Matej Medvecky

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.

Housing Systems Influence Gut Microbiota Composition of Sows but Not of Their Piglets.

Different housing systems can be used in pig production and little is known about their effect on gut microbiota composition. In this study we characterized fecal microbiota by sequencing the rRNA genes in sows kept during gestation in conventional pens with a slatted floor and in enriched pens with a floor covered with deep straw. After farrowing, microbiota of 1- and 4-day-old piglets were also monitored. Microbiota of sows from the enriched system contained significantly more Prevotella, Parabacteroides, CF231, Phascolarctobacterium, Fibrobacter, Anaerovibrio and YRC22 and significantly less Lactobacillus, Bulleidia, Lachnospira, Dorea, Ruminococcus and Oscillospira than microbiota of sows from the conventional system. The Firmicutes to Bacteroidetes ratio was 0.96 in the microbiota of sows kept in the enriched pens and this increased to 1.66 in the microbiota of sows kept in the conventional system. The production system therefore influenced microbiota composition, most likely due the ingestion of the straw. The microbiota of 1- and 4-day-old piglets differed from the microbiota of sows and sows therefore did not represent the most important source for their colonization in early days of life.

Characterization of the Complete Nucleotide Sequences of IncA/C 2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin

ABSTRACT A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C2 plasmids. pEc158 carried none of the blaCMY-2-like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn6317-like module or a Tn21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C2 plasmid lineages.

Antibiotic Resistance, Core-Genome and Protein Expression in IncHI1 Plasmids in Salmonella Typhimurium.

Conjugative plasmids from the IncHI1 incompatibility group play an important role in transferring antibiotic resistance in Salmonella Typhimurium. However, knowledge of their genome structure or gene expression is limited. In this study, we determined the complete nucleotide sequences of four IncHI1 plasmids transferring resistance to antibiotics by two different next generation sequencing protocols and protein expression by mass spectrometry. Sequence data including additional 11 IncHI1 plasmids from GenBank were used for the definition of the IncHI1 plasmid core-genome and pan-genome. The core-genome consisted of approximately 123 kbp and 122 genes while the total pan-genome represented approximately 600 kbp. When the core-genome sequences were used for multiple alignments, the 15 tested IncHI1 plasmids were separated into two main lineages. GC content in core-genome genes was around 46% and 50% in accessory genome genes. A multidrug resistance region present in all 4 sequenced plasmids extended over 20 kbp and, except for tet(B), the genes responsible for antibiotic resistance were those with the highest GC content. IncHI1 plasmids therefore represent replicons that evolved in low GC content bacteria. From their original host, they spread to Salmonella and during this spread these plasmids acquired multiple accessory genes including those coding for antibiotic resistance. Antibiotic-resistance genes belonged to genes with the highest level of expression and were constitutively expressed even in the absence of antibiotics. This is the likely mechanism that facilitates host cell survival when antibiotics suddenly emerge in the environment.

Transient and Prolonged Response of Chicken Cecum Mucosa to Colonization with Different Gut Microbiota.

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.

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-β-Lactamase

ABSTRACT 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-β-lactamases (MβLs), 15 produced the VIM-2 MβL, 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.

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).

First description in Czech Republic of emergence of an Enterobacter asburiae producing an IMI-2 carbapenemase

The acquired class A carbapenemase IMI-1 was originally described in an Enterobacter cloacae isolated in a Californian hospital in 1984 [1]. Since their first description, IMI-type carbapenemases have occasionally been detected in Enterobacteriaceae from the USA, Europe, the Far East and South Africa. Here we report the case of an IMI-2-producing Enterobacter asburiae identified in the Czech Republic. In 2016, E. asburiae Easb-36567cz was recovered from a patient admitted to a Czech hospital. Easb-36567cz was isolated from a rectal swab during routine screening for carbapenemase-producing Enterobacteriaceae (CPE). Easb-36567cz was resistant to aminopenicillins, aminopenicillin/sulbactam combinations, second-generation cephalosporins, aztreonam, carbapenems and colistin but was susceptible to piperacillin/tazobactam, cefotaxime, ceftazidime and various non-beta-lactam antibiotics (Table 1). Carbapenemase production was hypothesised by a positive result in the matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay [2]. Easb-36567cz tested negative by the ethylene diamine tetra-acetic acid (EDTA)–meropenem test, whilst the respective boronic acid–meropenem combined-disk test appeared positive indicating production of a class A carbapenemase. PCR and sequencing showed that Easb-36567cz carried blaIMI-2 [3].

First description in Europe of the emergence of Enterococcus faecium ST117 carrying both vanA and vanB genes, isolated in Greece

OBJECTIVES An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. METHODS Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. RESULTS E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. CONCLUSIONS This is the first detection in Greece of vanA-vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.

Complete Nucleotide Sequences of Two VIM-1-Encoding Plasmids from Klebsiella pneumoniae and Leclercia adecarboxylata Isolates of Czech Origin

ABSTRACT Two multidrug resistance (MDR) plasmids, carrying the VIM-1-encoding integron In110, were characterized. Plasmid pLec-476cz (311,758 bp), from a Leclercia adecarboxylata isolate, consisted of an IncHI1 backbone, a MDR region, and two accessory elements. Plasmid pKpn-431cz (142,876 bp), from a sequence type 323 (ST323) Klebsiella pneumoniae isolate, comprised IncFIIY-derived and pKPN3-like sequences and a mosaic region. A 40,400-bp sequence of pKpn-431cz was identical to the MDR region of pLec-476cz, indicating the en bloc acquisition of the VIM-1-encoding region from one plasmid by the other.