named erm(47), encoding a protein sharing 44%-48% amino acid identity with known Erm methylases. In both S. aureus and S. agalactiae, the introduction of pAT29Ωerm(47) conferred a significant increase (≥16-fold) in MICs of all macrolides and lincosamides tested, as well as a 4-fold increase in MICs of quinupristin (streptogramin B), confirming the MLSB resistance. The TSS identification revealed the presence of a short leader peptide, potentially implicated in a translational attenuation mechanism. It was also demonstrated that erm(47) was harboured by a 81 kb genomic island integrated into a chromosomal gene. CONCLUSIONS: This is the first description of a novel MLSB resistance determinant, named erm(47). The prevalence of this gene among Gram-positive cocci must be further investigated to determine its clinical significance.

Transfer between an Algerian and a French hospital of four multi-drug resistant bacterial strains together via a single patient.

A 5 years-old girl, seriously burnt with fire, was first hospitalized during four days in an hospital at Alger, and then transferred to our hospital at Paris. Admitted in our intensive care burns unit, she was third degree burnt on 78% of total body surface area, already treated with imipenem and vancomycin at her arrival. Clinical aggravation was rapidly observed and death occurred within 24 hours. Cultures of blood and multiple wound swabs yielded 3 multi-drug resistant bacterial strains: Acinetobacter baumannii with carbapenemase OXA-23, Pseudomonas aeruginosa serotype O11 with metallo-ß-lactamase VIM-4 and Klebsiella pneumoniae with CTX-M-15 extended-spectrum ß-lactamase. Culture of a rectal swab showed colonization by Enterococcus faecium with vanA glycopeptides resistance. Patients colonized with one or two multi-drug-resistant strains were not rare in our burns unit, especially those transferred from Algeria, but this case of a single patient harboring four multi-drug-resistant strains is exceptional.

Recurrent peritoneal dialysis-related peritonitis caused by Microbacterium resistens.

We report a case of a recurrent peritonitis due to Microbacterium resistens in a 71-year-old male patient undergoing peritoneal dialysis (PD). Importantly, this Gram-positive rod was intrinsically resistant to cephalosporins and vancomycin, classically used in PD-related peritonitis treatment. His infection resolved after several weeks of appropriate therapy (amoxicillin plus gentamicin) and PD catheter removal.

[Ecology and mechanisms of bacterial resistance to antibiotics in peritonitis]. / Écologie bactérienne et mécanismes de résistance aux antibiotiques lors des infections du liquide de dialyse péritonéale.

Peritonitis remains a common complication of peritoneal dialysis. The aim of our study is to describe the mechanisms of antibiotic resistance in bacteria isolated during peritonitis in peritoneal dialysis, to determine whether antibiotic therapy proposed by the International Society for Peritoneal Dialysis (ISPD) is adapted to the mechanisms of resistance. All causative microorganisms of peritonitis, isolated in 106 dialysis patients and reported 170 episodes of peritonitis, during the study period (01/01/2005 to 31/12/2010) were reviewed. According to the usual classification, twelve groups of microorganism were created. An interpretive reading of antibiograms was performed in each group to identify resistance phenotypes. The species most frequently isolated are coagulase-negative staphylococci (n=73) of which 46 had PBP2a (penicillin-binding protein). Many Enterobacteriaceae were also isolated (n=45), they are susceptible to third generation cephalosporins with the exception of Enterobacteriaceae producing an extended spectrum ß-lactamase (ESBL) or a cephalosporinase. Except for staphylococci, probabilistic antibiotic therapy recommended by the ISPD to treat peritonitis is effective. Indeed, many staphylococci producing a PBP2a, a first-generation cephalosporin cannot be administered in all cases. It is therefore necessary to identify patients with a strain of staphylococcus producing a PBP2a, it must be treated by vancomycin.

D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium.

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 µg/ml) but susceptible to teicoplanin (MIC, 0.5 µg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.

Changing trends in vancomycin-resistant enterococci in French hospitals, 2001-08.

OBJECTIVES: Unprecedented outbreaks of vancomycin-resistant enterococci (VRE) have occurred in French hospitals since 2004. The aim of this study was to provide a picture of the spread and control of VRE in France and to characterize the isolates. METHODS: Notification of VRE cases to Institut de Veille Sanitaire has been mandatory since 2001. Isolates of VRE were sent to the National Reference Centre for species and vancomycin-resistance gene identification. Isolates were tested for antimicrobial susceptibility and typed by PFGE and multilocus sequence typing. RESULTS: Five hundred and four VRE notifications from 195 hospitals were recorded, corresponding to 2475 cases of infection (n=243) or colonization (n=2232) and 74 episodes of clustered cases. Outbreaks were controlled by implementation of infection control measures, although the number of new hospitals reporting isolation of VRE was increasing. The majority of 902 VRE isolated from 2006 to 2008 were Enterococcus faecium (94.8%) with the vanA or vanB gene. No isolate was resistant to linezolid, tigecycline or fusidic acid. PFGE analysis showed 161 different patterns. Generally a few predominant clones and several minor clones spread in a single hospital. In a subset of 46 representatives of PFGE clones, 13 different sequence types were characterized, all belonging to clonal complex CC17, while the esp and hyl genes were inconsistently detected. CONCLUSIONS: The national mandatory notification of unusual nosocomial events allowed rapid identification of VRE outbreaks and early implementation of control measures that have proved effective. However, VRE continue to emerge in a growing number of hospitals.

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium.

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.

Rapid detection of vancomycin-resistant enterococci from rectal swabs by the Cepheid Xpert vanA/vanB assay.

The detection of vancomycin-resistant enterococci using a novel commercial multiplex real-time polymerase chain reaction assay (Xpert vanA/vanB, Cepheid, Sunnyvale, CA) was evaluated on 804 rectal swab specimens. Compared to enriched culture, sensitivity and negative predictive value of this method were 100%. Many false-positive results were recovered (sensitivity, 85.4%; positive predictive value, 8.7%), especially for the vanB gene.

In vitro stability of fortified ophthalmic antibiotics stored at -20 degrees C for 6 months.

PURPOSE: This study evaluates the in vitro stability and sterility of fortified ophthalmic antibiotic preparations of 5% solutions (50 mg/mL) made from commercially available powders of amikacin, ceftazidime, and vancomycin stored at -20 degrees C for 6 months. METHODS: Antibiotic solutions were prepared with aseptic techniques by reconstitution and dilution of the commercially available powders (amikacin, ceftazidime, and vancomycin) and stored at -20 degrees C. After various durations of storage, the stability of various physical and chemical properties was determined by a pH meter, an osmometer, and high-performance liquid chromatography. Microbiological activity was assayed according to the European Pharmacopoeia. RESULTS: Solutions of 5% amikacin or ceftazidime in 0.9% sodium chloride and vancomycin in 5% dextrose were found to be physically, chemically, and microbiologically stable after storage at -20 degrees C for 190 days. A time limitation of 6 months is proposed. CONCLUSION: Fortified ophthalmic antibiotic solutions prepared and supplied by a pharmacy can be stored in freezers at -20 degrees C for later use.

Emergence of rifampin-resistant Rhodococcus equi with several types of mutations in the rpoB gene among AIDS patients in northern Thailand.

The antimicrobial susceptibilities of 30 Rhodococcus equi isolates obtained from 30 patients between 1993 and 2001 in northern Thailand were investigated. The MICs showed a tendency toward resistance to various antibiotics but sensitivity to imipenem, minocycline, vancomycin, and teicoplanin (MICs, /=64 micro g/ml) to rifampin. PCR amplification and DNA sequencing of the rpoB gene and molecular typing by pulsed-field gel electrophoresis (PFGE) were performed for eight R. equi isolates from eight AIDS patients with pneumonia or lung abscess caused by R. equi between 1998 and 2001, including one low- and three high-level rifampin-resistant isolates. As a result, two high-level rifampin-resistant strains with PFGE pattern A had a Ser531Trp (Escherichia coli numbering) mutation, and one high-level rifampin-resistant strain with PFGE pattern B had a His526Tyr mutation, whereas one low-level rifampin-resistant strain with PFGE pattern C had a Ser509Pro mutation. Four rifampin-susceptible strains with PFGE patterns D and E showed an absence of mutation in the rpoB region. Our results indicate the presence of several types of rifampin-resistant R. equi strains among AIDS patients in northern Thailand.