Evaluation of the Reveal® rapid AST system to assess the susceptibility of Pseudomonas aeruginosa from blood cultures.

This study was set up to assess the performance of the Reveal® rapid AST system to determine the drug susceptibility of Pseudomonas aeruginosa strains directly from blood cultures. Two hundred fully sequenced clinical P. aeruginosa strains were selected for the evaluation, of which 26.5% (n = 53) produced transferable ß-lactamases, and 2.0 to 33.0% had susceptibility levels close to the EUCAST 2021 breakpoints of 11 commonly used antipseudomonal antibiotics. The Reveal® AST system was run with a commercial MIC microplate designed for fast-growing Gram-negative bacilli (Microscan Neg MDR MIC 1), and was compared to the manually operated GN6F MIC microdilution panel from Thermo Fisher, as a comparator method. The Reveal® AST system provided MIC results for the 11 antipseudomonal antibiotics tested within a mean time to result of 6 h 22 min. By comparison with the GN6F panel, the overall rates of categorical agreement (CA), very major errors (VME), major errors (ME), and minor errors (mE for meropenem only) were 96.1%, 1.6%, 4.2%, and 0.6%, respectively. The Specific Reveal® AST system appears to be a reliable and fast technology to determine the susceptibility of P. aeruginosa to antibiotics, including those with resistance levels near categorical breakpoints, directly from blood cultures.

Elizabethkingia anophelis outbreak in France.

OBJECTIVE: We report an outbreak of Elizabethkingia anophelis infections in France. To the best of our knowledge, this is the first outbreak described in Europe. METHODS: Each E. anophelis-positive microbiological sample was considered a case. All patients were hospitalized in an infectious diseases unit. Clinical, environmental, and microbiological investigations (MALDI-TOF mass spectrometry, PCR, E-test) were performed for each case. RESULTS: Twenty cases were reported from September 2020 to September 2021, mainly community-acquired infections, responsible for nine deaths. The phylogenetic analysis showed a clonal origin and excluded nosocomial transmission. Despite the analysis of multiple environmental specimens, no source of contamination was identified. All strains were highly resistant to cefotaxime, ceftazidime, and imipenem. CONCLUSIONS: Clinicians and microbiologists should be aware of this multidrug-resistant bacterium, capable of causing severe infections. Most strains showed the lowest minimum inhibitory concentration values for cotrimoxazole and ciprofloxacin, making them the best choice for empirical antibiotic therapy.

Emergence of plasmid-mediated colistin resistance (mcr-1) among Enterobacteriaceae strains: Laboratory detection of resistance and measures to control its dissemination.

The increasing use of colistin has contributed to the emergence of resistant bacteria and to an increase in the frequency of infections caused by naturally resistant Enterobacteriaceae strains such as Proteus, Providencia, Morganella, and Serratia. In August 2016, the French High Council for Public Health (French acronym HCSP) received a request from the Ministry of Health on the advice of the French National Public Health agency (Santé publique France) with regard to measures that should be taken to tackle the emergence of plasmid-mediated colistin resistance among Enterobacteriaceae strains. French healthcare facilities were asked to take the necessary measures as soon as possible, such as updating the definition of emerging highly resistant bacteria and defining the identification methods so as to take account of the evolving epidemiology of this type of resistance. This article describes the epidemiological context of the discovery of this emergence in France and worldwide, the resistance mechanisms, the microbiological methods of routine laboratory detection and the level of hygiene measures to implement in French facilities.

Detection of carbapenemase activity in Pseudomonas aeruginosa by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

A new MALDI-TOF MS imipenem hydrolysis assay was developed to optimize detection of carbapenemase-producing strains of Pseudomonas aeruginosa. The method correctly discriminated carbapenemase producers (n=74) from non-producers (n=95) in 99.4% of cases, and successfully detected 100% GES-5 strains (n=5).

Unexpected persistence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in the faecal microbiota of hospitalised patients treated with imipenem.

Imipenem is active against extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) but favours the intestinal emergence of resistance. The effects of imipenem on intestinal microbiota have been studied using culture-based techniques. In this study, the effects were investigated in patients using culture and metagenomic techniques. Seventeen hospitalised adults receiving imipenem were included in a multicentre study (NCT01703299, http://www.clinicaltrials.gov). Most patients had a history of antibiotic use and/or hospitalisation. Stools were collected before, during and after imipenem treatment. Bacterial and fungal colonisation was assessed by culture, and microbiota changes were assessed using metagenomics. Unexpectedly, high colonisation rates by imipenem-susceptible ESBL-E before treatment (70.6%) remained stable over time, suggesting that imipenem intestinal concentrations were very low. Carriage rates of carbapenem-resistant Gram-negative bacilli (0-25.0%) were also stable over time, whereas those of yeasts (64.7% before treatment) peaked at 76.5% during treatment and decreased thereafter. However, these trends were not statistically significant. Yeasts included highly diverse colonising Candida spp. Metagenomics showed no global effect of imipenem on the bacterial taxonomic profiles at the sequencing depth used but demonstrated specific changes in the microbiota not detected with culture, attributed to factors other than imipenem, including sampling site or treatment with other antibiotics. In conclusion, culture and metagenomics were highly complementary in characterising the faecal microbiota of patients. The changes observed during imipenem treatment were unexpectedly limited, possibly because the microbiota was already disturbed by previous antibiotic exposure or hospitalisation.

Mutations in ß-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins.

Mutation-dependent overproduction of intrinsic ß-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the ß-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on ß-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious ß-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic ß-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.

Alternatives to carbapenems in ESBL-producing Escherichia coli infections.

OBJECTIVES: The authors had for objective to assess the activity of a wide panel of antibiotics on extended-spectrum-ß-lactamase producing Escherichia coli isolates (ESBL-Ec), because of the sharp increase of their frequency, leading to an increased use of carbapenems. MATERIAL AND METHODS: We selected 100 ESBL-Ec in which ESBLs were identified by PCR and sequencing, between 2009 and 2010. We determined the MICs of amoxicillin-clavulanate, piperacillin-tazobactam, temocillin, mecillinam, cefoxitin, cefotaxime, ceftazidime, aztreonam, tigecycline, nitrofurantoin, and fosfomycin using reference methods. The susceptibility profiles were defined according to EUCAST 2012 recommendations. RESULTS: Fosfomycin, nitrofurantoin, and pivmecillinam were active against more than 90% of isolates and remain excellent choices for the oral treatment of urinary tract infections (UTIs). Temocillin and piperacillin-tazobactam are also good candidates for the treatment of pyelonephritis or bloodstream infections. Only 27, 23, and 8% of isolates were susceptible to ceftazidime, cefepime, and cefotaxime, respectively. CONCLUSION: Our study results prove that in many cases, there are non-carbapenem alternatives for the treatment of ESBL-Ec infections.

Molecular epidemiology of multidrug-resistant Pseudomonas aeruginosa in a French university hospital.

The aim of this study was to assess the incidence and molecular epidemiology of multidrug-resistant (MDR) Pseudomonas aeruginosa in our university hospital. Analysis included antimicrobial susceptibility profiling, bla gene identification and pulsed-field gel electrophoresis (PFGE). During the one-year study, 654 patients had at least one sample that tested positive for P. aeruginosa, of whom 38 (5.8%) were colonised or infected with an MDR isolate, giving an incidence of 0.1 patient per 1000 patient-days. The 38 non-duplicate isolates yielded 12 different PFGE patterns, three of which included isolates from four patients and one of which included isolates from 15 patients. Two isolates produced acquired extended-spectrum ß-lactamase (one OXA-14 and one OXA-28). Genotyping showed that cross-transmission was responsible for about 70% of MDR P. aeruginosa cases although spatio-temporal analysis failed to demonstrate when this might have occurred for most cases. The major epidemic and the three main micro-epidemic clones were already present in our hospital with a more susceptible phenotype. It is likely that some P. aeruginosa clones are endemic in our hospital and that, within these clones, MDR isolates emerge under antibiotic pressure. Our results indicate that cross-transmission plays a major role in the spread of MDR P. aeruginosa and suggest that priority should be given to the improvement of standard hygienic precautions.

Emergence of extensive-drug-resistant Pseudomonas aeruginosa in a French university hospital.

The aim of this study was to describe the molecular epidemiology and the mechanisms of resistance to beta-lactams of emerging extensive-drug-resistant Pseudomonas aeruginosa (XDRPA) in a tertiary-care university hospital over a three-year period. Analysis included antimicrobial susceptibility profiling and pulsed-field gel electrophoresis (PFGE). Resistance mechanisms to beta-lactams were identified: production of naturally occurring and acquired beta-lactamases, overproduction of MexAB-OprM and MexXY efflux systems and loss of porin OprD were assessed. Eighteen patients were colonised or infected with XDRPA which remained susceptible to colistin and, to a lesser extent, to rifampicin. beta-lactam resistance was, in most cases, due to the overproduction of AmpC, overproduction of the MexXY efflux system and loss of porin OprD. One isolate produced the class D extended-spectrum oxacillinase (OXA-ESBL) Oxa-28, but none produced metallo-beta-lactamase (MBL) or class A extended-spectrum beta-lactamase (ESBL). The XDRPA clustered in eight PFGE patterns and both the acquisition and loss of resistance determinants was observed within a single clone during its spread. The emergence of XDRPA isolates in our university hospital has been characterised by genotypic heterogeneity, variation of mechanisms of resistance to beta-lactams in a single clone and the predominance of chromosomally encoded resistance mechanisms.

Antibiotic susceptibility and mechanisms of beta-lactam resistance among clinical strains of Pseudomonas aeruginosa: first report in Algeria.

OBJECTIVE: Pseudomonas aeruginosa is a major causative agent of hospital infections. Studies on this subject being rare in Algeria, we determined the antibiotic susceptibility of P. aeruginosa and investigated the mechanisms of beta-lactam resistance and the spread of multidrug resistant strains in the university affiliated Hospital of Tlemcen (Algeria). DESIGN: One hundred and ninety-nine consecutive strains of P. aeruginosa were collected between November 2005 and February 2007. MICs of antibiotics were measured by the agar dilution method. The resistance mechanisms to beta-lactams were identified phenotypically or by molecular methods (isoelectrofocusing, PCR and sequencing). Strains expressing a secondary beta-lactamase were serotyped and genotyped (Random Amplified Polymorphic DNA). RESULTS: The proportion of susceptible isolates were: ticarcillin (56%), piperacillin-tazobactam (81%), ceftazidime (88%), cefepime (80%), aztreonam (64%), imipenem (65%), amikacin (83%), tobramycin (81%) and ciprofloxacin (97%) according to the French CASFM breakpoints. Resistance to beta-lactams was linked to the production of transferable beta-lactamases (16%), overproduction of cephalosporinase AmpC (12%) and/or non-enzymatic mechanisms such as the loss of porin OprD (35%) and overproduction of the active efflux system MexAB-OprM (24%). High level resistance to ticarcillin was due to the expression of beta- lactamase OXA-10 alone or associated with TEM-110. A genotypic analysis revealed the spread of a multidrug resistant epidemic clone expressing these two acquired beta-lactamases in the surgical ICU. CONCLUSIONS: This study shows that resistance to antibiotics, in particular to imipenem of P. aeruginosa, is becoming a cause of concern in the Hospital of Tlemcen.

Susceptibility of Pseudomonas aeruginosa to antimicrobials: a 2004 French multicentre hospital study.

OBJECTIVES: Pseudomonas aeruginosa is a major causative agent of hospital infections. The purpose of this study was to determine the antibiotic susceptibility of P. aeruginosa in a French multicentre study and to investigate the mechanisms of beta-lactam resistance. METHODS: Four hundred and fifty non-repetitive strains of P. aeruginosa were collected in 15 French university hospitals in 2004. MICs of antibiotics were measured by agar dilution methods. For all the strains with MICs of ticarcillin >16 mg/L, detection and identification of the beta-lactamases, quantitative determination of cephalosporinase and overproduction of the MexAB-OprM efflux pump were evaluated. RESULTS: The percentages of susceptible isolates were as follows: ticarcillin, 62%; ticarcillin + clavulanic acid, 61%; piperacillin, 78%; piperacillin + tazobactam, 80% (MICs

Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium.

Pseudomonas aeruginosa is a major cause of nosocomial infections. This organism shows a remarkable capacity to resist antibiotics, either intrinsically (because of constitutive expression of beta-lactamases and efflux pumps, combined with low permeability of the outer-membrane) or following acquisition of resistance genes (e.g., genes for beta-lactamases, or enzymes inactivating aminoglycosides or modifying their target), over-expression of efflux pumps, decreased expression of porins, or mutations in quinolone targets. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. Susceptibility testing is therefore crucial in clinical practice. Empirical treatment usually involves combination therapy, selected on the basis of known local epidemiology (usually a beta-lactam plus an aminoglycoside or a fluoroquinolone). However, therapy should be simplified as soon as possible, based on susceptibility data and the patient's clinical evolution. Alternative drugs (e.g., colistin) have proven useful against multiresistant strains, but innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been unsuccessful to date. Among broad-spectrum antibiotics in development, ceftobiprole, sitafloxacin and doripenem show interesting in-vitro activity, although the first two molecules have been evaluated in clinics only against Gram-positive organisms. Doripenem has received a fast track designation from the US Food and Drug Administration for the treatment of nosocomial pneumonia. Pump inhibitors are undergoing phase I trials in cystic fibrosis patients. Therefore, selecting appropriate antibiotics and optimising their use on the basis of pharmacodynamic concepts currently remains the best way of coping with pseudomonal infections.

In-vivo impact of the MexXY efflux system on aminoglycoside efficacy in an experimental model of Pseudomonas aeruginosa pneumonia treated with tobramycin.

Aminoglycosides are of major importance in treating Pseudomonas aeruginosa pneumonia (PAP). However, their efficacy may be compromised by low-level resistance caused by the inducible MexXY multidrug efflux pump. In the present study, the impact of the MexXY efflux pump was investigated in vivo in an experimental model of PAP in rabbits treated with intravenous tobramycin. Three strains were used to induce PAP in rabbits: PAO1 (wild-type strain; MIC 1 mg/L), mutant 11B (mexX::Tn501; no expression of MexXY; MIC 0.5 mg/L) and mutant MutGR1 (MexZ null; constitutive expression of MexXY; MIC 2 mg/L). Five hours after inoculation, treatment with tobramycin (10 mg/kg) was implemented (peak serum concentration 30 mg/L). The animals were killed humanely 48 h after inoculation, and the residual pulmonary bacterial concentration was determined. Selection of bacteria expressing MexXY was determined by plating lung homogenates on agar plates containing antibiotic. Mean bacterial counts (log(10) CFU/g) for treated vs. untreated rabbits were 6.26 and 8.13 (p < 0.0001), 6.00 and 8.38 (p < 0.001), and 7.25 and 8.79 (p 0.04) for PAO1, 11B and MutGR1, respectively, with an overall mortality rate of 0% vs. 8.9% (p < 0.01). MexXY-overexpressing bacteria were recovered from three (21%) treated rabbits. The C(max)/MIC ratio was the parameter that was best associated with tobramycin efficacy. The bacteria overexpressing MexXY, recovered from lung, occurred with a C(max)/MIC window of 19-26. It was concluded that the experimental PAP model highlights poor tobramycin bacteriological efficacy in vivo, contrasting with survival gain, and that the contribution of the MexXY system to this low level of tobramycin efficacy is modest. Finally, this model appears to be suitable for the investigation of new anti-pseudomonal therapeutic strategies.

[Surveillance of antimicrobial resistance and antimicrobial use in a university-affiliated hospital: implementation of a computerized system]. / Mise en place d'un outil informatisé de surveillance de la résistance bactérienne et de la consommation antibiotique dans un centre hospitalier universitaire.

UNLABELLED: After half a century of antibiotic use, the increasing problem of the emergence and spread of antimicrobial-resistant pathogens has created a problem of public health. The causes of this problem are multifactorial, but the excessive and inappropriate use of antimicrobials is the principal cause. The current guidelines for the control of antimicrobial resistance in hospitals recommend the implementation of a surveillance system of antimicrobial use and antimicrobial resistance data. AIM OF THE STUDY: The objective of our project was to develop a computerised tool to survey the antibiotic consumption data and the antimicrobial resistance. MATERIALS AND METHODS: We have collected antimicrobial resistance data from the software of the bacteriology laboratory, antibiotic use data from the pharmacy and demographical data from the hospital's admission department. These data were integrated in a database server and available with a web application. Antimicrobial resistance data of 15 major microorganisms were extracted and expressed as a frequency with elimination of repeats by using time criteria (7, 14 or 28 days). Antibiotic use data were converted into defined daily doses (DDD) and expressed per 1000 patient-days. RESULTS: Data are available for consultation in the form of tables or graphs per unit, type of units (medicine, surgery, pediatrics, intensive care units) or in the whole hospital. The system allows the confrontation on the same graph of antimicrobial resistance and antibiotic use data. CONCLUSION: Our surveillance system constitutes a needed prerequisite to the implementation of a global strategy of antibiotic use improvement in our hospital.

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.

[Low-level resistance to fluoroquinolones conferred by efflux overproduction in Pseudomonas aeruginosa: clinical significance and routine detection]. / Résistance de bas niveau aux fluoroquinolones par surexpression de l'efflux chez Pseudomonas aeruginosa: conséquences thérapeutiques et détection au laboratoire.

The aim of this study was (i) to assess the impact of stable overproduction of efflux systems MexAB-OprM and MexXY-OprM on the bacteriostatic activities of fluoroquinolones in clinical Pseudomonas aeruginosa strains and (ii) to find a convenient test for screening isolates with a low level resistance to fluoroquinolones. The minimal inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were determined for clinical isolates of P. aeruginosa overexpressing MexAB-OprM or MexXY-OprM. Efflux pumps derepression was associated with a modest two- to fourfold increase in resistance to the tested fluoroquinolones. Clinical significance of low level resistance conferred by the efflux mechanism was evaluated with a Monte Carlo simulation with various fluoroquinolone regimens. With this model, low levels of resistance to ciprofloxacin (MIC > or =0.25 mg/L) or levofloxacin (MIC > or =1 mg/L) such as those due to overproduced MexAB-OprM or MexXY-OprM were predicted to result in poor clinical outcomes. Altogether these data strongly suggest that when derepressed MexAB-OprM or MexXY-OprM provides P. aeruginosa with a resistance that may be sufficient to impair the efficacy of single therapy with highly potent fluoroquinolones such as ciprofloxacin and levofloxacin. Routine detection of clinical strains that displayed low-level resistance to fluoroquinolones with a Mueller Hinton agar containing 0.20 mg/L of ciprofloxacin will help clinician in his therapeutical choice.

Susceptibility of Escherichia coli to the amoxycillin-clavulanate combination: which recommendations should be used to provide relevant information to clinicians?

This study compared MIC distributions of amoxycillin-clavulanate obtained with NCCLS and French (Comite de l'Antibiogramme de la Societe Francaise de Microbiologie; CA-SFM) methodologies for Escherichia coli isolates from urine that were non-susceptible to amoxycillin-clavulanate by the disk diffusion method. With the NCCLS and CA-SFM methods, 74% and 13%, respectively, of these isolates were susceptible to amoxycillin-clavulanate. Therefore, the apparent relatively poor efficacy of amoxycillin-clavulanate against E. coli in French hospitals probably reflects a methodological difference rather than a localised resistance problem. This implies that amoxycillin-clavulanate could be used as an alternative to fluoroquinolones for treatment of E. coli urinary tract infections. Susceptibility tests for amoxycillin-clavulanate should be standardised worldwide.