Enterobacterial Repetitive Intergenic Consensus Sequence Research Articles

Labrys soli sp. nov., isolated from the rhizosphere of ginseng.

In this study, we describe strain DCY64T that was isolated from the rhizosphere of three-year-old Korean ginseng root. Cells were Gram-reaction negative, oxidase- and catalase-positive, strictly aerobic, capsulated, non-motile, non-sporulating and spherical to short rod-shaped. Multiplicative budding cells were produced. Vesicles covered the surface of cells. Phylogenetic analysis placed strain DCY64T within the genus Labrys with the highest similarity to Labrys monachus VKM B-1479T (97.6 % 16S rRNA gene sequence similarity), followed by Labrys okinawensis MAFF 210191T (97.5 %), Labrys miyagiensis G24103T (97.4) and Labrys portucalensis F11T (97.0 %). The genomic DNA G+C content was 63 mol%. The presences of summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C19 : 1 cyclo ω8c and C16 : 0 as major fatty acids; phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol as major polar lipids; ubiquinone Q-10 as the predominant quinone and sym-homospermidine as the dominant polyamine were found in strain DCY64T. These chemotaxonomic results were in accordance with those of members of the genus Labrys. However, the absence of C16 : 0 2-OH, C16 : 0 3-OH and C18 : 1 2-OH from the fatty acids profile and differences in minor polar lipids and phenotypic characteristics distinguished strain DCY64T from the closest type strains. The discrimination was also supported by unique enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) fingerprints, as well as DNA-DNA hybridization values ( ≤ 48 %) between strain DCY64T and related type strains. Therefore, we propose that strain DCY64T represents a novel species of the genus Labrys. The name Labrys soli sp. nov. is proposed, with DCY64T ( = KCTC 32173T = JCM 19895T) as the type strain.

In vitro probiotic profiling of novel Enterococcus faecium and Leuconostoc mesenteroides from Tunisian freshwater fishes.

Novel lactic acid bacteria isolated from different organs of freshwater fish were examined for their potential application as probiotics in raw and processed foods. Four isolates of Enterococcus faecium and Leuconostoc mesenteroides were identified at the molecular level by 16S rRNA sequencing and random amplification of polymorphic DNA - polymerase chain reaction, and their antimicrobial activity against a panel of pathogens and food-poisoning bacteria was investigated. The whole bacteriocins of the 4 isolates were characterized by enterobacterial repetitive intergenic consensus sequences in PCR. The isolates exhibited high inhibitory activities against food-borne pathogens and spoilage microbial species and have significant probiotic profiles, since they survived at pH 3.0 and in the presence of bile salts, pancreatin, and pepsin, without any detectable hemolytic activity. Further, moderate heat resistance, adhesion ability to steel surfaces, and sensitivity to clinically relevant antimicrobial agents were revealed for all the isolates. These results highlight the specific probiotic properties of the strains and give evidence for potential application in minimally processed foods subjected to moderate heat processing.

Prevalence and population analysis of Vibrio parahaemolyticus in aquatic products from South China markets.

Vibrio parahaemolyticus is a common foodborne pathogen in aquatic products. To investigate the prevalence of Vibrio parahaemolyticus in aquatic products in South China, 224 samples were collected from markets in four provinces (11 cities) from May 2013 to January 2014. One hundred and fifty isolates were isolated from 98 samples. All isolates were analyzed for the presence of thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) by PCR, antibiotic susceptibility analysis by disk diffusion method, serotyping by multiplex PCR and molecular typing by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing. Although all 150 isolates were negative for tdh, 61 strains were trh positive (40.67%). Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (88.67%), cefazolin (66.00%) and ampicillin (62.67%). All strains were susceptible to chloramphenicol. Forty percent of all isolates were O2 type. The 150 isolates were grouped into three clusters by ERIC-PCR typing. The results demonstrated the presence of V. parahaemolyticus in aquatic products from the retail market and this methodology can be used for microbiological risk assessment in China.

Molecular Characterization of Vibrio cholerae Isolated From Clinical Samples in Kurdistan Province, Iran.

Background:Vibrio cholerae causes diarrhoeal disease that afflicts thousands of people annually. V. cholerae is classified on the basis of somatic antigens into serovars or serogroups and there are at least 200 known serogroup. Two serogroups, O1 and O139 have been associated with epidemic diseases. Virulence genes of these bacteria are OmpW, ctxA and tcpA.Objectives:Due to the importance of V. cholerae infection and developing molecular diagnostics of this organism in medical and microbiology sciences, this study aimed to describe molecular characterization of V. cholerae isolated from clinical samples using a molecular method.Materials and Methods:In this study, 48 samples were provided during summer 2013 (late August and early September) by reference laboratory. Samples were assessed using biochemical tests initially. The primer of OmpW, ctxA and tcpA genes was used in Polymerase Chain Reaction (PCR) protocols. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and Repetitive Extragenic Palindromic (REP)-PCR methods were used to subtype V. cholerae.Results:In this study, from a total of 48 clinical stool samples 39 (81.2 %) were positive for V. cholerae in biochemical tests and bacteria culture tests. The PCR results showed that of 39 positive isolates 35 (89.7%), 34 (87.1%) and 37 (94.8%) were positive for ctxA, tcpA and OmpW gene, respectively. Also, in the REP-PCR method with ERIC primer strains were divided into 10 groups. In the REP-PCR method with REP primer, strains were divided into 13 groups.Conclusions:Polymerase chain reaction has specificity and accuracy for identification of the organism and is able to differentiate biotypes. Enterobacterial repetitive intergenic consensus sequence is one of the informative and discriminative methods for the analysis of V. cholerae diversity. The REP-PCR is a less informative and discriminative method compared to other methods for the analysis of V. cholerae diversity.

English

The bacterium causing cholera, Vibrio cholerae, is a marine organism and coastal waters are important reservoirs of the organism. There are more than 200 serogroups of V. cholerae, of which serogroups O1 and O139 are known to be the causative agent of the cholera. The main virulent factor in V. cholerae is cholera toxin gene (ctx) that is found from the epidemic O1 and O139 strains, but may also be found in some strains other than O1 and O139 (non-O1 and non-O139). In this study, 48 V. cholerae strains isolated from three estuaries of Tanzania and 20 stool isolates were characterized in terms of their serogroups and possession of ctx gene and then compared using two PCR based fingerprinting methods: Enterobacterial repetitive intergenic consensus (ERIC) sequences and repetitive extragenic palindromic (REP) sequences. All the stool isolates and twelve of the environmental isolates belonged to serogroup O1 while the remaining 36 environmental isolates were defined as non-O1/O139. The entire stool isolates and 21 of the environmental isolates had the cholera toxin gene (ctxA). Both ERIC and REP methods gave almost unique fingerprints for each strain and confirmed high genetic heterogeneity among the different cholera strains. Higher similarity was observed in REP-PCR (70-100%) than in ERIC-PCR (62-100%), indicating different discriminative power of these methods. Environmental isolates clustered together with clinical isolates at ≥90% similarity level suggesting their great potential of producing pathogenic strains that may be the causative agents for the frequent observed cholera outbreaks particularly along the coast. Key words: Vibrio cholerae, enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR, estuaries of Tanzania.

Operational taxonomic units (OTUs) of endophytic bacteria isolated from banana cultivars in the Amazon.

Endophytic microorganisms colonize plants, inhibit the growth of pathogens (by competing for nutrients and/or space), or produce antagonistic substances. Fifty-five endophytic bacteria were isolated from the leaf tissue of the FHIA 18 banana cultivar. Genetic diversity analyses were performed using the enterobacterial repetitive intergenic consensus sequence polymerase chain reaction method and BOX molecular markers. These analyses resulted in 33 and 21 polymorphic bands, respectively. The similarity data, obtained using the Dice coefficient based on the polyphasic analysis method, ranged from 22 to 100%. This indicated a high genetic diversity among the analyzed isolates. Sixty percent similarity was utilized as the cut-off criterion for the formation of operational taxonomic units (OTUs); this resulted in the identification of 32 possible OTUs, indicating a high number of potential species.

High prevalence of metallo-β-lactamase among carbapenem-resistant Klebsiella pneumoniae in a teaching hospital in China.

The aim of this study was to characterize the carbapenemases in carbapenem-resistant Klebsiella pneumoniae (CR-KP) from a Chinese teaching hospital. A total of 40 CR-KPs were screened for the presence of carbapenemases. Minimum inhibitory concentrations were determined by agar dilution. The modified Hodge test was used for the detection of carbapenemase production. Carbapenemase, extended-spectrum β-lactamase, and AmpC genes were detected using polymerase chain reaction (PCR) and sequencing. A conjugation test was performed using a broth culture mating method, transferred plasmids were typed by PCR-based replicon typing, and clonal relatedness was investigated by enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) and multilocus sequence typing (MLST). The results revealed that modified Hodge test was positive for 28 CR-KPs, and CR-KPs exhibited high resistance rates against various antibiotics, except colistin (5.0%) and tigecycline (22.5%). ERIC and MLST profiles showed no clonal outbreak. PCR demonstrated a high prevalence rate (55.0%, 22/40) of metallo-β-lactamases (MBLs) in CR-KPs. IMP-4, IMP-8, NDM-1, and KPC-2 were identified in 14 (35.0%), 7 (17.5%), 2 (5.0%), and 7 (17.5%) isolates, respectively. Notably, 2 CR-KPs coproduced 2 carbapenemases simultaneously (IMP-8/NDM-1 and IMP-4/KPC-2). In vitro transfer of carbapenem resistance was successful for 11 MBL-producing CR-KPs. The extended spectrum β-lactamase genes were detected in 30 (75.0%) of these CR-KPs. To the best of our knowledge, this is the first report focusing on carbapenem resistance in K. pneumoniae due to metalloenzymes in China. Screening and surveillance of MBLs in Enterobacteriaceae is urgently needed in this region to control and prevent the spread of these resistance determinants.

Molecular characterization, genetic diversity and antibacterial susceptibility of Escherichia coli encoding Shiga toxin 2f in domestic pigeons.

This study aimed to evaluate prevalence, characteristics, genotypic diversity and antibacterial susceptibility of Escherichia coli encoding Shiga toxin 2f in domestic pigeons in different provinces of Iran. A total of 117 faecal samples were collected from pigeons and were subjected to molecular detection of stx2f. In total, 20, 25·8, 21·4 and 9% of pigeons from Tehran, Ferdows, Garmsar and Babol cities carried stx2f+ isolates, respectively. Of the 460 E. coli isolates examined, 43 were stx2f+ and most also carried eae (95·3%) and astA (97·7%) genes. Some of the stx2f+ isolates harboured cnf (9·3%), but all were negative for stx1, stx2 (other subtypes) and ehly. Most Strains (90%) were assigned to B1 phylogroup and possessed Intimin-β. Fingerprinting of the stx2f+ isolates using either enterobacterial repetitive intergenic consensus sequences (ERIC) or random amplified polymorphic DNA (RAPD)-polymerase chain reaction revealed seven distinct profiles by each method, with one prevailing (65·1 and 46·5%, respectively). By the combination of methods, 10 profiles were recognized. Ten isolates from different profiles were shown to belong to O20, O78 and O115 serogroups, and eight were 100% identical in the stx2f gene sequence. The strains were consistently resistant to amoxicillin and lincospectin and commonly resistant to tetracycline (88·4%) and doxycycline (74·4%). Overall, the results indicate a limited degree of genetic diversity in stx2f-harbouring E. coli from pigeons. Significance and impact of the study: Carriage of stx2f gene tends to be underreported in pigeon Escherichia coli isolates because most routine genetic and phenotypic tests cannot efficiently target this gene or detect the toxin. Nevertheless, pigeons frequently carry E. coli strains that are stx2f-positive, and this situation is not limited to any distinct geographical area. The current results suggest that genetic background of stx2f-encoding E. coli is distinct from most Shiga toxin-producing E. coli strains. However, the factors that contribute to host preferences and pathogenicity remain unclear. These findings have public health significance that should be addressed in future research.

Multidrug-resistant nontuberculous mycobacteria isolated from cystic fibrosis patients.

Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.

Shiga-toxin genes and genetic diversity of Escherichia coli isolated from pasteurized cow milk in Brazil.

This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paraná state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied. Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.

Epidemic and virulence characteristic of Shigella spp. with extended-spectrum cephalosporin resistance in Xiaoshan District, Hangzhou, China

BackgroundShigellae have become increasingly resistant to the extended-spectrum cephalosporin (ESC) worldwide and pose a great challenge to anti-infection treatment options. The purpose of this study was to determine the resistance, cephalosporin resistance mechanisms, virulence characteristic and genotype of ESC-resistant Shigella.MethodsFrom 2008 to 2012, Shigella isolates collected from diarrhea patients were detected for antibiotics sensitivity by disk diffusion, cephalosporin resistance determinants and virulence genes using polymerase chain reaction (PCR) and genotyping through enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR).ResultsA total of 356 Shigella isolates were gathered, and 198 (55.6%, 58 S. flexneri and 140 S. sonnei) were resistant to ESC. All ESC-resistant isolates were susceptible to imipenem, and only 0.5% isolate was resistant to piperacillin/tazobactam. ESC-resistant S. flexneri showed high degrees of resistance to ampicillin (100%), ampicillin/sulbactam (96.6%), piperacillin (100%), trimethoprim/sulfamethoxazole (74.1%), ciprofloxacin (74.1%), levofloxacin (53.4%), ceftazidime (58.6%) and cefepime (58.6%). ESC-resistant S. sonnei exhibited high resistance rates to ampicillin (100%), piperacillin (100%) and trimethoprim/sulfamethoxazole (96.4%). Cephalosporin resistance genes were confirmed in 184 ESC-resistant isolates. blaCTX-M types (91.8%, mainly blaCTX-M-14, blaCTX-M-15 and blaCTX-M-57) were most prevalent, followed by blaOXA-30 (26.3%). Over 99.0% ESC-resistant isolates harbored virulence genes ial, ipaH, virA and sen. However, set1 were more prevalent in ESC-resistant S. flexneri isolates than in S. sonnei isolates. ERIC-PCR results showed that 2 and 3 main genotypes were detected in ESC-resistant S. flexneri and S. sonnei, respectively.ConclusionOur findings indicated that a high prevalence of ESC-resistant Shigella mediated mainly by blaCTX-M with stronger resistance and virulence, and the existence of specific clones responsible for these Shigella infection in the region studied.

Characterization of Weissella ceti infections in Brazilian rainbow trout, Oncorhynchus mykiss (Walbaum), farms and development of an oil‐adjuvanted vaccine

Weissella ceti is an emerging bacterial pathogen that affects rainbow trout, Oncorhynchus mykiss (Walbaum), farms. The aims of this study were to genotype W.ceti strains isolated from distinct geographical origins and to determine the efficacy of an oil-adjuvanted vaccine against the disease. Between 2010 and 2012, outbreaks were recorded in five Brazilian farms, and 34 W.ceti isolates were genetically characterized by repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequences PCR and pulsed-field gel electrophoresis. Two different W.ceti vaccines were tested: an aqueous-based whole-cell inactivated vaccine (bacterin) and oil-adjuvanted vaccine. Their efficacy was evaluated in rainbow trout at 30 and 60days post-vaccination (d.p.v.). W.ceti was found to be a highly homogeneous population in Brazil, with clonally related genotypes. Oil-adjuvanted vaccine exhibited the best (P<0.05) protection against disease, reaching relative percentage survival (RPS)values of 92% at 30 and 60d.p.v. Bacterin resulted in RPS values of 67% and 58% at day 30 and 60, respectively. The oil-adjuvanted vaccine provided effective protection against W.ceti infection in rainbow trout.

Molecular Typing of Cronobacter Strains from Food in China by Enterobacterial Repetitive Intergenic Consensus Sequence PCR (ERIC‐PCR) and Sequence Analysis of the gyrB Gene

AbstractThe occurrence of outbreaks of Cronobacter strains causing necrotizing meningitis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 43 Cronobacter isolates and five Enterobacteriaceae strains were used in this study. The strains were characterized using two molecular typing methods, including enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC‐PCR) and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these two molecular typing tests clearly showed that there was no close relationship between Cronobacter spp. and Enterobacteriaceae strains. Discriminatory index of ERIC‐PCR typing for the 43 Cronobacter isolates and five Enterobacteriaceae strains was high (D = 0.984) by Simpson's index of diversity based on the similarity value of more than 80% using BioNumerics version 5.0. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains. These results demonstrate that ERIC‐PCR combined with sequence analysis of the gyrB gene may be a reliable, rapid typing strategy for Cronobacter strains.Practical ApplicationsMolecular typing of foodborne pathogenic bacteria has been shown as a useful tool for tracking the source of infection and detection of virulent strains, as well as for determining the geographical and host distribution of possible variants. ERIC‐PCR provides rapid clustered results when a large number of Cronobacter spp. isolates need to be subtyped rapidly. Furthermore, partial sequence analysis of the gyrB gene was utilized to differentiate among the closely related isolates. The results of the current study suggest that ERIC‐PCR in conjunction with the gyrB sequence analysis would provide a reliable and accurate typing strategy for Cronobacter spp. strains. This combination of techniques can be used as a rapid means of comparing Cronobacter spp. isolates for epidemiological investigations.

ERIC-PCR genotyping and drug resistant analysis of pathogenic Aeromonas hydrophila from freshwater animals

The freshwater aquiculture industry has developed rapidly in China.As a result,China has become the largest producer of freshwater cultured fish in the world.However,production is threatened by Aeromonas hy-drophila,a widely distributed freshwater bacterial pathogen that is thought to cause disease in a range of aquatic animals.For example,A.hydrophila is thought to cause bacterial septicemia in fish and red abdominal shell dis-eases in soft-shelled turtles.Outbreaks of A.hydrophila have resulted in significant economic losses to the aqua-culture industry.In addition,A.hydrophila is a zoonotic pathogen,sometimes causing gastroenteritis and septice-mia in humans.Our objective was to document the epidemiological characteristics of pathogenic A.hydrophila to provide insight into potential measures to prevent and control infections.We genotyped pathogenic Aeromonas hydrophila strains collected from a variety of sources,determined their drug susceptibility,and evaluated the rela-tionship between genotype and antimicrobial resistance patterns.We used enterobacterial repetitive intergenic consensus sequence(ERIC)-PCR to genotype 50 strains of Aeromonas hydrophila collected from freshwater cul-ture sites in Shanghai,Zhejiang,Jiangsu,Jiangxi,Hubei,and Guangdong.ERIC-PCR is a more rapid,efficient,and accurate molecular typing method than the traditional serotyping methods.In addition,we evaluated the sus-ceptibility of each isolate to 12 antibacterial drugs and determined the relationship between the genotype and the antimicrobial resistance pattern.We identified 12 special genotypes(I–XII) from the 50 isolates.Among these,genotype VII was the dominant genotype,consisting of 14 strains,whereas genotype III and X contained only one strain.All strains were resistant to ampicillin and 98% of the strains were resistant to cefalexin.However,94% of the strains were susceptible to aminoglycosides and quinolones.We identified 9 patterns of drug-resistance: AMP,CX/AMP,CX/AMP/POL,CX/AMP/SMZ,CX/AMP/NM,CX/AMP/SMZ/POL,CX/AMP/SMZ/POL/GM,CX/AMP/ SMZ/LOM/ENR,and CX/AMP/SMZ/LOM/ENR/OF/LEV.The twelve strain genotypes shared a common pattern of drug resistance(CX/AMP/POL).Similarly,the majority of genotypes within strain II and XI shared a common pattern of drug resistance(CX/AMP/SMZ).The drug-resistant pattern of most genotypes within strains IV,VI,and VIII was CX/AMP and for strain X the pattern was CX/AMP/NM.Thus,our results suggest that there is likely a correlation between the genotype and drug-resistance among A.hydrophila strains.

Selection and Persistence of Antimicrobial-ResistantEscherichia coliIncluding Extended-Spectrum β-Lactamase Producers in Different Poultry Flocks on One Chicken Farm

Escherichia coli isolates (n=438) from six different broiler chicken flocks (all in, all out) with known consumption of antimicrobials were investigated for their antimicrobial resistance and the prevalence of extended-spectrum β-lactamase (ESBL) phenotypes. E. coli were isolated from chicken at the third and fifth week of age and tested for antimicrobial resistance during the course of fattening. Resistance to sulfamethoxazole+trimethoprim, which was used in four flocks within the first days of life, decreased significantly in all six flocks between the third and fifth week of broiler chicken's life (mean 65.9% vs. 54.3%). By contrast, resistance to spectinomycin increased significantly in all six flocks within the same period (mean 36.1% vs. 57.0%); doxycycline resistance increased significantly in five of six flocks (mean 19.2% vs. 41.7%), although both substances were not used for treatment. Of the sulfonamide resistance genes sul1, sul2, and sul3, sul2 was most frequently found (up to 60%). The prevalence of sul2 increased significantly between weeks 3 and 5, if the chicken were treated with sulfamethoxazole + trimethoprim in the first days of life. If sulfamethoxazole + trimethoprim was not used, then the prevalence of sul2 decreased significantly in the same period. The prevalence of sul1+qacEΔ1 (classical class 1 integrons) was significantly higher in E. coli from sulfamethoxazole + trimethoprim-treated flocks (9.63%), compared to untreated flocks (2.92%). The detection of phenotypes that potentially indicate plasmid-borne AmpC-β-lactamases was inversely associated with sulfamethoxazole + trimethoprim treatment. ESBL phenotypes were found without selective enrichment in four of six flocks. Of all isolated E. coli, 1.8% (n=8) had an ESBL phenotype. ESBL strains differed in their accompanying resistances and/or enterobacterial repetitive intergenic consensus sequences. In conclusion, clonal dissemination seems not to be a major cause of ESBL detection on a chicken farm with all-in all-out production mode.

Distribution and sequencing of enterobacterial repetitive intergenic consensus sequence elements in Streptococcus mutans serotype c

ObjectiveThe banding pattern of Streptococcus mutans strains generated using repetitive extragenic palindromic PCR (rep-PCR) with enterobacterial repetitive intergenic consensus (ERIC) primers has been shown previously. In the present study, the sequencing and analysis of gene sequences of the 6 most intense and commonly occurring amplicons from S. mutans serotype c was investigated. MethodsThe sequences of the amplicons from S. mutans serotype c strain by using rep-PCR with ERIC1R and ERIC2 primers were compared to the whole genome sequence of S. mutans UA159 and NN2025 strains. ResultsThe amplicons were found to contain partial gene sequences coding for proteins such as the hypothetical protein, glucan binding protein A, putative methylated-DNA-protein-cysteine S-methyltransferase, putative d-3-phosphoglycerate dehydrogenase, putative excinuclease ABC (subunit A) and putative GTP-binding protein. The locations of 5 of the 6 amplicons were found to be assembled downstream in the UA159 genome. The coding direction of the amplicons in the NN2025 genome was in reverse orientation relative to that in the UA159 strain, except in the 2170-bp amplicon. ConclusionsThe repeating sequence elements of ERIC in S. mutans serotype c are located on one side of the genome and have less frequency and similarity compared to those in enterobacteria.

English

isolates from healthy cattle while comparing two methods namely, PCR assay for enterobacterial repetitive intergenic consensus sequences (ERIC-PCR) and amplified fragment length polymorphism (AFLP). Our study showed that ERIC-PCR is an appropriate/efficient method for determining diversity of

Lactobacillusspecies isolated from vaginal secretions of healthy and bacterial vaginosis-intermediate Mexican women: a prospective study

BackgroundLactobacillus jensenii, L. iners, L. crispatus and L. gasseri are the most frequently occurring lactobacilli in the vagina. However, the native species vary widely according to the studied population. The present study was performed to genetically determine the identity of Lactobacillus strains present in the vaginal discharge of healthy and bacterial vaginosis (BV) intermediate Mexican women.MethodsIn a prospective study, 31 strains preliminarily identified as Lactobacillus species were isolated from 21 samples collected from 105 non-pregnant Mexican women. The samples were classified into groups according to the Nugent score criteria proposed for detection of BV: normal (N), intermediate (I) and bacterial vaginosis (BV). We examined the isolates using culture-based methods as well as molecular analysis of the V1–V3 regions of the 16S rRNA gene. Enterobacterial repetitive intergenic consensus (ERIC) sequence analysis was performed to reject clones.ResultsClinical isolates (25/31) were classified into four groups based on sequencing and analysis of the 16S rRNA gene: L. acidophilus (14/25), L. reuteri (6/25), L. casei (4/25) and L. buchneri (1/25). The remaining six isolates were presumptively identified as Enterococcus species. Within the L. acidophilus group, L. gasseri was the most frequently isolated species, followed by L. jensenii and L. crispatus. L. fermentum, L. rhamnosus and L. brevis were also isolated, and were placed in the L. reuteri, L. casei and L. buchneri groups, respectively. ERIC profile analysis showed intraspecific variability amongst the L. gasseri and L. fermentum species.ConclusionsThese findings agree with previous studies showing that L. crispatus, L. gasseri and L. jensenii are consistently present in the healthy vaginal ecosystem. Additional species or phylotypes were detected in the vaginal microbiota of the non-pregnant Mexican (Hispanic-mestizo) population, and thus, these results further our understanding of vaginal lactobacilli colonisation and richness in this particular population.

Development of an ERIC sequence typing scheme for Laribacter hongkongensis, an emerging pathogen associated with community-acquired gastroenteritis and travellers’ diarrhoea

Laribacter hongkongensis is a potential emerging pathogen, associated with community-acquired diarrhoea. For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing, have been developed for this pathogen. However, these methods require specialized equipment and costly reagents. More importantly, they are labour-intensive and time-consuming, which is not really suitable for foodborne disease outbreak investigations. In this study, we developed a rapid and reliable method using 22-mer primers specific for the enterobacterial repetitive intergenic consensus sequence (ERIC). PFGE was used for comparison, to evaluate this method. A total of 81 isolates of L. hongkongensis were examined: 79 isolates recovered from food of diverse origins and two strains derived from patients with L. hongkongensis-associated infection. Typing patterns and clustering analysis indicated that the 81 L. hongkongensis isolates were grouped into 21 and 13 genotypes by ERIC-PCR and PFGE, respectively. ERIC-PCR was found as reproducible as PFGE. A high percentage (70.4%) of isolates yielded distinguishable ERIC-PCR patterns, which were concordant with the results from PFGE. These results suggest that ERIC-PCR is valuable for use in the epidemiological investigation of L. hongkongensis.

Virulence and Multidrug Resistance Patterns ofVibrio choleraeO1 Isolates from Diarrheal Outbreaks of South India During 2006–2009

A total of 31 Vibrio cholerae O1 (4- Inaba and 27- Ogawa serotype) isolates collected during a three-year period (2006-2009) from acute diarrheal cases in Tamil Nadu, India were analyzed for antibiotic resistance profiling, virulence-associated factors, genetic profiling by enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC PCR), and biofilm-forming ability. Antibiotic resistance profile revealed that most of the strains have become multidrug-resistant strains. All the isolates are resistant to ampicillin and polymyxin B, 97% of the isolates are resistant to nalidixic acid, 90% to co-trimoxazole, 32.3% to norfloxacin and ciprofloxacin, 29% to doxycycline, 10% to gentamicin, whereas only 3% to chloramphenicol. Molecular characterization of virulence-associated genes by multiplex PCR revealed the presence of ace, ctxA, tcpA, toxR, and ompU as 93.5%, followed by ompW with 33.3%. The presence of zot was restricted to only one isolate and hlyA was not encountered in any of the strains. ERIC PCR produced more than 10 bands for each isolate and the dendrogram generated based on the cluster analysis showed the presence of 29 electrophoretic types among the 31 isolates. Isolates from different area or year of isolation are intermingled in all the clusters. With respect to biofilm formation, 24 isolates were found to be biofilm formers and eight of them produced strong biofilm. This study demonstrates the presence of critical virulence factors and antibiotic resistance in the diarrhea isolates, which signifies the importance of routine monitoring and proper treatment to prevent cholera outbreaks.