Joyce de Azavedo

Genetic locus for streptolysin S production by group A streptococcus

ABSTRACT 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 andsagB appears to regulate the amount of sagAtranscript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagAknockout mutant. Finally, heterologous expression of the intactsag 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 sagoperon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.

Prevalence and Mechanisms of Macrolide Resistance in Invasive and Noninvasive Group B Streptococcus Isolates from Ontario, Canada

ABSTRACT 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 ermmethylase 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 thelinB gene, previously identified only inEnterococcus 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.

A Nosocomial Outbreak of Fluoroquinolone-Resistant Streptococcus pneumoniae

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.

Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae: Contributions of Type II Topoisomerase Mutations and Efflux to Levels of Resistance

ABSTRACT 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 ofS. 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.

Description of Staphylococcus Serine Protease (ssp) Operon in Staphylococcus aureus and Nonpolar Inactivation of sspA-Encoded Serine Protease

ABSTRACT 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. aureusRN6390. The ssp gene encoding V8 protease is designated assspA, 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.

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.

Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in Canada during 2000.

ABSTRACT 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 (MIC90s) 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 MIC90s 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.

Streptococcus iniae Virulence Is Associated with a Distinct Genetic Profile

ABSTRACT Streptococcus iniae causes 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 102 CFU 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 108 CFU. 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 invasive S. iniaedisease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.

Interspecies Recombination Contributes Minimally to Fluoroquinolone Resistance in Streptococcus pneumoniae

ABSTRACT Analysis of 71 ciprofloxacin-resistant (MIC ≥ 4 μg/ml)Streptococcus pneumoniae clinical isolates revealed only 1 for which the quinolone resistance-determining regions of theparC, 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.

Decreased Susceptibility ofStreptococcus pneumoniaeto Fluoroquinolones in Canada

BACKGROUND Fluoroquinolones are now recommended for the treatment of respiratory tract infections due to Streptococcus pneumoniae, particularly when the isolates are resistant to beta-lactam antibiotics. Although pneumococci with reduced susceptibility to fluoroquinolones have been identified, their prevalence has not been determined in a defined population. METHODS We performed susceptibility testing on 7551 isolates of S. pneumoniae obtained from surveillance in Canada in 1988 and from 1993 to 1998. Pneumococci with reduced susceptibility to fluoroquinolones (defined as a minimal inhibitory concentration of ciprofloxacin of at least 4 microg per milliliter) were further characterized. We also examined antibiotic prescriptions dispensed in Canadian retail pharmacies. RESULTS Between 1988 and 1997, fluoroquinolone prescriptions increased from 0.8 to 5.5 per 100 persons per year. The prevalence of pneumococci with reduced susceptibility to fluoroquinolones increased from 0 percent in 1993 to 1.7 percent in 1997 and 1998 (P=0.01). Among adults, the prevalence increased from 1.5 percent in 1993 and 1994 combined to 2.9 percent in 1997 and 1998 combined. The prevalence was higher in isolates from older patients (2.6 percent among those 65 years of age or older vs. 1.0 percent among those 15 to 64 years of age, P<0.001) and among those from Ontario (1.5 percent, vs. 0.4 percent among those from the rest of Canada; P< 0.001). Fluoroquinolone use was greatest among the elderly and in Ontario. The 75 isolates (17 serotypes) of pneumococci with reduced susceptibility to fluoroquinolones were submitted by 40 laboratories in eight provinces. Reduced susceptibility to fluoroquinolones was associated with resistance to penicillin. CONCLUSIONS The prevalence of pneumococci with reduced susceptibility to fluoroquinolones is increasing in Canada, probably as a result of selective pressure from the increased use of fluoroquinolones.