Predominance of t355/ST152/SCCmec V clonal type among PVL-positive MRSA isolates in a tertiary care hospital in Belgrade, Serbia.

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continually changing. Frequency of genotypes typical for community-associated MRSA (CA-MRSA) is increasing in hospitals, as well as resistance to antimicrobial agents. Moreover, different clones predominate in different geographic regions, and temporal shifts occur in the predominant clonal type. The aim of this study was to estimate the prevalence of MRSA, CA-MRSA and PVL-positive MRSA isolates from patients hospitalised in the Military Medical Academy (MMA) and from outpatients, and to perform genotyping of PVL-positive MRSA isolates. MRSA isolates were obtained by standard microbiological techniques. PVL-positive MRSA were detected by single PCR. Determination of SCCmec types in MRSA isolates was done using multiplex PCR and genotyping of PVL-positive MRSA by PFGE, MLST and spa typing. The prevalence of MRSA among S. aureus isolates from different clinical specimens was 43.4%. In outpatients the prevalence of MRSA was 3.2%. SCCmec types specific for CA-MRSA were found in 26% of MRSA isolates from hospitalised patients. In groups, hospitalised patients and outpatients, the prevalence of PVL-positive MRSA isolates was 4%, and all of them harboured SCCmec type V genetic element. PFGE revealed minor differences between four groups of PVL-positive MRSA isolates, but all of them belonged to ST152, and all except one were of the t355 spa type. High prevalence of MRSA and CA-MRSA in MMA, especially the presence of PVL-positive CA-MRSA, represent a serious health threat for patients. Genotype t355/ST152/SCCmec V is the dominant MRSA clone among PVL-positive CA-MRSA.

Prevalence of Panton-Valentine leukocidin genes in community-associated methicillin-resistant Staphylococcus aureus in the District of Pomoravlje.

BACKGROUND/AIM: Community-associated methicillin-resistant Staphjlococcus aureus (CA-MRSA) strains appear to have rapidly disseminated among population in the community without established risk factors for MRSA worldwide. Panton-Valentine leukocidin (PVL) is a cytolytic toxin, encoded by the lukF-PV and lukF-PV genes. PVL may be the key toxin responsible for enhanced virulence of CA-MRSA. The aim of this study was to detect the genes encoding PVL in CA-MRSA isolates from healthy people from the District of Pomoravlje. METHODS: We took throat and nose swabs from healthy, employed persons with mandatory sanitary examinations and analyzed the presence of MRSA, between January 2011 and December 2012 in the District of Pomoravlje. Susceptibility of isolated strains to cefoxitin was investigated by using disc diffusion according to the recommendation of CLSI (Clinical Laboratory Standard Institute), and by E test. The presence of penicillin-binding protein 2a (PBP2a) in Stapbylococd was detected using latex agglutination Slidex@MRSA Detection test. The gold standard, polymerase chain reaction (PCR) assay, was used for detection of mecA gene and PVL gene, and typing of SCCmec region. RESULTS: Our investigation showed that staphylococcal carrier state was present in 2.58% of 52,910 throat and nasal swabs, and in 50 of them (3.67%) MRSA was isolated. Among these MRSA, 2 (4/6) isolates were PVL-positive. CONCLUSION: The prevalence of CA-MRSA and the presence of PVL gene among healthy, employed population in the District of Pomoravlje were low. The values obtained in this study show that, our region is not significantly different from the other parts of our country, nor from the other European countries.

High Prevalence and Resistance Patterns of Community-Associated Methicillin-Resistant Staphylococcus Aureus in the Pomoravlje Region, Serbia.

With a view to estimating the prevalence and resistance patterns of CA-MRSA in one region of Serbia, we performed an analysis of MRSA isolates from healthy people and hospitalised patients. The detection of CA-MRSA was carried out by SCCmec typing. In MRSA isolates from hospitalised patients SCCmec types IV and V were found in 76% of the strains. Similar percentage (80%) of CA-MRSA genotypes was present in healthy people. SCCmec type V harbouring MRSA was the most successful clone. Higher prevalence of type V in hospitalised patients to that in healthy people (70% vs 54%) may indicate nosocomial transmissions in at least some hospital units. All MRSA strains from hospitalised patients were resistant to one or more non-ß-lactam antibiotics while 52% were multi-resistant. In isolates from healthy people, 16% were sensitive to all non-ß-lactam antibiotics and 40% were multi-resistant. Similar percentage of multi-resistant CA- and HA-genotypes occurred in a particular environment (53% vs 50% in hospitalised patients, and 37.5% vs 37.5% in healthy people) indicating selective pressure of antibiotics as a leading force conferring antibiotic resistance. High prevalence of CA-MRSA and high resistance rate both in hospitals and the community suggest that this pathogen has been present in the Pomoravlje Region, central Serbia for years.

Antimicrobial susceptibility and ß-lactamase production in Bacillus cereus isolates from stool of patients, food and environment samples.

Background/Aim: Bacillus cereus (B. cereus) usually ingested by food can cause two types of diseases: vomiting due to the presence of emetic toxin and diarrheal syndrome, due to the presence of diarrheal toxins. Systemic manifestations can also occur. The severe forms of disease demand antibiotic treatmant. The aim of this study was to determine the differences in antibiotic susceptibility and ß-lactamase activity of B. cereus isolates from stools of humans, food and environment. Methods: Identification of B. cereus was performed with selective medium, classical biochemical test and polymerase chain reaction (PCR) with primers specific for bal gene. Thirty isolates from each group were analysed for antibiotic susceptibility using the disk-diffusion assay. Production of ß-lactamase was determined by cefinase test, and double-disc method. Results: All strains identified as B. cereus using classical biochemical test, yielded 533 bp fragment with PCR. Isolates from all the three groups were susceptible to imipenem, vancomycin, and erythromycin. All isolates were susceptible to ciprofloxacin but one from the environment. A statistically significant difference between the groups was confirmed to tetracycline and trimethoprim-sulphamethoxazole sensitivity. A total of 28/30 (93.33%) samples from the foods and 25/30 (83.33%) samples from environment were approved sensitive to tetracycline, while 10/30 (33.33%) isolates from stools were sensitive. Opposite to this result, high susceptibility to trimethoprim-sulphamethoxazole was shown in samples from stools (100%), while isolates from foods (63.33%) and from environment (70%) had low susceptibility. All samples produced ß-lactamases. Conclusion: The strains of B. cereus from all the three groups showed high rate of sensitivity to most tested antibiotics, except to tetracycline in samples from human stool and to trimethoprim-sulphamethoxazole in samples from food and environment. The production of ß-lactamases was confirmed in all the strains.

Two copies of blaNDM-1 gene are present in NDM-1 producing Pseudomonas aeruginosa isolates from Serbia.

New Delhi metallo-ß-lactamase producing Pseudomonas aeruginosa isolates are of special interest since P. aeruginosa is a major cause of nosocomial infections, the treatment of which could now be jeopardized, especially in developing countries. Six additional NDM-1 positive P. aeruginosa clinical isolates belonging to two different genotypes were shown to be plasmid-free. PFGE-hybridization experiments revealed the chromosomal location of the blaNDM-1 gene. Restriction analysis and hybridization revealed that two copies of the blaNDM-1 gene are present in the genomes of all tested isolates, as in previously characterized P. aeruginosa MMA83. Moreover, it was shown that increasing imipenem concentration did not have the effect on copy number of the blaNDM-1 gene in the genome of P. aeruginosa MMA83.

The clinical isolate Pseudomonas aeruginosa MMA83 carries two copies of the blaNDM-1 gene in a novel genetic context.

The genetic context of the blaNDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for the blaNDM-1 gene revealed the presence of two blaNDM-1 copies in the genome of P. aeruginosa MMA83 in a unique genetic environment. Additionally, mating assays, DNA-DNA hybridization, and an S1 nuclease assay strongly suggest that the blaNDM-1 gene in P. aeruginosa MMA83 is chromosome borne.

Shiga toxin-producing Escherichia coli O171:H25 strain isolated from a patient with haemolytic uraemic syndrome.

Shiga toxin-producing Escherichia coli (STEC) strains of O157:H7 serotype are a predominant cause of haemolytic uraemic syndrome (HUS) worldwide, but strains of non-O157 serotypes can also be associated with serious disease. Some of them are associated with outbreaks of HUS, others with sporadic cases of HUS, and some with diarrhoea but not with outbreaks or HUS. A large number of STEC serotypes isolated from ruminants and foods have never been associated with human disease. In this study we characterize a STEC strain belonging to serotype O171:H25 that is responsible for a case of HUS. This strain has a single Shiga toxin gene encoding Stx2 toxin, and hlyA gene, but is eae-negative.

Emergence of NDM-1 metallo-ß-lactamase in Pseudomonas aeruginosa clinical isolates from Serbia.

This work reports, for the first time, the presence of New Delhi metallo-ß-lactamase 1 (NDM-1) in Pseudomonas aeruginosa. Moreover, this is the first report of the NDM-1 presence in the Balkan region. Cosmid gene libraries of carbapenem-nonsusceptible Pseudomonas aeruginosa clinical isolates MMA83 and MMA533 were screened for the presence of metallo-ß-lactamases. Accordingly, both MMA83 and MMA533 carried the bla(NDM-1) gene. Pulsed-field gel electrophoresis (PFGE) analysis indicated that strains MMA83 and MMA533 belonged to different clonal groups. Five additional isolates from different patients clonally related to either MMA83 or MMA533 were found to be NDM-1 positive.

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in Belgrade area.

OBJECTIVE: Lyme borreliosis is vector-borne zoonosis. The causative agent of Lyme borreliosis is a spirochete of Borrelia burgdorferi (Bb) sensu lato complex, which is transmitted by ticks of the Ixodes ricinus complex. The aim of our paper is to estimate the prevalence of I. ricinus ticks, the level of their infectivity by Bb, and the prevalence of certain genospecies of Bb sensu lato in ixodide ticks inhabiting Belgrade. MATERIALS AND METHODS: An estimate of the tick population density was expressed by the value of flag/hour. For isolation and cultivation of Borrelia, selective Barbour-Stonner-Kelly II media was used. Typization of Borrelia was made by applying the species-specific polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism analysis. In statistical analysis, Chi(2) test was used. RESULTS: Values of flag/hour have varied in relation to year observed and type of habitat: The lowest values were recorded in the city parks (17.9). The values were higher in parks-woods (19.7 and 33.4, respectively). The highest values were detected in localities similar to wooded areas (48.0). The estimated average infestation of ticks with Bb was 21.9%, excluding statistically significant differences by years of investigation. We found the dominance of Borrelia afzelii (75%). Bb sensu stricto (22.2%) as well as Borrelia garinii (2.8%) was much less present. Statistically significant difference was established in the prevalence of the above-mentioned genospecies in relation to the examined localities. CONCLUSIONS: We have established the prevalence of all three genospecies in the city of Belgrade. Bb sensu lato was found, with the dominance of B. afzelii.

Multilocus sequence typing for investigation of diversity of Campylobacter jejuni strains from humans and environment in Norway.

This report describes the use of the Multilocus Sequence Typing (MLST) method for typing 32 strains previously identified as Campylobacter jejuni. This system identified a great diversity between the investigated strains. The majority of strains belonged to already existing alleles found on the Campylobacter MLST home page, and most of them represented clonal complexes ST-21 and ST-45, but there were some newly identified alleles as well.

[Reliability of Cobas Amplicor PCR test in detection of Mycobacterium tuberculosis in respiratory and nonorespiratory specimens].

BACKGROUND/AIM: Traditional methods for detection of mycobacteria, such as microscopic examination for the presence of acid-fast bacilli and isolation of the organism by culture, have either a low sensitivity and/or specificity, or take weeks before a definite result is available. Molecular methods, especially those based on nucleic acid amplification, are rapid diagnostic methods which combine high sensitivity and high specificity. The aim of this study was to determine the usefulness of the Cobas Amplicor Mycobacterium tuberculosis polymerase chain reaction (CA-PCR) assay in detecting the tuberculosis cause in respiratory and nonrespiratory specimens (compared to culture). METHODS: Specimens were decontaminated by the N-acetyl-L-cystein-NaOH method. A 500 microL aliquot of the processed specimen were used for inoculation of Löwenstein-Jensen (L-J) slants, a drop for acid-fast staining, and 100 microL for PCR. The Cobas Amplicor PCR was performed according to the manufacturer's instructions. RESULTS: A total of 110 respiratory and 355 nonrespiratory specimens were investigated. After resolving discrepancies by reviewing medical history, overall sensitivity, specificity, and positive and negative predictive values for CA-PCR assay compared to culture, were 83%, 100%, 100%, and 96.8%, respectively. In comparison, they were 50%, 99.7%, 87.5%, and 98%, respectively, for the nonrespiratory specimens. The inhibition rate was 2.8% for respiratory, and 7.6% for nonrespiratory specimens. CONCLUSION: CA-PCR is a reliable assay that enables specialists to start treatment promptly on a positive test result. Lower value for specificity in a group of nonrespiratory specimens is a consequence of an extremely small number of mycobacteria in some of them.

Identification of PER-1 extended-spectrum beta-lactamase producing Pseudomonas aeruginosa clinical isolates of the international clonal complex CC11 from Hungary and Serbia.

PER-1 extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa clinical isolates from Budapest, Hungary, and Belgrade, Serbia, were characterized by molecular methods. Two PER-1-positive isolates were recovered from sporadic cases in Budapest and a small cluster of PER-1-positive infections involving four patients were identified at a Belgrade hospital. A class 1 integron harbouring a bla(OXA-2)beta-lactamase gene and four other gene cassettes was detected in both the Budapest and the Belgrade isolates. The two P. aeruginosa isolates from Budapest also carried another class 1 integron containing bla(OXA-74), aac(6')-Ib-cr and cmlA7 genes in its variable region. The aac(6')-Ib-cr fluoroquinolone-acetylating aminoglycoside acetyltransferase gene is described here for the first time in P. aeruginosa. Multilocus sequence typing (MLST) revealed that the PER-1 positive P. aeruginosa isolates identified in this study display ST235, a sequence type that belongs to clonal complex CC11. Two bla(PER-1)-positive P. aeruginosa reference isolates from France and Belgium could also be assigned to complex CC11 by MLST. Our results underscore the role of complex CC11 in the dissemination of bla(PER-1) among P. aeruginosa clinical isolates.

Molecular characterization of vancomycin-resistant Enterococcus spp. clinical isolates from Hungary and Serbia.

The objectives of this work were to collect and characterize vancomycin-resistant Enterococcus faecium (VREF) clinical isolates from Hungary and Serbia and to analyse their genetic relatedness. VREF isolates were initially typed by PFGE. A selection of VREF isolates representing all participating hospitals was further examined by multiple-locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST). VanB VREF isolates (n=18) recovered from blood, urine and faecal cultures at a Budapest hospital between August 2003 and December 2004 were molecularly characterized. Macrorestriction analysis of the isolates revealed their monoclonal relatedness. A cluster of infections caused by 2 distinct VanA VREF clones recovered from 6 departments was identified in a Belgrade hospital in Serbia. The vanA resistance determinant was transferable by in vitro conjugation experiments. We also identified 2 vanA-positive E. gallinarum blood culture isolates in this Belgrade hospital. Molecular typing of representative VREF isolates from Hungary and Serbia by MLVA and MLST revealed that all tested isolates belonged to MLST complex CC17 and the corresponding MLVA cluster 1. Our results extend the documented occurrence of CC17 to a new region in Europe.

[Plasmid profile analysis in identification of epidemic strains of Salmonella enterica serovar Enteritidis].

BACKGROUND/AIM: As illness caused by Sallmonella enterica serovar Enteritidis (S. Enteritidis) occurs not only as sporadic cases but as outbreaks, to reveal the source and routes of spreading of infection it is necessary to identify epidemic strain by the use of some typing methods. To determine whether plasmid profile analysis, as genotyping method, could be applied for the investigation of epidemic strains, isolates of S. Enteritidis, recovered from patient's stools and food associated with outbreaks and those isolated from sporadic cases of diarrhea, were investigated. METHODS: Investigation of antibiotic resistance was performed by Kirby-Bauer disc-diffusion method. Isolation of plasmid DNA was carried out by Birnboim and Dolly alkaline lysis method, modified by Ish-Horovitz. RESULTS: Out of 276 izolates of S. Enteritidis 94 were isolated from patient's stools and food associated with outbreaks and 182 were isolated from sporadic cases of diarrhea. The presence of 12 plasmid profiles was established. An average correlation degree of plasmid profiles between the strains was 0.84, that implies high degree of similarity of plasmid profiles of epidemic and non epidemic strains isolated at our geographic region for the given period of time. CONCLUSION: The strains of S. Enteritidis, isolated in outbreaks of enterocolitis as well as from spordic cases of diarrhea in the same period of time and at the same area, frequently exhibit the same plasmid profile characterized by a single plasmid of 38 MDa. Therefore, in most cases plasmid profile analysis is not valuable in the identification of epidemic strains of S. Enteritidis. However, for this purpose plasmid profile analysis could be used when drug-resistant strains of S. Enteritidis are isolated, as they often possess additional resistant plasmids what increases discrimination power of this method.

Characterisation of the first VIM metallo-beta-lactamase-producing Pseudomonas aeruginosa clinical isolate in Serbia.

From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa strains were published from Croatia, Poland and Hungary. The aim of this study was to assess the contribution of MBL-production to carbapenem-resistance among P. aeruginosa clinical isolates in the Military Medical Academy (MMA) in Belgrade, Serbia between August 2004 and September 2007. Only one P. aeruginosa isolate with strain number 722 proved MBL-positive that harboured a novel class 1 integron with a bla(VIM-2)-like cassette in the first position, followed by orfD, a putative gene with unknown function. Our data indicate that MBL-producing strains occur at a prevalence of less than 1% among imipenem-nonsusceptible P. aeruginosa clinical isolates in this Belgrade hospital. The newly identified VIM MBL-producing P. aeruginosa strain 722 could be assigned to serotype O11, and it was panresistant to all antimicrobials tested. The isolate displayed sequence type ST235 by multilocus sequence typing which is the founder sequence type of the previously identified international clonal complex CC11 that already contains bla(VIM)-positive isolates from Italy, Greece, Sweden, Hungary and Poland. In conclusion, this is the first report of VIM MBL-producing P. aeruginosa from Serbia and also of the occurrence of such isolates belonging to the international clonal complex CC11 in this country.

Evaluation of usefulness of single-strand conformation polymorphism method for rapid detection of rifampicin-resistant Mycobacterium tuberculosis.

The aim was to evaluate the Single-Strand Conformation Polymorphism method as a potential tool for rapid detection of rifampicin-resistant strains by the use of 39 rifampicin-resistant Mycobacterium tuberculosis strains isolated in Serbia. SSCP analysis on acrylamide gel detected 56.4% of the rifampicin-resistant M. tuberculosis strains and showed the inability to detect one of the most frequent mutations, TCG-->TTG mutation in codon 531 of the rpoB gene, which was shown by automated sequencing.

Improved serodiagnosis of early Lyme borreliosis: immunoblot with local Borrelia afzelii strain.

To improve the serodiagnosis of Lyme borreliosis (LB) the performances of four tests were evaluated. An indirect immunofluorescent assay based on Borrelia burgdorferi s.s., enzyme-linked immunosorbent assay (ELISA) based on local isolates of Borrelia afzelii and B. burgdorferi s.s., and immunoblot (IB) of B. afzelii were prepared. The serum panels contained 214 serum samples: control group (n=120) and patients at different stages of LB (n=94). The specificity of IB was 96%, of in-house ELISA 93%, and of IFA 89%. In early LB the sensitivity of IFA was 36%, ELISA 67%, and IB 93%. In late-stage LB the sensitivity was: 72% for IFA, 80% for ELISA, and 94% for IB. Comparison of in-house and Behring ELISA showed that the sensitivity of the serological assay could be increased when the test was based on local Borrelia strains. IgM and IgG antibodies from sera of patients with early and late LB most frequently demonstrated reactivity to OspC. The other significant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p21 and p43 in IgG IB. Using IB based on local B. afzelii isolates improves the serodiagnosis of early LB in our geographical region.

[First documented case of enterocolitis in Yugoslavia caused by enterohemorrhagic Escherichia coli O157]. / Prvi dokazani slucaj enterokolitisa u Jugoslaviji uzrokovan enterohemoragijskom eserihijom koli O157.

A 'new' group of pathogenic agents, enterohemorrhagic Escherichia coli (EHEC) (particularly the strains of O157 serogroup), emerged in the last 20 years, causing an increased number of sporadic and epidemic diarrhoeal diseases with hemorrhagic enterocolitis as a most common clinical manifestation of the infection. As a consequence of the absorption and cytotoxic effect of the main virulence factor of these bacteria--verotoxin (shiga-toxin), in about 10% of the affected persons extraintestinal complications, most frequently hemolytic-uremic syndrome (HUS), occurred 7-14 days after an episode of diarrhoeal disease. The first case of hemorrhagic enterocolitis with the documented EHEC O157 infection in Yugoslavia is presented in this paper. Considering the existing expansion trend of these carriers, practitioners should be aware of them in case of the occurrence of diarrhoeal disease, (particularly hemorrhagic enterocolitis), and keep these patients under control during the reconvalescence period because of potential development of extraintestinal complications, such as HUS.