The two-component system sivS/R regulates virulence in Streptococcus iniae.

Streptococcus iniae causes invasive disease and death in fish, and to a lesser extent, sporadic cases of soft-tissue infections in humans. A two-component system termed sivS/R, which regulates capsule expression, was previously identified and characterized. In this study, it is shown that a sivS/R deletion-insertion mutant, termed 9117Deltasiv, causes transient bacteremia and reduced virulence compared with the parent strain when tested in a murine model of bacteremic infection. Furthermore, real-time PCR studies indicated that SivS/R regulates the expression levels of the streptolysin S structural gene, sagA, as well as the CAMP factor gene, cfi. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of S. iniae spheroplasts revealed downregulation of three surface proteins in the mutant strain compared with the parent strain. These proteins were identified by MS to be a putative lipoprotein, a hyaluronate-associated protein and a pyruvate kinase. This study demonstrates that SivS/R regulates virulence in vivo, and controls the expression of a number of genes in S. iniae.

Capsule expression regulated by a two-component signal transduction system in Streptococcus iniae.

Streptococcus iniae causes disease in fish and humans and the presence of capsule is associated with virulence. Tn917 transposon mutagenesis was performed to identify capsule-associated genes and a mutant was isolated, with an insertion in a genetic locus encoding a two-component signal transduction system (TCS), which we termed sivS/R. sivS and sivR encode a 506-amino-acid (aa) putative histidine kinase and a 223-aa putative response regulator, respectively. In order to investigate the role of sivS/R, a deletion-insertion mutant was constructed using a PCR ligation technique. Real-time PCR showed that transcription of cpsA, the first gene in the S. iniae capsule operon, was reduced in the mutant, indicating that sivS/R regulates expression of this gene at the transcriptional level. Whole human blood killing assays demonstrated that unlike the parent, the mutant was susceptible to phagocytosis. Transmission electron microscopy showed exopolysaccharide on the surface of the parent strain but not the mutant which showed aberrant asymmetric septae that resulted in clumps of abnormal-shaped cells. Exponential growth rates of the mutant and parent strain were similar, although the mutant exhibited a longer lag phase. We conclude that sivS/R regulates capsule expression, thus affecting the ability to evade phagocytosis.

Identification of group A Streptococcus antigenic determinants upregulated in vivo.

Group A Streptococcus (GAS) causes a range of diseases in humans, from mild noninvasive infections to severe invasive infections. The molecular basis for the varying severity of disease remains unclear. We identified genes expressed during invasive disease using in vivo-induced antigen technology (IVIAT), applied for the first time in a gram-positive organism. Convalescent-phase sera from patients with invasive disease were pooled, adsorbed against antigens derived from in vitro-grown GAS, and used to screen a GAS genomic expression library. A murine model of invasive GAS disease was included as an additional source of sera for screening. Sequencing DNA inserts from clones reactive with both human and mouse sera indicated 16 open reading frames with homology to genes involved in metabolic activity to genes of unknown function. Of these, seven genes were assessed for their differential expression by quantitative real-time PCR both in vivo, utilizing a murine model of invasive GAS disease, and in vitro at different time points of growth. Three gene products-a putative penicillin-binding protein 1A, a putative lipoprotein, and a conserved hypothetical protein homologous to a putative translation initiation inhibitor in Vibrio vulnificus-were upregulated in vivo, suggesting that these genes play a role during invasive disease.

Prevalence and characterization of invasive isolates of Streptococcus pyogenes with reduced susceptibility to fluoroquinolones.

Fluoroquinolone susceptibility testing was performed on invasive group A streptococcus isolates from 1992-1993 and 2003 from Ontario, Canada. None were nonsusceptible to levofloxacin. Two of 153 (1.3%) from 1992-1993 and 7 of 160 (4.4%) from 2003 had a levofloxacin MIC of 2 mug/ml; all nine had parC mutations, and eight were serotype M6.

Novel murine model of pneumococcal pneumonia: use of temperature as a measure of disease severity to compare the efficacies of moxifloxacin and levofloxacin.

Surface temperature measured by an infrared temperature-scanning thermometer was used to evaluate disease severity and predict imminent death in a murine model of pneumococcal pneumonia. We showed that a decrease in temperature was associated with increasing severity of disease and concomitant histological changes and also that a temperature of 30 degrees C or less was a predictor of death. Furthermore, viable bacterial counts in the lungs of mice euthanized at a temperature of < or = 30 degrees C were not significantly different from those seen in the lungs of mice allowed to die without intervention. These data support temperature change as a more subtle indicator of outcome than death and demonstrate that this could be used as a reliable end point for euthanasia. To test the utility of our model in a drug trial, we examined the efficacies of moxifloxacin and levofloxacin by using temperature as a measure of disease severity prior to and during treatment. Regardless of the antibiotic used, mice assessed as moderately ill (temperature > or = 32 degrees C) at the start of treatment had better clinical and bacteriological outcomes than mice assessed as severely ill (temperature < 32 degrees C). However, moxifloxacin offered better protection and greater bacterial clearance than did levofloxacin in all infected mice independent of disease severity. This model not only allows a more subtle evaluation of drug efficacy but also ensures a better degree of standardization and a more humane approach to drug efficacy studies involving animals.

Type II topoisomerase mutations in Bacillus anthracis associated with high-level fluoroquinolone resistance.

OBJECTIVES: To identify and characterize the mechanisms of high-level fluoroquinolone resistance in two strains of Bacillus anthracis following serial passage in increasing concentrations of fluoroquinolones. METHODS: Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strains were obtained following serial passage in the presence of increasing concentrations of four different fluoroquinolones. The quinolone-resistance-determining regions of the type II topoisomerase genes from the resistant strains were amplified by PCR and characterized by DNA sequence analysis. The MICs in the presence and absence of reserpine were determined using broth microdilution as a means of detecting active efflux. RESULTS: Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) and GrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common. A single amino acid substitution in GyrB (Asp-430-Asn) was also identified. Efflux only applied to isolates selected for by either levofloxacin or ofloxacin. CONCLUSIONS: Specific amino acid substitutions in the type II topoisomerase enzymes significantly contributed to the development of high-level fluoroquinolone resistance in B. anthracis. However, notable differences between the strains and the drugs tested were identified including the role of efflux and the numbers and types of mutations identified.

Identification of a streptolysin S-associated gene cluster and its role in the pathogenesis of Streptococcus iniae disease.

Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.

Activities of new fluoroquinolones, ketolides, and other antimicrobials against blood culture isolates of viridans group streptococci from across Canada, 2000.

The rates of nonsusceptibility to penicillin, erythromycin, and clindamycin of 191 blood culture isolates of viridans group streptococci collected from across Canada in 2000 were 36, 42, and 10%, respectively. Although 8% of the strains were resistant to ciprofloxacin (MIC >or= 4 microg/ml), the MICs of gemifloxacin, BMS 284756, telithromycin, and ABT 773 at which 90% of the strains were inhibited were 0.06, 0.06, 0.12, and 0.03 microg/ml, respectively.

Streptolysin S and necrotising infections produced by group G streptococcus.

BACKGROUND: We encountered three patients with severe necrotising soft tissue infections due to beta-haemolytic group G streptococcus. Due to strong clinical similarities with invasive infections produced by group A streptococcus, we investigated a potential link of shared beta-haemolytic phenotype to disease pathogenesis. METHODS: Hybridisation, DNA sequencing, targeted mutagenesis, and complementation studies were used to establish the genetic basis for group G streptococcus beta-haemolytic activity. The requirement of group G streptococcus beta-haemolysin in producing necrotising infection was examined in mice. FINDINGS: Each patient had an underlying medical condition. beta-haemolytic group G streptococcus was the sole microbial isolate from debrided necrotic tissue. The group G streptococcus chromosome contained a homologue of the nine-gene group A streptococcus sag operon encoding the beta-haemolysin streptolysin S (SLS). Targeted mutagenesis of the putative SLS structural gene sagA in group G streptococcus eliminated beta-haemolytic activity. Mice injected subcutaneously with wild-type group A streptococcus or group G streptococcus developed an inflammatory lesion with high bacterial counts, marked neutrophil infiltration, and histopathological evidence of diffuse tissue necrosis. These changes were not found in mice injected with the isogenic group A streptococcus or group G streptococcus SLS-negative mutants. INTERPRETATION: In patients with underlying medical conditions, beta-haemolytic group G streptococcus can produce necrotising soft tissue infections resembling those produced by group A streptococcus. The beta-haemolytic phenotype of group G streptococcus is produced by the exotoxin SLS, encoded by a functional homologue of the nine-gene group A streptococcus sag operon. SLS expression contributes to the pathogenesis of streptococcal necrotising soft tissue infection.

Quinolone Resistance: Older Concepts and Newer Developments.

New quinolone compounds have been recommended for use in the treatment of respiratory tract infections, particularly pneumonia caused by multi drug-resistant Streptococcus pneumoniae. Of concern, however, is the recent emergence of pneumococcal isolates with reduced susceptibilities to both old and new quinolone compounds. This necessitates the employment of quinolone-use strategies aimed at restricting the emergence of resistance, to extend the effectiveness of this very important class of antibacterial agents. This article provides a comprehensive review of the recent discoveries in type II topoisomerase/quinolone structure-function relationships. It also addresses new insights into the mechanisms of quinolone resistance, the predicted trends in quinolone resistance, and possible strategies for quinolone use against S. pneumoniae.