Pattern of infection and antibiotic activity among Streptococcus agalactiae isolates from adults in Mashhad,Iran

Background:

One of the main causes of sexually transmitted diseases is group B β- hemolytic streptococci (GBS) multiplying in the genital tracts. Penicillin is the most common drug for the treatment of infections caused by these bacteria, but in patients suffering from Penicillin allergy, Erythromycin and Clindamycin are used as alternative therapeutic drugs against GBS. Recently, resistance to these drugs has been reported more often. In this study, efforts have been made to determine the prevalence and antibiotic resistance of GBS.

Methods:

Modified Christie Atkins Munch-Petersen (CAMP) test was conducted on over 2400 samples of urine and discharge taken from vagina, urethra and prostate. The drug sensitivity was performed by double disk sensitivity tests to Bacitracin, Trimethoprim, and Sulfamethoxazole and then the resistant samples were investigated by E-test to determine the minimal inhibitory concentrations (MICs) value.

Results:

Twenty-three vaginal and 10 urethral discharge, 27urine and 6 prostatic secretion samples were GBS positive. The most symbiotic microorganisms with GBS were strains of Enterococci (90%), Staphylococcus saprophyticus (25%) and Candida albicans (6%). The disk diffusion method showed 18 cases with Penicillin resistance (MIC: 1.5 mg/ml).

Conclusion:

Taken together, GBS carriers’ rate in this study was found 20.65% (8.24% men and 12.4% women). Furthermore, findings showed high-level resistance to Erythromycin and Clindamycin.Key Words: Antibiotic resistance, Genitourinary system, Streptococcus agalactiae, minimal inhibitory concentration (MIC)     

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.     

High rates of macrolide resistance among clinical isolates of Streptococcus agalactiae in Tunisia

One hundred sixty non duplicate erythromycin resistant Streptococcus agalactiae isolates were collected in Tunisia from January 2005 to December 2007 They were investigated to determine their resistance level to different macrolides and the mechanisms involved. Most erythromycin resistant S. agalactiae isolates were isolated from urinary specimens (38.75%, 62/160). The constitutive MLSB phenotype (cMLS) showed in 84.3% (135/160) with high MICs of macrolides and lincosamides (MIC90>256 microg/mL) and 8.2% (13/160) inducible MLSB phenotype (iMLS) with high MICs of macrolides (MIC90>256 microg/mL) and moderately increased MICs of lincosamides (MIC90=8 microg/mL). The M phenotype showed in 7.5% (12/160) with moderately increased MICs of macrolides (MIC90=32 microg/mL) and low MICs of lincosamides (MIC90=0.75 microg/mL). All strains were susceptible to quinupristun-dalfopristin association and linezolid (MIC90: 05 and 0.38 microg/mL respectively). Strains with MLSB phenotype harboured erm(B) gene with 825% (n=132), erm(TR) gene with 8.12% (n=13) and erm(B) plus mef (A) with 1.88% (n=3). All strains categorized as M phenotype carried the mef(A) gene (75%, n=12). cMLSB phenotype conferring cross resistance to macrolides, lincosamides and streptogramins B with high level of resistance was the most prevalent.     

Occurrence of virulence genes in multidrug-resistant Escherichia coli isolates from Iberian wolves (Canis lupus signatus) in Portugal

While much evidence supports the view that the total consumption of antimicrobials is the critical factor in selecting resistance, the possibility of resistant isolates and/or genes encoding resistance being transferred among different living communities has raised serious concerns. In the present study, Escherichia coli isolates recovered from faecal samples (n?=?34) of Iberian wolves (Canis lupus signatus) were characterized for their antimicrobial drug susceptibility. Nearly two thirds of the isolates carried resistance to one or more antimicrobial drugs (in a panel of 19 antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. By screening a set of 20 multidrug-resistant E. coli for virulence genes, we found strains positive for cdt, chuA, cvaC, eaeA, paa and bfpA, which was the most common virulence trait. Phylogenetic analyses have shown that the majority of these E. coli strains fall into phylogenetic groups A and B1. In this study, the diversity of extended-spectrum β-lactamase-producing strains was expressed by both polymorphism of the pulsed-field gel electrophoresis patterns and the presence of various resistance and virulence genes profiles. Finding the specific implications of these multi-resistant bacteria (hosting several virulence factors) in wolf conservation is a challenging topic to be addressed in further investigations.     

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.     

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.     

The macrophage chemotactic activity of Streptococcus agalactiae and Streptococcus iniae extracellular products (ECP)

The ability of Streptococcus agalactiae and Streptococcus iniae to attract macrophages of Nile tilapia (Oreochromis niloticus) was investigated. The extracellular products (ECP) from S. agalactiae and S. iniae were tested in vitro for macrophage chemotaxis using blind-well chambers. The macrophages were obtained from the peritoneal cavity 4-5 days after intraperitoneal injection of squalene. Both macrophage chemotactic and chemokinetic activities were demonstrated using the S. agalactiae ECP. However, only chemotactic activity was shown for S. iniae ECP. High-pressure liquid chromatography fractionation revealed that semi-purified S. agalactiae and S. iniae ECPs had estimated molecular weights of 7.54 and 19.2kDa, respectively. The prominent chemotactic activities of ECP from S. agalactiae and S. iniae are likely to be involved in the proinflammatory responses of macrophages to S. agalactiae and S. iniae infections.     

The cyl genes of Streptococcus agalactiae are involved in the production of pigment

The cyl genes of Streptococcus agalactiae are required for the production of hemolysin. Based on the observation that nonhemolytic S. agalactiae mutants do not produce pigment, a close genetic linkage between hemolysin and pigment has been postulated. To investigate this genetic linkage and to identify genes involved in the production of the S. agalactiae pigment, we screened mutant libraries for nonpigmented clones. Four distinct mutants were isolated with a nonpigmented and nonhemolytic phenotype. The mutations had occurred either in known cyl genes or in two open reading frames located immediately downstream. These novel genes are cotranscribed with the cyl gene cluster and were designated cylF and cylI. Our data indicate that identical genes participate in the production of S. agalactiae hemolysin and pigment.     

The role of surface structures of Streptococcus agalactiae in adhesion to epithelial cells

Induced mutants of S. agalactiae which differed in surface structures were used for the study. The aim of using them was to try to correlate the presence of defined structures or surface properties with the ability of group B streptococci to attach to epithelial cells. The presence of protein antigen R conditioned strong binding of S. agalactiae cells to hydrophobic gel. Strains bearing clumping factor (CF) showed high surface hydrophobicity and presented compact growth in serum soft agar. However, there was no correlation between high surface hydrophobicity and the ability to adhere. Fibrinogen binding decreased the attachment to epithelial cells of CF-positive strains. Preincubation of bacterial cells with lectin (ConA) did not influence the attachment of S. agalactiae strains with protein surface antigen but increased the adhesion of the strains with polysaccharide antigen or untypable.     

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae.

A method for the analysis of lipoteichoic acid (LTA) by polyacrylamide gel electrophoresis (PAGE) is described. Purified LTA from Streptococcus agalactiae tended to smear in the upper two-thirds of a 30 to 40% linear polyacrylamide gel, while the chemically deacylated form (cdLTA) migrated as a ladder of discrete bands, reminiscent of lipopolysaccharides. The deacylated polymer appeared to separate in this system on the basis of size, as evident from results obtained from PAGE analysis of cdLTA subjected to limited acid hydrolysis and LTA that had been fractionated by gel filtration. A survey of cdLTA from other streptococci revealed similarities in molecular weight ranges. The polymer from Enterococcus hirae was of a higher molecular weight. This procedure was used to examine the effect of penicillin and chloramphenicol on the synthesis, turnover, and heterogeneity of LTA in S. agalactiae. Penicillin appeared to enhance LTA synthesis while causing the release of this polymer into the supernatant fluid. In contrast, chloramphenicol inhibited the synthesis of this molecule and resulted in its depletion from the cell surface. Penicillin did not alter the heterogeneity of this polymer, but chloramphenicol caused an apparent shift to a lower-molecular-weight from of the LTA, as determined by PAGE. This shift in the heterogeneity of LTA did not appear to be due to increased carbohydrate substitution, since chloramphenicol did not alter the electrophoretic migration profile of LTA from E. hirae. From a pulse-chase study, it was determined that LTA was released as a consequence of deacylation.     

Multiple interactions of FbsA, a surface protein from Streptococcus agalactiae, with fibrinogen: affinity, stoichiometry, and structural characterization

Streptococcus agalactiae is an etiological agent of several infective diseases in humans. We previously demonstrated that FbsA, a fibrinogen-binding protein expressed by this bacterium, elicits a fibrinogen-dependent aggregation of platelets. In the present communication, we show that the binding of FbsA to fibrinogen is specific and saturable, and that the FbsA-binding site resides in the D region of fibrinogen. In accordance with the repetitive nature of the protein, we found that FbsA contains multiple binding sites for fibrinogen. By using several biophysical methods, we provide evidence that the addition of FbsA induces extensive fibrinogen aggregation and has noticeable effects on thrombin-catalyzed fibrin clot formation. Fibrinogen aggregation was also found to depend on FbsA concentration and on the number of FbsA repeat units. Scanning electron microscopy evidentiated that, while fibrin clot is made of a fine fibrillar network, FbsA-induced Fbg aggregates consist of thicker fibers organized in a cage-like structure. The structural difference of the two structures was further indicated by the diverse immunological reactivity and capability to bind tissue-type plasminogen activator or plasminogen. The mechanisms of FbsA-induced fibrinogen aggregation and fibrin polymerization followed distinct pathways since Fbg assembly was not inhibited by GPRP, a specific inhibitor of fibrin polymerization. This finding was supported by the different sensitivity of the aggregates to the disruptive effects of urea and guanidine hydrochloride. We suggest that FbsA and fibrinogen play complementary roles in contributing to thrombogenesis associated with S. agalactiae infection.     

Conjugative R plasmids in Streptococcus agalactiae (group B).

Twenty-one drug-resistant clinical isolates of group B streptococci were investigated for drug-resistance transfer by conjugation. Six strains were resistant to tetracycline, two to chloramphenicol, one to both drugs, and twelve to macrolide antibiotics (erythromycin, oleandomycin and spiramycin), lincomycin, pristinamycin I, and/or chloramphenicol and tetracycline. Ten strains carried R plasmids which were transferable to group B and/or group D recipients by a conjugation-like phenomenon. Six plasmids were transferred at a high frequency (9 × 10^?2 to 4 × 10^?4) and four, at low frequency (5 × 10^?6 to 7 × 10^?8). The molecular weight of one plasmid (pIP501) was investigated after transfer into the new hosts and was found to be similar to that carried by the wild strain (19.8 × 10⁶).     

First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncatus)

The isolation and characterization of two bacterial species, Streptococcus agalactiae and Lactococcus garvieae, previously unreported in wild marine mammals are described from a freshly dead bottlenose dolphin, Tursiops truncatus, from Kuwait Bay, Kuwait, in September 2001. Conventional and rapid identification systems were used to determine that isolates from muscle and kidney were S. agalactiae and L. garvieae, respectively. The isolates were gram-positive, catalase-negative, oxidase-negative, nonhemolytic cocci. The S. agalactiae was serotyped to group antigen B, whereas the L. garvieae could not be assigned to any serogroup. These Kuwait isolates displayed considerable homogeneity with corresponding American Type Culture Collection (ATCC) type isolates. Although the dolphin S. agalactiae isolate was nonhemolytic, it was biochemically similar to S. agalactiae isolated from mullet sampled in the concurrent Kuwait Bay fish kill. Some biochemical heterogeneity was observed between the dolphin isolates and corresponding mammalian ATCC type isolates, especially with Voges Proskauer, alanine-phenylanaline-proline arylamidase, and alpha-galactosidase tests. Nile tilapia, Oreochromis niloticus, experimentally infected with the dolphin S. agalactiae and L. garvieae isolates experienced 90% and 0% mortalities, respectively. This is the first isolation of S. agalactiae and L. garvieae from a wild marine mammal, and the microbial characteristics established here provide pertinent information for the future isolation of these bacteria.     

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.     

Purification of the protein X of Streptococcus agalactiae with a monoclonal antibody

The protein X of Steptococcus agalactiae is a surface antigen included in the typing scheme of group B streptococci (GBS). We have developed a monoclonal antibody to the protein X and used it to purify this antigen by affinity chromatography. Electrophoresis in polyacrylamide, and immunoblotting using the monoclonal antibody or a rabbit antiserum raised with the affinity purified protein X, revealed a major band in the region of 200 kDa and a smaller one at 100 kDa. The isolated protein X will make possible investigations of its potential role in virulence and protection.     

Prevalence of IgA receptors in clinical isolates of Streptococcus pyogenes and Streptococcus agalactiae; Serologic distinction between the receptors by blocking antibodies

Abstract Group A and B streptococci ( Streptococcus pyogenes and Streptococcus agalactiae ) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding epitopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.