Metagenomic analysis of wastewater phageome from a University Hospital in Turkey.

Phage DNA analysis gives opportunity to understand living ecosystem of the environment where the samples are taken. In the present study, we analyzed phage DNA obtained from wastewater sample of university hospital sewage. After filtration, long high-speed centrifugation was done to collect phages. DNA was extracted from pellet by phenol chloroform extraction and used for NGS sequencing. The host profile, taxonomic and functional analyses were performed using MG-RAST, and ResFinder program was used for resistance gene detection. High amounts of reads belong to bacteriophage groups (~ 95%) from our DNA sample were obtained and all bacteriophage reads were found belonging to Caudovirales order and Myoviridae (56%), Siphoviridae (43%), and Podoviridae (0.02%) families. The most common host genera were Escherichia (88.20%), Salmonella (5.49%) and Staphylococcus (5.19%). SEED subsystems hits were mostly structural parts and KEGG Orthology hits were nucleotide- and carbohydrate metabolism-related genes. No anti-microbial resistance genes were detected. Our bacteriophage DNA purification method is favorable for phage metagenomic studies. Dominance of coliphages may explain infrequent Podoviridae. Dominancy of structural genes and auxiliary genes is probably due to abundance of lytic phages in our sample. Absence of antibiotic resistance genes even in hospital environment phages indicates that phages are not important carrier of resistance genes.

Molecular Detection of Virulence Associated Genes in Coagulase Negative Staphylococci Isolated from Blood Culture.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are one of the most important causes of infections. Unlike Staphylococcus aureus, less is known about their pathogenic mechanisms. In the present study, we aimed to evaluate the presence of virulence genes among 98 CoNS isolated from blood cultures of inpatients. METHODS: The isolates were identified by MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). PCR was performed to detect 29 virulence factors using specific primers for icaA, icaB, icaC, icaD, icaADB, aap, fbe, aae, sesI, atIE, hla, hlb, hld, gehC, gehD, sea, seb, sec, sed, see, seg, seh, sei, tst, eta, etb, etd, etx, and pvl genes. The VITEK2 system (bio-Merieux, France) and the BD Phoenix™ System (Becton Dickinson, USA) were used for antimicrobial susceptibility testing. RESULTS: Staphylococcus epidermidis was found to be the most virulent CoNS species. All isolates were negative for eta, etb, etd, sea, seb, sed, see, seg, sei, and pvl virulence genes. We detected up to 15 virulence genes in a single isolate. The most common gene was icaC (73.5%), followed by icaA (57.1%), icaD (56.1%), aap (55.1%), aae (52.0%), sesl (51.0%), gehC (50.0%), hld (50.0%), hlb (49.0%), fbe (44.9%), atIE (37.8%), icaADB (37.8%), gehD (34.7%), icaB (31.6%), hla (30.6%), etx (2.0%), sec (1.0%), seh (1.0%), and tst (1.0%). CONCLUSIONS: We determined high rates of genes encoding biofilm formation. Only four isolates did not possess either the ica operon or aap gene. Although we found low rates of toxin-related genes, our data indicates that apart from biofilm formation, the CoNS isolates could express various virulence genes similar to those of Staphylococcus aureus.

Carbapenem and Colistin Resistant Klebsiella Pneumoniae ST14 and ST2096 Dominated in Two Hospitals in Turkey.

BACKGROUND: The determination of clonal interactions between microorganisms is very important in epidemiological studies. In the present study, we aimed to evaluate the resistance mechanisms and genetic relationships of carbapenem and colistin resistant Klebsiella pneumoniae (K. pneumoniae) strains isolated from inpatients at two university hospitals in Turkey. METHODS: A total of 38 K. pneumoniae clinical isolates were included in the study. Identification of isolates was confirmed by 16S rRNA sequencing. Antimicrobial susceptibility testing was performed with VITEK-2 system (bio-Mérieux, France). Modified Hodge test was used for the detection of carbapenemase activity in isolates. Carbapenem resistance genes (blaOXA-48, blaNDM, blaKPC, blaIMP, blaVIM) and colistin resistance genes (mcr-1, mcr-2 and mcr-3) were investigated by PCR. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequence typing (MLST) analysis were used to determine the genetic relatedness among the isolates. RESULTS: We detected that 58% of isolates were positive for only blaOXA-48, 5% were only positive for blaNDM, and 34% were positive for both blaOXA-48 and blaNDM. blaKPC, blaIMP, blaVIM, mcr-1, mcr-2, and mcr-3 were not detected among the isolates. Only one carbapenem resistant isolate was negative for the carbapenemase genes tested. A total of nine profiles were found by ERIC-PCR, and MLST results showed seven different sequence types-ST14, ST16, ST79, ST101, ST1543, ST2096, and ST2832. The seven STs were grouped by PHYLOVIZ Online into four clonal complexes. The most common ST was ST14 (81%) in Center 1 and ST2096 (94%) in Center 2. CONCLUSIONS: We determined MLST types of carbapenem and colistin resistant K. pneumoniae isolates from two different centers. Although the most common ST types were different in these centers, both ST types were clustered in CC14. To the best of our knowledge this is the first report of ST14 and ST2096 outbreaks in Turkey.

Early Detection and Control of Methicillin resistant Staphylococcus aureus Outbreak in an Intensive Care Unit.

BACKGROUND: Although Methicillin resistant Staphylococcus aureus (MRSA) is one of the major pathogens of healthcare associated infections, we had only sporadic cases in our intensive care unit (ICU) for years. AIMS: To investigate the sudden increase in the number of MRSA cases in ICU. STUDY DESIGN: Descriptive study. METHODS: From the 5th December 2016 to 26th January 2017, we detected 11 new MRSA cases in ICU. Screening of 73 ICU healthcare workers (HCWs) and screening of 13 patients was performed for outbreak investigation. Nine clinical isolates available in stocks and eight screening MRSA isolates were included in molecular studies. PFGE, spa-mecA-mecC-PVL in-house multiplex PCR assay and spa typing, SCCmec typing were performed for all isolates. Sequence type of the representative strain was determined by Multi-Locus Sequence typing (MLST). RESULTS: All strains were mecA positive, PVL negative, and have the same antimicrobial susceptibility pattern except for two strains. All clinical, two patient screening and three nasal isolates of HCWs showed the same pulsotype, named clone A. The spa type of outbreak isolates is t030 and the SCCmec type is SCCmecIII; the MLST type of representative strain is ST239 (PFGE pulsotype A, ST239-SCCmecIII-t030). Unrelated three isolates had PFGE pulsotype B-SCCmecI-t030, PFGE pulsotype C-SCCmecIII-t459, PFGE pulsotype D-SCCmecIII. CONCLUSION: Molecular typing techniques are the cornerstones for the investigation of outbreaks. Infection control measures, such as enhancing cleaning procedures, promoting hand hygiene, should be enforced in the ICU unit. All patients, including those who have already been discharged to other departments, must be put on contact isolation. HCWs carrying the MRSA strains could be offered decolonization.

Cloned ermTR Gene Confers Low Level Erythromycin but High Level Clindamycin Resistance in Streptococcus pyogenes NZ131.

Objectives: The most common macrolide resistance mechanisms in streptococci are the presence of methylase encoding genes ermB and ermTR or the presence of efflux encoded by mef genes. In the present study we aimed to show the effects of the ermTR gene under isogenic conditions on the activities of macrolides and lincosamides in streptococci. Materials and Methods: Total DNA was extracted from Streptococcus pyogenes C1, and the ermTR gene was amplified with or without the regulatory region using modified primer with insertion of restriction sites to clone in to pUC18. Transformants were selected after electroporation of Escherichia coli DB10. The recombinant plasmids were purified and merged to pJIM2246 to transform Gram positive bacteria. Recombinant pJIM2246 plasmids with the ermTR gene were then introduced into S. pyogenes NZ131 by electroporation. Results: After transformation with ermTR without regulatory region the minimal inhibitory concentration (MIC) for erythromycin and clindamycin increased from ≤0.06 to ≤0.06 to 8 and >128 mg/L, respectively. Induction with erythromycin affected the MICs for clindamycin of S. pyogenes transformed with ermTR with the regulatory region. Double disk testing showed that induction with erythromycin and azithromycin for the S. pyogenes transformed with ermTR, and regulatory regions decreased the clindamycin inhibition zone but not telithromycin. The ermTR gene in isogenic conditions confers low level resistance to erythromycin and high level resistance to clindamycin. Conclusion: The different induction and resistance profiles of ermTR compared to other erm genes suggest that the methylation of ErmTR may be different than well studied methylases.

Virulence genes fliC, toxA and phzS are common among Pseudomonas aeruginosa isolates from diabetic foot infections.

BACKGROUND: Outcomes of antibiotic treatment of diabetic foot infections (DFIs) may depend not only on the antimicrobial susceptibility of the aetiologic agents, but also their ability to produce virulence factors. This study aimed to use polymerase chain reaction (PCR) with specific primers to investigate the presence of virulence genes among isolates of Pseudomonas aeruginosa isolates cultured from specimens from diabetic foot and other infections. METHODS: We examined 63 P. aeruginosa isolates from inpatients at two University Hospitals for the presence of 23 known bacterial virulence genes, including lasI, lasR, lasA, lasB, rhll, rhlR, rhlAB, aprA, fliC, toxA, plcH, plcN, ExoS, ExoT, ExoU, ExoY, phzI, phzII, phzM, phzS, pvdA, pilA and pilB. RESULTS: Seven virulence genes (lasl, lasR, lasB, rhll, rhlR, rhlABand Exo T) were present in each isolate. No isolate expressed or presented aprA gene. We found that fliC (p = .01), toxA (p = .041) and phzS (p < .001) were statistically and significantly more common in diabetic foot isolates, while plcH (p < .001) was significantly more common in other infections. CONCLUSIONS: Among clinical isolates of P. aeruginosa from patients with DFIs, three virulence genes that can play important roles in tissue penetration (fliC), tissue damage and survival under anaerobic condition (phzS) and cell death (toxA) were significantly more common than isolates from other infections. The Multilocus sequence typing (MLST) analysis of diabetic foot isolates failed to point/indicate the existence of a specific clone or was not able to characterize/identify a specific clone/clonal complex group. Development of new agents to inhibit the synthesis of these genes may improve outcomes in DFIs treatment.

[t030 is the most common spa type among methicillin-resistant Staphylococcus aureus strains isolated from Turkish hospitals]. / t030, Türkiye'deki Hastanelerden Izole Edilen Metisiline Dirençli Staphylococcus aureus Izolatlari Arasinda En Yaygin spa Tipidir.

Staphylococcus aureus is one of the most frequent agents causing hospital infections. S.aureus has a great ability to adapt itself to variety of conditions and successful clones can be epidemic and even pandemic by its ability spread from one continent to another. The aims of this study were to detect spa types of 397 methicillin-resistant S.aureus (MRSA) strains isolated from 12 centers in different geographical regions of Turkey from 2006 to 2008, and to investigate their clonality by PFGE and MLST typing. Additionally, 91 MRSA from four of those 12 centers isolated during 2011 were also studied for their spa types. PFGE profiles indicated the presence of a major pulsotype, namely pulsotype A with a rate of 91.4% (363/397), followed by pulsotype B (n= 18, 4.5%) and pulsotype C (n= 11, 2.8%). Among isolates tested 363 (91.4%) were SCCmec type III, 30 (7.6%) were SCCmec type IV. Sequence analysis of representative isolates revealed that ST239 (85.1%) was the most common MLST type followed by two MLST types ST737 (4%), and ST97 (2.8%), both SCCmec type IV. Two isolates were ST80 with SCCmec type IV. Of 397 isolates, 338 (85.1%) were t030, followed by t005 (2.5%) and t632 (2%). Among MRSA isolated during 2011, 64 (70.3%) of 91 were t030, 4 (4.4%) were t005, 2 (2.2%) were t015, and 2 (2.2%) were t1094. Among centers the t030 prevalence of 2006-2008 isolates ranged from 59-100%. The highest t030 prevalence was found in Ankara (100%) and lowest in Trabzon (59%) provinces which are located at central and northestern Anatolia, respectively. In Istanbul province, the prevalence of t030 was 94.5% among 2006-2008 isolates which decreased to 55.5% among 2011 isolates. Also a decrease in t030 rates was observed among samples from Konya and Trabzon but not from Aydin. Our results showed that the most common MRSA clone in Turkey is ST 239-SCCmec type III, t030 which persisted during the six years of the study period. Presence of PVL toxin gene was tested by PCR and 5 (3%) isolates found to be positive, of them two were SCCmec Type IV-ST80 and three were SCCmec Type III-ST239. This study is the largest epidemiological survey ever done in Turkey which showed presence of a hospital Turkish clone TR09 (ST239-SCCmecIII-t030) and a community clone TR10 (ST737-SCCmecIV-t005) largely disseminated in Turkey.

[MLST types of vancomycin-resistant Enterococcus faecium strains isolated from blood cultures]. / Kan kültürlerinden izole edilen vankomisine dirençli Enterococcus faecium suslarinin MLST tipleri.

Enterococci, particularly vancomycin-resistant enterococci (VRE), are important nosocomial pathogens with limited treatment options. Enterococci have low-level resistance to penicillins and aminoglycosides and are intrinsically resistant to cephalosporins. In addition, they can acquire high-level resistance to beta-lactam antibiotics, aminoglycosides and glycopeptides. The aim of this study was to determine glycopeptide resistance mechanisms and genetic relationships of vancomycin-resistant E.faecium strains isolated from blood cultures between 2003-2009 years by molecular epidemiologic methods. A total of 38 VRE strains isolated from blood cultures were included in this study. The isolates were identified by conventional methods and Phoenix 100 BD automated system (Becton Dickinson Diagnostic Systems, USA) and confirmed by sequence analysis of 16S rRNA amplicons. Antibiotic susceptibility tests were performed by the Kirby-Bauer disk diffusion method accor-ding to the CLSI standards. MIC values of vancomycin were determined in vancomycin resistant strains by E-test (AB Biodisk, Sweden) method. Vancomycin resistance genes included vanA, vanB, vanC, and vanD were investigated by polymerase chain reaction (PCR) method. Clonal relationship between strains was determined by pulsed-field gel electrophoresis (PFGE). Sequence analysis was performed for examples selected for multilocus sequence typing (MLST) of each pulsotype and subtype. Thirty eight strains of enterococci isolated from blood cultures were defined as E.faecium by phenotypic methods and confirmed by 16S rRNA sequence analysis. Vancomycin MIC values of strains were determined as > 256 µg/ml by E test. The vanA gene was detected in all isolates. Clonal relationship of 38 isolates E.faecium carrying the vanA gene was determined by PFGE and MLST methods. PFGE detected four pulsotypes (A-D) and one sporadic isolate. Twenty nine strains belonged to A pulsotype, three strains belonged to B pulsotype, two strains belonged to C pulsotype and three strains belonged to D pulsotype. Out of 29 isolates, eight strains were type A1, nine strains were type A2, six strains were type A3, two strains were type A4 and four strains were type A5. MLST identified four different sequence types (STs). Twenty nine A pulsotype and its subtypes belonged to ST117 (76.3%), three B pulsotype belonged to ST280 (7.9%), two C pulsotype belonged to ST18 (5.2%) and three D pulsotype belonged to ST17 (7.9%). In conclusion, bloodstream infections caused by VRE in our hospital arose from a dominant strain belonged to ST117. However, presence of different pulsotypes of this strain indicated that the strain had been present in the hospital for a long time and had accumulated genetic variations. In addition, infections caused by minor pulsotypes were also detected. Therefore for prevention and control of the spread of nosocomial infections caused by VRE, it is crucial to identify resistance patterns and clonal relationship of these organisms.

[Distribution of emm genotypes and antibiotic susceptibility of Streptococcus pyogenes strains: analogy with the vaccine in development]. / Streptococcus pyogenes suslarinin emm genotiplerinin dagilimi ve antibiyotik duyarliligi: Gelistirilmekte olan asi ile karsilastirma.

Streptococcus pyogenes is the most common bacterial pathogen causing pharyngotonsillitis, and also can lead to diseases such as otitis media, impetigo, necrotizing fasciitis, bacteremia, sepsis and toxic shock-like syndrome. M protein encoded by emm gene is an important virulence factor of S.pyogenes and it is used for genotyping in epidemiological studies. The aims of this study were to determine the M protein types of group A streptococci (GAS) by using emm gene sequence analysis method, to compare the M types in terms of analogy with the vaccine in development and to determine the antibiotic susceptibilities of the isolates. A total of 35 GAS strains isolated from various clinical specimens in our laboratory were included in the study. Strains growing in blood culture were considered as invasive, strains growing in throat and abscess cultures were considered as non-invasive. The isolates have been identified by conventional methods and 16S rRNA sequence analysis at species level. emm genotyping of strains identified as S.pyogenes, was performed by PCR method as proposed by the CDC. Amplicons were obtained and sequenced in 23 out of 35 isolates. The results were compared with CDC emm sequence database. Antibiotic susceptibility of the isolates was performed by agar dilution method and evaluated as recommended by CLSI. Twenty-three out of 35 isolates could be typed and 15 different emm genotypes were detected. The most common emm types were emm1 (22%), emm89 (13%), emm18 (9%) and emm19 (9%). The detection rate of other emm types (emm5, 12, 14, 17, 26, 29, 37, 74, 78, 92, 99) was 47%. Types emm1, 12, 19, 74, 89 and 99 were observed in strains isolated from blood cultures. It was detected that nine of the 15 (60%) emm types are within the contents of 26 valent vaccine (emm 1, 5, 12, 14, 18, 19, 29, 89, 92). It was also observed that 17 (74%) of the 23 cases were infected by vaccine types and the four emm types (emm1, 12, 19, 89) identified in blood samples were among the vaccine types. All of the strains were found susceptible to penicillin, ampicillin, erythromycin, lincomycin, gentamicin, chloramphenicol, vancomycin and linezolid, however six isolates were resistant to levofloxacin (MIC= 4 and 16 µg/ml) and one isolate was resistant to tetracycline (MIC= 16 µg/ml). In conclusion, this preliminary local study with limited number of invasive and non-invasive S.pyogenes isolates, emphasized the need for larger scale multi-center studies to determine the analogy and efficacy of the vaccine in development.

ICESp1116, the genetic element responsible for erm(B)-mediated, inducible resistance to erythromycin in Streptococcus pyogenes.

ICESp1116, responsible for erm(B)-mediated, inducible erythromycin resistance in Streptococcus pyogenes, was comprehensively characterized, and its chromosomal integration site was determined. It displayed a unique mosaic organization consisting of a scaffold, related to TnGallo1 from Streptococcus gallolyticus, with two inserted fragments separated by IS1216. One fragment, containing erm(B), displayed high-level identity to a portion of the S. pyogenes plasmid pSM19035; the other, containing a truncated tet(M) gene, displayed high-level identity to the right-hand portion of Clostridium difficile Tn5397.

Biological decolorization of textile dyes from isolated microfungi.

In this study, biological decolorization of two textile dyestuff (Benazol black ZN and Cibacron black W-NN) was comparatively studied using 22 microfungi strains isolated from polluted industrial soil areas. The initial dye concentrations in the medium were 250 and 500 mg l(-1). Benazol black ZN was the best decolorized by Haematonectria haematococca (HH1) (36.0%) and Cibacron black W-NN was the best decolorized by Aspergillus niger (AN1) (33.0%) at 250 mg l(-1) dye concentration. At 500 mg l(-1) dye concentration for two different dyes all microfungi strains used showed weak decolorization rates, maximum 13.0% for Benazol black ZN and 6.0% for Cibacron Black W-NN.

Phenotypic and genotypic characterization of bacteriocins in enterococcal isolates of different sources.

UNLABELLED: A collection of 57 enterococcal isolates from different origin (including river, treatment plant, spring and garbage water, soil, animal, and vegetables from Aydin) was screened for the production of bacteriocins. Enterococci were identified at species levels as Enterococcus faecium (34), E. hirae (6), E. casseliflavus (4), E. durans (4), E. faecalis (4), E. mundtii (3) and E. avium (2). Of the 57 isolates 40 of them inhibited the growth of at least one indicator bacterium. Based on our PCR results 54 strains possesed enterocin genes. The genes of entA and entB were the most frequently detected structural genes among the PCR positive strains (54 and 53 strains, respectively) and the entB gene was always associated with entA gene. The highest combination of enterocin genes (24 of 54 strains) detected was entA, entB, entP and entL50A/B. The enterocins AS-48 and CylL(LS) genes were not found. Three enterococcal isolates, 2 E. faecium and 1 E. hirae were not harbour any of tested enterocin genes. No correlation between the presence of enterocin structural genes and the origin of the strain was detected, also no relationship seemed to exist between the tested enterocin genes and the activity spectra of isolates. Genes encoding bacteriocins are widely disseminated among enterocci from different origin and more studies should be done for evaluate industrial potential of bacteriocins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12088-011-0143-0) contains supplementary material, which is available to authorized users.