Streptococcus pyogenes ("Group A Streptococcus"), a Highly Adapted Human Pathogen-Potential Implications of Its Virulence Regulation for Epidemiology and Disease Management.

Streptococcus pyogenes (group A streptococci; GAS) is an exclusively human pathogen. It causes a variety of suppurative and non-suppurative diseases in people of all ages worldwide. Not all can be successfully treated with antibiotics. A licensed vaccine, in spite of its global importance, is not yet available. GAS express an arsenal of virulence factors responsible for pathological immune reactions. The transcription of all these virulence factors is under the control of three types of virulence-related regulators: (i) two-component systems (TCS), (ii) stand-alone regulators, and (iii) non-coding RNAs. This review summarizes major TCS and stand-alone transcriptional regulatory systems, which are directly associated with virulence control. It is suggested that this treasure of knowledge on the genetics of virulence regulation should be better harnessed for new therapies and prevention methods for GAS infections, thereby changing its global epidemiology for the better.

A New Approach to Harness Probiotics Against Common Bacterial Skin Pathogens: Towards Living Antimicrobials.

In this study, the potential of certain lactic acid bacteria-classified as probiotics and known to be antimicrobially active against pathogens or food-poisoning microorganisms-was evaluated with respect to their activity against bacterial skin pathogens. The aim of the study was to develop a plaster/bandage for the application of inhibitory substances produced by these probiotics when applied to diseased skin. For this purpose, two Streptococcus salivarius strains and one Lactobacillus plantarum were tested for production of antimicrobials (bacteriocin-like substances) active against Gram-positive and Gram-negative pathogens using established methods. A newly designed membrane test ensured that the probiotics produce antimicrobials diffusible through membranes. Target organisms used were Cutibacterium acnes, Staphylococcus aureus, and Pseudomonas aeruginosa. Moreover, the L. plantarum 8P-A3 strain was tested against additional bacteria involved in skin disorders. The Lactobacillales used were active against all potential skin pathogens tested. These probiotics could be enclosed between polymer membranes-one tight, the other permeable for their products, preserved by vacuum drying, and reactivated after at least three months storage. Importantly, the reactivated pads containing the probiotics demonstrated antibacterial activity on agar plates against all pathogens tested. This suggests that the probiotic containing pads may be topically applied for the treatment of skin disorders without the need for a regular antibiotic treatment or as an adjunctive therapy.

Rapid detection of Streptococcus agalactiae from swabs by peptide nucleic acid fluorescence in situ hybridization.

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae [group B streptococci (GBS)] from swab samples was evaluated. Three swab-sample-processing protocols with different time-to-result (TTR) values were compared: (i) direct smearing of fresh swabs onto microscope slides (n=153, TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n=153, TTR 2.7 h), and (iii) short-term LIM broth enrichment culture incubation (7 h, 37 degrees C) of fresh swabs (n=120, TTR 9.5 h). The sensitivity, specificity, positive predictive value and negative predictive value for GBS PNA FISH for sample processing procedures, with TTR values of 2.5, 2.7 and 9.5 h, were 68, 100, 100 and 95 %; 91, 100, 100 and 98 %; and 100, 100, 100 and 100 %; respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Comparison of two chromogenic media for selective isolation of vancomycin-resistant enterococci from stool specimens.

Two chromogenic media (Chromagar VRE and chromID VRE [C-ID]) performed equally well in the direct detection of vancomycin-resistant enterococci (VRE) in stool specimens after an overnight enrichment step and a 48-h incubation period, with a sensitivity of 98.2% (56/57) for both and specificities of 96.5% (195/202) and 97.5% (197/202), respectively. However, assigning discriminatory colony color was sometimes difficult, especially on C-ID. In order to facilitate simple species identification, biochemical key reactions were implemented.

A new closed-tube multiplex real-time PCR to detect eleven superantigens of Streptococcus pyogenes identifies a strain without superantigen activity.

Superantigens (SAgs) are very potent microbial toxins that are involved in severe diseases such as necrotizing fasciitis and toxic shock syndrome. There are currently 11 different SAgs that have been identified from Streptococcus pyogenes. In the present study, two sets of multiplex PCRs were developed for detection of these 11 SAg genes. The first group comprises spea1-3+5, spec, speg, spej, spek, and spel. The second group consists of spea1-4, speh, spei, spem, ssa, and smez. The presence of Streptococcus pyogenes SAg genes can be immediately identified using a real-time method with SYBR-Green, thus providing an excellent tool in clinical diagnostics. After testing more than 300 clinical isolates, we identified one strain without any SAg gene. This finding contrasts with previous reports describing SAg genes located on every Streptococcus pyogenes genome. This SAg gene-negative strain also did not show any mitogenic activity. It is hypothesized that clinical isolates from patients may overrepresent bacterial strains with pathogenic factors, such as SAgs.

Epidemiology of Streptococcus agalactiae colonization in Germany.

Streptococcus agalactiae can cause severe pneumonia, sepsis and meningitis in neonates and remains one of the most prevalent causes of invasive neonatal infections. Maternal transmission of S. agalactiae during delivery can be prevented by prenatal screening and peripartal antibiotic prophylaxis. Implementation of CDC guidelines for group B streptococci (GBS) disease prevention resulted in a significant decline of invasive neonatal S. agalactiae infections in the USA. Similar national guidelines were issued in 2000 for Germany. However, the epidemiology of S. agalactiae colonization in Germany has not been investigated for more than 15 years and the impact these guidelines will have is therefore unknown. To assess colonization rates in Germany, we cultured vaginal and rectal swabs for S. agalactiae from pregnant and non-pregnant adult patients in the region of Aachen and Munich. Swabs were cultivated in selective broth medium for 24h and subsequently plated on blood agar plates according to the CDC recommendations. Colonies negative for catalase and pyrrolidonyl aminopeptidase were further differentiated by the CAMP test and a DNA probe specific for S. agalactiae. Rectal or vaginal colonization of S. agalactiae was found in 34 (16%) of 210 pregnant patients and in 41 (16%) of 250 non-pregnant women. S. agalactiae was found only in rectal swabs in 4% of pregnant and non-pregnant patients. For further characterization of the strains capsular serotypes and major surface protein antigens were determined by Ouchterlony immunodiffusion and PCR. Among the 75 different patient isolates serotype III was the most prevalent with 21 (28%) isolates, followed by 16 (21%) isolates of serotype II, 13 (17%) isolates of serotype Ia, 12 (16%) of serotype V, 11 (15%) of serotype Ib and only 2 (3%) isolates of serotype IV. The vast majority of all strains harbored genes for the major surface protein antigens, the alpha-C-protein or alpha-C-protein like antigens like Alp2-4, epsilon and Rib. These data show that S. agalactiae colonization is common in Germany and strict adherence to the guidelines for the preventions of GBS disease will result in peripartal antibiotic prophylaxis in up to 20% of all deliveries.

Lack of mitogenic activity of speG- and speG(dys)-positive Streptococcus dysgalactiae subspecies equisimilis isolates from patients with invasive infections.

In recent years, Streptococcus dysgalactiae subspecies equisimilis has been isolated with an increasing frequency as the cause of invasive streptococcal diseases. For 46 S. dysgalactiae subspecies equisimilis isolates from invasive infections and four isolates from superficial infections, the presence of emm/emmL genes and of genes encoding various different streptococcal superantigens was determined by polymerase chain reaction (PCR). Subsequently, PCR products were identified by DNA sequencing, and the expression of mRNA from superantigen genes was assessed by reverse transcriptase-PCR. The mitogenic activity of S. dysgalactiae subspecies equisimilis was assessed by [3H]thymidine incorporation into human lymphocytes and compared with that of Streptococcus agalactiae and Streptococcus pyogenes. All S. dysgalactiae subspecies equisimilis isolates studied harbored an emm/emmL gene. Only in six of the S. dysgalactiae subspecies equisimilis isolates from invasive infections, speG was detected by PCR, two of which were further identified as speGdys by sequencing of the PCR product. None of the S. dysgalactiae subspecies equisimilis isolates harbored any of the genes speA, speB, speC, speF, speH, speI, speJ, speK, speL, speM, smeZ, or ssa of S. pyogenes. In contrast to S. pyogenes, no expression of speG or speGdys mRNA, respectively, was detected in the reverse transcriptase-PCR assay for any of the speG- or speGdys-positive S. dysgalactiae subspecies equisimilis isolates. Moreover, S. dysgalactiae subspecies equisimilis and S. agalactiae revealed no or very low mitogenic activity, while S. pyogenes was a very powerful inducer of proliferative responses. These findings support the hypothesis that the pathogenicity of S. dysgalactiae subspecies equisimilis may be associated in part with the presence of emm/emmL genes, and suggest that the severity of S. dysgalactiae subspecies equisimilis invasive infections is not mediated by superantigen-induced mitogenicity.

Evaluation of postdischarge surveillance of surgical site infections after total hip and knee arthroplasty.

BACKGROUND: Artificial joint replacement of hip (HPRO) and knee (KPRO) are 2 of about 20 categories of operative procedures of the surveillance of surgical site infection (SSI) as stated by nosocomial infections surveillance systems in the United States and in Germany. Periprosthetic SSI can manifest itself after a long period. METHODS: Seven hundred fifty-six orthopedic patients from 2 centers were evaluated after HPRO (n = 508) or KPRO (n = 248). SSI was recorded during hospitalization and for 12 month postdischarge. The surveillance regimen was extended by also sending patients a questionnaire after 12 months postdischarge. All complaints were followed up by contacting the patients and any clinicians and general practitioners (GPs) involved. Stratified infection rates and standardized infection ratio (SIR) were calculated and compared with reference data of the national surveillance system. RESULTS: The total response rate to the postal questionnaire survey was 85.2%. SSI was recorded in 16 patients (3.15%) after HPRO; 12 were detected by predischarge surveillance, and the 4 cases found postdischarge were all organ/space SSI. In total, only 1 SSI was detected after KPRO before discharge and none after discharge (SSI rate 0.40%). Time between discharge and detection of SSI cases ranged from 8 days to 8 months. SIR of HPRO was 1.25 and SIR of KPRO was 0.36. CONCLUSION: Because 25% of SSIs after HPRO occurred after discharge and all were organ/space SSI, highlights the importance of postdischarge surveillance of nosocomial infections (NIs). Because all SSIs were reported already by current surveillance, the extended postdischarge surveillance appears to be unnecessary. The pursuit of shorter hospital stay after surgery may challenge the methods of surveillance systems in future.

Enterococcus raffinosus infection in an immunosuppressed patient: case report and review of the literature.

Enterococcus raffinosus rarely occurs in humans. We describe the case of a haematoma infected by E. raffinosus in a 72-year-old patient undergoing immunosuppressive therapy. This study confirms the results of previous reports about the pathogenic potential of this bacterium in humans.

Surveillance of nosocomial infections in a neurologic intensive care unit.

OBJECTIVE: To assess data on the epidemiology of nosocomial infection (NI) among neurologic intensive care patients. DESIGN: Prospective periodic surveillance study. SETTING: An 8-bed neurologic intensive care unit (ICU). PATIENTS: All those admitted for more than 24 hours during five 3-month periods between January 1999 and March 2003. METHODS: Standardized surveillance within the German infection surveillance system. RESULTS: Three hundred thirty-eight patients with a total of 2,867 patient-days and a mean length of stay of 8.5 days were enrolled during the 15-month study period. A total of 71 NIs were identified among 52 patients. Urinary tract infections (UTIs) were the most frequent NI (36.6%), followed by pneumonia (29.6%) and bloodstream infections (BSIs) (15.5%). The overall incidence and incidence density of NIs were 21.0 per 100 patients and 24.8 per 1,000 patient-days, respectively. Incidence densities were 9.8 UTIs per 1,000 urinary catheter-days (CI95, 6.4-14.4), 5.6 BSIs per 1,000 central venous catheter-days (CI9s, 2.8-10.0), and 12.8 cases of pneumonia per 1,000 ventilation-days (Cl95, 8.0-19.7). Device-associated UTI and pneumonia rates were in the upper range of national and international reference data for medical ICUs, despite the intensive infection control and prevention program in operation in the hospital. CONCLUSION: Neurologic intensive care patients have relatively high rates of device-associated nosocomial pneumonia and UTI. For a valid comparison of surveillance data and implementation of targeted prevention strategies, we would strongly recommend provision of national benchmarks for the neurologic ICU setting.

Evaluation of recombinant antigens for the measurement of antibodies to streptolysin O and streptococcal DNase B on the Beckman Coulter IMMAGE system and comparison with a method employing conventional antigens.

The performance of a newly devised method employing recombinant antigens for the determination of antibodies to streptolysin O and DNase B on the Beckman Coulter IMMAGE System was compared with a nephelometric assay using conventional antigens. Sera from 114 patients with or without evidence of streptococcal infection were analysed by the two techniques. The results obtained with the new method correlated well with those of the reference test. The sensitivity of a combination measurement of antibodies to streptolysin O and DNase B was higher than that obtained when measuring each parameter alone. Handling of the tests was easier on the Beckman Coulter IMMAGE System. In conclusion, the new test from Beckman Coulter for the measurement of antibodies to streptolysin O and DNase B performs well and equals measurements using conventional antigens on a nephelometer.

Antimicrobial resistance of Streptococcus pneumoniae isolates of outpatients in Germany, 1999-2000.

BACKGROUND: The aim of the study was to determine the prevalence of antimicrobial resistance among clinical isolates of Streptococcus pneumoniae during the winter of 1999-2000 in Germany. METHODS: Pneumococcal isolates were prospectively collected by 14 different clinical microbiology laboratories. Minimal inhibitory concentrations of penicillin G, erythromycin A, clarithromycin, roxithromycin, azithromycin, clindamycin, levofloxacin and telithromycin were determined by the broth microdilution method. RESULTS: Among 328 strains 4.6% were nonsusceptible to penicillin G (intermediate and resistant strains) and 9.5% were resistant to erythromycin A. Analysis of erythromycin-resistant strains for the underlying resistance determinants revealed that 12 (38.7%) belonged to the erm(B) and 19 (61.3%) to the mef(E) type of resistance. Among the macrolide-resistant strains, serotypes 19F (n = 9) and 14 (n = 8) were the predominant types. CONCLUSIONS: Macrolide resistance in Germany is of growing concern and mainly due to the high prevalence of pneumococci expressing the mef(E) type of resistance.

Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002-2003.

In the present study, a real-time PCR protocol was developed for the detection of macrolide resistance determinants and was validated in a nationwide study in Germany covering a total of 236 Streptococcus pyogenes and 241 Streptococcus pneumoniae strains collected from children < or = 16 years of age with community-acquired infections. Macrolide resistance was observed in 19.9% of pneumococcal strains and 14% of S. pyogenes isolates. Of the erythromycin A-resistant S. pyogenes strains, 93.9% showed the efflux type mef(A); 62.5% of the S. pneumoniae strains were mef(A)- and 37.5% erm(B)-positive. The correlation of the results of real-time PCR assay genotyping in the present study compared with those of conventional PCR genotyping and resistance phenotyping was 100%. Macrolide resistance is of growing concern in Germany. This highly sensitive and specific PCR assay to detect macrolide resistance has the potential to provide sufficiently rapid results to improve antibiotic treatment of streptococcal infections.

HMG-CoA reductase inhibitor simvastatin profoundly improves survival in a murine model of sepsis.

BACKGROUND: HMG-CoA reductase inhibitors, such as simvastatin, have been shown to exhibit pronounced immunomodulatory effects independent of lipid lowering but to date have not been used to treat severe inflammatory disease such as sepsis. We thus approached the question of whether treatment with simvastatin might improve cardiovascular function and survival in sepsis. METHODS AND RESULTS: Mice treated with simvastatin and rendered septic by cecal ligation and perforation (CLP) show a mean survival time close to 4 times the value found in untreated mice. This dramatic improvement is based on a complete preservation of cardiac function and hemodynamic status, which are severely impaired in untreated CLP mice [eg, 20 hours after CLP, cardiac output declined from 1.24+/-0.09 to 0.87+/-0.11 mL x min(-1) x g(-1) in untreated mice (P<0.005; n=12), while remaining unaltered (1.21+/-0.08 mL x min(-1) x g(-1) at baseline and 1.15+/-0.1 mL x min(-1) x g(-1) 20 hours after CLP, P=NS, n=12) in CLP mice treated with simvastatin]. Untreated CLP mice remained refractory to beta-stimulation, whereas the responsiveness to dobutamine was restored by treatment with simvastatin. Susceptibility of coronary flow to endothelial nitric oxide synthase (eNOS) stimulation by bradykinin was close to 3 times as pronounced in untreated CLP mice as in untreated sham-operated mice, indicating a high level of eNOS activation secondary to sepsis. In addition, treatment with simvastatin reversed inflammatory alterations in CLP mice, namely, increased monocyte adhesion to endothelium. CONCLUSIONS: Simvastatin, which is well established in the treatment of lipid disorders and coronary artery disease, might have the additional potential of being an effective agent in sepsis treatment.

Clonal relatedness of erythromycin-resistant Streptococcus pyogenes isolates in Germany.

In a nationwide study in Germany, a total of 381 Streptococcus pyogenes were collected. Erythromycin A-resistant strains were characterized for the underlying resistance genotype, showing 55.6% had the efflux type mef(A), 31.5% had erm(A), and 13.0% had erm(B). A total of 23 different multilocus sequence types were observed.

Characterization of German penicillin non-susceptible serotype 23F pneumococci using multilocus sequence typing.

Three nationwide multicentre studies (n = 5071) showed an increase in antibiotic resistance in pneumococci in Germany. Serotype 23F was the predominant serotype (n = 45, 22.4 %), followed by 6B (n = 30, 14.9 %) and 9V (n = 19, 9.5 %). Multilocus sequence typing was used to characterize 45 serotype 23F strains with reduced penicillin susceptibility. The Spanish(23F)-1 clone [profile 4-4-2-4-4-1-1, sequence type (ST) 81] contributes significantly to the emergence of penicillin resistance in Germany (n = 21, 46.7 % of all penicillin non-susceptible serotype 23F isolates). Isolates of ST 277 (profile 7-13-8-6-6-12-8), which has been found previously in the Netherlands, are also observed, particularly in western Germany (n = 8, 17.8 %). A high proportion of strains (n = 11, 24.4 %) have sequence types that have not been reported to date from other countries (STs 353-362). The major penicillin-resistant clones are present in Germany, a country with relatively low levels of beta-lactam resistance.

Ribosomal mutations conferring resistance to macrolides in Streptococcus pneumoniae clinical strains isolated in Germany.

Among a collection of 4281 pneumococcal isolates, 7 strains isolated in Germany had an unusual macrolide resistance phenotype. The isolates were found to have multiple mutations in the 23S rRNA and alterations in the L4 ribosomal protein. One strain had an amino acid alteration in the L22 ribosomal protein.

drs (Distantly related sic) gene polymorphisms among emm12-type Streptococcus pyogenes isolates.

Twenty-eight emm12-type Streptococcus pyogenes isolates from patients with invasive and noninvasive infections or from asymptomatic carriers were genetically typed. Sequencing of drs (distantly related sic [streptococcal inhibitor of complement]) genes identified two novel alleles and revealed a polymorphism for drs similar to that of sic. No association was observed between the five different drs alleles and the five restriction patterns of the vir regulon for the isolates studied. These data suggest that drs sequencing may be useful for further differentiation of S. pyogenes isolates with emm12 and identical vir regulon restriction patterns.

Macrolide-resistant Streptococcus pneumoniae and Streptococcus pyogenes in the pediatric population in Germany during 2000-2001.

In a nationwide study in Germany covering 13 clinical microbiology laboratories, a total of 307 Streptococcus pyogenes (mainly pharyngitis) and 333 Streptococcus pneumoniae (respiratory tract infections) strains were collected from outpatients less than 16 years of age. The MICs of penicillin G, amoxicillin, cefotaxime, erythromycin A, clindamycin, levofloxacin, and telithromycin were determined by the microdilution method. In S. pyogenes isolates, resistance rates were as follows: penicillin, 0%; erythromycin A, 13.7%; and levofloxacin, 0%. Telithromycin showed good activity against S. pyogenes isolates (MIC(90) = 0.25 micro g/ml; MIC range, 0.016 to 16 micro g/ml). Three strains were found to be telithromycin-resistant (MIC >/= 4 micro g/ml). Erythromycin-resistant strains were characterized for the underlying resistance genotype, with 40.5% having the efflux type mef(A), 38.1% having the erm(A), and 9.5% having the erm(B) genotypes. emm typing of macrolide-resistant S. pyogenes isolates showed emm types 4 (45.2%), 77 (26.2%), and 12 (11.9%) to be predominant. In S. pneumoniae, resistance rates were as follows: penicillin intermediate, 7.5%; penicillin resistant, 0%; erythromycin A, 17.4%; and levofloxacin, 0%. Telithromycin was highly active against pneumococcal isolates (MIC(90)

Streptococcus pyogenes meningitis complicating varicella in a 3-month-old child.

Varicella is a common, usually self-limited infectious disease, and complications are believed to be rare. Despite the dramatic increase in invasive Streptococcus pyogenes infections associated with varicella zoster virus infections in recent years, post-varicella S. pyogenes meningitis occurs very rarely. The third case in the literature is described here.