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16S ribosomal DNA sequence analysis distinguishes biotypes of Streptococcus bovis: Streptococcus bovis Biotype II/2 is a separate genospecies and the predominant clinical isolate in adult males.

We characterized 22 human clinical strains of Streptococcus bovis by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the S. bovis type strain ATCC 33317 (cluster 1); other closely related type strains were S. equinus and S. infantarius. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the S. bovis type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from S. gallolyticus. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar to S. bovis biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b as S. bovis biotype II/2, those of cluster 1 as S. bovis biotype II/1, and that of cluster 2a as S. bovis biotype I. There was an excellent correlation of biotype and genotype: S. bovis biotype II/2 isolates form a separate genospecies distinct from the S. bovis, S. gallolyticus, and S. infantarius type strains and are the most common isolates in adult males.

Adult↗

Compositions and characteristics of strains of Streptococcus bovis.

Streptococcus bovis strains JB1, 26, 581AXY2, 21096C, and 45S1 grew on glucose, maltose, starch, sucrose, cellobiose, and lactose. None of these strains grew on xylose or ribose, but arabinose was a suitable energy source for strains 2109C and K27FF4. All strains grew at 45 degrees C, but incubation at 50 degrees C prevented growth. Growth was permitted in 2% sodium chloride, but 6.5% sodium chloride was inhibitory. Doubling times ranged from 24 to 27 min, and final pH on glucose was approximately 4.6. None of the strains had a requirement for amino acids, and growth was rapid in media containing glucose salts and B vitamins. There was no ammonia production from arginine. All strains showed aminoendopeptidase activity, but there was considerable strain variation. Strain 7H4, reported as Streptococcus bovis, was noticeably different from the other six strains. It had a doubling time that was more than four times as long, and it grew poorly on starch or in the absence of an amino acid source. Six-and-a-half percent sodium chloride was not inhibitory, and it produced ammonia from arginine. Cell morphology was coccoid rather than ovoid. Based on these criteria, classification of strain 7H4 as Streptococcus bovis seemed doubtful. Other experiments with strain 7H4 indicated that Streptococcus bovis was devoid of diaminopimelic acid. In these experiments strain 7H4 contained significant diaminopimelic acid. The six Streptococcus bovis strains all contained diaminopimelic acid as well, but concentration varied.

Animals↗

Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular alpha-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium.

Amylases↗

Meningitis caused by Streptococcus bovis.

Streptococcus bovis was isolated from the CSF of a 66-year-old man with meningitis. His clinical appearance was unusual in that he lacked typical signs and symptoms of pyogenic meningitis. Streptococcus bovis was also recovered from his blood, which suggested that bacterial endocarditis was the source of his CNS infection. He was cured after four weeks of therapy with intravenous penicillin G potassium. This is the fourth reported case of meningitis caused by S bovis. The previous three patients also had endocarditis caused by S bovis. Because of the reported propensity of S bovis to infect heart valves and the frequent association of S bovis bacteremia with malignant gastrointestinal (GI) tract tumors, recovery of this organism form CSF should prompt a search for bacterial endocarditis and occult GI cancer.

Aged↗

Regulation and cloning of the gene encoding amylase activity of the ruminal bacterium Streptococcus bovis.

Streptococcus bovis is an important starch-degrading ruminal bacterium that has been implicated as being important in the etiology of a number of ruminal pathologies associated with diets high in grains. Previous studies with S. bovis have shown that amylase production was influenced by the growth substrate, but the nature of this regulation was not determined. The current study was conducted to better describe the regulatory phenomena and gain a better understanding of the molecular characteristics of this activity. Nutritional experiments demonstrated that the presence of starch or the starch-derived disaccharide maltose was required for maximum amylase production. Subsequent time-course experiments showed that amylase synthesis was induced by maltose and repressed by glucose, cellobiose, and fructose, while inulin and lactose had little effect on enzyme accumulation. The effects of the added antibiotics rifampin and tetracycline were consistent with transcriptional control of amylase synthesis. Analysis of S. bovis cells grown on glucose or maltose showed that they contained similar low levels of cyclic AMP, indicating that it was unlikely that regulation of amylase synthesis was mediated through a mechanism involving this nucleotide. The amylase gene from S. bovis JB1 was cloned and expressed in Escherichia coli. The amylase produced in E. coli was of lower molecular weight than that synthesized by S. bovis and had catalytic characteristics different from those of S. bovis amylase. When the gene was introduced back into S. bovis JB1, only one form of amylase activity was detected, indicating that the entire gene was present on this insert. The use of the amylase gene as a genetic probe for identification of S. bovis strains is discussed.

Amylases↗

Emergence of vancomycin resistance in the genus Streptococcus: characterization of a vanB transferable determinant in Streptococcus bovis.

Streptococcus bovis NEM760 was isolated from a stool swab collected on admission from a patient as surveillance for vancomycin-resistant enterococci. Strain NEM760 was identified as S. bovis by conventional biochemical methods and partial sequence analysis of its 16S rRNA. This strain was resistant to a low level of vancomycin (MIC, 64 micrograms/ml) but was susceptible to teicoplanin (MIC, 1 micrograms/ml), and vancomycin induced resistance to both glycopeptides. The presence of a vanB-related gene in NEM760 was demonstrated in a PCR assay which enabled specific amplification of a 635-hp internal segment of vanB. Sequence analysis of the corresponding PCR product revealed that it was highly homologous (96% identity) to the prototype vanB sequence of Enterococcus faecalis V583. The VanB resistance of determinant of S. bovis NEM760 was transferred by conjugation to E. faecalis and Enterococcus faecium at a similar frequency of 2 x 10(-5) per donor. SmaI-digested genomic DNAs of independently obtained transconjugants of E. faecalis and E. faecium were analyzed by pulsed-field gel electrophoresis and Southern hybridization with a vanB DNA probe. The electrophoretic and hybridization patterns obtained with all transconjugants of the same species were indistinguishable and revealed vanB-containing chromosomal insertions of approximately 100 kb. These results suggest that the genes mediating VanB-type resistance in S. bovis NEM760 are part of large transferable genetic elements. The results presented in the report demonstrate for the first time the role of streptococci in the dissemination of vancomycin resistance among gram-positive bacteria.

Anti-Bacterial Agents↗

Regulation of two aspartokinase isozymes in Streptococcus bovis.

Streptococcus bovis has been found to contain two distinct aspartokinases that can be separated by gel filtration chromatography. One of these isozymes elutes on Sephadex G-200 gel filtration at a molecular weight greater than 250,000. The molecular weight of the other isozyme is approximately 125,000. The earlier peak of aspartokinase activity is slightly inhibited by meso-diaminopimelate, while the second peak is sensitive to inhibition by lysine. The latter aspartokinase is not formed when the organism is grown in a medium containing more than 1 mM lysine. The level of lysine-sensitive aspartokinase is decreased during the growth cycle, whereas diaminopimelate-sensitive activity is little affected by the growth conditions. The regulatory properties of the two aspartokinases suggest that they may play different physiological roles.

Amino Acids↗

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis.

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)-1(min)-1. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (Ks) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a Ks of 0.20 mM, but the Ks of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport.

Bacteriological Techniques↗

Frothy feedlot bloat in cattle: production of extracellular polysaccharides and development of viscosity in cultures of Streptococcus bovis.

Streptococcus bovis was cultured in a synthetic medium with three concentrations of sucrose. Initial viscosity of the media was 1.5 centipoise (cp). After incubation for 8 h, the viscosity of the medium with 0.5% sucrose was unchanged, that with 3% sucrose had increased to 8 cp, and that with 6% sucrose to 112 cp. Similar results were found with a rumen fluid medium. A slimy material, responsible for increased viscosity of these cultures, was digested by dextranase. The material appeared as a complex system of intercellular fibers when viewed under the electron microscope after freeze-etching. With proteins and other polymers released from lysed bacteria, this slimy material may contribute directly to increased viscosity and foam formation. In addition to these intercellular fibers, each cell was surrounded by a fibrous capsule that was not digested by dextranase. This capsule stained with lead citrate and uranyl acetate, but not with ruthenium red. The amount of capsular material produced was similar whether the media contained 0.5, 3.0, or 6% sucrose.

Anaerobiosis↗

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius.

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.

Amino Acid Sequence↗

Identification of Streptococcus bovis and Streptococcus salivarius in clinical laboratories.

Streptococci identified as Streptococcus bovis, S. bovis variant, and Streptococcus salivarius were examined with respect to physiological and serological characteristics and cellular fatty acid content. Similarities in physiological reactions and problems encountered in serological analysis were noted, suggesting that an expanded battery of physiological tests is needed to definitively identify these streptococci. Cellular fatty acid analysis provided an accurate method for distinguishing S. salivarius from S. bovis and S. bovis variant.

Antigens, Bacterial↗

Bacteremia with Streptococcus bovis and Streptococcus salivarius: clinical correlates of more accurate identification of isolates.

Two biotypes of Streptococcus bovis can be identified by laboratory testing and can be distinguished from the phenotypically similar organism Streptococcus salivarius. We assessed the clinical relevance of careful identification of these organisms in 68 patients with streptococcal bacteremia caused by these similar species. S. bovis was more likely to be clinically significant when isolated from blood (89%) than was S. salivarius (23%). There was a striking association between S. bovis I bacteremia and underlying endocarditis (94%) compared with that of S. bovis II bacteremia (18%). Bacteremia with S. bovis I was also highly correlated with an underlying colonic neoplasm (71% of patients overall, 100% of those with thorough colonic examinations) compared with bacteremia due to S. bovis II or S. salivarius (17% overall, 25% of patients with thorough colonic examinations). We conclude that careful identification of streptococcal bacteremic isolates as S. bovis biotype I provides clinically important information and should be more widely applied.

Adolescent↗

Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis.

Streptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7% (w/v) tannic acid and 4% (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1% (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.

Carboxy-Lyases↗

Heterologous expression of an endoglucanase gene (endA) from the ruminal anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis.

The heterologous expression of a cloned endoglucanase gene (endA) from the ruminal bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DL1. The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.

Bacterial Proteins↗

Taxonomic dissection of the Streptococcus bovis group by analysis of manganese-dependent superoxide dismutase gene (sodA) sequences: reclassification of 'Streptococcus infantarius subsp. coli' as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis biotype 11.2 as Streptococcus pasteurianus sp. nov.

The taxonomic dissection of the Streptococcus bovis-Streptococcus equinus group was carried out upon obtaining sequences for the manganese-dependent superoxide dismutase gene (sodA) of the type strains of S. bovis, Streptococcus caprinus, S. equinus, Streptococcus gallolyticus, Streptococcus infantarius, Streptococcus macedonicus and Streptococcus waius. The sodA sequences of 29 streptococcal strains of animal and human origin that were related to S. bovis were also sequenced. A phylogenetic analysis of the sodA sequences revealed that the S. bovis-S. equinus group comprises five different clusters that correspond to five distinct species. The type strains of S. bovis and S. equinus were associated in the same cluster, corresponding to the species S. equinus. The type strains of S. caprinus, S. gallolyticus, S. macedonicus and S. waius were associated in the same cluster, which defined a single species containing S. gallolyticus and its junior synonym S. caprinus, and S. macedonicus and its junior synonym S. waius. The two subspecies thought to constitute the species S. infantarius, namely S. infantarius subsp. infantarius and 'S. infantarius subsp. coli', were located in two distinct clusters. One of these clusters defined the species S. infantarius and included the type strain of S. infantarius subsp. infantarius. The other cluster defined 'S. infantarius subsp. coli', leading to the proposal of its reclassification as the novel species Streptococcus lutetiensis (NEM 782T = CIP 106849T). The remaining cluster comprised all of the strains previously identified as belonging to S. bovis biotype 11.2, leading to the proposal to reassign these strains to the novel species Streptococcus pasteurianus (NEM 1202T = CIP 107122T). The results of the phylogenetic analysis were confirmed by DNA-DNA hybridization experiments, thus demonstrating that sequence databases of defined DNA targets, such as sodA, may constitute a valuable alternative approach for modern bacterial systematics.

Animals↗

Factors affecting the antibacterial activity of the ruminal bacterium, Streptococcus bovis HC5.

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h(-1)) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2-1.2 h(-1)) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h(-1)) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h(-1)) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml(-1)) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application.

Animals↗