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Increased activity of 16-membered lactone ring macrolides against erythromycin-resistant Streptococcus pyogenes and Streptococcus pneumoniae: characterization of South African isolates.

The susceptibility of 40 erythromycin-resistant isolates of Streptococcus pyogenes and 40 multiply-resistant isolates of Streptococcus pneumoniae to six macrolide antibiotics, representing 14-, 15- and 16-membered lactone ring structures, was tested. The genetic basis for macrolide resistance in the strains was also determined. Both erm and mef determinants were encountered in the 36 S. pneumoniae isolates tested, but only mef in the five S. pyogenes isolates tested. All isolates showed cross-resistance among the 14-membered macrolides erythromycin, clarithromycin and roxithromycin and the 15-membered macrolide, azithromycin. However, the erythromycin-resistant S. pyogenes isolates retained full susceptibility to spiramycin and josamycin (16-membered agents). These latter two antibiotics were also more active than the other macrolides against erythromycin-resistant S. pneumoniae isolates, especially josamycin which was 8-64 times more active than erythromycin; spiramycin was only two to eight times more active than erythromycin.

Anti-Bacterial Agents↗

Accumulation of ppGpp in Streptococcus pyogenes and Streptococcus rattus following amino acid starvation.

We have re-examined the stringent response of Streptococcus rattus and Streptococcus pyogenes, two organisms that had originally been reported not to accumulate ppGpp following amino acid deprivation. We conclude that ppGpp does accumulate when S. rattus and S. pyogenes are deprived of isoleucine by mupirocin addition. The kinetics of ppGpp accumulation was faster in S. pyogenes compared with S. rattus. Cell fractionation and analysis of in vitro ppGpp synthesis showed that in S. pyogenes most activity was associated with the S-100 ribosomal pellet, whereas in S. rattus the S-100 soluble fraction contained greater activity. The addition of 20% methanol or salt-washed ribosomes to the assay mixture did not stimulate the in vitro (p)ppGpp synthesis activity of fractions isolated from S. rattus or S. pyogenes. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that neither organism cross-reacted under conditions that detected RelA in E. coli CF1648. However, cross-reaction with anti-RelSeq antibody was observed in S. pyogenes but not S. rattus, suggesting that ppGpp synthesis is carried out by a putative SpoT protein in S. pyogenes and by a functionally unknown protein in S. rattus.

Amino Acids↗

Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates.

Resistance of streptococci to macrolide antibiotics is caused by target-site modification or drug efflux. The phenotypic expression of target-site modification can be inducible or constitutive. The prevalence of the three phenotypes among Belgian erythromycin-resistant Group A streptococci (GAS) and Streptococcus pneumoniae isolates was surveyed, their MICs for seven antibiotics were determined and the clonality of the isolates was explored. Of the 2014 GAS isolates tested 131(6.5%) were erythromycin resistant (MIC > 1 mg/L): 110 (84.0%) showed the M-resistance phenotype whereas the remaining 21 strains (16.0%) were constitutively resistant. No inducibly resistant strains were detected. Of 100 S. pneumoniae isolates, 33 were erythromycin resistant (MIC > 1 mg/L). In contrast to the GAS isolates, only 9.1% of the 33 erythromycin-resistant S. pneumoniae isolates showed the M-resistance phenotype. The presence of mefA/E and ermB genes in the M-resistant and constitutively and inducibly resistant strains, respectively, was confirmed by PCR analysis. Genomic analysis based on pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SfiI, revealed 54 different PFGE patterns among the 131 erythromycin-resistant GAS isolates, of which an M6 clone represented 16.0% of the strains; all other clones, exhibiting different M-types, represented <7% of the strains. The S. pneumoniae isolates also appeared to be polyclonally based, as determined by arbitrarily primed PCR. The macrolides miocamycin and rovamycin, the lincosamide clindamycin and the ketolide HMR 3647 showed excellent activity against the M-resistant GAS and S. pneumoniae strains.

Anti-Bacterial Agents↗

Genotype and phenotype relationship in MLSb resistance in Streptococcus pyogenes and Streptococcus agalactiae isolated in central Italy.

The aim of this work is to study a correlation between phenotype and genotype in clinical isolates of erythromycin-resistant Streptococcus spp. Among the 25 erythromycin-resistant S. pyogenes, we detected six strains with iMLSB, nine with cMLSB and two with M phenotypes. Among 14 erythromycin-resistant S. agalactiae, we detected five strains with iMLSB, seven with cMLSB and none with an M phenotype. Moreover, 8 S. pyogenes and 2 S. agalactiae showed a phenotype not matching the known ones described in literature, defining an unknown phenotype. Upon examination, the genetic profiles, erm(A), erm(B) and mef(A), of the clinical isolates did not easily correlate with a specific phenotype. Our findings highlighted that the whole matter of phenotypic diversity in macrolide-resistant S. pyogenes and S. agalactiae strains and the correlation with their genetic profiles should be submitted to a more careful analysis of phenotypic and genotypic characterization.

Anti-Bacterial Agents↗

[The morphology of microcolonies as a criterion for recognizing bacteria in the differentiation of Streptococcus pyogenes and Streptococcus group B].

The authors studied the diagnostic importance of the morphology of microcolonies of S. pyogenes and group B streptococci in comparison with the currently used tests for the differentiation of these two species: the bacitracin test, the hydrolysis of sodium hippurate and the CAMP test. The standard tests proved to be positive in 94-97% and microcolonies had typical morphology in 86-95.7%. The statistical indices of the diagnostic effectiveness of differential tests varied within 93.8-98.9%. The diagnostic value of the study of the morphology of colonies was characterized by the following data: the sensitivity and prognostic negative value of the study were 95% for S. pyogenes and 86-89% for group B streptococci, while its specificity and prognostic positive value were 100% due to the absence of false positive results.

Bacteriological Techniques↗

[In vitro activity of josamycin in a comparison of Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae].

The antibacterial effect of josamycin has been tested in vitro on strains of S. Aureus, Str. Pyogenes and Str. Pneumoniae isolated during 1981 from human liquor, blood and purulent secretions. It is confirmed that even modest doses of the antibiotic have this effect upon S. Aureus strains which are resistant to several antibiotics including erythromycin and lincomycin. In some cases, although bacteriostasis occurred, bacterial survival indices were fairly modest.

Dose-Response Relationship, Drug↗

[Evaluation of rapid diagnostic kits for the detection of group A streptococcus to Streptococcus pyogenes and Streptococcus spp. with Lancefield's group A antigen].

We studied the basic performance of eight rapid diagnostic kits for the detection of Group A streptococcus by immunochromatography under the same conditions. Kits were the; QuickVue Dipstick Strep A (Sumitomo Seiyaku Biomedical Co., Ltd.), TESTPACK Plus STREP A (ABBOTT JAPAN Co., Ltd), CLEAVIEW STREP A (Nihon Schering K. K.), QuickVue STREP A (Wako Pure Chemical Industries, Ltd), ImmunoCard STAT! STREP A (TFB, INC.), DIPSTICK 'Eiken' STREP A (Eiken Chemical Co., Ltd.), Rapid Testa Strep A (Daiichi Pure Chemical Co., Ltd.), and StatCheck Strep A (KAINOS Laboratories, Inc.). Four of these kits, i.e. QuickVue Dipstick Strep A, TESTPACK Plus STREP A, Rapid Testa Strep A, and StatCheck Strep A showed sensitivity at 1.0 x 10(5) CFU/mL (1.0 x 10(4)CFU/test) with all of S. pyogenes tested, while the Anginosus group and S. dysgalactiae subsp. equisimilis with Lancefield' s group A antigen showed sensitivity very similar to S. pyogenes. Of these strains, S. dysgalactiae subsp. equisimilis formed a beta-hemolytic colony resembling that of S. pyogenes on sheep blood agar, and was sensitive to bacitracin. It is thus indispensable to identify the colony using biochemical tests such as the PYR (pyrrolidonylarylamidase production) test. In using rapid diagnostic kits for the detection of Group A streptococcus, it is important to rule out the possibility of Group A streptococcus other than S. pyogenes in throats. Severe invasive group-G streptococcal infections are increasing recently. Concerning S. dysgalactiae subsp. equisimilis, it is especially important to conduct these identification tests.

Antigens, Bacterial↗

Argentinian collaborative study on prevalence of erythromycin and penicillin susceptibility in Streptococcus pyogenes. The Argentinian Streptococcus Study Group.

Two monthly studies on the prevalence of penicillin and erythromycin susceptibility of Streptococcus pyogenes were performed in May and October of 1994 in Argentina. A total of 58 centers from 27 cities participated in these studies. A total of 1072 isolates were tested by a diffusion method, although 595 isolates were tested both by the diffusion and an agar dilution method (n = 1767 isolates). No penicillin-resistant streptococci were found in our study (MIC100 = 0.03 microgram/ml). Only four isolates were confirmed as erythromycin resistant S. pyogenes (prevalence 0.14 and 0.28% in May and October 1994, respectively). Resistance in three isolates was due to an inducible mechanism, although in one strain a different phenotype was observed.

Anti-Bacterial Agents↗

Proapoptotic effect of proteolytic activation of matrix metalloproteinases by Streptococcus pyogenes thiol proteinase (Streptococcus pyrogenic exotoxin B).

Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.

Animals↗

[Antimicrobial susceptibility of Streptococcus pyogenes, Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis from community acquired respiratory infections in 2000].

The Viriato Study is a nationwide, multicenter prospective study of the antimicrobial susceptibility of bacterial pathogens commonly associated with community-acquired respiratory tract infections in Portugal. In 2000, 28 laboratories participated in the study with a total of 1071 strains, with testing undertaken in a central laboratory. Of the 213 Streptococcus pyogenes strains isolated from patients with acute tonsillitis, all were susceptible to penicillin, amoxicillin/clavulanate and cefuroxime, but 21.1% were resistant to erythromycin, clarithromycin, azithromycin and 16.4% to tetracycline. From patients with lower respiratory tract infection, 403 strains of Haemophilus influenzae, 366 of Streptococcus pneumoniae and 89 of Moraxella catarrhalis were studied. 13.1% of H. influenzae and 94.4% of M. catarrhalis produced beta-lactamase. Among S. pneumoniae isolates, 25.1% were resistant to penicillin (8.8% showing high-level resistance), 14.5% to tetracycline, 12.8% to erythromycin, clarithromycin and azithromycin, and 10.1% to cefuroxime. Overall, penicillin was the most active antimicrobial against S. pyogenes and amoxycillin/clavulanate the most active in vitro simultaneously against H. influenzae, S. pneumoniae and M. catarrhalis isolated from patients with community-acquired LRTI in Portugal.

Community-Acquired Infections↗

Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes strains.

Streptococcus pyogenes causes severe invasive diseases in humans, including necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome (STSS). We found that mice infected intramuscularly (i.m.) with S. pyogenes strains developed bacteremia and subsequent sudden death after at least 10 days of a convalescent period. Mostly, it occurred more than 21 days after muscle infection. We provisionally designate this phenomenon as "delayed death." Just after muscle infection, all the mice lost weight and activity, but recovered completely within 3 days. They had kept good activity and a fine coat of fur till one or two days before their death. Some of the dead mice were found to have soft-tissue necrosis. There was no correlation between the virulence leading to the delayed death and the severity of diseases from which strains were isolated. It was also found that the production of neither streptococcal pyrogenic exotoxin (SPE) A nor B correlated to the virulence leading to delayed death. The bacteria obtained from the organs of the mice with delayed death expressed capsule. We suggest that the mice with delayed onset of systemic bacterial dissemination and subsequent death after muscle infection with S. pyogenes are the animal models of STSS, because the pathophysiology is extremely similar to that of human STSS.

Animals↗

Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of Streptococcus pyogenes.

Many Streptococcus pyogenes immunoglobulin-binding proteins have structural similarities to the antiphagocytic M protein, including the well-known C repeats. One of these molecules is the immunoglobulin A-binding protein Arp, which is expressed by a serotype 4 strain for which no antiphagocytic M protein has yet been described. We expressed Arp4 in an S. pyogenes strain from which the structural gene for the M protein has been deleted and found that Arp4 is not sufficient to inhibit phagocytosis.

Antigens, Bacterial↗

Response of Streptococcus pyogenes to therapy with amoxicillin or amoxicillin-clavulanic acid in a mouse model of mixed infection caused by Staphylococcus aureus and Streptococcus pyogenes.

The response of Streptococcus pyogenes to amoxicillin or amoxicillin-clavulanic acid (Augmentin; Beecham Group) therapy of a mixed streptococcal-staphylococcal infection was studied in a surgical wound in mice. A superficial wound was produced on the backs of anesthetized mice, and a suture infected with S. pyogenes, Staphylococcus aureus, or a mixed inoculum of both organisms was inserted. Oral therapy was started 4 h after infection and continued for 3 days. Both amoxicillin and amoxicillin-clavulanic acid were effective in eliminating the streptococci from the pure wound infection. In contrast, amoxicillin failed to eliminate the streptococci from a mixed infection in which a beta-lactamase-producing strain of S. aureus was also present, wound counts reaching 10(7) streptococci per wound by 80 h, whereas amoxicillin-clavulanic acid reduced the count to less than 33 streptococci per wound by 24 h. Numbers of S. aureus were also reduced by amoxicillin-clavulanic acid therapy, controlling the infection, whereas amoxicillin was ineffective. Also of significance was the fact that successful therapy was achieved with blood and tissue concentrations of amoxicillin and clavulanic acid of the same order as those measured in humans. These results show that amoxicillin therapy failed to eliminate S. pyogenes from a wound infection in the presence of a beta-lactamase-producing strain of S. aureus and suggest the potential of amoxicillin-clavulanic acid in the treatment of mixed bacterial skin infections involving beta-lactamase-producing organisms.

Amoxicillin↗