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  • Author or Editor: Judit Pászti x
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We report an investigation on a collection of multidrug-resistant Enterobacter cloacae isolates from a Hungarian neonatal intensive care unit. All the isolates (n=142) were examined by antimicrobial susceptibility testing and ERIC-PCR. The seven ESBL positive isolates (derived from six patients) made up a separated group with regard to their patterns by antimicrobial susceptibility testing and ERIC-PCR and were further tested by class-1-integron PCR and plasmid electrophoresis. The ESBL isolates were found indistinguishable in each of these laboratory tests, one genetic clone were revealed in the background of ESBL cases by PFGE. The ESBL positive isolates were proven to harbour a ∼62 Md plasmid and two class-1 integrons (0.9 kb, 1.875 kb). With respect to their clinical relatedness and our laboratory findings there was a small outbreak caused by the ESBL clone. PCR-sequencing were performed on the outbreak strain and has revealed a bla SHV gene that encodes for an SHV-2a type ESBL enzyme. This is the first description of SHV-2a positive E. cloacae strains isolated in Hungary.

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Eighty isolates of Listeria monocytogenes cultured from human infections in Hungary between 2004 and 2012 were serotyped by the PCR technique of Doumith et al. [9] and characterised by pulsed-field gel electrophoresis (PFGE). Most of the isolates belonged to two serogroups: 53 isolates (66.3%) to serovar group 4b,4d,4e and 21 isolates (25.8%) to serogroup 1/2a,3a. Although many pulsotypes were identified a particular pulsotype proved highly excelling comprising of 31 isolates after digestion by both ApaI and AscI restriction enzymes. All strains from this pulsotype belonged to serovar group 4b,4d,4e. Interestingly 24% of isolates from invasive samples (cerebrospinal fluid, blood) belonged to two distinct pulsotypes in the less common serovar group 1/2a,3a. Several small clusters of cases caused by isolates with identical pulsotypes were identified.

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The integron content of 52 DT104/U302 phage type strains and 53 non-DT104/U302 strains of Salmonella enterica serotype Typhimurium (S. Typhimurium) was studied in PCR experiments using a 5'-CS/3'-CS primer pair (Lévesque et al., 1995). Forty-three out of 44 streptomycin- and/or ampicillin-resistant DT104 and related phage type strains were found to carry a 1 kb and/or 1.2 kb long integron. The other resistance markers did not affect the number and size of integrons; no integron-free multidrug-resistant (MDR) DT104 strains were found. The two large groups of DT104 strains (Felix-Callow's phage types 2 and 2c) proved to be identical in respect of integron patterns (IPs), supporting the views of those authors who consider DT104 a single clone. Strains of human and animal origin did not differ from each other in their IPs. Within the non-DT104 phage types, ampicillin- and/or streptomycin-resistant, integron-free MDR strains were also found. Based on amplicons varying between 290 and 3500 bp an IP system was suggested. The commonest amplicon sizes in non-DT104 strains were 1450 and 2050 bp. The IPs of DT104 strains and of non-DT104 strains containing an integron of 1 and 1.2 kb size were stable. In contrast, the IPs of other non-DT104 strains showed a varying degree of instability. Integron loss was frequently associated with spontaneous plasmid elimination and changes of R-type among the descendants of a given strain.

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By PCR using the ant(3”)-Ia primer pair the aadA gene was detected in 34 streptomycin- and spectinomycin-resistant Salmonella enterica serotype Typhimurium strains. Out of them 12 belonged to DT104 and 22 to non-DT104 phage type. Using different primer combinations it was demonstrated that this gene was integron-associated in all cases: in the DT104 strains it was generally contained by a 1 kb integron while in the majority of the non-DT104 strains by a 2.05 kb (less often by a 1.9 or 1 kb) integron. In the case of integrons carrying multiple cassettes the cassette containing the aadA gene was located closer to the 3' end of the integron. The aadA genes of DT104 and non-DT104 strains were different: in the former group the aadA2 gene, while in the latter group (constituted by strains of five different phages types as well as unclassifiable and untypable strains) the aadA1 gene could be identified. The RH50/RH51 primer pair described by Collis and Hall (1992) proved to be suitable for rapid discrimination between the aadA1 and aadA2 genes on the basis that the RH51 primer bound exclusively to the aadA2 gene.

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An account is given on the activity of the National Center for Phage Typing of Staphylococci in Hungary in the period between 1997 and 2000 related to methicillin resistant Staphylococcus aureus (MRSA) strains originating mainly from hospital infections and sporadic cases. The rate of multiresistant MRSA strains has decreased gradually from 98.1% in 1997 to 74.6% in 2000, accordingly the typability by phages showed a considerable improvement by the international basic phages. Resistance pattern of MRSA strains became narrower in the period of the examinations. With the exception of erythromycin the rate of resistance decreased probably as a consequence of the increased use of erythomycin. The typing method was completed with the phenotypic and genotypic characterization of macrolide resistance. Among 73 MRSA strains type A was the most frequent macrolide resistance group, while type B, C1 and C2 occurred rarely. Type A was frequent also among the few MSSA and CNS strains. Out of the 168 examined S. aureus strains ermA genes occurred in 81.5%; in MSSA and CNS strains ermC1 genes were frequent, both genes are responsible for the target modification. The msrA gene, encoding the increased efflux, occurred only in CNS strains. Comparing the results obtained by phenotyping (phage typing) and genotyping (AP-PCR) methods it is of note that MRSA strains which proved non-typable by phage typing gave suitable results by the AP-PCR.

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An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight  (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.

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Enterotoxigenic E. coli (ETEC) bacteria frequently cause watery diarrhoea in newborn and weaned pigs. Plasmids carrying genes of different enterotoxins and fimbrial adhesins, as well as plasmids conferring antimicrobial resistance are of prime importance in the epidemiology and pathogenesis of ETEC. Recent studies have revealed the significance of the porcine ETEC plasmid pTC, carrying tetracycline resistance gene tet(B) with enterotoxin genes. In contrast, the role of tet(A) plasmids in transferring resistance of porcine ETEC is less understood. The objective of the present study was to provide a comparative analysis of antimicrobial resistance and virulence gene profiles of porcine post-weaning ETEC strains representing pork-producing areas in Central Europe and in the USA, with special attention to plasmids carrying the tet(A) gene. Antimicrobial resistance phenotypes and genotypes of 87 porcine ETEC strains isolated from cases of post-weaning diarrhoea in Austria, the Czech Republic, Hungary and the Midwest USA was determined by disk diffusion and by PCR. Central European strains carrying tet(A) or tet(B) were further subjected to molecular characterisation of their tet plasmids. Results indicated that > 90% of the ETEC strains shared a common multidrug resistant (MDR) pattern of sulphamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. Tetracycline resistance was most frequently determined by the tet(B) gene (38%), while tet(A) was identified in 26% of all isolates with wide ranges for both tet gene types between some countries and with class 1 integrons and resistance genes co-transferred by conjugation. The virulence gene profiles included enterotoxin genes (lt, sta and/or stb), as well as adhesin genes (k88/f4, f18). Characterisation of two representative tet(A) plasmids of porcine F18+ ETEC from Central Europe revealed that the IncF plasmid (pES11732) of the Czech strain (~120 kb) carried tet(A) in association with catA1 for chloramphenicol resistance. The IncI1 plasmid (pES2172) of the Hungarian strain (~138 kb) carried tet(A) gene and a class 1 integron with an unusual variable region of 2,735 bp composed by two gene cassettes: estX-aadA1 encoding for streptothricin-spectinomycin/streptomycin resistance exemplifying simultaneous recruitment, assembly and transfer of multidrug resistance genes by the tet(A) plasmid of porcine ETEC. By this we provide the first description of IncF and IncI1 type plasmids of F18+ porcine enterotoxigenic E. coli responsible for cotransfer of the tet(A) gene with multidrug resistance. Additionally, the unusual determinant estX, encoding for streptothricin resistance, is first reported here in porcine enterotoxigenic E. coli.

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The proportion of Escherichia coli non-susceptible to 3rd 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.

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