Phenotypic and genotypic comparison of Escherichia coli from pristine tropical waters.

Nine fecal-coliform-positive strains were isolated from pristine sites in a tropical rain forest. These sites included nonpolluted rivers and water from bromeliads (epiphytes) which were 30 ft (ca. 910 cm) above the ground. Phenotypically, all of these isolates were identified as Escherichia coli. Their DNA was isolated and purified, and the base composition (G + C content) was determined and compared with that of E. coli B (ATCC 11303). The DNA from the environmental isolates was also hybridized to radiolabeled DNA from E. coli B. Eight strains had a DNA base composition similar to that of E. coli B and gave more than 75% homology with E. coli B. One strain had a different DNA base composition and a relatively low percentage of homology with the reference strain. The finding of E. coli in pristine tropical waters suggests that this bacterium could be a natural inhabitant in these environments and is not a reliable indicator of recent human fecal contamination in tropical waters. The indicators that are currently used in the tropics to test the biological quality of water should be reevaluated.     

Phenotypic and genotypic characterization of encapsulated Escherichia coli isolated from blooms in two Australian lakes

Escherichia coli has long been used as an indicator organism for water quality assessment. Recently there has been an accumulation of evidence that suggests some strains of this organism are able to proliferate in the environment, a characteristic that would detract from its utility as an indicator of faecal pollution. Phenotypic and genotypic characterization of E. coli isolated from blooms in two Australian lakes, separated by a distance of approximately 200 km, identified that the blooms were dominated by three E. coli strains. A major phenotypic similarity among the three bloom strains was the presence of a group 1 capsule. Genetic characterization of a conserved region of the cps gene cluster, which encodes group 1 capsules, identified a high degree of genetic variation within the bloom isolates. This differs from previously described encapsulated E. coli strains which are highly conserved at the cps locus. The phenotypic or genotypic profiles of the bloom strains were not identified in 435 E. coli strains isolated from vertebrates. The occurrence of these encapsulated strains suggests that some E. coli have evolved a free-living lifestyle and do not require a host in order to proliferate. The presence of the same three strains in bloom events in different geographical regions of a temperate climate, and at different times, indicates that free-living E. coli strains are able to persist in these water reservoirs. This study provides further evidence of circumstances where caution is required in using E. coli as an indicator organism for water quality.     

Phenotypic and genotypic analysis of pathogenic Escherichia coli virulence genes recovered from Riyadh,Saudi Arabia

The current study was carried out to evaluate the phenotypic and genotypic characterization of avian pathogenic Escherichia coli recovered from Riyadh, Saudi Arabia. During the period of 10th February–30th May 2015, 70 E. coli strains were isolated from chicken farms located in Riyadh, Saudi Arabia. All strains were tested phenotypically by standard microbiological techniques, serotyped and the virulence genes of such strains were detected by polymerase chain reaction (PCR). Most of the recovered strains from chickens belonged to serotype O111:K58 25 strains (35.7%), followed by serotype O157:H7 13 strains (18.57%), followed by serotype O114:K90 10 strains (14.29%), then serotype O126:K71 9 strains (12.9%), serotype O78:K80 8 strains (11.43%) and in lower percentage serotype O114:K90 and O119:K69 5 strains (7.14%). The virulence genotyping of E. coli isolates recovered from broilers revealed the presence of the uidA gene in all the field isolates (6 serovars) examined in an incidence of 100%, as well as the cvaC gene was also present in all field isolates (6 serovars), while the iutA gene and the iss gene were detected in 5 out of 6 field serovars in an incidence of 81.43% and 64.29%, respectively. Phenotypical examination of the other virulence factors revealed that 65 isolates were hemolytic (92.9%), as well as 15 isolates (21.42%) were positive for enterotoxin production. Meanwhile, 21 isolates (30%) were positive for verotoxin production, 58 isolates (82.86%) for the invasiveness and 31 isolates (44.29%) for Congo red binding activities of the examined serotypes.     

Specificity for field enumeration of Escherichia coli in tropical surface waters

In remote rural areas in developing countries, bacteriological monitoring often depends on the use of commercial field media. This paper evaluates a commercial field medium used for the enumeration of Escherichia coli in different surface waters under primitive field conditions in rural Pakistan. In order to verify the field kit, 117 presumptive E. coli isolates have been tested, finding a specificity of only 40%. By excluding some strains based on colony colours, the calculated specificity could be increased to 65%. Thus, it is suggested that prior to use in a tropical environment, the specificity of any commercial medium used should be tested with representative tropical isolates, in order to increase the specificity.     

In situ survival of plasmid-bearing and plasmidless Pseudomonas aeruginosa in pristine tropical waters

Two rare wild-type strains of Pseudomonas aeruginosa were mixed in membrane diffusion chambers and then introduced into a natural freshwater environment for 72 h. The plasmid-containing strain (R serotype 15) and the plasmidless strain (H serotype 5) had initial bacterial densities of 2 x 10(5) cells per ml. Samples collected from the chambers were analyzed for viable and direct counts and for acquired-resistance frequencies. Suspected transconjugant-to-donor ratios ranged from 0.5 to 1.3; transfer percentages ranged from 13 to 70%. [3H]thymidine uptake indicated DNA synthesis in both strains as well as in transconjugants. These studies indicate that rare wild-type bacterial strains with large plasmid loads can survive as well as can bacteria with low plasmid loads when exposed to the in situ conditions of a tropical freshwater habitat. These results also suggest that genetic modification of indigenous microbiota through conjugation or transformation is feasible when rare wild-type strains or genetically engineered microorganisms are released in large numbers in tropical aquatic ecosystems.     

Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.     

Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.     

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.     

In situ survival of plasmid-bearing and plasmidless Pseudomonas aeruginosa in pristine tropical waters.

Two rare wild-type strains of Pseudomonas aeruginosa were mixed in membrane diffusion chambers and then introduced into a natural freshwater environment for 72 h. The plasmid-containing strain (R serotype 15) and the plasmidless strain (H serotype 5) had initial bacterial densities of 2 x 10(5) cells per ml. Samples collected from the chambers were analyzed for viable and direct counts and for acquired-resistance frequencies. Suspected transconjugant-to-donor ratios ranged from 0.5 to 1.3; transfer percentages ranged from 13 to 70%. [3H]thymidine uptake indicated DNA synthesis in both strains as well as in transconjugants. These studies indicate that rare wild-type bacterial strains with large plasmid loads can survive as well as can bacteria with low plasmid loads when exposed to the in situ conditions of a tropical freshwater habitat. These results also suggest that genetic modification of indigenous microbiota through conjugation or transformation is feasible when rare wild-type strains or genetically engineered microorganisms are released in large numbers in tropical aquatic ecosystems.     

Cytotoxicity potential and genotypic characterization of Escherichia coli isolates from environmental and food sources

The presence of Escherichia coli isolates in the environment is a potential source of contamination of food and water supplies. Moreover, these isolates may harbor virulence genes that can be a source of new forms of pathogenic strains. Here, using multiplex PCR, we examined the presence of virulence gene markers (stx1, stx2, eaeA, hlyA) in 1,698 environmental isolates of E. coli and 81 isolates from food and clinical sources. The PCR analysis showed that approximately 5% (79 of 1,698) of the total environmental isolates and 96% (79 of 81) of the food and clinical isolates were positive for at least one of the genes. Of the food and clinical isolates, 84% (68 of 81 isolates) were positive for all four genes. Of the subset of environmental isolates chosen for further analysis, 16% (13 of 79 isolates) were positive for stx2 and 84% (66 of 79 isolates) were positive for eaeA; 16 of the latter strains were also positive for hlyA. The pathogenic potentials of 174 isolates (81 isolates from food and clinical sources and 93 isolates from environmental sources) were tested by using a cytotoxicity assay based on lactate dehydrogenase release from Vero cells. In general, 97% (79 of 81) of the food and clinical isolates and 41% (39 of 93) of the environmental isolates exhibited positive cytotoxicity. High cytotoxicity values correlated to the presence of stx genes. The majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. Isolates were also tested for sorbitol utilization and were genotyped by ribotyping and by repetitive extragenic palindromic PCR (REP-PCR) as potential means of quickly identifying virulent strains from the environment, but none of these methods could be used to distinguish cytotoxic environmental isolates. Only 31% of the isolates were negative for sorbitol fermentation, and none of the isolates had common ribotypes or REP-PCR fingerprints. This study suggests that overall higher cytotoxicity values correlated with the production of stx genes, and the majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. This study demonstrated that there is widespread distribution of potentially virulent E. coli strains in the environment that may be a cause of concern for human health.     

Phenotypic and molecular characteristics of Escherichia coli isolated from aquatic environment of Bangladesh

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.     

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

Phenotypic and genotypic diversity of yeasts isolated from water-buffalo Mozzarella cheese

Water-buffalo Mozzarella (WBM) cheese is one of the several 'pasta filata' or stretched curd cheeses that originated in southern Italy, traditionally manufactured from raw milk employing natural whey starter cultures. Lactose- and galactose-fermenting yeasts isolated from WBM were studied to evaluate their role in the ripening of this cheese. The kinetic parameters of the growth of the yeasts as well as their principal metabolic end-products showed a great variability depending on the species. Moreover, the genetic polymorphism of the yeasts was studied for their differentiation at species level by means of the polymerase chain reaction (PCR) fingerprinting and mitochondrial DNA (mtDNA) restriction analysis. While the differentiation based on metabolic traits was not able to discriminate Kluyveromyces marxianus, Candida kefyr and C. sphaerica, the PCR analysis with primers M13 and RF2 resulted in a reliable and rapid method for differentiating at species level Saccharomyces cerevisiae, K. marxianus, K. lactis and their anamorphic species. Furthermore, mtDNA analysis proved to be more discriminating at strain level.     

Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.     

Phenotypic and genotypic identification of staphylococci isolated from wild small mammals

229 strains of Staphylococcus isolated from the intestines of wild small mammals (Insectivora and rodents) were analysed by phenotypic tests and 16S-23S rDNA intergenic spacer PCR amplification (ITS-PCR). Based on the results of three methods (phenotypical tests, API numerical profiles and ITS-PCR) 65.5% of all staphylococcal strains could be identified. The remaining strains were compared by cluster analysis with reference and identified strains. It is quite possible that, a part of unidentified strains represent hitherto undescribed species, all the more as their ITS types were not found among other members of the genus Staphylococcus. Moreover, the results show that small mammals provides a suitable new habitat for certain staphylococci.     

Characteristic of saccharose fermentation by pathogenic Escherichia coli strains--phenotypic and genotypic elements

The purpose of the study was to characterize fermentation of sucrose by Escherichia coli strains and to answer why some of these strains doesn't utilize this disaccharide. Investigations included 16 E. coli strains. Only 5 of these strains utilized sucrose. Genotypic analysis demonstrated the presence of cscB gene (encoding the sucrase permease which catalyzes transport of sucrose through the plasma membrane of the cell) in 5 strains of E. coli and cscA gene (encoding an enzyme sucrase that catalyzes the utilization of sucrose) in 6 strains of E. coli. These 5 of E. coli strains which possessed a chromosomally encoded sucrose metabolic pathway utilized sucrose with a different time. 3 of them destroyed this disaccharide after 24 h and 2 of them destroyed it after 48 h. Ten of E. coli strains hadn't cscA gene and 11 of them had not cscB genes. The lack of these genes can be the prove that it is not possible for 11 of E. coli strains to synthesize sucrose permease and for 10 of them to synthesize sucrase and it may be the reason of not utilize disaccharide sucrose by these bacteria.     

Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Bangladesh

Background

Resistance to cephalosporins in Enterobacteriaceae is mainly due to the production of extended-spectrum beta-lactamase (ESBL). Little is known about ESBL-producing bacteria in Bangladesh. Therefore, the study presents results of phenotypic and molecular characterization of ESBL-producing Escherichia coli from hospitals in Bangladesh.

Methods

A total of 339 E. coli isolated from patients with urinary tract and wound infections attending three different medical hospitals in urban and rural areas of Bangladesh between 2003–2007 were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic susceptibility testing, PCR and sequencing of different β-lactamase and virulence genes, serotyping, and XbaI-macrorestriction followed by pulsed-field gel electrophoresis (PFGE).

Results

We identified 40 E. coli with ESBL phenotype. These isolates were resistant to ceftriaxone, ceftazidime, cefotaxime, aztreonam, cefepime, and nalidixic acid but remained susceptible to imipenem. All but one isolate were additionally resistant to ciprofloxacin, and 3 isolates were resistant to cefoxitin. ESBL genes of blaCTX-M-1-group were detected in all isolates; blaTEM-type and blaOXA-1-type genes were detected in 33 (82.5%) and 19 (47.5%) isolates, respectively. Virulence genes that are present in diarrhoeagenic E. coli were not found. Class-1 integron was present in 20 (50%) isolates. All the ESBL-producing E. coli isolates harbored plasmids ranging between 1.1 and 120 MDa. PFGE-typing revealed 26 different pulsotypes, but identical pulsotype showed 6 isolates of serotype O25:H4.

Conclusion

The prevalence of multidrug-resistant ESBL-producing E. coli isolates appears to be high and the majority of the isolates were positive for bla _CTX-M. Although there was genetic heterogeneity among isolates, presence of a cluster of isolates belonging to serotype O25:H4 indicates dissemination of the pandemic uropathogenic E. coli clone in Bangladesh.     

Phenotypic and genotypic characterization of Klebsiella pneumoniae isolates recovered from nonhuman primates

Klebsiella pneumoniae is a zoonotic, Gram-negative member of the family Enterobacteriaceae and is the causative agent of nosocomial septicemic, pneumonic, and urinary tract infections. Recently, pathogenic strains of K. pneumoniae sharing a hypermucoviscosity (HMV) phenotype have been attributed to multisystemic abscessation in both human and nonhuman primates. Although K. pneumoniae is a well-recognized zoonotic agent, there is a lack of general information including adequate diagnostic methods or treatments for nonhuman primates. In an effort to increase the body of knowledge of this enigmatic pathogen, K. pneumoniae isolates from African green monkeys (Chlorocebus aethiops sabaeus) on the island of St. Kitts, West Indies were genotypically and phenotypically characterized. Genetic fingerprints generated by PCR-mediated genomic fingerprinting, phenotypic characterization, and antimicrobial susceptibility all identified a high degree of similarity between the HMV and non-HMV K. pneumoniae isolates. The results obtained from this work will help establish a baseline for the development of efficacious diagnostic methods and treatment strategies for both human and nonhuman primates.