A retrospective observational cohort study of the clinical epidemiology of bloodstream infections due to carbapenem-resistant Klebsiella pneumoniae in an OXA-48 endemic setting.

This study aimed to characterize the epidemiology and clinical outcomes of patients with bloodstream infections (BSIs) due to carbapenem-resistant Klebsiella pneumoniae (CRKP) in an OXA-48-predominant environment. This was a retrospective single-centre cohort study including all consecutive patients with CRKP BSIs treated between 1 January 2014 and 31 December 2018. Multivariate analysis, subgroup analysis and propensity-score-matched analysis were employed to analyse 30-day mortality as the primary outcome. Clinical cure at day 14 was also analysed for the whole cohort. In total, 124 patients with unique isolates met all the inclusion criteria. OXA-48 was the most common type of carbapenemase (85.5%). Inappropriate therapy was significantly associated with 30-day mortality [70.6% vs 39.7%, adjusted odds ratio (aOR) 4.65, 95% confidence interval (CI) 1.50-14.40, P=0.008] and 14-day clinical failure (78.5% vs 56.2%, aOR 3.14, 95% CI 1.09-9.02, P=0.033) in multivariate analyses. Among those treated appropriately, the 30-day mortality rates were similar in monotherapy and combination therapy arms (OR 2.85, 95% CI 0.68-11.95, P=0.15). INCREMENT CPE mortality score (aOR 1.16, 95% CI 1.01-1.33, P=0.029), sepsis at BSI onset (aOR 2.90, 95% CI 1.02-8.27, P=0.046), and inappropriate therapy (aOR 4.65, 95% CI 1.50-14.40, P=0.008) were identified as independent risk factors for 30-day mortality. Colistin resistance in CRKP had no significant impact on 30-day mortality. These results were also confirmed in all propensity-score-matched analyses and sensitivity analyses. Appropriate regimens were associated with better clinical outcomes than inappropriate therapies for BSIs with CRKP predominantly possessing OXA-48.

Comparison of Conventional Methods, Automated Systems, and DNA Sequence Analysis Methods in the Identification of Corynebacterium afermentans and Corynebacterium mucifaciens Bacteria Isolated from Blood and Catheter Culture Samples.

The aim of this study is to compare different methods due to the difficulties in identifying coryneform bacteria to species level and to determine antibiotic resistance profiles. Isolates identified as Turicella otitidis (n:45) by VITEK 2 Compact and Corynebacterium mucifaciens (n:1) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), isolated from blood and catheter cultures between 2015 and 2017 were included in the study. For identification of the isolates, conventional tests and 16S rDNA sequence analysis were performed. Antibiotic susceptibilities of the isolates were determined by Etest. The isolates identified as T. otitidis with VITEK 2 Compact could not be identified by MALDI-TOF MS and described as C. mucifaciens/Corynebacterium afermentans spp. by 16S rDNA sequence analysis. One isolate identified as C. mucifaciens by MALDI-TOF MS could not be identified with VITEK 2 Compact and described as C. mucifaciens by 16S rDNA sequence analysis and conventional methods. All isolates (n:45) described as C. mucifaciens/C. afermentans spp. by 16S rDNA sequence analysis were identified as C. afermentans subsp. afermentans with conventional methods. All 45 isolates identified as C. afermentans subsp. afermentans were resistant to penicillin, erythromycin, and clindamycin and were susceptible to vancomycin and daptomycin, whereas 31 (69%) were resistant to trimethoprim-sulfamethoxazole (TMP-SXT). The isolate identified as C. mucifaciens was susceptible to penicillin, vancomycin, daptomycin, and TMP-SXT; it was resistant to erythromycin and clindamycin. In this study, we reported 45 C. afermentans isolates misidentified as T. otitidis in routine laboratory processes. To our knowledge, this is the first study to include the highest number of C. afermentans blood isolates.

A silent outbreak due to Klebsiella pneumoniae that co-produced NDM-1 and OXA-48 carbapenemases, and infection control measures.

OBJECTIVES: Infections due to carbapenemase-producing Klebsiella pneumoniae are associated with high morbidity and mortality. In this study, we report a hospital outbreak due to co-producing OXA-48 and NDM-1 K. pneumoniae clone. The aim of the study is to investigate the clonal relationship of strains, risk factors of outbreak and infection control measures. MATERIALS AND METHODS: Once an outbreak was suspected at the end of December 2017 in our intensive care unit (ICU), carbapenem resistance K. pneumoniae identified in patients' specimens. An outbreak analysis was begun to determine the risk factors and dissemination of the cases. A case-control study was conducted to determine the risk factors. To control the outbreak; tight contact prevention, good clean-up the medical devices and hospital environment, were done. Staff training programs such as hand hygiene, disinfection, wearing aprons, good cleaning were created. Carbapenem resistance genes determined by PCR. Clonal relationships of strains investigated by PFGE. RESULTS: We investigate 21 carbapenem-resistant K. pneumonia strains. Nine of them were found co-produced NDM-1 and OXA-48, 11 strains produced OXA-48, and one strain produced NDM-1. Seven strains of co-producing NDM-1 and OXA-48 were found clonally related with PFGE. We could not determine any risk factor except rectal colonization in the case-control study. CONCLUSION: The interventions that successfully controlled this outbreak were hand hygiene, tight contact prevention, good clean-up of the hospital environment and medical devices. As a result, we believe that it would be beneficial to take infection control measures to prevent the spread of these strains to the community and hospital settings.

[Co-production of OXA-48 and NDM-1 Carbapenemases in Providencia rettgeri: the first report]. / Providencia rettgeri'de OXA-48 ve NDM-1 karbapenemaz genlerinin birlikte üretimi: ilk bildirim.

Our country is the epicenter of the OXA-48-like carbapenemase-producing Klebsiella and Escherichia coli; and in the recent years, the concern has been increasing due to both spreading of this resistance to other members of Enterobacteriaceae family and acquiring other carbapenemases by the OXA-48-producing strains. In this study, OXA-48 and NDM-1 co-production was presented in Providencia rettgeri. Two P.rettgeri strains that were resistant to all antimicrobials except colistin and tigecyclin, were isolated from two patients in the burn unit of our hospital, including one from the urine sample of a 68 years female in April 2017, and the other from a burn wound swab of a 35 years old male, in November 2017. Minimal inhibitory concentrations (MICs) of the isolates for imipenem and meropenem were measured as ≥ 32 µg/ml; and for colistin and tigecyclin were 1 ve 0.5 µg/ml, respectively. Multiplex PCR analysis showed that both strains were carrying blaOXA-48 and blaNDM-1 carbapenemases, and blaTEM extended spectrum beta-lactamase genes. By using DNA sequence analysis, the TEM gene was typed as blaTEM-1. The Pulsed Field Gel Electrophoresis (PFGE) analysis indicated that these two strains which were consecutively isolated from two different patients in a single unit within about seven months were genetically indistinguishable. No significant data that could explain the spread of these isolates was obtained from our retrospective analysis of the medical records including the results of environmental surveillance cultures, and patients' history. Nevertheless, hospital infection control committee enforced the infection control measures in that unit, and no further isolation was observed within three months period following the last isolation, neither from environmental nor from clinical samples. With this study, it was emphasized that the co-production of OXA-48 and NDM-1 carbapenemases which was reported from only three Enterobacteriaceae species up to date was ongoing for spreading to other species by using horizontal route, and also showing a potential to be a growing problem in the hospitals, by clonal expansion (vertical route). Effectively using of the molecular epidemiological methods will provide useful data to better understand the transmission dynamics of such rare, but problematic species in hospitals.

Hospital Outbreak of a Colistin-Resistant, NDM-1- and OXA-48-Producing Klebsiella pneumoniae: High Mortality from Pandrug Resistance.

Colistin resistance causes substantial problems in the treatment of serious infections with carbapenem-resistant (CR) gram-negative bacteria. In this study, we report a fatal hospital outbreak from the spread of a pandrug-resistant Klebsiella pneumoniae clone. An outbreak investigation was conducted after consecutive isolation of nine CR-K. pneumoniae (CR-Kp) strains from eight patients in two intensive care units of a university hospital within 2 weeks. Carbapenem and colistin resistance genes were investigated with PCR, clonal relationships of isolates were studied with pulse-field gel electrophoresis, and multilocus sequence types were determined. The outcomes of the affected patients were analyzed. Genotyping showed a predominant CR-Kp clone consisting of seven strains from six patients. These strains were in ST11 type, an international high-risk clone. They were resistant to all antimicrobials, including colistin, and positive for NDM-1 and OXA-48 carbapenemases, but negative for plasmid-borne colistin resistance genes. One patient had colonization and the remaining five died due to the infection within mean 12 days. No environmental or staff links could be established, and the outbreak was stopped by augmenting infection-control measures. Colistin-resistant K. pneumoniae could clonally expand in the hospital setting, and this spread might be associated with high mortality due to the lack of an appropriate treatment option. Immediate implementation of infection-control measures may be the best way to limit fatal consequences of the spread of such incurable pathogens.

Clinical Strains of Chryseobacterium and Elizabethkingia spp. Isolated from Pediatric Patients in a University Hospital: Performance of MALDI-TOF MS-Based Identification, Antimicrobial Susceptibilities, and Baseline Patient Characteristics.

Our objective was to evaluate the performance of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of the Chryseobacterium and Elizabethkingia spp. isolated from pediatric patients at Hacettepe University Hospital using 16S rRNA gene sequencing as the gold standard and to determine the antimicrobial susceptibility patterns of the isolates and baseline characteristics of patients. All stored Chryseobacterium and Elizabethkingia spp. isolated from various clinical specimens (sputum, blood, and urine) of pediatric patients at Hacettepe University Hospital between 2012 and 2016 were included in this study. Minimum inhibitory concentrations of 10 antimicrobial agents were determined by Etest for all isolates. To determine the baseline characteristics of patients, medical records of all patients were retrospectively reviewed. In total, 18 isolates of Chryseobacterium spp. (16 C. indologenes, 2 C. gleum) and 5 isolates of Elizabethkingia spp. (3 E. meningoseptica, 2 E. anophelis) were identified by 16S rRNA sequencing. MALDI-TOF MS correctly identified 19 (82.6%) isolates to the species level. The quinolones (ciprofloxacin and levofloxacin), trimethoprim/sulfamethoxazole and piperacillin/tazobactam showed the highest spectrum of activity against the overall collection of isolates. Cystic fibrosis (CF) was the underlying disease in 81.8% of patients. To our knowledge, this study includes the largest number of Chryseobacterium spp. isolated from clinical specimens of pediatric patients in Turkey. In this study, we also report the first clinical isolate of E. anophelis in Turkey. Since, the majority of strains were isolated from patients with CF; larger, prospective clinical studies are needed to establish whether chryseobacteria could be considered as an emerging opportunistic pathogen in patients with CF.

[Femoral hemodialysis catheter-related bacteremia due to Globicatella sanguinis: challenges in species identification]. / Femoral hemodiyaliz kateteri ile iliskili Globicatella sanguinis bakteremisi: tür düzeyinde tanimlamada karsilasilan sorunlar.

In this case, catheter-related bacteremia due to Globicatella sanguinis in a 43 years old female patient undergoing hemodialysis with the diagnosis of diabetic nephropathy was presented and the methods in the laboratory diagnosis of the rare opportunistic pathogen, Globicatella cins, were nvestigated. A set of peripheral blood cultures and simultaneous catheter culture was obtained from the patient in third of May 2016. Biochemical tests, Phoenix (Becton Dickinson, USA) and MicroScan (Beckman Coulter, USA) automated systems and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) based Microflex MS (Bruker, Daltonics, Germany) and VITEK MS (database v2.0) (bioMérieux, France) systems were used for the identification of the cultured bacteria. Partial 16S rDNA sequencing was done by using specific p8FPL 5'-AGT TTG ATC ATG GCT CAG-3' and p806R 5'-GGA CTA CCA GGG TAT CTA AT-3' primers. Minimal inhibitory concentrations (MICs) for vancomycin, erythromycin, imipenem, cefotaxime and benzypenicillin were determined by agar gradient method. The blood and catheter cultures yielded the same type of bacterial colonies. Alfa-hemolytic, catalase negative colonies observed on blood agar plates after an over night incubation yielded gram-positive cocci on Gram staining. In Sisli Hamidiye Etfal Hospital, the isolate was identifed as G.sulfidifaciens (score value > 2) by Bruker MS system and as G.sanguinis by Phoenix automated system. In Inönü University, the isolate could not be identified by Microscan automated system while VITEK MS named the isolate as 99.9% G.sanguinis and 98.3% G.sulfidifaciens. The 16S rDNA sequencing identifed the isolate as 100% G.sanguinis (GenBank accessionno. KJ680157.1). The MIC values were 0.38 µg/ml, 1.5 µg/ml, 0.38 µg/ml, > 32 µg/ml and 64 µg/ml for vancomycin, eryrthromycin, imipenem, cefotaxime and benzylpenicillin, respectively. The patient was diagnosed as catheter-related bacteremia and vancomycin (1 x 1 g IV/72 h) was used for up to 10 days. No fever and bacterial growth in cultures were present in her control visits. As G.sanguinis is not among the commonly encountered pathogens and due to difficulties in laboratory diagnosis, it may be missedor mis-identified in clinical laboratories. BD Phoenix and Bruker MS data bases lack G.sulfidifaciens and G.sanguinis, respectively, while the Globicatella genus is not present in MicroScan database. The increased number of medical implementations and the increasing number of immunosuppressed patient populations in recenty ears will lead to the emergence of rare bacteria. Increasing the diagnostic power of clinical microbiology laboratories by conventional and molecular methods and renewal of the databases of commercial identification systems by expanding the pathogen spectrum are significantly important for the prevention and control of infections caused by these agents.

Systemic nocardiosis in a dog caused by Nocardia cyriacigeorgica.

BACKGROUND: Systemic nocardiosis due to Nocardia cyriacigeorgica has not been reported in dogs. CASE PRESENTATION: Light and electron microscopy, microbiological culture and molecular identification (PCR) were used to diagnose systemic nocardiosis caused by Nocardia cyriacigeorgica in a 3-month-old husky dog. The postmortem changes included multifocal to coalescing, sharply circumscribed pyogranulomatous inflammation and abscess formation in lungs, liver, myocardium, spleen, kidneys, brain, and hilar lymph nodes. The organism was isolated and sequencing of its 16S rRNA allowed its identification and speciation. Examination of the bacterial culture by scanning electron-microscope showed filamentous branching with fragmentation into widely bacillary and cocoid forms of the bacteria. There was no history of immunosupressive drug administration and infection by the immunosuppresive viral pathogens, canine distemper and parvovirus were excluded via PCR. CONCLUSION: N. cyriacigeorgica should be considered potential cause of systemic pyogranulomatous lesions in dogs. It is the first reported case of systemic nocardiosis due to N. cyriacigeorgica in a dog.

Evaluation of the Carbapenem Inactivation Method for Detection of Carbapenemase-Producing Gram-Negative Bacteria in Comparison with the RAPIDEC CARBA NP.

Timely detection of carbapenemases by both phenotypic and genotypic methods is essential for developing strategies to control the spread of infections by carbapenem-resistant isolates and related morbidity and mortality. The aim of this study was to compare the performance of a commercial kit, the RAPIDEC® CARBA NP, and an in-house technique, the carbapenem inactivation method (CIM), against a panel of 136 carbapenemase- and noncarbapenemase-producing Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates. RAPIDEC CARBA NP displayed 99% sensitivity and 100% specificity, whereas the sensitivity and specificity were 78% and 100% for the CIM test, respectively. A slight modification of the CIM test, a prolonged incubation time of 4 hours instead of two, increased the sensitivity of the test to 90% by diminishing false negativity particularly for A. baumannii. In conclusion, both tests possess a high performance and are practical for the detection of carbapenemases. Although RAPIDEC CARBA NP is a more rapid and reliable method, the CIM test may represent a useful tool for microbiology laboratories due to its simplicity and availability at any laboratory with low cost.

[Evaluation of the diagnostic performance of Xpert MTB/RIF test for the detection of Mycobacterium tuberculosis and rifampin resistance in clinical samples]. / Klinik örneklerde Mycobacterium tuberculosis'in saptanmasi ve rifampin direncinin tespitinde Xpert MTB/RIF testinin tanisal performansinin degerlendirilmesi.

Rapid and accurate detection of active tuberculosis (TB) cases is one of the most important goal of tuberculosis control programme. For this purpose, new methods are being developed to isolate, serotype and determine the drug resistance of the agent. Xpert MTB/RIF test (CepheidGeneXpert® System, USA) that has been recently developed, is a real-time polymerase chain reaction-based method which detects Mycobacterium tuberculosis complex and resistance of the strain to rifampicin (RIF) from the clinical sample directly within a couple of hours. However, there are not sufficient data about the performance of that test for extrapulmonary samples and pulmonary samples other than sputum. The aims of this study were to investigate the sensitivity, specificity, positive and negative predictive values of Xpert MTB/RIF test in detection of M. tuberculosis and the performance in the determination of rifampicin resistance of the isolates from pulmonary and extrapulmonary clinical samples. A total of 2160 clinical samples, in which 1141 (52.8%) were pulmonary and 1019 (47.2%) were extrapulmonary samples, sent to our laboratory between July 2013 to December 2014, were included in the study. Sixty seven of the evaluated samples (3.1%) were positive with microscopy (acid-fast stain; AFS), 116 samples (5.1%) were positive with culture and 98 samples (4.5%) were positive with Xpert MTB/RIF test. When the culture was considered as the reference method, the sensitivity and specificity of Xpert MTB/RIF test were determined as 73.3% and 99.3%, respectively for all samples; 77.5% and 99.5%, respectively for pulmonary samples and 63.9% and 99.2%, respectively for extrapulmonary samples. Among AFS positive samples, the sensitivity was 100% and specificity was 66.7%; whereas among AFS negative samples those values were 40.4% and 99.4%, respectively. Among all the samples involved in the study, RIF resistance was determined only in three samples with Xpert MTB/ RIF test and that was also proved phenotypically (100% concordance). According to mycobacterial culture results, positive and negative predictive values of Xpert MTB/RIF test were determined as 86.7% and 98.5%, respectively for all samples. Those were determined as 92.5% and 98.3%, respectively for extrapulmonary samples and were determined as 74.2 and 98.7%, respectively for pulmonary samples. According to the results obtained in our study, sensitivity of Xpert MTB/RIF test for extrapulmonary samples was found to be at moderate level; sensitivity of the test was found to be decreased especially in AFS negative samples with less bacilli load. Nonetheless, specificity of Xpert MTB/RIF test to the agent in all samples was found to be extremely high. In our study, although RIF-resistant strains were detected in few of the samples, Xpert MTB/ RIF test could differentiate all resistant and sensitive strains. Additionally, detection of M. tuberculosis and RIF resistance in our laboratory takes approximately 20.96 days with culture, this period decreases to a couple of hours with Xpert MTB/RIF test. Because of the advantages such as being practical, rapid and requiring minimal safety measures, it was concluded that Xpert MTB/RIF test may contribute to rapid diagnosis of TB also in extrapulmonary samples, with the confirmation of culture method.

The optimization of a rapid pulsed-field gel electrophoresis protocol for the typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp.

Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for subtyping of Acinetobacter baumannii, Escherichia coli and Klebsiella spp., which are commonly isolated from nosocomial infections in many hospitals. Reproducibility of our PFGE procedure was studied three times at 2- to 3-week intervals. Epidemiological concordance of the optimized PFGE procedure was tested on seven isolates of A. baumannii from a previous outbreak and seven A. baumannii isolates randomly selected among the clinical isolates. The optimized PFGE procedure was evaluated on a total of 174 clinical isolates including 62 A. baumannii, 50 E. coli, and 62 Klebsiella spp. The inter-laboratory reproducibility of the optimized protocol was tested at four laboratories. The optimized procedure is completed in 28 h after culturing. It is likely to be cost-effective, due to the reduction in the time, reagent volume and enzyme concentration needed. The procedure showed high concordance with epidemiological data. There were no non-typeable isolates among the tested bacteria. It is reproducible and versatile. This protocol can be used to identify outbreaks and monitor the spreading rate of nosocomial infections caused by the tested bacterial isolates. Furthermore, due to its high intra- and inter-laboratory reproducibility, the protocol has the potential to be useful for comparing PFGE fingerprinting profiles of the isolates from different settings.