Characterization of NDM-Encoding Plasmids From Enterobacteriaceae Recovered From Czech Hospitals

The aim of the present study was to characterize sporadic cases and an outbreak of NDM-like-producing Enterobacteriaceae recovered from hospital settings, in Czechia. During 2016, 18 Entrobacteriaceae isolates including 10 Enterobacter cloacae complex (9 E. xiangfangensis and 1 E. asburiae), 4 Escherichia coli, 1 Kluyvera intermedia, 1 Klebsiella pneumoniae, 1 Klebsiella oxytoca, and 1 Raoultella ornithinolytica that produced NDM-like carbapenemases were isolated from 15 patients. Three of the patients were colonized or infected by two different NDM-like producers. Moreover, an NDM-4-producing isolate of E. cloacae complex, isolated in 2012, was studied for comparative purposes. All isolates of E. cloacae complex, except the E. asburiae, recovered from the same hospital, were assigned to ST182. Additionally, two E. coli belonged to ST167, while the remaining isolates were not clonally related. Thirteen isolates carried blaNDM−4, while six isolates carried blaNDM−1 (n = 3) or blaNDM−5 (n = 3). Almost all isolates carried blaNDM-like-carrying plasmids being positive for the IncX3 allele, except ST58 E. coli and ST14 K. pneumoniae isolates producing NDM-1. Analysis of plasmid sequences revealed that all IncX3 blaNDM-like-carrying plasmids exhibited a high similarity to each other and to previously described plasmids, like pNDM-QD28, reported from worldwide. However, NDM-4-encoding plasmids differed from other IncX3 plasmids by the insertion of a Tn3-like transposon. On the other hand, the ST58 E. coli and ST14 K. pneumoniae isolates carried two novel NDM-1-encoding plasmids, pKpn-35963cz, and pEsco-36073cz. Plasmid pKpn-35963cz that was an IncFIB(K) molecule contained an acquired sequence, encoding NDM-1 metallo-β-lactamase (MβL), which exhibited high similarity to the mosaic region of pS-3002cz from an ST11 K. pneumoniae from Czechia. Finally, pEsco-36073cz was a multireplicon A/C2+R NDM-1-encoding plasmid. Similar to other type 1 A/C2 plasmids, the blaNDM−1 gene was located within the ARI-A resistance island. These findings underlined that IncX3 plasmids have played a major role in the dissemination of blaNDM-like genes in Czech hospitals. In combination with further evolvement of NDM-like-encoding MDR plasmids through reshuffling, NDM-like producers pose an important public threat

Plasmid-mediated colistin resistance among human clinical Enterobacterales isolates: national surveillance in the Czech Republic

The occurrence of colistin resistance has increased rapidly among Enterobacterales around the world. We performed a national survey of plasmid-mediated colistin resistance in human clinical isolates through a retrospective analysis of samples from 2009 to 2017 and a prospective sampling in 2018–2020. The aim of this study was to identify and characterize isolates with mcr genes from various regions of the Czech Republic using whole genome sequencing (WGS). Of all 1932 colistin-resistant isolates analyzed, 73 (3.8%) were positive for mcr genes. Most isolates carried mcr-1 (48/73) and were identified as Escherichia coli (n = 44) and Klebsiella pneumoniae (n = 4) of various sequence types (ST). Twenty-five isolates, including Enterobacter spp. (n = 24) and Citrobacter freundii (n = 1) carrying the mcr-9 gene were detected; three of them (Enterobacter kobei ST54) co-harbored the mcr-4 and mcr-9 genes. Multi-drug resistance phenotype was a common feature of mcr isolates and 14% (10/73) isolates also co-harbored clinically important beta-lactamases, including two isolates with carbapenemases KPC-2 and OXA-48. Phylogenetic analysis of E. coli ST744, the dominant genotype in this study, with the global collection showed Czech isolates belonged to two major clades, one containing isolates from Europe, while the second composed of isolates from diverse geographical areas. The mcr-1 gene was carried by IncX4 (34/73, 47%), IncHI2/ST4 (6/73, 8%) and IncI2 (8/73, 11%) plasmid groups. Small plasmids belonging to the ColE10 group were associated with mcr-4 in three isolates, while mcr-9 was carried by IncHI2/ST1 plasmids (4/73, 5%) or the chromosome (18/73, 25%). We showed an overall low level of occurrence of mcr genes in colistin-resistant bacteria from human clinical samples in the Czech Republic

Relative Strengths of the Class 1 Integron Promoter Hybrid 2 and the Combinations of Strong and Hybrid 1 with an Active P2 Promoter▿

The relative strengths of the uncommon promoters hybrid 2, hybrid 1 with an active P2 promoter (hybrid 1+P2), and strong+P2, which drive transcription of resistance genes in class 1 integrons, were evaluated using blaGES-1 as a reporter gene cassette. Hybrid 2 was stronger than hybrid 1. Coupling P2 with the strong promoter and with hybrid 1 caused a measurable increase in GES-1 expression

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

Extended-Spectrum Properties of CMY-30, a Val211Gly Mutant of CMY-2 Cephalosporinase▿

CMY-30, a Val211Gly mutant of CMY-2 cephalosporinase, was derived by mutagenesis. The hydrolytic efficiency of CMY-30 against expanded-spectrum cephalosporins was higher than that of CMY-2 due to increased kcat values. Findings indicate a role of the Ω loop residue 211 in determining the substrate specificities of CMYs also corroborated by modeling studies

Unravelling the Features of Success of VIM-Producing ST111 and ST235 Pseudomonas aeruginosa in a Greek Hospital

The objective of this study was to analyze the characteristics that contribute to the successful dissemination of VIM-producing Pseudomonas aeruginosa (P. aeruginosa), belonging to ST111 and ST235, in a Greek hospital. A total of 120 non-repetitive P. aeruginosa, which had meropenem minimal inhibitory concentrations (MICs) greater than 2 mg/L, were studied. VIM-encoding genes were amplified and sequenced within their integrons. Isolates were typed by multilocus sequence typing (MLST). Six VIM-producers, representative of different integron structures and sequence types (STs), were completely sequenced using Illumina platform. Sixty-one P. aeruginosa were confirmed to produce VIM-type carbapenemases. ST111 dominated (n = 34) among VIM-producers, while 15 VIM-producers belonged to ST235. The blaVIM-like genes were located in three integron types, including In59, In595 and In1760, which were integrated into P. aeruginosa chromosomes. Whole genome sequencing (WGS) data demonstrated that ST111 and ST235 MBL producers carried several resistance and virulence genes. Additionally, the presence of type I-C and type I-E clustered regularly interspaced short palindromic repeats (CRISPR)/Cas locus was observed in ST235 and ST395 isolates, respectively. In conclusion, our findings confirmed the clonal spread of ST111 P. aeruginosa, carrying the VIM-2-encoding integron In59, in the University Hospital of Larissa (UHL). In addition, they highlighted the important role of high-risk clones, ST111 and ST235, in the successful dissemination and establishment into hospital settings of clinically important pathogens carrying resistance determinants

Biochemical Characterization of VIM-39, a VIM-1-Like Metallo-β-Lactamase Variant from a Multidrug-Resistant Klebsiella pneumoniae Isolate from Greece

VIM-39, a VIM-1-like metallo-β-lactamase variant (VIM-1 Thr33Ala His224Leu) was identified in a clinical isolate of Klebsiella pneumoniae belonging to sequence type 147. VIM-39 hydrolyzed ampicillin, cephalothin, and imipenem more efficiently than did VIM-1 and VIM-26 (a VIM-1 variant with the His224Leu substitution) because of higher turnover rates

An Ertapenem-Resistant Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae Clone Carries a Novel OmpK36 Porin Variant▿

Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and β-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem- and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem