Comparison of Several Laboratory Media for Presumptive Identification of Enterococci and Group D Streptococci

Bile-esculin (Difco), modified bile-esculin (Difco), selective enterococcus (Pfizer Co.), and eosin-methylene blue agar media were evaluated for accuracy in identifying group D streptococci. The regular and modified bile-esculin media performed equally well, but the selective enterococcus and eosin-methylene blue agars did not accurately differentiate the group D from non-group D streptococci. A modified 6.5% NaCl broth was compared with unmodified 6.5% NaCl broth and Streptococcus faecalis (SF; Difco) broth for accuracy in differentiating enterococci from non-enterococci. The modified and unmodified broths worked equally well in the salt tolerance test, but the lot-to-lot variability of SF broth made this medium unusable as an indicator for enterococci. With all seven media, the number of strains giving positive tests decreased when the tests were incubated at 45 C as compared with 35 C, and the number of strains giving negative tests increased. Thus, the number of false-positive identifications decreased, but the number of false-negative identifications increased. Variability in the susceptibility of group D non-enterococcal streptococci to oxacillin and methicillin sensitivity disks limited the usefulness of these tests for presumptive identification of either enterococci or group D streptococci.     

Evaluation of a biochemical test scheme for identifying clinical isolates of Enterococcus faecalis and Enterococcus faecium

AIMS: To evaluate the full test scheme of Facklam and Sahm (1995) for the identification of clinical enterococcal isolates to genus and species level. METHODS AND RESULTS: Fifty-nine clinical isolates, previously provisionally classed as enterococci on the basis of just four biochemical tests of Facklam and Sahm and one other test, were subjected to genus and species identification using the full identification scheme of Facklam and Sahm; 98% of these strains were confirmed to be enterococci and of these, 69% were identified as Enterococcus faecalis and 31% as Enterococcus faecium. Six tests in the scheme (out of 24) gave anomalous or unreliable results for some strains, and two gave unexpected results for the majority of strains presumptively identified as Ent. faecium. CONCLUSIONS: Nine (out of 12) genus tests and nine (out of 12) species tests from the Facklam and Sahm scheme were reliable. Testing for the presence of the Lancefield antigen D was also useful. The majority of presumptive Ent. faecium strains gave different results for the sorbitol and raffinose tests from that expected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the level of reliability for each of the tests in a current enterococcal identification scheme for differentiating clinical isolates, and showed that two tests gave consistently different test results from those expected for Ent. faecium.     

Susceptibility to mercurials of clinical Pseudomonas aeruginosa isolated in México

Susceptibility to inorganic mercuric ions and to organomercurials of 237 Pseudomonas aeruginosa clinical strains isolated in Mexico was determined by agar dilution tests. Resistant strains fell into two classes: i) narrow-spectrum resistant strains (27% of total isolates) resistant only to mercuric ions and to merbromin, and most grouped in pyocin type 1; and ii) broad-spectrum resistant strains (5%) with additional resistances to thimerosal, phenylmercury, methylmercury and p-hydroxymercuribenzoate, that belonged mostly to pyocin type 10. Mercurial resistant isolates showed a higher proportion of resistance to antibiotics and metals than did mercurial sensitive isolates, and broad-spectrum resistant strains had the highest frequency of resistance to antibiotics and to tellurite and arsenate.     

Identification of enterococci isolated from cow's milk cheese: comparison of the classical methods and the API 20 STREP system (technical note)

A comparison of the results obtained using the classical methods with those of the API 20 Strep system was carried out in identifying 24 enterococci strains isolated from San Simón cow's milk cheese, a traditional Spanish variety. The results of both identification systems coincided exactly in 9 strains (37.5% of the strains studied). In one strain the results obtained using the classical methods did not coincide with those using the API 20 Strep method. 3 strains (12.5%) could not be identified using the API 20 Strep system. However, 11 strains (45%), that remained doubtful between both species E. faecalis and E. faecium on the basis of the classical methods, were identified using the API 20 Strep system. The API 20 Strep system does not include some biochemical tests of importance in identifying of foodborne enterococci and could not identify the atypical strains of Enterococcus. Moreover, this system is adapted to the identification of enterococci of clinical origin and their database does not include some species common in foods. However, it could have an application in combination with the classical methods in order to carry out a reasonably rapid and reliable identification of enterococci related to cheese.     

Application of PFGE to source tracking of faecal pollution in coastal recreation area: a case study in Aoshima Beach, Japan

Aims: The development of a microbial source tracking (MST) method is strongly desired to ensure public health and bacteriological safety in coastal recreation areas. We try to specify the source of faecal pollution by applying pulsed‐field gel electrophoresis (PFGE) to the study of the aquatic environment on Aoshima Beach, Japan. Methods and Results: Enterococcus faecium, an enterococcus, was used as a faecal indicator bacterium in this study. Enterococcus faecium strains were isolated and identified from each water sample collected from Aoshima Beach and five rivers (Oyodo, Kiyotake, Kaeda, Chifuku and Tsukunami Rivers) that might be potential sources of faecal pollution. Enterococcus faecium strains collected from water samples were analysed using PFGE. The similarities of all the PFGE types of the Ent. faecium strains were compared using dendrogram analysis. The PFGE types of the strains isolated from Aoshima Beach showed a high similarity to those of the strains isolated from the Oyodo River at a 0·9 similarity level. It was suggested that the Oyodo River is the source of faecal pollution on Aoshima Beach. Conclusions: The PFGE analysis using enterococci is a potential tool for the MST of faecal indicator bacteria that can be applied to the study of the coastal environment. Significance and Impact of the Study: This is one of the studies that PFGE was applied to the coastal environment. The approach using PFGE could estimate the river that is source of faecal pollution in Aoshima Beach. By applying PFGE as a tool of MST method, detailed information of faecal pollution in coastal area can be provided.     

Differential diagnosis of enterococci]

The most stable differential signs of enterococci are: growth in the medium at pH 10.2, growth in broth containing 40% bile, citrate utilization, resistance to 0,05% potassium tellurite, 2, 3, 5-triphenyltetrazolium chloride (TTC) reduction, the staining of colonies (plaques) on a medium with manganese, iron and zinc salts, glycerine fermentation under anaerobic conditions, mannite fermentation, the presence of hemolysin, of the proteolytic enzyme, and mobility. Combined differential-diagnostic nutrient medium permits to determine simultaneously 5 enterococci signs--resistance to nalidixic acid and to crystal violet, TTC reduction, hemolytic and proteolytic activity. The suggested scheme of enterococci laboratory diagnosis including a set of hard nutrient media poured into multisection Petri dish is simple reliable and accessible for any bacteriological laboratory.     

Use of a methylene blue azide medium for isolation of enterococci

A methylene blue azide medium (MBA), developed by Schaedler, Dubos, and Costello to isolate enterococci from the gastrointestinal tract of animals, was evaluated. This was done by comparing the isolation of enterococci from feces and saliva on the medium. Fifty-two catalase-negative, gram-positive cocci from human feces isolated from MBA were classified as enterococci. All strains grew in S F, 6.5% NaCl, and streptomycin broths, and all fermented mannitol. The isolates were provisionally subdivided into Streptococcus faecalis and S. faecium groups. S. faecalis-like strains fermented glycerol and pyruvate aerobically and produced acid in Snyder's medium (initial pH, 4.8). The S. faecium group fermented raffinose. Among all strains, several tests were variable. These included growth at 45 C, in 0.1% tellurite and in methylene blue milk. Three methods were employed to isolate and identify enterococci from the oral cavity. Direct streaking of MBA with saliva failed to produce any growth on the medium. Two other methods, with the use of various selective broths to promote the recovery of oral enterococci, failed to produce any bacteria capable of growing on MBA. The MBA-isolated fecal strains and oral viridans streptococci were generally indistinguishable on Mitis-Salivarius and K F agars. In experiments with fecal material, no gram-negative bacilli were found among the isolates selected. The MBA medium was judged as a high selectivity-low yield medium, and may provide a means of separating fecal and nonfecal enterococci.     

Prevalence of bacteriocin activities and bacteriocin-encoding genes in enterococcal clinical isolates in Iran

The aim of the project was to isolate and characterize bacteriocin-producing enterococci, as well as determine the prevalence of enterocin structural genes in 187 enterococcal clinical isolates from the northwest of Iran. The isolates were screened for antibacterial activity against 15 different indicator strains. The proteinaceous nature of the antimicrobial substances was confirmed by sensitivity to proteinase K; their stability to heat treatment was tested at 60 °C and 100 °C for 20 and 10 min, respectively. The PCR method was applied to detect previously identified enterocin genes. Our results showed that 38 (20.3%) of the enterococcal isolates were considered to be potential bacteriocinogenic strains. Furthermore, genes encoding diverse bacteriocin are highly distributed among clinical enterococci, and the strains with multi-bacteriocin genes displayed high antimicrobial activity. Enterocin A, enterolysin A, and enterocin L50A/B were the most abundant structural genes detected in bacteriocinogenic strains. This work is the first survey on the prevalence of bacteriocin genes among clinical enterococci in Iran that has isolated a strain with high antimicrobial activity and sensitivity to clinically relevant antibiotics.     

Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method.     

The relationships between salmonellas and faecal indicator concentrations in two pools in the Australian wet/dry tropics.

The relationships between total coliform, faecal coliform, enterococci and salmonella concentrations were investigated at Berry and Howard Pools in the Australian wet/dry tropics. Both pools have catchments with minimal human activity and no major point source of faecal pollution. Forty-five indicator and salmonella enumerations were made from each pool over a 1 year period. Salmonellas were isolated from 69% and 96% of samples collected from Howard and Berry Pools respectively, the maximum (MPN) concentration was 110/100 ml. Native fauna were the primary salmonella source. Spearman rank correlations between indicator organisms and salmonella at Howard Pool were significant at the 5% level and approximated 0.6. At Berry Pool, total coliform and enterococci Spearman rank correlations with salmonella were also statistically significant, approximating 0.3; faecal coliforms and salmonella rankings, however, were unrelated. The higher correlation coefficients at Howard Pool were attributed to its small catchment (4 km2) and the more recent nature of faecal contamination compared with Berry Pool which has a catchment of 130 km2. The results highlight the spatial variability of the indicator/pathogen numerical relationship. Total coliform and enterococci counts, as indicators of faecal pollution, were similar and more consistent than faecal coliforms.     

Antibacterial activity of enterococci strains against Vibrio cholerae

Thirty-seven strains of enterococci isolated from milk and milk products from Santa Fe (Argentina) region were tested for antagonistic activity against Vibrio cholerae 01 and non-01. Seven of 17 strains of Enterococcus faecalis , five of 10 strains of Enterococcus faecium and four of 10 strains of Enterococcus durans produced inhibition zones against the indicator species. The activity of the antibacterial compounds was completely destroyed by treatment with trypsin and pronase E in most cases (only the supernatant fluids of a few strains remained weakly active after the treatment), but was resistant to heat treatment at 100°C during 10 and 30 min. When the 10-fold concentrated supernatant fluids were added to a fresh culture of sensitive cells it produced a rapid inactivation. According to these preliminary tests, different strains of enterococci produced compounds with slightly different antivibrio properties, and these compounds were heat-resistant and had a predominantly proteinaceous nature.     

The interchangeability of siderophores in the Enterococcus genus

The functional interchangeability of siderophores was tested among 62 strais belonging to 12 species of genus Enterococcus. Most investigated strains were from E. faecalis and E. faecium species. The majority of examined enterococcal strains appeared highly resistant to EDDA (ethylene-di-amine-di-ortho-hydroxyphenylacetic acid), therefore the group of sensitive strains involved only 11 used as indicator strains. The determination of interchangeability of siderophores within enterococcal strains was performed using EDDA-agar media into which the indicator strains were included. Test colonies (donor strains) were applied to the surface of the media to determine whether the indicator organisms could obtain the required iron for growth by utilizing chelators from the test colony. Only two strains: E. solitarius DSM 5634 and E. pseudoavium DSM 5632 did not demonstrate the ability to utilize siderophores synthesized by all investigated strains. The other tested indicator strains appeared to be recipients of siderophores from 20-52 donor enterococcal strains. The ability to exchange siderophores in enterococci was found as the feature characterizing individual strains.     

Volatilization and Precipitation of Tellurium by Aerobic, Tellurite-Resistant Marine Microbes

Microbial resistance to tellurite, an oxyanion of tellurium, is widespread in the biosphere, but the geochemical significance of this trait is poorly understood. As some tellurite resistance markers appear to mediate the formation of volatile tellurides, the potential contribution of tellurite-resistant microbial strains to trace element volatilization in salt marsh sediments was evaluated. Microbial strains were isolated aerobically on the basis of tellurite resistance and subsequently examined for their capacity to volatilize tellurium in pure cultures. The tellurite-resistant strains recovered were either yeasts related to marine isolates of Rhodotorula spp. or gram-positive bacteria related to marine strains within the family Bacillaceae based on rRNA gene sequence comparisons. Most strains produced volatile tellurides, primarily dimethyltelluride, though there was a wide range of the types and amounts of species produced. For example, the Rhodotorula spp. produced the greatest quantities and highest diversity of volatile tellurium compounds. All strains also produced methylated sulfur compounds, primarily dimethyldisulfide. Intracellular tellurium precipitates were a major product of tellurite metabolism in all strains tested, with nearly complete recovery of the tellurite initially provided to cultures as a precipitate. Different strains appeared to produce different shapes and sizes of tellurium containing nanostructures. These studies suggest that aerobic marine yeast and Bacillus spp. may play a greater role in trace element biogeochemistry than has been previously assumed, though additional work is needed to further define and quantify their specific contributions.     

The relationships between salmonellas and faecal indicator concentrations in two pools in the Australian wet/dry tropics

S.A. TOWNSEND. 1992. The relationships between total coliform, faecal coliform, enterococci and salmonella concentrations were investigated at Berry and Howard Pools in the Australian wet/dry tropics. Both pools have catchments with minimal human activity and no major point source of faecal pollution. Forty-five indicator and salmonella enumerations were made from each pool over a 1 year period. Salmonellas were isolated from 69% and 96% of samples collected from Howard and Berry Pools respectively, the maximum (MPN) concentration was 110/100 ml. Native fauna were the primary salmonella source. Spearman rank correlations between indicator organisms and salmonella at Howard Pool were significant at the 5% level and approximated 0.6. At Berry Pool, total coliform and enterococci Spearman rank correlations with salmonella were also statistically significant, approximating 0.3; faecal coliforms and salmonella rankings, however, were unrelated. The higher correlation coefficients at Howard Pool were attributed to its small catchment (4 km²) and the more recent nature of faecal contamination compared with Berry Pool which has a catchment of 130 km². The results highlight the spatial variability of the indicator/pathogen numerical relationship. Total coliform and enterococci counts, as indicators of faecal pollution, were similar and more consistent than faecal coliforms.     

Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals

The production of antimicrobial activities as well as the presence of bacteriocin structural genes (entA, entB, entP, entQ, cylL, entAS-48, bac31, and entL50A/B) were studied in 140 non-selected faecal enterococcal isolates recovered from wild animals. Eight different indicator strains (including Listeria monocytogenes, Pediococcus pentosaceus, and different enterococcal species) were used for antimicrobial activity detection. Twenty-five of the 140 enterococci (18%) showed antimicrobial activity against L. monocytogenes and 33 additional isolates (24%) showed antimicrobial activity against other indicator strains, but Listeria. At least one bacteriocin structural gene was detected in 17 of the 25 enterococci with antimicrobial activity against L. monocytogenes and different combinations of entA, entB, entP, entQ, entL50A/B, and cylL genes were detected; entA and entB were the most prevalent detected genes, and they were generally associated. Bacteriocin structural genes were detected in 10 of 33 isolates with antimicrobial activity against indicator strains other than Listeria, and the cylL gene was the most prevalent one, especially in E. faecalis isolates.     

Genetic Diversity of Escherichia coli Isolated from Urban Rivers and Beach Water

Repetitive element anchored PCR was used to evaluate the genetic profiles of Escherichia coli isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of E. coli obtained from host sources. Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources. Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively. In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types. River water, which is expected to contain E. coli strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days. However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone. Beach sites with known gull fecal contamination contained E. coli most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains. These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location. Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters. Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology.     

Genetic diversity of Escherichia coli isolated from urban rivers and beach water

Repetitive element anchored PCR was used to evaluate the genetic profiles of Escherichia coli isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of E. coli obtained from host sources. Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources. Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively. In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types. River water, which is expected to contain E. coli strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days. However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone. Beach sites with known gull fecal contamination contained E. coli most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains. These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location. Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters. Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology.     

Comparison of Tellurite Resistance and Tetracycline Resistance among the Enterococci

A correlation was found to exist between tellurite and tetracycline resistance in the enterococci.     

Bacteriocin-like activity production and resistance in selected enterococci and streptococci of animal origin

Antimicrobial proteins and peptides produced by bacteria, termed bacteriocins, are widely acknowledged to be important contributors to their producer organism survival. Enterocin A, enterocin B, enterocin P and enterolysin A belong to the best studied enterocins, i.e., bacteriocins produced by enterococci and streptococci. Twenty-one enterococcal and seven streptococcal isolates were analysed for bacteriocin-like activity production and resistance by overlay test. Up to 50% of tested strains showed antibacterial activity at least against one indicator strain. The occurrence of enterocin B structural gene in several isolates was confirmed by PCR method. The results of this study should broaden knowledge of bacteriocin-like activity production and resistance among gram-positive bacteria.     

Bacteriocin-like activity production and resistance in selected enterococci and streptococci of animal origin

Antimicrobial proteins and peptides produced by bacteria, termed bacteriocins, are widely acknowledged to be important contributors to their producer organism survival. Enterocin A, enterocin B, enterocin P and enterolysin A belong to the best studied enterocins, i.e., bacteriocins produced by enterococci and streptococci. Twenty-one enterococcal and seven streptococcal isolates were analysed for bacteriocin-like activity production and resistance by overlay test. Up to 50% of tested strains showed antibacterial activity at least against one indicator strain. The occurrence of enterocin B structural gene in several isolates was confirmed by PCR method. The results of this study should broaden knowledge of bacteriocin-like activity production and resistance among gram-positive bacteria.