Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.

Identification and characterization of OXA-48 producing, carbapenem-resistant Enterobacteriaceae isolates in Turkey.

Carbapenem resistance in Enterobacteriaceae isolates has been reported from Turkey and is most often mediated by OXA-48 type carbapenemases. We report the identification and characterization of four carbapenem-resistant isolates (three Klebsiella pneumoniae and one Escherichia coli) among 515 clinical Enterobacteriaceae isolates collected during a 7-month study period in Ankara, Turkey. The four isolates were recovered from blood and urine specimens in patients with varied clinical manifestations. They had distinct pulsed-field gel electrophoresis patterns and harbored a variety of ß-lactamases including bla(TEM-1), bla(SHV-12) genes, bla(SHV-11), and/or bla(CTX-M-15). PCR and sequencing analysis revealed that the bla(OXA-48) gene was present in all four isolates. Our data indicated that the OXA-48-type carbapenemase was the only mechanism for carbapenem resistance in our hospital.

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay.

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.

Triplex real-time polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resistance directly from positive blood culture bottles.

We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf, nuc, and mecA genes in a single tube and had a detection limit of 10(5) CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus, 22 methicillin-susceptible S. aureus, and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf, nuc, and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.

Analysis of an outbreak of Salmonella enteritidis by repetitive-sequence-based PCR and pulsed-field gel electrophoresis.

OBJECTIVE: The aim of this study was to investigate a large food-borne outbreak associated with eggs contaminated by Salmonella Enteritidis in a military unit using pulse field gel electrophoresis (PFGE) and the Repetitive-sequence-based PCR (rep-PCR) employing the DiversiLab system. MATERIALS AND METHODS: In mid-January 2008, a food-borne outbreak associated with S. Enteritidis occurred in a military unit located in the city centre of Isparta. A total of 2,469 patients were registered to six hospitals with gastrointestinal disease symptoms such as diarrhea and abdominal pain. Of those registered, 445 were hospitalized. S. Enteritidis was isolated from 276 stool samples and a blood sample of the hospitalized patients and from a food item. The PFGE patterns after XbaI digestion and rep-PCR profiles produced by the DiversiLab system were determined for eight randomly selected stool isolates, one blood isolate and one food isolate. RESULTS: The PFGE patterns of all isolates were identical. The Rep-PCR profiles produced by using the DiversiLab system showed that all isolates were indistinguishable. The PFGE and rep-PCR interpretations were concordant for the S. Enteritidis isolates. All stool isolates, one blood isolate and one food isolate were susceptible to all antibiotics tested. CONCLUSION: This data suggest that the DiversiLab system may be a reasonable alternative to PFGE for investigation and control of S. Enteritidis outbreaks, since it is easy to use, rapid and does not require highly skilled operators.

Staphylococcal cassette chromosome mec (SCCmec) characterization and panton-valentine leukocidin gene occurrence for methicillin-resistant Staphylococcus aureus in Turkey, from 2003 to 2006.

Methicillin-resistant Staphylococcus aureus (MRSA) cause serious community-acquired and nosocomial diseases all over the world. We determined the SCCmec types and occurrence of the PVL gene by using TaqMan real-time PCR method, and correlated these with phenotypic antibiotic susceptibility patterns for MRSA strains collected from Gulhane Military Medical Academy Hospital (GMMAH) during 4 years study period. To our knowledge, this is the first report from Turkey of molecular SCCmec typing analysis of MRSA stains. A total of 385 clinical MRSA isolates collected in the clinical and Microbiology Laboratory at GMMAH between 2003 and 2006 were included in the study. Overall, SCCmec types-I, II, III, IV, V, nontypeable and PVL occurrence were detected in 11 (2.8%), 3 (0.8%), 316 (82.1%), 20 (5.1%), 20 (5.1%), 15 (3.9%) and 5 (1.3%) isolates, respectively. A total of 330 (85.5%) were SCCmec-I/II/III and 40 (10.3%) were SCCmec IV/V. SCCmec-I/II/III isolates were recovered more from patients with serious infections in surgical departments especially those with intensive care units than the SCCmec-IV/V isolates (chi(2) = 13.560, P < 0.001). SCCmec-I/II/III MRSA strains were predominantly recovered from blood stream (53.0%, P = 0.014), while SCCmec-IV/V strains were predominately isolated from skin and soft tissue and abscess (55.0%, P < 0.001). The PVL gene was detected in 10.0% of SCCmec-IV/V isolates in contrast to 0.3% in SCCmec-I/II/III (chi(2) = 25.164, P < 0.001). SCCmec-I/II/III MRSA strains were more resistant to clindamycin (chi(2) = 5.078, P = 0.024), amoxicillin-clavulanate (chi(2) = 84.912, P < 0.001), erythromycin (chi(2) = 4.651, P = 0.031), gentamicin (chi(2) = 24.869, P < 0.001), and rifampin (chi(2) = 18.878, P < 0.001) than SCCmec-IV/V MRSA strains. This data indicates that SCCmec-III MRSA strains that do not carry the PVL gene are the predominant MRSA strains in our hospital setting in Ankara, capital of Turkey and that SCCmec-I/II/III MRSA strains may cause serious infections in surgical departments especially those with intensive care units.

Molecular characterization of methicillin-resistant Staphylococcus aureus nasal isolates from Turkey.

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 mug oxacillin and 30 mug cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.

Macrolide-lincosamide-streptogramin B resistant phenotypes and genotypes for methicillin-resistant Staphylococcus aureus in Turkey, from 2003 to 2006.

Methicillin-resistant Staphylococcus aureus (MRSA) strains with inducible macrolide-lincosamide-streptogramin B (iMLS(B)) resistance phenotype may lead to clinical failure during clindamycin (CLI) therapy. The aim of this study was to determine the incidence of MLS(B) phenotypes by using D-test method and genotypes by using multiplex real-time PCR method in MRSA strains. A total of 265 MRSA strains were obtained from clinical samples from hospitalized and outpatients. Of the MRSA isolates, 225 (84.9%) were resistant to erythromycin (ERT), and 170 (64.1%) to CLI. Among the 225 ERT-resistant MRSA strains, the constitutive MLS(B) (cMLS(B)) rate was found in 49.3%, iMLS(B) in 39.1% and the M phenotype in 11.5%. Overall, ermA, ermC, ermA+ermC, msrA, ermC+msrA, and ermA+ermC+msrA genes were detected in 85 (37.7%), 60 (26.6%), 42 (18.6%), 26 (11.5%), 11 (4.8%), and 1 (0.4%) isolates, respectively. Most prevalent resistance determinant in MRSA strains was ermA, which was detected in 37.7% of the isolates. The 26 MRSA strains with M phenotype harboured only msrA gene. In conclusion, due to aware of the potential of CLI treatment failure, D-test should be performed and reported in MRSA strains in clinical laboratories. The multiplex real-time PCR method is easy to perform, fast and reliable method for the detection of MLS(B) resistance genotypes.

Nosocomial outbreak of Sphingomonas paucimobilis bacteremia in a hemato/oncology unit.

Nosocomial Sphingomonas paucimobilis infections can arise from contaminated water and the contaminated hands of hospital staff. Within a 1-month period, we isolated six S. paucimobilis strains, including four from blood cultures of four patients and two from hospital environment specimens including tap water and a bathtub in a hemato/oncology unit. We described here these strains' molecular epidemiological analyses by pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibilities by E-test. Although clinical and environmental isolates yielded three different antibiotic resistances and PFGE patterns, all four clinical strains had an identical pattern by both methods. Thus, the isolated clinical strain clone could be traced neither to health care workers nor to environmental samples. It was concluded that S. paucimobilis strains can cause outbreaks in hemato/oncology units. We did not demonstrate genetic relatedness between clinical and environmental isolates by PFGE, but did find PFGE a useful identification technique for epidemiological investigation.

Clustering of invasive Aspergillus ustus eye infections in a tertiary care hospital: a molecular epidemiologic study of an uncommon species.

Aspergillus infections are being increasingly recognized as an important cause of morbidity and blindness. We report here the first cluster of Aspergillus ustus endophthalmitis cases which occurred in a large tertiary care hospital during the period October 2003 to June 2004. In three of the cases, the patients required enucleation following cataract surgery, while the fourth involved a fatal infection in a pediatric patient hospitalized for osteopetrosis. Patient charts from the four cases were reviewed retrospectively and indicated that postoperative signs of fungal endophthalmitis developed in the patients 1-11 weeks after surgery. The molecular characterization of the isolates and their epidemiological relatedness were evaluated by Random Amplification of Polymorphic DNA (RAPD). A source investigation of this mini outbreak was performed by environmental sampling, but no isolates of A. ustus were recovered from these studies. All A. ustus strains isolated from three patients with fungal endophthalmitis had the same RAPD pattern suggesting a common source. The strain from the pediatric patient differed from the ophthalmic isolates in five electrophoretic loci. The latter was included solely as an outbreak, unrelated control to evaluate the discriminatory power of the molecular typing method employed in the analysis of the ophthalmic strains. These cases illustrate the potential for uncommon species like A. ustus to cause high morbidity and mortality in some clinical settings. Aspergillus ustus endophthalmitis is a serious and devastating complication of ocular surgery. It is unknown whether ongoing hospital construction may have contributed to this cluster of cases. Random amplification of polymorphic DNA may give valuable clues about the clonality of A. ustus strains.

Susceptibility of bacterial isolates from Turkey--a report from the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program.

The study monitored the susceptibility of nosocomial pathogens to meropenem and comparator antimicrobial agents isolated as part of the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program from Turkish university hospitals. In terms of minimum inhibitory concentration 90% (MIC(90)) values, meropenem was two- and eight-fold more active than imipenem against Escherichia coli and Klebsiella pneumoniae, respectively. 40.5% of K. pneumoniae, 23.1% of Klebsiella oxytoca and 15.3% of E. coli isolates were extended-spectrum beta-lactamase (ESBL) producers. Piperacillin/tazobactam was the most active agent against isolates of Pseudomonas aeruginosa, followed by meropenem and imipenem. Against Acinetobacter baumannii isolates, meropenem and imipenem were the most active agents. Continued surveillance by the MYSTIC Program appears to be prudent to help focus on effective empiric treatment regimens.

Inhibition of inducible nitric oxide synthase reduces bacterial translocation in a rat model of acute pancreatitis.

INTRODUCTION: Translocation of bacteria from the gut into pancreatic necrosis is an important factor in the development of septic complications and mortality in acute pancreatitis. S-methylisothiourea (SMT) is an inducible nitric oxide synthase inhibitor that has been shown to decrease bacteria] translocation in sepsis and thermal injury. AIM: To investigate whether SMT could affect bacterial translocation in acute necrotizing pancreatitis. METHODOLOGY: Forty-five Sprague-Dawley rats were studied. Acute pancreatitis was induced in Group I and Group II by injection of taurocholate and trypsin into the common biliopancreatic duct. Group III underwent laparotomy with the manipulation (but not cannulation) of the pancreas and received saline injection. Group I rats received normal saline as a placebo, and Group II rats received SMT after surgery for 2 days. At 48 hours, blood was drawn for serum amylase determinations. Bacterial translocation to mesenteric lymph nodes and distant sites (pancreas, liver, and peritoneum) were examined. A point scoring system of histologic features was used to evaluate the severity of pancreatitis. RESULTS: Plasma amylase levels and pancreatic histologic score were significantly reduced in Group II rats given SMT compared with those in Group I rats given saline (p < 0.01, p < 0.05, respectively). All Group I rats had bacterial translocation to mesenteric lymph nodes compared with 7 of 12 rats in Group II (p < 0.05). There was no difference in bacterial translocation to distant organs between the two groups, although rates tended to be lower in Group II compared with Group I (p > 0.05). Bacterial counts in the pancreas were significantly reduced in Group II rats compared with those in Group I rats (p < 0.05). CONCLUSION: Treatment with SMT appears to have ameliorated the course of acute pancreatitis; however, mortality was not affected.

Effect of hyperbaric oxygen and penicillin in a murine model of streptococcal myositis.

We investigated the effect of hyperbaric oxygen (HBO2) and penicillin therapy in a murine model of group A streptococcal myositis. The thighs of mice were inoculated with Streptococcus pyogenes. Four groups were evaluated: 1) control (n = 13), 2) HBO2 treatment (n = 15), 3) penicillin treatment (n = 12), and 4) penicillin and HBO2 treatment (n = 13). Histologic methods were utilized to prove the existence of myositis and histologic changes in tissues following experimental intramuscular inoculation of mice with Streptococcus pyogenes. Mortality (day of death) and the number of colony forming units (cfu) were measured. Microscopic sections of the left thighs revealed extensive necrosis of muscle with acute inflammatory infiltrate in all groups. Penicillin significantly lowered cfu count in comparison to the control (P < 0.01). Cfu's in group 4 were significantly lower than in group 3 (P < 0.01). Survival was significantly longer in the penicillin group compared to the control (P < 0.01). Survival in the combined treatment group was significantly longer than penicillin alone (P < 0.01). These results suggest that 1) HBO2 treatment alone does not decrease mortality significantly in vivo, 2) penicillin therapy alone improves outcome significantly, and 3) the combined treatment of penicillin and HBO2 exerts synergistic effects in both decreasing bacterial counts in vivo and increasing survival in this model.