Enterococcus hirae and Barnesiella intestinihominis Facilitate Cyclophosphamide-Induced Therapeutic Immunomodulatory Effects.

The efficacy of the anti-cancer immunomodulatory agent cyclophosphamide (CTX) relies on intestinal bacteria. How and which relevant bacterial species are involved in tumor immunosurveillance, and their mechanism of action are unclear. Here, we identified two bacterial species, Enterococcus hirae and Barnesiella intestinihominis that are involved during CTX therapy. Whereas E. hirae translocated from the small intestine to secondary lymphoid organs and increased the intratumoral CD8/Treg ratio, B. intestinihominis accumulated in the colon and promoted the infiltration of IFN-γ-producing γδT cells in cancer lesions. The immune sensor, NOD2, limited CTX-induced cancer immunosurveillance and the bioactivity of these microbes. Finally, E. hirae and B. intestinihominis specific-memory Th1 cell immune responses selectively predicted longer progression-free survival in advanced lung and ovarian cancer patients treated with chemo-immunotherapy. Altogether, E. hirae and B. intestinihominis represent valuable "oncomicrobiotics" ameliorating the efficacy of the most common alkylating immunomodulatory compound.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

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.

High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of ß-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the ß-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance lysis of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.

Impact of Enterococcus faecalis Endocarditis Treatment on Risk of Relapse.

BACKGROUND: Enterococcus faecalis infective endocarditis (EFIE) is characterized by a higher frequency of relapses than other infective endocarditis. The role of the treatment on its occurrence remains poorly understood. The aim of this study was to investigate whether the antibiotic regimen could impact the risk of relapse in EFIE. MATERIALS: This was a multicenter retrospective study of patients diagnosed with definite EFIE between 2015 and 2019 in 14 French hospitals. The primary endpoint was the occurrence of relapses within the year following endocarditis diagnosis. As death was a competing risk for relapse, Fine and Gray models were used for studying risk factors and impact of treatment. RESULTS: Of the 279 patients included, 83 (29.7%) received the amoxicillin-gentamicin (A-G) combination, 114 (40.9%) amoxicillin-ceftriaxone (A-C), 63 (22.6%) A-G and A-C (A-G/A-C) sequentially, 9 (3.2%) amoxicillin (A), and 10 received other treatments. One-year-relapse rate was 9.3% (26 patients). Relapse occurred after a median delay of 107 days from EFIE diagnosis; 6 occurred after 6 months, and 6 were diagnosed by blood cultures in asymptomatic patients. In multivariate analysis, surgery during treatment was a protective factor against one-year relapse and death.The cumulative incidence of relapse 1 year after endocarditis was 46.2% for patients treated with amoxicillin, 13.4% with A-G, 14.7% with A-C, and 4.3% with A-G/A-C (P≥.05 in multivariate analysis). CONCLUSIONS: Relapses after treatment of EFIE are frequent, frequently asymptomatic, and may occur more than 6 months after the initial episode.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward ß-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

Differential response to antibiotic therapy in staphylococcal infective endocarditis: contribution of an ex vivo model.

OBJECTIVES: Staphylococcal infective endocarditis (IE) remains a hard-to-treat infection with high mortality. Both the evaluation of new innovative therapies and research on alternative models mimicking human IE are therefore urgently needed to improve the prognosis of patients with diagnosed IE. Dalbavancin is a novel anti-staphylococcal lipoglycopeptide but there are limited data supporting its efficacy on biofilm infections. This antibiotic could be an alternative to current therapies for the medical treatment of IE but it needs to be further evaluated. METHODS: Here we developed an original ex vivo model of Staphylococcus aureus IE on human heart valves and assessed biofilm formation on them. After validating the model, the efficacy of two antistaphylococcal antibiotics, vancomycin and dalbavancin, was compared by measuring and visualizing their respective ability to inhibit and eradicate late-formed biofilm. RESULTS: Determination of the minimum biofilm inhibitory (MbIC) and eradicating (MbEC) concentrations in our ex vivo model identified dalbavancin as a promising drug with much lower MbIC and MBEC than vancomycin (respectively <0.01 versus 28 mg/L and 0.03 versus 32 mg/L). CONCLUSIONS: These data highlight a strong bactericidal effect of dalbavancin, particularly on an infected heart valve compared with vancomycin. Dalbavancin could be a realistic alternative treatment for the management of staphylococcal IE.

Defining standard and high dosages for ß-lactam agents administered by intermittent, prolonged or continuous infusion: a PK/PD simulation study.

BACKGROUND: The new definitions of antimicrobial susceptibility categories proposed by EUCAST in 2020 require the definition of standard and high dosages of antibiotic. For injectable ß-lactams, standard and high dosages have been proposed for short-infusion regimens only. OBJECTIVES: To evaluate dosages for ß-lactams administered by prolonged infusion (PI) and continuous infusion (CI). METHODS: Monte Carlo simulations were performed for seven injectable ß-lactams: aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, piperacillin and temocillin. Various dosage regimens based on short infusion, PI or CI were simulated in virtual patients. Pharmacokinetic (PK) profiles and PTAs were obtained based on reference population PK models, as well as PK/pharmacodynamic targets and MIC breakpoints proposed by EUCAST. Alternative dosage regimens associated with PTA values similar to those of recommended dosages up to the breakpoints were considered acceptable. RESULTS: Adequate PTAs were confirmed for most EUCAST short-infusion dosage regimens. A total of 9 standard and 14 high dosages based on PI (3 to 4 h) or CI were identified as alternatives. For cefepime and aztreonam, only PI and CI regimens could achieve acceptable PTAs for infections caused by Pseudomonas spp.: 2 g q8h as PI of 4 h or 6 g/24 h CI for cefepime; 2 g q6h as PI of 3 h or 6 g/24 h CI for aztreonam. CONCLUSIONS: These alternative standard and high dosage regimens are expected to provide antibiotic exposure compatible with new EUCAST definitions of susceptibility categories and associated MIC breakpoints. However, further clinical evaluation is necessary.

Simple purification and characterization of soluble and homogenous ABC-F translation factors from Enterococcus faecium.

The family of ATP-binding cassette F proteins (ABC-F) is mainly made up of cytosolic proteins involved in regulating protein synthesis, and they are often part of a mechanism that confers resistance to ribosome-targeting antibiotics. The existing literature has emphasized the difficulty of purifying these recombinant proteins because of their very low solubility and stability. Here, we describe a rapid and efficient three-step purification procedure that allows for the production of untagged ABC-F proteins from Enterococcus faecium in the heterologous host Escherichia coli. After four purified ABC-F proteins were produced using this protocol, their biological activities were validated by in vitro experiment. In conclusion, our study provides an invaluable tool for obtaining large amounts of untagged and soluble ABC-F proteins that can then be used for in vitro experiments.

Effectiveness of a multimodal intervention to improve blood culture collection in an adult emergency department.

We investigated the impact of a multimodal intervention to improve the compliance of BC collections as a composite outcome, taking into account both blood volume collected and absence of solitary BC. We performed a quasi-experimental study using a before-after design (5 months for pre- and post-intervention evaluation) in an adult emergency department at a tertiary care hospital that showed that a multimodal intervention was associated with a dramatic increase in the proportion of blood cultures that were collected as recommended per national guidelines, from 17.3% (328/1896) to 68.9% (744/1080), P < 0.0001. The implementation of such intervention in other settings could improve the diagnosis of bloodstream infections and reduce irrelevant costs.

Web-based prediction of antimicrobial resistance in enterococcal clinical isolates by whole-genome sequencing.

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics.

A small regulatory RNA alters Staphylococcus aureus virulence by titrating RNAIII activity.

Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.

Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species.

The Enterobacter cloacae complex (ECC) is a group of diverse environmental and clinically relevant bacterial species associated with a variety of infections in humans. ECC have emerged as one of the leading causes of nosocomial infections worldwide. The purpose of this paper is to evaluate the activity of NOSO-502 and colistin (CST) against a panel of ECC clinical isolates, including different Hoffmann's clusters strains, and to investigate the associated resistance mechanisms. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). MIC50 and MIC90 of NOSO-502 against ECC are 1 µg/mL and 2 µg/mL, respectively, with a MIC range from 0.5 µg/mL to 32 µg/mL. Only strains belonging to clusters XI and XII showed decreased susceptibility to both NOSO-502 and CST while isolates from clusters I, II, IV, and IX were only resistant to CST. To understand this phenomenon, E. cloacae ATCC 13047 from cluster XI was chosen for further study. Results revealed that the two-component system ECL_01761-ECL_01762 (ortholog of CrrAB from Klebsiella pneumoniae) induces NOSO-502 hetero-resistance by expression regulation of the ECL_01758 efflux pump component (ortholog of KexD from K. pneumoniae) which could compete with AcrB to work with the multidrug efflux pump proteins AcrA and TolC. In E. cloacae ATCC 13047, CST-hetero-resistance is conferred via modification of the lipid A by addition of 4-amino-4-deoxy-l-arabinose controlled by PhoPQ. We identified that the response regulator ECL_01761 is also involved in this resistance pathway by regulating the expression of the ECL_01760 membrane transporter.

High Prevalence of OXA-23 Carbapenemase-Producing Proteus mirabilis among Amoxicillin-Clavulanate-Resistant Isolates in France.

In this multicentric study performed in 12 French hospitals, we reported that 26.9% (14/52) of the amoxicillin-clavulanate-resistant Proteus mirabilis isolates produced the OXA-23 carbapenemase. We found that an inhibition zone diameter of <11 mm around the amoxicillin-clavulanate disc was an accurate screening cutoff to detect these OXA-23 producers. We confirmed by whole-genome sequencing that these OXA-23-producers all belonged to the same lineage that has been demonstrated to disseminate OXA-23 or OXA-58 in P. mirabilis.

Diffusion of amoxicillin into heart valves from infective endocarditis patients.

OBJECTIVES: Amoxicillin is the drug of choice in the management of streptococcal and enterococcal infective endocarditis (IE) but little is known regarding amoxicillin diffusion into infected heart valves. Herein, we assessed amoxicillin valvular distribution and related pharmacokinetic/pharmacodynamic (PK/PD) target attainment in IE patients undergoing heart valve surgery. PATIENTS AND METHODS: In this 2-year prospective study, patients with IE treated by continuous infusion of amoxicillin and undergoing a surgical valve replacement were included. Both amoxicillin plasma and tissue concentrations were measured the day of surgery. Amoxicillin concentration in plasma and crushed heart valves were measured by a validated liquid chromatography method coupled with ultra-violet and tandem mass spectrometry, respectively. MIC and MBC of amoxicillin were determined for all available isolates. The rate of achievement of PK/PD efficacy parameters were assessed. RESULTS: Twenty-two heart valves were removed from 20 patients. Bacterial aetiology was streptococcal (n = 17) and enterococcal (n = 3). Amoxicillin mean daily dose was 12 ±â€Š3 g/24 h, mean plasma concentration was 29 ±â€Š21 mg/L (n = 15), mean tissue concentration was 23 ±â€Š15 mg/L (n = 22). Median diffusion rate was 62%. Patients reached a plasma concentration target >4XCMI (n = 13). Tissue concentrations were bactericidal for all streptococcal IE but not for enterococcal IE. CONCLUSIONS: Amoxicillin intravalvular measurements in IE treated patients showed significant penetration into the infectious site. These data are reassuring that in situ bactericidal concentrations can be largely achieved in the management of streptococcal IE and support the need for combination antibiotic therapy for enterococcal IE.

Cerebral nocardiosis in a patient treated with pembrolizumab: a first case report.

BACKGROUND: Checkpoints inhibitors (CPIs) are increasingly used for the treatment of several malignancies. The most common side effects are Immune Related Adverse Events, while infectious complications are rare, especially cerebral nocardiosis. CASE PRESENTATION: Here, we report the first clinical case of a cerebral nocardiosis revealed after seizure in a patient treated by pembrolizumab for a metastatic lung cancer, in the absence of any additional immunosuppressive therapy or risk factors for cerebral nocardiosis. The extended evaluation including a brain CT-scan did not reveal any lesion before pembrolizumab. Nevertheless, the 3-month delay between the start of Pembrolizumab and the diagnosis of cerebral nocardiosis suggests that the infection occurred prior to the CPI. Unfortunately, the patient died during treatment for cerebral nocardiosis, while the lung cancer tumor mass had decreased by 80% after the sixth cycle of pembrolizumab. CONCLUSIONS: This case report emphasizes that clinicians should consider diagnoses other than metastasis in a patient with a brain mass and metastatic cancer treated with CPI, such as opportunistic infections or IRAE.

Genetic features of the poxtA linezolid resistance gene in human enterococci from France.

OBJECTIVES: To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments. METHODS: All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments. RESULTS: Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids. CONCLUSIONS: The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use.

Activity of the combination of colistin and fosfomycin against NDM-1-producing Escherichia coli with variable levels of susceptibility to colistin and fosfomycin in a murine model of peritonitis.

BACKGROUND: Alternative treatments are needed against NDM-1-producing Escherichia coli. Colistin (COL) and fosfomycin (FOS) often remain active in vitro but selection of resistant mutants is frequent if used separately. We determined whether the combination of colistin and fosfomycin may be useful to treat infections with NDM-1-producing E. coli with varying levels of resistance. METHODS: Isogenic derivatives of E. coli CFT073 with blaNDM-1 and variable levels of resistance to colistin and fosfomycin (CFT073-NDM1, CFT073-NDM1-COL and CFT073-NDM1-FOS, respectively) were used. The combination (colistin + fosfomycin) was tested in vitro and in a fatal peritonitis murine model. Mortality and bacterial loads were determined and resistant mutants detected. RESULTS: Colistin MICs were 0.5, 16 and 0.5 mg/L and fosfomycin MICs were 1, 1 and 32 mg/L against CFT073-NDM1, CFT073-NDM1-COL and CFT073-NDM1-FOS, respectively. In time-kill curves, combining colistin with fosfomycin was synergistic and bactericidal against CFT073-NDM1 and CFT073-NDM1-FOS, with concentrations of 4× MIC (for both drugs), but not against CFT073-NDM1-COL (concentrations of colistin = 0.5× MIC), due to regrowth with fosfomycin-resistant mutants. Mice died less and bacterial counts were lower in spleen with the combination compared with monotherapy against all strains; the combination prevented selection of resistant mutants except for CFT073-NDM1-COL where fosfomycin-resistant mutants were found in all mice. CONCLUSIONS: Combining colistin and fosfomycin was beneficial in vitro and in vivo against NDM-1-producing E. coli, even with strains less susceptible to colistin and fosfomycin. However, the combination failed to prevent the emergence of fosfomycin-resistant mutants against colistin-resistant strains. Combining colistin and fosfomycin constitutes an alternative for treatment of NDM-1 E. coli, except against colistin-resistant strains.