Lipopolysaccharide Stimulates the Growth of Bacteria That Contribute to Ruminal Acidosis.

Lipopolysaccharide (LPS) has been reported to contribute to a ruminal acidosis of cattle by affecting ruminal bacteria. The goal of this study was to determine how LPS affects the growth of pure cultures of ruminal bacteria, including those that contribute to ruminal acidosis. We found that dosing LPS (200,000 EU) increased the maximum specific growth rates of four ruminal bacterial species (Streptococcus bovis JB1, Succinivibrio dextrinosolvens 24, Lactobacillus ruminis RF1, and Selenomonas ruminantium HD4). Interestingly, all the species ferment sugars and produce lactate, contributing to acidosis. Species that consume lactate or ferment fiber were not affected by LPS. We found that S. bovis JB1 failed to grow in LPS as the carbon source in the media; growth of S. bovis JB1 was increased by LPS when glucose was present. Growth of Megasphaera elsdenii T81, which consumes lactate, was not different between the detoxified (lipid A delipidated) and regular LPS. However, the maximum specific growth rate of S. bovis JB1 was greater in regular LPS than detoxified LPS. Mixed bacteria from a dual-flow continuous culture system were collected to determine changes of metabolic capabilities of bacteria by LPS, and genes associated with LPS biosynthesis were increased by LPS. In summary, LPS was not toxic to bacteria, and lipid A of LPS stimulated the growth of lactate-producing bacteria. Our results indicate that LPS not only is increased during acidosis but also may contribute to ruminal acidosis development by increasing the growth of lactic acid-producing bacteria.IMPORTANCE Gram-negative bacteria contain lipopolysaccharide (LPS) coating their thin peptidoglycan cell wall. The presence of LPS has been suggested to be associated with a metabolic disorder of cattle-ruminal acidosis-through affecting ruminal bacteria. Ruminal acidosis could reduce feed intake and milk production and increase the incidence of diarrhea, milk fat depression, liver abscesses, and laminitis. However, how LPS affects bacteria associated with ruminal acidosis has not been studied. In this study, we investigated how LPS affects the growth of ruminal bacteria by pure cultures, including those that contribute to acidosis, and the functional genes of ruminal bacteria. Thus, this work serves to further our understanding of the roles of LPS in the pathogenesis of ruminal acidosis, as well as providing information that may be useful for the prevention of ruminal acidosis and reducetion of economic losses for farmers.

An Overview on Streptococcus bovis/Streptococcus equinus Complex Isolates: Identification to the Species/Subspecies Level and Antibiotic Resistance.

Streptococcus bovis/Streptococcus equinus complex (SBSEC), a non-enterococcal group D Streptococcus spp. complex, has been described as commensal bacteria in humans and animals, with a fecal carriage rate in humans varying from 5% to over 60%. Among streptococci, SBSEC isolates represent the most antibiotic-resistant species-with variable resistance rates reported for clindamycin, erythromycin, tetracycline, and levofloxacin-and might act as a reservoir of multiple acquired genes. Moreover, reduced susceptibility to penicillin and vancomycin associated with mobile genetic elements have also been detected, although rarely. Since the association of SBSEC bacteremia and colon lesions, infective endocarditis and hepatobiliary diseases has been established, particularly in elderly individuals, an accurate identification of SBSEC isolates to the species and subspecies level, as well as the evaluation of antibiotic resistance, are needed. In this paper, we reviewed the major methods used to identify SBSEC isolates and the antimicrobial resistance rates reported in the scientific literature among SBSEC species.

Presencia de Streptococcus bovis en muestras de orina de sujetos con síntomas de infección del aparato urinario / Presence of Streptococcus bovis in urine samples from patients experiencing symptoms of urinary tract

Dada la importancia de la correcta validación clínica de los aislamientos de Streptococcus bovis, nos planteamos la revisión de su presencia en muestras de orina con el objetivo de conocer su frecuencia relativa y su patrón de sensibilidad antibiótica. Se revisó retrospectivamente la sensibilidad a los antibióticos de 91 aislados de S. bovis recuperados de muestras de orina durante un período de 4 años (2012-2015). La media de la edad de los pacientes fue de 55 años y en su mayoría fueron mujeres (81%). El 37,4% eran pacientes hospitalizados con enfermedades urológicas (61%). La sensibilidad a penicilina, vancomicina y teicoplanina fue del 97,8%. Aunque S. bovis puede ser poco común en los aislamientos de orina, su presencia en sujetos con enfermedades de base justifica la realización de estudios de patogenicidad que demuestren la verdadera capacidad de producir enfermedad de este grupo de bacterias.

Given the relevance of proper clinical validation of Streptococcus bovis, we here consider revising its presence in urine samples in order to determine its relative frequency and the pattern of antibiotic susceptibility. The susceptibility to antibiotics of 91 isolates of S. bovis from urine samples was retrospectively reviewed over a period of 4 years (2012-2015). The mean age of patients was 55 years, 81% of whom were women and 37.4% were hospitalized patients suffering from urological diseases (61%). Susceptibility to penicillin, vancomycin and teicoplanin was 97.8%. Due to the fact that S. bovis can be infrequent in urine isolates and given its presence in patients suffering from urological diseases, further pathogenic studies, showing the true ability of this group of bacteria to produce disease, are required.

[Presence of Streptococcus bovis in urine samples from patients experiencing symptoms of urinary tract]. / Presencia de Streptococcus bovis en muestras de orina de sujetos con síntomas de infección del aparato urinario.

Given the relevance of proper clinical validation of Streptococcus bovis, we here consider revising its presence in urine samples in order to determine its relative frequency and the pattern of antibiotic susceptibility. The susceptibility to antibiotics of 91 isolates of S. bovis from urine samples was retrospectively reviewed over a period of 4 years (2012-2015). The mean age of patients was 55 years, 81% of whom were women and 37.4% were hospitalized patients suffering from urological diseases (61%). Susceptibility to penicillin, vancomycin and teicoplanin was 97.8%. Due to the fact that S. bovis can be infrequent in urine isolates and given its presence in patients suffering from urological diseases, further pathogenic studies, showing the true ability of this group of bacteria to produce disease, are required.

Clinical interest of Streptococcus bovis isolates in urine.

OBJECTIVE: Streptococcus bovis includes variants related to colorectal cancer and non-urinary infections. Its role as urinary pathogen is unknown. Our objective was to assess the presence of urinary infection by S. bovis, analysing the patients and subsequent clinical course. METHODS: Observational study, with longitudinal data collection, performed at our centre between all the cultures requested between February and April 2015. Clinical course of the patients and response to treatment were analysed. RESULTS: Two thousand five hundred and twenty urine cultures were analysed, of which 831 (33%) had a significant microbial count. S. bovis was isolated in 8 patients (0.96%). In 75% of these cases the urine culture was requested because of urinary tract infection symptoms; the remaining 25% because of fever of uncertain source; during the follow-up period no evidence of cancer or endocarditis was detected. S. gallolyticus subspecie pasteurianus was the only variant observed (100%). The clinical response to initial treatment was favourable in all cases. CONCLUSIONS: S. bovis bacteriuria may have clinical significance, especially when S. gallolyticus subspecies pasteurianus is isolated in cases with underlying urinary tract disease.

Is Streptococcus bovis a urinary pathogen?

The Streptococcus bovis group (SBG) comprises several microorganisms associated with human infections. They have been associated with bacteremia, endocarditis, biliary tract infection, meningitis, and colorectal cancer, but their role as urinary pathogens is not well known. The objective of this investigation was to discover the incidence and clinical significance of the bacteriuria associated with this complex. A retrospective analysis of all adult patients with bacteriuria caused by SBG during the period 1995-2012 was carried out. During the study period, SBG was isolated in 153 adult patients, who had a mean age of 67 years, most of them being women (80%). Most of our patients (65%) had some underlying disease, with urologic disease being the most common (37%), followed by diabetes mellitus (27%) and neurologic disease (25%). Among the 88 patients in whom we were able to correctly assess symptoms, 45% had asymptomatic bacteriuria, 35% had lower urinary tract infection, and 20% had upper urinary tract infection. In 14 cases (9%), SBG was also isolated in blood cultures. Most of the isolates of SBG (72%) were S. gallolyticus subsp. pasteurianus. All isolates were susceptible to penicillin, 98% to nitrofurantoin, and 77% to fosfomycin. Although SBG bacteriuria is uncommon, it should not always be taken as a contaminant, mainly when S. pasteurianus is isolated, because it may cause urinary tract infections and, occasionally, sepsis, whereas when S. gallolyticus is isolated from urine, it may be a marker of underlying endocarditis and colorectal cancer.

Inhibition of fructan-fermenting equine faecal bacteria and Streptococcus bovis by hops (Humulus lupulus L.) ß-acid.

AIMS: The goals of this study were to determine if ß-acid from hops (Humulus lupulus L.) could be used to control fructan fermentation by equine hindgut micro-organisms, and to verify the antimicrobial mode of action on Streptococcus bovis, which has been implicated in fructan fermentation, hindgut acidosis and pasture-associated laminitis (PAL) in the horse. METHODS AND RESULTS: Suspensions of uncultivated equine faecal micro-organisms produced fermentation acids when inulin (model fructan) was the substrate, but ß-acid (i.e. lupulone) concentrations ≥9 ppm inhibited lactate production and mitigated the decrease in pH. Inulin-fermenting Strep. bovis was isolated from the ß-acid-free suspensions after enrichment with inulin. The isolates were sensitive to ß-acid, which decreased the viable number of streptococci in faecal suspensions, as well as growth, lactate production and the intracellular potassium of Strep. bovis in pure culture. CONCLUSIONS: These results are consistent with the hypothesis that hops ß-acid prevented the growth of fructan-fermenting equine faecal bacteria, and that the mechanism of action was dissipation of the intracellular potassium of Strep. bovis. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial hindgut fermentation of grass fructans has been linked to PAL and other metabolic disorders in horses. Hops ß-acid is a potential phytochemical intervention to decrease the growth of bacteria responsible for PAL.

Daptomycin activity against uncommonly isolated streptococcal and other gram-positive species groups.

A total of 1,356 clinical isolates were tested against daptomycin by broth microdilution methods. Daptomycin was active against seven groups of viridans group streptococci (MIC50 and MIC90 values ranging from ≤0.06 and ≤0.06 µg/ml [Streptococcus bovis and Streptococcus dysgalactiae] to 0.5 and 1 µg/ml [Streptococcus mitis, Streptococcus oralis, and Streptococcus parasanguinis], respectively), beta-hemolytic streptococci serogroups C, F, and G (MIC50 and MIC90, ≤0.06 to 0.25 and 0.12 to 0.25 µg/ml, respectively), Corynebacterium spp. (MIC50 and MIC90, ≤0.06 and 0.12 µg/ml, respectively), and Micrococcus spp. (MIC50 and MIC90, ≤0.06 and 0.25 µg/ml, respectively). Listeria monocytogenes exhibited higher daptomycin MICs (MIC50 and MIC90, 2 and 4 µg/ml, respectively) than other tested organisms.

[Streptococcus bovis, taxonomic status, clinical relevance and antimicrobial susceptibility]. / Streptococcus bovis, situación taxonómica, relevancia clínica y sensibilidad antimicrobiana.

Streptococcus bovis is a large bacterial complex of facultative anaerobic Gram-positive cocci that includes distinct, genetically-related species. Traditionally, S. bovis was classified into three biotypes: I (mannitol fermentation-positive), II/1 (mannitol-negative and ß-glucuronidase-negative), and II/2 (mannitol-negative and ß-glucuronidase-positive). The introduction of molecular techniques in the last few decades has led to proposals for a genetic classification of this complex: S. bovis biotype I belongs to Streptococcus gallolyticus subsp. gallolyticus, S. bovis biotype II/1 is, in fact, Streptococcus infantarius subsp. infantarius, designated as Streptococcus lutetiensis, and S. bovis biotype II/2 is Streptococcus gallolyticus subsp. pasteurianus, commonly designated as Streptococcus pasteurianus. Although this modern taxonomy is currently accepted, many clinicians remain unfamiliar with these terms. The importance of correct identification lies in the strong association between bacteriemia, endocarditis and/or colon cancer and the various subspecies. In general, S. bovis is more susceptible to antimicrobial agents than other streptococci, but high levels of resistance to macrolides and tetracycline have been described.

Early in vitro and in vivo development of high-level daptomycin resistance is common in mitis group Streptococci after exposure to daptomycin.

The development of high-level daptomycin resistance (HLDR; MIC of ≥ 256 mg/liter) after exposure to daptomycin has recently been reported in viridans group streptococcus (VGS) isolates. Our study objectives were as follows: to know whether in vitro development of HLDR after exposure to daptomycin was common among clinical isolates of VGS and Streptococcus bovis; to determine whether HLDR also developed during the administration of daptomycin to treat experimental endocarditis caused by the daptomycin-susceptible, penicillin-resistant Streptococcus mitis strain S. mitis 351; and to establish whether combination with gentamicin prevented the development of HLDR in vitro and in vivo. In vitro studies were performed with 114 VGS strains (mitis group, 92; anginosus group, 10; mutans group, 8; and salivarius group, 4) and 54 Streptococcus bovis strains isolated from 168 consecutive patients with infective endocarditis diagnosed between 1995 and 2010. HLDR was only observed after 24 h of exposure to daptomycin in 27% of the mitis group, including 27% of S. mitis isolates, 47% of S. oralis isolates, and 13% of S. sanguis isolates. In our experimental model, HLDR was detected in 7/11 (63%) and 8/12 (67%) isolates recovered from vegetations after 48 h of daptomycin administered at 6 mg/kg of body weight/24 h and 10 mg/kg/24 h, respectively. In vitro, time-kill experiments showed that daptomycin plus gentamicin was bactericidal against S. mitis 351 at tested concentrations of 0.5 and 1 times the MIC and prevented the development of HLDR. In vivo, the addition of gentamicin at 1 mg/kg/8 h to both daptomycin arms prevented HLDR in 21 out of 23 (91%) rabbits. Daptomycin plus gentamicin was at least as effective as vancomycin plus gentamicin. In conclusion, HLDR develops rapidly and frequently in vitro and in vivo among mitis group streptococci. Combining daptomycin with gentamicin enhanced its activity and prevented the development of HLDR in most cases.

The relationship between the new taxonomy of Streptococcus bovis and its clonality to colon cancer, endocarditis, and biliary disease.

BACKGROUND: The nomenclature of Streptococcus bovis has changed. The study aims were to examine and compare the clinical characteristics and outcomes of infections based on the new taxonomy and the genetic relatedness of strains. METHODS: Bacteremic cases from 2004 to 2010 at Assaf Harofeh Medical Center were reviewed. VITEK 2 later confirmed with polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was used for subspecies identification. VITEK 2 later confirmed with Etests was used for minimal inhibitory concentration (MIC) testing. Repetitive extragenic palindromic polymerase chain reaction (rep-PCR) was used to determine the genetic relatedness of strains. RESULTS: Twenty-four bacteremia cases were included. The median age of patients was 81 years (range 1 day to 91 years), two were neonates, three were pregnant, and 18 were elderly (≥ 65 years of age). The Charlson's combined conditional age-related score was 8.2 ± 2.9, and 11 (58 %) patients were immunosuppressed. There were 13 patients who had S. gallolyticus subsp. pasteurianus, six had S. gallolyticus subsp. gallolyticus, four had S. infantarius subsp. coli, and one had S. infantarius subsp. infantarius. Ten of 19 non-pregnant adult patients had colon adenoma or carcinoma, three had acute biliary disease, and five had endocarditis. Two patients died in the hospital. rep-PCR revealed polyclonality. There were no significant associations between subspecies or genotypes and the various clinical characteristics or outcomes. CONCLUSION: S. bovis bacteremia is a serious disease that affects elderly immunosuppressed individuals. Infection is strongly associated with colon pathology and endocarditis, regardless of the new taxonomy or clone complex. The identification of S. bovis is of paramount importance, and microbiology laboratories should differentiate its processing from that of other S. viridans.

Molecular characterization of a novel mosaic tet(S/M) gene encoding tetracycline resistance in foodborne strains of Streptococcus bovis.

The presence of antibiotic-resistance (AR) genes in foodborne bacteria of enteric origin represents a relevant threat to human health in the case of opportunistic pathogens, which can reach the human gut through the food chain. Streptococcus bovis is a human opportunistic pathogen often associated with infections in immune-compromised or cancer patients, and it can also be detected in the environment, including fermented foods. We have focused on the molecular characterization of a tetracycline (Tet)-resistance gene present in 39 foodborne isolates of S. bovis phenotypically resistant to this drug. The gene was identified as a novel tet(S/M) fusion, encoding a mosaic protein composed of the N-terminal 33 amino acids of Tet(S), in-frame with the Tet(M) coding sequence. Heterologous expression of the mosaic gene was found to confer Tet resistance upon Escherichia coli recipients. Moreover, the tet(S/M) gene was found to be transcriptionally inducible by Tet under the endogenous tet(S) promoter in both S. bovis and E. coli. Nucleotide sequencing of the surrounding genomic region of 16.2 kb revealed large blocks of homology with the genomes of Streptococcus infantarius and Lactococcus lactis. A subregion of about 4 kb containing mosaic tet(S/M) was flanked by two copies of the IS1216 mobile element. PCR amplification with primers directed outwards from the tet(S/M) gene identified the presence of a 4.3 kb circular form corresponding to the intervening chromosomal region between the two IS1216 elements, but lacking a replication origin. The circular element shared extensive overall homology with a region of the multidrug-resistance plasmid pK214 from Lc. lactis, containing tet(S), as well as the IS1216 transposase-containing element and intervening non-coding sequences. Linear reconstruction of the insertion events likely to have occurred within this genomic region, inferred from sequence homology, provides further evidence of the chromosomal rearrangements that drive genomic evolution in complex bacterial communities such as the gut and food microbiota.

[Diseases associated with bloodstream infections caused by the new species included in the old Streptococcus bovis group]. / Cuadros clínicos asociados a bacteriemia causada por las nuevas especies incluidas en el antiguo grupo Streptococcus bovis.

OBJECTIVE: We sought to identify possible diseases associated with bloodstream infections caused by new species of S. bovis group isolated in blood cultures and by studying patient records METHODS: Forty-four consecutive blood culture isolates initially designated S. bovis were further characterised using phenotypic methods Patient records were examined. RESULTS: We identified 15 Streptococcus gallolyticus subsp. gallolyticus, 24 Streptococcus gallolyticus subsp. pasteurianus, and 5 Streptococcus infantarius isolates in 44 BSI episodes. CONCLUSIONS: The association between S. bovis bacteraemia and endocarditis and/or colon carcinoma is highly dependent on the causative species. Streptococcus gallolyticus subsp. gallolyticus is a surrogate for endocarditis and/or bowel disease, whereas Streptococcus gallolyticus subsp. pasteurianus is a surrogate for hepato-biliary disease.

Effects of a combination of hinokitiol (ß-thujaplicin) and an organic acid mixture on ruminal fermentation in heifers fed a high-grain diet.

This study evaluated the effects of hinokitiol (a natural antibacterial compound extracted from Thujopsis dolabrata var. hondai) and an organic acid mixture (citrate content 50%) on ruminal fermentation. Antibacterial properties were examined by measuring minimal inhibitory concentration. Hinokitiol at 1.56µg/mL or an organic acid mixture at 1600µg/mL inhibited Streptococcus bovis growth. The combination of 0.78µg/mL hinokitiol and 200µg/mL of an organic acid mixture also inhibited S. bovis growth. Both hinokitiol and the hinokitiol and an organic acid mixture combination showed strong antibacterial properties on Gram-positive bacteria such as S. bovis, but relatively weak antibacterial activities on Gram-negative bacteria such as Megasphaera elsdenii. Three ruminally cannulated heifers were fed a bloat-producing diet containing barley, pelleted alfalfa meal, soybean meal and salt without long-cut roughage to investigate the ruminal characteristics in vivo. Feeding to heifers a bloat-producing diet containing 7.8mg/kg hinokitiol and 0.2% of an organic acid mixture significantly decreased the increase in stable ingesta volume. Hinokitiol or an organic acid mixture did not affect ruminal volatile fatty acids, protozoa and bacteria. These results suggest that a combination of hinokitiol and an organic acid mixture might reduce frothy bloat in cattle fed high-grain diets.

Streptococcus bovis meningitis associated with colonic diverticulosis and hearing impairment: a case report.

A 75-year-old woman presented to the Tropical Diseases Hospital, Goiania, Brazil, with a two-day history of fever and chills followed by headache and vomiting over the last 24 hours. The cerebrospinal fluid (CSF) showed 270 leukocytes/mmc (30 percent neutrophils); 20 red cells/mmc; undetectable levels of glucose and 232 mg/dL of protein. The Gram stain revealed several Gram-positive cocci, and CSF culture yielded Streptococcus bovis. A colonoscopy showed diverticula in descendent and transverse colon. After a 14-day course of penicillin G, the patient was discharged in a good state of health, with only mild hearing impairment.

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria.

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria (Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic.

The activity and stability of cell-associated activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

Streptococcus bovis HC5 cultures released a broad spectrum lantibiotic, bovicin HC5, into the cell-fee culture supernatant after they reached stationary phase, but most of the antibacterial activity remained cell-associated. Cell-associated bovicin HC5 was more resistant to degradation by Pronase E than the cell-free activity. Acidic NaCl (pH 2.0, 100 mM) did not release all of the cell-associated activity, and cells that were sequentially treated with acidic NaCl and Pronase E still had antibacterial activity. Cell-associated activity retained after acidic NaCl treatment was still able to catalyze potassium efflux from S. bovis JB1, a sensitive strain. These results indicate that cell-associated bovicin HC5 is more active and stable than cell-free bovicin HC5.