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.

Reduced Chlorhexidine Susceptibility Is Associated with Tetracycline Resistance tet Genes in Clinical Isolates of Escherichia coli.

Chlorhexidine is a widely used antiseptic in hospital and community health care. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and health care-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutants from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal that two different evolutionary paths lead to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provide cross-resistance to colistin. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.

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.

Analysis of Paradoxical Efficacy of Carbapenems against Carbapenemase-Producing Escherichia coli in a Murine Model of Lethal Peritonitis.

The clinical benefit of carbapenems against carbapenemase-producing Enterobacteriaceae (CPE) remains in question. MICs of imipenem (IMP) and ertapenem (ERT) against isogenic derivatives of the wild-type strain Escherichia coli CFT073 producing KPC-3, OXA-48, or NDM-1 were 0.25, 2, 16, and 64 mg/liter for IMP and 0.008, 0.5, 8, and 64 mg/liter for ERT, respectively. Swiss ICR-strain mice with peritonitis were treated for 24 h with IMP or ERT. Despite a limited duration of time during which free antibiotic concentrations were above the MIC (down to 0% for the NDM-1-producing strain), IMP and ERT significantly reduced bacterial counts in spleen and peritoneal fluid at 24 h (P < 0.005) and prevented mortality. Several possible explanations were investigated. Addition of 4% albumin or 50% normal human serum did not modify IMP activity. Bacterial fitness of resistant strains was not altered and virulence did not decrease with resistance. In the presence of subinhibitory concentrations of ERT, growth rates of OXA-48, KPC-3, and NDM-1 strains were significantly decreased and filamentation of the NDM-1 strain was observed. The expression of blaNDM-1 was not decreased in vivo compared to in vitro No zinc depletion was observed in infected mice compared with Mueller-Hinton broth. In conclusion, a paradoxical in vivo efficacy of IMP and ERT against highly resistant carbapenemase-producing E. coli was confirmed. Alternative mechanisms of antibacterial effects of subinhibitory concentrations of carbapenems may be involved to explain in vivo activity. These results are in agreement with a potential clinical benefit of carbapenems to treat CPE infections, despite high carbapenem MICs.

Unexpected Activity of Oral Fosfomycin against Resistant Strains of Escherichia coli in Murine Pyelonephritis.

Fosfomycin tromethamine activity is well established for oral treatment of uncomplicated lower urinary tract infections, but little is known about its potential efficacy in pyelonephritis. Ascending pyelonephritis was induced in mice infected with 6 strains of Escherichia coli (fosfomycin MICs, 1 µg/ml to 256 µg/ml). The urine pH was 4.5 before infection and 5.5 to 6.0 during infection. Animals were treated for 24 h with fosfomycin (100 mg/kg of body weight subcutaneously every 4 h), and the CFU were enumerated in kidneys 24 h after the last fosfomycin injection. Peak (20.5 µg/ml at 1 h) and trough (3.5 µg/ml at 4 h) levels in plasma were comparable to those obtained in humans after an oral dose of 3 g. Fosfomycin treatment significantly reduced the bacterial loads in kidneys (3.65 log10 CFU/g [range, 1.83 to 7.03 log10 CFU/g] and 1.88 log10 CFU/g [range, 1.78 to 5.74 log10 CFU/g] in start-of-treatment control mice and treated mice, respectively; P < 10-6). However, this effect was not found to differ across the 6 study strains (P = 0.71) or between the 3 susceptible and the 3 resistant strains (P = 0.09). Three phenomena may contribute to explain this unexpected in vivo activity: (i) in mice, the fosfomycin kidney/plasma concentration ratio increased from 1 to 7.8 (95% confidence interval, 5.2, 10.4) within 24 h in vitro when the pH decreased to 5, (ii) the fosfomycin MICs for the 3 resistant strains (64 to 256 µg/ml) decreased into the susceptible range (16 to 32 µg/ml), and (iii) maximal growth rates significantly decreased for all strains and were the lowest in urine. These results suggest that local fosfomycin concentrations and physiological conditions may favor fosfomycin activity in pyelonephritis, even against resistant strains.

Neurologic Complications of Infective Endocarditis: A Joint Model for a Septic Thromboembolism and Inflammatory Small Vessel Disease.

OBJECTIVES: Embolic events from vegetations are commonly accepted as the main mechanism involved in neurologic complications of infective endocarditis. The pathophysiology may imply other phenomena, including vasculitis. We aimed to define the cerebral lesion spectrum in an infective endocarditis rat model. DESIGN: Experimental model of Staphylococcus aureus or Enterococcus faecalis infective endocarditis. Neurologic lesions observed in the infective endocarditis model were compared with three other conditions, namely bacteremia, nonbacterial thrombotic endocarditis, and healthy controls. SETTING: Research laboratory of a university hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: Brain MRI, neuropathology, immunohistochemistry for astrocyte and microglia, and bacterial studies on brain tissue were used to characterize neurologic lesions. MEASUREMENTS AND MAIN RESULTS: In the infective endocarditis group, MRI revealed at least one cerebral lesion in 12 of 23 rats (52%), including brain infarctions (n = 9/23, 39%) and cerebral microbleeds (n = 8/23, 35%). In the infective endocarditis group, neuropathology revealed brain infarctions (n = 12/23, 52%), microhemorrhages (n = 10/23, 44%), and inflammatory processes (i.e., cell infiltrates including abscesses, vasculitis, meningoencephalitis, and/or ependymitis; n = 11/23, 48%). In the bacteremia group, MRI studies were normal and neuropathology revealed only hemorrhages (n = 2/11, 18%). Neuropathologic patterns observed in the nonbacterial thrombotic endocarditis group were similar to those observed in the infective endocarditis group. Immunochemistry revealed higher microglial activation in the infective endocarditis group (n = 11/23, 48%), when compared with the bacteremia (n = 1/11, 9%; p = 0.03) and nonbacterial thrombotic endocarditis groups (n = 0/7, 0%; p = 0.02). CONCLUSIONS: This original model of infective endocarditis recapitulates the neurologic lesion spectrum observed in humans and suggests synergistic mechanisms involved, including thromboembolism and cerebral vasculitis, promoted by a systemic bacteremia-mediated inflammation.

Temocillin breakpoints in pyelonephritis: evaluation in a murine model due to ESBL-producing Escherichia coli clinical isolates.

BACKGROUND: Due to a spectrum restricted to Enterobacteriaceae and stability against ESBL and AmpC enzymes, temocillin is of major interest for the treatment of pyelonephritis. But there are still uncertainties about the optimal regimen and clinical breakpoints. OBJECTIVES: To study in a murine model of pyelonephritis the activity of temocillin against Escherichia coli isolates with different MICs in order to evaluate clinical breakpoints. METHODS: Four clinical uropathogenic E. coli isolates with temocillin MICs of 8 mg/L (Ec8), 16 mg/L (Ec16), 32 mg/L (Ec32) and 64 mg/L (Ec64) were evaluated. Antibiotic 24 h T>MIC achieved in humans was reproduced in mice with either intravenous temocillin (2 g q12h or 2 g q8h) or intravenous imipenem (1 g q8h). Efficacy was assessed by bacterial count in kidneys. RESULTS: Compared with controls, temocillin at 2 g q12h was highly efficient against Ec8 (-3.32 log10 cfu/g and negative cultures in 93% of mice; P < 0.001); imipenem gave similar results. Temocillin at 2 g q12h also induced high reduction of bacterial count against Ec16 (-2.92 log10 cfu/g; P < 0.001), albeit cultures were negative in only 48% of mice. In contrast, no significant effect was observed in mice infected by Ec32 (-0.01 log10 cfu/g; P = 0.981) or Ec64 (-0.55 log10 cfu/g; P = 0.523). Even temocillin at 2 g q8h failed to control Ec32 infection (-1.55 log10 cfu/g; P = 0.197). CONCLUSIONS: This model suggests a clinical breakpoint up to 16 mg/L for non-severe pyelonephritis treated with temocillin at 2 g q12h, a value consistent with the few previous available data.

Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections.

Ceftriaxone promotes the emergence of AmpC-overproducing Enterobacteriaceae in gut microbiota from hospitalized patients.

Epidemiological data suggest that ceftriaxone may promote the emergence of commensal AmpC-overproducing Enterobacteriaceae because of a high biliary excretion. We tested this hypothesis in hospitalized patients either treated by ceftriaxone alone or receiving no antibiotics. Hospitalized patients with no previous antibiotics or hospitalization in the last 3 months, treated only with ceftriaxone, were prospectively included. For each ceftriaxone-treated patient, a control patient receiving no antibiotics was included. Clinical data and stools were collected at T0 (before antibiotics) and T1 (at the end of ceftriaxone treatment or at discharge) and T2 (3-6 months after T1) for the ceftriaxone-treated patients and at T0 and T1 for control patients. Third-generation cephalosporin-resistant Enterobacteriaceae were detected, identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and characterized genetically. Clonal relatedness was evaluated by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Fifteen ceftriaxone and 22 control patients were included. Patients' characteristics did not differ. At T0, 2/15 ceftriaxone-treated versus 1/22 control patients carried third-generation cephalosporin-resistant Enterobacteriaceae (p = 0.6). At T1, 4/15 (27%) ceftriaxone-treated patients carried AmpC producers versus 0/22 control patients (p = 0.02). Additionally, two and three subjects carried extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in the ceftriaxone and control groups, respectively (p = 1). At T2, three ceftriaxone-treated patients still carried AmpC-producing Enterobacteriaceae with the same RAPD profile as at T1. In hospitalized subjects with no other selective pressure, treatment by ceftriaxone alone promotes the gut colonization by AmpC-overproducing Enterobacteriaceae in over a quarter of patients, with a persistent carriage after the end of antibiotic exposure. The ecological impact of ceftriaxone should not be underestimated.

Selection of Resistance to Clarithromycin in Mycobacterium abscessus Subspecies.

Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm(41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm(41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl, erm(41), whiB, rpIV, and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm(41) T28 sequevar and strain M. abscessus subsp. bolletii, which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm(41) C28 sequevar and M. abscessus subsp. massiliense, not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV, encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm(41) C28 and M. abscessus subsp. massiliense, whereas it is less common for M. abscessus subsp. abscessus erm(41) T28 sequevar and M. abscessus subsp. bolletii, where other mechanisms may be responsible for failure.

Ecological impact of ciprofloxacin on commensal enterococci in healthy volunteers.

Background: The ecological impact of ciprofloxacin on commensal enterococci is unknown. Methods: Forty-eight healthy volunteers received ciprofloxacin from day (D) 0 to D14; stools were collected on D7, D14 and D42. Fluoroquinolone-susceptible and -resistant enterococci (FQ-SE and FQ-RE) were detected and quantified by culture, and identified by MALDI-TOF MS. The relative abundance of FQ-RE over FQ-SE was determined. The genetic basis of fluoroquinolone resistance was deciphered by partial sequencing of gyrA and parC genes. Clonal relatedness was determined by random amplification of polymorphic DNA PCR. Clinical trial no.: NCT00190151. Results: Enterococci were carried by 47/48 (98%) subjects. Total counts were reduced during ciprofloxacin therapy (4.0 and 3.9 log cfu/g on D7 and D14 versus 5.9 log cfu/g before and 6.9 log cfu/g after treatment; P < 0.05). Twenty-one out of 48 (44%) carried FQ-RE; among them, 21/21 carried Enterococcus faecium , 19 carried Enterococcus faecalis and 11 carried other species. Five out of 48 (10%) harboured FQ-RE (ciprofloxacin MIC >4 mg/L) before treatment (all E. faecium ), 6 on D7 (3 E. faecium and 3 E. faecalis ), 8 on D14 (4 E. faecium and 4 E. faecalis ) and 10 (21%) on D42 (9 E. faecium and 1 E. faecalis ). The relative abundance of FQ-RE increased from 44% on D0 to 73% and 75% on D7 and D14, respectively. No acquisition of fluoroquinolone resistance among endogenous D0 strains was evidenced. All (14/14) distinct Fluoroquinolone-resistant E. faecalis clones were gyrA / parC double mutants with high-level resistance (ciprofloxacin MIC >64 mg/L). In contrast, 34/35 E. faecium exhibited low-level resistance (ciprofloxacin MIC 4-32 mg/L) with no gyrA / parC mutation, but overexpressed the chromosomal Efm qnr gene. As compared with Fluoroquinolone-susceptible strains, Fluoroquinolone-resistant E. faecium were more frequently ampicillin resistant and Fluoroquinolone-resistant E. faecalis were more highly resistant to gentamicin. Conclusions: Although intrinsically poorly susceptible to fluoroquinolones, gut populations of enterococci are highly impacted both quantitatively and qualitatively by ciprofloxacin.

Prospective Cohort Study of the Relative Abundance of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli in the Gut of Patients Admitted to Hospitals.

A total of 458 patients were prospectively included at hospital admission and screened for extended-spectrum-beta-lactamase-producing (ESBL) Escherichia coli carriage in 2007 and in 2010 to 2012. A 4-fold increase in ESBL carriage (3% to 12%), a 5-fold increase in numbers of community patients among ESBL carriers, and a higher number of multiple ESBL strains was found in the 2010 to 2012 period. ESBL E. coli represented the dominant E. coli strain (relative abundance, >50%) in 10/32 (31%) of ESBL carriers. This represents a major threat in terms of infectious risk and dissemination.

qnrA6 genetic environment and quinolone resistance conferred on Proteus mirabilis.

OBJECTIVES: To determine the genetic location and environment of the qnrA6 gene in Proteus mirabilis PS16 where it was first described and to characterize the quinolone resistance qnrA6 confers. METHODS: Transformation experiments and Southern blotting were performed for plasmid and genomic DNA of P. mirabilis PS16 to determine the qnrA6 location. Combinatorial PCRs with primers in qnrA6 and genes usually surrounding qnrA genes were used to determine the genetic environment. The qnrA6 coding region, including or not the promoter region, was cloned into vectors pTOPO and pBR322 and the MICs of six quinolones were measured for transformants of Escherichia coli TOP10 and P. mirabilis ATCC 29906 Rif(R). RESULTS: qnrA6 was shown to be chromosomally encoded in P. mirabilis PS16 and its genetic environment was 81%-87% similar to that of qnrA2 in the Shewanella algae chromosome. The 5138 bp region up- and downstream of qnrA6 contained an IS10 sequence surrounded by two ISCR1. This resulted in qnrA6 being displaced 1.9 kb from its native promoter but supplied a promoter present in ISCR1. qnrA6 cloned into pTOPO and pBR322 conferred a 4-32-fold increase in fluoroquinolone MICs when expressed in E. coli but only 2-3-fold in P. mirabilis. When including the promoter region, a further increase in resistance was observed in both species, reaching MIC values above clinical breakpoints for only P. mirabilis. CONCLUSIONS: qnrA6 is the first chromosomally located qnrA gene described in Enterobacteriaceae. The quinolone resistance conferred by qnrA6 depends on the proximity of an efficient promoter and the host strain where it is expressed.

Emergence of quinolone resistance in the microbiota of hospitalized patients treated or not with a fluoroquinolone.

BACKGROUND: Quinolone resistance is a major global clinical problem. It primarily emerges in microbiota under selective pressure. Studies evaluating the incidence and risk factors for carrying quinolone-resistant bacteria in hospitalized patients treated with fluoroquinolones (FQs) are lacking. METHODS: We prospectively included hospitalized patients treated with FQs. Nasal, throat and rectal swabs were performed before FQ treatment, at the end of FQ treatment and 30 days later. A 'reference group' of patients not receiving FQs was also included to determine the rates of quinolone resistance acquisition not linked to FQ treatment. Prevalence and incidence of quinolone-resistant strains of nasal coagulase-negative staphylococci (CoNS) and Staphylococcus aureus, pharyngeal α-haemolytic streptococci and faecal Escherichia coli, and risk factors for emergence of quinolone resistance in FQ-treated patients were assessed. RESULTS: Four-hundred and fifty-one FQ-treated patients were included, as well as 119 subjects in the 'reference group'. Emergence of quinolone resistance occurred in 110/213 (51.6%), 50/336 (14.9%), 53/290 (18.3%) and 46/336 (13.7%) of FQ-treated patients for CoNS, S. aureus, α-haemolytic streptococci and E. coli, respectively, significantly more than for reference patients for CoNS (23/65; P < 0.05), S. aureus (5/91; P < 0.02) and E. coli (4/84; P < 0.05), but not for α-haemolytic streptococci (15/70; P = 0.55). Emergence of resistance was not associated with the type of FQ received, the duration of therapy or the duration of hospital stay, but was associated with host factors such as immunosuppression and altered performance status. CONCLUSIONS: FQs received during hospitalization account for high rates of emergence of resistance to FQs in clinically relevant bacteria from human microbiota, reflecting the important ecological impact of FQs. Host factors outweighed treatment or hospitalization characteristics as risk factors for carrying quinolone-resistant strains.

Variability in cell division among anatomical sites shapes Escherichia coli antibiotic survival in a urinary tract infection mouse model.

Urinary tract infection (UTI), mainly caused by Escherichia coli, are frequent and have a recurrent nature even after antibiotic treatment. Potential bacterial escape mechanisms include growth defects, but probing bacterial division in vivo and establishing its relation to the antibiotic response remain challenging. Using a synthetic reporter of cell division, we follow the temporal dynamics of cell division for different E. coli clinical strains in a UTI mouse model with and without antibiotics. We show that more bacteria are actively dividing in the kidneys and urine compared with the bladder. Bacteria that survive antibiotic treatment are consistently non-dividing in three sites of infection. Additionally, we demonstrate how both the strain in vitro persistence profile and the microenvironment impact infection and treatment dynamics. Understanding the relative contribution of the host environment, growth heterogeneity, non-dividing bacteria, and antibiotic persistence is crucial to improve therapies for recurrent infections.

Predominant role of host proteases in myocardial damage associated with infectious endocarditis induced by Enterococcus faecalis in a rat model.

Infective endocarditis (IE) remains a life-threatening infectious disease with high morbidity and mortality. The objectives of the present study are to assess the host proteolytic activities of the vegetations and their cytotoxic potential in a rat model of experimental IE. Rats were infected with a strain of Enterococcus faecalis of particularly low virulence and weak protease expression. We tested the presence of proteases released by infiltrated leukocytes (matrix metalloproteinases and elastase) or produced in situ within the septic vegetation, such as those linked to the fibrinolytic system (plasmin and plasminogen activators). We also assessed the tissue damage induced by the infective thrombus in vitro and ex vivo. The model of IE was characterized by larger and more extensive vegetations in infected than in nonseptic rats and by an intense neutrophil infiltrate interfacing with the injured underlying tissue. Neutrophil extracellular DNA was shown to trap bacteria and to produce increased levels of cell-free DNA in plasma. Matrix metalloproteinase-9, elastase, and plasminogen activators were increased in septic versus nonseptic vegetations (as shown by zymography and immunohistology). Finally, proteolysis of the extracellular matrix and apoptosis were shown to be associated with host proteases. Bacteria exhibited no detectable proteolytic activity or direct cytotoxic effects. Bacterial membranes/dead bacteria were sufficient to induce leukocyte recruitment and activation that could promote vegetation formation and growth. Our results suggest that, despite the lack of bacterial proteases, the continuous attractant signals coming from bacterial colonies may lead to a chronic and deleterious aggression toward myocardial/valvular tissues by host proteases.

Diversity of individual dynamic patterns of emergence of resistance to quinolones in Escherichia coli from the fecal flora of healthy volunteers exposed to ciprofloxacin.

BACKGROUND: Emergence of quinolone-resistant Escherichia coli (QREC) is an increasing clinical challenge mostly originating in fecal microbiota. The dynamics of the emergence of QREC in feces from individuals exposed to ciprofloxacin is unknown. METHODS: A total of 48 healthy volunteers received oral ciprofloxacin for 14 days. Fecal specimens were collected on days 0, 8, 14, and 42. Subpopulations of QREC were detected on selective agar, genetically characterized, and compared with quinolone-susceptible E. coli (QSEC) strains collected on different days. RESULTS: On day 42, 34 subjects carried QSEC, and 14 carried QREC. Of the 14 who carried QREC, 9 carried quinolone-susceptible E. coli on day 0, 1 carried E. coli with a lower level of quinolone resistance on day 0, and 4 carried E. coli with similar levels of resistance and RAPD-genotypes on days 0 and 42. No plasmid acquisition and no selection of resistant mutants from the initial microbiota was evidenced in any case. CONCLUSIONS: In QREC emerging under ciprofloxacin pressure in the fecal microbiota, no proof of selection of quinolone-resistant mutants from the initial microbiota was evidenced, suggesting that QREC strains on day 42 were either present at undetectable levels in the initial microbiota or that exogenous acquisition of QREC strains occurred. Clinical Trials Registration. NCT00190151.

Synergistic Activity of Pep16, a Promising New Antibacterial Pseudopeptide against Multidrug-Resistant Organisms, in Combination with Colistin against Multidrug-Resistant Escherichia coli, In Vitro and in a Murine Peritonitis Model.

Colistin is a drug of last resort to treat extreme drug-resistant Enterobacterales, but is limited by dose-dependent toxicity and the emergence of resistance. A recently developed antimicrobial pseudopeptide, Pep16, which acts on the cell membrane, may be synergistic with colistin and limit the emergence of resistance. We investigated Pep16 activity against Escherichia coli with varying susceptibility to colistin, in vitro and in a murine peritonitis model. Two isogenic derivatives of E. coli CFT073 (susceptible and resistant to colistin) and 2 clinical isolates (susceptible (B119) and resistant to colistin (Af31)) were used. Pep16 activity, alone and in combination with colistin, was determined in vitro (checkerboard experiments, time-kill curves, and flow cytometry to investigate membrane permeability). Toxicity and pharmacokinetic analyses of subcutaneous Pep16 were performed in mice, followed by the investigation of 10 mg/kg Pep16 + 10 mg/kg colistin (mimicking human concentrations) in a murine peritonitis model. Pep16 alone was inactive (MICs = 32-64 mg/L; no bactericidal effect). A concentration-dependent bactericidal synergy of Pep16 with colistin was evidenced on all strains, confirmed by flow cytometry. In vivo, Pep16 alone was ineffective. When Pep16 and colistin were combined, a significant decrease in bacterial counts in the spleen was evidenced, and the combination prevented the emergence of colistin-resistant mutants, compared to colistin alone. Pep16 synergizes with colistin in vitro, and the combination is more effective than colistin alone in a murine peritonitis by reducing bacterial counts and the emergence of resistance. Pep16 may optimize colistin use, by decreasing the doses needed, while limiting the emergence of colistin-resistant mutants.

Faecal carriage of extended-spectrum ß-lactamase-producing Escherichia coli in a remote region of Niger.

OBJECTIVE: Whole genome sequencing (WGS) of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-E. coli) in developing countries is lacking. Here we describe the population structure and molecular characteristics of ESBL-E. coli faecal isolates in rural Southern Niger. METHODS: Stools of 383 healthy participants were collected among which 92.4% were ESBL-Enterobacterales carriers. A subset of 90 ESBL-E. coli containing stools (109 ESBL-E. coli isolates) were further analysed by WGS, using short- and long-reads. RESULTS: Most isolates belonged to the commensalism-adapted phylogroup A (83.5%), with high clonal diversity. The blaCTX-M-15 gene was the major ESBL determinant (98.1%), chromosome-integrated in approximately 50% of cases, in multiple integration sites. When plasmid-borne, blaCTX-M-15 was found in IncF (57.4%) and IncY plasmids (26.2%). Closely related plasmids were found in different genetic backgrounds. Genomic environment analysis of blaCTX-M-15 in closely related strains argued for mobilisation between plasmids or from plasmid to chromosome. CONCLUSIONS: Massive prevalence of community faecal carriage of CTX-M-15-producing E. coli was observed in a rural region of Niger due to the spread of highly diverse A phylogroup commensalism-adapted clones, with frequent chromosomal integration of blaCTX-M-15. Plasmid spread was also observed. These data suggest a risk of sustainable implementation of ESBL in community faecal carriage.