Vendula Študentová

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.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH4HCO3, a Reliable Tool for Direct Detection of Carbapenemase Activity

ABSTRACT A comparison of a matrix-assisted laser desorption ionization–time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH4HCO3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers.

Complete Nucleotide Sequences of Two NDM-1-Encoding Plasmids from the Same Sequence Type 11 Klebsiella pneumoniae Strain

ABSTRACT The sequence type 11 Klebsiella pneumoniae strain Kpn-3002cz was confirmed to harbor two NDM-1-encoding plasmids, pB-3002cz and pS-3002cz. pB-3002cz (97,649 bp) displayed extensive sequence similarity with the blaNDM-1-carrying plasmid pKPX-1. pS-3002cz (73,581 bp) was found to consist of an IncR-related sequence (13,535 bp) and a mosaic region (60,046 bp). A 40,233-bp sequence of pS-3002cz was identical to the mosaic region of pB-3002cz, indicating the en bloc acquisition of the NDM-1-encoding region from one plasmid by the other.

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.

Characterization of pKP-M1144, a Novel ColE1-Like Plasmid Encoding IMP-8, GES-5, and BEL-1 β-Lactamases, from a Klebsiella pneumoniae Sequence Type 252 Isolate

ABSTRACT IMP-8 metallo-β-lactamase was identified in Klebsiella pneumoniae sequence type 252 (ST252), isolated in a Portuguese hospital in 2009. blaIMP-8 was the first gene cassette of a novel class 3 integron, In1144, also carrying the blaGES-5, blaBEL-1, and aacA4 cassettes. In1144 was located on a ColE1-like plasmid, pKP-M1144 (12,029 bp), with a replication region of limited nucleotide similarity to those of other RNA-priming plasmids, such as pJHCMW1. In1144 and pKP-M1144 represent an interesting case of evolution of resistance determinants in Gram-negative bacteria.

Isolation from a Nonclinical Sample of Leclercia adecarboxylata Producing a VIM-1 Metallo-β-Lactamase

Leclercia adecarboxylata belongs to the family Enterobacteriaceae and is rarely isolated from clinical material, especially from immunocompromised patients. This bacterium is usually susceptible to most commonly used antibiotics, including beta-lactams. However, a few cases of antibiotic-resistant L

Report on a transborder spread of carbapenemase-producing bacteria by a patient injured during Euromaidan, Ukraine

Spread of carbapenemase-producing bacteria has been described all over the world. This phenomenon may be accelerated by many factors, including wars and natural disasters. In this report, we described an NDM-1-producing Klebsiella pneumonia ST11 recovered from a patient injured during the Maidan revolution in Ukraine. To our knowledge, this is the first report of a carbapenemase-producing Enterobacteriaceae in Ukraine and one of several reports describing wound colonization/infection of humans injured during war.