Genetic typing of Enterococcus faecium VRE strains isolated in three hospitals in Warsaw and Siedlce in 2015-2016

INTRODUCTION: Since the first report of vancomycin-resistant enterococci (VRE) in Poland, in 1996, these strains have spread in Polish hospitals, mainly due to selective pressure associated with increased use of vancomycin in the treatment of infections caused by methicillin-resistant staphylococci and Clostridium difficile. At the beginning of 2016 a growing number of patients colonized with VRE in the gastrointestinal tract was observed in the Children's Memorial Health Institute (IPCZD). Some of these patients were transferred from other hospitals, and VRE colonization was found on admission. AIM: To analyze genetic similarity of VRE strains isolated from patients hospitalized in IPCZD and two other hospitals in Mazovian district, genetic typing by pulsed field gel electrophoresis (PFGE) was performed. MATERIALS AND METHODS: VRE strains were isolated from rectal swabs, and other clinical samples such as blood, cerebrospinal fluid, other body fluids, and environmental samples. A total of 56 VRE strains from IPCZD, 20 strains from Siedlce and 4 strains from patients from Grochowski Hospital in Warsaw were typed by PFGE. RESULTS: PFGE typing revealed 4 VRE clones containing several strains with identical restriction patterns. Among VRE strains isolated from neonates hospitalized in IPCZD, two clones with 24 and 20 identical strains were found. Respectively, 16 (67%) and 12 (60%) isolates were originated from rectal swabs from patients at admission to the hospital. Clonal strains were identified in all three hospitals included in the study. CONCLUSIONS: Our results showed that VRE strains had spread in the region. Isolation of clonal strains on admission to the hospital suggested independent VRE introductions from environment or other hospitals. Identification of clonal strains obtained from rectal swabs and other clinical samples during hospitalization indicated horizontal transmission.

Emergence of macrolide-resistant Campylobacter strains in chicken meat in Poland and the resistance mechanisms involved.

In this study, we investigated the molecular mechanisms involved in erythromycin resistance in the first resistant Campylobacter strains isolated from chicken meat in Poland, and analyzed their genetic relatedness. A total of 297 samples of raw chicken meat and giblets from retail trade in the Warsaw area collected between 2006 and 2009 were examined. Among 211 Campylobacter strains (52 C. jejuni and 159 C. coli), 10 C. coli isolates (4.7%) were resistant to erythromycin. All the C. jejuni strains were susceptible. Among the high-level macrolide-resistant isolates, two different point mutations within the domain V of the 23S rRNA gene were observed. Eight of the strains had adenine→guanine transitions at position 2075, two other isolates at position 2074. Sequence analysis of ribosomal proteins L4 (rplD) and L22 (rplV) indicated that ribosomal protein modifications did not contribute to macrolide resistance. A mutation in the inverted repeat in the cmeR and cmeABC intergenic region was found in a single resistant strain. The genetic relatedness of Campylobacter isolates showed that two resistant strains obtained from the same production plant in a 2-month interval were genetically identical. The risk of transmission of resistant strains via the food chain highlights the need for constant monitoring of resistance in Campylobacter isolates of human and animal hosts.

[Epidemiologic analysis of increased frequency of isolation of multidrug-resistant Acinetobacter sp. using PFGE technique]. / Analiza epidemiologiczna zwiekszonej czestosci izolacji wielolekoopornych Acinetobacter sp z zastosowaniem techniki PFGE.

From November 2007 to November 2008, 25 multidrug resistant Acinetobacter baumanii/haemolyticus strains were isolated from 21 patients treated in the Pulmonary Diseases and Tuberculosis Hospital in Bystra. Seven cases were regarded as nosocomial infections. Most of the patients were hospitalized several times and/or admitted from other hospitals. The aim of this study was to find the reason for increasing colonization and infections with this microorganism. PFGE analysis showed high, exceeding 90% relationship among tested strains suggesting their clonal spread among patients in several hospitals in our region. Despite excessive search, no environmental reservoir of Acinetobacter was found, so cross-contamination via staff's hands was suspected the most likely source of this spread. Therefore, hand hygiene and contact isolation seemed the most relevant infection control measures.

[Phenotype and genotype characterization of Candida albicans strains isolated from patients hospitalized at the Children's Memorial Health Institute]. / Fenotypowa i genotypowa charakterystyka szczepów Candida albicans izolowanych od pacjentów hospitalizowanych w instytucie "Pomnik-Centrum Zdrowia Dziecka".

Phenotype and genotype analysis was prepared from Candida strains causing colonization and fungal infections in children hospitalized at The Children's Memorial Health Institute intensive care unit and oncology unit. We totally analyzed 117 C. albicans strains cultured from different clinical specimens received from 51 patients--oncology unit (26) and intensive care unit (25). Enzymatic activity was assessed by API ZYM bioMerieux. Strains were biotyped according to Williamson, Kurnatowska and Kurnatowski classification. Candida albicans isolates were RAPD typed. C. albicans strains causing colonization and fungal infections on those units released such enzymes like esterase lipasis, waline arylamidasis and phosphohydrolasis. The most frequent biotypes in group of patients oncological unit with fungal infections were biotype E (64.7%) and biotype C (26.6%) while in group of patients with colonized mucosa dominated biotypes: E (28.6%) and A (28.5%). Comparing with intensive care unit group patients, the most frequent biotype isolated among patients with fungal infection symptoms was biotype E (71.7%), while in mucosa colonization group C (35.5%) occurred as a dominating biotype. This study demonstrated clonal character of fungal infections among patients hospitalized in two units and considerable polymorphism among yeast strains: 23 pattern stripes in oncology unit and 28 pattern stripes in intensive care unit. What is more, yeast biotype determination allows showing frequent occurrence of virulence character of isolates received from infected patients in comparison with those received from colonized patients.

Distribution of carbapenem resistance mechanisms in Pseudomonas aeruginosa isolates among hospitalised children in Poland: Characterisation of two novel insertion sequences disrupting the oprD gene.

This study aimed to analyse the distribution of carbapenem resistance mechanisms among Pseudomonas aeruginosa clinical isolates. Fifty-five P. aeruginosa isolates, resistant both to imipenem and meropenem, from children hospitalised in 2009-2010 were studied. All strains were genotyped by pulsed-field gel electrophoresis (PFGE). Mutations in the oprD gene and the occurrence of insertion sequences (ISs) were determined by DNA sequencing. Mex efflux systems were determined by analysis using the efflux pump inhibitor Phe-Arg ß-naphthylamide. Metallo-ß-lactamase (MBL) production was determined with Etest MBL strips and PCR for blaVIM and blaIMP. PFGE show high genetic diversity among the isolates. Mutations inactivating the oprD gene were detected in 44 strains (80%). Frameshift mutations detected in 20 isolates were the most common cause of inactivation of the oprD gene. Point mutations leading to premature stop codons were found in 12 isolates, and various substitutions were found in 6 isolates. Disruption of the coding sequence of oprD by ISs was found in six isolates. Two novel ISs (ISPa51 and ISPa52) were detected. Increased activity of different Mex systems was observed in 27 isolates (49%). Ten isolates simultaneously overexpressed two (n=3) or three (n=7) types of Mex efflux system. Seven (13%) P. aeruginosa strains were found to have minimum inhibitory concentrations (MICs) of >64mg/L both for imipenem and meropenem (two VIM-4, four VIM-2 and one IMP-1). These results show a significant diversity of P. aeruginosa strategies for resistance development. Noteworthy, a variety of ISs were found to disrupt the oprD gene.

Molecular Epidemiology and Drug Resistance of Acinetobacter baumannii Isolated from Hospitals in Southern Poland: ICU as a Risk Factor for XDR Strains.

The objectives of the present study were to investigate the carbapenemase and metallo-beta-lactamase genes of Acinetobacter baumannii clinical isolates by polymerase chain reaction (PCR) and real time PCR and to determine the molecular epidemiology of the strains using the DiversiLab tool. From these data, correlations between drug resistance, resistance genes, and epidemiological clones may be revealed. The study was conducted on 125 A. baumannii collected over the 2013 year. The majority of the isolates from both intensive care unit (ICU) and non-ICU cases originated from pneumonia infections (79.2%), isolates from blood infections accounted for 17.6% and 3.2% were from meningitis infections. In the ICU cases compared with the non-ICU cases, bloodstream infections were more frequently diagnosed (19.2% vs. 11.5%). Sixty percent of A. baumannii strains were resistant to all the antimicrobials tested with the exception of colistin. All strains were susceptible to colistin and polymyxin B. Extensively drug-resistant (XDR) strains accounted for 80.8% of the isolates tested and these XDR strains were more frequently isolated from ICU cases than from non-ICU cases (93.9% vs. 30.8%). Among the 101 isolates of A. baumannii exhibiting the XDR pattern of resistance, 80 possessed the blaOXA-24 gene and 29 had the blaOXA-23 gene. Only two isolates possessed the blaVIM gene. The presence of the ISAba1element was confirmed among 10 strains from patients hospitalized in the ICU. Using repetitive extragenic palindromic sequence PCR (DiversiLab typing), six clones and 12 unique strains were identified, of which two clones dominated. Most isolates belonging to clone 1 (66.7%) and clone 2 (85.5%) were susceptible only to colistin. In summary, it is clear from our findings and those of other studies that carbapenem resistance among A. baumannii strains presents a serious clinical problem worldwide. Furthermore, the presence of XDR international clone II in ICUs poses a potential risk for future outbreaks of A. baumannii infection and controlling A. baumannii infections in hospitals presents a serious challenge.

Carriage of genes for various extended-spectrum beta-lactamases: a novel resistance strategy of Klebsiella pneumoniae in Poland.

Among 110 randomly sampled strains from a collection of 247 extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae collected from hospitalised children in three paediatric hospitals in Poland, 64 strains (58.2%) with multiple ESBLs were found, including five non-clonal strains (4.5%) harbouring bla genes for ESBLs of three families (CTX-M, SHV and TEM). This is the first report of the emergence of triple ESBL-producing K. pneumoniae in Poland. In addition, K. pneumoniae strains harbouring bla genes for TEM-130 and TEM-132 ESBLs were detected in Poland for the first time. Epidemiological analysis of the multiple ESBL-producing K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) revealed a relatively high genetic diversity between isolates producing the same combination of enzymes. Clonally related strains were uncommon.

[Antimicrobial drug resistant pathogens in nosocomial urinary tract infections--selected aspects]. / Lekooporne drobnoustroje w szpitalnych zakazeniach dróg moczowych--wybrane aspekty.

The article discusses the etiology of hospital urinary tract infections focusing on spread of antimicrobial-resistant pathogens. It also describes the most important biochemical and genetic mechanisms of resistance to beta-lactams, aminoglycosides and fluoroquinolones among hospital isolates.

Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001).

OBJECTIVES: During 1997-2001, 151 isolates of imipenem-resistant Pseudomonas aeruginosa were obtained from clinical specimens taken from children hospitalized in Warsaw, Poland. These strains were investigated further to determine the mechanism of resistance. METHODS: The strains were analysed by a combination of genotyping and PCR-based strategies. RESULTS: Eleven of these strains were found to contain the metallo-beta-lactamase (M beta L) gene bla(VIM-4). The first strain appeared in 1998, and P. aeruginosa strains harbouring this M beta L have become endemic in this hospital since then. All P. aeruginosa strains belonged to serotype O:6, and PFGE analysis revealed four different patterns and three sub-types. All 11 M beta L-producing strains contained an identical class 1 integron with the usual 5' and 3' conserved sequences. The integron included two resistance cassettes, aacA4 in the first position and the bla(VIM-4) cassette in the second position. The bla(VIM-4) gene included an unusual direct repeat of 169 bp of the 3' portion of the bla(VIM-4) gene. CONCLUSIONS: An unusual bla(VIM-4) M beta L has become endemic in P. aeruginosa isolates infecting Polish children hospitalized on surgical wards. The formation of this unusual bla(VIM-4) gene cassette could be explained by a mechanism involving deletion of a segment of an ancestral tandem repeat of bla(VIM-4) via slipped strand replication, mediated by a combination of polymerase and integrase.