Christine Lascols

Laboratory Detection of Enterobacteriaceae That Produce Carbapenemases

ABSTRACT A study was designed to evaluate the modified Hodge test (MHT), Mastdiscs ID inhibitor combination disks (MDI), Rosco Diagnostica Neo-Sensitabs (RDS), metallo-β-lactamase (MBL) Etest, and in-house multiplex PCR for the detection of well-characterized carbapenemase-producing Enterobacteriaceae. One hundred forty-two nonrepeat clinical isolates of carbapenemase-producing Enterobacteriaceae (including Klebsiella spp., Escherichia coli, Citrobacter freundii, and Enterobacter spp.) obtained from the SMART worldwide surveillance program during 2008 to 2009 were included. These included 49 KPC-, 27 NDM-, 19 VIM-, 14 OXA-48-like enzyme-, and 5 IMP-producing isolates and 28 carbapenem-resistant, carbapenemase-negative isolates. The manufacturers instructions were followed for MDI, RDS, and MBL Etest and CLSI guidelines for MHT. A multiplex PCR was designed to detect KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Overall, the sensitivity and specificity were 78% and 93% for MDI, 80% and 93% for RDS, 58% and 93% for MHT, and 55% and 100% for MBL Etest, respectively. The PCR had 100% sensitivity and specificity. MDI and RDS performed well for the detection of KPCs and NDMs but poorly for VIMs, IMPs, and OXA-48-like enzymes. MHT performed well for KPCs and OXA-48-like enzymes but poorly for NDMs, VIMs, and IMPs. MDI and RDS were easy to perform and interpret but lacked sensitivity for OXA-48-like enzymes, VIMs, and IMPs. MHT and MBL Etest were often difficult to interpret. We recommend using molecular tests for the optimal detection of carbapenemase-producing Enterobacteriaceae.

Increasing prevalence and dissemination of NDM-1 metallo-β-lactamase in India: data from the SMART study (2009)

OBJECTIVES To investigate the β-lactamase background of ertapenem non-susceptible isolates for the presence of the most commonly detected carbapenemase genes, bla(KPC), bla(OXA-48) and bla(VIM), and the newly described bla(NDM-1). METHODS Two hundred and thirty-five ertapenem-non-susceptible (MIC ≥ 0.5 mg/L) isolates of Enterobacteriaceae from the worldwide Study for Monitoring Antimicrobial Resistance Trends (SMART) 2009 programme were screened using a multiplex PCR for the presence of bla(KPC), bla(OXA-48), bla(VIM) and bla(NDM-1) genes. Extended-spectrum β-lactamase (ESBL) and AmpC genes (bla(ESBL) and bla(AmpC)) were identified using the Check-MDR CT101 microarray. DNA sequencing was performed to identify the bla(ESBL), bla(KPC) and bla(NDM-1) genes. Molecular typing was also performed to genetically characterize these isolates. RESULTS Sixty-six isolates (28%) had a carbapenemase gene, with bla(NDM-1) identified in 33 isolates including 2 isolates carrying both bla(NDM-1) and bla(OXA-48); other carbapenemase genes found included bla(KPC) (n = 23), bla(VIM) (n = 7) and bla(OXA-48) (n = 3). All bla(NDM-1)-carrying isolates were from patients in India and comprised five different species. With the exception of one isolate carrying only bla(NDM-1), all bla(NDM-1) carbapenemase-possessing isolates carried additional β-lactamases in various combinations: bla(ESBL) and bla(AmpC) (n = 18); bla(ESBL) (n = 10); bla(ESBL), bla(AmpC) and bla(OXA-48) (n = 2); and bla(AmpC) (n = 2). Except for bla(OXA-48)-carrying isolates, novel multilocus sequence types or enterobacterial repetitive intergenic consensus PCR patterns were observed along with clonal dissemination within and among sites. CONCLUSIONS A range of carbapenemase genes, associated with diverse ESBLs and/or AmpC backgrounds, were found among Enterobacteriaceae isolated during the study. Many of these ertapenem non-susceptible strains were clonally related and carried various combinations of β-lactamases.

Single and Double Mutations in gyrA but Not in gyrB Are Associated with Low- and High-Level Fluoroquinolone Resistance in Helicobacter pylori

ABSTRACT In one French hospital the rate of resistance to ciprofloxacin in Helicobacter pylori was 3.3% (2 of 60 strains) in 1999. The six resistant clinical strains (four from 1996 and two from 1999) and three ciprofloxacin-selected single-step mutants studied carried one gyrA mutation but none in gyrB. Clinafloxacin and garenoxacin were the most active fluoroquinolones against these mutants. Occurrence of a second gyrA mutation was associated with high MICs of all fluoroquinolones tested.

Surveillance and Molecular Epidemiology of Klebsiella pneumoniae Isolates That Produce Carbapenemases: First Report of OXA-48-Like Enzymes in North America

ABSTRACT A study was designed to characterize nonrepeat isolates of carbapenemase-producing K. pneumoniae obtained from the SMART worldwide surveillance program during 2008 and 2009. Characterization was done by PCR and sequencing for blaVIM, blaIMP, blaNDM, blaOXA, blaKPC, and plasmid-mediated quinolone resistance and virulence factors (VFs). Genetic relatedness was determined with pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequence typing. A total of 110 isolates were included; 47 possess genes that encode K. pneumoniae carbapenemases (KPCs), 26 NDMs, 19 VIMs, 13 OXA-48-like, and 5 imipenems (IMPs). We identified 3 different major sequence types (STs) among 65% of the isolates (i.e., ST11 [n = 11], ST147 [n = 23], and ST258 [n = 38]). ST11 and ST147, producing OXA-48-like and NDMs, were found in Argentina, Turkey, Greece, Italy, and India; ST258, producing KPCs, was present in the United States, Israel, Greece, and Puerto Rico. The major STs consisted of up to 4 different pulsotypes, and each pulsotype had a specific geographical distribution. A new ST, named ST903, with blaIMP-26, was identified in the Philippines, while two blaOXA-48-positive isolates were detected in the United States. There were no significant differences in the distribution of the VFs between the isolates; all were positive for fimH, mrkD, wabG, and ureA. This is the first report of OXA-48-like enzymes in North America. Our study highlights the importance of surveillance programs using molecular techniques as powerful tools to identify the importance of international sequence types.

Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of Antibiotic Resistance in Helicobacter pylori

ABSTRACT The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.

Fast and Accurate Quantitative Detection of Helicobacter pylori and Identification of Clarithromycin Resistance Mutations in H. pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR

ABSTRACT Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.

Using Nucleic Acid Microarrays To Perform Molecular Epidemiology and Detect Novel β-Lactamases: a Snapshot of Extended-Spectrum β-Lactamases throughout the World

ABSTRACT The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008–2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla ESBL (bla SHV, bla TEM, and bla CTX-M genes of groups 1, 2, 9, and 8/25) and bla KPC genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla ESBL and bla KPC genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla ESBL gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla TEM (n = 5), bla SHV (n = 11), bla CTX-M (n = 19), and bla KPC (n = 3) were detected. A new bla SHV variant, bla SHV-129, and a new bla KPC variant, bla KPC-11, were also identified. The most common bla genes found in this study were bla CTX-M-15, bla CTX-M-14, and bla SHV-12. Using nucleic acid microarrays, we obtained a “molecular snapshot” of bla ESBL genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla SHV-129 and bla KPC-11.

Susceptibility of Klebsiella pneumoniae Isolates from Intra-Abdominal Infections, and Molecular Characterization of Ertapenem-Resistant Isolates

ABSTRACT A total of 2,841 clinical isolates of Klebsiella pneumoniae from intra-abdominal infections worldwide were collected in the Study for Monitoring Antimicrobial Resistance Trends (SMART) during 2008 and 2009. Overall, 22.4% of isolates had extended-spectrum β-lactamases (ESBLs). The most active antibiotics among the 11 tested were imipenem, amikacin, and ertapenem, though even these, like all other comparators, were less consistently active against ESBL-positive isolates than against ESBL-negative isolates. Globally, 6.5% of isolates were ertapenem resistant based on the June 2010 clinical breakpoints published by the Clinical and Laboratory Standards Institute, with MICs of ≥1 μg/ml. Molecular characterization of 43 isolates with ertapenem MICs of ≥4 μg/ml showed that they variously produced CTX-M or SHV ESBLs combined with altered impermeability and/or had KPC (n = 28), OXA-48 (n = 3), or VIM (n = 1) carbapenemases. Further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates are needed.

Susceptibility of European Escherichia coli clinical isolates from intra-abdominal infections, extended-spectrum β-lactamase occurrence, resistance distribution, and molecular characterization of ertapenem-resistant isolates (SMART 2008–2009)

A total of 3160 clinical isolates of Escherichia coli from intra-abdominal infections were collected during 2008-2009 from 13 European countries. The frequency of extended-spectrum β-lactamase (ESBL)-producing isolates in Europe was 11%. The most active antibiotics tested were typically imipenem, ertapenem, and amikacin, although the activity of all non-carbapenem antibiotics was lower when tested against ESBL-positive isolates than when tested against ESBL-negative isolates. Ertapenem exhibited 99.3% susceptibility with all isolates, and 96.8% susceptibility with ESBL-positive isolates. With application of the ertapenem CLSI clinical breakpoint for resistance (MIC ≥1 mg/L), only six isolates (0.2%) were ertapenem-resistant, and only three of these were available for molecular characterization. Of those three, only one was ESBL-positive (CTX-M-14), and two were carbapenemase-positive (OXA-48). All three were negative for, VIM, NDM and KPC carbapenemases. Although the level of ertapenem resistance in E. coli is very low, further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates is needed.

The Pentapeptide Repeat Proteins MfpAMt and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex

MfpA(Mt) and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA(Mt) gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA(Mt) on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 microM, MfpA(Mt) inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 microM. We showed that the D87 residue in GyrA has a major role in the MfpA(Mt)-gyrase interaction, as D87H and D87G substitutions abolished MfpA(Mt) inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA(Mt) and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 microM as described for other Qnr proteins, but MfpA(Mt) did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA(Mt) also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA(Mt) and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.