Dominique Decré

Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae

OBJECTIVESnTo develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases.nnnMETHODSnFollowing the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes.nnnRESULTSnUnder optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1).nnnCONCLUSIONSnWe report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.

wzi Gene Sequencing, a Rapid Method for Determination of Capsular Type for Klebsiella Strains

ABSTRACT Pathogens of the genus Klebsiella have been classified into distinct capsular (K) types for nearly a century. K typing of Klebsiella species still has important applications in epidemiology and clinical microbiology, but the serological method has strong practical limitations. Our objective was to evaluate the sequencing of wzi, a gene conserved in all capsular types of Klebsiella pneumoniae that codes for an outer membrane protein involved in capsule attachment to the cell surface, as a simple and rapid method for the prediction of K type. The sequencing of a 447-nucleotide region of wzi distinguished the K-type reference strains with only nine exceptions. A reference wzi sequence database was created by the inclusion of multiple strains representing K types associated with high virulence and multidrug resistance. A collection of 119 prospective clinical isolates of K. pneumoniae were then analyzed in parallel by wzi sequencing and classical K typing. Whereas K typing achieved typeability for 81% and discrimination for 94.4% of the isolates, these figures were 98.1% and 98.3%, respectively, for wzi sequencing. The prediction of K type once the wzi allele was known was 94%. wzi sequencing is a rapid and simple method for the determination of the K types of most K. pneumoniae clinical isolates.

Rectal Carriage of Extended-Spectrum Beta-Lactamase-Producing Gram-Negative Bacilli in Community Settings in Madagascar

Background Extended-spectrum ß-lactamase-producing Enterobacteria (ESBL-PE) emerged at the end of the 1980s, causing nosocomial outbreaks and/or hyperendemic situations in hospitals and long-term care facilities. In recent years, community-acquired infections due to ESBL-PE have spread worldwide, especially across developing countries including Madagascar. Objectives This study aimed to determine the prevalence and risk factors of intestinal carriage of ESBL-PE in the community of Antananarivo. Methods Non-hospitalized patients were recruited in three health centers in different socio economic settings. Fresh stool collected were immediately plated on Drigalski agar containing 3 mg/liter of ceftriaxone. Gram-negative bacilli species were identified and ESBL production was tested by a double disk diffusion (cefotaxime and ceftazidime +/− clavulanate) assay. Characterization of ESBLs were perfomed by PCR and direct sequencing . Molecular epidemiology was analysed by Rep-PCR and ERIC-PCR. Results 484 patients were screened (sex ratio u200a=u200a1.03, median age 28 years). 53 ESBL-PE were isolated from 49 patients (carrier rate 10.1%). The isolates included Escherichia coli (31), Klebsiella pneumoniae (14), Enterobacter cloacae (3), Citrobacter freundii (3), Kluyvera spp. (1) and Pantoae sp.(1). In multivariate analysis, only the socioeconomic status of the head of household was independently associated with ESBL-PE carriage, poverty being the predominant risk factor. Conclusions The prevalence of carriage of ESBL in the community of Antananarivo is one of the highest reported worldwide. This alarming spread of resistance genes should be stopped urgently by improving hygiene and streamlining the distribution and consumption of antibiotics.

Outbreak of Klebsiella pneumoniae producing KPC-2 and SHV-12 in a French hospital

M et al. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. 2 Clermont O, Lavollay M, Vimont S et al. The CTX-M-15-producing Escherichia coli diffusing clone belongs to a highly virulent B2 phylogenetic subgroup. 3 Karisik E, Ellington MJ, Livermore DM et al. Virulence factors in Escherichia coli with CTX-M-15 and other extended-spectrum b-lactamases in the UK. 4 Deschamps C, Clermont O, Hipeaux MC et al. Multiple acquisitions of CTX-M plasmids in the rare D 2 genotype of Escherichia coli provide evidence for convergent evolution. 7 Ender PT, Gajanana D, Johnston B et al. Transmission of an extended-spectrum-b-lactamase-producing Escherichia coli (sequence type ST131) strain between a father and daughter resulting in septic shock and emphysematous pyelonephritis. A et al. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum b-lactamase CTX-M-15. Sir, The emergence and dissemination of Klebsiella pneumoniae strains harbouring carbapenemases is a serious concern. Klebsiella pneu-moniae carbapenemase (KPC) enzymes belong to molecular class A and are able to hydrolyse most b-lactams including carbapenems. Since the initial report of a KPC b-lactamase from a strain of K. pneu-moniae in 1996, KPC producers have been reported from various geographical regions. Current reports indicate that KPC-producing isolates are widespread in China, Israel, Greece, South America and the USA, where the epidemiology of KPC in the hospital setting is changing. 1 Fortunately, these strains are still rare in western and northern Europe, but their detection remains difficult. 2 Since 2003, patients hospitalized in the surgical ward of our hospital have been systematically screened on admission and weekly thereafter for intestinal carriage of bacteria producing extended-spectrum b-lactamases (ESBLs) and carbapenemases by plating rectal swabs on Drigalski agar containing 0.5 mg/L cefotaxime and MacConkey agar containing 2 mg/L ceftazidime (AES Labora-toire, Combourg, France). We report here four patients with K. pneu-moniae producing KPC-2 and SHV-12. The first case was a patient transferred in July 2009 from Crete for treatment of recurrent angiocholitis on a biliary stent. The patient was negative on the day of admission, but 3 days later a further stool sample grew with ESBL-producing K. pneumoniae. This first isolate was not suspected to produce a carbapenemase since testing for susceptibility to imipenem using a disc diffusion method showed a diameter of 24 mm and an MIC of 1.5 mg/L by Etest (Bio-Rad, Marne la Coquette, France), both considered as susceptible according to the national recommendations of the Antibiogram …

Evaluation of the βLacta Test, a Rapid Test Detecting Resistance to Third-Generation Cephalosporins in Clinical Strains of Enterobacteriaceae

ABSTRACT For decades, third-generation cephalosporins (3GC) have been major drugs used to treat infections due to Enterobacteriaceae; growing resistance to these antibiotics makes the rapid detection of such resistance important. The βLacta test is a chromogenic test developed for detecting 3GC-resistant isolates from cultures on solid media within 15 min. A multicenter prospective study conducted in 5 French and Belgian hospitals evaluated the performance of this test on clinical isolates. Based on antibiotic susceptibility testing, strains resistant or intermediate to cefotaxime or ceftazidime were classified as 3GC resistant, and molecular characterization of this resistance was performed. The rates of 3GC resistance were 13.9% (332/2,387) globally, 9.4% in Escherichia coli (132/1,403), 25.6% in Klebsiella pneumoniae (84/328), 30.3% in species naturally producing inducible AmpC beta-lactamases (109/360), and 5.6% in Klebsiella oxytoca and Citrobacter koseri (7/124). The sensitivities and specificities of the βLacta test were, respectively, 87.7% and 99.6% overall, 96% and 100% for E. coli and K. pneumoniae, and 67.4% and 99.6% for species naturally producing inducible AmpC beta-lactamase. False-negative results were mainly related to 3GC-resistant strains producing AmpC beta-lactamase. Interestingly, the test was positive for all 3GC-resistant extended-spectrum beta-lactamase-producing isolates (n = 241). The positive predictive value was 97% and remained at ≥96% for prevalences of 3GC resistance ranging between 10 and 30%. The negative predictive values were 99% for E. coli and K. pneumoniae and 89% for the species producing inducible AmpC beta-lactamase. In conclusion, the βLacta test was found to be easy to use and efficient for the prediction of resistance to third-generation cephalosporins, particularly in extended-spectrum beta-lactamase-producing strains.

Complete Nucleotide Sequence of the First KPC-2- and SHV-12-Encoding IncX Plasmid, pKpS90, from Klebsiella pneumoniae

ABSTRACT We report the complete nucleotide sequence of the pKpS90 plasmid, carrying the blaKPC-2 and blaSHV-12 genes. This plasmid was isolated from a sequence type 258 (ST258) Klebsiella pneumoniae strain responsible for an outbreak in a French university hospital in 2009. pKpS90 is a 53,286-bp plasmid that belongs to the IncX incompatibility group. pKpS90 consists of a backbone from IncX plasmids, in which the KPC-2-encoding Tn4401 transposon and a blaSHV-12-encoding region have been inserted.

Emergence of NDM-1 in Association with OXA-48 in Klebsiella pneumoniae from Tunisia

The carbapenemase New Delhi metallo-β-lactamase-1 (NDM-1), initially identified in Escherichia coli and Klebsiella pneumoniae in 2008 in a Swedish patient who was repatriated to Sweden from India ([1][1]), is spreading rapidly worldwide except in Central and South America. Most of the reported

Complete nucleotide sequence of the large conjugative pTC2 multireplicon plasmid encoding the VIM-1 metallo-β-lactamase

OBJECTIVESnTo determine the complete nucleotide sequence of the VIM-1-encoding plasmid pTC2, which was isolated from a Greek Providencia stuartii multiresistant strain.nnnMETHODSnThe pTC2 plasmid was extracted and sequenced using shotgun and 3 kb paired-end DNA libraries and a 454 sequencing approach. Following assembly into a unique scaffold, gaps were closed by PCR followed by Sanger DNA sequencing. Gene predictions and annotations were performed using the CAAT-Box tool and the Conserved Domain Search service. Sequence comparisons were performed using the Artemis Comparison Tool.nnnRESULTSnPlasmid pTC2 (180,184 bp) was found to be a multireplicon plasmid (IncA/C and IncR), with a large IncA/C backbone and a mosaic multidrug resistance (MDR) region, in which was inserted a 13 kb IncR fragment. Gene annotation allowed the identification of a complete IncA/C-type transfer system and of several putative maintenance modules, both on the IncA/C backbone and on the IncR fragment. The complex MDR region contained 9 insertion sequences (7 IS26, 1 IS1 and 1 IS6100), 10 resistance genes and a mercury resistance operon integrated into unit transposons, composite transposons or integrons.nnnCONCLUSIONSnThe pTC2 combines a broad host range, transfer and maintenance capacities, plasticity of the MDR region and a wide variety of resistance genes, properties that may contribute to the spreading of resistance determinants.

Complete Nucleotide Sequence of Two Multidrug-Resistant IncR Plasmids from Klebsiella pneumoniae

ABSTRACT We report here the complete nucleotide sequence of two IncR replicons encoding multidrug resistance determinants, including β-lactam (blaDHA-1, blaSHV-12), aminoglycoside (aphA1, strA, strB), and fluoroquinolone (qnrB4, aac6′-1b-cr) resistance genes. The plasmids have backbones that are similar to each other, including the replication and stability systems, and contain a wide variety of transposable elements carrying known antibiotic resistance genes. This study confirms the increasing clinical importance of IncR replicons as resistance gene carriers.

Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae

Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called plasmid relaxase gene typing (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice.