Extensive Genetic Commonality among Wildlife, Wastewater, Community, and Nosocomial Isolates of Escherichia coli Sequence Type 131 (H30R1 and H30Rx Subclones) That Carry blaCTX-M-27 or blaCTX-M-15.

Escherichia coli sequence type 131 (ST131) is currently one of the leading causes of multidrug-resistant extraintestinal infections globally. Here, we analyzed the phenotypic and genotypic characteristics of 169 ST131 isolates from various sources (wildlife, wastewater, companion animals, community, and hospitals) to determine whether wildlife and the environment share similar strains with humans, supporting transmission of ST131 between different ecological niches. Susceptibility to 32 antimicrobials was tested by disc diffusion and broth microdilution. Antibiotic resistance genes, integrons, plasmid replicons, 52 virulence genes, and fimH-based subtypes were detected by PCR and DNA sequencing. Genomic relatedness was determined by pulsed-field gel electrophoresis (PFGE). The genetic context and plasmid versus chromosomal location of extended-spectrum beta-lactamase and AmpC beta-lactamase genes was determined by PCR and probe hybridization, respectively. The 169 ST131 study isolates segregated predominantly into blaCTX-M-15H30Rx (60%) and blaCTX-M-27H30R1 (25%) subclones. Within each subclone, isolates from different source groups were categorized into distinct PFGE clusters; genotypic characteristics were fairly well conserved within each major PFGE cluster. Irrespective of source, the blaCTX-M-15H30Rx isolates typically exhibited virotype A (89%), an F2:A1:B- replicon (84%), and a 1.7-kb class 1 integron (92%) and had diverse structures upstream of the blaCTX-M region. In contrast, the blaCTX-M-27H30R1 isolates typically exhibited virotype C (86%), an F1:A2:B20 replicon (76%), and a conserved IS26-ΔISEcp1-blaCTX-M-like structure. Despite considerable overall genetic diversity, our data demonstrate significant commonality between E. coli ST131 isolates from diverse environments, supporting transmission between different sources, including humans, environment, and wildlife.

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-ß-lactamase (MßL), 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.

Bacterial DNA detected on pathologically changed heart valves using 16S rRNA gene amplification.

Nowadays, dental diseases are one of the most common illnesses in the world. Some of them can lead to translocation of oral bacteria to the bloodstream causing intermittent bacteraemia. Therefore, a potential association between oral infection and cardiovascular diseases has been discussed in recent years as a result of adhesion of oral microbes to the heart valves. The aim of this study was to detect oral bacteria on pathologically changed heart valves not caused by infective endocarditis. In the study, patients with pathologically changed heart valves were involved. Samples of heart valves removed during heart valve replacement surgery were cut into two parts. One aliquot was cultivated aerobically and anaerobically. Bacterial DNA was extracted using Ultra-Deep Microbiome Prep (Molzym GmbH, Bremen, Germany) followed by a 16S rRNA gene PCR amplification using Mastermix 16S Complete kit (Molzym GmbH, Bremen, Germany). Positive PCR products were sequenced and the sequences were analyzed using BLAST database ( http://www.ncbi.nlm.nih/BLAST ). During the study period, 41 samples were processed. Bacterial DNA of the following bacteria was detected in 21 samples: Cutibacterium acnes (formerly Propionibacterium acnes) (n = 11; 52.38% of patients with positive bacterial DNA detection), Staphylococcus sp. (n = 9; 42.86%), Streptococcus sp. (n = 1; 4.76%), Streptococcus sanguinis (n = 4; 19.05%), Streptococcus oralis (n = 1; 4.76%), Carnobacterium sp. (n = 1; 4.76%), Bacillus sp. (n = 2; 9.52%), and Bergeyella sp. (n = 1; 4.76%). In nine samples, multiple bacteria were found. Our results showed significant appearance of bacteria on pathologically changed heart valves in patients with no symptoms of infective endocarditis.

Emergence of sequence type 252 Enterobacter cloacae producing GES-5 carbapenemase in a Czech hospital.

ST252 Enterobacter cloacae, producing GES-5 carbapenemase, was isolated in a Czech hospital. blaGES-5 was part of a novel class 1 integron, In1406, which also included a new allele of the aadA15 gene cassette. In1406 was located on a ColE2-like plasmid, pEcl-35771cz (6953bp).

Validation of a novel automatic deposition of bacteria and yeasts on MALDI target for MALDI-TOF MS-based identification using MALDI Colonyst robot.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) -based identification of bacteria and fungi significantly changed the diagnostic process in clinical microbiology. We describe here a novel technique for bacterial and yeast deposition on MALDI target using an automated workflow resulting in an increase of the microbes' score of MALDI identification. We also provide a comparison of four different sample preparation methods. In the first step of the study, 100 Gram-negative bacteria, 100 Gram-positive bacteria, 20 anaerobic bacteria and 20 yeasts were spotted on the MALDI target using manual deposition, semi-extraction, wet deposition onto 70% formic acid and by automatic deposition using MALDI Colonyst. The lowest scores were obtained by manual toothpick spotting which significantly differ from other methods. Identification score of semi-extraction, wet deposition and automatic wet deposition did not significantly differ using calculated relative standard deviation (RSD). Nevertheless, the best results with low error rate have been observed using MALDI Colonyst robot. The second step of validation included processing of 542 clinical isolates in routine microbiological laboratory by a toothpick direct spotting, on-plate formic acid extraction (for yeasts) and automatic deposition using MALDI Colonyst. Validation in routine laboratory process showed significantly higher identification scores obtained using automated process compared with standard manual deposition in all tested microbial groups (Gram-positive, Gram-negative, anaerobes, and yeasts). As shown by our data, automatic colony deposition on MALDI target results in an increase of MALDI-TOF MS identification scores and reproducibility.

Molecular Characterization of Carbapenemase-Producing Pseudomonas aeruginosa of Czech Origin and Evidence for Clonal Spread of Extensively Resistant Sequence Type 357 Expressing IMP-7 Metallo-ß-Lactamase.

The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-ß-lactamases (MßLs), 15 produced the VIM-2 MßL, and the remaining isolates expressed the GES-5 enzyme. The blaIMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. blaVIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). blaGES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.

Comparison of imipenem and meropenem antibiotics for the MALDI-TOF MS detection of carbapenemase activity.

A comparison of carbapenem molecules for the detection of carbapenemase-producing bacteria by MALDI-TOF MS showed that imipenem exhibited higher sensitivity (97%) and specificity (100%) scores for Pseudomonas aeruginosa than meropenem. However, meropenem was more efficient (98% sensitivity and 100% specificity) against Enterobacteriaceae.

Molecular Characterization of OXA-48-Like-Producing Enterobacteriaceae in the Czech Republic and Evidence for Horizontal Transfer of pOXA-48-Like Plasmids.

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.

Characterisation of IncA/C2 plasmids carrying an In416-like integron with the blaVIM-19 gene from Klebsiella pneumoniae ST383 of Greek origin.

The complete nucleotide sequences of three multidrug resistance (MDR) IncA/C-like plasmids from Enterobacteriaceae isolates carrying the VIM-type carbapenemase-encoding integrons In4863 (blaVIM-19-aacA7-dfrA1-ΔaadA1-smr2) or In4873 (blaVIM-1-aacA7-dfrA1-ΔaadA1-smr2) were determined, which are the first In416-like elements identified in Greece. Plasmids pKP-Gr642 and pKP-Gr8143 were from Klebsiella pneumoniae ST383 isolates, whereas plasmid pEcl-Gr4873 was from an Enterobacter cloacae ST88 isolate. Sequencing showed that pKP-Gr642 (162787bp) and pKP-Gr8143 (154395bp) consisted of the type 1 IncA/C2 conserved backbone, the blaCMY-2-like gene-containing region, and the ARI-B (with the sul2 gene) and ARI-A (with a class 1 integron) resistance islands, like the plasmid pUMNK88_161 from the USA. The third plasmid, pEcl-Gr4873 (153958bp), exhibited extensive similarity with the type 2 IncA/C2 plasmid pR55 from France. pEcl-Gr4873 carried only one resistance island of a hybrid transposon structure inserted in a different location to ARI-A in type 1 A/C2 plasmids. In all three plasmids, the In416-like integrons In4863 or In4873 were identified within non-identical class II transposon structures. All three In416-like-carrying regions presented significant similarities with the MDR region of the IncA/C2 plasmid pCC416 from Italy, carrying the prototype In416 integron (blaVIM-4-aacA7-dfrA1-ΔaadA1-smr2). These findings provided the basis for speculations regarding the evolution of IncA/C2 plasmids with In416-like integrons, and confirmed the rapid evolution of some IncA/C2 plasmid lineages. Considering the broad host range of IncA/C2 molecules, it seems that pKP-Gr642, pKP-Gr8143 and pEcl-Gr4873 plasmids might support the diffusion of In416-like integrons among Enterobacteriaceae.