Enumeration and speciation of group D streptococci from above and below a sewer outfall,their susceptibilities to six antibiotics and a comparison with clinical isolates

Isolates of group D streptococci from above and below a sewer outfall and a series of clinical isolates have been speciated to sub-species level. From below the sewer outfall, Streptococcus faecalis var. faecalis predominates whereas above the outfall, S. faecium var. casseliflavus predominates. S. faecalis var. faecalis, S. faecalis var. liquefaciens and S. faecalis var. zymogenes were the predominant sub-species in the clinical isolates where S. faecium var. casseliflavus was virtually absent. S. faecalis var. liquefaciens and S. faecalis var. zymogenes were uncommon in the environmental isolates. S. faecium and S. durans were more abundant in the environmental than in the clinical isolates. The use of group D streptococci as indicators of faecal pollution would seem more suited to higher, rather than lower, levels of pollution. A statistically significant increase in resistance to antibiotics (ampicillin, penicillin, streptomycin, gentamicin, erythromycin and tetracycline) was seen in isolates from below the outfall compared with those from above and a further significant increase was seen in the clinical isolates compared with the former. Resistance to tetracycline was most common and ampicillin was the only antibiotic tested to which no resistance was detected. Multiple antibiotic resistance was rare in the environmental isolates. Even in moderately polluted water, there is not a large pool of antibiotic resistance.     

Recognition of Group D Streptococcal Species of Human Origin by Biochemical and Physiological Tests

The speciation of 262 strains of group D streptococci isolated from human sources is described. One hundred forty-two isolates from blood cultures were included; 96 of these were submitted as isolates from clinical cases of subacute bacterial endocarditis. The results show that 98 Streptococcus faecalis, 29 S. faecalis var. zymogenes, 44 S. faecalis var. liquefaciens, 27 S. faecium, 13 S. durans, 44 S. bovis, and 7 unspeciated S. bovis-like group D isolates were identified. No S. faecium var. casseliflavus, S. equinus, or S. avium (group Q streptococci) were identified among the human isolates. The speciation procedures and techniques are detailed. The procedures and limitations of the tests used are discussed. Ninety-eight percent of the 262 strains were speciated by a spectrum of tests that allowed us to recognize atypical as well as typical strains within species.     

Numerical taxonomy of Streptococcus

A numerical taxonomic study of strains of Streptococcus, together with representatives of allied genera, showed 28 reasonably distinct phenons. The major areas, with their phenons, were: (a) enterococcal species group (S. faecalis, S. faecium, 'S. avium' and a proposed new species 'S. gallinarum'); (b) paraviridans species group (S. bovis, S. equinus, S. salivarius, 'S. casseliflavus', S. mutans, S. raffinolactis and an unidentified Oral Group I); (c) lactic species group (S. lactis including S. cremoris); (d) thermophilic species group (S. thermophilus); (e) viridans species group (S. mitis, S. sanguis, a proposed new species 'S. oralis' and 'S. milleri'); (f) pyogenic species group (S. agalactiae, S. pyogenes, S. equi, 'S. equisimilis' including 'S. zooepidemicus, and a cluster of Lancefield Group B strains of human origin); (g) parapyogenic species group (S. uberis, 'S. dysgalactiae', and a cluster of strains of Lancefield Groups R, S and T). Species of Aerococcus, Gemella, Leuconostoc and Pediococcus are very closely related to the streptococci.     

The distribution of isoprenoid quinones in streptococci of serological groups D and N.

The isoprenoid quinone contents of streptococci of serological groups D and N were investigated. Streptococcus faecalis, S. faecalis subsp. liquefaciens and S. faecalis subsp. zymogenes strains contained demethylmenaquinones with nine isoprene units as their major isoprenologues. Menaquinones with eight isoprene units predominated in S. faecium subsp. casseliflavus and S. faecium subsp. mobilis whereas menaquinones with nine isoprene units constituted the major components in strains of S. cremoris, S. cremoris subsp. alactosus, S. lactis and S. lactis subsp. diacetylactis. Strains of S. avium, S. bovis, S. durans, S. equinus, S. faecium, S. raffinolactis and S. suis contained neither menaquinones nor ubiquinones. The isoprenoid quinone data correlate well with other kinds of data on these organisms and are of value in the classification of these bacteria.     

Incidence and relationship of group D streptococci with other indicator organisms in meats.

Raw and processed meats were analyzed for presumptive group D streptococci using KF streptococcus agar. Counts were compared with coliform, presumptive Escherichia coli, and Enterobacteriaceae counts but no meaningful relationships were observed. Results indicated that group D streptococci and E. coli type I were principally contaminants from the packing plant, rather than at retail level. The predominating group D streptococcus in both beef and pork cuts was Streptococcus faecalis, while in processed meat (bologna), the predominating group D streptococci were Streptococcus faecium var. durans and Streptococcus faecium. Streptococcus bovis was not detected among the isolates from any meat samples. Marked differences were noted in numbers of group D streptococci in processed meat from different manufacturers. The results did not support the use of group D streptococci as alternative indicator organisms for meats. However, the association of group D streptococci with packing plant contamination may prove to be of value.     

Studies on the mechanism of intrinsic resistance to beta-lactam antibiotics in group D streptococci

Six penicillin-binding proteins (PBPs) were detected in clinical isolates of each one of three group D streptococci: Streptococcus bovis, S. faecalis and S. faecium. When examined in whole organisms, the PBPs of S. faecium, the most penicillin-resistant species of group D streptococci, generally had lower affinities for the antibiotic than those of S. faecalis (intermediate penicillin resistance), which in turn were of lower affinity than those of S. bovis (penicillin-sensitive). On the other hand, no quantitative correlation could be established between the binding of penicillin to any one PBP or group of PBPs, and the penicillin MIC value for the corresponding micro-organism. Examination of the amounts of antibiotic bound and the rates of binding to PBPs of equal numbers of protoplasts and whole bacteria of S. faecalis and S. faecium, indicated that there was no permeability barrier to benzylpenicillin in the cell walls of these species. The lower antibacterial effectiveness of cephalothin compared with ampicillin in group D streptococci was paralleled by the higher concentrations of cephalothin needed in competition assays to inhibit the lower molecular size PBPs of these bacteria.     

Cores, Microbial Organelles Possibly Specific to Group D Streptococci

A long, thin, approximately cylindrical core spans the interior of cells of 24-hr cultures of all group D streptococci that were examined, five strains of Streptococcus faecalis, single strains of S. faecalis subsp. zymogenes and S. durans, and three strains of Streptococcus spp. In one strain of S. faecalis, serial section electron microscopy showed that most cells possess a core. The core is 0.10 to 0.16 mum thick and consists of a matrix and an axial array of ribosomelike particles. It resembles one of two types of cores present in a stable protoplast form of one of the S. faecalis strains. Cores were not present in single strains of S. pyogenes (beta-hemolytic group A), S. agalactiae (group B), S. dysgalactiae (group C), S. equisimilis (group C), and S. mitis (viridans group) that were examined; nor were cores observed in single strains of Staphylococcus aureus, Escherichia coli, and Bacillus megaterium. Cores may be useful, therefore, in identification of group D streptococci. For preservation and rapid recognition of cores, a glutaraldehyde-osmium tetroxide sequence of fixation appears superior to the osmium tetroxide method often employed in processing bacteria for electron microscopy.     

Differentiation of Some Enterococci by Gas Chromatography

Relative fatty acid compositions of 37 enterococci were examined by gas chromatography. Streptococcus faecalis, S. faecium, and S. faecium var. durans yielded similar fatty acid patterns. Strains of S. faecium var. casseliflavus, and a motile yellow-pigmented streptococcus, contained very low levels of C(19:0) cyclopropane fatty acid and four unidentified components, compared to the other strains of enterococci examined. There were no significant differences in the fatty acid patterns of enterococci grouped according to plant, animal, or human source.     

Enterococci in Insects

Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10(3) to 3 x 10(7)/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution.     

Peptidoglycan composition and taxonomy of group D, E, and H streptococci, and Streptococcus mutans

The qualitative and quantitative composition of the peptidoglycan from the cell wall of groups D, E, and H streptococci, and Streptococcus mutans, was determined. In group D, S. faecalis and the closely related species S. liquefaciens and S. zymogenes were separated from S. faecium and the closely related species S. durans on the basis of their peptidoglycan composition. A relationship among S. bovis, S. equinus, and some strains of S. mutans was indicated by the presence in each of a similar peptidoglycan containing threonine. Threonine was released from the S. mutans polymer as a threonine-lysine dipeptide. Hydrolysis of the dipeptide at 100 C for 24 hr in 6 n HC1 was required to break the peptide bond. Motile group D streptococci possessed a peptidoglycan of the same composition as S. faecium. Group E and H strains were also similar in the composition of their peptidoglycan. The results demonstrate that peptidoglycan composition can be used to (i) aid in the division of members of an immunological group into subgroups, and (ii) indicate a relationship between members of the same genus which are not related on an immunological basis.     

Studies on faecal streptococci in the river Tigris

Six hundred water samples collected from the river Tigris at Mosul City were investigated for faecal streptococci. Human faecal streptococci were predominant, and animal faecal streptococci were also detected. Eight species and varieties were identified, viz Streptococcus faecalis, atypical Streptococcus faecalis, Streptococcus bovis, Streptococcus equinus, Streptococcus faecalis var. liquefaciens, Streptococcus faecalis var. zymogenes, Streptococcus durans and Streptococcus faecium. The incidence of these species and varieties were 43.32%, 13.18%, 11.47%, 11.30%, 9.76%, 5.30%, 3.76% and 1.88%, respectively.     

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans , Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.     

Characterization of 'faecal streptococci' as indicators of faecal pollution and distribution in the environment

The recent revision of the taxonomy of 'faecal streptococci' prompted us to verify the importance of identifying the species of this group of cocci. During a study carried out to assess the hygienic quality of environmental samples from a variety of sources, we isolated 198 strains named faecal streptococci on the basis of conventional international tests (EVA broth multiple tube test) used for Public Health purposes. The predominant species were Enterococcus faecalis (39%) and Ent. faecium (29%), followed by Ent. durans/hirae, Ent. casseliflavus/gallinarum, Ent. raffinosus, with a different prevalence of the species depending on the source. Eighty-four per cent of isolates were true faecal species. Only one isolate was identified as belonging to the Streptococcus genus. The authors stress the opportunity to identify the species. This may help to clarify the ecological and epidemiological characteristics of intestinal enterococci and streptococci in the environment, in drinking and recreational waters and their meaning as indicators of faecal pollution. All isolates were tested for their susceptibility to some antimicrobial agents widely used in medical therapy and the pattern was compared with the pattern of isolates from clinical specimens.     

Differentiation of enterococci from other group D streptococci by means of a specific monoclonal antibody

We report on the specificity of a monoclonal antibody which reacts with autoclaved extracts of four species of enterococci but does not react to the same extent with similar extracts from two non-enterococcal group D streptococci. The monoclonal antibody also reacts specifically with purified lipoteichoic acid from Streptococcus faecalis but not significantly with purified lipoteichoic acid from the non-enterococcal species Streptococcus bovis and Streptococcus equinus. The specific antigen detected with this antibody could correlate with the definition of the enterococcus subgroup of the streptococci which would provide further evidence that this sub-group is taxonomically distinct from the other group D streptococci.     

Virulence factors and<Emphasis Type="Italic">in Vitro</Emphasis> adherence of<Emphasis Type="Italic">Enterococcus</Emphasis> strains to urinary catheters

The ability to adhere in vitro to urinary catheters and the presence of enterococcal virulence factors was determined in 30 Enterococcus urinary isolates (12 E. faecalis, 12 E. faecium, 3 E. casseliflavus, 3 E. gallinarum). Silicone, siliconized latex and polyvinyl chloride (PVC) were examined by sonication quantitative culture technique and scanning electron microscope. As compared to E. faecalis and E. faecium, E. casseliflavus and E. gallinarum displayed lower adhesion to all synthetic materials. All the tests performed showed higher adherence of all tested strains to siliconized latex and silicone than to PVC. Biofilmforming ability was observed in 5 E. faecalis but in none of the remaining strains. The gene coding enterococcal surface protein (Esp) was detected in 7 E. faecalis and 6 E. faecium strains. Gelatinase was found in 1 E. faecalis, 2 E. faecium and hemolysins were found in 6 E. faecalis and 1 E. faecium strains. All E. casseliflavus and E. gallinarum strainswere negative for these traits. Hydrophobic type of cell surface (measured by its affinity for n-hexadecane) was shown in a few isolates. Bacterial adherence was not significantly associated with the above pathogenic factors.     

The phylogenetic position of Streptococcus and Enterococcus

Streptococcus pyogenes, S. equinus, S. bovis, S. salivarius, S. sanguis, S. mutans, S. rattus, S. cricetus, S. lactis, S. raffinolactis and Enterococcus faecalis have been characterized by oligonucleotide cataloguing of their 16S ribosomal RNA. All the organisms form a loose but coherent group that is phylogenetically equivalent to those of lactobacilli, bacilli, the Brochothrix and Listeria group, and related taxa that constitute one of several sublines within the 'Clostridium' branch of Gram-positive eubacteria. Within the Steptococcus-Enterococcus group, organisms fall into three moderately related clusters defined by Enterococcus, the lactic acid streptococci and streptococci of the pyogenic and oral groups, respectively.