Tamara Bergerova

Carbapenemase Activity Detection by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.

Detection of NDM-1, VIM-1, KPC, OXA-48, and OXA-162 carbapenemases by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a potentially useful tool for the detection of antimicrobial resistance, especially that conferred by β-lactamases. Here we describe a modification of a previously reported MALDI-TOF MS meropenem hydrolysis assay. The modified method was validated on 108 carbapenemase-producing members of the Enterobacteriaceae, two NDM-1-producing Acinetobacter baumannii isolates, and 35 carbapenem-resistant enterobacteria producing no carbapenemase. The detection of carbapenemases by MALDI-TOF MS seems to be a powerful, quick, and cost-effective method for microbiological laboratories.

Clinical and microbiological efficacy of continuous versus intermittent application of meropenem in critically ill patients: a randomized open-label controlled trial

IntroductionMeropenem bactericidal activity depends on the time when the free drug concentrations remain above the minimum inhibitory concentration of pathogens. The goal of this study was to compare clinical and bacteriological efficacy of continuous meropenem infusion versus bolus administration in critically ill patients with severe infection, and to evaluate the safety of both dosing regimens.MethodsPatients admitted to the interdisciplinary Intensive Care Unit (ICU) who suffered from severe infections and received meropenem were randomized either in the Infusion group (n = 120) or in the Bolus group (n = 120). Patients in the Infusion group received a loading dose of 2 g of meropenem followed by a continuous infusion of 4 g of meropenem over 24 hours. Patients in the Bolus group were given 2 g of meropenem over 30 minutes every 8 hours. Clinical and microbiological outcome, safety, meropenem-related length of ICU and hospital stay, meropenem-related length of mechanical ventilation, duration of meropenem treatment, total dose of meropenem, and ICU and in-hospital mortality were assessed.ResultsClinical cure at the end of meropenem therapy was comparable between both groups (83.0% patients in the Infusion vs. 75.0% patients in the Bolus group; P = 0.180). Microbiological success rate was higher in the Infusion group as opposed to the Bolus group (90.6% vs. 78.4%; P = 0.020). Multivariate logistic regression identified continuous administration of meropenem as an independent predictor of microbiological success (OR = 2.977; 95% CI = 1.050 to 8.443; P = 0.040). Meropenem-related ICU stay was shorter in the Infusion group compared to the Bolus group (10 (7 to 14) days vs. 12 (7 to 19) days; P = 0.044) as well as shorter duration of meropenem therapy (7 (6 to 8) days vs. 8 (7 to 10) days; P = 0.035) and lower total dose of meropenem (24 (21 to 32) grams vs. 48 (42 to 60) grams; P < 0.0001). No severe adverse events related to meropenem administration in either group were observed.ConclusionsContinuous infusion of meropenem is safe and, in comparison with higher intermittent dosage, provides equal clinical outcome, generates superior bacteriological efficacy and offers encouraging alternative of antimicrobial therapy in critically ill patients.

International Clones of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum β-Lactamases in a Czech Hospital

ABSTRACT A 2-month survey of extended-spectrum β-lactamase (ESBL) producers was performed in a Czech hospital. Klebsiella pneumoniae produced SHV-2, -5, or -12, Escherichia coli produced CTX-M-9 or -15, and other species produced TEM-92 or -132. All K. pneumoniae and E. coli isolates belonged to sequence types (STs) or clonal complexes (CCs) spread across the world (K. pneumoniae clonal complex 11 [CC11], CC14, and sequence type 101 [ST101] and E. coli CC31, CC73, CC131, and CC405) and carried various plasmids (mainly with A/C- and FII-type replicons).

Carbapenem-nonsusceptible strains of Klebsiella pneumoniae producing SHV-5 and/or DHA-1 β-lactamases in a Czech hospital

Resistance to carbapenems in enterobacteria is mediated by the production of several types of carbapenemases or by the decreased permeability of the outer membrane, combined with the expression of extended-spectrum beta-lactamases (ESBLs) or AmpC-like cephalosporinases. The objective of this study was to characterize carbapenem-nonsusceptible (C-NS) isolates of Klebsiella pneumoniae in the University Hospital in Plzen (Czech Republic) and compare them with carbapenem-susceptible (C-S) K. pneumoniae isolates from the same patients. Six C-NS K pneumoniae isolates from different patients were collected between January 2007 and June 2008, and from three of these patients, C-S isolates were available for the study as well. The isolates were typed by pulsed-field gel electrophoresis and multilocus sequence typing. beta-Lactamases were analyzed by isoelectric focusing, bioassay, and PCR and sequencing of bla genes. Major porin channels, OmpK35 and OmpK36, were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot; porin genes were amplified and sequenced, and their expression was assessed by reverse transcriptase-PCR. The C-NS isolates belonged to three pulsotypes and to the clone ST11, produced the SHV-5 ESBL and/or DHA-1 AmpC-type cephalosporinase, did not express OmpK36, and had a reduced expression of OmpK35. The C-S isolates differed from their C-NS counterparts only by porin expression profiles.

Identification of a New Delhi metallo-β-lactamase-4 (NDM-4)-producing Enterobacter cloacae from a Czech patient previously hospitalized in Sri Lanka.

In recent two years, a global spread of NDM-type metallobeta-lactamase-producing bacteria has been observed. NDM enzymes are mainly produced by Enterobacteriaceae, but have also been detected in Acinetobacter baumannii and Vibrio spp. as well (Tzouvelekis et al. 2012; Walsh et al. 2011). Two cases of the new variant, NDM-4, have been recently described in isolates recovered from patients previously hospitalized in India and in Cameroon (Nordmann et al. 2012; Dortet et al. 2012). In Czech Republic, only a case of an NDM-1-producing A. baumannii isolated from a Czech citizen repatriated from Egypt has been observed (Hrabak et al. 2012a). Here, we describe an NDM-4producing Enterobacter cloacae strain recovered from a patient previously hospitalized in Sri Lanka. We also present data on the localization and the genetic environment of the blaNDM-4 gene. The patient, a 62-year-old female, who traveled to Sri Lanka in August 2012, was hospitalized for 28 days in three different hospitals in Sri Lanka for sepsis of a putative wound origin. The patient was treated with amoxicillin/clavulanic acid, followed by a combination therapy with meropenem, clindamycin, and doxycycline as an empiric therapy because no infectious agent was identified. After cultivation of Streptococcus pyogenes from wound swab, clindamycin was changed to penicillin. The patient was mechanically ventilated for 11 days. In September, the patient was repatriated to the Czech Republic. Immediately after repatriation to Czech Republic, screening for carbapenemase-producing Enterobacteriaceae was performed according to the national guidelines. Rectal swab and another clinical material standardly collected from patients hospitalized in ICUs (e.g., sputum/bronchoalveolar lavage, nose swabs, throat swabs) were inoculated on the selective plates for the detection of cephalosporin-resistant isolates (ChromIDTM, ESBL Agar, bioMérieux, Paris, France). Colonies grown on the media after 24 h cultivation were identified to the species level. Susceptibility to imipenem and meropenem was determined according to the EUCAST recommendation (EUCAST 2003). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MALDI Biotyper; Bruker DaltoniK, GmbH, Bremen, Germany) identified an E. cloacae clinical isolate (Encl-922), which exhibited elevated minimum inhibitory concentrations to carbapenems (Table 1). Encl-922 was resistant to all β-lactams, including meropenem, as determined by broth dilution method (EUCAST 2003). The isolate was also resistant to various non-β-lactam antibiotics (Table 1). Carbapenemase production was verified byMALDITOF MS hydrolysis assay (Hrabák et al. 2011, 2012b). Additionally, ethylenediaminetetraacetic acid–meropenem combined disk test confirmed metallo-β-lactamase production, while boronic acid-based tests using meropenem appeared negative. PCR and sequencing showed that Encl-922 carried C. C. Papagiannitsis :V. Studentova : E. Chudackova : T. Bergerova : J. Hrabak Department of Microbiology, Faculty of Medicine and University Hospital, Charles University, Plzen, Czech Republic

Molecular characterization of OXA-48-like-producing Enterobacteriaceae in the Czech Republic: evidence for horizontal transfer of pOXA-48-like plasmids

ABSTRACT The aim of this study was to characterize the first cases and outbreaks of OXA-48-like-producing Enterobacteriaceae recovered from hospital settings in the Czech Republic. From 2013 to 2015, 22 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, and 1 Enterobacter cloacae isolate producing OXA-48-like carbapenemases were isolated from 20 patients. Four of the patients were colonized or infected by two or three different OXA-48-like producers. The K. pneumoniae isolates were classified into nine sequence types (STs), with ST101 being predominant (n = 8). The E. coli isolates were of different STs, while the E. cloacae isolate belonged to ST109. Twenty-four isolates carried blaOXA-48, while two isolates carried blaOXA-181 or blaOXA-232. Almost all isolates (n = 22) carried blaOXA-48-positive plasmids of a similar size (∼60 kb), except the two isolates producing OXA-181 or OXA-232. In an ST45 K. pneumoniae isolate and an ST38 E. coli isolate, S1 nuclease profiling plus hybridization indicated a chromosomal location of blaOXA-48. Sequencing showed that the majority of blaOXA-48-carrying plasmids exhibited high degrees of identity with the pOXA-48-like plasmid pE71T. Additionally, two novel pE71T derivatives, pOXA-48_30715 and pOXA-48_30891, were observed. The blaOXA-181-carrying plasmid was identical to the IncX3 plasmid pOXA181_EC14828, while the blaOXA-232-carrying plasmid was a ColE2-type plasmid, being a novel derivative of pOXA-232. Finally, sequencing data showed that the ST45 K. pneumoniae and ST38 E. coli isolates harbored the IS1R-based composite transposon Tn6237 containing blaOXA-48 integrated into their chromosomes. These findings underlined that the horizontal transfer of pOXA-48-like plasmids has played a major role in the dissemination of blaOXA-48 in the Czech Republic. In combination with the difficulties with their detection, OXA-48 producers constitute an important public threat.

Detection of OXA-48-type carbapenemase-producing Enterobacteriaceae in diagnostic laboratories can be enhanced by addition of bicarbonates to cultivation media or reaction buffers

Carbapenemase-mediated resistance to carbapenems in Enterobacteriaceae has become the main challenge in the treatment and prevention of infections recently. The partially unnoticed spread of OXA-48-type carbapenemase producers is usually assigned to low minimum inhibitory concentrations (MICs) of carbapenems that OXA-48-producing isolates often display. Therefore, there is an urgent need of specific and sensitive methods for isolation and detection of OXA-48 producers in clinical microbiology diagnostics. The influence of bicarbonates on carbapenem MICs against carbapenemase-producing Enterobacteriaceae was tested. We also checked whether the addition of bicarbonates to liquid media supplemented with meropenem may facilitate the selective enrichment of various carbapenemase producers in cultures. Furthermore, the sensitivity of carbapenemase confirmation by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) and spectrophotometric hydrolysis assays upon the addition of NH4HCO3 was examined. The addition of NaHCO3 significantly increased MICs of ertapenem and meropenem for OXA-48 producers. Furthermore, liquid media supplemented with NaHCO3 and meropenem were reliable for the selective enrichment of carbapenemase producers. The presence of NH4HCO3 in buffers used in the spectrophotometric and MALDI-TOF MS carbapenemase detection increased the sensitivity of that assay. Our results demonstrate that bicarbonates in media or reaction buffers can enhance the sensitivity of screening methods and diagnostic tests for carbapenemase producers.

A case of imported Clostridium difficile PCR-ribotype 027 infection within the Czech Republic which has a high prevalence of C. difficile ribotype 176.

The first case of Clostridium difficile RT027 infection in the Czech Republic (CZ) was identified. The patient had been hospitalised in Germany prior to moving to CZ. Multiple-Locus Variable number tandem repeat Analysis revealed a genetic relatedness between the patients isolate and RT027 isolate collected in the German hospital.

DHA-1-Producing Klebsiella pneumoniae in a Teaching Hospital in the Czech Republic

Thirty-eight AmpC-producing Klebsiella pneumoniae isolates identified from January to October 2006 in a large teaching hospital in the Czech Republic were analyzed. The AmpC cephalosporinase was identified as DHA-1, encoded by a plasmid-located complex class 1 integron, previously observed in a K. pneumoniae isolate from the Parisian region. The DHA-1 expression was inducible, and although in two isolates with higher resistance, the induction effect was masked at the phenotypic level. All of the isolates belonged to the international K. pneumoniae clone sequence type 11, split into two disseminated pulsed-field gel electrophoresis types. This is the first report on enterobacteriaceae with acquired AmpCs in the Czech Republic and possibly the first description of organisms with DHA-1 in the Central and Eastern Europe.