Can Imirzalioglu

Culture-Independent Identification of Pathogenic Bacteria and Polymicrobial Infections in the Genitourinary Tract of Renal Transplant Recipients

ABSTRACT Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.

Predictors of surgical site infections among patients undergoing major surgery at Bugando Medical Centre in Northwestern Tanzania

BackgroundSurgical site infection (SSI) continues to be a major source of morbidity and mortality in developing countries despite recent advances in aseptic techniques. There is no baseline information regarding SSI in our setting therefore it was necessary to conduct this study to establish the prevalence, pattern and predictors of surgical site infection at Bugando Medical Centre Mwanza (BMC), Tanzania.MethodsThis was a cross-sectional prospective study involving all patients who underwent major surgery in surgical wards between July 2009 and March 2010. After informed written consent for the study and HIV testing, all patients who met inclusion criteria were consecutively enrolled into the study. Pre-operative, intra-operative and post operative data were collected using standardized data collection form. Wound specimens were collected and processed as per standard operative procedures; and susceptibility testing was done using disc diffusion technique. Data were analyzed using SPSS software version 15 and STATA.ResultsSurgical site infection (SSI) was detected in 65 (26.0%) patients, of whom 56 (86.2%) and 9 (13.8%) had superficial and deep SSI respectively. Among 65 patients with clinical SSI, 56(86.2%) had positive aerobic culture. Staphylococcus aureus was the predominant organism 16/56 (28.6%); of which 3/16 (18.8%) were MRSA. This was followed by Escherichia coli 14/56 (25%) and Klebsiella pneumoniae 10/56 (17.9%). Among the Escherichia coli and Klebsiella pneumoniae isolates 9(64.3%) and 8(80%) were ESBL producers respectively. A total of 37/250 (14.8%) patients were HIV positive with a mean CD4 count of 296 cells/ml. Using multivariate logistic regression analysis, presence of pre-morbid illness (OR = 6.1), use of drain (OR = 15.3), use of iodine alone in skin preparation (OR = 17.6), duration of operation ≥ 3 hours (OR = 3.2) and cigarette smoking (OR = 9.6) significantly predicted surgical site infection (SSI)ConclusionSSI is common among patients admitted in surgical wards at BMC and pre-morbid illness, use of drain, iodine alone in skin preparation, prolonged duration of the operation and cigarette smoking were found to predict SSI. Prevention strategies focusing on factors associated with SSI is necessary in order to reduce the rate of SSI in our setting.

Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses

BackgroundBlack elderberries (Sambucus nigra L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus.MethodsThe antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays.ResultsFor the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of Streptococcus pyogenes and group C and G Streptococci, and the Gram-negative bacterium Branhamella catarrhalis in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses.ConclusionRubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product.

Hidden pathogens uncovered: metagenomic analysis of urinary tract infections.

Urinary tract infections (UTIs) are the most common kidney and urologic diseases in industrial nations and are usually caused through faecal contamination of the urinary tract. In this study, we have examined 1449 urine specimens both by culture and by PCR. The majority of UTIs examined were caused by Escherichia coli (35.15%), followed by miscellaneous bacteria (23.03%), and by Enterococcus faecalis (19.39%). A large fraction of fastidious and anaerobic bacteria (22.43%) was not detected under culture conditions but only by using PCR. This group of bacteria evade the standard culture conditions used in routine diagnostic laboratories examining urine specimens. The molecular approach used broad‐range 16S rDNA PCR, denaturing high‐performance liquid chromatography analysis, sequencing, and bioinformatic analysis to uncover these ‘hidden’ pathogens and is recommended in particular when examining leukocyte esterase‐positive and culture‐negative urinary tract specimens.

Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX-M-15 in a tertiary hospital in Tanzania.

The molecular epidemiology of 32 non-duplicate, CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains, isolated from clinical samples, was investigated. Multilocus sequence typing revealed multiple sequence type clonal complexes: ST131 (12), ST405 (4), ST638 (3), ST38 (2), ST827 (2), ST224 (1), ST648 (1), ST46 (1) and two new sequence type clonal complexes (1845 and 1848) in 22 pulsed field gel electrophoresis clusters. The bla(CTX-M-15) gene was located on conjugative IncF plasmids. This is the first report of the worldwide emerging clonal complex ST131 linked to bla(CTX-M-15) in Tanzania and demonstrates the need for constant surveillance in developing countries to prevent the spread of these multiresistant isolates.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: An approach to quantify the distribution of ESBL types between different reservoirs

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Multiresistant extended-spectrum β-lactamase- producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany

BackgroundMultiresistant Gram-negative bacteria producing extended-spectrum β-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of β-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses.ResultsIn this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac(‘6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates.ConclusionsIsolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.

Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany

BackgroundMulti-drug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital.MethodsWe analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays.ResultsExamination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb.ConclusionOur data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.

Multidrug-resistant bacteria in geriatric clinics, nursing homes, and ambulant care – Prevalence and risk factors

Colonization/infection with multidrug-resistant bacteria (MDRB) such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, is an increasing problem not only in hospitals but also in long-term care facilities. The aim of this study was to determine the prevalence as well as the risk factors of colonization/infection with MRSA, VRE, and ESBL producing Enterobacteriaceae in geriatric clinics, nursing homes, and ambulant care in Frankfurt am Main, Germany. 288 patients from 2 geriatric clinics (n=46), 8 nursing homes (n=178), and 2 ambulant care facilities (n=64) as well as 64 staff members were screened for MDRB in the time period from October 2006 to May 2007. 58 patients (20.1%) and 4 staff members (6.2%) were colonized with MDRB. Among patients, 27 (9.4%) were colonized with MRSA, 11 (3.8%) were screened positive for VRE, and 25 (8.7%) were found to be colonized with ESBL producing Enterobacteriaceae. Prevalence of MDRB in geriatric clinics, nursing homes, and ambulant care facilities were 32.6%, 18.5%, and 15.6%, respectively. Significant risk factors for MDRB were immobility (OR: 2.7, 95% CI: 1.5-4.9; p=0.002), urinary catheter (OR: 3.1, 95% CI: 1.7-5.9; p<0.001), former hospitalization (OR: 2.1, 95% CI: 1.1-4.0; p=0.033), and wounds/decubiti (OR: 2.3, 95% CI: 1.5-4.9; p=0.03). Finally, the high level of MDRB in geriatric clinics, nursing homes, and ambulant care points to the importance of these institutions as a reservoir for dissemination.

Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania

BackgroundKlebsiella pneumoniae strains expressing ESBLs are a predominant cause of hospital acquired infections. Here we describe the molecular epidemiology of these isolates in a tertiary hospital in Tanzania, as potential pathogens for neonatal infections.MethodsBetween April 2009 and March 2010 all Klebsiella pneumoniae isolates with phenotypic expression Extended Spectrum Beta Lactamase (ESBL) were collected and characterized. Identification was done using in house biochemical tests in case of ambiguous results confirmation was done using API 20E. Susceptibility testing was determined using the disc diffusion method followed by specific PCR and sequencing to determine ESBL genes. Phylogenetic analysis, Pulse field gel electrophoresis (PFGE) and Multi-Locus sequence typing (MLST) to PFGE clusters representative isolates were performed to determine clones of the isolates. Conjugation and hybridization were performed to determine the location of blaCTX-M-15 gene.ResultsA total of 92 non- repetitive ESBL producing K. pneumoniae representing 50.3% of Klebsiella pneumoniae isolates were characterized. These isolates were from blood 61 (66%), wound swab 13 (14%), urine 12 (13%) and pus 6 (7%) were analyzed. Most blood culture strains originated from neonatal unit 39/61(64%) and 22 (36%) of the blood culture isolates were from neonatal ICU. All isolates were resistant to gentamicin and 54% were resistant to ciprofloxacin. Using a similarity index of 80%, the isolates were assigned to thirteen clusters based on PFGE patterns and contained sub-clusters with identical strains indicating clonal outbreaks. Cluster X5, X7 and X8, and X9 were grouped into ST48, ST14 and ST348 respectively. Based on gyrA PCR- RFLP phylogenetic analysis all isolates were grouped as KpI. The predominant ESBL allele detected was blaCTX-M-15 which was found in 76% of isolates, followed by blaTEM-104 (19%), blaSHV-11 (3.2%) and blaTEM-176 (2%). The blaCTX-M-15 gene was located in multiple conjugative IncF plasmids ranging from 25 kb-485 kb in size.ConclusionThe high prevalence of blaCTX-M-15 observed among ESBL producing K. pneumoniae in Tanzania, is possibly due to the spread of a common IncFII 145 kb plasmid and of certain clones such as ST14 and ST48. Furthermore the 485 kb plasmid detected is the largest plasmid reported to carry blaCTX-M-15 todate.