Resistance to amoxicillin-clavulanate and its relation to virulence-related factors in Yersinia enterocolitica biovar 1A.

Recent studies have reported that the virulence factors (VFs) were detected more frequently in amoxicillin-clavulanate (AMC) susceptible clinical isolates of Escherichia coli. Here, we have evaluated the relationship between VFs and AMC-resistance phenotype in clinical isolates of Y. enterocolitica biovar 1A. The presence/absence of VFs was compared with their minimum inhibitory concentrations for AMC in strains of two serovars. We observed that the strains of the serovar O: 6, 30-6, 31 showed a similar relationship between the number of VFs and resistance to clavulanic acid as in E. coli but not of serovar O: 6, 30. Variations in the promoters/complete coding sequences (CCDSs) of ß-lactamase gene (bla A) or the serological characteristics could not account for unusual susceptibility to AMC displayed by the strains of the serovar O: 6, 30. Therefore, we speculate that since the clinical strains of serovar O: 6, 30-6, 31 originated from the environment they were less exposed to antibiotics compared to clinical strains of serovar O: 6, 30. Thus, AMC susceptibility seems to be influenced by factors other than serotypes or promoters/CCDS of ß-lactamase genes.

Detection of Yersinia enterocolitica in food: an overview.

Yersinia enterocolitica is a gastrointestinal pathogen which causes yersiniosis, an illness characterized by diarrhea, ileitis, and mesenteric lymphadenitis. Y. enterocolitica is transmitted via the feco-oral route by the consumption of contaminated food or water. Several phenotypic and genotypic methods have been developed to reliably detect Y. enterocolitica in food. However, the source of infection of many recently reported foodborne outbreaks remains obscure. The detection of this pathogen in food is a challenging task, since it shares similarities with other enteric bacteria. The presence of other microorganisms in the food samples makes it even more difficult to identify this slow-growing pathogen. Therefore, the present-day emphasis is on the development of sensitive, easily automated methods suitable for in-situ detection, allowing quick and cost-effective characterization of food samples. This review summarizes and compares the currently available cultural, immunological, and molecular methods, particularly in relation to their specific merits or demerits when implemented for the detection of Y. enterocolitica in food.

Identification and distribution of putative virulence genes in clinical strains of Yersinia enterocolitica biovar 1A by suppression subtractive hybridization.

AIMS: To detect putative virulence genes in clinical strains of Yersinia enterocolitica biovar 1A by suppression subtractive hybridization between two closely related strains of clinical and nonclinical origin having the same serotype (O:6,30-6,31). METHODS AND RESULTS: Suppression Subtractive Hybridization (SSH) was used to identify genomic differences between clinical (serotype O:6,30-6,31, from diarrhoeic human stools) and nonclinical (serotype O:6,30-6,31, from wastewater) strains of Y. enterocolitica biovar 1A. Following genomic subtraction and DNA sequencing, nine DNA sequences that were present only in clinical biovar 1A strains were identified. The sequences identified using SSH showed similarity to conserved hypothetical proteins, proteins related to iron acquisition and haemin storage, type 1 secretion proteins, flagellar hook proteins, exported protein and ABC transport system. All these sequences showed high similarity with Y. enterocolitica 8081 (biovar 1B). The distribution of these genes was further analysed using PCR in 26 clinical strains of Y. enterocolitica biovar 1A. The results revealed that the distribution of these genes was not uniform. CONCLUSIONS: Genes related to iron acquisition and storage, and flagellar proteins might be responsible for virulence of some of the clinical strains of Y. enterocolitica biovar 1A. SIGNIFICANCE AND IMPACT OF THE STUDY: Genes identified in this study might be useful in understanding the pathogenic potential of clinical strains of Y. enterocolitica biovar 1A.

Whole cell protein profiling reiterate phylogenetic relationships among strains of Yersinia enterocolitica biovar 1A as discerned earlier by different genotyping methods.

AIM: To evaluate whole cell protein profiling vis-à-vis genotyping to discern phylogenetic relationships among strains of Y. enterocolitica biovar 1A. METHODS AND RESULTS: Whole cell protein profiling of Y. enterocolitica biovar 1A strains was performed using SDS-PAGE. Twenty-one distinct protein profile types were obtained among a collection of 81 strains isolated from clinical and nonclinical sources. Whole cell protein profiling exhibited discriminatory index (DI) of 0·80 and clustered the strains into two distinct clonal groups. The clinical and the aquatic serotype O:6,30-6,31 strains were clustered into two discrete subgroups. CONCLUSIONS: Whole cell protein profiling displayed sufficient diversity among strains of Y. enterocolitica biovar 1A, and the phylogenetic relationships obtained were in good agreement with those established earlier by genotyping techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Whole cell protein profiling was as discriminatory as some of the genotyping methods and has the potentiality to be used as an adjunct tool to study epidemiology of Y. enterocolitica.

Multilocus variable number tandem repeat analysis as a tool to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A.

AIMS: To identify variable number tandem repeat (VNTR)-containing loci, and to use multilocus VNTR (MLVA) to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A isolated from diverse sources. METHODS AND RESULTS: The whole genome sequence of Y. enterocolitica 8081 was analysed and eight VNTR loci with repeat sizes between 4 and 9 bp, and each containing more than four repeat copies were selected for MLVA typing of 88 strains of Y. enterocolitica. Of these, four loci were polymorphic and generated 26 MLVA genotypes among 81 strains of Y. enterocolitica biovar 1A. MLVA was found to be quite discriminatory (DI = 0.87). Cluster analysis and population modelling using minimum spanning tree (MST) clearly clustered Y. enterocolitica biovar 1A into two major groups. CONCLUSIONS: The MLVA is easy to perform and can be used to discern clonal relationship among strains of Y. enterocolitica. Also the phylogenetic relationships obtained with MLVA genotypes were in good agreement with those established by other typing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The MLVA method reported is relatively more discriminatory than the other genotyping methods and has the potential to be used as an epidemiological tool for the study of strains of Y. enterocolitica biovar 1A.

Detection and assay of beta-lactamases in clinical and non-clinical strains of Yersinia enterocolitica biovar 1A.

OBJECTIVES: To detect beta-lactamases (A & B) and extended-spectrum beta-lactamases (ESBLs) in clinical and non-clinical isolates of Yersinia enterocolitica biovar 1A, and to determine their activity in the presence of specific lactamase inhibitors. METHODS: The presence of beta-lactamases and ESBLs was detected by disc diffusion in 219 (36 clinical, 183 non-clinical) isolates. beta-Lactamase activity was assayed spectrophotometrically in all 36 clinical and 10 representative non-clinical isolates using nitrocefin as the substrate. Inhibition of beta-lactamases was studied by clavulanic acid, aztreonam and cloxacillin. RESULTS: Of the 219 isolates, all except two non-clinical isolates indicated the presence of beta-lactamase A (Bla-A) based on the smaller (2-8 mm) radius of the inhibition zone around the ticarcillin disc. Synergy between ticarcillin and co-amoxiclav discs was, however, observed in only 34% of isolates of non-clinical origin. beta-Lactamase B (Bla-B) was found to be consistently positive among all the clinical and non-clinical isolates, as indicated by its characteristic appearance of flattening of the zone of inhibition around the cefotaxime disc adjacent to an imipenem disc. Bla-B was induced more strongly in clinical than in non-clinical isolates. Inhibition of enzyme A by clavulanic acid, aztreonam and cloxacillin was found to be similar, whereas enzyme B was inhibited more strongly by aztreonam and cloxacillin. None of the isolates showed the unequivocal presence of ESBL. CONCLUSION: This is the first report on beta-lactamases of Yersinia enterocolitica biovar 1A from Asia. Y. enterocolitica biovar 1A expressed both Bla-A and Bla-B. Heterogeneity was, however, discerned in the expression of Bla-A and by induction of Bla-B among clinical and non-clinical isolates of Y. enterocolitica biovar 1A.

Rhizobacterial diversity in India and its influence on soil and plant health.

The rhizosphere or the zone of influence around roots harbors a multitude of microorganisms that are affected by both abiotic and biotic stresses. Among these are the dominant rhizobacteria that prefer living in close vicinity to the root or on its surface and play a crucial role in soil health and plant growth. Both free-living and symbiotic bacteria are involved in such specific ecological niches and help in plant matter degradation, nutrient mobilization and biocontrol of plant disease. While the rhizosphere as a domain of fierce microbial activity has been studied for over a century, the availability of modern tools in microbial ecology has now permitted the study of microbial communities associated with plant growth and development, in situ localization of important forms, as well as the monitoring of introduced bacteria as they spread in the soil and root environment. This interest is linked to environmental concerns for reduced use of chemicals for disease control as well as an appreciation for utilization of biologicals and organics in agriculture. Indian researchers have studied the diversity of rhizobacteria in a variety of plants, cereals, legumes and others along with assessment of their functionality based on the release of enzymes (soil dehydrogenase, phosphatase, nitrogenase, etc.), metabolites (siderophores, antifungals, HCN, etc.), growth promoters (IAA, ethylene) and as inducers of systemic disease resistance (ISR). Based on such primary screening protocols, effective rhizobacteria have been field tested with success stories from various agroecological zones of the country, as reflected in the control of root- and soil-borne diseases, improved soil health and increased crop yields. Several commercial formulations, mostly based on dry powder (charcoal, lignite, farmyard manure, etc.) have been prepared and field tested, however, problems of appropriate shelf-life and cell viability are still to be solved. Also, inherent in such low cost technologies are the problems of variability in field performance and successful establishment of introduced inoculants in the root zone. In addition, most products available in the market are not properly monitored for quality before they reach the farmer. As a consequence, the acceptance of rhizobacterial formulations in the country is limited. However, several laboratories have now developed protocols for the rapid characterization of effective isolates based on molecular fingerprinting and other similar tools. Also, the use of molecular markers (gus, lux, gfp, etc.) makes it easy to monitor introduced inoculants in situ in soil and rhizosphere environments. The government initiative in integrated nutrient management and pest management systems has provided additional incentives to relate rhizobacterial science to other ongoing activities so that the benefit of this research leads to technologies that are environmentally and socially acceptable.

Emerging water-borne pathogens.

Emerging water-borne pathogens constitute a major health hazard in both developed and developing nations. A new dimension to the global epidemiology of cholera-an ancient scourge-was provided by the emergence of Vibrio cholerae O139. Also, water-borne enterohaemorrhagic Escherichia coli ( E. coli O157:H7), although regarded as a problem of the industrialized west, has recently caused outbreaks in Africa. Outbreaks of chlorine-resistant Cryptosporidium have motivated water authorities to reassess the adequacy of current water-quality regulations. Of late, a host of other organisms, such as hepatitis viruses (including hepatitis E virus), Campylobacter jejuni, microsporidia, cyclospora, Yersinia enterocolitica, calciviruses and environmental bacteria like Mycobacterium spp, aeromonads, Legionella pneumophila and multidrug-resistant Pseudomonas aeruginosa have been associated with water-borne illnesses. This review critically examines the potential of these as emerging water-borne pathogens. It also examines the possible reasons, such as an increase in the number of immunocompromised individuals, urbanization and horizontal gene transfer, that may underlie their emergence. Further, measures required to face the challenge posed by these pathogens are also discussed.

Arsenite-induced multiple antibiotic resistance phenotype in environmental isolates of Yersinia enterocolitica.

Minimal inhibitory concentrations (MICs) of five antibiotics namely amikacin, gentamicin, tetracycline, ciprofloxacin, and nitrofurantoin for pork isolates of Yersinia enterocolitica increased two- to eightfold after bacteria were grown in the presence of 5 mm arsenite. For Y. enterocolitica isolates obtained from wastewater (sewage effluents), an unequivocal increase in MICs was seen with amikacin and gentamicin. No change was discernible in the outer-membrane proteins after isolates were grown in the presence of arsenite.

Virulence plasmid (pYV)-associated susceptibility of Yersinia enterocolitica to chlorine and heavy metals.

This study was aimed at elucidating the role of virulence plasmid (pYV) in the susceptibility of Yersinia enterocolitica to bactericidal agents such as chlorine and heavy metals. Plasmid-bearing (pYV+) Y. enterocolitica was less susceptible to the antimicrobial action of chlorine and heavy metals compared with the isogenic plasmidless (pYV-) derivative. This difference was, however, observed only with bacteria cultured at 25 degrees C. pYV-associated susceptibility apart, cells cultured at 37 degrees C were also found to be less susceptible to the antimicrobial action of these agents. The results indicate that the susceptibility of Y. enterocolitica to these agents was influenced both by the presence of the virulence plasmid and the temperature at which the cells were cultured.

Arsenic and cadmium resistance in environmental isolates of Yersinia enterocolitica and Yersinia intermedia.

Environmental strains of Yersinia enterocolitica representing biotype 1A lack virulence plasmid (pYV) and are regarded as non-pathogenic. Though these occupy a diverse range of environmental niches, nothing is known about their resistance to heavy metals. The minimal inhibitory concentrations (MICs) of various metal ions, namely Ag+, Cu2+, Zn2+, Cd2+, As5+, and As3+, for strains of Yersinia enterocolitica (biotype 1A) and Yersinia intermedia (biotypes 1, 2, and 4), isolated from sewage effluents or pork, were determined. All isolates were resistant (MICs 2.5-5 mM) to Cd2+. The MICs of arsenic varied with bacterial strain and the chemical species of the arsenic used. For the majority of the strains, however, it was between 5-10 mM of Na2HAsO4.7H2O and NaAsO2, and 0.625-2.5 mM of As2O3. Except for one isolate, MICs of Ag+, Cu2+, and Zn2+ for these strains were in the range of 0.3-0.625 mM.

Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis.

Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30-55 degrees C) and pH (3-7.5). Among various divalent cations Mn2+ and Hg2+ completely inhibited the purified enzyme while beta-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.

Chemical analysis of post-lithotripsy stone fragments: a critical evaluation.

A scheme for the chemical microanalysis of renal stone fragments recovered from urine voided immediately after lithotripsy has been developed and evaluated. The analytical procedure includes assay of calcium, magnesium, phosphate, oxalate and urate and has been applied to 78 such urine samples. Problems relating to co-existing crystalluria and blood and urine contaminants have been recognised and overcome. However, significant loss of all stone components due to fragment dissolution in urine prior to recovery was found to occur and was investigated. The distribution of stone components found in these analyses was similar to that seen in previous surveys of intact stones.

Prognostic value of renal venous involvement in renal carcinoma.

A series of 108 renal tumour nephrectomies carried out between 1975 and 1984 was studied to determine the prognostic statistical significance of the relationship between venous involvement and various pathological features. Tumour size, spread, histological grade and lymph node involvement were compared between V0 tumours (58%), V1 (32%) and V2 tumours (10%). Actuarial 5-year survival rates revealed a poor prognosis with venous involvement (V0 66%, V1 27%, V2 33%). Tumours larger than 10 cm with perirenal spread and of higher histological grade were significantly related to venous involvement. Survival between renal vein involvement and inferior vena caval extension was statistically similar, but it was influenced by tumour size and higher grade. Perirenal spread and nodal involvement were poor indicators.

'Solitary' Ta-T1 G1 bladder tumour--history and long-term prognosis.

Natural history of 'solitary', histological grade 1, stage Ta-T1 transitional cell carcinoma of the bladder was studied in 198 patients retrospectively over a period from 1975 to 1987. Three patterns of tumour behaviour were evident. In 56% of patients the tumour did not recur following the initial resection. Twenty-one percent developed recurrences localised to the site of the original tumour. This group became tumour-free by 5 years and remained so thereafter. The remaining 23% continued to produce recurrent tumours up to 10 years at different sites in the bladder. The actuarial percentage of patients who remained continuously free of tumour after the initial resection was 53% at 5 years and 51% at 10 years. The results of this study suggest that cytoscopic follow-up may be discontinued in patients who remain continuously tumour-free for 5 years.

Long-term follow-up of intravesical Epodyl therapy for superficial bladder cancer.

The records of 111 patients with multiple superficial grade 1-2 transitional cell tumours of the bladder treated with intravesical Epodyl and followed up for a mean period of 6.4 years were retrospectively analysed. The study showed that 65% of patients responded completely after 12 weekly instillations and 46% of these remained continuously free of disease for 5 years. An initial complete response to therapy and lower pathological grade seemed to indicate a long-term successful response to treatment. In patients who failed to respond by 1 year, further therapy was of no benefit. Clearance of bladder disease by 1 year carried a 14% risk of subsequent tumour invasion by 5 years. This risk increased to 75% in those who failed to respond by 1 year.