Carriage and Genetic Diversity of Methicillin-Resistant Staphylococcus aureus among Patients and Healthcare Workers in a Serbian University Hospital.

OBJECTIVES: There is a paucity of data on methicillin-resistant Staphylococcus aureus (MRSA) epidemiology among Balkan countries. The aim of our study was to determine the prevalence of nasal and pharyngeal carriages and diversity of MRSA among patients and healthcare workers (HCWs) in the major referral centre in Serbia, and to evaluate performance of three different media for MRSA screening. METHODS: Nasal and pharyngeal swabs were obtained from 195 patients and 105 HCWs in Emergency Department (ED), Surgical Department (SD) and Medical Department (MD). After broth enrichment, samples were inoculated onto MRSA-ID, ORSA and oxacillin-MSA and incubated for 24/48 hours. Characterisation of isolated MRSA strains was determined by MLVA, spa, SCCmec and agr typing, PVL genes detection and antimicrobial susceptibility. RESULTS: MRSA carriage prevalence was 11.8% in patients and 7.6% in HCWs. Introduction of pharyngeal swabs in screening procedure increased MRSA carriage rate by over 30%. Variable found to be independently associated with an increased risk for MRSA carriage was ED (odd ratio (OR) = 4.45, 95% confidence interval (CI) 1.78-11.14). A higher risk of multidrug-resistant MRSA carriage was observed among patients (OR = 22; 95% CI 1.92-251.54). CC5-MRSA-SCCmecI was the dominant clone among patients and HCWs in ED and MD, while high genetic diversity of community-associated MRSA (CA-MRSA) was shown in SD especially among HCWs. MRSA-ID was superior to the other tested media with a sensitivity/specificity of 95.2% and 99.6% after 48 hours of incubation. CONCLUSIONS: These results indicate high MRSA carriage rate in the hospital and emergence of CA-MRSA through HCWs in these settings. MRSA-ID was the optimal available choice for MRSA screening.

[Nasal carriage of methicillin-resistant Staphylococcus aureus among medical students of Belgrade University].

INTRODUCTION: Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) present the growing problem in the whole world. Carriage of MRSA is most frequent in the nose, and medical students come in contact both with patients and different persons in the community. Therefore, they may be significant for the transmission of MRSA from hospitals to out-of-hospital communities and vice versa. OBJECTIVE: The aim of this study was to establish the carriage rate among students of the second, third and fourth year of study at the School of Medicine in Belgrade and to analyze their genotypic and phenotypic characteristics. METHODS: In total 533 nasal samples were taken. The samples were incubated inTrypcase-soy broth supplemented with 6.5% NaCl, and thereafter the swabs were inoculated on mannitol salt agar supplemented with 2 microg/mL of oxacillin. The presence of nuc, mecA and Panton-Valentine leukocidin genes was examined by PCR.The characteristics of the MRSA strains were determined using: antibiotic susceptibility testing by Vitek2 System, SCCmec, agr typing and MLST. RESULTS: MRSA was isolated from two of 533 investigated samples (0.37%). MRSA were isolated from the students of the second and third year of study. Profiles of strains were: ST80 (SCCmec type IV, agr type 3) and ST152 (SCCmec type V, agr type 1). MRSA strains were multiresistant. CONCLUSION: The nasal carriage rate of MRSA in population of medical students of the first year of study in Belgrade is low. Genotypic and phenotypic characteristics of MRSA strains indicate their community origin. MLST typing revealed that isolates belong to ST80 and ST152.

Staphylococcus stepanovicii sp. nov., a novel novobiocin-resistant oxidase-positive staphylococcal species isolated from wild small mammals.

Five novobiocin-resistant and oxidase-positive staphylococcal strains were isolated from wild small mammals. Phenotypic studies and phylogenetic analysis based on 16S rRNA, rpoB and dnaJ gene sequences revealed that these strains were members of the Staphylococcus sciuri cluster group and were similar to Staphylococcus fleurettii. DNA-DNA hybridisation with closely related staphylococcal species suggested that the strains represented a novel species. The name Staphylococcus stepanovicii is proposed, and the type strain is 196(T) (=PCM 2693(T) =CCM 7717(T)).

Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism analysis of dnaJ gene.

A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

Identification and antimicrobial susceptibility testing of Staphylococcus vitulinus by the BD phoenix automated microbiology system.

This study evaluated the performance of the BD Phoenix system for the identification (ID) and antimicrobial susceptibility testing (AST) of Staphylococcus vitulinus. Of the 10 S. vitulinus isolates included in the study, 2 were obtained from the Czech Collection of Microorganisms, 5 from the environment, 2 from human clinical samples, and 1 from an animal source. The results of conventional biochemical and molecular tests were used for the reference method for ID, while antimicrobial susceptibility testing performed in accordance with Clinical and Laboratory Standards Institute recommendations and PCR for the mecA gene were the reference for AST. Three isolates were incorrectly identified by the BD Phoenix system; one of these was incorrectly identified to the genus level, and two to the species level. The results of AST by the BD Phoenix system were in agreement with those by the reference method used. While the results of susceptibility testing compared favorably, the 70% accuracy of the Phoenix system for identification of this unusual staphylococcal species was not fully satisfactory.

Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci.

The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.

Aminoglycoside resistance in members of the Staphylococcus sciuri group.

This study investigated the prevalence of aminoglycoside resistance and genes encoding aminoglycoside-modifying enzymes in members of the Staphylococcus sciuri group. A total of 304 S. sciuri group member isolates (284 S. sciuri, 12 S. lentus, and 8 S. vitulinus) from humans (n = 34), animals (n = 133), and environmental sources (n = 137; out-hospital and hospital environment, food) were examined for their susceptibility to amikacin, gentamicin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, streptomycin, and tobramycin. The overall prevalence of resistance to aminoglycosides was low at 12.1%. Resistance to single aminoglycosides ranged from 0% to 7.2%. The aac(6')-Ie/aph(2"), ant(4')-Ia, and aph(3')-IIIa genes, either alone or in combination, were found in 16 out of 19 isolates showing resistance to nonstreptomycin aminoglycosides. Among the 22 isolates that showed resistance to streptomycin, the genes str and ant(6)-Ia were identified in 18 and 4 isolates, respectively.

Supplementary biochemical tests useful for the differentiation of oxidase positive staphylococci.

Differentiation of the oxidase positive staphylococci, Staphylococcus sciuri, Staphylococcus lentus, Staphylococcus vitulinus and Staphylococcus fleurettii, based on tributyrin, urease, caseinase, gelatinase and DNase activity is described. These tests may be used for preliminary identification of oxidase positive isolates of staphylococci resulting in more accurate identification of these species.

Surgical wound infection associated with Psychrobacter phenylpyruvicus-like organism.

A case of surgical wound infection caused by Psychrobacter phenylpyruvicus-like organism is described. The strain showed phenotypic characteristics typical of P. phenylpyruvicus, but 16S rRNA sequencing showed 98.2% relatedness to Moraxella phenylpyruvica strain 752/52 and only 94.8% with P. phenylpyruvicus type strain ATCC 23333(T). The results of molecular analysis suggest that the strain we isolated may represent a new species within the genus Psychrobacter.

Resistance to macrolides, lincosamides, streptogramins, and linezolid among members of the Staphylococcus sciuri group.

This study aimed to characterize the resistance profiles of the Staphylococcus sciuri group members to macrolides, lincosamides, streptogramins (MLS antibiotics), and linezolid upon analysis of large series of isolates that included 162 S. sciuri isolates, nine S. lentus, and one S. vitulinus. The evaluation of their susceptibility by disk diffusion and agar dilution methods, along with PCR detection of the resistance genes erm(A), erm(B), erm(C), mef(A), lnu(A), and lnu(B), were performed. Resistance to macrolides was detected in 10 (5.8%) tested strains, with three and six isolates exhibiting constitutive and inducible MLS(B) resistance phenotypes, respectively. Resistance mediated by active efflux was detected in one strain. The presence of genes conferring resistance, namely erm(B) or erm(C), was detected in two strains. All tested strains were susceptible to pristinamycin and linezolid. Of 172 tested strains, 70.9% were resistant and 26.2% had intermediary resistance to lincomycin, whereas 1.7% were resistant and 50% had intermediary resistance to clindamycin. The lnu(A) gene was detected in two strains only. The great majority of the tested S. sciuri strains (153 out of 162; 94.4%) presumably exhibited LS(A) phenotype because they did not carry lnu genes nor displayed constitutive MLSB resistance, but still showed intermediate resistance or resistance to lincomycin (MICs of 4, 8, 16, and 32 microg/ml). The results obtained indicate that S. sciuri may be naturally resistant to lincomycin. Expression of a novel type of inducible resistance to lincosamides, induced by erythromycin in erythromycinsusceptible strains, was observed in the S. sciuri group isolates.

Evaluation of phenotypic and molecular methods for detection of oxacillin resistance in members of the Staphylococcus sciuri group.

In this paper we report on an experimental evaluation of phenotypic and molecular methods as means for the detection of oxacillin resistance in members of the Staphylococcus sciuri group. A total of 109 S. sciuri group member isolates (92 S. sciuri isolates, 9 S. lentus isolates, and 8 S. vitulinus isolates) were tested by the disk diffusion method, the agar dilution method, the oxacillin salt-agar screening method, slide latex agglutination for PBP 2a, and PCR assay for mecA as the reference method. The mecA gene was detected in 29 S. sciuri isolates, and the true-positive and true-negative results of the other tests were defined on the basis of the presence or the absence of the mecA gene. For the different methods evaluated, the sensitivities and specificities were as follows: for the disk diffusion test with a 1-microg oxacillin disk, 100% and 55.9%, respectively; for the disk diffusion test with a 30-mug cefoxitin disk, 93.5% and 100%, respectively; for the agar dilution method, 100% and 50%, respectively; for the oxacillin salt-agar screen test (with 6 microg of oxacillin per ml and 4% NaCl) 100% and 100%, respectively; and for the slide latex agglutination test for PBP 2a, 100% and 100%, respectively. The disk diffusion test with various beta-lactam antibiotics was performed to evaluate their use for the prediction of oxacillin resistance. The results indicate that meropenem, cefazolin, cefamandole, cefuroxime, cefotetan, cefoperazone, cefotaxime, ceftriaxone, moxalactam, cefaclor, and cefprozil may be used as surrogate markers of oxacillin resistance, although further studies of their use for the detection of oxacillin resistance are required.

Survey of genes encoding staphylococcal enterotoxins, toxic shock syndrome toxin 1, and exfoliative toxins in members of the Staphylococcus sciuri group.

Genes encoding staphylococcal enterotoxins (sea to see, seg, and seh), toxic shock syndrome toxin 1 (tst), and exfoliative toxins (eta and etb) were not detected in a large panel of 48 Staphylococcus sciuri group isolates tested. This strongly suggests that production of the staphylococcal exotoxins by these bacteria is highly unlikely.

A comparative evaluation of phenotypic and molecular methods in the identification of members of the Staphylococcus sciuri group.

Members of the Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5-100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.

Isolation and molecular characterization of Staphylococcus sciuri in the hospital environment.

Staphylococcus sciuri is a principally animal-associated bacterial species, but its clinical relevance for humans is increasing. Our study aimed to provide the first insight into the prevalence of this bacterium in a hospital environment. A 3-month surveillance was conducted in a hospital located in Belgrade, Serbia, and 1,028 samples taken from hands of medical personnel, medical devices, and various hospital surfaces were screened for S. sciuri presence. In total, 108 isolates were obtained, which resulted in a relatively high rate of colonization (10.5%). These isolates, along with 7 S. sciuri strains previously isolated in the same hospital (n = 115), were phenotypically and genotypically characterized. Antimicrobial susceptibility testing revealed that 73% of the strains were resistant to one or more antibiotics, with 4.3% strains displaying multiresistance. Examination of 16S-23S ribosomal DNA intergenic spacer length polymorphism identified the strains at the subspecies level, and 74 (64.3%) strains of S. sciuri subsp. sciuri, 37 (32.2%) strains of S. sciuri subsp. rodentium, and 4 (3.5%) strains of S. sciuri subsp. carnaticus were established. Pulsed-field gel electrophoresis (PFGE) analysis showed 21 distinct pulsotypes, including 17 main types and 4 subtypes. One dominant cluster with 62 strains was found, while 19 (90.5%) of the PFGE types and subtypes identified had 5 or fewer strains. The predominance of small PFGE clusters suggests that the ubiquitous presence of S. sciuri in the outside environment presents the continuous source for colonization of the hospital environment. The presence of one dominant PFGE cluster of strains indicates that some S. sciuri strains may be capable for adaptation to hospital environment conditions and continuous existence in this environment.

Brain abscess due to Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a constituent of the oral flora, is a rare cause of brain abscesses. We report the case of a 47-year-old male who presented with multiple brain abscesses due to this organism, presumably originating from his poor dentition. Problems met in isolating and identifying A. actinomycetemcomitans suggest that its true rate of isolation from non-oral samples may have been underestimated.

Identification and characterization of clinical isolates of members of the Staphylococcus sciuri group.

A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.