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The aim of this study was to evaluate the antibacterial efficacy of active irrigation techniques: Er, Cr:YSGG laser-activated irrigation (LAI), passive ultrasonic irrigation (PUI), RinsEndo®, and conventional syringe irrigation against intracanal Enterococcus faecalis (E. faecalis). Root canals of 100 human extracted teeth were instrumented, sterilized in plasma, contaminated with E. faecalis and incubated for ten days. The samples were randomly distributed into five experimental groups (n=20 each): I: LAI by Er, Cr:YSGG (1, 25W, 20Hz) for 4x5 s ; II. Passive ultrasonic irrigation (PUI) for 60 s ; III. RinsEndo® system for 60 s, IV. 30-gauge syringe irrigation for 60 s, and one positive control group (n=10). In all experimental groups, 2.5% sodium hypochlorite (NaOCl) was used as an irrigant while the positive controls were rinsed with saline. The root canals were sampled by flushing with saline solution before and after the treatments, serially diluted and cultured. The presence or the absence of E. faecalis in the root canals was checked by polymerase chain reaction. There were no difference between three active irrigation techniques (P > 0.05), which were more effective than the conventional syringe irrigation (P

Since the first carbapenem-resistant Klebsiella pneumoniae strain was isolated in 2008, Enterobacteriaceae with reduced susceptibility to one or more carbapenems have emerged sporadically in different geographical regions in Croatia. These observations gave rise to a multicenter study on carbapenem resistance in Enterobacteriaceae from Croatia. Fifty-seven carbapenem-non-susceptible strains of Enterobacteriaceae were collected during 2011–2012 from four large hospital centres in Croatia. Overall, 36 strains produced VIM-1 β-lactamase, three produced NDM-1, and one produced KPC-2. A high degree of clonal relatedness was observed in Enterobacter cloacae and Citrobacter freundii strains, in contrast to K. pneumoniae strains. Bla VIM genes were located within class1 integron which contained genes encoding resistance to aminoglycosides (aac A4 ). The study found strong association between bla VIM and qnr B6 and between bla NDM and qnr A6 genes.

One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the bla VIM-2 gene located in a novel class 1 integron composed in its variable part of the bla VIM-2 - bla oxa-10 -Δ qacF-aacA4 genes. Metallo-β-lactamase-producing strains belonged to sequence types ST235 and ST111.

Background: Some of the genes belonging to cag pathogenicity island ( cag PAI) in Helicobacter pylori were found to be associated with an increased severity of gastric mucosal inflammation that might lead to the development of gastroduodenal disease. Aim: The aim of our study was to define a group of patients based on the frequency of virulence genes of cag PAI island and comparison with pathohistological alterations of gastric mucosa who need to be subjected to further eradication therapy after previous unsuccessful eradication therapy and in spite of benign endoscopic findings. Material and methods : In total 103 H. pylori isolates were analysed. Genes encoding virulence factors were detected by PCR with primers for 10 loci in cag PAI: Ap cag (cagA promotor region), cagA1 , cagA2 , cagA3 , cagM , cagT , cagE , LEC , tnpA and tnpB . The patients who provided isolates were classified into three clinical categories: non-ulcer dyspepsia (n=69), erosio/ulcus ventriculi (n=22) and erosio/ulcus duodeni (n=12). Results: 16 strains (15.5%) were negative for all tested genes. 87 (84.5%) of the isolates had parcially deleated cag PAI. None of the isolates possessed all 10 genes. The frequency of single cag PAI genes were as follows: Apcag 63.1%, cagA1 71.8%, cagA2 69.9%, cagA3 5.8%, cagM 71.8%, cagE 75,7%, cagT 68% tnpA 9.7%, tnpB 7.8% i LEC 48.5%. No statistically significant difference was observed between the presence of any cag PAI genes and endoscopic diagnosis (p>0.16). The presence of CagA2 , Apcag and cagM showed statistically significant correlation with higher level of patohistological parameters of gastritis (p

Background Lasers have been recommended in final root canal disinfection protocol, however, there is no clear evidence about their efficacy against bacteria in biofilms. The aim of the study was to evaluate and compare the disinfection effect of antimicrobial photodynamic therapy (aPDT), Nd:YAG laser and QMiX solution against Enterococcus faecalis biofilm. Methods The study sample consisted of 65 dentine slices, which were inoculated with E. faecalis and incubated anaerobically for three weeks. The dentine discs were randomly allocated to one of the following experimental groups: aPDT (100 mW, 10 mg/ml phenothiazinium chloride, 1 min), Nd:YAG laser (2 W, 15 Hz, 4 × 5 s), QMiX solution (1 min). Positive controls did not receive any treatment and negative controls were treated with 5.25% NaOCl. To harvest surviving adherent cells, each dentine sample was transffered to a test tube containing of TSB, serial ten-fold dilutions were made and aliquot of 1 ml was plated onto blood agar plates and incubated for 48 h. Colony forming units grown were counted and transformed into actual counts based on the dilution factor. The remaining viable cells after each protocol were analysed by FISH. Results The aPDT and the QMiX solution were equally effective, with the reduction rate of E. faecalis CFUs of 98.8% and 99.3% respectively ( p = 1.107). The Nd:YAG laser caused 96% reduction of E. faecalis ( P Conclusion The aPDT and the QMiX solution showed similar antibacterial efficacy against old E. faecalis biofilm, followed by Nd:YAG irradiation.

Background: In the last few years, Klebsiella pneumoniae strains producing K. pneumoniae carbapenemase (KPC) enzymes have emerged as important multidrug-resistant pathogens in hospitalized patients. This report describes KPC-producing isolates collected through the Croatian antimicrobial resistance surveillance program in the early stage of their dissemination in Croatia. Materials and Methods: Forty-eight KPC-producing K. pneumoniae isolates, collected during a period from February 2011 to August 2013, were analyzed in this study. Antimicrobial susceptibility profiles were determined using disk diffusion and E-test. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. Identification of β-lactamase genes and associated antibiotic resistance mechanisms was performed by polymerase chain reaction and positive products were sequenced. Localization of blaKPC was investigated by S1 PFGE and Southern hybridization. Results: Of 40 participating centers in Croatia, KPC isolates were recorded in 9 of them. They all had multidrug-resistant phenotype, but showed varying levels of resistance to carbapenems. All isolates displayed ST258, and PFGE showed that all but one were closely related. All isolates harbored blaKPC-2. Isolate with a unique PFGE pattern produced TEM-1, while others produced TEM-116. All isolates harbored blaSHV-11, but were negative for blaCTX-M and blaAmpC genes. All isolates contain one KPC-harboring plasmid, ranging in size from ∼60 to ∼210 kb, characterized as FIIs and IncR. Conclusion: This report describes that the early stage of KPC-producing K. pneumoniae dissemination in Croatia is associated with a prolific PFGE type belonging to ST258. So far, the spread of an outbreak strain is limited to the northwest region of the country.

Vancomycin and ciprofloxacin were often used in the therapy of infections associated with Bacillus cereus.Four B. cereus food and clinical isolates were chosen for determination of time-kill curves and postantibiotic effects (PAE) of ciprofloxacin and vancomycin.According to the minimum inhibition concentration (MIC), breakpoints defined by CLSI for Staphylococcus spp. were all four strains intermediate for vancomycin (MIC = 4 μg/ml) and sensitive to ciprofloxacin (MIC = 0.2 μg/ml) except the strain Bc63 resistant to the last antimicrobial (MIC = 1.6 μg/ml). The lowest CFU values of tested strains were reached after 3-5 hours of exposure to 4 × MIC of vancomycin, and after 6-7 hours exposure to 10 × MIC of ciprofloxacin. The maximum reduction of the CFU in the presence of vancomycin and ciprofloxacin was about 2.46 log10 and 2.48 log10, respectively. The average duration of the PAE of vancomycin and ciprofloxacin was 0.94 and 1.60 hours, respectively. The statistically significant differences between PAEs induced with 3 × MIC, 4 × MIC and 8 × MIC of vancomycin were observed (P 0.05). Both antibiotics did not affect the sporulation of tested bacterial strains.The differences in PAE duration were strain and antimicrobial dependent.

The aim of this study was to compare the sensitivity and specificity of six different beta-lactam antibiotics using five phenotypical tests for detection of extended spectrum beta-lactamases (ESBLs) based on synergism of beta-lactam antibiotics and clavulanate. Experiments were performed on a set of 80 Klebsiella pneumoniae strains and 105 Escherichia coli strains with previously characterized ESBLs (SHV, TEM and CTX-M). ESBLs were detected by five different phenotypical methods: MIC (minimum inhibitory concentration) determination of beta-lactam antibiotics with and without clavulanate, double-disk synergy test (DDST), inhibitor-potentiated disk-diffusion test (IPDDT), CLSI-Clinical and Laboratory Standard Institution (former NCCLS) combined-disk-test, and modified MAST-disk-diffusion test (MAST-DD-test). Seven antibiotics were tested as indicators of ESBL production: ceftazidime, cefotaxime, ceftriaxone, aztreonam, ceftibuten, cefpodoxime and cefepime. Ceftazidime and aztreonam were the best indicators for SHV-5, SHV-12 and TEM beta-lactamases whereas cefotaxime and ceftriaxone were the most sensitive in detection of SHV-2 and CTX-M beta-lactamases in DDST, IPDDT and CLSI test. MIC determination of beta-lactam antibiotics with and without clavulanate was the most sensitive method. DDST was the least sensitive test. Double-disk synergy test, which is the most frequently used test for detection of ESBLs in routine laboratories, was the least sensitive independently of the indicator antibiotic. Since MIC determination is a very laborious and time consuming method, we would recommend the NCCLS combined disk test or IPDD test for detection of ESBLs in routine laboratories with 5 mm zone augmentation breakpoint.

The aim of the study was to assess the antibacterial efficacy of photon-initiated photoacoustic streaming (PIPS) using an Er:YAG laser and sonic activated irrigation combined with QMiX irrigant or sodium hypochlorite against Enterococcus faecalis intracanal biofilm. Root canals of 91 human extracted teeth with single straight canal were instrumented, sterilized in plasma, contaminated with Enterococcus faecalis and incubated at 37°C for 15 days. The infected teeth were then randomly distributed into six experimental groups: G1: PIPS/Er:YAG laser (wavelength: 2940 nm, pulse energy: 20 mJ, 15 Hz, pulse duration: 50 μs, 3x20 s) with the QMiX irrigant ; G2: PIPS/Er:YAG laser activated 2.5% NaOCl ; G3: sonic activated irrigation (EndoActivator system) for 60 s with the QMiX irrigant ; G4: sonic activated irrigation for 60 s with 2.5% NaOCl ; G5: 30-gauge needle irrigation with the QMiX irrigant ; G6: 30-gauge needle irrigation with 2.5% NaOCl. The positive control group was rinsed with sterile saline solution. The root canals were sampled by flushing with saline solution at baseline and after the treatments, serially diluted and cultured. The number of bacteria in each canal was determined by plate count. The presence and the absence of Enterococcus faecalis in root canals were also demonstrated by polymerase chain reaction (PCR) and the pattern of the bacteria colonization after 15 days was visualized by scanning electron microscopy. The results were analysed by the Wilcoxon test (intragroup analysis) and the Mann-Whitney U test (intergroup analysis). There was significant reduction in the bacterial population for all groups (p

The aims of this study were to induce in vitro metronidazole resistance in nim-negative Bacteroides fragilis group strains and to determine the lactate dehydrogenase (LDH) activity of the induced strains. A collection of B. fragilis group strains were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution technique. The presence of nim genes was screened by PCR. A sample of 52 nim-negative metronidazole-susceptible strains were selected at random and were exposed to metronidazole in the resistance induction experiment. LDH activity was measured by spectrophotometry. Of the 52 selected strains, 12 (23.1%) acquired resistance to metronidazole. MICs ranged from 8mg/L to 96mg/L. Eight of the twelve induced strains displayed decreased LDH activity, whilst only one expressed a significant increase in LDH activity with LDH values of 49.1U/mg and 222.0U/mg, respectively. In conclusion, in vitro induction of metronidazole resistance could be selected in nim-negative B. fragilis group strains. A statistically significant decrease in LDH activity was in contrast to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of pyruvate-ferredoxin oxidoreductase (PFOR). These findings could be explained if the induction caused only physiological and not genetic changes. We believe that genetic mutations in the B. fragilis strain that demonstrated an emergent increase in LDH activity were responsible for the increased activity.

Background: Acinetobacter spp. is an opportunistic pathogen that has demonstrated increasing relevance in nosocomial infections. Carbapenem-resistant strains have been reported worldwide. Methods: Since 2014, screening for metallo-β-lactamases (MBLs) in all Acinetobacter spp. isolates using phenotypic methods and PCR has been implemented at the University Hospital Center Zagreb. Results: The bacterial strain was isolated from the drain of a child hospitalized in a paediatric intensive care unit and identified as Acinetobacter guillouiae using a MALDI TOF automated system. The strain was resistant to meropenem, ceftazidime, cefotaxime, ceftriaxone, cefepime, sulbactam/ampicillin, gentamicin and ciprofloxacin, intermediately susceptible to piperacillin/tazobactam and imipenem, and susceptible to amikacin and colistin. The Hodge test and combined disk test with EDTA were positive. The MICs of meropenem and imipenem were not reduced by cloxacillin, but a small reduction of two dilutions was observed following the addition of sodium chloride, which indicated that OXA-58 was produced. PCR and sequencing of chromosomal DNA from boiled colonies revealed blaOXA-58 and blaNDM-1 genes. Conclusion: This is the first report of NDM-1 in Acinetobacter spp. in Croatia. The early detection of these genes will aid in the prevention and in the achievement of adequate infection control by limiting the spread of these organisms.

OBJECTIVES: The objectives of this study were (i) to investigate the genetic background of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from Croatia and (ii) to monitor the prevalence of Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) among these isolates.METHODS: Eighty-two hospital-acquired MRSA bloodstream isolates, collected in 2001 and 2002 in Croatia, were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). The presence of genes encoding PVL and TSST-1 was investigated by real-time PCR.RESULTS: All strains were multiresistant and were distributed among 16 different similarity groups as determined by PFGE. Two of the groups, groups H and K, harboured the majority of the MRSA strains with 52 and 12%, respectively. The predominant SCCmec type found among the isolates was type I (89%). Eleven per cent of the strains harboured a modified SCCmec type III, which contained, in contrast to the regular type III, an additional dcs region. One strain harboured a novel SCCmec type, containing the ccrC gene in combination with the mecI gene, the dcs region, the locus between pI258 and Tn554 (locus E) and the locus between Tn554 and orfX (locus F). MLST showed the presence of ST111-MRSA-I and ST247-MRSA-I among Croatian MRSA isolates. All isolates were negative for both PVL and TSST-1.CONCLUSIONS: These results indicate the emergence of ST111-MRSA-I and ST247-MRSA-I in Croatia among MRSA bloodstream isolates. The virulence factors PVL and TSST-1 were not present among these isolates.

Plasmid-encoded resistance to broad-spectrum cephalosporins and aztreonam is becoming a widespread phenomenon in clinical medicine. These antibiotics are inactivated by an array of different extended-spectrum beta-lactamases (ESBLs) which have evolved by point mutations of parental TEM or SHV beta-lactamases. In a previous study conducted during 1994-1995, SHV-2, SHV-2a and SHV-5 beta-lactamases were found among Klebsiella pneumoniae isolates in Dubrava University Hospital. High prevalence of ESBLs among K. pneumoniae strains in this hospital (20%) required further investigation. In this investigation, beta-lactamases from 42 K. pneumoniae strains collected in 1997 and 15 in 2004 from Dubrava University Hospital, were characterized in order to study the evolution of plasmid-encoded resistance to extended-spectrum cephalosporins and aztreonam in that hospital over a prolonged study period. Susceptibility to antibiotics was determined by disk-diffusion and broth microdilution method. beta-lactamases were characterized by isoelectric focusing, determination of hydrolysis of beta-lactam substrates, polymerase chain reaction and sequencing of bla(SHV) genes. All K. pneumoniae strains and their Escherichia coli transconjugants produced beta-lactamase with an isoelectric point of 8.2. Based on sequencing of bla(SHV) genes enzymes of all transconjugants were identified as SHV-5 beta-lactamase which conferred on the producing isolates high level of ceftazidime and aztreonam resistance. In this study, an outbreak of nosocomial infections caused by SHV-5 producing K. pneumoniae was described in 1997 which evolved to endemic spread of SHV-5 producing K. pneumoniae due to multiple plasmid transfer in the Dubrava University Hospital. The strains from 1997 and 2004 were not clonally related. Hospital hygiene measures should be applied in order to control the spread of epidemic strains through the hospital wards and the consumption of the broad-spectrum cephalosporins needs to be restricted to reduce the selection pressure which enables the proliferation of ESBL producers in hospital.

The purpose of the study was to evaluate the efficacy of antimicrobial photodynamic therapy (aPDT) used as an adjunct to the endodontic re- treatment in the eradication of microorganisms from previously filled root canals. The study sample consisted of 21 randomly selected patients with root filled and infected root canal system with chronic apical periodontitis on incisors or canines, who have had previously endodontic treatment, more than two years ago. Microbiological samples from the root canals were collected after accessing the canal, following the endodontic re-treatment and after the aPDT procedure. During instrumentation, the root canals were irrigated with 2, 5% sodium hypochlorite (NaOCl), and the final irrigation protocol included 17% ethylenediaminetetraacetic acid followed by NaOCl and saline solution. Root canals were filled with a photosensitizing agent and irradiated with a diode laser (100 mW) for 1 min. Microbiological samples from the root canals were cultivated, and the identification was done by micromorphology, macromorphology and different API strips as well as bacterial counts (colony forming units). Fourteen bacteria species were isolated from the root canals, with a mean value of 4.57 species per canal. There was statistically significant reduction in the number of bacteria species and bacterial counts per canal after each step of the root canal treatment (p

Background Residents of nursing homes (NHs) are often hospitalized and could present a potential reservoir for methicillin-resistant Staphylococcus aureus (MRSA). The aim of the study was to determine the prevalence for MRSA carriage in residents and staff in Croatian NHs and to characterize MRSA strains using genotyping techniques. Methods A cross-sectional study was performed among 877 residents and staff of 7 NHs representing 3 major Croatian regions. Nasal swabs from residents and staff and other samples from residents with invasive devices were obtained. Identified isolates were submitted to susceptibility testing and genotyping with SCC mec typing, S aureus protein A (spa) locus typing, and pulsed-field gel electrophoresis (PFGE). Results The overall prevalence of MRSA colonization was 7.1% (95 confidence interval, 5.4%-8.8%), ranging from 0% to 28.8%. Four MRSA isolates were found in NH staff. All MRSA isolates were negative for Panton-Valentine leukocidin–encoding genes. SCC mec type II was found in 32 MRSA strains; SCC mec IV, in 27 strains; SCC mec I, in 3 strains. The predominant spa type was t008, found in 49 strains; PFGE analysis revealed 2 major clonal groups. Conclusions MRSA strains were found to be colonizing residents and staff of 7 NHs in Croatia. Our study demonstrates the spread of 2 clones within and among Croatian NHs. The data presented here provide an important baseline for future surveillance of MRSA in NH.

Carbapenems are potent β-lactam antibiotics used to treat serious infections in hospital settings. In comparison to penicillins, cephalosporins or β-lactam/β-lactamase inhibitor they have broad antimicrobial spectrum that includes Gram-positive (e.g. imipenem, doripenem) and Gram-negative bacteria (e.g. meropenem, ertapenem). Imipenem and meropenem have better activity against P. aeruginosa while imipenem and doripenem have better activity than meropenem against Acinetobacter baumannii. Doripenem has the lowest MIC against P. aeruginosa and A. baumannii in comparison to imipenem and meropenem, and it is least susceptible to hydrolysis by carbapenemases. To act on PBPs, carbapenems have to enter the wall of Gram-negative bacteria through outer membrane proteins (porins). Binding to different PBPs they inhibit the synthesis of cell wall finally leading to the death of bacterium [1]. Carbapenem resistance in Gram-negative bacteria can be the consequence of the production of a β-lactamase, expression of efflux pumps, porin loss and alterations in PBPs. Since β-lactams, including carbapenem-like compounds, are natural products of several environmental bacteria and fungi, it is supposed that other bacteria started to produce their intrinsic β-lactamase to give them selective advantage for survival. Thus, several genes encoding different carbapenemases can be found in environmental bacteria like Bacillus anthracis, Serratia fonticola, Pseudomonas cepacia or Acinetobacter spp. as part of their chromosome [1, 2]. Further step in this evolution of resistance was the escape of carbapenemase encoding genes to mobile genetic elements (plasmids, transposons) providing possibility of successful horizontal spread of resistance genes even between different genera [3]. Since this discovery, carbapenemases became global problem. According to the Ambler classification (based on structural similarities) they belong to the class A, B and D [1]. Class A carbapenemases contain serine at their active site and are capable of hydrolyzing all β-lactams, including aztreonam. In this group of carbapenemases, Sme (Sme-1 to Sme-3), IMI (IMI-1-3), NmcA and SFC-1 enzymes are mostly chromosomally encoded, while KPC (KPC- 2 to KPC-13) and GES (GES 1-GES-20) are plasmid encoded. Dominant carbapenemase from this group is KPC, identified in 1996 in North Caroline, USA, now causing many regional outbreaks, with endemicity in northeastern part of the USA, Israel, China, Porto Rico, Colombia, Israel, Greece, and becoming more and more prevalent throughout the Europe [4]. Beside K. pneumoniae, represented by a predominant clone (ST258), it has been found in other Enterobacteriaceae, as well as in P. aeruginosa and A. baumannii–calcoaceticus complex. It is sometimes difficult to be recognized since MICs to carbapenems are in many cases lower than the breakpoints [2, 5]. Class B carbapenemases are also known as metallo-β-lactamase (MBL) since they contain metal ion(s) in their active site. Beside those chromosomally located in environmental bacteria (Bacillus cereus-BCI, BCII, Aeromonas spp-CphA, S. maltophilia-L1), acquired MBL encoding genes are often located in gene cassettes within integron, being part of a plasmid or chromosome. Firstly described acquired MBLs were in Japan in 1991, so called IMP-enzymes (there are now more than 30 derivatives) and are still dominant MBLs in Asian continent causing mainly sporadic outbreaks [6]. VIM-enzymes (there are now more than 30 derivatives) were firstly described in P. aeruginosa but later arisen to Enterobacteriaceae as well, and fastly spread over whole Europe, causing outbreaks in many Mediterranean countries (like Greece, Italy, Turkey). VIM metallo-β-lactamase are now the most prevalent carbapenemase spreading globally, and although largely connected to P. aeruginosa, now reported more often from Enterobacteriacea from Mediterranean countries, particulary Greece and Turkey, with the description of many panresistant strains [6, 7]. Another worrisome metallo-enzyme, arose from India in 2008, namely New-Delhi MBL (NDM-1 ; until now more than ten variants are described), and spread fastly over Indian subcontinet in next few years. NDM-enzymes are mostly associated with nonclonally related isolates of K. pneumoniae and E. coli, but also described in P. aeruginosa and A. baumannii [8]. Beside proven facts that those enzymes exist in isolates spreading in environment, and are carried in general population by enteric flora, the magnitude of the problem potentiates the huge population reservoir from Indian subcontinet and Middle Asia that moves across the world spreading further the resistance genes [9, 10, 11]. Another new source of those enzymes could be the Balkan region [12, 13]. Oxacillinases from molecular class D demonstrating carbapenemase activity are often found in Acinetobacter spp. They are divided into the most globally spread OXA-23 group, found also in environmental isolate of Acinetobacter spp. suggesting the possible natural and not nosocomial source of these genes, OXA-24 group, not so widespread as OXA-23, mostly described in Europe and USA, and OXA-58 group, described in several outbreaks all over the world [14]. The problem became more global with the discovery of OXA-48 in Enterobacteriaceae, particulary in K. pneumoniae and to lesser extent in E. coli, spreading all around the world but specifically in countries close to the Mediterranean Sea [14-16]. Carbapenemase producing Gram-negative bacteria can cause a wide spectrum of infections including bacteraemia, nosocomial pneumonia, wound infections, endocarditis, urinary tract infections. Those infections are often associated with treatment failures, long hospital stay and high mortality rates ; for example attributable mortality for carbapenem resistant P. aeruginosa infections ranged between 51.2 and 95% [17, 18].

The aim of the present study was to investigate whether real-time polymerase chain reaction (PCR) has a low identification rate for samples with low acid-fast bacilli (AFB) grades, including those obtained using bronchoscopy. When 50 smear-positive samples were compared by AFB score and PCR result, PCR had was 100% successful in identifying AFB 2ţ and AFB 3ţ samples. However, only 14 of 26 (52%) AFB 1ţ samples were identified. In paucibacillary smear-positive samples, PCR is not reliable enough to exclude tuberculosis, but in smear-positive patients with high AFB grades PCR can immediately change the clinical management of the disease.

Helicobacter pylori resistance to macrolides is possibly an important factor for the failure of macrolide therapy for H. pylori infection. The aim of this study was to assess the propensity of H. pylori to develop in vitro resistance to azithromycin. In 73 clinical isolates taken from patients before starting antimicrobial therapy of H. pylori infection, MIC was determined using an agar dilution method (Müller-Hinton agar with 7.5% unlysed horse blood, pH = 7.2, at 35°C, during 72 h in a humid microaerobic atmosphere). Each strain was first cultivated at half minimal inhibitory concentration (MIC) then in doubling concentrations until growth arrest. All experiments for induction of resistance were performed on the same media, incubation temperature, atmosphere and time of MIC determination. MIC interpretative standards for sensitivity, intermediate sensitivity and resistance of H. pylori to azithromycin were ≤2, 4 and ≥8 mg/l, respectively. Of 73 strains, 5 died during the experiments, and in the remaining 68 strains, serial passage with increasing azithromycin concentrations resulted in the development of resistance in 19 (26.9%) strains. Two strains had an MIC of 16 mg/l azithromycin. Thirty-three (48.5%) strains kept the same MIC or doubled their MIC, 16 (23.5%) strains had 4- to 16-fold MIC but still remained sensitive, 2 resistant strains had 128-fold MICs and 17 resistant strains had increased their MICs more than 128 times. Seventeen highly resistant strains (MIC >128 mg/l) were kept frozen at –70°C for 3 months in a brain-heart infusion broth with 15% glycerol. MIC was assessed again to determine the stability of resistance. Eleven strains kept MICs >128 mg/l, 2 became sensitive and 1 intermediate, but reverted easily, after only 2 passages, to an MIC of >128 mg/l azithromycin. Although macrolides are very active against H. pylori, the propensity to develop resistance in a high proportion of strains has a clear impact on the choice of the right combinations of macrolides with other agents as well as the dosage of the macrolide antibiotics.

Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a longitudinal global surveillance study to monitor in vitro data on microbial susceptibility in centers that prescribe meropenem. Results of the six years period (2002-2007) for the antimicrobial efficacy of meropenem compared to other broad-spectrum agents against Gram-negative and Gram-positive isolates collected at pediatric intensive care units of the University Hospital Center Zagreb in Croatia were reported.A total of 110 Gram-negative and 43 Gram-positive pathogens from pediatric specimens were tested. The minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI.There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. High resistance rates of K. pneumoniae to ceftazidime and gentamicin (50%) are a raising concern. Pseudomonas aeruginosa was the most resistant Gram-negative species with two (12%) of the strains resistant to meropenem, three (18%) to imipenem, 10 (47%) to gentamicin and six (35%) to piperacillin/tazobactam and ciprofloxacin. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria in pediatric population.The results indicate that meropenem has excellent potency and spectrum of activity despite being prescribed for a long time for the treatment of seriously ill patients, and still appears to be a reliable option for the initial empirical therapy of serious nosocomial infections in children. However, later studies have shown the emergence of carbapenem-resistant Gram-negative bacteria after 2008.

Uvod: Učinkovitost PCR metoda za direktnu identifikaciju mikobakterija postaje vrlo zanimljivo područje. Novija istraživanja utvrdila su smanjenu uspješnost identifikacije kada se PCR metoda primjenjuje na uzorcima iskašljaja s malim brojem prisutnih acidorezistentnih bacila (1+) u mikroskopskom preparatu. Cilj ovog istraživanja bio je utvrditi da li bi PCR bilo opravdano raditi samo na mikroskopski pozitivnim kliničkim uzorcima visokog pozitiviteta (2+ i 3+). Metode: Istraživanje je provedeno u Kliničkom bolničkom centru Zagreb. Real-time PCR metoda koja razlikuje Mycobacterium tuberculosis complex (MTBC) i ostale Mycobacterium spp. napravljena je na mikroskopski pozitivnim kliničkim uzorcima. Dobiveni rezultati evaluirani su u odnosu na intenzitet pozitiviteta mikroskopskog preparata (1+, 2+ i 3+). Osim molekularnih korištene su i konvencionalne metode (bojanje aureminom, te kultivacija mikobakterija na krutim i tekućim hranjivim podlogama). Results: Od ukupno pedeset dva mikroskopski pozitivna klinička uzorka (33 iskašljaja, 12 bronhoalveolarnih lavata, 6 bronhalnih aspirata i 1 pleuralni izljev), u 51 uzorku je u kulturi porastao MTBC, a u 1 netuberkulozna mikobakterija (NTM). PCR je imao 100%-tnu uspješnost identifikacije kod uzoraka s mikroskopskim pozitivitetom 2+ i 3+, dok je identifikacija bila uspješna kod svega 14 od 27 (52%) uzoraka s pozitivitetom 1+. Zaključak: Točna identifikacija mikobakterija u kliničkim uzorcima bila je manje uspješna kod uzoraka s niskim mikroskopskim pozitivitetom. Naše istraživanje potvrdilo je zapažanja da uspješnost PCR metode ovisi o količini mikobakterija prisutnih u kliničkom uzorku. Stoga PCR nije dovoljno pouzdan da bi se njime isključila dijagnoza tuberkuloze kod paucibacilarne plućne tuberkuloze, ali ga svakako treba koristiti kod kliničkih uzoraka s visokim pozitivitetom zbog brzog utvrđivanja dijagnoze. Potrebna su daljnja istraživanja kako bi se potvrdilo da za optimalnu učinkovitost (odnos cijenakorist) PCR metode treba raditi samo na kliničkim uzorcima visokog pozitiviteta (2+ i 3+).

Mycoplasma genitalium is considered the smallest self-replicating cell. It was first isolated in 1981, from 2 of 13 men with urethritis. Mycoplasma genitalium causes urethrtis, cervicitis and pelvic inflammatory disease. Because of difficulties in cultivation, the diagnosis is based exclusively on PCR methodology. The recommended therapy for Mycoplasma genitalium infections is azithromycin or doxycycline. Developement of macrolide resistance was shown to correlate with treatment failure. Key words: Mycoplasma genitalium,urethritis, molecular diagnosis, therapy

Zbog starenja stanovništva broj starijih osoba kojima je potrebna skrb ukroničnim zdravstvenim ustanovama (LTCF) diljem Europe i drugdje, i dalje raste značajno. Korisnici tih ustanova često imaju visok rizik za stjecanje i razvoj infekcija povezanih s zdravstvenim sustavom (HAI) . Oni su osjetljiviji zbog imunokompromitiranosti uzrokovane prirodnim starenjem, a uzrok može biti i (npr. šećerna bolest, demencija) i fizioloških ograničenja. Osim toga, oni često dobivaju antimikrobne lijekove za akutne i kronične infekcije i češće borave u bolnici što im povećava rizik da akviriraju multirezistentni mikroorganizam. Od 2009-2011, Europski centar za prevenciju i kontrolu bolesti (ECDC) financira projekt HALT. Cilj ovog projekta je razviti i implementirati protokol za nadzor HAI, uporabe antibiotika I praćenje rezistencije kako bi se utvrdili osnovne stope i prioritete za poboljšanje. Studija prevalencije (PPS) provedena je u 722 LTCFs u preko 25 europskih zemalja. Nije bilo ograničavanja minimalni broj ustanova koji je potreban za sudjelovanje. Studija prevalencije provedena je u razdoblju od svibnja do rujna 2010. Republika Hrvatska je sudjelovala u pps studiji 2010.

The aim of this study was to determine the incidence of fungal infections detected on autopsy in a group of 40 patients with hematologic malignancies treated with intensive chemotherapy or bone marrow transplantation, and to evaluate the risk factors for fungal infections. A control group included 38 patients with nonhematologic diseases and without granulocytopenia but with at least one of the known risk factors for fungal infections. Standard histopathological and microbiological methods were used. A higher incidence of invasive fungal infections was found in patients with hematologic malignancies as compared to the control group (p < 0.01). The predominant causes of fungal infections were Candida albicans and Aspergillus spp. The incidence of fungal infections caused by Aspergillus was higher (p < 0.05) in patients with hematologic malignancies than in the control group. The independent risk factors for fungal infections were fungal colonization, number of antibiotics and duration of antibiotic therapy, duration of fever and skin rash. A higher proportion of fungal infections was diagnosed on autopsy than during the patients' life (p < 0.01).

In February 2011, a 78-year-old male patient was admitted to Clinical Hospital Center Zagreb with subdural haematoma. Klebsiella pneumoniae with reduced susceptibility to carbapenems was isolated. PCR revealed the presence of bla(KPC), bla(TEM), and bla(SHV) genes. Sequencing of bla(KPC) gene identified K. pneumoniae carbapenemase (KPC)-2 beta-lactamase. The strain belonged to ST37 clone by multilocus sequence typing. Infection control efforts limited the spread of KPC-producing clone of K. pneumoniae in our hospital so far. To our knowledge, this is the first report of a KPC-producing K. pneumoniae in Croatia.

During 2010-2011, six Providencia spp. (five Providencia stuartii and one Providencia rettgeri) urine isolates with unusual resistance phenotype were collected from various hospital units at the University Hospital Split in Croatia. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins.The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method according to CLSI guidelines. A double-disk-synergy test (DDST) was performed to detect ESBLs. The transferability of cefotaxime resistance was determined by conjugation. The presence of genes encoding ESBLs was determined by PCR while genotyping of the isolates was performed by PFGE.All strains were positive for ESBL production by DDST. They were uniformly resistant to amoxycillin alone and combined with clavulanate, cefazoline, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, gentamicin and ciprofloxacin. P. stuartii strains transferred cefotaxime resistance to E. coli recipient strain with frequency ranging from 10-5 to 5x10-4. Five P. stuartii strains were positive for TEM and CTX-M β-lactamases while P. rettgeri was positive only for TEM β-lactamases. Five CTX-M producing isolates were shown to be clonally related.Continuous surveillance in tracking CTX-M-15- producing P. stuartii in the hospitals is necessary to prevent their spread to other hospitals and community. Global spread of ESBL positive Providencia spp all over the world is of great clinical concern.

To evaluate the antimicrobial effect of a diode laser irradiation, photo-activated disinfection (PAD), conventional and sonic activated irrigation with 2.5% sodium hypochlorite (NaOCl) on Enterococcus faecalis.Root canals of 120 human extracted teeth with single straight canals were prepared with ProTaper files, sterilized, contaminated with an E. faecalis suspension and incubated for 7 days. They were then randomly distributed into six groups: G1, diode laser irradiation (2 W, 3 × 20 s); G2, PAD (100 mW, 60 s); G3, PAD with 3D Endoprobe (100 mW, 60 s); G4, 30-gauge syringe irrigation with NaOCl (60 s); G5, sonic agitation of NaOCl with the EndoActivator system (60 s); G6, 30-gauge syringe irrigation with NaCl (60 s). The pattern of colonization was visualized by scanning electron microscopy. The root canals were sampled by flushing with saline solution at baseline and after the treatments. The number of bacteria in each canal was determined by plate count. The presence and the absence of E. faecalis in root canals were also demonstrated by polymerase chain reaction (PCR).There was a significant reduction in the bacterial population after all treatments (P0.001). The PAD, using both laser systems, and the sonic activated NaOCl irrigation were significantly more effective than diode irradiation and single NaOCl irrigation in reducing CFUs (P0.05). High-power diode laser and single NaOCl irrigation had an equal antibacterial effect (P0.05).The PAD and EndoActivator system were more successful in reducing the root canal infection than the diode laser and NaOCl syringe irrigation alone.

Objectives Acquired resistance to carbapenems in uncommon in Enterobacteriaceae although extended-spectrum β-lactamase resistance in these organisms is well established. However, β-lactamase mediated resistance to carbapenems has been reported in Klebsiella pneumoniae, mostly due to the expression of class A KPC β-lactamases susceptible to the inhibition by clavulanic acid, class B metallo-β-lactamases (IMP or VIM) or OXA-48 β-lactamase belonging to the class D β-lactamases. Furtermore, carbapenem resistance can be mediated by hyperproduction of ESBLs or plasmid-mediated AmpC β-lactamases combined with porin loss. Material and methods In February 2011. a 78 old male patient was admitted to Clinical Hospital Center Zagreb with subdural haematoma. He was previously diagnosed with acute myeloblastic leukemia. After surgical removal of haematoma he developed purulent meningtis. K. pneumoniae with reduced susceptibility to carbapenems was isolated. The antibiotic therapy that he received prior to isolation of resistant strain included: vankomycin, ceftazidime, gentamicin, meropenem, penicillin and piperacillin/tazobactam. The patient died from intracerebral bleeding in April 2011. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method in Mueller-Hinton broth and 96 well microtiter plates according to CLSI guidelines. A double-disk-synergy test was performed to detect ESBLs. Modified Hodge Test (MHT) was used to screen for production of carbapenemases. MBL E-test was used to screen for production of metallo-β-lactamases . The transferability of meropenem resistance was determined by conjugation (broth mating method) employing E. coli A15R- strain resistant to rifampicin. Transconjugant was selected on the combined plates containing meropenem (1 mg/L) and rifampicin (128 mg/mL). The frequency of conjugation was expressed relatively to the number of donor cells. The presence of genes encoding ESBLs (blaSHV, blaTEM, blaCTX-M), plasmid mediated ampC beta-lactamases and carbapenemases blaKPC, blaOXA-48, blaOXA-NDM, blaVIM and blaIMP was determined by PCR using protocols and conditions as desribed previously. Sequences were analyzed using BioEdit v.7.0.9. (Ibis Biosciences) program. Designation of bla genes based on identified mutations was done according to: Bush K, Jacoby GA. Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant β-lactamases. Published by the Lahey Clinic ; 2002. Available from: http:// www.lahey.org/studies/). Genotyping of the strain was performed by MLST. Results The isolate showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, β-lactam combinations with inhibitors, carbapenems and gentamicin but remained susceptible only to ciprofloxacin and colistin. Modified Hodge test was consistent with the activity of carbapenemases. The MBL test for metallo-β-lactamase was negative indicating the absence of metallo β-lactamase. Imipenem resistance was not transferred to E. coli recipient strain by conjugation. PCR revealed the presence of blaKPC, blaTEM genes and blaSHV genes. Sequencing of blaKPC gene revealed the presence of KPC-2 beta-lactamase. The strain also possessed intrinsic SHV-1 β-lactamase of K. pneumoniae. Neither plasmid-mediated ampC β-lactamase nor OXA-48 β-lactamase were found. The strain was found belong to ST37 clone by MLST. Infection control efforts limited the spread of KPC-producing clone of K. pneumoniae in our hospital so far. The patient was successfully treated with ciprofloxacin. Conclusions To our knowledge this is the first report of a KPC-producing K. pneumoniae in Croatia . KPC-2 beta-lactamase with similar properties was previously reported from USA, United Kingdom, Israel and Greece. This report demonstrates the need to monitor both hospitalized patients for the further emergence of carbapenem resistance in K. pneumoniae. Continuos surveillance in tracking KPC-producing K. pneumoniae in the the hospitals is necessary to prevent their spread to other hospitals and community.

In 80 adult patients with community acquired pneumonia (CAP) conventional microbiological methods, polymerase chain reaction (PCR) and serum C-reactive protein (CRP) levels were performed and the appropriateness of the empirical antimicrobial treatment was evaluated according to bacterial pathogen detected. The aetiology was determined in 42 (52.5%) patients, with Streptococcus pneumoniae as the most common pathogen. PCR applied to bronchoalveolar lavage (BAL) provided 2 and PCR on sputum samples 1 additional aetiological diagnosis of CAP The mean CRP values in the S. pneumoniae group were not significantly higher than in the group with other aetiological diagnoses (166.89 mg/L vs. 160.11 mg/L, p = 0.457). In 23.8% (10/42) of patients with determined aetiology, the empirical antimicrobial treatment was inappropriate. PCR tests need further investigation, particularly those for the atypical pathogens, as they are predominant in inappropriately treated patients. Our results do not support the use of CRP as a rapid test to guide the antimicrobial treatment in patients with CAP.

It has been suggested that certain viruses such as human papillomaviruses (HPV) and Epstein-Barr virus (EBV) might have a role in oral squamous cell carcinoma (OSCC). However, results of the published studies are controversial and are dependent on the geographic distribution and methods of sampling and sample analysis. The aim of this study was to determine the prevalence of HPV 16 and EBV in OSCC patients. In 24 patients with OSCC (mean age 59.6±8.8) and 30 controls (mean age 49.1±8.3), 5 mL of blood was collected to determine the prevalence of EBV by serologic methods. In addition, swabs were obtained to analyze the presence of HPV 16 and EBV by use of polymerase chain reaction. Statistical analysis was performed by use of Mann Whitney test, χ2-test and Spearman correlation test. The results of this study showed that there were no significant differences between OSCC patients and control subjects according to the presence of EBV or HPV 16. Therefore, it can be concluded, Čini se kako određeni virusi poput humanih papilomavirusa (HPV) i Epstein-Barrova virusa (EBV) mogu imati ulogu u nastanku karcinoma pločastih stanica u usnoj šupljini (OPCK). Ipak, rezultati objavljenih istraživanja na tu temu su kontroverzni i ovise o geografskoj distribuciji i metodama uzimanja uzoraka odnosno raščlambi uzoraka. Cilj ovoga istraživanja je bio odrediti učestalost HPV 16 i EBV u osoba s OPCK. U 24 osobe s OPCK (srednje dobi 59,6±8,8) i u 30 kontrolnih ispitanika (srednje dobi 49,1±8,3) je uzeto 5 mL krvi kako bi se odredio EBV serološkim metodama. Uz to su uzeti i brisevi kako bi se odredila prisutnost HPV 16 i EBV uz pomoć reakcije lančane polimeraze. Statistička analiza je napravljena pomoću Mann-Whitneyeva testa, χ2-testa i Spearmanova korelacijskog testa. Rezultati ovoga istraživanja pokazuju kako nije bilo znakovitih razlika između osoba s OPCK i kontrolnih ispitanika s obzirom na prisutnost HPV 16 ili EBV. Može se zaključiti kako ovi virusi u našoj populaciji oboljelih od OPCK vjerojatno nemaju veliku ulogu.

Helicobacter pylori infection is associated with numerous gastroduodenal diseases. The oral cavity could be a potential extragastric reservoir for H. pylori, and oral H. pylori might cause gastric reinfection after the eradication therapy. The aim of the study was to evaluate the presence of H. pylori in oral cavity of patients with gastric H. pylori infection and to examine the effectiveness of the eradication therapy against H. pylori in stomach and in the oral cavity.Fifty-six patients with chronic periodontitis and gastric H. pylori were enrolled in the study. Gastric H. pylori infection was determined using (13) C-urea breath test before and 3 months after eradication therapy. The presence of the oral H. pylori was assessed using polymerase chain reaction before and 3 months after eradication therapy. The 1-week eradication therapy consisted of amoxycilin 1 g, clarithromycin 500 mg, and proton pump inhibitor 20 mg twice a day.Of 56 subjects with gastric infection, 23 (41.1%) harbored H. pylori in the oral cavity. Eradication rate in stomach was 78.3%, whereas in the oral cavity, H. pylori was not detected from any sample after the eradication therapy.Almost half of the patients with gastric H. pylori harbored the bacterium in the oral cavity. After the eradication therapy, H. pylori was not detected in the oral cavity, what suggests high effectiveness of the therapy protocol in the oral cavity, or it is possible that oral H. pylori is of a transient character.

Background and aim The aim of the study was to analyze antibiotic susceptibilities of Gram- negative and Gram-positive bacterial isolates from pediatric intensive care units included in Mystic programe. Methods The isolates were collected from various clinical specimens at pedriatic intensive care units of the Clinical Hospital Center Zagreb in Croatia. The minimum- inhibitory concentrations (MIC) were determined by broth microdilution method according to CLSI. Extended-spectrum β-lactamases were detected by double-disk synergy test. Results There was no resistance to either imipenem or meropenem observed for E. coli, K. pneumoniae and P. mirabilis. 37% of E. coli isolates showed resistance to gentamicin and 12% to ciprofloxacin. High rates of resistance of K. pneumoniae to ceftazidime and gentamicin (40%) are raising concern (Table 1). Prevalence of ESBLs was 37% in E. coli and 50% in K. pneumoniae. According to MIC90 carbapenems were the most potent antibiotics against Enterobacter cloacae whereas ceftazidime was the least potent antibiotic tested (Table 2). All A. baumannii strains were resistant to gentamicin (Table 3). 11% of P. aeruginosa strains were resistant to carbapenems and 22% to gentamicin, piperacillin/tazobactam and ciprofloxacin (Table 4). Among Gram-positive pathogens staphylococci showed susceptibility to most tested antibiotics (Table 5) contrary to enterococci which showed 85% resistance to gentamicin (Table 6). All enterococci were resistant to cephalosporins due to their intrinsic resistance. Conclusions • These data provide evidence that despite the continued use of meropenem, carbapenem resistance is not increasing among those species tested except for P.aeruginosa, in pediatric ICU, and suggest that clinicians can still administer carbapenems as reliable and effective choice in managing serious nosocomial infections in children. • Low resistance rates to ciprofloxacin are probably due to the fact the fluoroquinolones are not prescribed in children. • Meropenem showed better activity against Gram-negative bacteria while imipenem was more efficient against Gram-positive cocci. • Instrinsic resistance of enterococci to cephalosporins is atributed to the lack of autolytic enzyme in the bacterial cell wall.

In the summer of 1998, an increased number of S. maltophilia isolates from clinical specimens was recorded at our hospital. We tried to identify the sources and mode of spread of S. maltophilia strains in the hospital.

The meropenem yearly Susceptibility Test Information Collection (MYSTIC) programme is a global, longitudinal resistance surveillance network that monitors the activity of meropenem and compares its activity with other broadspectrum antimicrobial agents. We now report the antimicrobial efficacy of meropenem compared to other broad-spectrum agents within the selective Gram-negative pathogen groups from two Croatian Hospitals investigated between 2002-2007. A total of 1510 Gram-negative pathogens were tested and the minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI.There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in both medical centers. High resistance rates of K. pneumoniae to ceftazidime (18%), cefepime (17%) and gentamicin (39%) are raising concern. Acinetobacter baumannii turned out to be the most resistant Gram-negative bacteria with 81% resistant to ceftazidime, 73% to cefepime, 69% to gentamicin and 71% to ciprofloxacin. Almost 20% of Pseudomonas aeruginosa strains were resistant to imipenem, 13% to meropenem, 69% to gentamicin and 38% to ciprofloxacin.The prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli was 10% and in K. pneumoniae 49%. PCR and sequencing of the amplicons revealed the presence of SHV-5 in nine E. coli strains and additional tem-1 beta-lactamase five strains. Five K. pneumoniae strains were positive for bla(SHV-5 )gene. Eight ESBL positive Enterobacter spp. strains were found to produce tem and CtX-m beta-lactamases. Plasmid-mediated AmpC beta-lactamases were not found among K. pneumoniae, E. coli and Enterobacter spp. Three A. baumannii strains from Zagreb University Center were identified by multiplex PCR as OXA-58 like producers. Six A. baumannii strains from Split University Center were found to possess an ISAba1 insertion sequence upstream of bla(OXA-51 )gene. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria. These data indicate that despite continued use of meropenem, carbapenem resistance is not increasing among species tested, except for A. Baumannii, in the two study hospitals and suggest that clinicians can still administer carbapenems as a reliable and effective choice in managing serious nosocomial infections.

Background: The study compares the eradication success of standard first-line triple therapies of different durations (7, 10, and 14 days). Materials and Methods: A total of 592 naive Helicobacter pylori -positive patients were randomized to receive pantoprazole, amoxicillin, and clarithromycin or metronidazole for 14 days (PACl14 or PAM14), 10 days (PACl10 or PAM10), or 7 days (PACl7 or PAM7). H. pylori eradication was assessed by histological, microbiological, and rapid urease examination. Results: The intention-to-treat (ITT) and per-protocol (PP) analyses have shown no overall statistically significant differences between the eradication success of PACl and PAM treatment groups (ITT p = .308, PP p = .167). Longer treatment duration has yielded statistically significant increase in eradication success for clarithromycin (ITT p = .004; PP p = .004) and metronidazole (ITT p = .010; PP p = .034) based regimens. Namely, PACl10, PACl14, and PAM14 protocols resulted in eradication success exceeding 80% in ITT and 90% in PP analysis. Primary resistance to clarithromycin and metronidazole equals 8.2% and 32.9%, respectively. Prolonging the metronidazole-based treatment duration in patients with resistant strains resulted in statistically significant higher eradication success. Conclusions: For all antimicrobial combinations, 14 days protocols have led to a significant increase of H. pylori eradication success when compared to 10 and 7 days, respectively. Prolonging the treatment duration can overcome the negative effect of metronidazole resistance. Only PAM14, PACl10 protocols achieved ITT success > 80% and should be recommended as the first line eradication treatment in Croatia.

The aim of this study was to investigate the effect of the macrolide antibiotic azithromycin on mucosal changes and colonic bacterial load in a murine model of colitis.Colitis was induced in CD1 mice using enema of 0.2% solution of dinitrofluorobenzene, combined with skin sensitization. Four experimental groups of animals (N = 10 per group) were treated with 50 mg/kg/day azithromycin (AZ) or metronidazole (MN) perorally, starting 24 h before (AZ-1, MN-1) or 6 h after (AZ+1, MN+1) induction of colitis and for consecutive 5 days. Additional experimental mice group was treated with 10 mg/kg/day methylprednisolone intraperitoneally after induction of experimental colitis in the same manner (MP). Two control groups consisted of healthy animals (C) that received the challenge enema with phosphate-buffered saline (PBS) and animals with experimental colitis (chall) treated with equivolume of PBS perorally. Clinical score (0-5) and histopathologic score (0-30) were used to assess inflammatory changes, and colon washings were used to determine changes in bacterial load.The anti-inflammatory effect of azithromycin did not differ from the effect of methylprednisolone, when compared with control group with experimental colitis. Metronidazole did not show a significant anti-inflammatory effect. Number of colonic bacteria did not differ significantly between control and experimental groups of animals.We documented the anti-inflammatory effect of azithromycin in a murine model of acute colitis, suggesting that effects were targeted to oxidative burst and on mucosal/bacterial interface, independent of luminal bacterial load. Further studies should be focused on effect of azithromycin on the role of bacterial biofilm in perpetuation of chronic intestinal inflammation.

Comparison between detection of pneumococcal antigen from urine and PCR for detection of S. pneumoniae from blood is done in group of patients with pneumonia. Results are also compared according to severity of pneumonia.

The aim of this study was to determine the virulence characteristics and resistance pattern of the extended-spectrum b lactamases (ESBLs)-producing Escherichia coli strains isolated from urine of outpatients in the Zagreb region during a five-month period, and to compare them with the non ESBLs-producing E. coli strains isolated in the same period. Out of 2,451 E. coli strains isolated from urine of nonhospitalized patients with significant bacteriuria, a total of 39 ESBLsproducing strains (1.59%) were detected by a double-disk diffusion technique and by the broth-dilution minimal inhibitory concentration reduction method. The 45 non ESBLs-producing strains were randomly chosen, and phenotype of the two groups of strains was characterized and compared. Serogroup O4, hemolysin production, expression of P- and type 1 fimbriae as well as resistance to gentamicin and amikacin were significantly more prevalent characteristics among the ESBLs-producing strains than among non ESBLs-producing strains (p0.05). Chromosomal DNA analysis by pulsed-field gel electrophoresis exhibited a great genomic similarity among ESBLs-producing strains and revealed that those highly virulent and resistant E. coli strains isolated from urine of outpatients in the Zagreb region had a clonal propagation.

Bacterial contamination of blood products cause significant patient morbidity and mortality. Contaminated platelet transfusion ia a frequent cause of bacteremia and sepsis because of the storage conditions of platelets. A fatal case of Morganella morganii platelet transfusion associated with sepsis is described, along with procedures traced back to the isolation of M.morganii from a donors stool. Molecular typing was performed (Pulse Field Gel Electrophoresis-PFGE) and the same M. morganii strain was found in blood and post mortem organ cultures of platelet recipient and platelet bag and in the donor's stool. The route of contamination is unknown. The contamination could be due to either insufficient venipuncture site disinfection or the donor's transient bacteriemia. Patient died five days after transfusion.