Comparative genomics and the role of lateral gene transfer in the evolution of bovine adapted Streptococcus agalactiae

In addition to causing severe invasive infections in humans, Streptococcus agalactiae, or group B Streptococcus (GBS), is also a major cause of bovine mastitis. Here we provide the first genome sequence for S. agalactiae isolated from a cow diagnosed with clinical mastitis (strain FSL S3-026). Comparison to eight S. agalactiae genomes obtained from human disease isolates revealed 183 genes specific to the bovine strain. Subsequent polymerase chain reaction (PCR) screening for the presence/absence of a subset of these loci in additional bovine and human strains revealed strong differentiation between the two groups (Fisher exact test: p < 0.0001). The majority of the bovine strain-specific genes (∼85%) clustered tightly into eight genomic islands, suggesting these genes were acquired through lateral gene transfer (LGT). This bovine GBS also contained an unusually high proportion of insertion sequences (4.3% of the total genome), suggesting frequent genomic rearrangement. Comparison to other mastitis-causing species of bacteria provided strong evidence for two cases of interspecies LGT within the shared bovine environment: bovine S. agalactiae with Streptococcus uberis (nisin U operon) and Streptococcus dysgalactiae subsp. dysgalactiae (lactose operon). We also found evidence for LGT, involving the salivaricin operon, between the bovine S. agalactiae strain and either Streptococcus pyogenes or Streptococcus salivarius. Our findings provide insight into mechanisms facilitating environmental adaptation and acquisition of potential virulence factors, while highlighting both the key role LGT has played in the recent evolution of the bovine S. agalactiae strain, and the importance of LGT among pathogens within a shared environment.     

Clinical Laboratory Perspective on Streptococcus halichoeri,an Unusual Nonhemolytic,Lancefield Group B Streptococcus Causing Human Infections

Streptococcus halichoeri is a relatively newly identified species of pyogenic streptococci that causes zoonotic infection in humans. S. halichoeri was first described in 2004 as indigenous to seals, and only 8 reports of human S. halichoeri infection have been published. S. halichoeri grows as small, white, nonhemolytic colonies and may be strongly catalase-positive on routine blood agar media, which can lead to isolates being misidentified as coagulase-negative staphylococci. S. halichoeri tests positive for Lancefield group B antigen, like S. agalactiae, but can be identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry or partial 16S rRNA sequencing. We describe 3 cases of S. halichoeri bone and joint infections in patients in the United States with underlying health conditions. In addition, we examine the microbiologic characteristics of S. halichoeri and discuss the importance of fully identifying this organism that might otherwise be disregarded as a skin commensal.     

Recent advances in defining the cariogenicity of mutans streptococci: Molecular genetic approaches

The application of molecular genetic approaches with oral mutans streptococci has resulted in the isolation of several genes which may be involved in the cariogenicity of these organisms. Among these are genes coding for cell surface proteins, sucrose metabolizing enzymes, and glycosyltransferase activities. The isolated genes have been utilized to create specific mutants of Streptococcus mutans to assess the potential roles of the gene products in cariogenicity both in vitro and in vivo. These approaches should prove useful in answering some still unresolved questions at the molecular level regarding the cariogenic properties of the organisms.     

Genomic insights on DNase production in Streptococcus agalactiae ST17 and ST19 strains

Streptococcus agalactiae evasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence of S. agalactiae ST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. All S. agalactiae ST17 strains, with the exception of 2211–04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203–05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases in S. agalactiae revealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(−), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(−) ST17 2211–04 and the DNase(+) ST19 1203–05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology in S. agalactiae and a transposon homologous to TnGBS2.3 in ST17 DNase(−) 2211–04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(−) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(−) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes in S. agalactiae.     

IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge

Staphylococcus aureus is the most frequent cause of bacteremia and hospital-acquired infection, however a vaccine that prevents staphylococcal disease is currently not available. Two sortase-anchored surface proteins, IsdA and IsdB, have been identified as subunit vaccines that, following active immunization, protect experimental animals against intravenous challenge with staphylococci. Here we investigate the molecular basis of this immunity and report that, when passively transferred to naïve mice, purified antibodies directed against IsdA or IsdB protected against staphylococcal abscess formation and lethal intravenous challenge. When added to mouse blood, IsdA- or IsdB-specific antibodies did not promote rapid opsonophagocytic killing of wild-type staphylococci. Antibodies directed against IsdA interfered with heme-binding and IsdB antibodies perturbed the ability of this surface protein to bind hemoglobin. As the structural genes for isdA and isdB are required for heme-iron scavenging during the pathogenesis of infection, we hypothesize that IsdA and IsdB antibodies may at least in part provide protection against staphylococci by interfering with the pathogen's heme-iron scavenging mechanisms.     

农村井水中耐碳青霉烯类铜绿假单胞菌及耐药基因分子流行病学特征

目的 分析农村井水中耐碳青霉烯类铜绿假单胞菌（PA）及耐药基因的分子流行病学特征。方法 依据《饮用天然矿泉水检验方法》（GB 8538-2016）对山东省巨野县农村井水采集112份水样进行检测,并对检出的PA分别进行PFGE分型和药物敏感性试验。PCR鉴定碳青霉烯类耐药基因后,采用S1-PFGE和Southern杂交确定耐药基因的位置,并用结合实验判断基因的可转移性。结果 农村井水中PA检出率为54.46％（61/112）。61株菌分为56个PFGE型,100.00%一致带型最多的有2株菌,无明显的优势带型。药敏实验结果显示,多重耐药菌占93.44％（57/61）,2株菌携带bla_VIM-2耐药基因,均位于质粒上,且均可随质粒发生水平转移。结论 农村井水中检出耐碳青霉烯类PA及其耐药基因,此类耐药基因存在水平传播的可能性。     

Localization of Staphylococcus inside the vacuole of Candida albicans by immunodetection and FISH

In our previous study, two bacteria B1 and B2 were excised from two amphotericin B-treated Candida albicans Y1 and Y2, respectively. Bacteria were identified as B1: Staphylococcus hominis and B2: Staphylococcus haemolyticus according to their biochemical characteristics and detection and sequencing of Staphylococcus-specific genes. In this study the intracellular origin of staphylococci inside the vacuole of yeast was examined. Polyclonal antibodies against S. hominis and S. haemolyticus were raised in rabbit and used for detection of staphylococcal proteins in protein pool of yeasts by western blotting (WB). Fluorescein-isothiocyanate (FITC)-conjugated antibodies were used for bacterial localization inside yeast's vacuole by direct immunofluorescence (DIF). Fluorescent in situ hybridization (FISH) with Staphylococcaceae –specific probe was performed for validation of immunodetection results. WB results showed occurrence of several proteins in protein pool of yeasts that were similar to staphylococcal proteins such as those with molecular weight of 57.5 and 66 kDa. Fluorescent microscopy showed interactions of FITC-antibodies with intracellular staphylococci which appeared as green spots. Hybridization of staphylococcal- specific probe with bacteria inside yeasts' vacuole confirmed immunodetection results. Detection of staphylococcal proteins and genes inside Candida albicans yeast indicates existence of intracellular bacteria inside the vacuole of yeast. These results suggest C. albicans as the potential reservoir of medically important bacteria.     

Misidentification of alpha-hemolytic streptococci by routine tests in clinical practice

Accurate species-level identification of viridans group streptococci (VGS) is very important for understanding of their pathogenicity and virulence. However, an extremely high level of the similarity between VGS, especially Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis and Streptococcus pseudopneumoniae, often results in misidentification of these organisms, so there is an urgent need of novel approaches to species identification.A set of 50 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (alpha-hemolysis, colony morphology, Gram stain and optochin susceptibility). Modern proteomic and genetic approaches – the direct bacterial profiling (DBP) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and multilocus sequence analysis (MLSA) scheme (http://viridans.emlsa.net/) – were applied for the accurate species identification. After that all isolates were stored at −70 °C. Later they were re-inoculated, and a number of additional tests (bile solubility, latex agglutination by commercial “Slidex® pneumo-kit” and repeated optochin test) were performed.A considerable discrepancy was discovered in the results of the different approaches. Looking in the future, one could say that MLSA-like schemes based on the analysis of the nucleotide sequences of seven or more loci of the bacterial genome, appeared to be the most useful instrument in the VGS discrimination, in contrast to the numerous one-target identification schemes, which have been introduced into practice by now.     

The antimicrobial activity of cationic proteins isolated from the cells in bulk milk samples

Cationic proteins isolated from the cells in bulk milk samples were shown to inhibit the growth of two pathogenic strains of staphylococci and also Streptococcus agalactiae S 13. Polyacrylamide gel disk electrophoresis studies on these proteins revealed the presence of at least 9 components some of which had isoelectric pH''s between 7·0 and 9·0. Trace amounts of the isolated protein had isoelectric pH''s greater than 9·0. Staphylococci incubated with milk-cell cationic proteins absorbed the protein, thereby allowing the organism to be stained with the anionic dye Fast Green FCF. Protein-treated staphylococci in isotonic solutions autoagglutinated. This autoagglutination was more marked in hypo- and hypertonic solutions. Lysozyme was not demonstrated in the isolated protein fractions in assays involving incubation with Micrococcus lysodeikticus for 90 min. The antimicrobial activity of the cationic proteins isolated from the bulk milk samples was not destroyed after heating to temperatures up to 70° C for 30 min., whereas at higher temperatures the activity diminished and was almost completely lost at 100° C.     

Development of a single-dose recombinant CAMP factor entrapping poly(lactide-co-glycolide) microspheres-based vaccine against Streptococcus agalactiae

Streptococcus agalactiae is an important contagious bovine mastitis pathogen. Although it is well controlled and even eradicated in most Northern European and North American dairy herds, the prevalence of this pathogen remains very high in China. However, research on development of a vaccine against S. agalactiae mastitis is scarce. The aims of the present study were to: (1) develop a single-dose vaccine against S. agalactiae based on poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) encapsulated CAMP factor, a conserved virulent protein encoded by S. agalactiae’s cfb gene; and (2) evaluate its immunogenicity and protective efficacy in a mouse model. The cfb gene was cloned and expressed in a recombinant Escherichia coli strain Trans1-T1. The CAMP factor was tested to determine a safe dose range and then encapsulated in MS of PLGA (50:50) to assess its release pattern in vitro and immune reaction in vivo. Furthermore, a mouse model and a histopathological assay were developed to evaluate bacterial burden and vaccine efficacy. In the low dosage range (<100 μg), CAMP factor had no obvious toxicity in mice. The release pattern in vitro was characterized by an initial burst release (44%), followed by a sustained and slower release over 7 wk. In mice immunized with either pure CAMP factor protein or PLGA-CAMP, increased antibody titers were detected in the first 2 wk, whereas only PLGA-CAMP immunization induced a sustained increase of antibody titers. In mice vaccinated with PLGA-CAMP, mortality and bacteria counts were lower (compared to a control group) after S. agalactiae challenge. Additionally, no pathological lesions were detected in the vaccinated group. Therefore, PLGA-CAMP conferred protective efficacy against S. agalactiae in our mouse model, indicating its potential as a vaccine against S. agalactiae mastitis. Furthermore, the slow-release kinetics of PLGA MS warranted optimism for development of a single-dose vaccine.     

Characterization and antibiotic susceptibility of Streptococcus agalactiae isolates causing urinary tract infections

Streptococcus agalactiae (GBS) has been implicated in urinary tract infections but the microbiological characteristics and antimicrobial susceptibility of these strains are poorly investigated. In this study, 87 isolates recovered from urine samples of patients who had attended the Spedali Civili of Brescia (Italy) and had single organism GBS cultured were submitted to antimicrobial susceptibility testing, molecular characterization of macrolide and levofloxacin resistance, PCR-based capsular typing and analysis of surface protein genes. By automated broth microdilution method, all isolates were susceptible to penicillin, cefuroxime, cefaclor, and ceftriaxone; 80%, 19.5% and 3.4% of isolates were non-susceptible to tetracycline, erythromycin, and levofloxacin, respectively. Macrolide resistance determinants were iMLS_B (n = 1), cMLS_B (n = 10) and M (n = 5), associated with ermTR, ermB and mefA/E. Levofloxacin resistance was linked to mutations in gyrA and parC genes. Predominant capsular types were III, Ia, V, Ib and IX. Type III was associated with tetracycline resistance, while type Ib was associated with levofloxacin resistance. Different capsular type-surface protein gene combinations (serotype V-alp2, 3; serotype III-rib; serotype Ia-epsilon) were detected. A variety of capsular types are involved in significant bacteriuria. The emergence of multidrug resistant GBS may become a significant public health concern and highlights the importance of careful surveillance to prevent the emergence of these virulent GBS.     

Sensitivity of a Standard Host Resistance Assay Using Streptococcus agalactiae for Assessing Exposure to Immunotoxicants in Wild Cotton Rats (Sigmodon hispidus)

Resident small mammals have been used for in situ biomonitoring of contaminated waste sites containing suspected immunotoxicants. Host resistance assays, which involve challenging animals with an actual pathogen, allow for testing of overall immune system function in animals. Because such assays have not been evaluated for use with wild rodent species, it was our objective to assess the efficacy of Streptococcus agalactiae as a pathogenic model for use in a host resistance assay for detecting alterations in immune system function in wild cotton rats (Sigmodon hispidus). The ability of the assay to detect immunosuppression was evaluated by inducing immunosuppression chemically (cyclophosphamide or dexamethasone) and by protein malnutrition. The estimated lethal dose of S. agalactiae that killed 50% of challenged animals (LD₅₀) was 5.76 × 10⁷ colony-forming units (CFUs). Although bacterial agglutination titers indicated that animals developed an antibody response when immunized, immunization was not sufficient to adequately protect animals from a subsequent pathogenic challenge. Sensitivity of the host resistance assay was only suitable for detecting substantial immunosuppression, such as that induced by protein malnutrition or dexamethasone administration. Received: 28 July 1999/Accepted: 30 November 1999     

Species identification and detection of oxacillin resistance in coagulase-negative Staphylococcus blood isolates from neutropenic patients

One hundred coagulase-negative staphylococcal isolates from septicemic neutropenic patients with hematologic malignancies were identified to a species level by means of the French API STAPH strip system and by the Automicrobic VITEK system. According to these two methods, which concurred in 95% of cases, S. epidermidis (80–82% of the isolates) was the most frequently identified species, followed by S. haemolyticus (6–7% of the isolates). The susceptibility to oxacillin was also evaluated by macrodilution MIC, Automicrobic VITEK system and agar screen, and 76,78 and 79 of the 100 isolates, respectively, were found resistant to this antibiotic. All oxacillin-resistant isolates according to Automicrobic VITEK were confirmed resistant by agar screen. A 48h incubation was required to determine oxacillin resistance in 11 of 79 isolates with agar screen and in 10 of 76 isolates with macrodilution MIC.Automicrobic VITEK system may represent a useful method for rapid identification to a species level and early recognition of oxacillin resistance in coagulase-negative staphylococci.Corresponding author.     

Transformation as a tool for studying the epidemiology of tet determinants in Streptococcus pneumoniae.

Transformation of pneumococcus was used to detect homology among tetracycline resistance determinants of clinical isolates of Streptococcus pneumoniae. A strain of pneumococcus containing a mutated tet determinant (tet-3), of class M, integrated into the chromosome was used as a recipient in transformation experiments, where donor DNA was from the tetracycline resistant isolates. 34/34 strains appeared to have tet determinants homologous to tet-3 (i.e. tet M).Still using transformation it was possible to determine that the tet-3 transforming activity of DNA from Tn916 and S. pneumoniae BM6001 was contained in a 5 kb HincII fragment. For this purpose a transformation technique where donor DNA was directly taken from low melting point agarose gels was standardized and used.Corresponding author.     

A prospective study of swimming-related illness. II. Morbidity and the microbiological quality of water

A prospective cohort epidemiological-microbiological study was carried out at 10 beaches in Ontario, Canada. Lake water and sediment samples collected at the beaches were analyzed for fecal coliforms, fecal streptococci, heterotrophic bacteria, Pseudomonas aeruginosa, and total staphylococci. Mean fecal coliform levels in the surface water of the lakes were within accepted guidelines. Bacterial densities were found to be approximately 10 times higher in the sediment than in the corresponding surface water samples. Morbidity among swimmers was shown to be related to staphylococcal counts, to fecal coliform levels, and, somewhat less strongly, to fecal streptococcal counts. Total staphylococci appeared to be more consistent indicators for predicting total morbidity rates among swimmers.     

Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases

The enzyme glyceraldehyde-3-P-dehydrogenase (GAPDH) has been identified as having other properties in addition to its key role in glycolysis. The ability of GAPDH to bind to numerous extracellular matrices, modulation of host-immune responses, a role in virulence and surface location has prompted numerous investigators to postulate that GAPDH may be a good vaccine candidate for protection against numerous pathogens. Although immune responses against GAPDH have been described for many microorganisms, vaccines containing GAPDH have been successfully tested in few cases including those against the trematode—Schistosoma mansoni, the helminth—Enchinococcus multilocularis; the nematode filaria— Litomosoides sigmodontis; fish pathogens such as Aeromonas spp., Vibrio spp., Edwarsiella spp., and Streptococcus iniae; and environmental streptococci, namely, Streptococcus uberis and Streptococcus dysgalactiae. Before GAPDH-based vaccines are considered viable options for protection against numerous pathogens, we need to take into account the homology between the host and pathogen GAPDH proteins to prevent potential autoimmune reactions, thus protective GAPDH epitopes unique to the pathogen protein must be identified.     

Synthesis, spectroscopic characterization and antibacterial activity of new cobalt(II) complexes of unsymmetrical tetradentate (OSN2) Schiff base ligands

Cobalt ion complexes with the Schiff bases, (4-X-2-{[2-(2-pyridine-2-yl-ethylsulfanyl)ethylimino]methyl}phenol (X = methoxy (OMe), phenylazo (N₂Ph), bromo (Br), nitro (NO₂)),were synthesized and investigated by several techniques using elemental analysis (C, H, N), FTIR, electronic spectra and molar conductivity. The thermal stability of free ligands and related cobalt complexes were studied by using differential scanning calorimetry (DSC) and thermogravimetric analyses (TGA). Cyclic voltammetry indicates that the investigated cobalt complexes, under the experimental conditions, have irreversible redox behavior. The synthesized compounds have antibacterial activity against the four Gram-positive bacteria: Streptococcus pyogenes, Streptococcus agalactiae, Staphylococcus aureus and Bacillus anthracis and also against the two Gram-negative bacteria: Klebsiella pneumoniae and Pseudomonas aeruginosa. The activity data show that the parent Schiff bases are more potent antibacterials than the cobalt complexes.     

Diversity of resistance phenotypes and plasmid analysis in multi-resistant 0:12 Pseudomonas aeruginosa

Antibiotic resistance phenotypes, plasmid content and ability of conjugal transfer of antibiotic resistance genes of 35 multi-resistant Pseudomonas aeruginosa strains were examined. The strains were isolated in 12 Greek hospitals and the majority of them (80%) belonged to serotype 0:12. The isolates were distributed to a variety of different antibiotic resistance phenotypes. Plasmid analysis showed that 10 isolates harboured plasmids ranging in size from 20 to 100 Mda. Among these strains, four carried plasmids of 100 Mda, two strains had 60 Mda plasmid each while in three strains the plasmids detected were 65, 25 and 20 Mda, respectively. One strain harboured two plasmids of 100 and 60 Mda. All strains containing plasmids belonged to 0:12 serotype, except the one harbouring the 25 Mda plasmid, which belonged to serotype 0:6. Using a P. aeruginosa recipient resistant to rifampicin and ciprofloxacin, conjugal transfer was achieved in two occasions. These plasmids, 100 Mda in size, encoded high-level resistance to both gentamicin and tobramycin whereas resistance to other drugs was not transferable. Interestingly, all 100 Mda plasmids, including the self-tansferable ones, were found to share a certain degree of homology as judged by restriction analysis. It is suggested that both resistance phenotypes and analysis of plasmid content might be useful in subdividing 0:12 multi-resistant P. aeruginosa.     

Molecular epidemiology of two genes encoding 3-N-aminoglycoside acetyltransferases AAC(3)I and AAC(3)II among gram-negative bacteria from a Spanish Hospital

The molecular epidemiology of the aacC1 and aacC2 genes, encoding 3-N-aminoglycoside acetyltransferases AAC(3)I and AAC(3)II, respectively, was studied by DNA-DNA hybridization. The sample included 315 gentamicin-resistant Gram-negative bacilli collected over a six-month period from patients attending a Spanish Hospital. The aminoglycoside resistance phenotype of these strains was also determined. The aacC1 probe hybridized with 39 strains, the aacC2 probe with 146 strains and both probes hybridized with 26 strains. The aacC1 gene was most frequently detected in Pseudomonas aeruginosa whereas the aacC2 gene was most frequently detected in enterobacteria and Acinetobacter spp. Strains harbouring aacC genes were isolated from both in-and outpatients with different infectious diseases, mainly urinary tract infections. As inferred from the results of Southern hybridization, both genes showed a wide horizontal dispersion among plasmids and bacteria.Corresponding author.