The efficacy of photon-initiated photoacoustic streaming and sonic-activated irrigation combined with QMiX solution or sodium hypochlorite against intracanal E. faecalis biofilm.

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 single-canal teeth were instrumented, sterilized, contaminated with E. faecalis and incubated 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, energy density 2.06 J/cm(2), 3 × 20 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 E. faecalis in root canals were demonstrated by polymerase chain reaction (PCR), and the pattern of the bacteria colonization was visualized by scanning electron microscopy. There was significant reduction in the bacterial population for all groups (p < 0.001). The best antibacterial efficacy was recorded after sonic-activated irrigation with both NaOCl (99.999 %) and QMiX (99.999 %) and after PIPS with QMiX (99.999 %), which were more effective than conventional irrigation with NaOCl (99.998 %) and the PIPS with the NaOCl (99.966 %). Also, the PIPS with QMiX solution provided the highest number of sterile samples (five). There was no difference in the bacteria reduction between the active irrigation techniques, regardless of the irrigant used. Although the laser activation did not improve the antimicrobial action of the NaOCl nor QMiX, the fact that it generated the greatest number of sterile samples warrants further investigation.

Direct identification of mycobacteria from smear-positive samples using real-time polymerase chain reaction.

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 was 100% successful in identifying AFB 2+ and AFB 3+ samples. However, only 14 of 26 (52%) AFB 1+ 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.

Diversity of carbapenemases in clinical isolates of Enterobacteriaceae in Croatia--the results of a multicentre study.

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 ß-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. BlaVIM genes were located within class1 integron which contained genes encoding resistance to aminoglycosides (aacA4 ). The study found strong association between blaVIM and qnrB6 and between blaNDM and qnrA6 genes.

Antimicrobial efficacy of a high-power diode laser, photo-activated disinfection, conventional and sonic activated irrigation during root canal treatment.

AIM: 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. METHODOLOGY: 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). RESULTS: There was a significant reduction in the bacterial population after all treatments (P < 0.001). 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 (P < 0.05). High-power diode laser and single NaOCl irrigation had an equal antibacterial effect (P > 0.05). CONCLUSIONS: The PAD and EndoActivator system were more successful in reducing the root canal infection than the diode laser and NaOCl syringe irrigation alone.

Antimicrobial susceptibility and beta-lactamase production of selected gram-negative bacilli from two Croatian hospitals: MYSTIC study results.

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.

Sensitivity and specificity of various beta-lactam antibiotics and phenotypical methods for detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases.

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.

Epidemic and endemic spread of Klebsiella pneumoniae producing SHV-5 beta-lactamase in Dubrava University Hospital, Zagreb, Croatia.

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.

Morganella morganii causing fatal sepsis in a platelet recipient and also isolated from a donor's stool.

Bacterial contamination of blood products causes significant patient morbidity and mortality. Contaminated platelet transfusion is a frequent cause of bacteraemia 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 donor's stool. Molecular typing was performed, 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 bacteraemia. Patient died 5 days after the transfusion.

[Stenotrophomonas maltophilia as a cause of hospital infections--typing of clinical isolates using pulsed field gel electrophoresis] ]. / Stenotrophomonas maltophilia kao uzrocnik bolnickih infekcija--tipizacija klinickih izolata metodom elektroforeze u pulzirajucem polju (PFGE).

Stenotrophomonas maltophilia (S. maltophilia) has been recognised now as an important cause of hospital infections. As S. maltophilia is resistant to many antibiotics, attempts have been made to identify the sources of S. maltophilia infection and route of its transmission. From July till October 1998, 22 isolates of S. maltophilia were obtained from 20 patients hospitalised at eight different wards. Strain typing was performed by macrorestriction analysis of chromosomal DNA by use of PFGE (XbaI and SpeI enzymes, in a CHEF DR III drive module). PFGE analysis of 22 S. maltophilia isolates revealed 9 different types designated by letters A to I. The source and route of the spread of infection could not be identified. These results may indicate that we had clusters of S. maltophilia infection in cardiosurgery ward and ICU by types A, B, C and D; in neurosurgical ICU by type E and in urology ICU by type H.

Helicobacter pylori: in vitro induction of resistance to azithromycin.

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 degrees 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 < or = 2, 4 and > or = 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 degrees 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.

[Seroprevalence of Helicobacter pylori infection in health personnel in 3 hospitals in Zagreb]. / Seroprevalencija infekcije Helicobacterom pylori u zdravstvenih djelatnika triju zagrebackih bolnica.

By this study we wanted to investigate the seroprevalence of H. pylori infection in 175 health care workers of three Zagreb city hospitals. The obtained results were compared with those of 2492 volunteer blood donors. The influence of age, education, socioeconomic status and length of service at specific hospital working places were investigated in relation to the frequency of H. pylori seropositivity. The blood samples were tested by commercial kits of immunoenzyme assay (ELISA) and complement fixation (CF), according to manufacturers instructions. The mean seroprevalence of infection was 58.6% in the group of blood donors and 53.7% in the group of health care workers (NS). Statistically significant difference was found between physicians (29%) and all other health care workers: nurses (58.6%; p < 0.005), laboratory technicians (60.6%; p < 0.005), clerical workers (66.6%; p < 0.005) and auxiliary workers (82.6%; p < 0.001). Concerning the age, the infection seroprevalence was higher in workers aged more than 40 years than in those younger, and that difference was of statistical significance among nurses and laboratory technicians. Physicians under 29 yrs were of the lowest seropositivity (14.8%). Among health care workers with less than 20 working years, physicians expressed the lowest rate of infection (17.9%) in comparison with nurses (48.5%) and laboratory technicians (53.3%). In all health care workers with more than 20 working years there was significant increase of infection prevalence, particularly among nurses. The employees in gastrointestinal endoscopy laboratories were more often serologically positive than medical workers in other medical departments (58.3% versus 35.0%; p < 0.05).

An autopsy study of systemic fungal infections in patients with hematologic malignancies.

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).