Detection of the antibiotic resistance genes content of intestinal Bacteroides, Parabacteroides and Phocaeicola isolates from healthy and carbapenem-treated patients from European countries.

BACKGROUND: Bacteroides fragilis group (BFG) species are the most significant anaerobic pathogens and are also the most antibiotic-resistant anaerobic species. Therefore, surveying their antimicrobial resistance levels and investigating their antibiotic resistance mechanisms is recommended. Since their infections are endogenous and they are important constituents of the intestinal microbiota, the properties of the intestinal strains are also important to follow. The aim of this study was to investigate the main antibiotic gene content of microbiota isolates from healthy people and compare them with the gene carriage of strains isolated from infections. RESULTS: We detected 13, mainly antibiotic resistance determinants of 184 intestinal BFG strains that were isolated in 5 European countries (Belgium, Germany, Hungary, Slovenia and Turkey) and compared these with values obtained earlier for European clinical strains. Differences were found between the values of this study and an earlier one for antibiotic resistance genes that are considered to be mobile, with higher degrees for cfxA, erm(F) and tet(Q) and with lower degrees for msrSA, erm(B) and erm(G). In addition, a different gene prevalence was found depending on the taxonomical groups, e.g., B. fragilis and NBFB. Some strains with both the cepA and cfiA ß-lactamase genes were also detected, which is thought to be exceptional since until now, the B. fragilis genetic divisions were defined by the mutual exclusion of these two genes. CONCLUSIONS: Our study detected the prevalences of a series of antibiotic resistance genes in intestinal Bacteroides strains which is a novelty. In addition, based on the current and some previous data we hypothesized that prevalence of some antibiotic resistance genes detected in the clinical and intestinal BFG strains were different, which could be accounted with the differential composition of the Bacteroides microbiota and/or the MGE mobilities at the luminal vs. mucosal sites of the intestine.

In Vitro Evaluation of Planktonic Growth on Experimental Cement-Retained Titanium Surfaces.

BACKGROUND The purpose of this study was to compare the effects of selected cements, or their combination with titanium, on the growth of two periodontopathic bacteria: Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn). MATERIAL AND METHODS This study was comprised of several experimental groups: 1) Dental luting cements (glass ionomer cement, methacrylate-based resin cement, zinc-oxide eugenol cement, eugenol-free zinc oxide cement; 2) titanium discs; and 3) titanium combination cement discs. The disks were submerged in bacterial suspensions of either Fn or Pi. Planktonic bacterial growth within the test media was measured by determining the optical density of the cultures (OD600). Mean and standard deviations were calculated for planktonic growth from three separate experiments. RESULTS Intergroup comparison of all experimental groups revealed increased growth of Pi associated with cement-titanium specimens in comparison with cement specimens. Regarding the comparison of all groups for Fn, there was an increased amount of bacterial growth in cement-titanium specimens although the increase was not statistically significant. CONCLUSIONS The combination of cement with titanium may exacerbate the bacterial growth capacity of Pi and Fn in contrast to their sole effect.

[The first metronidazole-resistant Bacteroides species isolated at Marmara University Hospital: Bacteroides thetaiotaomicron]. / Marmara Üniversitesi Hastanesi'nde Izole Edilen Ilk Metronidazole Dirençli Bacteroides Kökeni: Bacteroides thetaiotaomicron.

Bacteroides species, the predominant constituents of the human intestinal microbiota can cause serious intraabdominal and postoperative wound infections and bacteremia. Moreover, these bacteria are more resistant to antimicrobial agents than the other anaerobes. The limited number of the antimicrobials, such as carbapenems, beta-lactam/beta-lactamase inhibitors and nitroimidazoles are highly effective in eliminating Bacteroides. However, a few metronidazole-resistant isolates have been reported from several countries recently. The nim genes (nim A-G) are suggested to be responsible for the majority of the metronidazole resistance. Here, we describe a metronidazole-resistant Bacteroides thetaiotaomicron isolated from a blood culture. A gram-negative obligate anaerobic rod was isolated from the postoperative 5th day blood culture of a 62-year-old male patient with adenocarcinoma of the pancreas head. The strain was identified as B.thetaiotaomicron by using a combination of conventional tests and commercially available biochemical kits. Antimicrobial susceptibility testing was performed by agar dilution method. The resistance genes were investigated by means of PCR using specific primer pairs for nim gene. The purified PCR product was sequenced and analyzed by comparison of the consensus sequences with GenBank sequences. The MIC for metronidazole was 16 mg/L. Although the strain was intermediate according the CLSI criteria, it was resistant (> 4 mg/L) according to EUCAST criteria. The isolate was nim gene positive, and nucleotide sequencing of the PCR product shared 100% similarity with nimE gene (emb |AM042593.1 |). On the other hand the isolate was susceptible to carbapenems and sulbactam-ampicillin. Following administration of ampicillin-sulbactam, the patient's fever disappeared after 24 hours. The clinical condition improved considerably and he was discharged at day 8. The patient was followed up at the medical oncology clinic; however he died due to disease progression six months after surgery. Since anaerobic bacteremia is associated with high mortality rate, prompt diagnosis and proper management are critical. The studies on Bacteroides bacteremia have revealed adverse outcomes in patients receiving antibiotics to which the bacterium was resistant. In the present case, the metronidazole-resistant organism would be reported as susceptible according to CLSI breakpoint value and on account of this result the treatment might lead to clinical failure. Therefore EUCAST MIC values seem to be more rational in case of Bacteroides antibiotic susceptibility testing.

Disseminated Nocardia farcinica Infection and Treatment Response on F-18 Fludeoxyglucose Positron Emission Tomography Computed Tomography.

We report a rare case of nocardiosis with increased F-18 Fludeoxyglucose (F-18 FDG) uptake in widespread abscess foci of Nocardia farcinica infection in an immunocompromised patient on positron emission tomography computed tomography (PET/CT) imaging. A relatively infrequent cause of nocardiosis, N. farcinica is an opportunistic infection that may present with clinically aggressive disseminated disease. Whole-body F-18 FDG-PET/CT allows identifying the extent of disease, as well as monitoring response to therapy in patients with nocardiosis especially the disseminated form.

[Investigation of toxin genes of Clostridium difficile strains isolated from hospitalized patients with diarrhoea at Marmara University Hospital]. / Marmara Üniversitesi Hastanesinde Yatan Ishalli Hastalardan Izole Edilen Clostridium difficile Kökenlerinde Toksin Genlerinin Arastirilmasi

Toxigenic Clostridium difficile strains cause a spectrum of antibiotic-associated diseases ranging from self-limited diarrhea to severe life-threatening colitis. Pathogenesis primarily involves the action of two important cytotoxins, namely toxin A and toxin B. However, epidemics of C.difficile-associated disease due to the novel, highly virulent strains of C.difficile (binary toxin positive and toxin A variant) have been recognised in hospitals of some countries. The aim of this study was to investigate the toxin gene profiles of C.difficile strains isolated from hospitalized patients with diarrhea. The stool specimens collected from 633 inpatients at Marmara University Hospital, Istanbul, Turkey, between September 2006-March 2008, were included to the study. The presence of C.difficile toxins in the samples has been screened by a commercial enzyme immunoassay kit (ImmunoCard Toxins A&B EIA; Meridian Diagnostics, Belgium). Stool samples were also cultivated on cycloserin-cefoxitin-fructose agar (CCFA; BioMerieux, France) at anaerobic conditions, and the isolates were identified by conventional methods and Rapid ID 32A (BioMerieux, France) system. Toxin production of C.difficile strains isolated from stool cultures have been detected by commercially available "Triage C.difficile Panel" (Biosite Diagnostics, Italy) and "ImmunoCard Toxins A&B EIA" (Meridian Diagnostics, Belgium) kits. In-house polymerase chain reaction (PCR) was performed to investigate the presence of genes for toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB). Stool specimens from 50 (7.9%) patients (age range: 2-> 65 years; mean age: 35.9 ± 27.6 years; 26 were male) yielded C.difficile in culture. All of 50 isolates were found positive for glutamate dehydrogenase enzyme and 28 (56%) were found positive for toxin A with "Triage C.difficile Panel" kit. Toxin positivity rate was detected as 4.7% (30/633) with EIA test performed in stool samples directly, however this rate was 5.7% (36/633) in culture filtrates of the isolates (n= 50), with the same test. Since EIA test yielded false negative results in six samples, the sensitivity of this test was estimated as 85.7% by means of the detection of toxin in direct stool samples. All of the 36 toxin-producing C.difficile isolates were found positive for toxin A and toxin B genes (tcdA+/tcdB+), however there were neither variant strains (tcdA-/tcdB+) nor binary toxin gene positive isolates among tested bacteria. Our results have also indicated that 77.8% (28/36) of patients who harbored toxigenic C.difficile strains have the history of beta-lactam antibiotic (penicillin, cephalosporin and imipenem) use. It was thought that the data of this study would constitute a database on the toxin gene profiles of C.difficile in hospitalized patients with diarrhea both in our hospital and Turkey. The current data have indicated that for the time being there were no risk for isolates producing new toxin variants or binary toxin, however, continuous monitorization of such C.difficile strains is of crucial importance in order to detect the emergence of those strains and establish necessary control and preventive measures.

[Can meropenem E-test be used to estimate the presence of carbapenem resistance gene cfiA among Bacteroides fragilis strains?]. / Meropenem E-test, bacteroides fragilis suslarinda karbapenem direnç geni cfiA varligini tahmin etmede kullanilabilir mi?

Bacteroides fragilis, which is found in normal colon flora, is the most commonly encountered pathogen in anaerobic infections and more resistant to antimicrobial agents than the other anaerobes. Limited number of antibiotics; such as carbapenems, beta-lactam/beta-lactamase inhibitors and nitroimidazoles are the most effective antibiotics against Bacteroides, however resistant isolates to these antimicrobials have been reported recently. Resistance against carbapenems occurs due to a metallo-beta-lactamase enzyme expressed by cfiA gene. While agar dilution method is used to test the antimicrobial susceptibility of anaerobic organisms, E-test is recommended for susceptibility testing of anaerobes associated with life-threatening infections with high mortality and morbidity. In this study, meropenem E-test was used to determine the carbapenem resistance of B.fragilis strains and to estimate the presence of cfiA gene. A total of 63 B.fragilis strains that were previously isolated from clinical samples (of which 16 were from stool samples) in our laboratory, were enrolled in the study. Minimum inhibitory concentration (MIC) values were determined by meropenem E test (AB Biodisk, Sweden) and presence of cfiA genes were investigated by in-house polymerase chain reaction. The MIC ranges of meropenem were < 0.002 - > 32 µg/ml and the resistance rate was 9.5% (6/63). Thirty-three percent (21/63) of strains harboured cfiA gene. A statistically significant relation (p< 0.0001) was determined between presence of cfiA gene and high MIC value (MIC 0.5 µg/ml). The proportion of cfiA-positive isolates detected in this study was substantially higher than that reported in other countries. This might be attributed to the frequent use of carbapenems in our hospital. The results of this study indicated that meropenem E-test method could be useful to estimate the presence of cfiA gene in B.fragilis strains and thus to detect the resistant strains.

Rapid reporting of urine culture results: impact of the uro-quick screening system.

This study evaluated the impact of the Uro-Quick (UQ) screening system (Alifax, Italy) for a rapid and accurate reporting of urine cultures, and whether it can provide bacterial yield to be used in identification and susceptibility testing. A total of 1480 urine samples collected between October 2006 and July 2008 were tested by conventional culture (CC) methods and UQ simultaneously. Sediments of positive UQ vials were used as bacterial yields for identification and susceptibility testing procedures. Of the 1480 samples, 999 revealed bacteria and/or leukocytes in direct microscopy. Among these 999 samples, positive growth was detected in 420 (42%) and 433 (43.3%) by UQ and CC, respectively. However, only 0.6% of samples without bacteria and leukocytes exhibited positive growth. When compared to CC, UQ demonstrated high levels of positive predictive value (95.9%), negative predictive value (94.8%), sensitivity (93%) and specificity (96.9%). Both CC isolates and UQ bacteria showed 81.3% concordance in identification results. Susceptibility testing of UQ bacteria displayed >90% agreement, when compared with standardized disk diffusion test. Our results indicate that UQ can reliably be used in routine laboratories giving microbial growth results in 3 hours. The most significant part of the study is that bacterial yields of UQ positive samples can be used in identification and susceptibility testing, allowing a rapid, same-day reporting of urine cultures.

[Neonatal Candida infections and the antifungal susceptibilities of the related Candida species]. / Neonatal kandida enfeksiyonlari ve etkenlerinin antifungal duyarliliklari

Among nosocomial infections in the newborns, the incidence of fungal infections has been rising over the last decades. Fluconazole has been a new option for treatment however, expanded use of the drug brought up the development of resistance. In this study, species of the Candida isolates from neonates with candida infections, their antifungal susceptibilities and the effectiveness of the therapy were evaluated. All the species of Candida isolates from blood, urine and sterile body fluids of 54 neonates and their antifungal susceptibilities were evaluated retrospectively over the 13-year period. Demographic characteristics, risk factors, infection foci, Candida species causing infection and their in vitro susceptibilities for fluconazole (FCZ) and amphotericin B (AMB) and treatment responses were analyzed. The antifungal susceptibility testing of isolates was performed by microdilution technique. The median birth weight and gestational age of the study groups were 1735 (660-3990) g and 33 (24-40) weeks, respectively. Among the patients, 19 (35%) were term, while 35 (65%) were preterm [< 32 weeks n = 20 (37%), < 28 weeks n = 7 (13%)]. The percentage of low birth weight infants was 65% (42% was < 1500 g, 13% was < 1000 g). Candida spp. were isolated mostly from blood samples (63%), followed by urine (46%), cerebrospinal fluid (CSF; 5%), peritoneal fluid (3%) and endotracheal aspirate (2%). Multifocal growth was determined in 10 (18%) cases. The isolated species were C.albicans (n =36) as being the most common isolate followed by C.parapsilosis (n = 12), C.tropicalis (n = 1), C.kefyr (n = 1), C.lusitaniae (n = 1), C.pelluculosa (n = 1) and Candida spp. (n = 2). Prior antibiotic use, long term hospitalization, total parenteral nutrition and use of lipid solutions, prematurity and catheter use were determined as the most frequently associated factors causing candidal infections. A congenital abnormality, mainly myeloschisis and hydrocephaly, was detected in 18 (33%) of the cases. Overall FCZ resistance rate was 5.5% and the rate of resistance according to the species was 2.8% for C.albicans and 11% for non-albicans isolates. No resistance was observed to AMB. Initial treatment was FCZ for 78% and AMB for 22% of the newborns. The treatment was switched to AMB in 15 (28%) cases because of no clinical or laboratory response to FCZ although only three of these babies showed resistance to FCZ (MIC ≥ 64 mcg/ml). Among the cases with no clinical/microbiological response, C.albicans was the most frequently (66%) isolated species followed by non-albicans species (33%). All of the isolates in the study group were susceptible to AMB and the rate of FCZ resistance was 5.5%. However, it was noted that the clinical treatment failure was higher than the resistance rate when FCZ was considered. Although antifungal susceptibility tests are helpful for guiding the therapy, in vivo and in vitro differences should be taken into account in case of treatment failure encountered with the use of in vitro effective agents.

A rare and emerging pathogen: Raoultella planticola identification based on 16S rRNA in an infant.

Raoultella planticola is rarely associated with clinical infection, and a limited number of pediatric cases have been reported. Herein we report a case of bacteremia presumptively secondary to bilateral conjunctivitis in an infant caused by R. planticola which was successfully treated with piperacillin-tazobactam. It should be kept in mind that R. planticola can be a pathogen in pediatric age groups.

Surveillance of Haemophilus influenzae among respiratory tract samples of Turkish children.

We conducted three prospective studies of Haemophilus influenzae in different groups of children. Pharyngeal swab samples were taken (i). from 1382 healthy infants and children between 0 and 10 years of age (group 1), attending well child clinics (n=438), day care centres (n=440) and elementary schools (n=504), and (ii). from 322 children aged 2-10 years (group 2), clinically diagnosed as having upper respiratory tract infection. Pharyngeal swab samples and sinus aspirates were obtained from 49 children between 2 and 9 years of age (group 3), clinically diagnosed as having sinusitis. H. influenzae was isolated in similar rates from 315 (22.7%) of children in group 1, 72 (22.3%) of children in group 2 and 12 (24.4%) of children in group 3. Serotype b comprised 7, 5.2 and 2% of all H. influenzae isolates for group 1, 2 and 3, respectively. Production of beta-lactamase was detected in 1.0% of H. influenzae type b isolates in group 1, 1.2 and 6.1% of all isolates in group 2 and 3, respectively. There were no beta-lactamase negative ampicillin-resistant strains.

Pharyngeal colonization with Haemophilus influenzae type b among healthy Turkish infants and children.

BACKGROUND: An absence of Haemophilus influenzae type b (Hib) disease surveillance and epidemiological data on the pharyngeal carriage of Turkish children causes delay in the introduction of conjugated Hib vaccination into proposed national vaccination programs. METHODS: Oropharyngeal cultures were obtained from 1404 healthy infants and children. Six healthy child clinic (HCC), 11 day-care centers (DCC) and seven elementary schools (ES) were randomly selected in seven different counties at the Anatolian side of Istanbul between January and April 2000. RESULTS: Haemophilus influenzae was isolated from 315 (22.8%) of all participants and 98 (31%) isolates were serotype b. The carriage rate for Hib was higher in children at DCC (43 out of 448, 9.6%) and ES (46 out of 504, 9.1%) compared to infants 0-24 months of age (nine out of 430, 2.1%) presented to HCC. All Hib isolates were susceptible to azithromycin, chloramphenicol and cefotaxime. Beta-lactamase production was detected in only one isolate. Trimethoprim-sulfamethoxazole resistance was found in 8.5% of Hib isolates. Multivariate analysis showed that DCC and ES attendance were independent predictors of Hib carriage. CONCLUSION: A significant proportion of healthy Turkish children was shown to be colonized with Hib. The burden of invasive Hib infections should be determined in order to evaluate the Hib conjugated vaccine as a part of a routine immunization program in Turkey.