Group A Streptococcus (GAS) has acquired an arsenal of virulence factors, promoting life-threatening invasive infections such as necrotizing fasciitis. Current therapeutic regimens for necrotizing fasciitis include surgical debridement and treatment with cell wall-active antibiotics. Addition of clindamycin (CLI) is recommended, although clinical evidence is lacking. Reflecting the current clinical dilemma, an observational study showed that only 63% of the patients with severe invasive GAS infection received CLI. This work thus aimed to address whether CLI improves necrotizing fasciitis outcome by modulating virulence factors of CLI-susceptible and CLI-resistant GAS in vitro and in vivo. Treatment with CLI reduced extracellular DNase Sda1 and streptolysin O (SLO) activity in vivo, whereas subinhibitory CLI concentrations induced expression and activity of SLO, DNase, and Streptococcus pyogenes cell envelope protease in vitro. Our in vivo results suggest that CLI should be administered as soon as possible to patients with necrotizing fasciitis, while our in vitro studies emphasize that a high dosage of CLI is essential.

Increasing antibiotic resistances and a lack of new antibiotics render the treatment of Gram-negative bacterial infections increasingly difficult. Therefore, additional approaches are being investigated. Macrolides are not routinely used against Gram-negative bacteria due to lack of evidence of in vitro effectiveness. However, it has been shown that Pseudomonas spp. are susceptible to macrolides in liquid RPMI-1640 and clinical data suggest improvement in patients’ outcomes. So far, these findings have been hardly applicable to the clinical setting due to lack of routine low-complexity antimicrobial susceptibility testing (AST) for macrolides. We therefore optimized and compared broth microdilution and disk diffusion AST. Multidrug-resistant Gram-negative bacteria (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa) were tested for azithromycin susceptibility by disk diffusion and broth microdilution in Mueller–Hinton and RPMI-1640 media. Azithromycin susceptibility of Enterobacteriaceae and a subgroup of P. aeruginosa increased significantly on RPMI-1640 agar compared to Mueller–Hinton agar. Further, a significant correlation (Kendall, τ, p) of zone diameters and minimal inhibitory concentrations (MICs) was found on RPMI-1640 agar for E. coli (−0.4279, 0.0051), E. cloacae (−0.3783, 0.0237) and P. aeruginosa (−0.6477, <0.0001). Performing routine disk diffusion AST on RPMI-1640 agar may lead to the identification of additional therapeutic possibilities for multidrug-resistant bacterial infections in the routine clinical diagnostic setting.

The clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.

We report a 67-year-old man with recurrent advanced oropharyngeal squamous cell carcinoma who developed aseptic meningitis, with first symptoms arising approximately 9hours after the first administration of cetuximab, and review the literature to identify key signs and symptoms of this condition. Cetuximab is a monoclonal antibody targeting the epidermal growth factor receptor which has been rarely associated with aseptic meningitis. Besides the case description, a MEDLINE search was performed. In five patients identified in the literature and our patient, the leading signs and symptoms included headache, neck stiffness and high fever developing within a few hours of the first cetuximab administration. Cerebrospinal fluid (CSF) analysis revealed severe pleocytosis (range: 528-2300/ l) with dominance of neutrophils ( 87%). Clinical recovery within 1-2weeks was accompanied by normalization of CSF cell count within 4-7days. Re-challenge with cetuximab at a reduced dose caused recurrent aseptic meningitis in one of three patients. In summary, aseptic meningitis is a rare complication after first cetuximab exposure that the clinician should be aware of. CSF analysis is the key to diagnosis and recovery is usually complete within days to weeks after withdrawal of the drug. Re-challenge may be considered but bears the risk of recurrence.

AIMS OF THE STUDY Invasive streptococcal infections affect more than half a million patients worldwide every year and have a high lethality. Little is known about the epidemiology and microbiological characteristics of streptococcal infections in Switzerland. This case series study aims to describe the demographics, known risk factors for streptococcal skin and soft tissue infections, clinical presentations, treatment and outcomes of patients admitted to the University Hospital Zurich between 2000 and 2014 with invasive streptococcal infections caused by Streptococcus pyogenes (group A Streptococcus), Streptococcus dysgalactiae ssp. equisimilis or the Streptococcus anginosus group, as well as the microbiological characteristics of the clinical isolates. METHODS Data collected retrospectively from patients hospitalised between 2000 and 2014 with invasive streptococcal infections were analysed. M protein gene (emm) typing of the bacterial clinical isolates was carried out according to the Centers for Disease Control and Prevention guidelines. RESULTS A total of 86 patients with invasive beta-haemolytic streptococcal infections were included in this study, of which 49% presented with necrotising fasciitis. The median age was 44 years and half were female. The most common risk factor was acute skin lesions. C-reactive protein levels were significantly higher in patients with necrotising fasciitis, as were acute renal failure and distributive shock. Beta-lactam antibiotics were given to most patients, and intravenous immunoglobulins were given to 18% of patients within the first 24 hours. All patients suffering from necrotising fasciitis underwent surgery. The overall case fatality rate was 8.1% at 30 days post admission. All Group A Streptococcus strains were susceptible to penicillin and clindamycin, and we found resistance to tetracycline in 11.9% of strains. The most common emm-type isolated was emm1 (44.4%). CONCLUSIONS Invasive beta-haemolytic streptococcal infections, the most severe presentation of which is necrotising fasciitis, remain a serious clinical issue and require rapid diagnosis and treatment. This is the first representative analysis monitoring clinical and microbiological characteristics of patients with a severe invasive beta-haemolytic streptococcal infection and treated in Zurich, Switzerland. In addition to the detailed reporting of various clinical and microbiological characteristics, we show that C-reactive protein levels, acute renal failure and distributive shock were higher in the patients with necrotising fasciitis. We also found a low case fatality rate compared to other reports. The detailed clinical data and microbiological characteristics depicted in this study will lead to a better understanding of regional differences in severe invasive streptococcal infections.

Beta-lactam antibiotics are widely used to treat infections caused by the important human pathogen Staphylococcus aureus. Resistance to beta-lactams, as found in methicillin-resistant S. aureus (MRSA), renders effective treatment difficult. The second messenger cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) promotes beta-lactam resistance in clinical S. aureus isolates. C-di-AMP plays a crucial role in the regulation of cellular processes such as virulence, cell wall homeostasis and resistance to beta-lactams in many bacterial species. In S. aureus, c-di-AMP synthesis is mediated by the diadenylate cyclase DacA, while its degradation is carried out by the phosphodiesterases GdpP and Pde2. In this work, we assessed the effect of altered c-di-AMP levels due to mutations in cacA, gdpP or gdpP/pde2 on virulence determinants. We report that a previously described growth defect in bacteria producing high c-di-AMP levels is mainly attributable to smaller cell size. High c-di-AMP levels also led to decreased survival upon oxidative stress, reduced production of the antioxidant staphyloxanthin, increased oxacillin and fosfomycin resistance and increased cell wall thickness. While resistance to ceftaroline was not affected, high c-di-AMP levels promoted tolerance to this antibiotic. In response to cell wall stress induced by antibiotics, the three-component regulatory system VraTSR mediates an increase in cell wall synthesis via the cell wall stress stimulon (CWSS). Increased c-di-AMP levels led to an activation of the CWSS. Upon deletion of vraR, resistance to oxacillin and fosfomycin as well as cell wall thickness diminished in the ΔgdpP mutant, indicating a contribution of the VraTSR system to the cell wall related phenotypes.

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Objectives: Group A Streptococcus (GAS) necrotizing fasciitis (NF) is a difficult-to-treat bacterial infection associated with high morbidity and mortality despite extensive surgery and targeted antibiotic treatment. Bacteria surviving prolonged antibiotic exposure without displaying genetic resistance, so-called persisters, are associated with difficult-to-treat infections, such as GAS-NF. In the present study, we investigated the presence of persistent GAS in tissue freshly debrided from three NF patients and examined more in depth the persisters phenomenon in GAS-NF clinical isolates. Methods: Time-lapse imaging of freshly isolated GAS-NF clinical isolates, the image analysis software ColTapp and antibiotic challenge-based persisters assays were used to assess the presence of persisters. Results: We show for the first time that GAS recovered directly from freshly debrided NF patient tissue are characterized by increased colony appearance time heterogeneity, indicating the presence of persisters. Acidic pH or nutrient stress exposure, mimicking the NF-like environment in vitro, similarly leads to phenotypic heterogeneity resulting in enhanced antibiotic survival and confirming the presence of GAS persisters. Conclusions: GAS persisters are present in the tissue freshly debrided from GAS-NF patients and might be one explanation for antibiotic treatment failure and surgery requirement in GAS-NF. Tailored treatment options, including the use of persisters-targeting drugs, need to be developed to increase GAS-NF therapy success.