High prevalence of CTX-M-15 and first report of CTX-M-3, CTX-M-22, CTX-M-28 and plasmid-mediated AmpC beta-lactamase producing Enterobacteriaceae causing urinary tract infections in Bosnia and Herzegovina in hospital and community settings.

AIM: To investigate molecular epidemiology of extended-spectrum ß-lactamase/ESBL and plasmid-mediated AmpC ß-lactamase/pAmpC producing Gram-negative bacteria causing urinary tract infections (UTIs) in Zenica-Doboj Canton, Bosnia and Herzegovina, in the period Decembar 2009-May 2010. METHODS: Antibiotic susceptibility was determined by disc diffusion and broth microdilution according to CLSI guidelines. Double-disk synergy test was performed in order to screen for ESBLs/pAmpC beta-lactamases. PCR was used to detect bla(ESBL)/bla(ampC)/bla(carb) genes. Genetic relatedness of the strains was determined by pulsed-field-gel-electrophoresis (PFGE). RESULTS: Among 85 patients with UTIs caused by ESBL producing isolates, 44 (51.8%) were from in-patients and 41 (48.2%) from outpatients. Klebsiella spp. was the most frequently isolated from in-patients, in 28 (63.6%) cases. Among outpatients, Klebsiella spp./Escherichia coli were the most frequently isolated, in 19 (46.3%)/16 (39.0%) cases. Twenty-one (75.0%) from hospital and nine (47.4%) from outpatient Klebsiella spp. isolates were positive for blaTEM, whereas 27 (96.4%) from in-patients and 6 (31.6%) from outpatient were bla(CTX-M) positive (18 hospital and five outpatient isolates were encoding bla(CTX-M-15)). One Klebsiella oxytoca and one Enterobacter cloacae inpatient isolates were positive for blaCTX-M-28. One Klebsiella pneumoniae outpatient isolate were positive for bla(CTX-M-22) and one E. coli for bla(CTX-M-3). One hospital Proteus mirabilis strain was positive for bla(CMY-2) and two Klebsiella spp. strains for blaDHA-1, whereas two E. coli, one K. oxytoca and one Proteus vulgaris outpatient strains were positive for bla(CMY-2). CONCLUSION: Identification of bla(CTX-M-3), bla(CTX-M-22) and bla(CTX-M-28) among Enterobacteriaceae is uncommon. In this study we report the emergency of CMY-2 and DHA-1 plasmid-mediated beta-lactamases.

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.

Detection of anti-SARS-CoV-2 antibodies and its seroprevalence in Zavidovici municipality of Zenica-Doboj Canton, Bosnia and Herzegovina.

Objectives: Improved serological detection of specific antibodies against SARS-CoV-2 could help estimate the true number of infections. Methods: A total of 443 serum samples provided by unvaccinated patients of all ages with unknown COVID-19 status that were originally submitted for routine screening or clinical management from outpatient laboratory during the March-April 2021 (third wave) were collected. Seroprevalence of IgM/IgG antibodies was determined by lateral flow immunoassay (Tigsun, Beijing, China). Results: Among 443 serum samples, 186 (42.0%) were positive (incidence of 5.2/1000) with slight predominace of females, 104 (55.9%), highest seropositivity in 25-50 and 51-64 years age groups, 61 (32.8%) and 57 (30.6%), respectively (P < 0.05); rural population was more prevalent, 101 (54.3%) (P < 0.05) and active workers, 86 (41.1%). Almost equal number of patients was with or without symptoms, 48.4% and 51.6%, respectively. For the comparison, in the same period it was registered 296 (out of 855; 34.6%) PCR SARS-CoV-19 positive persons (incidence of 8.2/1000) with the higher gender (females) and the highest age prevalence in 51-64 years age group (36.8%). In the period March 2020-June 2021, it was registered 804 (out of 3323; 24.2%) (incidence of 22.3/1000) PCR SARS-CoV-19 positive persons with no significant gender and significant age difference (25-50 and 51-64 years group, respectively). Conclusion: In the regions with high prevalence/incidence of SARS-CoV-2 in the general population (Bosnia and Herzegovina is on the World top on the number of deaths) seroprevalence measuring can help tracking the spread of disease.

Detection and characterisation of extended-spectrum and plasmid-mediated AmpC ß-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina.

Extended-spectrum ß-lactamases (ESBLs) hydrolyse extended-spectrum cephalosporins (ESC) and aztreonam. As ESBL-producing organisms have been identified in food producing animals, the aim of our study was to detect and analyse such Escherichia coli isolates from poultry. Antibiotic susceptibility of the isolates was determined with disk-diffusion and broth microdilution methods. ESBLs were detected with the double-disk synergy and inhibitor-based test with clavulanic acid. The transferability of cefotaxime resistance was determined with conjugation experiments, and genes encoding ESBLs, plasmid-mediated AmpC ß-lactamases, and quinolone resistance determinants identified by polymerase chain reaction. The study included 108 faecal samples (cloacal swabs) from 25 different poultry farms in the Zenica-Doboj Canton, Bosnia and Herzegovina. Of these, 75 (69.4 %) were positive for E. coli, of which 27 were resistant to cefotaxime, amoxicillin, cefazoline, and cefriaxone, and susceptible to imipenem, meropenem, ertapenem, and amikacin. All 27 cefotaxime-resistant isolates were positive in double-disk synergy and combined disk tests. Eighteen isolates transferred cefotaxime resistance to E. coli recipient. Twenty-one isolates were positive for the bla CTX-M-1 cluster genes and seven for bla CTX-M-15. Fourteen were positive for the bla TEM genes. The most frequent plasmid incompatibility group was IncFIB, whereas IncFIA and Inc HI1 were present in only a few isolates. Two different sequence types (STs) were identified: ST117 and ST155. The emergence of ESBL-producing E. coli in farm animals presents a public health threat, as they can colonise the intestine and cause infections in humans.

Antibiotic resistance in Enterobacter cloacae strains with derepressed/partly derepressed/inducible AmpC and extendedspectrum beta-lactamases in Zenica-Doboj Canton, Bosnia and Herzegovina.

Aim To investigate the prevalence of derepressed/partly derepressed/inducible and ESBL/AmpC-producing Enterobacter cloacae isolates and treatment options for infections associated with those isolates. Methods Antibiotic susceptibility was determined by disc diffusion and broth microdilution according to CLSI guidelines. Doubledisk synergy test (DDST) was performed in order to screen for ESBLs and combined disk test with phenylboronic acid to detect AmpC ß -lactamases. PCR was used to detect blaESBL/blacarb genes. Genetic relatedness of the strains was determined by pulsed-fieldgel-electrophoresis (PFGE). Results Among 14 isolates with the ESBL positive E. cloaceae producing isolates, four (28.6%), nine (64.3%) and one (7.1%) isolates were derepressed/partly derepressed and inducible AmpC producers. Eleven (out of 14) isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, aminoglycosides and fluoroquinolones. All isolates were susceptible to imipenem and meropenem, 79% to cefepime. Five (out of 14; 35.7%) isolates (four derepressed and one inducible AmpC carrying E. cloaceae) were negative in phenotypic test for ESBLs, but positive for broad spectrum TEM-1 ß-lactamase. One (out of four derepressed) also produced CMY-2 ß-lactamase. Four (out of nine) partly derepressed isolates were positive with the DDST, but did not yield PCR products with primers targeting TEM, SHV and CTX-M beta-lactamases. Four positive partly derepressed isolates carried a blaCTX-M-1 gene, two blaOXA-1 one blaCTX-M-15, OXA-1 and one blaCTX-M-28, OXA-1 (n=1). Conclusion Microbiology laboratories must be able to detect and recognize AmpC-carrying isolates in a timely manner, especially those that are falsely susceptible in vitro to drugs that may be consideredfor therapy of infected patients.

AUTHOR'S CORRECTION: Molecular epidemiology and antimicrobial susceptibility of AmpC- and/or extended-spectrum (ESBL) ß-lactamaseproducing Proteus spp. clinical isolates in Zenica-Doboj Canton, Bosnia and Herzegovina.

Volume 13, no. 2, p. 103-112, 2016. Page 103: The byline should appear as shown above.

Molecular characteristics and antibiotic resistance of Acinetobacter baumanniibeta-lactamase-producing isolates, a predominance of intrinsic blaOXA-51, and detection of TEM and CTX-M genes.

BACKGROUND/AIM: The aim of this study was to determine the molecular characteristics and antibiotic resistance of 13 (10 inpatient and three outpatient) Acinetobacter baumannii beta-lactamase-producing isolates collected in Bosnia and Herzegovina between December 2009 and May 2010. MATERIALS AND METHODS: Susceptibility testing was performed by disk diffusion and broth microdilution methods. The modified Hodge and combined disk test with EDTA/phenylboronic acid was used to screen for carbapenemase production. Production of extended spectrum beta-lactamases (ESBLs) was determined by double-disk synergy test. PCR was used to detect blaESBL/blacarb genes. RESULTS: Ten (22.2%) inpatient and three (13.6%) outpatient isolates produced beta-lactamases, ESBLs, or oxacillinases. More than 50% of the isolates showed multidrug resistance. Resistance rates to gentamicin and ciprofloxacin of the inpatients and outpatients were 80.0%, 60.0%, 75.0%, and 25.0%, respectively. MICs of carbapenems for resistant isolates ranged from 32 to >256 µg/mL. All imipenem-resistant Acinetobacter baumannii strains contained blaOXA-51. Three of the 10 inpatient isolates and one outpatient isolate containing blaOXA-51 additionally produced other beta-lactamases (TEM/CTX-M/OXA-1). None of the inpatient or outpatient isolates were positive for other carbapenemases, especially acquired oxacillinases (blaOXA-23/blaOXA-24/blaOXA-58/blaOXA-143). CONCLUSION: Production of blaOXA-51 presents an emerging threat in imipenem-resistant Acinetobacter spp. from Bosnia and Herzegovina.

Molecular epidemiology and antimicrobial susceptibility of AmpC- and/or extended-spectrum (ESBL) ß-lactamaseproducing Proteus spp. clinical isolates in Zenica-Doboj Canton, Bosnia and Herzegovina.

Aim To investigate prevalence, antimicrobial susceptibility, molecular characteristics, and genetic relationship of AmpC- and/or extended spectrum beta lactamase (ESBL)- producing Proteus spp. clinical isolates in Zenica-Doboj Canton, Bosnia and Herzegovina. Methods Antibiotic susceptibility was determined by disc diffusion and broth microdilution methods according to CLSI guidelines. Double-disk synergy test was performed in order to screen for ESBLs, and combined disk test with phenylboronic acid to detect AmpC ß -lactamases. PCR was used to detect blaESBL/blacarb genes. Genetic relatedness of the strains was determined by pulsed-fieldgel-electrophoresis (PFGE). Results Eleven ESBL-producing isolates were included in the study (six inpatients and five outpatients). Susceptibility rate to amoxicillin-clavulanic acid, imipenem and meropenem was 100%. Resistance rate to cefuroxime was 100%, gentamicine 90.9%, piperacillin/tazobactam 81.8%, cefotaxim, ceftriaxone and ceftazidime 72.7%, cefoxitine and ciprofloxacine 63.6% and to cefepime 45.5%. In five (out of 11) isolates multi-drug resistance (MDR) to cephalosporins, cefamicines, amynocligosides and fluoroquinolones was detected. Besides TEM-1 which was detected in all isolates, CTX-M+OXA-1 ß-lactamases were detected in seven (out of 11; 63.6%) isolates (five blaCTX-M-1 and two blaCTX-M-15 genes), and CMY-2 ß-lactamase in two isolates. PFGE showed no genetic relatedness. Conclusion Because of high prevalence of MDR strains in epidemiologically unrelated patients with AmpC- and/or ESBL producing Proteus spp. infection, further surveillance is needed. Molecular characterization and strain typing, or at least phenotypic test for AmpC/ESBL production is important for appropriate therapy and the detection of sources and modes of spread, which is the main step in order to design targeted infection control strategies.

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.