Molecular characterisation of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in inpatients and outpatients in Bosnia and Herzegovina.

The aim of this study was to investigate the genetic background of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) obtained from clinical specimens of inpatients and outpatients. Methicillin resistance was confirmed by the presence of the mecA gene by PCR. The genetic characterisation was performed using spa typing and the algorithm based upon repeat pattern (BURP). Staphylococcus aureus was isolated from 68 and 79 inpatient and outpatient samples, 31 (46 %) and 14 (18 %) of which were MRSA, respectively. Among 37 inpatients and 65 outpatients with MSSA, 22 and 38 spa types were clustered into seven and eight spa-CCs, respectively. The main MSSA spa-CC of inpatients and outpatients was spa-CC015 (multilocus sequence typing (MLST) CC45). Most MRSA were associated with spa-CC355/595 (MLST CC152). MRSA-related background was found in 32 % of inpatients and 43 % of outpatients with MSSA, suggesting that MRSA did not arise from predominant MSSA clones.

mecA-positive methicillin-sensitive Staphylococcus aureus clinical isolates in Zenica-Doboj Canton, Bosnia and Herzegovina.

Forty-four mecA-positive and eight mecA-negative Staphylococcus aureus isolates confirmed by PCR were further tested by disc-diffusion (DD) oxacillin and cefoxitin, oxacillin Epsilon (E)-test, and oxacillin and cefoxitin minimal inhibitory concentration (MIC) Strip methicillin-resistant phenotype in S. aureus (MRSA) tests. Among 44 mecA-positive S. aureus isolates, two (4·5%) were detected as MRSA by DD-oxacillin, 17 (38·6%) by DD-cefoxitin test, and seven (15·9%) by the E-test. In the cefoxitin MIC Strip MRSA test, 19 (43·2%) isolates were resistant. In the oxacillin MIC Strip MRSA test, 18 (40·9%) isolates were resistant and 26 (59·1%) were sensitive, i.e. oxacillin-sensitive MRSA (OS-MRSA) (MIC range 0·25-≤0·25 mg/l). Fifteen out of 26 OS-MRSA (57·7%) belonged to spa-CC 355/595, 78% of which belonged to the largest PFGE clone. Some discrepancies between the phenotypic methods for MRSA identification obtained in this study were caused by large proportion of OS-MRSA. Misidentification of OS-MRSA as MSSA might result in an appearance of highly resistant MRSA in patients treated with beta-lactam antibiotics.

Methicillin-resistant S. aureus (MRSA), extended-spectrum (ESBL)- and plasmid-mediated AmpC ß-lactamase -producing Gram-negative bacteria associated with skin and soft tissue infections in hospital and community settings.

AIM: To investigate the characteristics of meticillin-resistant S. aureus (MRSA), extended-spectrum (ESBL), and plasmid-mediated AmpC beta-lactamase producing Gram-negative bacteria causing skin and soft tissue infections (SSTIs) in hospital and outpatient settings of Zenica-Doboj Canton, Bosnia and Herzegovina. METHODS: Antibiotic susceptibility was determined by disc-diffusion and broth microdillution methods according to CLSI guidelines. MecA gene was detected by PCR, and genetic characterization of MRSA was performed using spa-typing and the algorithm based upon repeat patterns (BURP). Double-disk-synergy test was used to screen for ESBLs. PCR was used to detect blaESBL alleles. Genetic relatedness of the strains was tested by PFGE. RESULTS: Seventeen in-patients with MRSA, 13 with ESBL-producing Gram-negative bacteria and three patients co-infected with both, were detected. Five MRSA and 16 ESBL-producing Gram-negative bacteria were found in outpatient samples. Klebsiella spp. was isolated in 11 in- and seven outpatients. MLST CC152 was the most prevalent MRSA. Seven (38.9%) Klebsiella spp. yielded amplicons with primers specific for SHV, TEM-1 and CTX-M group 1 ß-lactamases. Eight K. pneumonia (44.4%) and 16 (64%) MRSA (including the in- and outpatient) strains were clonally related. CONCLUSION: The presence of MRSA and ESBL-producing organisms causing SSTIs in the community poses a substantial concern, due to the high morbidity and mortality associated with possible consequent hospital infections.

Emergency (clonal spread) of methicillin-resistant Staphylococcus aureus (MRSA), extended spectrum (ESBL)--and AmpC beta-lactamase-producing Gram-negative bacteria infections at Pediatric Department, Bosnia and Herzegovina.

PURPOSE: Aim of this study was to investigate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum (ESBL) and plasmid-mediated AmpC beta-lactamase producing Gram-negative bacteria in children. METHODS: Antibiotic susceptibility of MRSA and beta-lactamase producing Gram-negative bacteria was determined by disc diffusion and broth microdilution methods according to CLSI guidelines. Methicillin resistance was confirmed by the presence of mecA gene by PCR. The genetic characterization of S. aures was performed using spa-typing and the algorithm based upon repeat pattern (BURP). Double-disk synergy test was used to screen for ESBL production. PCR was used to detect bla ESBL alleles. Genetic relatedness of the strains was tested by pulsed-field gel electrophoresis (PFGE). RESULTS: Among 23 MRSA, 12 (52.2 %) were obtained from newborns. MLST CC152 (spa-CC 355-595) (Balkan clone) was the most prevalent, 20 (87 %) cases. Among 24 beta-lactamase producing Gram-negative bacteria, 10 (41.7 %) were obtained from each newborns and one-year-old children; 14 (58.3 %) were from urine. Among 11 Klebsiella strains isolated from urine eight (73 %) produced CTX-M-15, and one CTX-M-3 beta-lactamase. Twenty (83 %) of CTX-M producers were coproduced by other types of beta-lactamases. Fifteen (65.2 %) MRSA isolates were clonally related. Five clones among 13 K. pneumoniae isolates were detected by PFGE suggesting clonal spread of ß-lactamase producing Gram-negative bacteria. CONCLUSION: Pediatric infections caused by clonal spread of MRSA and beta-lactamase-producing Gram-negative bacteria are of major concern. Proper infection control measures should be implemented in order to avoid the transmission and major outbreaks.

Inducible clindamycin resistance in methicillin-susceptible and methicillin-resistant Staphylococcus aureus of inpatient, outpatient and healthy carriers in Bosnia and Herzegovina.

AIM: To investigate the iMLSB prevalence in 142 methicillin-sensitive (MSSA) and 48 methicillin-resistant (MRSA) in-patient (65), outpatient (75), and healthy carrier (150) Staphylococcus aureus isolates in Zenica-Doboj Canton, Bosnia and Herzegovina. METHODS: Disk diffusion testing by placing clindamycin (CLI) and erythromycin (ERY) disks 15 mm apart (edge to edge) on a Mueller-Hinton agar, as per CLSI guideline was performed. Two distinct induction phenotypes labelled as D and D+, and three noninduction phenotypes designated as Neg, R (constitutive, cMLSB), and S (susceptible). Methicillin-resistance was confirmed by the presence of mecA gene by PCR. The genetic characterization was performed using spa-typing and the algorithm based upon repeat patterns (BURP). RESULTS: iMLSB was detected in six (2.1%) isolates, of which five (3.5%) (two outpatients and three carriers) were MSSA, and one (2.1%) (outpatient) MRSA. One of them, D+ phenotype (iMLSB) was obtained from a carrier (MSSA). None of the inpatients had iMLSB. HD phenotype was not detected. One (MRSA) isolate has shown negative phenotype. Two strains with iMLSB originated from skin and soft tissue (MRSA) and eye infection (MSSA) belonged to the same MLST CC8, with different spa-types (t451 and t008, respectively). R phenotype (cMLSB) was detected in two (inpatient) isolates (0.7%). CONCLUSION: D test identified 2% of wrongly reported isolates as clindamycin sensitive. Despite low prevalence of S. aureus with iMLSB , it is a significant finding that they were mostly MSSA, and all were isolated from outpatients or carriers. D-test becomes an imperative part of routine antimicrobial susceptibility test for all S. aureus isolates.

Antimicrobial resistance and molecular typing of Staphylococcus aureus bloodstream isolates from hospitals in Peru.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infections are of worldwide concern. The present study describes the antimicrobial resistance and molecular typing of methicillin-resistant and methicillin-susceptible S. aureus (MSSA) bloodstream isolates in Peru. METHODS: Consecutive non-duplicate S. aureus bloodstream isolates were collected over a 15-month period (2008-2009) from seven hospitals in Lima and Callao, two contiguous cities in Peru. Detection of mecA gene, spa typing and Staphylococcal Chromosomal Cassette (SCC)mec typing were performed. Antimicrobial resistance was assessed by disk diffusion. RESULTS: Of 338 isolates, MRSA rate was 50.0%. Among MRSA isolates (n = 169), 81.7% were associated to MLST CC5, 68.8% had spa t149/SCCmec I, and more than 85% were co-resistant to ciprofloxacin, clindamycin, erythromycin and gentamicin; 8.9% (n = 15) were associated to MLST CC8, 14 of them had spa t148/SCCmec IV, and more than 70% were co-resistant to ciprofloxacin, clindamycin and erythromycin. Among MSSA isolates (n = 169), there was a higher diversity of spa types (n = 56) compared to MRSA isolates (n = 17), 27.2% were associated to MLST CC8, 23.7% were resistant to erythromycin and clindamycin resistance exceeded 20%. CONCLUSIONS: MRSA rate among bloodstream isolates in Peru was 50%, with MLST CC5/t149/SCCmec I representing the most frequent clone.

The Staphylococcus aureus lineage-specific markers collagen adhesin and toxic shock syndrome toxin 1 distinguish multilocus sequence typing clonal complexes within spa clonal complexes.

Spa typing/based upon repeat pattern (BURP) sometimes cannot differentiate multilocus sequence typing (MLST) clonal complexes (CCs) within spa-CCs. It has been observed previously that virulence factors, such as collagen adhesin (CNA) and toxic shock syndrome toxin 1 (TSST-1), are associated with certain Staphylococcus aureus lineages. Analysis of methicillin-sensitive and methicillin-resistant S. aureus by spa typing/BURP and detection of CNA and TSST-1 observed an association between CNA and MLST CC1, 12, 22, 30, 45, 51, and 239 and between TSST-1 and MLST CC30. In spa-CC 012, associated with MLST CC7, CC15, and CC30, MLST CC30 could be distinguished from MLST CC7 and CC15 with CNA and TSST-1 as lineage-specific markers. Lineage-specific markers can overcome clustering of nonrelated MLST CCs into 1 spa-CC.

A rapid, 2-well, multiplex real-time polymerase chain reaction assay for the detection of SCCmec types I to V in methicillin-resistant Staphylococcus aureus.

For us to assess the spread of methicillin-resistant Staphylococcus aureus (MRSA), typing of the staphylococcal cassette chromosome mec (SCCmec) is a valuable addition to existing typing methods, such as multilocus sequence typing (MLST). Traditional SCCmec typing assays, that is, that of Oliveira et al. and Ito et al., are polymerase chain reaction (PCR) based, requiring electrophoresis. We introduce a rapid, 2-well, multiplex real-time PCR assay that can be used directly on bacterial suspensions and is able to characterize SCCmec type I to V based on the detection of the ccr genes and the mec complex. The assay was evaluated on 212 clinical MRSA isolates from various countries, associated with MLST clonal complexes (CC) 1, 5, 8, 22, 30, and 45, as well as pig-associated CC398. When comparing the real-time PCR assay with traditional methods, the correct SCCmec element was identified in 209 (99%) of the 212 MRSA isolates. The new assay enables high-throughput analyses for SCCmec on large strain collections.