Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

Chromosomally and extrachromosomally mediated high-level gentamicin resistance in Streptococcus agalactiae

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in non-pregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to loss of a synergistic effect. We therefore performed a multicentre study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centres in four countries, 1128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. Though, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon, designated Tn3706. In the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing a plasmid mediated HLGR in GBS. Thus, in our clinical GBS isolates HLGR is mediated both chromosomally and extrachromosomally.

Cross-sectional study of Streptococcus species in quarter milk samples of dairy cows in the canton of Bern, Switzerland

A total of 2538 quarter milk samples from 638 lactating dairy cows from 47 farms in the canton of Bern, Switzerland, were investigated for streptococci. A novel, simple and inexpensive laboratory method was used for the differentiation of Streptococcus species, and a risk factor analysis was carried out. The prevalence in the quarter milk samples was 0.2 per cent for Streptococcus agalactiae, 1.3 per cent for Streptococcus uberis, 1.3 per cent for Streptococcus dysgalactiae, 0.1 per cent for Enterococcus species and 2.9 per cent for minor Streptococcus species (designated Streptococcus-Lactococcus-Enterococcus [SLE] group). Based on the somatic cell count (SCC), S uberis and S dysgalactiae were classified as 'major' pathogens and the bacteria in the SLE group as 'minor' pathogens. For S uberis, S dysgalactiae and bacteria in the SLE group, the most significant risk factor was an intramammary infection (IMI) of a neighbouring quarter by the same pathogen. Other significant risk factors for S uberis infection were a positive California Mastitis Test (CMT) result and a SCC of more than 100,000 cells/ml. Significant risk factors for IMI with S dysgalactiae were a positive CMT result, teat injury and palpable abnormalities in the udder. Infection with bacteria in the SLE group was significantly associated with a SCC of more than 100,000 cells/ml, a lactation number of more than 2, the right rear quarter (as the location of infection) and a positive CMT result.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).

[Meningitis (II)--acute bacterial meningitis]

Acute meningitis is a medical emergency, particularly in patients with rapidly progressing disease, mental status changes or neurological deficits. The majority of cases of bacterial meningitis are caused by a limited number of species, i.e. Streptococcus pneumoniae, Neisseria meningitis, Listeria monocytogenes, group B Streptococci (Streptococcus agalactiae), Haemophilus influenzae and Enterobacteriaceae. Many other pathogens can occasionally cause bacterial meningitis, often under special clinical circumstances. Treatment of meningitis includes two main goals: Eradication of the infecting organism, and management of CNS and systemic complications. Empiric therapy should be initiated without delay, as the prognosis of the disease depends on the time when therapy is started. One or two blood cultures should be obtained before administering the first antibiotic. Empiric therapy is primarily based on the age of the patient, with modifications if there are positive findings on CSF gram stain or if the patient presents with special risk factors. It is safer to choose regimens with broad coverage, as they can usually be modified within 24-48 hours, when antibiotic sensitivities of the infecting organism become available. Adjunctive therapy with dexamethasone is also administered in severely ill patients concomitantly with the first antibiotic dose. In patients who are clinically stable and are unlikely to be adversely affected if antibiotics are not administered immediately, including those with suspected viral or chronic meningitis, a lumbar puncture represents the first step, unless there is clinical suspicion of an intracerebral mass lesion. Findings in the CSF and on CT scan, if performed, will guide the further diagnostic work-up and therapy in all patients.

Microbiological Findings in Subjects With Asymptomatic Oral Lichen Planus: A Cross-Sectional Comparative Study

Background: The bacterial colonization of the oral mucosa was evaluated in patients with asymptomatic oral lichen planus (OLP) and compared to the microbiologic status in mucosally healthy subjects. Methods: Bacteria from patients with clinically and histopathologically diagnosed OLP from the Stomatology Service, Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Bern, were collected with a non-invasive swab system. Samples were taken from OLP lesions on the gingiva and from non-affected sites on the contralateral side of the mouth. The control population did not have OLP and was recruited from the student clinic. All samples were processed with the checkerboard DNA-DNA hybridization method using well-defined bacterial species for the analysis. Results: Significantly higher bacterial counts of Bacteroides ureolyticus (P = 0.001), Dialister species (sp.) (P = 0.006), Staphylococcus haemolyticus (P = 0.007), and Streptococcus agalactiae (P = 0.006) were found in samples taken from OLP lesions compared to sites with no clinical evidence of OLP. Significantly higher bacterial counts were found for Capnocytophaga sputigena, Eikenella corrodens, Lactobacillus crispatus, Mobiluncus curtisii, Neisseria mucosa, Prevotella bivia, Prevotella intermedia, and S. agalactiae at sites with lesions in subjects with OLP compared to sites in control subjects (P <0.001). Conclusions: Microbiologic differences were found between sites with OLP and sites in subjects without a diagnosis of OLP. Specifically, higher counts of staphylococci and S. agalactiae were found in OLP lesions.

The impact of the stone age diet on gingival conditions in the absence of oral hygiene

BACKGROUND: The objective of this study was to assess the oral microbiota and clinical data in subjects without access to traditional oral hygiene methods and who ate a diet available in the Stone Age. METHODS: Ten subjects living in an environment replicating the Stone Age for 4 weeks were enrolled in this study. Bleeding on probing (BOP), gingival and plaque indices, and probing depth (PD) were assessed at baseline and at 4 weeks. Microbiologic samples were collected at the mesio-buccal subgingival aspects of all teeth and from the dorsum of the tongue and were processed by checkerboard DNA-DNA hybridization methods. RESULTS: No subject had periodontitis. Mean BOP decreased from 34.8% to 12.6% (P <0.001). Mean gingival index scores changed from 0.38 to 0.43 (not statistically significant) and mean plaque scores increased from 0.68 to 1.47 (P <0.001). PD at sites of subgingival sampling decreased (mean difference: 0.2 mm; P <0.001). At week 4, the total bacterial count was higher (P <0.001) for 24 of 74 species, including Bacteroides ureolyticus, Eikenella corrodens, Lactobacillus acidophilus, Capnocytophaga ochracea, Escherichia coli, Fusobacterium nucleatum naviforme, Haemophilus influenzae, Helicobacter pylori, Porphyromonas endodontalis, Staphylococcus aureus (two strains), Streptococcus agalactiae, Streptococcus anginosis, and Streptococcus mitis. Bacterial counts from tongue samples were higher at baseline (P <0.001) for 20 species, including Tannerella forsythia (previously T. forsythensis), Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans; serotype a), and Streptococcus spp. CONCLUSIONS: The experimental gingivitis protocol is not applicable if the diet (e.g., Stone Age) does not include refined sugars. Although plaque levels increased, BOP and PD decreased. Subgingival bacterial counts increased for several species not linked to periodontitis, whereas tongue bacterial samples decreased during the study period.

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.

Intramammary infections with the contagious Staphylococcus aureus genotype B in Swiss dairy cows are associated with low prevalence of coagulase-negative staphylococci and Streptococcus spp

The association between the contagious Staphylococcus aureus genotype B (GTB) and the presence of coagulase-negative staphylococci (CNS) and Streptococcus spp. (non-agalactiae streptococci), was investigated, and the identification of problem herds without genotyping was evaluated. Milk samples from 10 herds with Staph. aureus GTB herd problems (PH cases) were compared with samples from 19 herds with at least one Staph. aureus isolate of non-B genotype (CH cases). All samples were bacteriologically analysed and Staph. aureus genotyping carried out using a ribosomal spacer-PCR. Cow and quarter prevalences of Staph. aureus, CNS and Streptococcus spp. differed significantly between PH and CH groups. PH cases were highly associated with decreased cow prevalences of CNS and Streptococcus spp. These altered prevalences also contributed significantly to the identification of problem herds without resorting to genotyping. Common herd-level risk factors did not explain the difference between the prevalences in PH and CH cases.

In vitro evaluation of surface roughness, adhesion of periodontal ligament fibroblasts, and Streptococcus gordonii following root instrumentation with Gracey curettes and subsequent polishing with diamond-coated curettes

OBJECTIVES: The objective of the study was to evaluate the efficacy of an additional usage of a diamond-coated curette on surface roughness, adhesion of periodontal ligament (PDL) fibroblasts, and of Streptococcus gordonii in vitro. MATERIALS AND METHODS: Test specimens were prepared from extracted teeth and exposed to instrumentation with conventional Gracey curettes with or without additional use of diamond-coated curettes. Surface roughness (Ra and Rz) was measured before and following treatment. In addition, the adhesion of PDL fibroblasts for 72 h and adhesion of S. gordonii ATCC 10558 for 2 h have been determined. RESULTS: Instrumentation with conventional Gracey curettes reduced surface roughness (median Ra before: 0.36 μm/after: 0.25 μm; p < 0.001; median Rz before: 2.34 μm/after: 1.61 μm; p < 0.001). The subsequent instrumentation with the diamond-coated curettes resulted in a median Ra of 0.31 μm/Rz of 2.06 μm (no significance in comparison to controls). The number of attached PDL fibroblasts did not change following scaling with Gracey curettes. The additional instrumentation with the diamond-coated curettes resulted in a two-fold increase in the number of attached PDL fibroblasts but not in the numbers of adhered bacteria. CONCLUSIONS: Treatment of root surfaces with conventional Gracey curettes followed by subsequent polishing with diamond-coated curettes may result in a root surface which provides favorable conditions for the attachment of PDL fibroblasts without enhancing microbial adhesion. CLINICAL RELEVANCE: The improved attachment of PDL fibroblasts and the limited microbial adhesion on root surfaces treated with scaling with conventional Gracey curettes followed by subsequent polishing with diamond-coated curettes may favor periodontal wound healing.

Glycan-binding specificities of Streptococcus mutans and Streptococcus sobrinus lectin-like adhesins

Since the adhesion of bacteria to the tooth surface is a prerequisite for dental plaque and subsequent caries development, a promising caries preventive strategy could be to block the lectin-glycan-mediated adherence of cariogenic bacteria. The aim of the study was to evaluate potential differences in glycan-binding specificities of two Streptococcus mutans strains (DSM 20523 and DSM 6178) and Streptococcus sobrinus (DSM 20381). A competitive enzyme-linked lectin-binding assay was used to identify the binding specificities of isolated bacterial surface lectins. Blotting of the microbial proteins on neoglycoprotein-coated PVP membranes enabled a qualitative protein analysis of all specific bacterial lectins. Different glycan-binding sites could be identified for the S. mutans strains in comparison to S. sobrinus. An earlier reported glycan-binding specificity for terminal galactose residues could be confirmed for the S. mutans strains. For the S. sobrinus strain, more than one glycan-binding specificity could be found (oligomannose and terminal sialyl residues). Each of the tested strains showed more than one surface lectin responsible for the specific lectin-binding with varying molecular weight (S. mutans, 90/155 kDa and S. sobrinus, 35/45 kDa). The established experimental setup could be used as future standard procedure for the identification of bacterial lectin-derived binding specificities. The findings from this study might serve as basis for the design of an individual 'glycan cocktail' for the competitive inhibition of lectin-mediated adhesion of mutans streptococci to oral surfaces.

Capsule type of Streptococcus pneumoniae determines growth phenotype

The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.