The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

Development of an ultrafast PCR to detect clinically relevant acquired vancomycin-resistance genes from cultured enterococci.

BACKGROUND: VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. METHODS: Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. RESULTS: All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). CONCLUSIONS: The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.

Impact of the phenotypic expression of temocillin resistance in Escherichia coli on temocillin efficacy in a murine peritonitis model.

BACKGROUND: Temocillin is a narrow spectrum ß-lactam active against MDR Enterobacterales. Mechanisms of acquired resistance to temocillin are poorly understood. We analysed resistance mechanisms in clinical isolates of Escherichia coli and evaluated their impact on temocillin efficacy in vitro and in a murine peritonitis model. METHODS: Two sets of isogenic clinical E. coli strains were studied: a susceptible isolate (MLTEM16S) and its resistant derivative, MLTEM16R (mutation in nmpC porin gene); and temocillin-resistant derivatives of E. coli CFT073: CFT-ΔnmpC (nmpC deletion), CFTbaeS-TP and CFTbaeS-AP (two different mutations in the baeS efflux-pump gene).Fitness cost, time-kill curves and phenotypic expression of resistance were determined. Temocillin efficacy was assessed in a murine peritonitis model. RESULTS: MICs of temocillin were 16 and 64 mg/L for MLTEM16S and MLTEM16R, respectively, and 8, 128, 256 and 256 mg/L for E. coli-CFT073, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP, respectively. No fitness cost of resistance was evidenced. All resistant strains showed heteroresistant profiles, except for CFTbaeS-AP, which displayed a homogeneous pattern. In vitro, temocillin was bactericidal against MLTEM16R, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP at 128, 256, 512 and 512 mg/L, respectively. In vivo, temocillin was as effective as cefotaxime against MLTEM16R, CFT-ΔnmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality). CONCLUSIONS: Heteroresistant NmpC porin alteration and active efflux modification do not influence temocillin efficacy despite high MIC values, unfavourable pharmacokinetic/pharmacodynamic conditions and the absence of fitness cost, whereas homogeneously expressed BaeS efflux pump alteration yielding similar MICs leads to temocillin inefficacy. MIC as sole predictor of temocillin efficacy should be used with caution.

Fatal Clostridium septicum gas gangrene complicating ECMO: case report and review of literature.

Clostridium septicum gas gangrene is a severe and deadly infection caused by an anaerobic, spore-forming, Gram-positive bacillus. As previously described, two entities are observed: traumatic and spontaneous (or non-traumatic) forms. In this report, we aim to describe the case of a fulminant and ultimately fatal C. septicum myonecrosis occurring in a patient who was first admitted for refractory cardiac arrest and placed on veino-arterial extracorporeal membrane oxygenation (ECMO). Building upon prior studies that have documented cases of spontaneous gas gangrene caused by C. septicum, we provide an updated compilation, focusing on microbiological characteristics of C. septicum, along with the diagnostic and therapeutic challenges associated with spontaneous gas gangrene. Additionally, the specific clinical situation of our case illustrates the seriousness of this infectious complication that combined both spontaneous and traumatic gas gangrene risk factors. We thus, discuss the antibiotic coverage prior to the initiation of ECMO procedure.

Intracellular activity and in vivo efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against Staphylococcus aureus clinical isolates.

Objectives: Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both in vitro and in vivo for the treatment of septic arthritis caused by Staphylococcus aureus. Methods: Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the in vivo efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg). Results: MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2 × MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (∼10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h. Conclusions: Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

RNAIII is linked with the pentose phosphate pathway through the activation of RpiRc in Staphylococcus aureus.

Staphylococcus aureus RNAIII is a dual-function regulatory RNA that controls the expression of multiple virulence genes and especially the transition from adhesion to the production of exotoxins. However, its contribution to S. aureus central metabolism remains unclear. Using MS2-affinity purification coupled with RNA sequencing, we uncovered more than 50 novel RNAIII-mRNA interactions. Among them, we demonstrate that RNAIII is a major activator of the rpiRc gene, encoding a regulator of the pentose phosphate pathway (PPP). RNAIII binds the 5' UTR of rpiRc mRNA to favor ribosome loading, leading to an increased expression of RpiRc and, subsequently, of two PPP enzymes. Finally, we show that RNAIII and RpiRc are involved in S. aureus fitness in media supplemented with various carbohydrate sources related to PPP and glycolysis. Collectively, our data depict an unprecedented phenotype associated with the RNAIII regulon, especially the direct implication of RNAIII in central metabolic activity modulation. These findings show that the contribution of RNAIII in Staphylococcus aureus adaptation goes far beyond what was initially reported. IMPORTANCE: Staphylococcus aureus is a major human pathogen involved in acute and chronic infections. Highly recalcitrant to antibiotic treatment, persistent infections are mostly associated with the loss of RNAIII expression, a master RNA regulator responsible for the switch from colonization to infection. Here, we used the MS2 affinity purification coupled with RNA sequencing approach to identify novel mRNA targets of RNAIII and uncover novel functions. We demonstrate that RNAIII is an activator of the expression of genes involved in the pentose phosphate pathway and is implicated in the adjustment of bacterial fitness as a function of carbohydrate sources. Taken together, our results demonstrate an unprecedented role of RNAIII that goes beyond the knowledge gained so far and contributes to a better understanding of the role of RNAIII in bacterial adaptation expression and the coordination of a complex regulatory network.