Molecular identification and phylogenetic analysis of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> from human blood samples in Egypt

Lymphatic filariasis (LF) is a serious vector-borne health problem, and Wuchereria bancrofti (W.b) is the major cause of LF worldwide and is focally endemic in Egypt. Identification of filarial infection using traditional morphologic and immunological criteria can be difficult and lead to misdiagnosis. The aim of the present study was molecular detection of W.b in residents in endemic areas in Egypt, sequence variance analysis, and phylogenetic analysis of W.b DNA. Collected blood samples from residents in filariasis endemic areas in five governorates were subjected to semi-nested PCR targeting repeated DNA sequence, for detection of W.b DNA. PCR products were sequenced; subsequently, a phylogenetic analysis of the obtained sequences was performed. Out of 300 blood samples, W.b DNA was identified in 48 (16%). Sequencing analysis confirmed PCR results identifying only W.b species. Sequence alignment and phylogenetic analysis indicated genetically distinct clusters of W.b among the study population. Study results demonstrated that the semi-nested PCR proved to be an effective diagnostic tool for accurate and rapid detection of W.b infections in nano-epidemics and is applicable for samples collected in the daytime as well as the night time. PCR products sequencing and phylogenitic analysis revealed three different nucleotide sequences variants. Further genetic studies of W.b in Egypt and other endemic areas are needed to distinguish related strains and the various ecological as well as drug effects exerted on them to support W.b elimination.     

Genotyping of <Emphasis Type="Italic">Nocardia farcinica</Emphasis> with multilocus sequence typing

Nocardia are aerobic Gram-positive saprophytes that are widely distributed in nature, but some species cause nocardiosis, especially opportunistic infections that affect immunocompromised patients mostly. In this study, we developed a multilocus sequence typing (MLST) scheme using seven housekeeping genes (gyrB, hsp65, secA1, rpoB, rpoA, recA, and trpB) for genotyping the most common clinical species, Nocardia farcinica (37 clinical isolates from the patients with nocardiosis and seven from animals in China and 15 reference strains). The results showed that using these loci could perform accurate identification among different species, and high discriminative power within the N. farcinica species. Of the 59?N. farcinica isolates, 44 sequence types have been identified; 32 STs covering 46 isolates could be assigned to six clonal complexes that encompassed most of the collected strains. The results showed that these strains displayed a sufficiently informative population structure using this method. Our study also provided a suitable approach for epidemiological studies of N. farcinica. A large clonal complex comprising 16 strains was identified, and was notable for its wide distribution and host adaptation. This complex should be monitored closely and merits further study.     

Low occurrence of the new species <Emphasis Type="Italic">Staphylococcus argenteus</Emphasis> in a <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> collection of human isolates from Belgium

Staphylococcus argenteus is a novel Staphylococcus species closely related to Staphylococcus aureus that has been recently described. In this study, we investigated the proportion and the characteristics of S. argenteus recovered from humans in Belgium. S. aureus. human isolates collected in Belgium from 2006 to 2015 (n?=?1,903) were retrospectively characterised via the presence of non-pigmented colonies on chocolate agar, spa typing and rpoB sequencing to determine if some of them were in fact S. argenteus. Out of 73 strains non-pigmented on chocolate plates, 3 isolates (0.16 %) showed rpoB sequences, in addition to spa and sequence types (ST2250/t5787, ST2250/t6675, ST3240/t6675), related to S. argenteus. Two of them were methicillin-resistant, harbouring a SCCmec type IV. The three S. argenteus isolates carried genes (sak, scn) of the immune evasion cluster. This first Belgian nationwide analysis showed a low occurrence of S. argenteus. Further studies should be conducted to identify the distribution range and the clinical impact of this new species.     

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.     

The Phenomenon of the Formation of Biofilms by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. in the Presence of Clinical Strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The formation of biofilms by M. tuberculosis on Shkolnikova’s medium (synthetic medium, analogue of Sauton’s medium) has been researched. We studied 150 clinical and 20 laboratory strains of M. tuberculosis. None of the 150 strains isolated from human beings produced biofilms (pellicle), but all yielded abundant planktonic growth. Twenty reference strains of M. tuberculosis produced both biofilms (pellicle) and planktonic growth. The phenomenon of biofilm formation by mixed cultures was observed when inoculating sputum treated with NALC-NaOH from patients with tuberculosis. We obtained 63 mixed biofilms. In 30.2% (19/63) of cases, biofilms contained the DNA of the causative agent of tuberculosis. The RV-PCR method was used to select six samples with the highest concentration of mycobacterial DNA. Molecular cloning and sequencing of a fragment of the 16S rRNA gene from one of the biofilms was carried out. The nucleotide sequence had 99% homology with the Bacillus thermoamylovorans species. From the mixed biofilms obtained, three strains of spore-forming bacilli were isolated. Strains are identified by Sanger’s sequencing of the 16S rRNA gene, one as Bacillus licheniformis, and the other two as Brevibacillus spp. A study of the resistance of isolated strains of spore bacilli against 12 antituberculosis drugs of the first and second series was carried out. All three strains were resistant to maximum concentrations of isoniazid, streptomycin, ethionamide, and ethambutol. Strains of Brevibacillus spp. were additionally resistant to para-aminosalicylic acid (PAS) and kanamycin. In a model experiment, the possibility of cogrowth of clinical strains of M. tuberculosis and B. licheniformis was demonstrated with prolonged co-incubation in Shkolnikova’s medium. In the first few days of growth, B. licheniformis produced a biofilm that remained stable for the entire observation period of 45 days. The hypothesis suggesting the possibility of a short-term persistence of some “saprophytic” species of bacilli in the caseous contents of necrosis foci in the late stages of pulmonary tuberculosis has been postulated.     

Hypervirulent <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> induced ventilator-associated pneumonia in mechanically ventilated patients in China

The purpose of this study was to investigate the clinical characteristics of hypervirulent K. pneumoniae (hvKP) induced ventilator-associated pneumonia (VAP) and the microbiological characteristics and epidemiology of the hvKP strains. A retrospective study of 49 mechanically ventilated patients with K. pneumoniae induced VAP was conducted at a university hospital in China from January 2014 to December 2014. Clinical characteristics and K. pneumoniae antimicrobial susceptibility and biofilm formation were analyzed. Genes of capsular serotypes K1, K2, K5, K20, K54 and K57 and virulence factors plasmid rmpA(p-rmpA), iroB, iucA, mrkD, entB, iutA, ybtS, kfu and allS were also evaluated. Multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) analyses were used to study the clonal relationship of the K. pneumoniae strains. Strains possessed p-rmpA and iroB and iucA were defined as hvKP. Of 49 patients, 14 patients (28.6 %) were infected by hvKP. Antimicrobial resistant rate was significantly higher in cKP than that in hvKP. One ST29 K54 extended-spectrum-beta-lactamase (ESBL) producing hvKP strain was detected. The prevalence of K1 and K2 in hvKP was 42.9 % and 21.4 %, respectively. The incidences of K1, K2, K20, p-rmpA, iroB, iucA, iutA, Kfu and alls were significantly higher in hvKP than those in cKP. ST23 was dominant among hvKP strains, and all the ST23 strains had identical RAPD pattern. hvKP has become a common pathogen of VAP in mechanically ventilated patients in China. Clinicians should increase awareness of hvKP induced VAP and enhance epidemiologic surveillance.     

Identification of genus <Emphasis Type="Italic">Campylobacter</Emphasis> up to species level using internal features of 16S rRNA gene sequences

Introduction: 16S rRNA sequencing of novel isolates is one of the preliminary steps in characterization of bacteria, especially when the isolates are of medical relevance. The genus Campylobacter belongs to Class ε-proteobacteria under the Phylum Proteobacteria. It represents economically important species which are gastrointestinal pathogens in humans and livestock animals. Currently, more than 400 16S rDNA sequences belonging to 28 species of this genus are present in the RDP database. But heterogeneity has led to the misplacement of many of these sequences within wrong species. Also, various sequences belonging to Campylobacter have been deposited as orphans. The current study aimed to explore the internal features of 16S rRNA gene sequences in order to develop methods for identification of Campylobacter up to species level. Methods: 428 16S rRNA sequences of 28 species of Campylobacter were analyzed. 392 sequences (>1200 nucleotides, nts) of 16 species were considered for (i) phylogenetic framework analysis and (ii) in silico restriction digestion. 28 uncharacterized sequences present in the database were also investigated in the present study. Results: Phylogenetic framework analysis allowed the identification of genetic variability within Campylobacter species and helped to segregate certain uncharacterized sequences up to species level. 12 out of the 16 species under study showed homogenous behavior, but heterogeneity was observed between C. jejuni and C. coli and C. helveticus and C. upsaliensis respectively. Unique restriction enzymes were identified for six species. Conclusions: The present approach clearly showed that internal features of 16S rRNA is a useful tool for characterization of novel isolates up to the species level. Studies have revealed that niche overlap and consequent increase in the horizontal gene transfer between C. coli and C. jejuni, due to anthropogenic factors, maybe the reason for their heterogeneous nature. This explains the difficulties faced in segregation of the members of these species. 16S rRNA gene proved to be a viable and excellent marker for characterizing the uncharacterized Campylobacter strains leading to a significant diminution in database redundancy. Further, the approaches used in the study might assist in easier identification of the various Campylobacter sequences present in the database.     

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.     

A method for differentiation of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> strains and phylogenetically related species based on determination of the structural differences between chromosomal genes for biosynthesis of flagellin and methionine

Nucleotide sequence analysis of several genes responsible for definitive properties of the anthrax pathogen—motility and penicillinase activity—determined a chromosomal locus promising for interspecies differentiation. We demonstrated that the fliC gene encoding flagellin synthesis contains an extended region distinguishing B. anthracis strains from the majority of nonpathogenic and opportunistic bacilli. A novel method for anthrax pathogen indication and identification based on determination of the differences in the fliC and hom2 chromosomal genes structure has been proposed. A total of 60 strains of different Bacillus spp. (B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. megaterium, B. subtilis, etc.) were tested using two chromosomal DNA targets. The algorithm developed in this work permits detection of the pathogenic microorganism and reliably differentiation of it from other Bacillus spp. representatives. The introduction of primers complementary to specific sequences of pXO1 and pXO2 plasmids into multiplex PCR makes it possible to obtain additional information on the proposed virulence of the isolate.     

Whipworm diversity in West African rodents: a molecular approach and the description of <Emphasis Type="Italic">Trichuris duplantieri</Emphasis> n. sp. (Nematoda: Trichuridae)

Whipworms were collected from rodents (Muridae) from six West African countries: Burkina-Faso, the Islamic Republic of Mauritania, and the Republics of Benin, Guinea, Mali and Senegal. Molecular sequences (ITS-1, 5.8S and ITS-2 of the ribosomal DNA gene) and morphometric characters were analysed in Trichuris (Nematoda: Trichuridae) specimens found in seven host species: Arvicanthis niloticus, Gerbilliscus gambianus, Gerbillus gerbillus, G. tarabuli, Mastomys erythroleucus, M. huberti and M. natalensis. Phylogenetic analyses revealed three clades, one recognised as Trichuris mastomysi, previously recorded in M. natalensis from Tanzania, and the other two previously undescribed. A new species named Trichuris duplantieri n. sp., found in Gerbillus spp. from Mauritania, was characterised using molecular and morphometric methods.     

Outbreak of NDM-1-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> ST76 and ST37 isolates in neonates

The purpose of this study was to investigate the epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) in Shanghai Children’s Hospital in China. Twenty-two non-duplicate CRKP strains were collected from pediatric patients between March and June in 2014. Antimicrobial susceptibility testing was conducted by the agar dilution method. Beta-lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of bla _NDM-1 was investigated by conjugation experiment. The plasmids bearing antibiotic resistance genes were characterized by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization. Clonal relatedness was evaluated by PFGE and multilocus sequence typing (MLST). The clinical data of patients were retrospectively reviewed. The 22 CRKP strains were resistant to most of the antimicrobial agents tested, except tigecycline and colistin. Overall, 59, 77, and 100 % of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. The bla _NDM-1 was positive in 77.3 % (17/22) of the CRKP strains, of which the 16 isolates from inpatients were designated as ST37 (n?=?9) and ST76 (n?=7) and one isolate from an outpatient belonged to ST846. The 17 bla _NDM-1-positive isolates belonged to PFGE type A (n?=?9), type C (n?=?7), or type B (n?=?1). The plasmids bearing bla _NDM-1 could be transferred into recipient Escherichia coli J53 through conjugation in 88.2 % (15/17) of the strains. The hybridization results showed that the plasmids carrying the bla _NDM-1 gene were approximately 50–240 kb in size. This is the first report of an outbreak caused by NDM-1-producing K. pneumoniae ST76 and ST37 among neonates.     

Clinical characteristics of infections caused by <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> P1 genotypes in children

Mycoplasma pneumoniae (M. pneumoniae) isolates can be classified into two major genetic groups, P1 type 1 (MP1) and P1 type 2 (MP2), based on the DNA sequence of the P1 adhesion protein gene. The aim of our study was to determine if M. pneumoniae P1 genotype is associated with disease manifestation and severity of acute M. pneumoniae infection. We compared epidemiological and clinical data of children infected with either MP1 or MP2. In addition, we separately analysed data of patients presenting with individual manifestations of M. pneumoniae infection. Data of 356 patients infected with MP1 were compared with those of 126 patients infected with MP2. MP2-infected children presented with higher median baseline C-reactive protein levels and were admitted to the hospital more often. The distribution of P1 genotype varied among groups of patients with different manifestations of M. pneumoniae infection. MP2 was more common than MP1 among patients with neurological and cardiovascular manifestations, whereas MP1 was more prevalent in other manifestations. The results from our large cohort indicate that the two P1 subtypes may have different pathogenic potential and that infections with MP2 strains could be more virulent than those with MP1 strains.     

Structural organization of intact and damaged by <Emphasis Type="Italic">IS</Emphasis>100 element porin genes adjacent to the PGM region in <Emphasis Type="Italic">Yersinia pestis</Emphasis> and <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> strains

The porin gene, which is adjacent to the pigmentation region (pgm), is usually damaged by IS100 element in highly virulent Yersinia pestis strains. In addition, the pgm region, which carries the genes responsible for virulence (high pathogenicity island) and biofilm generation (hms-operon), is flanked by direct IS100 copies (causing its destabilization). The study of distribution of intact and truncated porin genes was conducted among 240 Y. pestis strains from 39 natural foci of Russia and countries of the near abroad and 68 Yersinia pseudotuberculosis strains from different geographical regions. Most highly virulent Y. pestis strains and some phylogenetic Y. pseudotuberculosis lines of O:1 serotype contain truncated porin genes. At the same time, deletion of the pgm region by flanked IS100 in Y. pseudotuberculosis is impossible, since IS100 is integrated in the porin gene in an orientation opposite to that of Y. pestis. The intact porin gene is carried by all Y. pestis strains with low epidemic significance and certain phylogenetic lines of highly virulent Y. pestis strains from desert foci and Caspian sandy focus, as well as most Y. pseudotuberculosis strains of O:1 serotype. A continuous deletion, which includes the porin gene and a part of the astE gene, was detected in less virulent Y. pseudotuberculosis strains of O:3 serotype. The nucleotide sequence of porin genes is identical in Y. pestis and Y. pseudotuberculosis strains from different geographical regions. Three porin gene allele only differ by IS100 integration site and orientation or absence of its integration. The nucleotide sequence of IS100 introduced in the porin gene of Yersinia has small differences only for two Y. pestis strains isolated in America. The correlation of low frequency of Hms-mutants with the intact porin gene state in Y. pestis and the absence of such a correlation in Y. pseudotuberculosis were established.     

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, <Emphasis Type="Italic">Mythimna unipuncta</Emphasis>, contains two copies of the <Emphasis Type="Italic">lef</Emphasis>-<Emphasis Type="Italic">7</Emphasis> gene

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2–5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.     

Gene <Emphasis Type="Italic">sak0192</Emphasis> of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> contains direct repeats and spacers serving as genetic markers characterizing the strains

A new method of strain differentiation in Streptococcus agalactiae is proposed in this work. It is based on revealing the polymorphism of structure of gene sak0192 encoding a hypothetical protein, which in different strains of Str. agalactiae contains a different number of regularly alternating repeats and spacer sites having sizes of 16 and 44 bp, respectively. The sequences of forward repeats are mostly identical, while spacers are characterized with expressed heterogeneity. In general, the structure of gene sak0192 is similar to one of sites with direct repeats and spacers in genomes of Mycobacterium tuberculosis and Str. pyogenes. The possibility of using polymorphism of sak0192 in both identification of species affiliation and intraspecific differentiation of Str. agalactiae strains is demonstrated.     

Identification of a new turncurtovirus in the leafhopper <Emphasis Type="Italic">Circulifer haematoceps</Emphasis> and the host plant species <Emphasis Type="Italic">Sesamum indicum</Emphasis>

Turncurtoviruses (family: Geminiviridae; genus: Turncurtovirus) appear to have a high degree of genetic variation in Iran. Leafhoppers of the species Circulifer haematoceps (Mulsant and Rey, 1855) (family: Cicadellidae) were collected in 2014 from three geographical regions in south-eastern Iran (Orzoeyeh, Jiroft and Sirjan; Kerman province) and screened for the presence of turncurtoviruses using a combination of PCR and rolling circle amplification (RCA) methods. Eleven genomes of turncurtovirus were recovered and sequenced. Leafhoppers were sampled off sesame (S. indicum L.) and turnip (Brassica rapa sub sp. rapa). Thus, we identified three symptomatic sesame plants (yellowing, boat-shaped leaf curling, vein swelling on the lower leaf surfaces) from sesame farms in Jiroft. In these samples, we identified the same turncurtovirus as in the leafhoppers and have named it sesame curly top virus (SeCTV). Collectively, these SeCTV share?>?98% genome-wide pairwise identity and ~?87.3% to a recently identified turncurtovirus (sesame yellow mosaic virus; SeYMV) from sesame in Pakistan (GenBank accession MF344550). The SeCTV and SeYMV sequences share?<?70% genome-wide pairwise identity with isolates of Turnip curly top virus and Turnip leaf roll virus, the two species in the genus Turncurtovirus. Based on the pairwise identities and phylogenetic analysis, SeCTV (n?=?12) and SeYMV (n?=?1) represent two strains of a new species in the genus Turncurtovirus.     

Increasing incidence of carbapenemase-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in Belgian hospitals

Carbapenemase-producing Enterobacteriaceae are increasingly reported worldwide. The aim of the study was to determine the incidence and molecular epidemiology of carbapenemase-producing (CP) Escherichia coli and Klebsiella pneumoniae (CP-E/K) in Belgium. Eleven hospital-based laboratories collected carbapenem non-susceptible (CNS) isolates of E. coli and K. pneumoniae detected in clinical specimens from January 2013 to December 2014. All CNS strains were tested for carbapenemase production and typed by multilocus sequence typing (MLST) for a 6-month period as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE) structured survey. In addition, an equal number of carbapenem-susceptible isolates collected were preserved as a control group for risk factor analysis. The overall incidence rate of CP-E/K isolates in hospitals increased from 0.124 in 2013 to 0.223 per 1000 admissions in 2014. From November 2013 to April 2014, 30 CP K. pneumoniae [OXA-48 (n?=?16), KPC (n?=?13), OXA-427 (n?=?1)] and five CP E. coli [OXA-48 (n?=?3), NDM (n?=?1), OXA-427 (n?=?1)] isolates were detected in ten hospitals. The 16 OXA-48-producing K. pneumoniae strains were distributed into eight sequence types (STs), while the 13 KPC-producing K. pneumoniae clustered into three STs dominated by ST512 (n?=?7) and ST101 (n?=?5). Compared to controls, we observed among CP-E/K carriers significantly higher proportion of males, respiratory origins, previous hospitalization, nosocomial setting, and a significantly lower proportion of bloodstream infections. Our study confirms the rapid spread of CP-E/K in Belgian hospitals and the urgent need for a well-structured and coordinated national surveillance plan in order to limit their dissemination.     

<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.     

<Emphasis Type="Italic">Helicobacter pylori vacA i</Emphasis> region polymorphism but not <Emphasis Type="Italic">babA2</Emphasis> status associated to gastric cancer risk in northwestern Iran

Helicobacter pylori-specific genotypes have been strongly associated with an increased risk of gastric cancer (GC). The aim of the present work was to study the associations of H. pylori virulence factors, vacA i region polymorphisms and babA2 status with GC risk in Azerbaijan patients. The DNA extracted from gastric biopsy specimens was used to access the babA2 and vacA genotypes. Overall, babA2 was present in 85.39 % (76/89) of H. pylori strains: 19 out of 24 (79.16 %) strains from GC, 16 out of 17 (94.14 %) strains from peptic ulcer disease (PUD) and 41 out of 48 (85.14 %) strains from chronic gastritis. No significant association was found between babA2 genotype and clinical outcomes (P > 0.05). i1 vacA polymorphism was detected in 46/89 (51.68 %) strains: in 21/24 (87.5 %), 6/17 (35.29 %) and 19/48 (39.58 %) patients with GC, PUD and chronic gastritis, respectively. i2 allele was detected in 43 (48.31 %) out of all 89 strains examined: 3 (14.28 %) of 24 strains from GC, 11 (64.71 %) of 17 from PUD, and 29 (60.42 %) of 48 strains from chronic gastritis. In this study, multiple linear regression analysis confirmed the strong association of i1 allele with GC (partial regression correlation 0.455 ± 0.101; P = 0). Results of multiple logistic regression analysis showed that vacA i1 genotype was significantly associated with GC compared with a control group (gastritis) (odds ratio 13.142, 95 % CI 3.116–55.430; P = 0). Findings from the measurement of H. pylori babA2 and vacA genotypes indicate a strong correlation between the vacA i1 allele and GC risk in the Azerbaijan area of Iran.     

High prevalence of the PER-1 gene among carbapenem-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> in Riyadh,Saudi Arabia

The prevalence of carbapenem-resistant Acinetobacter baumannii in Saudi Arabia and their resistance genetic mechanisms are yet to be identified. We studied the prevalence and genetic diversity of extended-spectrum beta-lactamase genes, particularly the PER-1 gene, among carbapenem-resistant A. baumannii strains from patients at a tertiary care hospital in Riyadh, Saudi Arabia between 2006 and 2014. Fresh subcultured samples were tested for antimicrobial susceptibility minimum inhibitory concentration (MIC). Total genomic DNA was extracted from each isolate and further used for polymerase chain reaction (PCR) genotyping, sequence-based typing (SBT) of PER-1 and OXA-51-like gene, and multilocus sequence typing (MLST) of positive isolates. Randomly selected clinical isolates (n = 100) were subjected to MLST. A total of 503 isolates were characterized as multidrug-resistant (MDR) using the MIC. Isolates were further PCR tested for bla _-TEM and bla _-PER-1 resistance genes (n = 503). The genotyping results showed that 68/503 (14 %) isolates were positive to bla TEM. The genotyping results of PER-1-like genes showed that 384/503 (76.3 %) were positive among MDR Acinetobacter isolates. Based on SBT, the majority of these isolates were clustered into three main groups including isolates harboring PER-1: AB11 (bla _-PER-1), isolate AB16 (bla _-PER-1), and, finally, the plasmid pAB154 (bla _-PER-7). Remarkably, many isolates were concealing the PER-1 gene and harboring the TEM resistance genes as well. MLST results for selected isolates (n = 100) identified four main sequence types (STs: 2, 19, 20, and 25) and four novel isolates (ST 486–489). We report 76.3 % prevalence of the PER-1 resistance gene among Acinetobacter clinical isolates from Riyadh, Saudi Arabia. Further work is needed to explore the clinical risks and patient outcome with such resistance related to healthcare-associated infections and investigate the genetic and molecular mechanisms that confer the MDR phenotype.