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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.

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.

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.

Received 20 January 2004; returned 9 March 2004; revised 19 April 2004; accepted 21 April 2004Objectives: To identify and characterize the mechanisms of high-level fluoroquinolone resistancein two strains of Bacillus anthracis following serial passage in increasing concentrations offluoroquinolones.Methods: Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strainswere obtained following serial passage in the presence of increasing concentrations of four differentfluoroquinolones. The quinolone-resistance-determining regions of the type II topoisomerase genesfrom the resistant strains were amplified by PCR and characterized by DNA sequence analysis. TheMICs in the presence and absence of reserpine were determined using broth microdilution as a meansof detecting active efflux.Results: Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) andGrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common. A single amino acid substitution inGyrB (Asp-430-Asn) was also identified. Efflux only applied to isolates selected for by either levofloxa-cin or ofloxacin.Conclusions: Specific amino acid substitutions in the type II topoisomerase enzymes significantly con-tributed to the development of high-level fluoroquinolone resistance in B. anthracis. However, notabledifferences between the strains and the drugs tested were identified including the role of efflux and thenumbers and types of mutations identified.Keywords: anthrax, DNA gyrase, topoisomerase IV

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in innate immunity. Recent in vitro studies have shown that TNF-alpha may also serve as a growth factor for some bacteria. We examined the physiologic relevance of this phenomenon both in vitro and in vivo. Recombinant mouse TNF-alpha increased in vitro proliferation of Escherichia coli but not Pseudomonas aeruginosa in a concentration-dependent manner, and this effect was attenuated by anti-TNF-alpha antibodies. However, in vivo, TNF-alpha gene-deficient (TNF-alpha-/-) mice showed higher mortality than wild-type (TNF-alpha+/+) mice after inoculation of intranasal bacteria. An impaired bacterial clearance in TNF-alpha-/- mice was associated with decreased systemic concentrations of chemokine macrophage inflammatory protein-2, reduced pulmonary neutrophil recruitment, and depressed expression of neutrophil CD11b and CD16/CD32, suggesting that the effect of TNF-alpha on E. coli growth was outweighed by the recruited neutrophils. We also demonstrated that neutropenic TNF-alpha+/+ mice had approximately 100-fold higher E. coli counts in their lungs than TNF-alpha-/- mice, although survival rates in both groups were similar. We conclude that TNF-alpha augments E. coli growth in vitro and in vivo. However, in vivo, this effect becomes only apparent in neutropenic animals. The relevance of these findings for immune compromised patients remains to be investigated.

The emergence of Streptococcus pneumoniae resistance to β-lactam and macrolide antimicrobial agents has led to recommendations that fluoroquinolones with increased activity against S. pneumoniae, such as levofloxacin, moxifloxacin, and gatifloxacin, be used to treat patients at risk for infection caused by such multidrug-resistant strains (1–6). Fluoroquinolone resistance in S. pneumoniae is primarily due to mutations in the genes encoding the target topoisomerase enzymes, namely parC, which encodes the A subunit of DNA topoisomerase IV, and gyrA, which encodes the A subunit of DNA gyrase (7). Mutations in parE and gyrB have been reported, but to a lesser extent (8–10). Most pneumococcal isolates with reduced susceptibilities to fluoroquinolones have amino acid substitutions in either ParC alone or ParC and GyrA (11–14). Resistance can also be mediated by active efflux (15), although the role of efflux in contributing to resistance by the newer fluoroquinolones is unclear (16). The MIC of an antimicrobial agent is a value that has been used to determine breakpoints that predict the probability of clinical success, detect resistant populations, or both (17). Clinical breakpoints are dependent on the antimicrobial activity and pharmacology of the drug; such breakpoints are ascertained with the goals of eradicating the infection and ultimately achieving clinical success with the antimicrobial agent. In contrast, microbiologic breakpoints are established to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. These microbiologic breakpoints are therefore useful in monitoring the emergence of resistance. The current National Committee for Clinical Laboratory Standards (NCCLS) guidelines make no distinction between these two interpretations of MIC, with clinical breakpoints used to characterize most antimicrobial agents, including the fluoroquinolones. Levofloxacin has been used as a surrogate marker to predict fluoroquinolone susceptibility in clinical laboratories and surveillance studies (18). To establish whether current levofloxacin breakpoints are also able to function as microbiologic breakpoints, we determined the percentage of S. pneumoniae isolates with first-step parC mutations that would go undetected by using the current NCCLS breakpoints for levofloxacin (19).

A total of 2,245 clinical isolates of Streptococcus pneumoniae were collected from 63 microbiology laboratories from across Canada during 2000 and characterized at a central laboratory. Of these isolates, 12.4% were not susceptible to penicillin (penicillin MIC, ≥0.12 μg/ml) and 5.8% were resistant (MIC, ≥2 μg/ml). Resistance rates among non-β-lactam agents were the following: macrolides, 11.1%; clindamycin, 5.7%; chloramphenicol, 2.2%; levofloxacin, 0.9%; gatifloxacin, 0.8%; moxifloxacin, 0.4%; and trimethoprim-sulfamethoxazole, 11.3%. The MICs at which 90% of the isolates were inhibited (MIC 90 s) of the fluoroquinolones were the following: gemifloxacin, 0.03 μg/ml; BMS-284756, 0.06 μg/ml; moxifloxacin, 0.12 μg/ml; gatifloxacin, 0.25 μg/ml; levofloxacin, 1 μg/ml; and ciprofloxacin, 1 μg/ml. Of 578 isolates from the lower respiratory tract, 21 (3.6%) were inhibited at ciprofloxacin MICs of ≥4 μg/ml. None of the 768 isolates from children were inhibited at ciprofloxacin MICs of ≥4 μg/ml, compared to 3 of 731 (0.6%) from those ages 15 to 64 (all of these >60 years old), and 27 of 707 (3.8%) from those over 65. The MIC 90 s for ABT-773 and telithromycin were 0.015 μg/ml for macrolide-susceptible isolates and 0.12 and 0.5 μg/ml, respectively, for macrolide-resistant isolates. The MIC of linezolid was ≤2 μg/ml for all isolates. Many of the new antimicrobial agents tested in this study appear to have potential for the treatment of multidrug-resistant strains of pneumococci.

Macrolide resistance has been demonstrated in group B streptococcus (GBS), but there is limited information regarding mechanisms of resistance and their prevalence. We determined these in GBS obtained from neonatal blood cultures and vaginal swabs from pregnant women. Of 178 isolates from cases of neonatal GBS sepsis collected from 1995 to 1998, 8 and 4.5% were resistant to erythromycin and clindamycin, respectively, and one isolate showed intermediate penicillin resistance (MIC, 0.25 μg/ml). Of 101 consecutive vaginal or rectal/vaginal isolates collected in 1999, 18 and 8% were resistant to erythromycin and clindamycin, respectively. Tetracycline resistance was high (>80%) among both groups of isolates. Of 32 erythromycin-resistant isolates, 28 possessed the erm methylase gene (7 ermB and 21 ermTR/ermA ) and 4 harbored the mefA gene; one isolate harbored both genes. One isolate which was susceptible to erythromycin but resistant to clindamycin (MIC, 4 μg/ml) was found to have the linB gene, previously identified only in Enterococcus faecium . The mreA gene was found in all the erythromycin-resistant strains as well as in 10 erythromycin-susceptible strains. The rate of erythromycin resistance increased from 5% in 1995–96 to 13% in 1998–99, which coincided with an increase in macrolide usage during that time.

Studies of the antimicrobial activity of neutrophil defensins have mostly been carried out in microbiological media, and their effects on the host defense in physiological conditions are unclear. We examined 1) the antibacterial activity of defensins in physiological media with and without lung tissue present, 2) the effect of defensins on hydrogen peroxide (H2O2) production by lung tissue that had been exposed to bacteria, and 3) the effect of diphenyleneiodonium (DPI), an inhibitor of reactive oxygen species formation, on the antibacterial activity of defensins in the presence of lung tissue. Defensins were incubated with Escherichia colior Pseudomonas aeruginosa in the absence or presence of primary cultured mouse lung explants. Defensins reduced bacterial counts by ∼65-fold and ∼25-fold, respectively, at 48 h; bacterial counts were further decreased by ∼600-fold and ∼12,000-fold, respectively, in the presence of lung tissue. Defensins induced H2O2production by lung tissue, and the rate of killing of E. coli by defensins was reduced by ∼2,500-fold in the presence of 10 μM DPI. We conclude that defensins exert a significant antimicrobial effect under physiological conditions and that this effect is enhanced in the presence of lung tissue by a mechanism that involves the production of reactive oxygen species.

Over the course of a 20-month period, in a hospital respiratory ward in which ciprofloxacin was often used as empirical antimicrobial therapy for lower respiratory tract infections (LRTIs), 16 patients with chronic bronchitis developed nosocomial LRTIs caused by penicillin- and ciprofloxacin-resistant Streptococcus pneumoniae (serotype 23 F). The minimum inhibitory concentration (MIC) of ciprofloxacin for all isolates from the first 9 patients was 4 microg/mL, in association with a parC mutation. Isolates from the subsequent 7 patients all had a ciprofloxacin MIC of 16 microg/mL, in association with an additional mutation in gyrA. The MICs for this isolate were 8 microg/mL of levofloxacin (resistant), 2 microg/mL of moxifloxacin and gatifloxacin (intermediately resistant), and 0.12 microg/mL of gemifloxacin. This outbreak demonstrates the ability of S. pneumoniae to acquire multiple mutations that result in increasing levels of resistance to the fluoroquinolones and to be transmitted from person to person.

Streptococcus iniaecauses meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsed-field gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 102CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (107) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 108CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasiveS. iniaedisease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.

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.

We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of S. pneumoniae for which the ciprofloxacin MIC is ≥4 μg/ml and 28 isolates for which the ciprofloxacin MIC is ≤2 μg/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 μg/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 μg/ml) and high-level (MICs, 64 μg/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.

Streptococcus iniae is a cause of septicemia, meningoencephalitis, and death in farmed fish and of cellulitis in human beings. A set of nested oligonucleotide PCR primers that specifically amplified a 373-bp subunit from a variety of clinical isolates from farmed fish and human patients were constructed from a 524-bp consensus sequence of the S. iniae 16S-23S ribosomal DNA intergenic spacer.

Of 500 group A streptococci isolated from pharyngeal swabs, 72 (14.4%) were macrolide resistant, compared to 2.1% in 1997. Of these, 66 (92%) were of the M phenotype and 6 (8.3%) were of the MLS phenotype. Pulsed-field gel electrophoresis found that two clones, with patterns identical to those of serotypes M1 and M4, accounted for 19.4 and 68.1% of the macrolide-resistant isolates, respectively.

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.

Streptolysin S (SLS) is responsible for the hallmark Phemolytic phenotype seen around colonies of Group A Streptococcus (GAS), also known as Streptococcus pyogenes, when cultured on blood agar. It has proven difficult to characterize due to the difficulty of obtaining purified preparations since additional factors are required for synthesis and a carrier molecule is needed to provide stability in solution. However, attempts at N-terminal sequencing of the peptide proved to be unsuccessful, and the amino acid structure remains a mystery despite more recent genetic information as to the structural gene for SLS. Although the cytolytic capacity of SLS has been well documented, its contribution to the pathogenesis ofGAS disease was not clear until the use of SLS-deficient mutants demonstrated a role in lesion formation and tissue necrosis. Recent work also provides evidence that the SLS RNA transcript might act as a regulator for other GAS determinants, in which case its role could prove to be complex. This chapter summarizes early work and focus on the more recent findings, such as its genetic organization, regulation, and possible role(s) in the pathophysiology of streptococcal infections. The amino acid structure of the SLS peptide has not been elucidated, nor have the theoretical predictions regarding its processing been verified. Whether the sagA transcript alone acts as a regulator or whether SLS may also function as a quorum-sensing molecule deserves further investigation.

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 μg/ml; all nine had parC mutations, and eight were serotype M6.

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°C or less was a predictor of death. Furthermore, viable bacterial counts in the lungs of mice euthanized at a temperature of ≤ 30°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 ≥ 32°C) at the start of treatment had better clinical and bacteriological outcomes than mice assessed as severely ill (temperature < 32°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.

This report describes four patients with pneumococcal pneumonia in whom empirical treatment with levofloxacin failed. In these patients, the isolates of Streptococcus pneumoniae were resistant to levofloxacin, and in two of them the resistance appeared to have been acquired during the current course of treatment with fluoroquinolones.

Summary Background We encountered three patients with severe necrotising soft tissue infections due to β-haemolytic group G streptococcus. Due to strong clinical similarities with invasive infections produced by group A streptococcus, we investigated a potential link of shared β-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 β-haemolytic activity. The requirement of group G streptococcus β-haemolysin in producing necrotising infection was examined in mice. Findings Each patient had an underlying medical condition.β-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 β-haemolysin streptolysin S (SLS). Targeted mutagenesis of the putative SLS structural gene sag A in group G streptococcus eliminated β-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 Gstreptococcus SLS-negative mutants. Interpretation In patients with underlying medical conditions, β-haemolytic group G streptococcus can produce necrotising soft tissue infections resembling those produced by group A streptococcus. The β-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.

Analysis of 71 ciprofloxacin-resistant (MIC ≥ 4 μg/ml) Streptococcus pneumoniae clinical isolates revealed only 1 for which the quinolone resistance-determining regions of the parC , parE , and gyrB genes were genetically related to those of viridans group streptococci. Our findings support the occurrence of interspecies recombination of type II topoisomerase genes; however, its contribution to the emergence of quinolone resistance among pneumococci appears to have been minimal.

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA , and is followed by sspB , which encodes a 40.6-kDa cysteine protease, and sspC , which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA , encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671–1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project ( www.genome.ou.edu/strep.html ), we have now identified a contiguous nine-gene locus ( sagA to sagI ) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis . We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.

Antimicrobial resistance was characterized for 14 strains of Streptococcus mitis . Hin fI restriction fragment length mapping of gyrA PCR amplicons from three ciprofloxacin-resistant isolates correlated with mutations associated with such resistance in other organisms. By using PCR, seven erythromycin-resistant strains were found to possess either the mef or ermB gene. Hybridization revealed tet (M) in seven tetracycline-resistant isolates.

Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn 916 insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn 916 donor strain Enterococcus faecalis CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn 916 insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated sagA , rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, gained weight during the first 24 h (+1.15 versus −1.16 g; P < 0.05) and had fewer necrotic lesions (0 versus 7; P = 0.0007). Animals infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus −0.66 g; P < 0.05) and produced fewer necrotic lesions (1 versus 8; P = 0.001). Revertants of the mutants in which Tn 916 had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn 916 mutations, are markedly less virulent than their isogenic parents.

S. pneumoniae is a major cause of meningitis, otitis media and pneumonia. Treatment of these infections is becoming a challenge since resistance to penicillin continues to rise worldwide. Penicillin resistance in clinical isolates was first reported in 19655 and has increased rapidly since then, particularly in eastern Europe, with some countries such as Hungary reporting rates of up to 70%8. Resistance to multiple antibiotics emerged in 19784 and also appears to be increasing. In Canada, the first multidrug-resistant strain was reported in 19836 and in a survey carried out in 1989, 11 of 551 (1.5%) isolates were penicillin- resistant with only a few demonstrating reduced susceptibility to multiple agents9. In a recent study, the prevalence of penicillin-resistant S. pneumoniae isolated from community and teaching hospitals across Canada, had increased to 9.5%7.