Antimicrobial resistance in viridans group streptococci among patients with and without the diagnosis of cancer in the USA, Canada and Latin America

Objective   To investigate antimicrobial resistance in viridans group streptococci (VGS) among patients with and without the diagnosis of cancer in the USA, Canada and Latin America.
Methods   All bloodstream isolates of VGS collected from SENTRY centers in the Western Hemisphere between January 1997 and December 1999 were tested by reference broth microdilution methods (NCCLS). Results for isolates from patients with cancer were compared to those from other patient populations.
Results   Overall, 438 unique patient bloodstream isolates of VGS were collected during the study. Percentage susceptible/MIC₉₀ (mg/L) values for antimicrobials tested were as follows: penicillin, 66/1; erythromycin, 60/4; clindamycin, 92/0.12; cefepime, 86/1; trimethoprim–sulfamethoxazole, 80/2; ciprofloxacin, 44/> 2; gatifloxacin, 98/0.5; and vancomycin, 100/1. Of these isolates, 70 (16%) were confirmed to be from cancer patients. VGS isolates from cancer patients were less susceptible to most antimicrobials tested than were isolates from non-cancer patients. The greatest differences in susceptibility rates for cancer- versus non-cancer-associated VGS isolates were seen for ciprofloxacin (34% versus 46%, P  = 0.07) and trimethoprim–sulfamethoxazole (64% versus 83%, P  < 0.001), two agents which are often used for prophylaxis or as presumptive therapy in cancer patients.
Conclusions   Susceptibility rates for VGS isolates from cancer patients are lower than those for isolates from patients without a cancer diagnosis. These differences are greatest for agents that have seen widespread prophylactic and empirical use. Ongoing surveillance of VGS infections in this patient population is important and should help to guide therapy decisions.     

Differentiation between Streptococcus pneumoniae and other viridans group streptococci by matrix-assisted laser desorption/ionization time of flight mass spectrometry

ObjectivesMatrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is becoming the method of choice for bacterial identification. However, correct identification by MALDI-TOF of closely related microorganisms such as viridans streptococci is still cumbersome, especially in the identification of S. pneumoniae. By making use of additional spectra peaks for S. pneumoniae and other viridans group streptococci (VGS). We re-identified viridans streptococci that had been identified and characterized by molecular and phenotypic techniques by MALDI-TOF.MethodsVGS isolates (n = 579), 496 S. pneumoniae and 83 non-S. pneumoniae were analysed using MALDI-TOF MS and the sensitivity and specificity of MALDI-TOF MS was assessed. Hereafter, mass spectra analysis was performed. Presumptive identification of proteins represented by discriminatory peaks was performed by molecular weight matching and the corresponding nucleotides sequences against different protein databases.ResultsUsing the Bruker reference library, 495 of 496 S. pneumoniae isolates were identified as S. pneumoniae and one isolate was identified as non-S. pneumoniae. Of the 83 non-S. pneumoniae isolates, 37 were correctly identified as non-S. pneumoniae, and 46 isolates as S. pneumoniae. The sensitivity of the MALDI-TOF MS was 99.8% (95% confidence interval (CI) 98.9–100) and the specificity was 44.6% (95% CI 33.7–55.9). Eight spectra peaks were mostly present in one category (S. pneumoniae or other VGS) and absent in the other category and inversely. Two spectra peaks of these (m/z 3420 and 3436) were selected by logistic regression to generate three identification profiles. These profiles could differentiate between S. pneumoniae and other VGS with high sensitivity and specificity (99.4% and 98.8%, respectively).ConclusionsSpectral peaks analysis based identification is a powerful tool to differentiate S. pneumoniae from other VGS species with high specificity and sensitivity and is a useful method for pneumococcal identification in carriage studies. More research is needed to further confirm our findings. Extrapolation of these results to clinical strains need to be deeply investigated.     

Genetic variation in group A streptococci

Group A streptococcus (GAS) is responsible for a range of human diseases that vary in their clinical manifestations and severity. While numerous virulence factors have been described, the way these factors interact to promote different streptococcal diseases is less clear. In order to identify multifactorial relationships between GAS and the human host, novel high-throughput techniques such as microarrays are necessary. We have performed comparative studies using custom-designed virulence arrays to enhance our understanding of the high degree of genotypic variation that occurs in streptococci. This study has pointed to mobile genetic elements as the major agents that promote variation. Our results show that multiple combinations of genes might bring about similar clinical pictures. This adds further complexity to the intricate relationship between pathogen and host.     

Increasing erythromycin resistance among group A streptococci causing tonsillitis in a tertiary care hospital in southern India

Group A streptococci (GAS) (n = 1313) isolated from patients with clinical symptoms of pharyngitis or tonsillitis attending a tertiary care hospital in southern India during 1986-2002 were tested for susceptibility to penicillin and erythromycin. The overall erythromycin resistance rate was 2.7% (n = 36). During 1986-1993, erythromycin resistance was observed in only one (2%) isolate in 1987, but reappeared in 1994 (2.7%), increased to 5.8% in 1999, and reached a maximum frequency of 13.8% in 2002. All isolates were susceptible to penicillin. The data indicate the need for continued surveillance of susceptibility patterns among GAS isolates in order to monitor the development of antibiotic resistance.     

Molecular characterisation and clonal analysis of group A streptococci causing pharyngitis among paediatric patients in Palermo, Italy

Group A streptococci (n = 123), isolated consecutively from paediatric patients with pharyngitis from Palermo, Italy, were analysed. The emm and sof genes were sequenced, the presence of the speA and speC genes was investigated, and the macrolide resistance phenotypes and genotypes were determined. A limited number of emm/sof genotypes was found, and the most prevalent types were different from those found in a previous study from Rome. Macrolide resistance was found in the most prevalent clones, suggesting that the spread of mobile antibiotic resistance genes among the fittest clones in the community was the main mechanism influencing macrolide resistance rates in different emm types.     

Biotypes of group A streptococci from patients with pharyngitis and soft tissue infections

This study determined the biotypes of group A streptococci (GAS) isolated from 66 pharyngeal and 62 skin and soft-tissue infections. Among all GAS isolates tested, the most common biotypes were 1 and 3, irrespective of the isolation source and the severity of clinical symptoms. However, compared with the pharyngeal group, a more heterogeneous distribution of biotypes was observed among the cutaneous group of isolates, including seven isolates that were non-typeable but had an identical biotype pattern, suggesting that they may represent a new biotype.     

Erythromycin susceptibility of viridans streptococci from the normal throat flora of patients treated with azithromycin or clarithromycin

Objective   To study the emergence of macrolide resistance in throat flora following treatment with clarithromycin or azithromycin.
Methods   Throat samples were collected before and after treatment and plated as a lawn on Columbia blood agar with an erythromycin E test strip. Minimum inhibitory concentrations (MICs) of erythromycin, clarithromycin and azithromycin were determined against isolates of distinct morphology with erythromycin E test MIC results equal to or greater than 2 mg/L. Polymerase chain reaction techniques were used to determine the genetic mechanisms of resistance.
Results   There were 749 resistant isolates of which 474 (63%) were streptococci. Only a quarter of the patients had no resistant streptococci before treatment started. There were increases in the numbers of resistant isolates and in the number of patients carrying a resistant flora during and after treatment. The most common genes identified were mef A/E in isolates with low-level resistance and erm A/M in isolates with high-level resistance.
Conclusions   There is a pool of streptococci carrying genes associated with macrolide resistance in the normal respiratory flora of generally healthy adults. Differences between the patients treated with clarithromycin and those treated with azithromycin were difficult to assess because of the large number of patients in each group with macrolide-resistant streptococci before treatment. Although there were some differences these were not statistically significant.     

Reduced susceptibility to penicillin of viridans group streptococci in the oral cavity of patients with haematological disease

The occurrence of oral penicillin-resistant viridans group streptococci (VGS) was studied in 50 patients with either newly diagnosed acute leukaemia or autologous peripheral stem cell transplants. One patient was excluded because of Staphylococcus aureus growth in the stem cell harvest. VGS were isolated from the oral cavity of 48 of the remaining 49 patients. Of these 48 patients, 12 (25%) yielded VGS resistant (MIC > 2 mg/L) to penicillin. These 12 patients had a higher frequency of septicaemia (p 0.04) and more days of treatment with trimethoprim-sulphamethoxazole (p 0.04) than patients who harboured susceptible or intermediately resistant VGS (MIC 2 mg/L). There were no other statistically significant differences between the two groups. It is important to be aware of the high level of penicillin resistance in oral VGS in patients with haematological disease, and this parameter should be considered when selecting antibiotic therapy for cases of septicaemia caused by VGS in immunocompromised patients.     

Predominance of two M-types among erythromycin-resistant group A Streptococci from Greek children

In order to investigate the potential relationship between erythromycin resistance and specific M-serotypes among clinical isolates of Streptococcus pyogenes from children in Greece, we randomly selected a total of 49 erythromycin-resistant (EryR) and 21 erythromycin-susceptible (EryS) isolates from the 1158 S. pyogenes isolates from the two main children's hospitals of Athens during the period October 1997 to October 1998. The isolates were further characterized by M-serotyping, examined for their susceptibility to penicillin, vancomycin and clindamycin, and categorized into resistance phenotypes. A total of 248 (21%) S. pyogenes isolates in the two main children's hospitals of Athens during the study period were resistant to erythromycin. All 49 EryR and 21 EryS isolates were susceptible to penicillin and vancomycin. With respect to erythromycin and clindamycin resistance, phenotypes M and IR MLS_B dominated, with 30 and 17 isolates, respectively, two isolates belonged to the CR MLS_B phenotype. Among the erythromycin resistant isolates, two M serotypes were dominant: M22 (30%) and M84 (41%). More specifically, M22 and M84 were most prevalent in resistance phenotypes IR MLS_B (65%) and M (63%), respectively. In the susceptible group, no isolate belonged to these two M-serotypes, nor was a predominant serotype found. In contrast to susceptible isolates, two distinct M-serotypes were highly represented among EryR S. pyogenes isolates and predominantly associated with two distinct phenotypes.     

Prevalence and mechanisms of erythromycin and clindamycin resistance in clinical isolates of β-haemolytic streptococci of Lancefield groups A, B, C and G in Seville, Spain

Susceptibility to erythromycin and clindamycin was determined in 860 consecutive clinical isolates of β-haemolytic streptococci belonging to groups A (GAS, n  =   134), B (GBS, n  =   689), C (GCS, n  =   19) and G (GGS, n  =   18). Erythromycin resistance was 26.1% in GAS, 15.7% in GBS, 5.3% in GCS and 33.3% in GGS. The highest rate of clindamycin resistance (33.3%) was in GGS, followed by GBS (15.8%), GCS (15.8%) and GAS (5.2%). The M phenotype was predominant in GAS (80%), the constitutive MLS_B phenotype was predominant in GBS (75%), and all GGS isolates showed the inducible MLS_B phenotype. The uncommon erythromycin-susceptible and clindamycin-resistant phenotype was found in four GBS and two GCS isolates.     

Improved methods for typing nontypeable isolates of group B streptococci

Group B streptococci (GBS) are classified by capsular polysaccharide (CPS) type and by cell surface-expressed proteins (c and R). Isolates lacking detectable CPS are considered nontypeable (NT) although they frequently express surface proteins. Immunological and genetic methods were used to study 91 NT GBS isolates collected during surveillance studies for invasive disease or colonization in pregnant or non-pregnant women and neonates less than seven days of age. CPS production was upregulated by the addition of glucose and sodium phosphate to Todd-Hewitt broth (THB) and cells were extracted using hot HCl or mutanolysin. Extracts were tested with antisera for specific CPS types Ia, Ib, and II - VIII by double immunodiffusion (DD) in agarose. By mutanolysin extraction, 12 (13.2%) of the 91 isolates were typeable. In contrast, only four of these 12 newly typeable isolates tested positive for CPS with the HCl extracts of cells grown in modified THB. DNA was analyzed by pulsed-field gel electrophoresis (PFGE) using SmaI restriction with NT isolates grouped by protein profile to facilitate analysis. PFGE results of the NT isolates were compared to DNA profiles of typeable isolates and were correlated with the DD results. The DNA profiles of the newly typeable isolates were similar to profiles of isolates with corresponding defined CPS type. Of the remaining 78 NT isolates digested by SmaI, 63 (80.8%) had DNA profiles that resembled those of specific types of GBS. These approaches will be useful for classification of NT isolates in continued epidemiological surveillance associated with GBS vaccine trials.     

Cysteine protease SpeB expression in group A streptococci is influenced by the nutritional environment but SpeB does not contribute to obtaining essential nutrients

Group A streptococcal (GAS) cysteine protease is a major virulence factor involved in the pathogenesis of purulent and invasive infections. The secreted enzyme cleaves a number of different bacterial and host proteins which could contribute to different stages of the infective processes. It has been proposed that, among these functions, SpeB plays a role in obtaining nutrients during late growth phases. In the present study, speB mutants of various GAS serotypes were found to exhibit unaltered growth characteristics in several complex and chemically defined media (CDM). When amino acid-depleted CDM was prepared, neither SpeB activity on whole proteins added to the medium during incubation nor the addition of SpeB-digested proteins was able to support bacterial growth. SpeB also was unable to liberate iron from iron-containing protein sources added to iron-deficient CDM. However, SpeB levels in culture supernatants changed in response to the protein and glucose content of the media. Using a speB promoter-luciferase reporter, speB expression levels were found to correspond to peptide concentrations in the culture media. The effect appeared to be specific for peptides since addition of peptides derived from various proteins had an affect on expression, while addition of the whole proteins had no effect. Addition of glucose to CDM had no effect on speB expression, while glucose addition to complex medium decreased speB expression. Overall, SpeB did not appear to be directly involved in providing the bacteria with nutritional factors but expression of the speB gene responded to ratios of peptides and carbohydrates in the culture medium. Received: 10 April 1999     

Prevalence of erythromycin and clindamycin resistance among Streptococcus agalactiae isolates in Germany

The antimicrobial susceptibilities of 338 clinical Streptococcus agalactiae isolates from two geographical regions in Germany were determined by agar dilution. All isolates were susceptible to penicillin, cefotaxime and vancomycin. The overall frequencies of erythromycin and clindamycin resistance were 11% and 4.7%, respectively. Determination of resistance phenotypes among the 37 erythromycin-resistant isolates revealed constitutive and inducible MLS(B) resistance in 40.6% and 37.8% of isolates, respectively, and susceptibility to clindamycin in 21.6% of isolates. Only 14.3% of isolates with inducible MLS(B) resistance were identified as clindamycin-resistant by determination of clindamycin MICs. Pulsed-field gel electrophoresis suggested a clonal distribution pattern among the erythromycin-resistant isolates.     

Circulating serotypes and antimicrobial sensitivity of Streptococcus pyogenes isolates from children in Cyprus

The most common T-serotypes among group A streptococci (n = 88) isolated from pharyngeal samples of children referred to a tertiary hospital in Cyprus for pharyngitis or scarlet fever during a 14-month period (2003-2004) were T28 (25%), T8/25/Imp19 (22.7%) and T12 (9.1%). All 88 isolates were sensitive to penicillin and clindamycin, but 1.1% and 18.2% of isolates were resistant to erythromycin and tetracycline, respectively. Macrolide consumption was estimated at 1.7 defined daily doses/1000 inhabitants/day. The low percentage of resistance to macrolides may have been related, at least in part, to the low consumption of macrolides.     

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK

Objective  To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK.
Methods  During 1995–97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone.
Results  Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mef A gene was detected in seven isolates (five serotype 14 and two serotype 19F), erm B in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone.
Conclusions  The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.     

Comparison of LightCycler PCR and culture for detection of group B streptococci from vaginal swabs

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. New rapid, sensitive and specific methods for detection of GBS in pregnant women are needed in order to provide timely treatment of neonates. The sensitivity, specificity and cost of a LightCycler PCR method was compared with selective culture for the detection of GBS from 400 vaginal swabs. In addition, two DNA extraction methods (simple boiling and automated DNA extraction by Roche MagNA Pure LC) were compared for a subgroup of 100 clinical samples. The sensitivity of the LightCycler PCR assay for the detection of GBS from vaginal swabs was significantly higher than that of culture. There were no culture-positive, LightCycler PCR-negative cases. The efficiencies of the two DNA extraction procedures were not significantly different. The detection of GBS from vaginal swabs by the molecular method (including simple boiling extraction) required the same hands-on time, but the procedure was completed in 1.5 h, compared with c. 48 h for the culture-based approach. Disadvantages of the molecular method are the increased costs (45%) and the absence of antibiogram data. The LightCycler PCR is a promising tool for sensitive, specific and rapid detection of GBS directly from clinical specimens of pregnant women.     

Macrolide resistance trends in beta-hemolytic streptococci in a tertiary Korean hospital

PURPOSE: Erythromycin-resistant beta-hemolytic streptococci (BHS) has recently emerged and quickly spread between and within countries throughout the world. In this study, we evaluate the antimicrobial susceptibility patterns and erythromycin resistance mechanisms of BHS during 2003-2004. MATERIALS AND METHODS: The MICs of seven antimicrobials were determined for 204 clinical isolates of BHS from 2003 to 2004. Resistance mechanisms of erythromycin-resistant BHS were studied by the double disk test as well as by polymerase chain reaction (PCR). RESULTS: Compared with our previous study, resistance among Streptococcus pyogenes isolates to a variety of drugs decreased strikingly: from 25.7% to 4.8% in erythromycin; 15.8% to 0% in clindamycin; and 47.1% to 19.0% in tetracycline. The prevalent phenotypes and genotypes of macrolide-lincosamide-streptograminB (MLSB) resistance in Streptococcus pyogenes isolates have been changed from the constitutive MLSB phenotype carrying erm(B) to the M phenotype with mef(A) gene. In contrast with Streptococcus pyogenes, resistance rates to erythromycin (36.7%), clindamycin (43.1%), and tetracycline (95.4%) in Streptococcus agalactiae isolates did not show decreasing trends. Among the Streptococcus dysgalactiae subsp. equisimilis isolates (Lancefield group C, G), resistance rates to erythromycin, clindamycin, tetracycline and chloramphenicol were observed to be 9.4%, 3.1%, 68.8%, and 9.4%, respectively. Conclusion: Continual monitoring of antimicrobial resistance among large-colony-forming BHS is needed to provide the medical community with current data regarding the resistance mechanisms that are most common to their local or regional environments.     

Enhancement of growth of group A β-hemolytic streptococci in mixed infections with aerobic and anaerobic bacteria

Objective: To evaluate the potential for mutual enhancement of growth of group A β-hemolytic streptococci (GABHS), and 11 commonly associated aerobic and anaerobic microorganisms frequently isolated in tonsillar infections.
Methods: Enhancement was assessed by measuring the relative increase in colony-forming units (CFU) of GABHS and each of the 11 microorganisms inducing a subcutaneous abscess in mice.
Results: Of the 11 combinations of GABHS and aerobe or anaerobe, GABHS was enhanced in 10 cases and the other microorganism in 5, namely, Staphylococcus aureus, Haemophilus influenzae type b, Klebsiella pneumoniae, Prevotella melaninogenica and Bacteroides fragilis.
Conclusions: These findings confirm the mutual symbiotic enhancement of growth of GABHS in the presence of other aerobic and anaerobic bacteria.