Evaluating the occurrence of Escherichia albertii in chicken carcass rinses by PCR, Vitek analysis, and sequencing of the rpoB gene.

Escherichia albertii is a recently described species that has been associated with gastroenteritis in humans and with healthy and ill birds. Most recently, it has been identified as the causative agent in a food-borne outbreak in Japan. The distribution and clinical importance of E. albertii are not well studied because its importance is unclear. Culture methods for clinical isolation frequently miss E. albertii or incorrectly identify it as Shigella spp., Escherichia coli, or Hafnia alvei. This study was designed to determine if E. albertii could be recovered from chicken carcass rinses collected at slaughter during a 1-year period from November 2009 until October 2010. Colonies were isolated from chicken carcass rinses and tested by PCR for the presence or absence of clpX, lysP, mdh, intimin (eae), Shiga toxins 1 and 2 (stx1, stx2, and stx2f), heat-stable enterotoxin A (staA), and cytolethal distending toxins 1 and 2 (cdtB) genes. Sixty-five isolates were analyzed by sequencing a section of the rpoB gene. Analysis of the rpoB gene sequences revealed 14 fixed differences between E. albertii and other, closely related organisms. The fixed differences found in the rpoB gene could aid in future discrimination of E. albertii from closely related bacteria.

Analysis of antimicrobial resistance genes detected in multiple-drug-resistant Escherichia coli isolates from broiler chicken carcasses.

Multi-drug-resistant (MDR) bacteria in food animals are a potential problem in both animal and human health. In this study, MDR commensal Escherichia coli isolates from poultry were examined. Thirty-two E. coli isolates from broiler carcass rinses were selected based on their resistance to aminoglycosides, ß-lactams, chloramphenicols, tetracyclines, and sulfonamide antimicrobials. Microarray analysis for the presence of antimicrobial resistance and plasmid genes identified aminoglycoside [aac(6), aac(3), aadA, aph, strA, and strB], ß-lactam (bla(AmpC), bla(TEM), bla(CMY), and bla(PSE-1)), chloramphenicol (cat, flo, and cmlA), sulfamethoxazole (sulI and sulII), tetracycline [tet(A), tet(C), tet(D), and tetR], and trimethoprim (dfrA) resistance genes. IncA/C plasmid core genes were detected in 27 isolates, while IncHI1 plasmid genes were detected in one isolate, indicating the likely presence of these plasmids. PCR assays for 18 plasmid replicon types often associated with MDR in Enterobacteriaceae also detected one or more replicon types in all 32 isolates. Class I integrons were investigated by PCR amplification of the integrase I gene, intI1, and the cassette region flanked by conserved sequences. Twenty-five isolates were positive for the intI1 gene, and class I integrons ranging in size from ~1,000 to 3,300 bp were identified in 19 of them. The presence of class I integrons, IncA/C plasmid genes, and MDR-associated plasmid replicons in the isolates indicates the importance of these genetic elements in the accumulation and potential spread of antimicrobial resistance genes in the microbial community associated with poultry.

Gene expression analysis of Salmonella enterica Enteritidis Nal(R) and Salmonella enterica Kentucky 3795 exposed to HCl and acetic acid in rich medium.

In the United States, serovar Kentucky has become one of the most frequently isolated Salmonella enterica serovars from chickens. The reasons for this prevalence are not well understood. Phenotypic comparisons of poultry Salmonella isolates belonging to various serovars demonstrated that serovar Kentucky isolates differed from those of most other serovars in their response to acid. Microarray and qPCR analyses were performed with aerated exponentially growing poultry isolates, Salmonella enterica serovar Kentucky 3795 and Enteritidis Nal(R), exposed for 10 min to tryptic soy broth (TSB) adjusted to pH 4.5 with HCl and to pH 5.5 with HCl or acetic acid. Data obtained by microarray analysis indicated that more genes were up- or down-regulated in strain Kentucky 3795 than in Enteritidis Nal(R) under acidic conditions. Acid exposure in general caused up-regulation of energy metabolism genes and down-regulation of protein synthesis genes, particularly of ribosomal protein genes. Both strains appear to similarly utilize the lysine-based pH homeostasis system, as up-regulation of cadB was observed under the acidic conditions. Expression of regulatory genes (rpoS, fur, phoPQ) known to be involved in the acid response showed similar trends in both isolates. Differences between Kentucky 3795 and Enteritidis Nal(R) were observed with respect to the expression of the hdeB-like locus SEN1493 (potentially encoding a chaperone important to acid response), and some differences in the expression of other genes such as those involved in citrate utilization and motility were noted. It appears that the early stages of the transcriptional response to acid by isolates Kentucky 3795 and Enteritidis Nal(R) are similar, but differences exist in the scope and in some facets of the response. Possibly, the quantitative differences observed might lead to differences in protein levels that could explain the observed differences in the acid phenotype of serovar Kentucky and other Salmonella serovars.

Related antimicrobial resistance genes detected in different bacterial species co-isolated from swine fecal samples.

A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), ß-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ², p < 0.0000001). These data suggest that there may have been horizontal exchange of AR genes between these bacteria or there may be a common source of AR genes in the swine environment for E. coli and Salmonella.

Development of a DNA microarray to detect antimicrobial resistance genes identified in the National Center for Biotechnology Information database.

To understand the mechanisms and epidemiology of antimicrobial resistance (AR), the genetic elements responsible must be identified. Due to the myriad of possible genes, a high-density genotyping technique is needed for initial screening. To achieve this, AR genes in the National Center for Biotechnology Information GenBank database were identified by their annotations and compiled into a nonredundant list of 775 genes. A DNA microarray was constructed of 70mer oligonucelotide probes designed to detect these genes encoding resistances to aminoglycosides, beta-lactams, chloramphenicols, glycopeptides, heavy metals, lincosamides, macrolides, metronidazoles, polyketides, quaternary ammonium compounds, streptogramins, sulfonamides, tetracyclines, and trimethoprims as well as resistance transfer genes. The microarray was validated with two fully sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and Typhi CT18 (multidrug resistance [MDR]). All resistance genes encoded on the MDR plasmid, pHCM1, harbored by CT18 were detected in that strain, whereas no resistance genes were detected in LT2. The microarray was also tested with a variety of bacteria, including MDR Salmonella enterica serovars, Escherichia coli, Campylobacter spp., Enterococcus spp., methicillin-resistant Staphylococcus aureus, Listeria spp., and Clostridium difficile. The results presented here demonstrate that a microarray can be designed to detect virtually all AR genes found in the National Center for Biotechnology Information database, thus reducing the subsequent assays necessary to identify specific resistance gene alleles.

Seroprevalence and genital DNA prevalence of HPV types 6, 11, 16 and 18 in a cohort of young Norwegian women: study design and cohort characteristics.

BACKGROUND: Long-term efficacy evaluations of a quadrivalent HPV type 6/11/16/18 vaccine are ongoing in the Nordic region. As there are limited epidemiological data on HPV infection in Norway, we determined prevalence and identified sociobehavioural correlates of HPV 6/11/16/18 infection in young Norwegian women. METHODS: Norwegian (n=898) women, aged 1624 years, were enrolled in a 4-year prospective study. At enrolment and at 6-month intervals thereafter, an interview on behavioural data and a gynaecological examination were undertaken. Genital samples were tested for the L1,E6 and E7 genes of HPV-6/11/16/18, and serum anti-HPV-6/11/16/18 levels were measured using a competitive Luminex immunoassay (cLIA). Results. DNA and seroprevalence of HPV 6, 11, 16 or 18 ranged from 0.9 to 16.3% and 2.6 to 16.2%,respectively; and most infected women (approximately 75%) were infected with only 1 type. Of the HPV DNA positive cases, 54.3, 50.0,47.3 and 38.5% had detectable HPV 6, 11, 16 or 18 antibodies, respectively. More than 50% of the high-grade cervical intraepithelial neoplasia (CIN) cases were HPV-16 or HPV-18 DNA positive. Lifetime number of partners was the strongest and only predictor of sero- and DNA-positivity across the 4 HPV types. CONCLUSION: Given the high prevalence of HPV infection among young women with mostly single-type infection, and the fact that type-specific HPV screening is not recommended prior to the administration of the quadrivalent HPV vaccine, our data suggest the importance of widespread,rather than targeted, immunisation.

Performance of risk indices for identifying low bone density in postmenopausal women.

OBJECTIVE: To examine the ability of 4 published osteoporosis risk indices to identify women with low bone density. SUBJECTS AND METHODS: Subjects included postmenopausal women 45 years and older consecutively recruited from US clinics, women from general practice centers in The Netherlands (age range, 50-80 years), women in the Rotterdam Study (The Netherlands) 55 years and older, and women aged 55 to 81 years old screened for a clinical trial of alendronate. Bone mineral density (BMD) was measured at the femoral neck or lumbar spine; T scores represent the number of SDs below the mean for young healthy women. One risk index was calculated from age and weight; the other risk indices included up to 4 additional variables obtained by questionnaire. We calculated the sensitivity and specificity for identifying women with BMD T scores of -2.5 or less or -2.0 or less in the US clinic sample and created 3 risk categories, using each of the 4 indices. RESULTS: Data were available for 1102 women from the US clinic sample, 3374 women in the Rotterdam Study, 23,833 women screened for a clinical trial of alendronate, and 4204 women from general practice centers in The Netherlands. Specificity for identifying BMD T scores of -2.5 or less ranged from 37% to 58% (depending on risk index) when sensitivity was approximately 90%. The prevalence of osteoporosis (defined as T scores < or = -2.5) differed widely across the 3 risk categories, ranging from 2% to 4% for the low-risk category to 47% to 61% for the high-risk category in the US clinic sample. For spine BMD in the US clinic sample, the prevalence of T scores of -2.5 or less ranged from 7% (low risk) to 38% (high risk). The large differences in prevalence across risk categories were consistent across the other 3 samples of postmenopausal women in the United States and The Netherlands for all 4 risk indices. CONCLUSIONS: We recommend measuring BMD in women who are classified as having an increased risk of osteoporosis by using any of these risk indices because all 4 indices appear to predict low bone mass equally well. The Osteoporosis Self-assessment Tool index is easiest to calculate and therefore may be most useful in clinical practice.