Genomic Epidemiology of C2/H30Rx and C1-M27 Subclades of Escherichia coli ST131 Isolates from Clinical Blood Samples in Hungary.

Extended-spectrum ß-lactamase-producing Escherichia coli ST131 has become widespread worldwide. This study aims to characterize the virulome, resistome, and population structure of E. coli ST131 isolates from clinical blood samples in Hungary. A total of 30 C2/H30Rx and 33 C1-M27 ST131 isolates were selected for Illumina MiSeq sequencing and 30 isolates for MinION sequencing, followed by hybrid de novo assembly. Five C2/H30Rx and one C1-M27 cluster were identified. C1-M27 isolates harbored the F1:A2:B20 plasmid in 93.9% of cases. Long-read sequencing revealed that blaCTX-M-27 was on plasmids. Among the C2/H30Rx isolates, only six isolates carried the C2-associated F2:A1:B- plasmid type. Of 19 hybrid-assembled C2/H30Rx genomes, the blaCTX-M-15 gene was located on plasmid only in one isolate, while in the other isolates, ISEcp1 or IS26-mediated chromosomal integration of blaCTX-M-15 was detected in unique variations. In one isolate a part of F2:A1:B- plasmid integrated into the chromosome. These results suggest that CTX-M-15-producing C2/H30Rx and CTX-M-27-producing C1-M27 subclades may have emerged and spread in different ways in Hungary. While blaCTX-M-27 was carried mainly on the C1/H30R-associated F1:A2:B20 plasmid, the IncF-like plasmids of C2/H30Rx or its composite transposons have been incorporated into the chromosome through convergent evolutionary processes.

Virulence Characteristics and Molecular Typing of Carbapenem-Resistant ST15 Klebsiella pneumoniae Clinical Isolates, Possessing the K24 Capsular Type.

Klebsiella pneumoniae is an opportunistic pathogen that frequently causes nosocomial and community-acquired (CA) infections. Until now, a limited number of studies has been focused on the analyses of changes affecting the virulence attributes. Genotypic and phenotypic methods were used to characterise the 39 clinical K. pneumoniae isolates; all belonged to the pan-drug resistant, widespread clone ST 15 and expressed the K24 capsule. PFGE has revealed that the isolates could be divided into three distinct genomic clusters. All isolates possessed allS and uge genes, known to contribute to the virulence of K. pneumoniae and 10.25% of the isolates showed hypermucoviscosity, 94.87% produced type 1 fimbriae, 92.3% produced type 3 fimbriae, and 92.3% were able to produce biofilm. In vivo persistence could be supported by serum resistance 46.15%, enterobactin (94.87%) and aerobactin (5.12%) production and invasion of the INT407 and T24 cell lines. Sequence analysis of the whole genomes of the four representative strains 11/3, 50/1, 53/2 and 53/3 has revealed high sequence homology to the reference K. pneumoniae strain HS11286. Our results represent the divergence of virulence attributes among the isolates derived from a common ancestor clone ST 15, in an evolutionary process that occurred both in the hospital and in the community.

Comparison of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates From Rooks (Corvus frugilegus) and Contemporary Human-Derived Strains: A One Health Perspective.

During winter, a large number of rooks gather and defecate at the park of a university clinic. We investigated the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in these birds and compared recovered isolates with contemporary human isolates. In 2016, fecal samples were collected from 112 trap-captured rooks and investigated for presence of ESBL producers using eosin methylene blue agar supplemented by 2 mg/L cefotaxime; 2,455 contemporary human fecal samples of patients of the clinics sent for routine culturing were tested similarly. In addition, 42 ESBL-producing E. coli isolates collected during the same period from inpatients were also studied. ESBL genes were sought for by PCR and were characterized by sequencing; E. coli ST131 clones were identified. Epidemiological relatedness was determined by pulsed-field gel electrophoresis and confirmed using whole genome sequencing in selected cases. Thirty-seven (33%) of sampled rooks and 42 (1.7%) of human stools yielded ESBL-producing E coli. Dominant genes were bla CTX-M-55 and bla CTX-M-27 in corvid, bla CTX-M-15 and bla CTX-M-27 in human isolates. ST162 was common among rooks. Two rook-derived E. coli belonged to ST131 C1-M27, which was also predominant (10/42) among human fecal and (15/42) human clinical isolates. Another potential link between rooks and humans was a single ST744 rook isolate grouped with one human fecal and three clinical isolates. Despite possible contact, genotypes shared between rooks and humans were rare. Thus, rooks are important as long-distance vectors and reservoirs of ESBL-producing E. coli rather than direct sources of infections to humans in our setting.

New geographical area on the map of Crimean-Congo hemorrhagic fever virus: First serological evidence in the Hungarian population.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease that is endemic in Africa, Asia, the Middle East, and the Balkan region of Europe; the disease is spreading northwards following widespread distribution of the main vector, Hyalomma marginatum, which was first found in Hungary in 2011. The aim of this pilot sero-surveillance study was to assess CCHF seroprevalence in Hungary. A total of 2700 serum samples obtained from healthy volunteer blood donors were screened using an in-house immunofluorescence assay and a commercially available ELISA kit. We found ten (0.37 %) seropositive donors. The western and central regions proved to be the most affected areas, with a prevalence of 2.97 %. Higher positivity was found among male donors (0.55 %) and younger donors (18-34 years; 0.78 %). Based on these results, a more extended surveillance focusing on specific at-risk populations and animals is advised. The results should also raise the awareness of clinicians and other high-risk populations, such as foresters and hunters, about the emerging threat of CCHF in Hungary.

Characterization of a rare bla VIM-4 metallo-ß-lactamase-producing Serratia marcescens clinical isolate in Hungary.

A carbapenem-resistant S. marcescens isolate was recovered from a patient with an inflammed pacemaker inplantation pocket from a Cardiac Surgery ward in a Hungarian University Hospital. Phenotypic tests and polymerase chain reaction (PCR) confirmed a very rare gene responsible for production of a carbapenemase ( bla VIM-4 ), which was further characterized by Sanger-sequencing. The characterization of this S. marcescens strain emphasizes the ongoing emergence of novel or rare carbapenemases. Strains expressing a weak carbapenemase like this strain might go unrecognized by routine diagnostics due to low minimum inhibitory concentrations (MICs) for the bacterial strains producing such enzymes.

Multiple Benefits of Plasmid-Mediated Quinolone Resistance Determinants in Klebsiella pneumoniae ST11 High-Risk Clone and Recently Emerging ST307 Clone.

International high-risk clones of Klebsiella pneumoniae are among the most common nosocomial pathogens. Increased diversity of plasmid-encoded antimicrobial resistance genes facilitates spread of these clones causing significant therapeutic difficulties. The purpose of our study was to investigate fluoroquinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing strains, including four K. pneumoniae and a single K. oxytoca, isolated from blood cultures in Hungary. Whole-genome sequencing and molecular typing including multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed in selected strains. Gene expression of plasmid-mediated quinolone resistance determinants (PMQR) was investigated by quantitative-PCR. MLST revealed that three K. pneumoniae strains belonged to ST11 and one to ST307 whereas K. oxytoca belonged to ST52. The isolates harbored different ß-lactamase genes, however, all K. pneumoniae uniformly carried bla CTX-M-15. The K. pneumoniae isolates exhibited resistance to fluoroquinolones and carried various PMQR genes namely, two ST11 strains harbored qnrB4, the ST307 strain harbored qnrB1 and all K. pneumoniae harbored oqxAB efflux pump. Levofloxacin and moxifloxacin MIC values of K. pneumoniae ST11 and ST307 clones correlated with qnr and oqxAB expression levels. The qnrA1 carrying K. oxytoca ST52 exhibited reduced susceptibility to fluoroquinolones. The maintained expression of qnr genes in parallel with chromosomal mutations indicate an additional protective role of Qnr proteins that can support dissemination of high-risk clones. During development of high-level fluoroquinolone resistance, high-risk clones retain fitness thus, enabling them for dissemination in hospital environment. Based on our knowledge this is the first report of ST307 clone in Hungary, that is emerging as a potential high-risk clone worldwide. High-level fluoroquinolone resistance in parallel with upregulated PMQR gene expression are linked to high-risk K. pneumoniae clones.

Successful elimination of extended-spectrum beta-lactamase (ESBL)-producing nosocomial bacteria at a neonatal intensive care unit.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are highly dangerous to neonates. At our Neonatal Intensive Care Unit (NICU), the presence of these bacteria became so threatening in 2011 that immediate intervention was required. METHODS: This study was conducted during a nearly two-year period consisting of three phases: retrospective (9 months), educational (3 months) and prospective (9 months). Based on retrospective data analysis, a complex management plan was devised involving the introduction of the INSURE protocol, changes to the antibiotic regimen, microbiological screening at short intervals, progressive feeding, a safer bathing protocol, staff hand hygiene training and continuous monitoring of the number of newly infected and newly colonized patients. During these intervals, a total of 355 patients were monitored. RESULTS: Both ESBL-producing Enterobacter cloaceae and Klebsiella pneumoniae were found (in both patients and environmental samples). In the prospective period a significant reduction could be seen in the average number of both colonized (26/167 patients; P=0.029) and infected (3/167 patients; P=0.033) patients compared to data from the retrospective period regarding colonized (72/188 patients) and infected (9/188 patients) patients. There was a decrease in the average number of patient-days (from 343.72 to 292.44 days per months), though this difference is not significant (P=0.058). During the prospective period, indirect hand hygiene compliance showed a significant increase (from the previous 26.02 to 33.6 hand hygiene procedures per patient per hospital day, P<0.001). CONCLUSION: Colonizations and infections were rolled back successfully in a multi-step effort that required an interdisciplinary approach.

Countrywide dissemination of a DHA-1-type plasmid-mediated AmpC ß-lactamase-producing Klebsiella pneumoniae ST11 international high-risk clone in Hungary, 2009-2013.

The first plasmid-mediated AmpC ß-lactamase-producing Klebsiella pneumoniae (pAmpC KP) isolate was detected in December 2009 in Hungary. Hungarian microbiological laboratories were asked to send all KP strains showing cefoxitin resistance and decreased susceptibility or resistance to any third-generation cephalosporins to the Reference Laboratories at the National Center for Epidemiology. Investigation was conducted in order to outline spatio-temporal distribution and genetic characterization of pAmpC-KP isolates in Hungary. Between December 2009 and December 2013, 312 consecutive KP clinical isolates were confirmed as producing pAmpCs. All isolates showed resistance to third-generation cephalosporins, aminoglycosides and fluoroquinolones, and 77 % were non-susceptible to at least one carbapenem. Analysis of ß-lactamase genes showed blaDHA-1 in all and additionally blaCTX-M-15 in 90 % of isolates. PFGE typing revealed 12 pulsotypes; of these, KP053 (262/312) and KP070 (38/312) belonged to sequence type ST11 and comprised 96 % of the isolates. The blaDHA-1 and blaCTX-M-15 co-producing KP053/ST11 clone affected 234 patients and spread to 55 healthcare centres across Hungary during the study period. Three KP053 isolates were also resistant to colistin. In two of these, the mgrB gene was truncated by IS10R, while in the third isolate, insertional inactivation of mgrB by ISKPn14 was identified. Hungary is the first European country showing endemic spread of blaDHA-1 facilitated by the international high-risk clone ST11. The rapid countrywide spread of this multidrug-resistant clone seriously endangers Hungarian healthcare facilities and warrants strengthening of infection control practices and prudent use of carbapenems and colistin.

Emergence of OXA-162-producing Klebsiella pneumoniae in Hungary.

In August 2012, 2 carbapenem-resistant Klebsiella pneumoniae isolates from the University of Szeged were submitted to the National Reference Laboratory at the National Centre for Epidemiology to confirm the carbapenem resistance mechanism. PCR assays and sequencing revealed that the isolates harboured the blaOXA-162 carbapenemase gene, a very recently described variant of OXA-48, and the blaCTX-M-15 extended-spectrum ß-lactamase gene. The isolates had indistinguishable PFGE patterns and belonged to sequence type ST15. To the best of our knowledge, this is the first description of OXA-48-like carbapenemase-producing bacteria in Hungary and of an OXA-162-type carbapenemase gene in the K. pneumoniae ST15 clone.

First description of bla(NDM-1), bla(OXA-48), bla(OXA-181) producing Enterobacteriaceae strains in Romania.

We report the first isolation and characterization of several Enterobacteriaceae strains harboring bla(NDM-1), bla(OXA-48) and/or bla(OXA-181) genes in a Romanian emergency teaching hospital. Between January 2010 and September 2012 nine carbapenemase-producing Enterobacteriaceae strains were identified. The bla(NDM-1) gene was present in two Enterobacter cloacae strains, an Escherichia coli and two Klebsiella pneumoniae strains. One of these K. pneumoniae strains also harbored the bla(OXA-181) gene. Three other K. pneumoniae strains and one Serratia marcescens carried bla(OXA-48).

Characterization of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli strains isolated from animal and human clinical samples in Hungary in 2006-2007.

The proportion of Escherichia coli non-susceptible to 3(rd) generation cephalosprins from invasive clinical samples has risen in Hungary from 5.1 per cent in 2006 to 15.5 per cent in 2011. The prevalence of ESBL-production in E. coli of animal origin remains unknown. During the first stage of a probe forty-five human and 18 animal ESBL-producing E. coli strains isolated in 2006-2007 were investigated. The human strains were representatively selected from a collection of 113 ESBL-producing isolates sent to the national reference center from local laboratories across the country. A variety of ESBLs were detected (SHV-2, -5, -12, CTX-M-32) with CTX-M-15 being the most common in human and CTX-M-1 the dominant in animal isolates. Genetic characterization revealed that thirty-six human isolates (80 per cent) belonged to either the phylogenetic group (PG) B2 or D. Conversely, 15 animal isolates (83 per cent) proved to be members of the A and B1 commensal PGs. Furthermore 46 per cent of human isolates (21/45) from 12 centres belonged to the international O25-ST131/B2 clone while nine isolates from seven centers showed the O15 serotype. Pulsed-field gel electrophoresis (PFGE) detected 22 and 11 diverse pulsotypes among 45 human and 18 animal isolates, respectively. The human and animal strains did not share any pulsotypes.

Synergistic antibiotic combinations for colistin-resistant Klebsiella pneumoniae.

In this study antibiotic combinations for multidrug-resistant Klebsiella pneumoniae strains were investigated. The study included a colistin-susceptible and a colistin-resistant KPC-2 producing K. pneumoniae ST258 strains isolated in 2008 and 2009 during an outbreak in Hungary. Antibiotic combinations were analyzed by checkerboard technique and fractional inhibitory concentration indices were calculated. The following antibiotics were tested: ceftazidime, cefotaxime, ceftriaxone, ampicillin, imipenem, ertapenem, amikacin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, polymyxin B and colistin. Combinations including 0.25 µg/ml colistin plus 1 µg/ml rifampicin, 0.25 µg/ml polymyxin B plus 1 µg/ml rifampicin, 1 µg/ml imipenem plus 2 µg/ml tobramycin, were found synergistic.These in vitro synergistic combinations suggest potential therapeutical options against infections caused by KPC-2 producing, multidrug-resistant K. pneumoniae ST258.

Emergence of VIM-4- and SHV-12-producing Enterobacter cloacae in a neonatal intensive care unit.

In order to reveal colonization with multidrug-resistant bacteria early, routine screening is done on samples of all patients of the neonatal intensive care units at Semmelweis University, Hungary. Due to the extended-spectrum ß-lactamase (ESBL) screening examinations, emergence of multidrug-resistant Enterobacter cloacae isolates was found with suspicion of clonal transmission, therefore active microbiological surveillance was initiated. The aim of our study was to characterize 60 E. cloacae isolates recovered in a 7-month period in 2010. MIC values of antibiotics were determined and ESBL and carbapenemase production was tested. Metallo-ß-lactamase (MBL) genes, ESBL genes, and class-1 integrons were characterized, and the possible clonal relationship between isolates was investigated. The isolates showed increased MIC values for carbapenems and cephalosporins. All 60 E. cloacae strains recovered from 16 neonates proved to be VIM-4 MBL producers. Fifty-three strains were SHV-12 ESBL producers also. In all cases, the bla(VIM-4) gene was a part of class-1 integron, In238a. XbaI-macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) revealed identical patterns for the isolates. Our study supports the importance of active microbiological surveillance as well as molecular epidemiology at the NICUs as a part of infection control.

Molecular epidemiology and antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli isolates of poultry, swine, and cattle origin collected from slaughterhouses in Hungary.

Campylobacter spp. are the most common cause of bacterial enteritis in Hungary, and the aim of this study was to identify the distribution, genotypes, and antimicrobial susceptibility of Campylobacter species in the most important food-producing animals at the time of slaughter during 2008 and 2009. Of 1,110 samples, 266 were identified as Campylobacter coli (23.9%) and 143 as C. jejuni (12.9%) by real-time PCR. Resistance to enrofloxacin-ciprofloxacin and nalidixic acid was significant, especially in C. jejuni (73.3%) and C. coli (77.2%) from broilers. Higher erythromycin (P = 0.043) and tetracycline (P = 1.865e-14) resistance rates were found among C. coli isolates (9.7 and 74.1%, respectively) than among C. jejuni isolates (3.1 and 36.6%, respectively). A total of 47 fla short variable region sequences were identified among 73 selected C. coli and C. jejuni isolates, with 35 fla types detected only once. At the nucleotide level, fla types A66 and A21 were the most common. Using the pulsed-field gel electrophoresis method, 66% of strains exhibited unique profiles after Sma I digestion. Forty-two isolates assigned to 18 Sma I clusters were further typed by Kpn I, and of these, 24 were assigned to 10 Kpn I clusters. For isolates in five Kpn I clusters, epidemiological links were observed. Stable C. jejuni and C. coli clones were detected, indicating that further studies involving broiler and human isolates need to be conducted to elucidate the importance of these stable clones in human infections.

Molecular characterization of Campylobacter lanienae strains isolated from food-producing animals.

During 2008 and 2009, within the framework of the Hungarian monitoring program of antibiotic resistance of zoonotic agents from food-producing animals, a significant number (43 strains) of Campylobacter lanienae were detected for the first time in Hungary. The isolates were genotyped using partial 16S rRNA gene sequencing and pulsed-field gel electrophoresis using three different restriction enzymes. The antimicrobial resistance of the isolates was determined by microtiter broth dilution. C. lanienae isolation was successful only from swine but not from other animal species. According to phylogenetic analysis, clustering of the isolates shows the same extensive genetic diversity as other Campylobacter species. Sequence analysis of the partial 16S rRNA gene showed that additional variations exist in variable regions Vc2 and Vc6. SmaI restriction enzyme proved to be the most efficient for pulsed-field gel electrophoresis analysis of C. lanienae. A significant tetracycline resistance (60.9%) and the presence of erythromycin-, enrofloxacin-, and multiresistant C. lanienae strains were found. Although the pathogenic potential of C. lanienae in humans is currently unknown, this study demonstrates that C. lanieanae is common in pigs in the country, provides further details on the genotypic and phenotypic properties of C. lanienae, and offers a genotyping method for use in source tracing.

Investigation of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae outbreaks in Hungary between 2005 and 2008.

Fourteen outbreaks in Hungary between 2005 and 2008 caused by extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) were epidemiologically investigated and the isolated pathogens were characterized by molecular techniques. Ten of the fourteen outbreaks occurred in adult wards and four in neonatal units affecting a total number of 73 patients. The 54% [40] of the patients developed bloodstream infections and 21.9%-21.9% [16] pneumonia and surgical site infections, respectively. The overall rate of mortality proved high: 36.9% [27]. Outbreaks in adults affected more patients, had higher attack rates, were more prolonged in duration and had a 6.9-fold higher mortality rate than outbreaks observed in neonates. The outbreaks in neonates were caused by SHV-type ESBL-producing klebsiellae, while in the "adult outbreaks" exclusively CTX-M-type ESBL-KP strains were involved. While the outbreak strains isolated from neonatal units could be assigned to a variety of pulsotypes, the previously described K. pneumoniae epidemic clones, ST15 and ST147, could be identified among the pathogens causing outbreaks in adult units.

Virulence genes and molecular typing of different groups of Escherichia coli O157 strains in cattle.

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.

Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type beta-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005--the new 'MRSAs'?

OBJECTIVES: To investigate the molecular epidemiology of ciprofloxacin-resistant CTX-M-15-producing Klebsiella pneumoniae epidemic clones (ECs) isolated from six nosocomial outbreaks and sporadic cases during 2005 in Hungary. METHODS: Two hundred and eighty-one extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae clinical isolates collected from 41 centres were submitted to the National ESBL Reference Laboratory for further investigations. Of the 281 strains, 75 isolates proved to be SHV producers, whereas 6 isolates were ciprofloxacin-susceptible CTX-M-type ESBL producers. One hundred and ninety-six ciprofloxacin-resistant CTX-M-type beta-lactamase-producing isolates collected from 35 centres were subjected to macrorestriction profile analysis. Furthermore, molecular typing was performed by PCR and sequencing of several antibiotic resistance genes, plasmid profile analysis, transfer of resistance determinants and multilocus sequence typing (MLST). RESULTS: PFGE revealed the existence of three genetic clusters defined as ECs, where 129 isolates belonged to the previously described Hungarian EC (HEC), 46 isolates to epidemic clone II (EC II) and 21 isolates to epidemic clone III (EC III), respectively. All isolates harboured plasmids ranging from 2.0 to 230 kb. PstI digestion of plasmid DNA from transconjugants/transformants revealed diverse restriction patterns from distinct ECs. Sequence analysis of beta-lactamase genes from 19 selected isolates detected bla(CTX-M-15) and bla(OXA-1) in strains from all three ECs and bla(TEM-1) in EC III isolates located on large plasmids. ISEcpI associated with CTX-M-15 was detected only on a 50 kb non-conjugative plasmid from EC III. MLST identified three allelic profiles: ST 15 (HEC), ST 11 (EC III) and the novel ST 147 (EC II), which correspond to the PFGE clusters, respectively. CONCLUSIONS: In 2005, 97% of all CTX-M-producing K. pneumoniae isolates detected across Hungary were highly ciprofloxacin-resistant CTX-M-15 producers and represented just three stable genetic clones.