Corinne Ruppen

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved.

In vitro activity of gentamicin as an adjunct to penicillin against biofilm group B Streptococcus.

Objectives: Group B Streptococcus (GBS) increasingly causes invasive disease in non-pregnant adults, particularly in elderly persons and those with underlying diseases. Combination therapy with penicillin plus gentamicin has been suggested for periprosthetic joint infection. The postulated synergism of this combination is based on experiments with planktonic bacteria. We aimed to assess the efficacy of this combination against sessile bacteria. Methods: Four different GBS strains were used. We compared results of MICs with those of minimal biofilm eradication concentrations (MBECs), applied chequerboard assays to the MBEC device and calculated the fractional inhibitory concentration index. Synergism was evaluated with time–kill assays against bacteria adherent to cement beads, using penicillin (0.048, 0.2 and 3 mg/L), gentamicin (4 and 12.5 mg/L) and a combination thereof. Results were evaluated via colony counting after sonication of beads and scanning electron microscopy. Results: MBEC/MIC ratios were 2000–4000 for penicillin and 1–4 for gentamicin. In chequerboard assays, synergism was observed in all four isolates. In time–kill assays, penicillin and 12.5 mg/L gentamicin showed synergism in two isolates. In the other two isolates 12.5 mg/L gentamicin alone was as efficient as the combination therapy. Conclusions: These in vitro investigations show activity of 12.5 mg/L gentamicin, alone or as an adjunct to penicillin, against four strains of biofilm GBS. This concentration cannot be achieved in bone with systemic administration, but can be reached if administered locally. The combination of systemic penicillin plus local gentamicin indicates a potential application in orthopaedic-device-associated GBS infections. Studies with a larger number of strains are required to confirm our results.

Is Penicillin Plus Gentamicin Synergistic against Clinical Group B Streptococcus isolates?: An In vitro Study

Group B Streptococcus (GBS) is increasingly causing invasive infections in non-pregnant adults. Elderly patients and those with comorbidities are at increased risk. On the basis of previous studies focusing on neonatal infections, penicillin plus gentamicin is recommended for infective endocarditis (IE) and periprosthetic joint infections (PJI) in adults. The purpose of this study was to investigate whether a synergism with penicillin and gentamicin is present in GBS isolates that caused IE and PJI. We used 5 GBS isolates, two clinical strains and three control strains, including one displaying high-level gentamicin resistance (HLGR). The results from the checkerboard and time-kill assays (TKAs) were compared. For TKAs, antibiotic concentrations for penicillin were 0.048 and 0.2 mg/L, and for gentamicin 4 mg/L or 12.5 mg/L. In the checkerboard assay, the median fractional inhibitory concentration indices (FICIs) of all isolates indicated indifference. TKAs for all isolates failed to demonstrate synergism with penicillin 0.048 or 0.2 mg/L, irrespective of gentamicin concentrations used. Rapid killing was seen with penicillin 0.048 mg/L plus either 4 mg/L or 12.5 mg/L gentamicin, from 2 h up to 8 h hours after antibiotic exposure. TKAs with penicillin 0.2 mg/L decreased the starting inoculum below the limit of quantification within 4–6 h, irrespective of the addition of gentamicin. Fast killing was seen with penicillin 0.2 mg/L plus 12.5 mg/L gentamicin within the first 2 h. Our in vitro results indicate that the addition of gentamicin to penicillin contributes to faster killing at low penicillin concentrations, but only within the first few hours. Twenty-four hours after antibiotic exposure, PEN alone was bactericidal and synergism was not seen.

Is gentamicin necessary in the antimicrobial treatment for group B streptococcal infections in the elderly? An in vitro study with human blood products

Abstract Background: According to expert opinions, gentamicin should be administered as an adjunct to penicillin against severe group B streptococcal (GBS) infections. Whether the adjunct is important is of particular interest for elderly patients. Not only is the risk of aminoglycoside nephrotoxicity higher in elderly persons, but their immune defence to bacterial infections may also be impaired. Method: Time-kill assays with human blood products, such as serum, neutrophilic granulocytes (opsonophagocytic assays) and whole blood from healthy, elderly volunteers were performed to evaluate the effect of gentamicin in combination with penicillin. Results: In time-kill assays with human serum and in opsonophagocytic assays, we saw a trend for faster killing with the penicillin–gentamicin combination therapy. This effect was seen 4 and 6 h after antibiotic exposure but not at time points evaluated at ≥8 h. In whole blood killing assays, no difference in killing rates was observed with adjunctive gentamicin therapy. Conclusion: The criteria for synergism were not fulfilled when the effect of penicillin–gentamicin combinations was compared with that of penicillin monotherapy. Rapid killing of GBS within the first few hours was observed in time-kill assays with human blood products. Considering that elderly people are prone to gentamicin nephrotoxicity and that in severe GBS infection a high penicillin dose is administered every 4–6 h, the prolonged use of adjunctive aminoglycosides in these infections requires caution.

Is Penicillin Plus Gentamicin Synergistic Against Sessile Group B Streptococcal Isolates? An in Vivo Study With an Experimental Model of Foreign-Body Infection

The rate of invasive group B Streptococcus (GBS) infections is steadily increasing, particularly in older persons and in adults with diabetes and other comorbidities. This population includes persons with a foreign body (e.g., who have undergone arthroplasty). In a rat tissue cage model, we evaluated the efficacy of adjunctive gentamicin (GEN) administered systemically (5 mg/kg body weight) every 24 h, or locally (12.5 mg/L tissue cage concentration) every 24 or 72 h, in combination with penicillin (PEN) administered systemically (250,000 IU/kg body weight three times per day). The efficacy was evaluated on two different sessile forms of GBS: transition (i.e., in between planktonic and biofilm) and biofilm. After 3 days of treatment, the mean bacterial load reduction of transition-form GBS was greater in all PEN–GEN combination groups than in the PEN monotherapy group (P ≤ 0.03). The 6-day regimen decreased the bacterial load significantly in comparison to the 3-day regimen, irrespective of growth form and adjunctive GEN (P < 0.01). After 6 days of treatment, the mean reduction in transition-form GBS was greater with PEN plus GEN administered locally every 24 h than with PEN monotherapy (P = 0.03). These results were not confirmed with biofilm GBS. The difference in mean bacterial load reduction between all PEN–GEN and PEN monotherapy groups was <100 CFU/mL. Hence, synergy criteria were not fulfilled. Adjunctive systemic GEN consists of potential side effects and showed poor efficacy in this study. Combining systemic PEN and local GEN has a potential application in the treatment of streptococcal implant-associated infections.

Osteoarticular and skin and soft-tissue infections caused by Streptococcus agalactiae in elderly patients are frequently associated with bacteremia

Older persons (≥65 years) are at risk for invasive group B streptococcal (GBS) infections. The most frequent clinical syndromes in 174 infection episodes were osteoarticular (40%) and skin and soft-tissue infections (30%). In 36% of episodes, a companion microorganism was isolated, and in 45%, blood culture results were positive. Antibiotics were streamlined after species identification in 29% of monomicrobial infections. These findings have clinical and therapeutic implications for GBS infections in the elderly.

A 10-year observational study of Streptococcus dysgalactiae bacteraemia in adults: frequent occurrence among female intravenous drug users.

Beta-haemolytic streptococci of groups C and G have become increasingly recognized as causes of invasive human infections. We reviewed clinical and molecular characteristics of Streptococcus dysgalactiae isolates that caused bacteraemia in adults from 2006 to 2015. Among 67 episodes, skin and soft-tissue infections (43%) and emm types stG62647.0 (26%) were the most frequent clinical manifestation and emm type, respectively. Nineteen (28%) episodes occurred in intravenous drug users (75% women). Our observational study shows similarities to but also differences from other reports. The former include the most frequent clinical presentations, and the most frequently found emm types. This report highlights a relatively high proportion of female intravenous drug users among S. dysgalactiae bacteraemia episodes.