Phylogenetic analysis of the mycoplasma isolated from the Javanese macaque (<Emphasis Type="Italic">M. fascicularis</Emphasis>)

A nucleotide sequence of the 16S-rRNA mycoplasma fragment was detected. The fragment was identified using the PCR method in a urogenital scrape of the Javanese macaque (M. fascicularis). A sequenced fragment of M. fascicularis mycoplasma was compared to well-known sequences of the mycoplasma of mammals. Comparative and phylogenetic analyses performed with a sequenced DNA fragment have shown the mycoplasma to be classified M. primatum in the same cluster as M. hominis. The mycoplasma M. primatum was first revealed in M. fascicularis and M. primatum monkeys. The pathogenicity of this mycoplasma species with respect to monkeys is being studied.     

Phylotyping and ColV plasmid-associated virulence genotyping of <Emphasis Type="Italic">E. coli</Emphasis> isolated from broiler chickens with colibacillosis in Iran

Avian pathogenic Escherichia coli (APEC) strains are responsible for significant losses to the poultry industry in many parts of the world. Although, many serotypes of E. coli are non pathogen, a relatively few number of serotypes carry virulence genes which make them damaging for chicken and other poultry. Previously, high prevalence of virulence genes carried on plasmid and/or chromosomal DNA has demonstrated in APECs obtained from different countries. The aim of the present study is characterization of E. coli isolated from broiler chickens with pericarditis and femoral head necrosis (FHN) associated with colibacillosis based on the phylogenetic typing and ColV plasmid-carried virulence genotyping. In present study, 290 E. coli isolates from pericardial sac (n?=?120) and lesions of femur (n?=?100) of chickens exhibiting clinical and necropsy findings of colibacillosis from poultry farms in Fars province, Iran, besides 70 isolates from the intestinal content of apparently healthy birds were phylotyped and examined for the presence of seven virulence genes (ompT, hly, iss, iutA, iron, tsh, and cvaC) carried on ColV plasmid by multiplex PCR. Phylotyping analysis showed that the most of the APEC isolates fell into the phylogenetic group A and subgroup A₁. Also, virulence genotyping indicated that ColV plasmid-carried virulence genes were significantly higher in APEC compared to avian fecal E. coli (AFEC). Based on the results of this study, having several virulence genes of ColV plasmid and belonging to the phylogenetic group A might provide useful characteristics for the better identifying Iranian APECs. However, the link between avian E. coli pathogenicity and different phylotypes and genotypes needs more future works involving a larger number of samples to be confirmed.     

Molecular xenomonitoring (MX) and transmission assessment survey (TAS) of lymphatic filariasis elimination in two villages,Menoufyia Governorate,Egypt

Lymphatic filariasis (LF) is focally endemic in Egypt where the female mosquito, Culex pipiens, is responsible for its transmission. The aim of the study was to investigate the impact of implementation of the 13th round of MDA in two Egyptian villages in the Menoufyia Governorate area after failing the transmission assessment survey (TAS) in 2005 using two methods, and to decide whether it is safe to stop MDA in these, as well as in similar implementation units (IUs). To achieve this aim, both the immunochromatographic card test (ICT) and molecular xenomonitoring (MX) techniques were employed. A cross-sectional study was carried out in the villages in 2014 with two sections:

• Section (1): a school-based survey where all the primary school entrants (6–7) years of age were tested by ICT.
• Section (2): a mosquito-based survey where a total of 152 mosquito pools collected from Samalay and 167 from Kafr El-Tarainah were tested for the presence of the gDNA of Wuchereria bancrofti microfilaria by real-time PCR assays.

The results revealed that all primary school children in both villages were 100% negative for antigenemia. Also, all mosquito pools were 100% negative for the microfilarial gDNA.

     

Isolation and characterization of a novel,T7-like phage against <Emphasis Type="Italic">Aeromonas veronii</Emphasis>

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6–10) and temperatures (4–45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.     

Morphological characterization and <Emphasis Type="Italic">HSP70</Emphasis>-, <Emphasis Type="Italic">IGS</Emphasis>-based phylogenetic analysis of two microsporidian parasites isolated from <Emphasis Type="Italic">Antheraea pernyi</Emphasis>

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 μm, and the other type was short-axis-oval, measuring 3.64 × 2.17 μm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 μm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.     

Genomic insights into the TTSS island of enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> and its conjugational transfer

Whole genome sequences of the non-pathogenic K12 and pathogenic O127:H6 strains of Escherichia coli were analyzed using Mauve software. The genomes showed 80% similarity with few desert regions including a 35.99 kb region in pathogenic strain. This region contained Locus of Enterocyte Effacement (LEE) and Type III Secretion System (TTSS) genes. Whole genome alignment of this E. coli pathogenic strain and Salmonella enterica subsp. enterica serovar Newport str. SK254 showed 40% homology. Intimin protein coding escU gene of E. coli and ssaU gene of S. enterica were analyzed by BLASTn and ClustalW and cross referred with Pathogenicity Island Database (PDI DB) to check similarity with other foodborne bacterial species. The ssaU gene of Salmonella was found to be designated as escU in E. coli database. Comparison of these genes at nucleotide and amino acid sequence level revealed possible codon redundancies. Six clinical isolates of E. coli (designated as SBANU 1 to 6) were screened for salt aggregation and hemolysin production abilities followed by PCR analysis of escU gene. The E. coli isolate SBANU 6 was further used in induced conjugation assay with S. enterica as donor. PCR analysis and sequencing of the amplified DNA in E. coli transconjugant cells revealed the possible acquiring of ssaU gene from S. enterica.     

Association study of <Emphasis Type="Italic">Demodex</Emphasis> bacteria and facial dermatoses based on DGGE technique

The role of bacteria is unclear in the facial skin lesions caused by Demodex. To shed some light on this issue, we conducted a case-control study comparing cases with facial dermatoses with controls with healthy skin using denaturing gradient gel electrophoresis (DGGE) technique. The bacterial diversity, composition, and principal component were analyzed for Demodex bacteria and the matched facial skin bacteria. The result of mite examination showed that all 33 cases were infected with Demodex folliculorum (D. f), whereas 16 out of the 30 controls were infected with D. f, and the remaining 14 controls were infected with Demodex brevis (D. b). The diversity analysis showed that only evenness index presented statistical difference between mite bacteria and matched skin bacteria in the cases. The composition analysis showed that the DGGE bands of cases and controls were assigned to 12 taxa of 4 phyla, including Proteobacteria (39.37–52.78%), Firmicutes (2.7–26.77%), Actinobacteria (0–5.71%), and Bacteroidetes (0–2.08%). In cases, the proportion of Staphylococcus in Firmicutes was significantly higher than that in D. f controls and D. b controls, while the proportion of Sphingomonas in Proteobacteria was significantly lower than that in D. f controls. The between-group analysis (BGA) showed that all the banding patterns clustered into three groups, namely, D. f cases, D. f controls, and D. b controls. Our study suggests that the bacteria in Demodex should come from the matched facial skin bacteria. Proteobacteria and Firmicutes are the two main taxa. The increase of Staphylococcus and decrease of Sphingomonas might be associated with the development of facial dermatoses.     

Uncommonly isolated clinical <Emphasis Type="Italic">Pseudomonas</Emphasis>: identification and phylogenetic assignation

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.     

Telomere length in non-neoplastic gastric mucosa and its relationship to <Emphasis Type="Italic">H</Emphasis>. <Emphasis Type="Italic">pylori</Emphasis> infection,degree of gastritis,and NSAID use

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. We measured average telomere length using quantitative real-time PCR in non-neoplastic gastric mucosa and assessed its relationship to H. pylori-related gastritis, DNA methylation, ulcer disease, and nonsteroidal anti-inflammatory drug (NSAID) usage. Gastric biopsies were obtained from 151 cancer-free subjects including 49 chronic NSAID users and 102 nonusers. Relative telomere length in genomic DNA was measured by real-time PCR. H. pylori infection status, histological severity of gastritis, and serum pepsinogens (PGs) were also investigated. E-cadherin (CDH1) methylation status was determined by methylation-specific PCR (MSP). Average relative telomere length of H. pylori-infected subjects was significantly shortened when compared to H. pylori-negative subjects (p = 0.002) and was closely associated with all histological parameter of gastritis (all p values <0.01) and CDH1 methylation (p = 0.0002). In H. pylori-negative subjects, NSAID users presented significantly shorter telomere length than nonusers (p = 0.028). Shorter telomere length was observed in duodenal and gastric ulcer patients compared with non-ulcer subjects among NSAID users. Telomere shortening is closely associated with severity of H. pylori-induced gastritis and CDH1 methylation status. Also, telomere shortening is accelerated by NSAID usage especially in H. pylori-negative subjects.     

Serological reactivity to <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in neoehrlichiosis patients

The tick-borne bacterium Candidatus (Ca.) Neoehrlichia (N.) mikurensis is a cause of “fever of unknown origin” because this strict intracellular pathogen escapes detection by routine blood cultures. Case reports suggest that neoehrlichiosis patients may display serological reactivity to Anaplasma (A.) phagocytophilum. Since Anaplasma serology is part of the diagnostic work-up of undetermined fever in European tick-exposed patients, we wanted to investigate (1) the prevalence of A. phagocytophilum seropositivity among neoehrlichiosis patients, (2) the frequency of misdiagnosed neoehrlichiosis patients among A. phagocytophilum seropositive patients, and (3) the frequency of A. phagocytophilum and Ca. N. mikurensis co-infections. Neoehrlichiosis patients (n?=?18) were analyzed for A. phagocytophilum IgM and IgG serum antibodies by indirect immunofluorescence assay. Serum samples from suspected anaplasmosis patients (n?=?101) were analyzed for bacterial DNA contents by singleplex PCR specific for A. phagocytophilum and Ca. N. mikurensis, respectively. One fifth of the neoehrlichiosis patients (4/18) were seropositive for IgM and/or IgG to A. phagocytophilum at the time of diagnosis. Among the patients with suspected anaplasmosis, 2% (2/101) were positive for Ca. N. mikurensis by PCR whereas none (0/101) had detectable A. phagocytophilum DNA in the serum. To conclude, patients with suspected anaplasmosis may in fact have neoehrlichiosis. We found no evidence of A. phagocytophilum and Ca. N. mikurensis co-infections in humans with suspected anaplasmosis or confirmed neoehrlichiosis.     

The first molecular genetic identification of the tularemia pathogen in <Emphasis Type="Italic">Ixodes trianguliceps</Emphasis> Bir. ticks in Russia

A real-time PCR (RT PCR) assay for Francisella tularensis DNA in ixodes ticks Ixodes trianguliceps (140 mature individuals and 211 nymph pools) found on small mammals from the Middle Ural forests (Chusovskoi district, Perm Region) has been performed for the first time. Francisella DNA was detected in 12 mature ticks and 4 nymph pools when the 16SrRNA gene (amplicon size 1165–1170 bp) was used as the target. Amplification of a shorter fragment of the same gene (221–222 bp) resulted in identification of additional positive samples among mature ticks (17 of 128) and nymph pools (16 of 89). All 49 RT-PCR positive samples were identified as F. tularensis DNA using primers and probes complementary to a fragment of the lpnA (tul4) gene and the ISFtu2 element. The data obtained are indicative of the possible involvement of I. trianguliceps ticks in tularemia pathogen circulation in natural foci of forest type.     

Cloning,molecular characterization,and expression analysis of the unc45 myosin chaperone b(<Emphasis Type="Italic">unc45b</Emphasis>)gene of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

Unc45 myosin chaperone b(unc45b)gene is a molecular chaperone that mediates the folding, assembly and accumulation of thick-filament myosin in the formation of sarcomere, which plays an important role in the development of striated muscle and the stability of sarcomere. In this study, the complete cDNA sequence of unc45b gene of grass carp was obtained by rapid amplification of cDNA ends (RACE), and the characteristics of the unc45b protein predicted from gene sequence was analyzed by bioinformatics methods. The differential expression pattern in tissues was also detected by quantitative real-time PCR. The results showed that the full-length of unc45b gene of grass carp is 3163 bp, which contains a 60 bp 5′UTR, a 298 bp 3′UTR, and a 2865 bp open reading frame (ORF) encoding a 934 amino acid peptide. The deduced unc45b protein exhibits a homology of 92, 86, 86 % with the protein of zebrafish (Danio rerio), channel catfish (Ietalurus punctatus) and tilapia (Oreochromis niloticus) respectively, and the protein contains UCS myosin head binding domain and TPR peptide repeat domain. The protein is a hydrophilic and non-secretory protein with a molecular mass and isoeletronic point of 103,699.8 and 7.39 Da. The structural elements of the protein includes α-helixes and loops, and the unc45b gene highly expresses in skeletal muscle and heart in grass carp. This study laid a foundation for further research in explaining the myofibril accumulation in crisped grass carp.     

High frequency of occupied <Emphasis Type="Italic">attB</Emphasis> regions in Norwegian <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates supports a two-step MRSA screening algorithm

Rapid nucleic acid amplification tests for methicillin-resistant Staphylococcus aureus (MRSA) diagnostics commonly target the mec resistance gene, genes specific for S. aureus, and the integration site for the SCCmec resistance cassette, orfX. Due to poor specificity when these target genes are used individually, additional culture is required to verify positive results. The combination of these targets is useful, but the optimal algorithm may depend on the presence of the genetic markers in S. aureus isolates, as well as the prevalence of MRSA in a population. The aim of the present study was to identify a rapid, low-cost, and functional screening algorithm in order to reduce the response time for MRSA diagnostics. An in-house orfX-SCCmec polymerase chain reaction (PCR) assay was established and evaluated. The results were compared with an existing mec/nuc PCR assay and traditional culture. Methicillin-sensitive S. aureus (MSSA) that tested false-positive in the orfX-SCCmec PCR assay were further investigated with full genome sequencing using the Ion PGM? System to verify results and causality. Based on these data, a two-step screening algorithm with initial mec/nuc PCR followed by orfX-SCCmec PCR on positive samples was suggested and tested on 1443 patient samples. 22.5 % of MSSA isolates tested false-positive with the orfX-SCCmec PCR. Full genome sequencing of these isolates identified genetic variation in the attB region of S. aureus, including empty cassette variants and non-mec SCC. The suggested two-step MRSA screening algorithm allowed us to report MRSA results for 95.6 % of all samples and 99 % of MRSA-negative samples after one day.     

Preparation of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> DNA from respiratory samples for real-time PCR by commercial kits

Bordetella pertussis is increasingly detected by real-time PCR, but most kits for extracting Bordetella-DNA from respiratory samples are not validated for this material. Respiratory clinical materials were spiked with Bordetella pertussis cells. Four ion-exchange chromatography methods from one manufacturer were used for DNA preparation. Two real-time PCRs detecting the IS481 of Bordetella pertussis and based either on a hybridisation probes format (LightCycler®) or on a TaqMan® format were used. All kits effectively prepared DNA for Bordetella pertussis real-time PCR. The procedures were linear over a broad range, and the lower level of sensitivity was similar. Sensitivities measured as CT values were different. Inter-assay CVs were between 6.8% and 17.3%. Two kits did not effectively remove inhibitory substances from the respiratory samples. Commercial kits are useful for preparing Bordetella pertussis DNA from respiratory samples, but even kits from one manufacturer show significant differences in effectiveness and removal of inhibitory substances.     

Diversity of plasmids and Tn<Emphasis Type="Italic">1546</Emphasis>-type transposons among VanA <Emphasis Type="Italic">Enterococcus faecium</Emphasis> in Poland

The objective of this study was to investigate the antimicrobial resistance, Tn1546 transposon variability and plasmid diversity among Polish vancomycin-resistant Enterococcus faecium (VREfm) isolates of VanA phenotype in the context of their clonal structure. Two hundred sixteen clinical VREfm isolates collected between 1997 and 2010 were studied by antimicrobial susceptibility testing, MLST, MLVA and detection of IS16, esp _Efm, pilA, intA and plasmid-specific genes by PCR. Tn1546 structure was revealed by overlapping PCR and sequencing. Selected isolates were subjected to PFGE-S1 and Southern hybridization analyses. The vast majority of the isolates (95.8 %) belonged to lineages 17/18 (during the whole study period 1997–2010) and 78 (mostly in 2006–2010) of hospital-adapted meroclone of E. faecium. All isolates displayed a multi-drug resistance phenotype. Twenty-eight Tn1546 types (including 26 novel ones) were associated with eight different ISs (IS1216, IS1251, ISEfa4, ISEfa5, ISEfm2, ISEf1, IS3-like, ISEfm1-like). The vanA-determinant was typically located on plasmids, which most commonly carried rep2_pRE25, rep17_pRUM, rep18_pEF418, rep1_pIP501, ω-ε-ζ and axe-txe genes. VanA isolates from 1997–2005 to 2006–2010 differed in clonal composition, prevalence of gentamicin- and tetracycline-resistance and plasmidome. Our analysis revealed high complexity of Tn1546-type transposons and vanA-plasmids, and suggested that diverse genetic events, such as conjugation transfer, recombination, chromosomal integration and DNA mutations shaped the structure of these elements among Polish VREfm.     

Investigation of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> in Iranian camels

Coxiella burnetii is a zoonotic, obligate intracellular bacterium that caused Q fever. Antibodies to this organism have been reported in a wide range of animals including mammals, reptiles, amphibians, and birds. This study is aimed to detect C. burnetii in camel by polymerase chain reaction (PCR). Blood samples from 130 camels were collected between August and September 2011 then examined in laboratory conditions. Detection of the presence of C. burnetii DNA was carried out using a PCR assay with specific primers (Coc-F and Coc-R) targeting the 16S ribosomal RNA gene (242 bp). In this study, a total of 14 (10.76 %) camel blood samples were found PCR positive for C. burnetii. This result proves that camels are an important reservoir of C. burnetii infection. This study showed relatively high positivity of C. burnetii in Iranians camels, and accordingly, it seems necessary to evaluate the prevalence for this microorganism in Iran.     

Development of a method for identification and genotyping of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> and <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> bacteria using polymerase chain reaction and phylogenetic analysis of bacterial cultures isolated from cattle

The results of developing the identification and genotyping method for the Pasteurella multocida bacteria of five capsule groups and the Mannheimia haemolytica A 1 bacteria, using the multiplex polymerase chain reaction (PCR) with the electrophoretic detection are reported. The diagnostic sensitivity of the developed method came to 10³ CFU/mL in the pure culture studies and 10⁵ CFU/g in the biological material studies. The analysis revealed 50% of P. multocida and 11.2% of M. haemolytica in all 260 tested samples of biological material from the infected animals. Circulation of bacteria of P. multocida capsule groups B and E among the susceptible animals was not determined. Group A bacteria were found in the majority of the samples; bacteria of group D were infrequently identified; in one case, group F bacteria were detected. The circulation of capsule group A P. multocida bacteria of two genetic types was determined using phylogenetic analysis.     

Development of <Emphasis Type="Italic">T</Emphasis><Subscript><Emphasis Type="Italic">m</Emphasis></Subscript>-shift genotyping method for detection of cat-derived <Emphasis Type="Italic">Giardia lamblia</Emphasis>

To develop T _m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5′-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T _m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T _m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10^?5 and 4.88 × 10^?5 ng/μL samples of assemblage A and F standard plasmids. The T _m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T _m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T _m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.     

<Emphasis Type="Italic">Burkholderia cepacia</Emphasis> complex in Serbian patients with cystic fibrosis: prevalence and molecular epidemiology

The Burkholderia cepacia complex (Bcc) organisms remain significant pathogens in patients with cystic fibrosis (CF). This study was performed to evaluate the prevalence, epidemiological characteristics, and presence of molecular markers associated with virulence and transmissibility of the Bcc strains in the National CF Centre in Belgrade, Serbia. The Bcc isolates collected during the four-year study period (2010–2013) were further examined by 16 s rRNA gene, pulsed-field gel electrophoresis of genomic DNA, multilocus sequence typing analysis, and phylogenetic analysis based on concatenated sequence of seven alleles. Fifty out of 184 patients (27.2 %) were colonized with two Bcc species, B. cenocepacia (n?=?49) and B. stabilis (n?=?1). Thirty-four patients (18.5 %) had chronic colonization. Typing methods revealed a high level of similarity among Bcc isolates, indicating a person-to-person transmission or acquisition from a common source. New sequence types (STs) were identified, and none of the STs with an international distribution were found. One centre-specific ST, B. cenocepacia ST856, was highly dominant and shared by 48/50 (96 %) patients colonized by Bcc. This clone was characterized by PCR positivity for both the B. cepacia epidemic strain marker and cable pilin, and showed close genetic relatedness to the epidemic strain CZ1 (ST32). These results indicate that the impact of Bcc on airway colonization in the Serbian CF population is high and virtually exclusively limited to a single clone of B. cenocepacia. The presence of a highly transmissible clone and probable patient-to-patient spread was observed.     

Incidence of antibiotic resistance and virulence determinants in <Emphasis Type="Italic">Enterococcus faecium</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> strains,isolated from traditional cheeses in Turkey

Enterococcus faecalis (n = 15) and Enterococcus faecium (n = 33) strains isolated from traditional Turkish cheeses were tested for susceptibility to 11 different antimicrobial agents and for the presence of selected genes encoding resistance and 13 genes encoding virulence factors using PCR. Furthermore, the plasmid profile of enterococci was examined. All E. faecium and E. faecalis isolates were resistant to nalidixic acid and kanamycin. The percentages of other resistant E. faecium and E. faecalis isolates, respectively, were 97 and 100% to streptomycin, 15.1 and 20% to ampicillin, 9.1 and 26.7% to gentamycin, 12.1 and 46.6% to chloramphenicol, 12.1 and 60% to tetracycline, 75.7 and 93.3% to rifampicin, 84.8 and 80% to vancomycin, 97 and 100% to erytromycin and 72.7 and 60% to ciprofloxacin. efaA _fm (100%) and ccf (90.1%) genes were the most common virulence genes identified among E. faecium isolates while efaA _fs (100%), cpd (100%), ccf (93.3%) and cob (86.7%) genes among E. faecalis isolates. Cytolysin determinants (cylM, cylB, cylA) were not detected among tested strains. Plasmid profile analysis of Enterococcus spp. revealed plasmid DNA bands ranging in size from 2.4 to 35.8 kb.