Diversity of strains of Salmonella enterica serotype Enteritidis from English poultry farms assessed by multiple genetic fingerprinting

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella enterica serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was PstI-SphII ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XhaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of Salmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.

Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype typhimurium DT104 isolates

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC --> AAC) mutation, an Asp-87-to-Gly (GAC --> GGC) mutation, and a Ser-83-to-Phe (TCC --> TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC --> TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC --> TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI. while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Comparative genomic indexing reveals the phylogenomics of Escherichia coli pathogens

The Escherichia coli O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing E. coli O26 strains represent the same clonal lineage and that W-expressing E. coli O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the E. coli species and identified regions of variation from the E. coli K-12 chromosomal backbone.

Attaching-effacing bacteria in animals

Enteric bacteria with a demonstrable or potential ability to form attaching-effacing lesions, so-called attaching-effacing (AE) bacteria, have been found in the intestinal tracts of a wide variety of warm-blooded animal species, including man. In some host species, for example cattle, pigs, rabbits and human beings, attaching-effacing Escherichia coli (AEEC) have an established role as enteropathogens. In other host species, AE bacteria are of less certain significance. With continuing advances in the detection and typing of AE strains, the importance of these bacteria for many hosts is likely to become clearer. The pathogenic effects of AE bacteria result from adhesion to the intestinal mucosa by a variety of mechanisms, culminating in the formation of the characteristic intimate adhesion of the AE lesion. The ability to induce AE lesions is mediated by the co-ordinated expression of some 40 bacterial genes organized within a so-called pathogenicity island, known as the "Locus for Enterocyte Effacement". It is also believed that the production of bacterial toxins, principally Vero toxins, is a significant virulence factor for some A-EEC strains. Recent areas of research into AE bacteria include: the use of Citrobacter rodentium to model human AEEC disease; quorum-sensing mechanisms used by AEEC to modulate virulence gene expression; and the potential role of adhesion in the persistent colonization of the intestine by AE bacteria. This review of AE bacteria covers their molecular biology, their occurrence in various animal species, and the diagnosis, pathology and clinical aspects of animal diseases with which they are associated. Reference is made to human pathogens where appropriate. The focus is mainly on natural colonization and disease, but complementary experimental data are also included. (C) 2004 Elsevier Ltd. All rights reserved.

Use of miniaturized protein arrays for Escherichia coli O serotyping

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

Sources and spread of thermophilic Campylobacter spp. during partial depopulation of broiler chicken flocks

The practice of partial depopulation or ‘thinning’, i.e. early removal of a proportion of birds from a commercial broiler flock, is a reported risk factor for Campylobacter colonization of residual birds because of the difficulty in maintaining biosecurity during the process. Therefore, the effect of this practice was studied in detail for 51 target flocks, each at a different growing farm belonging to one of seven major poultry companies throughout the United Kingdom. On 21 of these farms, the target flock was already colonized by Campylobacter and at slaughter all cecal samples examined were positive, with a mean of log10 8 cfu / g. A further 27 flocks became positive within 2 – 6 days of the start of thinning and had similarly high levels of cecal carriage at slaughter. Just prior to the thinning process, Campylobacter could be isolated frequently from the farm driveways, transport vehicles, equipment and personnel. Strains from seven such farms on which flocks became colonized after thinning were examined by PFGE typing. The study demonstrated an association between strains occurring at specific sampling sites and those isolated subsequently from the thinned flocks. There were also indications that particular strains had spread from one farm to another, when the farms were jointly company-owned and served by the same bird-catching teams and / or vehicles. The results highlighted the need for better hygiene control in relation to catching equipment and personnel, and more effective cleaning and disinfection of vehicles, and bird-transport crates.

Contrasting arbuscular mycorrhizal communities colonizing different host plants show a similar response to a soil phosphorus concentration gradient

High soil phosphorus (P) concentration is frequently shown to reduce root colonization by arbuscular mycorrhizal (AM) fungi, but the influence of P on the diversity of colonizing AM fungi is uncertain. We used terminal restriction fragment length polymorphism (T-RFLP) of 18S rDNA and cloning to assess diversity of AM fungi colonizing maize (Zea mays), soybean (Glycene max) and field violet (Viola arvensis) at three time points in one season along a P gradient of 10280mgl1 in the field. Percentage AM colonization changed between sampling time points but was not reduced by high soil P except in maize. There was no significant difference in AM diversity between sampling time points. Diversity was reduced at concentrations of P > 25mgl1, particularly in maize and soybean. Both cloning and T-RFLP indicated differences between AM communities in the different host species. Host species was more important than soil P in determining the AM community, except at the highest P concentration. Our results show that the impact of soil P on the diversity of AM fungi colonizing plants was broadly similar, despite the fact that different plants contained different communities. However, subtle differences in the response of the AM community in each host were evident.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

A haplotype at the UCP1 gene locus contributes to genetic risk for type 2 diabetes in Asian Indians (CURES-72)

BACKGROUND: The gene encoding for uncoupling protein-1 (UCP1) is considered to be a candidate gene for type 2 diabetes because of its role in thermogenesis and energy expenditure. The objective of the study was to examine whether genetic variations in the UCP1 gene are associated with type 2 diabetes and its related traits in Asian Indians. METHODS: The study subjects, 810 type 2 diabetic subjects and 990 normal glucose tolerant (NGT) subjects, were chosen from the Chennai Urban Rural Epidemiological Study (CURES), an ongoing population-based study in southern India. The polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms, namely -3826A-->G, an A-->C transition in the 5'-untranslated region (UTR) and Met229Leu, were not associated with type 2 diabetes. However, the frequency of the A-C-Met (-3826A-->G-5'UTR A-->C-Met229Leu) haplotype was significantly higher among the type 2 diabetic subjects (2.67%) compared with the NGT subjects (1.45%, P < 0.01). The odds ratio for type 2 diabetes for the individuals carrying the haplotype A-C-Met was 1.82 (95% confidence interval, 1.29-2.78, P = 0.009). CONCLUSIONS: The haplotype, A-C-Met, in the UCP1 gene is significantly associated with the increased genetic risk for developing type 2 diabetes in Asian Indians.

Association of the PPARGC1A gene polymorphism with diabetic nephropathy in an Asian Indian population (CURES-41)

BACKGROUND: The aim of this study was to evaluate the association of polymorphisms of the peroxisome proliferator-activated receptor gamma (PPARG) gene and peroxisome proliferators-activated receptor gamma co-activator 1 alpha (PPARGC1A) gene with diabetic nephropathy (DN) in Asian Indians. METHODS: Six common polymorphisms, 3 of the PPARG gene [-1279G/A, Pro12Ala, and His478His (C/T)] and 3 of the PPARGC1A gene (Thr394Thr, Gly482Ser, and +A2962G) were studied in 571 normal glucose-tolerant (NGT) subjects, 255 type 2 diabetic (T2D) subjects without nephropathy, and 141 DN subjects. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing. Logistic regression analysis was performed to assess the covariables associated with DN. RESULTS: Among the 6 polymorphisms examined, only the Gly482Ser of the PPARGC1A gene was significantly associated with DN. The genotype frequency of Ser/Ser genotype of the PPARGC1A gene was 8.8% (50/571) in NGT subjects, 7.8% (20/255) in T2D subjects, and 29.8% (42/141) in DN subjects. The odds ratios (ORs) for DN for the susceptible Gly/Ser and Ser/Ser genotype after adjusting for age, sex, body mass index, and duration of diabetes were 2.14 [95% confidence interval (CI), 1.23-3.72; P = 0.007] and 8.01 (95% CI, 3.89-16.47; P < 0.001), respectively. The unadjusted OR for DN for the XA genotype of the Thr394Thr polymorphism was 1.87 (95% CI, 1.20-2.92; P = 0.006) compared to T2D subjects. However, the significance was lost (P = 0.061) when adjusted for age, sex, BMI, and duration of diabetes. The +A2962G of PPARGC1A and the 3 polymorphisms of PPARG were not associated with DN. CONCLUSION: The Gly482Ser polymorphism of the PPARGC1A gene is associated with DN in Asian Indians.

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (-11377C-->G and -11282T-->C), one intronic SNP (+10211T-->G) and one exonic SNP (+45T-->G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T-->G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T-->G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07-1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3-1.8; P < 10(-7)) and that for GG genotype, 2.10 (95% CI 1.3-3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T-->G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.

Association of lipoprotein lipase gene polymorphisms with obesity and type 2 diabetes in an Asian Indian population

AIMS: Lipoprotein lipase (LPL), a pivotal enzyme in lipoprotein metabolism, catalyzes the hydrolysis of triglycerides of very low-density lipoproteins and chylomicrons. Assuming that the variants in the promoter of the LPL gene may be associated with changes in lipid metabolism leading to obesity and type 2 diabetes, we examined the role of promoter variants (-T93G and -G53C) in the LPL gene in an urban South Indian population. METHODS: The study subjects (619 type 2 diabetic and 731 normal glucose-tolerant (NGT) subjects) were chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The polymorphisms were genotyped using polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP). Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The two polymorphisms studied were not in LD. The -T93G was not associated with type 2 diabetes but was associated with obesity. 11.5% of the obese subjects (62/541) had the XG(TG+GG) genotype compared with 6.4% of the nonobese subjects (52/809; P=0.001). The odds ratio for obesity for the XG genotype was 1.766 (95% CI: 1.19-2.63, P=0.005). Subjects with XG genotype also had higher body mass index and waist circumference compared with those with TT genotype. With respect to G53C, subjects with the XC(GC+CC) genotype had 0.527 and 0.531 times lower risk for developing type 2 diabetes and obesity, respectively. CONCLUSIONS: Among Asian Indians, the -T93G SNP of the LPL gene is associated with obesity but not type 2 diabetes, whereas the -G53C SNP appears to be protective against both obesity and type 2 diabetes.

Effect of polymorphisms in the PPARGC1A gene on body fat in Asian Indians

OBJECTIVE: To evaluate whether polymorphisms in the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PPARGC1A) gene were related to body fat in Asian Indians. METHODS: Three polymorphisms of PPARGC1A gene, the Thr394Thr, Gly482Ser and +A2962G, were genotyped on 82 type 2 diabetic and 82 normal glucose tolerant (NGT) subjects randomly chosen from the Chennai Urban Rural Epidemiology Study using PCR-RFLP, and the nature of the variants were confirmed using direct sequencing. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies using an expectation-maximization algorithm. Visceral, subcutaneous and total abdominal fat were measured using computed tomography, whereas dual X-ray absorptiometry was used to measure central abdominal and total body fat. RESULTS: None of the three polymorphisms studied were in LD. The genotype (0.59 vs 0.32, P=0.001) and allele (0.30 vs 0.17, P=0.007) frequencies of Thr394Thr polymorphism were significantly higher in type 2 diabetic subjects compared to those in NGT subjects. The odds ratio for diabetes (adjusted for age, sex and body mass index) for the susceptible genotype, XA (GA+AA) of Thr394Thr polymorphism, was 2.53 (95% confidence intervals: 1.30-5.04, P=0.009). Visceral and subcutaneous fat were significantly higher in NGT subjects with XA genotype of the Thr394Thr polymorphism compared to those with GG genotype (visceral fat: XA 148.2+/-46.9 vs GG 106.5+/-51.9 cm(2), P=0.001; subcutaneous fat: XA 271.8+/-167.1 vs GG 181.5+/-78.5 cm(2), P=0.001). Abdominal (XA 4521.9+/-1749.6 vs GG 3445.2+/-1443.4 g, P=0.004), central abdominal (XA 1689.0+/-524.0 vs GG 1228.5+/-438.7 g, P<0.0001) and non-abdominal fat (XA 18763.8+/-8789.4 vs GG 13160.4+/-4255.3 g, P<0.0001) were also significantly higher in the NGT subjects with XA genotype compared to those with GG genotype. The Gly482Ser and +A2962G polymorphisms were not associated with any of the body fat measures. CONCLUSION: Among Asian Indians, the Thr394Thr (G --> A) polymorphism is associated with increased total, visceral and subcutaneous body fat.

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians

AIMS: The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-gamma co-activator-1 alpha (PGC-1alpha) gene with Type 2 diabetes in Asian Indians. METHODS: The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype frequencies were estimated using an expectation-maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264-2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. CONCLUSIONS: The A allele of Thr394Thr (G --> A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population.