Analysis of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> clinical isolates circulating in European countries during the period 1998–2012

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998–2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.     

Evolution of French Bordetella pertussis and Bordetella parapertussis isolates: increase of Bordetellae not expressing pertactin

Bordetella pertussis and Bordetella parapertussis are closely related bacterial agents of whooping cough. Whole-cell pertussis (wP) vaccine was introduced in France in 1959. Acellular pertussis (aP) vaccine was introduced in 1998 as an adolescent booster and was rapidly generalized to the whole population, changing herd immunity by specifically targeting the virulence of the bacteria. We performed a temporal analysis of all French B. pertussis and B. parapertussis isolates collected since 2000 under aP vaccine pressure, using pulsed-field gel electrophoresis (PFGE), genotyping and detection of expression of virulence factors. Particular isolates were selected according to their different phenotype and PFGE type and their characteristics were analysed using the murine model of respiratory infection and in vitro cell cytotoxic assay. Since the introduction of the aP vaccines there has been a steady increase in the number of B. pertussis and B. parapertussis isolates collected that are lacking expression of pertactin. These isolates seem to be as virulent as those expressing all virulence factors according to animal and cellular models of infection. Whereas wP vaccine-induced immunity led to a monomorphic population of B. pertussis, aP vaccine-induced immunity enabled the number of circulating B. pertussis and B. parapertussis isolates not expressing virulence factors to increase, sustaining our previous hypothesis.     

Comparative study of all <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis strains isolated from food and food animals in Greece from 2008 to 2010 with clinical isolates

The aim of the present work was to study the epidemiology of Salmonella enterica serovar Enteritidis (S. Enteritidis) in Greece, comparing all the food and food animal isolates during a 3-year period with clinical isolates. Submission of the generated data to the PulseNet Europe database was carried out in order to study the population structure of this particular serovar and indicate possible connections with European strains. One hundred and sixty-eight (168) S. Enteritidis strains of human, animal, and food origin, isolated during the period 2008–2010 in Greece, were studied. Strains were characterized by phenotypic (antibiotic resistance) and molecular [pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)] methods. PFGE revealed 39 XbaI, 48 BlnI, and 80 XbaI–BlnI distinct pulsotypes, suggesting several clones circulating through the food chain and multiple sources of transmission. Submission to the PulseNet Europe database indicated that PFGE profile SENTXB.0001, the most common PFGE profile in Europe, was also predominant in Greece (33.3 %). MLST showed that all the strains studied shared the same sequence type (ST11), representing the most common ST in Europe. High rates of resistance to nalidixic acid were observed among human and poultry isolates (~25 %), indicating the potential fluoroquinolone treatment failure. Our data suggest that strains originating from multiple reservoirs circulated in Greece through the food chain during the study period. Predominant profiles in Greece were common to PulseNet Europe profiles, indicating similarities between the S. Enteritidis populations in Greece and Europe.     

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.     

A proteoliposome formulation derived from <Emphasis Type="Italic">Bordetella pertussis</Emphasis> induces protection in two murine challenge models

Whooping cough remains a health problem despite high vaccination coverage. It has been recommended that development of new strategies provide long-lasting immunity. The aim of this work was to evaluate the potential of proteoliposomes (PL) extracted from Bordetella pertussis as a vaccine candidate against whooping cough. The size of the B. pertussis PL was estimated to be 96.7±50.9 nm by Scanning Correlation Spectroscopy and the polydispersity index was 0.268. Western blots using monoclonal antibodies revealed the presence of pertussis toxin, pertactin, and fimbriae 3. The Limulus Amebocyte Lisate (LAL) assay showed endotoxin levels lower than those reported for whole cell pertussis licensed vaccines, while the Pyrogen Test indicated 75 ng/mL/Kg. The PL showed high protection capacity in mouse challenge models. There was 89.7% survival in the intracerebral challenge and total reduction of the number of CFU in the intranasal challenge. No significant differences (p>0.05) were observed between mice immunized with B. pertussis PL and the Cuban DTwP vaccine, whichever challenge model used. These results encouraged us to continue the development of the B. pertussis PL as a component of a new combined vaccine formulated with tetanus and diphtheria toxoids or as a booster dose for adolescents and adults.     

Characterisation of VIM-2-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolates from lower tract respiratory infections in a Spanish hospital

To analyse the antimicrobial phenotype, carbapenem mechanisms, integrons, virulence factors and molecular typing of 164 Pseudomonas aeruginosa isolates recovered from lower tract respiratory samples in a Spanish hospital (1 year) as well as the patients’ clinical data. Susceptibility testing to 12 antipseudomonal agents was determined by microdilution and metallo-beta-lactamase (MBL) phenotype by double disc method. The oprD gene was studied by PCR, sequencing and comparison with P. aeruginosa PAO1 sequence. Detection and characterisation of MBLs, class 1, 2 and 3 integrons, and virulence genes were studied by PCR and sequencing. The prevalence of carbapenem-resistant P. aeruginosa (CRPA) was 26.8%. MBL phenotype was detected in 52.3% CRPA, and all of them were disseminated throughout the intensive care unit. Most of the MBL-carrying patients presented respiratory disease, mechanical ventilation, tracheostomy, bacteraemia, ≥?30 hospitalisation days and previous treatment with carbapenems and/or ≥?3 different antimicrobial families. The bla_VIM-2 gene was the unique MBL encoding gene and was detected inside class 1 integrons. The class 1 integrons detected in 39 strains (23.8%) were associated with aminoglycosides (aadB, aadA1, aadA6, aacA4, aac(3)-I) and carbapenems resistance genes (bla_VIM-2). The aac(3)-I?+?aadA1 and bla_VIM-2 arrangements were the most prevalent ones. Thirty-one different PFGE patterns and 4 STs (ST175, ST235, ST253, ST973) were detected among the 39 intI1-positive isolates, being ST235 the most frequent. CRPA showed a great variety of alterations in oprD gene. The exoU⁺/exoS^? genotype was detected in 82.6% of bla_VIM-2-producing strains (ST235) and the exoU^?/exoS⁺ in the remaining 17.4% (ST973).     

Direct molecular typing of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> from nasopharyngeal specimens in China in 2012–2013

Data on the molecular epidemiology of Bordetella pertussis are limited in developing countries where whole-cell pertussis vaccines (WCVs) have been used. The aim of this study was to determine the genotypes of circulating B. pertussis in China by direct molecular typing of clinical specimens. DNA extracts of 122 nasopharyngeal swabs (NPs) positive for B. pertussis by polymerase chain reaction (PCR) (targeting IS481 and ptx-Pr) from 2012 to 2013 were used for typing using the multiple-locus variable number tandem repeat analysis (MLVA) and also by PCR-based multilocus sequence typing (MLST) of B. pertussis virulence genes (ptxP, prn, and fim3). One hundred and eight DNA extracts (89 %) generated a complete MLVA type (MT). Among the 18 MTs obtained, MT55 (52 %) and MT104 (13 %) were the most common. MT27, which is linked to the ptxP3 allele and is prevalent in many developed countries using acellular pertussis vaccines (ACVs), was only found in 7 (6 %) DNA extracts. Eighty-seven DNA extracts (71 %) produced a complete multiantigen sequence typing (MAST) type. Of them, 77 (89 %) had the ptxP1/prn1/fim3-1 allele profile. Four DNA extracts (5 %) had the ptxP3/prn2/fim3-2 profile and 3 (4 %) had the ptxP3/prn1/fim3-2 allele profile. These seven DNA extracts also harbored MT27. Our result shows that B. pertussis circulating in China was different from those found in countries where ACVs have been in use, supporting the notion that selection pressure induced by WCVs and ACVs on the bacterial population differs.     

Emergence of Panton-Valentine leucocidin-positive ST8-methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (USA300 clone) in Korea causing healthcare-associated and hospital-acquired bacteraemia

Panton-Valentine leucocidin (PVL)-positive sequence type (ST)8-MRSA-SCCmec IVa (USA300) is the epidemic strain of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in North America. USA300 is extremely rare in South Korea, and PVL-negative ST72 SCCmec type IVc is the predominant CA-MRSA clone. In a multicentre, prospective cohort study of S. aureus bacteraemia, we identified PVL-positive ST8-MRSA isolates by performing multilocus sequence typing and PCR for PVL. We analyzed the clinical characteristics of patients with PVL-positive ST8-MRSA bacteraemia, and performed SCCmec, spa, and agr typing, PCR for arginine catabolic mobile element (ACME), virulence gene profiling, and pulsed-field gel electrophoresis (PFGE). Among a total of 818 MRSA isolates, we identified ten isolates of PVL-positive ST8-MRSA (USA300) (3 from Hospital D, 4 from Hospital G, and 3 from Hospital A), all of which involved exclusively healthcare-associated (5 isolates) and hospital-acquired bacteraemia (5 isolates). This strain accounted for 8~10 % of the hospital-acquired MRSA bacteraemia in Hospitals D and G. Bacteraemia of unknown origin was the most common type of infection followed by pneumonia. All the isolates were SCCmec type IVa, spa type t008, and agr group I. Eight of the isolates harboured ACME. In a PFGE analysis, four isolates were identical to the USA300 control strain, five differed by a single band, and the remaining one differed by two bands. All the isolates were pulsed-field type USA300. This is the first report of healthcare-associated and hospital-acquired bacteraemia caused by USA300 in South Korea. USA300 seems to be an emerging hospital clone in this country.     

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.     

Prevalence and molecular diversity of invasive <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> in a German tertiary care medical centre

Prevalence of invasive ß-haemolytic streptococci (BHS) at a tertiary care hospital and molecular diversity of S. pyogenes and S. dysgalactiae was studied. Between 2012 and 2016, all blood culture sets (n?=?55,839), CSF (n?=?8413) and soft tissue (n = 20,926) samples were analysed for BHS positivity using HYBASE software. Molecular profiles of 99 S. pyogenes and S. dysgalactiae were identified by sequencing of M protein genes (emm types) and multiplex PCR typing of 20 other virulence determinants. Streptococci contributed to 6.2% of blood, 10.7% of CSF and 14.5% of soft tissue isolates, being among the most common invasive isolates. The overall rates of invasive S. pyogenes, S. agalactiae, S. dysgalactiae and S. pneumoniae were 2.4, 4.4, 2.1, and 5.3%. Whereas S. pneumoniae was 1.5% more common in CSF samples, BHS isolates were 2-fold and 11-fold higher in bacteraemia and invasive soft tissue infections. Genetic BHS typing revealed wide molecular diversity of invasive and noninvasive group A and group G BHS, whereas one emm-type (stG62647.0) and no other virulence determinants except scpA were detected in invasive group C BHS. BHS were important invasive pathogens, outpacing S. pneumoniae in bacteraemia and invasive soft tissue infections. The incidence of S. dysgalactiae infections was comparable to that of S. pyogenes even with less diversity of molecular virulence. The results of this study emphasise the need for awareness of BHS invasiveness in humans and the need to develop BHS prevention strategies.     

New Mutations in Ciprofloxacin Resistant Strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Guilan Province,Northern Iran

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative bacterium that continues to be a major cause of opportunistic nosocomial infections. In P. aeruginosa, several fluoroquinolones resistance mechanisms have been proposed such as mutations in the target enzymes GyrA and ParC and upregulation of efflux pump MexAB-OprM. Fluoroquinolones inhibit the target enzymes DNA gyrase and topoisomerase IV, each comprised of two subunits: GyrA and GyrB, and ParC and ParE, respectively. In addition, fluoroquinolones were blocked by overexpression of MexAB-OprM induced through mutations in the regulatory genes mexR and nalC.

Materials and methods

In this study, 44 P. aeruginosa strains were isolated from different clinical samples of burn and infection from patients in some hospitals and laboratories in Guilan, which were identified by biochemical tests. The antibiotic resistance and susceptibility of strains was determined by Kirby Bauer method and microdilution method, and then PCR-sequencing was carried out to assess mutation in several genes involved in ciprofloxacin resistance.

Results

Of 44 isolates, 14 isolates were fluoroquinolone resistant. All 14 strains were nalidixic acid resistant with MIC = 1024 μg/mL. While ciprofloxacin resistance was showed in resistant isolates with MIC to be 32–1024 μg/mL. PCR-sequencing analysis showed that all 14 fluoroquinolone resistant isolates had one or two mutations in gyrA mutation. Mutations in parC, mexR and nalC were shown in some ciprofloxacin resistant isolates. Moreover, three strains had new mutation in mexR gene (111InsC and 262-263delAG).

Conclusions

It seems that high resistance to ciprofloxacin can simultaneously be the result of mutation in several genes such as topoisomerase enzymes and negative regulatory genes implicated in the expression of efflux pump systems in Guilan Province.

     

<Emphasis Type="Italic">Streptococcus suis</Emphasis> in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance

The purpose of this study was to perform an analysis of Streptococcus suis human invasive isolates, collected in Poland by the National Reference Centre for Bacterial Meningitis. Isolates obtained from 21 patients during 2000–2013 were investigated by phenotypic tests, multilocus sequence typing (MLST), analysis of the TR9 locus from the multilocus variable number tandem repeat (VNTR) analysis (MLVA) scheme and pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA. Determinants of virulence and antimicrobial resistance were detected by polymerase chain reaction (PCR) and analysed by sequencing. All isolates represented sequence type 1 (ST1) and were suggested to be serotype 2. PFGE and analysis of the TR9 locus allowed the discrimination of four and 17 types, respectively. Most of the isolates were haemolysis- and DNase-positive, and around half of them formed biofilm. Genes encoding suilysin, extracellular protein factor, fibronectin-binding protein, muramidase-released protein, surface antigen one, enolase, serum opacity factor and pili were ubiquitous in the studied group, while none of the isolates carried sequences characteristic for the 89K pathogenicity island. All isolates were susceptible to penicillin, cefotaxime, imipenem, moxifloxacin, chloramphenicol, rifampicin, gentamicin, linezolid, vancomycin and daptomycin. Five isolates (24 %) were concomitantly non-susceptible to erythromycin, clindamycin and tetracycline, and harboured the tet(O) and erm(B) genes; for one isolate, lsa(E) and lnu(B) were additionally detected. Streptococcus suis isolated in Poland from human invasive infections belongs to a globally distributed clonal complex of this pathogen, enriched in virulence markers. This is the first report of the lsa(E) and lnu(B) resistance genes in S. suis.     

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.     

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.     

Involvement of the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> plasticity region and <Emphasis Type="Italic">cag</Emphasis> pathogenicity island genes in the development of gastroduodenal diseases

Infection by Helicobacter pylori is associated with the development of several gastroduodenal diseases, including gastritis, peptic ulcer disease (gastric ulcers and duodenal ulcers), and gastric adenocarcinoma. Although a number of putative virulence factors have been reported for H. pylori, there are conflicting results regarding their association with specific H. pylori-related diseases. In this work, we investigated the presence of virB11 and cagT, located in the left half of the cag pathogenicity island (cagPAI), and the jhp917–jhp918 sequences, components of the dupA gene located in the plasticity zone of H. pylori, in Brazilian isolates of H. pylori. We also examined the association between these genes and H. pylori-related gastritis, peptic ulcer disease, and gastric and duodenal ulcers in an attempt to identify a gene marker for clinical outcomes related to infection by H. pylori. The cagT gene was associated with peptic ulcer disease and gastric ulcers, whereas the virB11 gene was detected in nearly all of the samples. The dupA gene was not associated with duodenal ulcers or any gastroduodenal disease here analyzed. These results suggest that cagT could be a useful prognostic marker for the development of peptic ulcer disease in the state of São Paulo, Brazil. They also indicate that cagT is associated with greater virulence and peptic ulceration, and that this gene is an essential component of the type IV secretion system of H. pylori.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

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.     

Mutations in Hotspot Regions of <Emphasis Type="Italic">ERG11</Emphasis> Gene in Fluconazole Resistant Isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> in Guilan Province,Northern Iran

Background

Widespread use of azoles has resulted in rapid development of azole resistance in Candida albicans strains. Mutations in ERG11, a target enzyme of azoles, alter the binding ability of azoles to this enzyme and result in the development of resistant strains. In this study, we evaluated ERG11 mutations in fluconazole resistant isolates of C. albicans.

Materials and methods

In this study, 60 clinical samples were isolated from Guilan hospitals. Then differential tests were used to identify C. albicans strains. Disc diffusion and MIC tests were used to the analyze fluconazole susceptibility. Then, the resistant isolates were evaluated by PCR and sequencing methods for ERG11 mutations.

Results

Of 60 clinical samples, 40 C. albicans strains were identified through specific symptoms. Susceptibility tests showed that four C. albicans strains were resistant to high dose fluconazole (≥512 μg/mL). In all resistant isolates was found missense mutations such as K291N, C470G and Q474R and three isolates had premature nonsense mutation (Y477stop).

Discussion

Our study indicates that the level of fluconazole resistance in C. albicans strains is high in Guilan province and other drugs should be used in resistant infections. It seems that missense mutations in four isolates play role in azole resistance. However in three isolates premature stop codon may be involved in high dose resistance. And it is suggested that in fourth isolates another mechanisms introduce increase of resistance dose in combination with missense mutation in ERG11. Results of this study suggest that in patients by high dose of resistance do not use azole because of mutations that decrease azole effects.

     

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.