Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia

Aims

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP‐PCR) techniques.

Methods and Results

A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP‐PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP‐PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.

Conclusions

Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.

Significance and Impact of the Study

Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.     

Determination of epidemiological relationships of Streptococcus agalactiae isolated from bovine mastitis

In the present study 79 streptococcal cultures isolated from subclinical mastitis of 54 cows from seven dairy farms (A-G) in Hesse, Germany, were comparatively investigated using conventional and molecular methods. The isolates could be identified as Streptococcus agalactiae, belonging to Lancefield's serological group B by determination of cultural, biochemical and serological properties and by polymerase chain reaction (PCR)-mediated amplification of species-specific parts of the 16S ribosomal DNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor gene cfb. The investigated group B streptococci were further characterized serologically for specific polysaccharide and protein antigens. Serotyping the isolates revealed a predominance of surface protein antigen X, either alone or in combination with polysaccharide antigen Ia. This could be observed for 39 isolates of farms A, B and C. Six group B streptococci from farm E displayed the serotype pattern III/Rib, two isolates from farm G showed the serotype pattern Ib/calpha. The remaining cultures from farms D and F (n=32) were non-typable. The occurrence of protein Rib could be confirmed by PCR amplification of the gene rib. The two isolates with serotype pattern Ib/calpha also reacted positively for the cbeta-encoding gene bag. Additional properties which allowed a phenotypic characterization of the S. agalactiae were the degree of pigmentation, growth properties in fluid media and soft agar, the surface hydrophobicity, the ability to hemagglutinate rabbit erythrocytes and their resistance reactions to tetracycline and minocycline. The isolates of the seven farms showed identical or almost identical characteristics. The 79 group B streptococci were additionally investigated by macrorestriction analysis of their chromosomal DNA using the restriction endonucleases SmaI, ApaI and SalI. The restriction patterns obtained by pulsed-field gel electrophoresis displayed identical or closely related patterns for the cultures of the various farms. The pheno- and genotypic characteristics of the 79 group B streptococci of the present study revealed that a single S. agalactiae strain or at least closely related subtypes of this strain were responsible for the mastitis situation of the seven farms.     

A Streptococcus uberis transposon mutant screen reveals a negative role for LiaR homologue in biofilm formation

Deinococcus spp are among the most radiation‐resistant micro‐organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro‐organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.     

Probiotic Lactobacillus sp. inhibit growth,biofilm formation and gene expression of caries‐inducing Streptococcus mutans

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real‐time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH‐neutralized, catalase‐treated or trypsin‐treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH‐dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide‐dependent antimicrobial activities. All biofilm‐forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.     

Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium

Photoautotrophic biofilms play an important role in various aquatic habitats and are composed of prokaryotic and/or eukaryotic organisms embedded in extracellular polymeric substances (EPS). We have isolated diatoms as well as bacteria from freshwater biofilms to study organismal interactions between representative isolates. We found that bacteria have a strong impact on the biofilm formation of the pennate diatom Achnanthidium minutissimum. This alga produces extracellular capsules of insoluble EPS, mostly carbohydrates (CHO), only in the presence of bacteria (xenic culture). The EPS themselves also have a strong impact on the aggregation and attachment of the algae. In the absence of bacteria (axenic culture), A. minutissimum did not form capsules and the cells grew completely suspended. Fractionation and quantification of CHO revealed that the diatom in axenic culture produces large amounts of soluble CHO, whereas in the xenic culture mainly insoluble CHO were detected. For investigation of biofilm formation by A. minutissimum, a bioassay was established using a diatom satellite Bacteroidetes bacterium that had been shown to induce capsule formation of A. minutissimum. Interestingly, capsule and biofilm induction can be achieved by addition of bacterial spent medium, indicating that soluble hydrophobic molecules produced by the bacterium may mediate the diatom/bacteria interaction. With the designed bioassay, a reliable tool is now available to study the chemical interactions between diatoms and bacteria with consequences for biofilm formation.     

Molecular serotype identification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.     

Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Inhibitory effects of 7-epiclusianone on glucan synthesis, acidogenicity and biofilm formation by Streptococcus mutans

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.