Sandrine Bernabeu

NDM-1-producing Klebsiella pneumoniae isolated in the Sultanate of Oman

OBJECTIVES To analyse the mechanisms responsible for multidrug resistance in two carbapenem-resistant Klebsiella pneumoniae isolates recovered from patients hospitalized in Oman. METHODS PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of each isolate. Clonal relationships were evaluated by PFGE. RESULTS Both isolates carried the bla(NDM-1) carbapenemase gene. Isolate 601 was recovered from a patient who was transferred from India, whereas isolate 419 was from an Omani patient who had not travelled abroad. The two isolates were clonally unrelated, and belonged to ST14 (isolate 601) and ST340 (isolate 419). In addition to NDM-1, the ST14 isolate expressed β-lactamases CTX-M-15, SHV-28, OXA-1, OXA-9 and TEM-1, and the aminoglycoside resistance methylase ArmA. The ST340 isolate expressed β-lactamases SHV-11, OXA-1 and ArmA. In both isolates, the bla(NDM-1) gene was located on plasmids that were of similar size (170 kb), but of different incompatibility groups. CONCLUSION This is the first description of NDM-1 producers in the Arabian peninsula and in the Middle East.

Evaluation of a DNA Microarray, the Check-Points ESBL/KPC Array, for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum β-Lactamases and KPC Carbapenemases

ABSTRACT Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ß-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ß-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ß-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.

Molecular analysis of NDM-1-producing enterobacterial isolates from Geneva, Switzerland

OBJECTIVES To analyse the mechanisms responsible for decreased susceptibility or resistance to carbapenems in several enterobacterial isolates recovered in 2009-10 in Geneva University Hospitals, Switzerland. METHODS PCR and sequencing were used to identify β-lactamases, 16S RNA methylases and plasmid-mediated quinolone resistance genes. The transferable properties of the plasmids were analysed, as well as their plasmid type. The strains were typed by multilocus sequence typing. RESULTS Three patients were found to be positive for NDM-1-producing enterobacterial isolates (one with Escherichia coli and Klebsiella pneumoniae, one with K. pneumoniae only and one with Proteus mirabilis), where NDM-1 stands for New Delhi metallo-β-lactamase-1. The bla(NDM-1) carbapenemase gene was detected in all isolates in addition to genes encoding narrow-spectrum β-lactamases (TEM-1, SHV-11, OXA-1, OXA-9 and OXA-10), extended-spectrum β-lactamases (CTX-M-15, CMY-16 and CMY-30), ArmA and quinolone resistance determinants (Qnr). The bla(NDM-1) gene was located on conjugative IncA/C- or IncF-type plasmids. Upstream of the bla(NDM-1) gene, part of ISAba125, previously identified in NDM-1-negative Acinetobacter baumannii, was found. Downstream of the bla(NDM-1) gene, variable sequences were found. CONCLUSIONS This work constitutes the first identification of NDM-1 producers in Switzerland. Interestingly, patients from whom these NDM-1-producing isolates were recovered had a link with the Indian subcontinent or the Balkans.

Multicopy blaOXA-58 Gene as a Source of High-Level Resistance to Carbapenems in Acinetobacter baumannii

ABSTRACT The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the blaOXA-58 gene showed genetic variability, with the presence in several isolates of multiple copies of the blaOXA-58 gene; this extra copy number was likely related to an IS26-mediated transposition or recombination process.

A novel IncQ plasmid type harbouring a class 3 integron from Escherichia coli

OBJECTIVES To determine the genetic structures at the origin of the mobilization of the extended-spectrum beta-lactamase (ESBL) bla(GES-1) gene in an Escherichia coli clinical isolate. METHODS ESBL-encoding genes and class 1 or class 3 integron-specific motifs were screened. Conjugation experiments were performed to determine whether the plasmid-carrying bla(GES-1) gene was self-transferable. Plasmid sequencing was achieved by a primer-walking approach. RESULTS The bla(GES-1) gene was located in a class 3 integron. That unusual genetic structure was itself located on an approximately 9 kb plasmid, pQ7, which was not self-transferable. Sequence analysis revealed that plasmid pQ7 belonged to a novel subtype of the IncQ group. CONCLUSIONS This study identified for the first time the bla(GES-1) gene in E. coli and in Switzerland. It describes a novel IncQ-type plasmid subgroup that possesses original features, in particular iteron sequences that constitute a hot spot for integration of foreign DNA.

NDM-1-Producing Klebsiella pneumoniae in Mauritius

The carbapenemase NDM-1 initially identified in Escherichia and Klebsiella pneumoniae in Sweden from a patient transferred from India (15) has been now identified in many enterobacterial species and isolates from patients mainly in the United Kingdom, India, and Pakistan (6) but also from many different countries in Europe, Asia, Africa, and North America (10). Most of these reports indicated a link with the Indian subcontinent, corresponding to either hospital or community acquisitions. Balkan countries have been also recently considered an additional reservoir for NDM producers (8). Here we report an isolate from a 39-year-old male patient who was admitted to the surgery department of the Victoria Hospital, city of Quatre Bornes, Mauritius, in 2009. Urine samples grew a multidrug-resistant K. pneumoniae strain, and susceptibility testing performed and interpreted according to the updated CLSI guidelines (3) showed that it was resistant to all -lactams, including carbapenems, to all aminoglycosides, and to fluoroquinolones, nitrofurantoin, chloramphenicol, and trimethoprim-sulfamethoxazole. It was susceptible only to tigecycline and colistin (MICs of 0.5 g/ml for both). According to phenotypic test results (synergy between aztreonam and clavulanate), K. pneumoniae CL produced an extended-spectrum -lactamase (ESBL). Metallo-lactamase (MBL) detection performed by using the Etest combining imipenem and EDTA (AB bioMérieux, Solna, Sweden) gave a positive result. MICs of imipenem, ertapenem, doripenem, and meropenem determined by Etest for K. pneumoniae isolate CL were, respectively, 4, 12, 3, and 4 g/ml. According to the CLSI updated guidelines (3), the isolate could therefore be considered resistant to those molecules, except for having intermediate susceptibility to doripenem. Unfortunately no information was available on the treatment and outcome of this patient. PCR, sequencing, and plasmid analysis revealed that K. pneumoniae CL harbored the blaNDM-1 carbapenemase gene (9), in addition to the ESBL gene blaCTX-M-15, both of which were located on different plasmids (120 and 160 kb in size, respectively) (13). Screening for additional -lactamase genes (13) and for 16S RNA methylase genes as reported previously (1) showed that K. pneumoniae CL was coharboring the blaCMY-6, blaOXA-1, blaSHV-28, and blaTEM-1 genes (a total of five -lactamase genes) and the rmtC gene, encoding high-level resistance to all aminoglycosides. Mating-out assays performed as described previously (14) allowed us to obtain an E. coli transconjugant expressing NDM-1, exhibiting resistance to all -lactams, although remaining susceptible to aztreonam and with reduced susceptibility to carbapenems (MICs of 3, 1, and 0.75 g/ml for imipenem, ertapenem, and meropenem, respectively), as well as to all sulfonamides and all aminoglycosides. This transconjugant harbored a single 120-kb plasmid that was of the IncA/C type, as identified by PCR-based replicon typing (2), and that carried the blaCMY-6 and rmtC genes in addition to blaNDM-1. Interestingly, the same resistance determinants had been identified on an IncA/C type and 120-kb plasmid from K. pneumoniae in Kenya (14), suggesting that both plasmids could be related. Multilocus sequence typing was performed as described previously (4), and the results were analyzed by eBURST (http: //pubmlst.org). The results showed that isolate CL belonged to the ST231 sequence type that corresponds to the ST of one NDM-1positive K. pneumoniae isolate recently identified from India (7), but not to that of the NDM-1-producing K. pneumoniae isolates from Kenya (14). Nevertheless, it does not correspond to the most common STs identified from NDM-1-positive K. pneumoniae, which are ST14 and ST147 (7, 11, 13). It may be speculated that isolate CL would have a link with India, considering the geographical and cultural links between the two countries, the Indian diaspora being quite numerous in Mauritius. Corresponding medical authorities in Mauritius therefore have to be alerted about the threat of such multidrug-resistant strains and should implement adequate measures in order to control their spread. This study further underlines the occurrence of NDM-1 producers in countries from the African continent, after the recent identifications made in Kenya (14), Egypt (5), and Morocco (12).

Nosocomial spread of ESBL-positive Enterobacter cloacae co-expressing plasmid-mediated quinolone resistance Qnr determinants in one hospital in France

Service de Bacteriologie–Virologie, INSERM U914 ‘Emerging Resistance to Antibiotics’, Hopital de Bicetre, Assistance Publique/Hopitaux de Paris, Faculte de Medecine et Universite Paris-Sud, 78 rue du General Leclerc, 94275 K.-Bicetre cedex, France; Service de Reanimation Medicale, Hopital de Bicetre, Assistance Publique/Hopitaux de Paris, Faculte de Medecine et Universite Paris-Sud, 78 rue du General Leclerc, 94275 K.-Bicetre cedex, France

Emergence of the 16S rRNA methylase RmtG in an extended-spectrum-β-lactamase-producing and colistin-resistant Klebsiella pneumoniae isolate in Chile.

While carbapenemases are disseminating worldwide in Enterobacteriaceae, and particularly in Escherichia and Klebsiella pneumoniae ([1][1]), therapeutic options increasingly rely on very few choices ([2][2]). Although colistin and tigecycline are now considered last-resort antibiotics for treating

SME-2-Producing Serratia marcescens Isolate from Switzerland

Carbapenemases in Serratia marcescens have been rarely reported, being related either to metallo-β-lactamases IMP-1, IMP-6, and VIM-2 (10) or to SME-type Ambler class A β-lactamases (6). SME-1 had been identified first from the carbapenem-resistant S. marcescens strain S6, isolated in London in 1982 (5), and then in carbapenem-resistant S. marcescens strains isolated in 1999 in the United States (1). It hydrolyzes penicillins, aztreonam, cephalosporins, and carbapenems and is inhibited by clavulanic acid (4). The blaSME-1 gene was chromosome encoded in isolate S6 (5). The SME-2 and SME-3 variants have been identified (being just point mutant analogues) from S. marcescens isolates recovered from the United States and United Kingdom (8, 9). Our study was initiated by the isolation in May 2006 at the University Hospital of Lausanne, Switzerland, of a carbapenem-resistant S. marcescens isolate. This isolate was from an exudate obtained after a parotidectomy of a 62-year-old patient who received a prophylaxis containing amoxicillin-clavulanate. Three days after surgery, the patient suffered from a pulmonary deficiency and received an imipenem-containing treatment. Two days later, culture of the exudate gave S. marcescens strain AW, which was resistant to penicillins and imipenem. The MICs of imipenem, meropenem, and ertapenem were 32, 8, and 4 μg/ml, respectively. It was also resistant to cefoxitin and aztreonam, whereas it remained susceptible in vitro to expanded-spectrum cephalosporins. The results for double-disk synergy testing, performed as described previously (2), were slightly positive with the clavulanate-imipenem combinations, indicating that S. marcescens AW produced a carbapenemase that was inhibited by clavulanic acid. PCR amplification using various primers, including blaSME-specific primers and whole-cell DNA of S. marcescens AW, followed by sequencing performed as described previously (7), identified a gene encoding SME-2. No other similar isolate was recovered from the same hospital during the same period of time, and no history of travel abroad or previous hospitalization was identified. The clonal relationship between isolate AW and isolate S6 from London was evaluated by pulsed-field gel electrophoresis as described previously (8), showing that isolate AW was not clonally related to isolate S6. By analyzing the sequences of the rpoB genes as reported previously (3), we found that the sequences obtained from isolates AW and S6 and also from two randomly selected carbapenem-susceptible and blaSME-negative S. marcescens isolates were identical. This observation likely rules out a possible identity for SME producers as part of a given subspecies. Conjugation, electrotransformation, and plasmid analysis, performed as described previously (5, 7), failed to identify a plasmid-borne location for the blaSME-2 gene, suggesting a likely chromosomal location for this gene. Further PCR mapping showed the presence of the LysR-type regulatory gene smeR upstream of blaSME-2, as previously identified (4). This was in accordance with the observation of a slight antagonism observed between the cefoxitin- and imipenem-containing disks. This report underlines the possible identification of SME-type S. marcescens producers worldwide. Along with other clavulanic acid-inhibited carbapenemases (NMC-A, IMI, KPC), SME-type enzymes may confer clinically significant resistance to carbapenems. Interestingly, SME producers may confer a lower level of resistance to ertapenem than to the other carbapenems.

Evaluation of the β-CARBA™ test, a colorimetric test for the rapid detection of carbapenemase activity in Gram-negative bacilli

Objectives There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we have evaluated a novel colorimetric test (the β-CARBA™ test; Bio-Rad) to detect carbapenemase-producing Gram-negative bacilli from cultured colonies. Methods The performance of the β-CARBA™ test was compared with that of the Carba NP test (or the CarbAcineto NP test) and RAPIDEC ® CARBA NP (bioMérieux) using a collection of 290 isolates with characterized β-lactamase content. This collection included 199 carbapenemase producers (121 Enterobacteriaceae, 36 Pseudomonas and 42 Acinetobacter baumannii ) and 91 non-carbapenemase producers (55 Enterobacteriaceae, 20 Pseudomonas and 16 A. baumannii ). Results The β-CARBA™ test correctly detected 84.9% of the carbapenemase producers, including all KPC and IMP, 96.4% of VIM, 85.3% of NDM, 80.5% of OXA-48-like and 91.2% of A. baumannii -related OXA carbapenemases (OXA-23, OXA-40, OXA-58, OXA-143 and overexpressed OXA-51). All rare metallo-β-lactamases (SPM, AIM, GIM, DIM and SIM) were detected. Importantly, all non-KPC Ambler class A carbapenemases were not detected, including GES variants with carbapenemase activity ( n  = 6), IMI ( n  = 3), NMC-A ( n  = 1), SME ( n  = 2), FRI-1 ( n  = 1) and BIC-1 ( n  = 1). All non-carbapenemase producers gave a negative result except with OXA-163-, OXA-405- and one TEM-3-producing Citrobacter freundii . The overall sensitivity and specificity of the β-CARBA™ test were 84.9% and 95.6%, respectively. This test is easy to perform and to interpret by non-specialized staff members. Conclusions Despite lack of specificity towards non-KPC Ambler class A and OXA-48-like carbapenemases, the β-CARBA™ test could complete the existing panel of tests available for the confirmation of carbapenemases in Gram-negatives.