Blocking GM-CSF receptor α with mavrilimumab reduces infiltrating cells, pro-inflammatory markers and neoangiogenesis in ex vivo cultured arteries from patients with giant cell arteritis.

BACKGROUND: Effective and safe therapies are needed for the treatment of patients with giant cell arteritis (GCA). Emerging as a key cytokine in inflammation, granulocyte-macrophage colony stimulating factor (GM-CSF) may play a role in promoting inflammation in GCA. OBJECTIVES: To investigate expression of GM-CSF and its receptor in arterial lesions from patients with GCA. To analyse activation of GM-CSF receptor-associated signalling pathways and expression of target genes. To evaluate the effects of blocking GM-CSF receptor α with mavrilimumab in ex vivo cultured arteries from patients with GCA. METHODS: Quantitative real time PCR, in situ RNA hybridisation, immunohistochemistry, immunofluorescence and confocal microscopy, immunoassay, western blot and ex vivo temporal artery culture. RESULTS: GM-CSF and GM-CSF receptor α mRNA and protein were increased in GCA lesions; enhanced JAK2/STAT5A expression/phosphorylation as well as increased expression of target genes CD83 and Spi1/PU.1 were observed. Treatment of ex vivo cultured GCA arteries with mavrilimumab resulted in decreased transcripts of CD3ε, CD20, CD14 and CD16 cell markers, and reduction of infiltrating CD16 and CD3ε cells was observed by immunofluorescence. Mavrilimumab reduced expression of molecules relevant to T cell activation (human leukocyte antigen-DR [HLA-DR]) and Th1 differentiation (interferon-γ), the pro-inflammatory cytokines: interleukin 6 (IL-6), tumour necrosis factor α (TNFα) and IL-1ß, as well as molecules related to vascular injury (matrix metalloprotease 9, lipid peroxidation products and inducible nitric oxide synthase [iNOS]). Mavrilimumab reduced CD34 + cells and neoangiogenesis in GCA lesions. CONCLUSION: The inhibitory effects of mavrilimumab on multiple steps in the GCA pathogenesis cascade in vitro are consistent with the clinical observation of reduced GCA flares in a phase 2 trial and support its development as a therapeutic option for patients with GCA.

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.

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.

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.