Damien Fournier

Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa

BACKGROUND Pseudomonas aeruginosa can become resistant to carbapenems by both intrinsic (mutation-driven) and transferable (β-lactamase-based) mechanisms. Knowledge of the prevalence of these various mechanisms is important in intensive care units (ICUs) in order to define optimal prevention and therapeutic strategies. METHODS A total of 109 imipenem-non-susceptible (MIC >4 mg/L) strains of P. aeruginosa were collected in June 2010 from the ICUs of 26 French public hospitals. Their resistance mechanisms were characterized by phenotypic, enzymatic, western blotting and molecular methods. RESULTS Single or associated imipenem resistance mechanisms were identified among the 109 strains. Seven isolates (6.4%) were found to produce a metallo-β-lactamase (one VIM-1, four VIM-2, one VIM-4 and one IMP-29). Porin OprD was lost in 94 (86.2%) strains as a result of mutations or gene disruption by various insertion sequences (ISPa1635, ISPa1328, IS911, ISPs1, IS51, IS222 and ISPa41). Thirteen other strains were shown to be regulatory mutants in which down-regulation of oprD was coupled with overexpressed efflux pumps CzcCBA (n = 1), MexXY (n = 9) and MexEF-OprN (n = 3). The lack of OprD was due to disruption of the oprD promoter by ISPsy2 in one strain and alteration of the porin signal sequence in another. CONCLUSIONS Imipenem resistance in ICU P. aeruginosa strains may result from multiple mechanisms involving metallo-β-lactamase gene acquisition and genetic events (mutations and ISs) inactivating oprD, turning down its expression while increasing efflux activities or preventing insertion of porin OprD in the outer membrane. This diversity of mechanisms allows P. aeruginosa, more than any other nosocomial pathogen, to rapidly adapt to carbapenems in ICUs.

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-β-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.

Carbapenem resistance in cystic fibrosis strains of Pseudomonas aeruginosa as a result of amino acid substitutions in porin OprD

The aim of this work was to investigate the impact of single amino acid substitutions occurring in specific porin OprD on carbapenem resistance of cystic fibrosis (CF) strains of Pseudomonas aeruginosa. A PAO1ΔoprD mutant was complemented with the oprD genes from five carbapenem-resistant CF strains exhibiting very low amounts of mutated OprD porins in their outer membrane despite wild-type levels of oprD transcripts. Compared with wild-type porin from strain PAO1, single amino acid substitutions S403P (in periplasmic loop 8), Y242H, S278P and L345P (in β-sheets 10, 12 and 14, respectively) were found to result in reduced amounts of OprD in the outer membrane, increased carbapenem resistance, and slower growth in minimal medium containing gluconate, an OprD substrate, as the sole source of carbon and energy. This indicates that in CF strains of P. aeruginosa, loss of porin OprD may not only result from mutations downregulating the expression of or disrupting the oprD gene, but also from mutations generating deleterious amino acid substitutions in the porin structure.

A convenient method to screen for carbapenemase-producing Pseudomonas aeruginosa

ABSTRACT A combined-disk method using imipenem and cloxacillin was evaluated to discriminate between carbapenemase-producing (n = 56) and nonproducing (n = 62) strains of Pseudomonas aeruginosa. With a cloxacillin load of 4,000 μg/disk, this very simple method showed a sensitivity and a specificity of 100%, irrespective of the type of carbapenemase produced.

Outbreak of metallo-β-lactamase VIM-2-positive strains of Pseudomonas aeruginosa in the Ivory Coast

Sir, Acquired metallo-b-lactamases (MBLs) are increasingly recognized as important determinants ofb-lactam resistance in Pseudomonas aeruginosa. To date, seven types of MBL enzyme (IMP, VIM, NDM, SPM, GIM, AIM and FIM) have been identified in this opportunistic pathogen. Since its first description in France in 1996, the epidemiologically successful enzyme VIM-2 has been traced in resistant P. aeruginosa strains from various European countries (e.g. Spain, Italy, Croatia, Greece, the Netherlands and Turkey) and more recently in strains from northern (Tunisia and Algeria) and southern (South Africa) Africa. – 6 One study also mentioned its occurrence in Kenya. Illustrating the spread of the VIM-2-encoding gene, blaVIM-2, in sub-Saharan Africa countries, we found here that P. aeruginosa strains from the Ivory Coast harboured an atypical class I integron previously described in sporadic isolates from other continents. Twelve P. aeruginosa isolates that were non-susceptible to imipenem according to the EUCAST interpretation rules (imipenem MIC .4 mg/L) were recovered from separate patients hospitalized in the intensive care unit of the teaching hospital in Abidjan, Ivory Coast, between February 2009 and November 2011. A double-disc (10 mg of imipenem, 2.5 mmol of EDTA) synergy test and an MBL Etest (imipenem+EDTA; bioMerieux, Craponne, France) were performed and found to be positive with seven of these isolates, suggesting the production of an Ambler class B b-lactamase (MIC of imipenem ≥256 mg/L; MIC of imipenem+EDTA 32 mg/L). PCR and DNA sequencing experiments revealed the presence of the gene blaVIM-2 in these isolates. All were highly resistant to carbapenems (imipenem MIC ≥128 mg/L, meropenem MIC ≥32 mg/L and doripenem MIC ≥32 mg/L), ceftazidime (MIC ≥64 mg/L), aminoglycosides (tobramycin MIC ≥128 mg/L) and ciprofloxacin (MIC ≥16 mg/L), but remained susceptible to aztreonam (MIC ≤1 mg/L) and colistin (MIC ≤4 mg/L). Six isolates were responsible for bloodstream (n1⁄43), urinary tract (n1⁄42) and pulmonary (n1⁄41) infections; the last one was considered to be a cathetercontaminant. Theirgenetic relatednesswas investigatedbymultiple-locusvariablenumber tandem-repeat analysis using eight variable-number tandem repeats, as described previously. All the VIM-2-positive isolates turned out to belong to the same epidemic clone (serotype O6), which was assigned to a new sequence type (ST), ST1488, after multilocus sequence typing (MLST) analysis. Interestingly, the closest ST found in the PubMLST database (http://pubmlst.org/paeruginosa), ST233, relates to a P. aeruginosa strain (serotype O6) identified in Sweden in 2006 from a patient repatriated from Ghana. Nucleotide sequence alignments demonstrated that this strain isolated from Ghana carried the same blaVIM-2-containing integron, In559, as ST1488 (Figure 1). Since Ghana and the Ivory Coast are neighbouring countries, this suggests that closely related multidrug-resistant genotypes are present in this region of the African continent. Screening for additional b-lactamase genes by PCR (blaTEM, blaSHV, blaPER, blaGES, blaVEB, blaIMP, blaNDM, blaSPM, blaPSE and blaOXA including blaOXA-198) revealed that the seven isolates except one (12.871) harboured blaOXA-4, which codes for the narrow-spectrum oxacillinase OXA-4. The gene was located on a class 1 integron upstream of the aadA2 and cmlA1 cassettes, which code for the aminoglycoside-modifying enzyme ANT(3′′) (streptomycin and spectinomycin resistance) and for an active efflux pump accommodating chloramphenicol, respectively. PCR experiments using primers specific to 5′ and 3′ conserved sequences (5′CS and 3′CS) failed to detect a class 1 integron associated with blaVIM-2 in the strains. To characterize the genetic environment of blaVIM-2, a BamHI library of strain 12.871 DNA was cloned into the BamHIrestricted plasmid pK18 (Km). Transformants of E. coli DH5a were selected on Mueller–Hinton agar plates containing 100 mg/L ampicillin and 30 mg/L kanamycin. Compared with strain DH5a, carrying the empty pK18 vector, all the recombinant clones tested displayed high levels of resistance to b-lactams except aztreonam. Sequence analysis of the 6224 kb insert from one of these clones led to the identification of a class 1 integron with a 5′CS sequence and gene tniCat its 3′ end. This genetic structure wasfound to carry the following gene cassettes downstream of the integrase gene intI1: aacA7 (encoding acetyltransferase AAC6′-Il, which confers amikacin resistance), blaVIM-2, dhfrB5 (encoding a dihydrofolate reductase, providing trimethoprim resistance) and aacC5b (encoding an uncharacterized aminoglycoside acetyltransferase). Interestingly, this genetic structure was identical to that of blaVIM-2-containing integrons characterized in P. aeruginosa from the USA (AY943084), Taiwan, Russia (AM749810), India (HQ005291) and the Indian Ocean (AM296017), but was different from integrons reported so far in African countries other than Ghana (Figure 1). Interestingly, upstream of the intI1 gene, we could identify the transposase gene tnpA from ISPa58 (ISfinder database, http://www-is.biotoul.fr). The sequence was 100% identical to that of tnpA belonging to the Tn3/ Tn21 family (JX194160). Repeated assays to visualize a plasmid by

Clonal Dissemination of Pseudomonas aeruginosa Isolates Producing Extended-Spectrum β-Lactamase SHV-2a

ABSTRACT From January to December 2011, 24 Pseudomonas aeruginosa strains producing the extended-spectrum β-lactamase SHV-2a were identified in 13 hospitals in France. With one exception, all the strains belonged to the same clone. Double-disk synergy tests with cefepime and clavulanate were able to detect all the SHV-2a-positive isolates.

Which non-carbapenem antibiotics are active against extended-spectrum β-lactamase-producing Enterobacteriaceae?

In this study, the activity of 18 non-carbapenem antibiotics was evaluated against 100 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-Ec) and 50 ESBL-producing Klebsiella pneumoniae (ESBL-Kp) isolated from urinary tract infections and bacteraemia in 2016. Minimum inhibitory concentrations (MICs) were determined using reference methods and the susceptibility profiles were defined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2017 recommendations. All of the ESBL-Ec isolates were susceptible to ceftazidime/avibactam and a great majority of them were susceptible to fosfomycin (98%), piperacillin/tazobactam (97%), amikacin (97%) and nitrofurantoin (96%). Mecillinam, cefoxitin and ceftolozane/tazobactam remained active against 92%, 83% and 78% of the ESBL-Ec isolates, respectively. Moreover, 100%, 94% and 90% of the ESBL-Kp tested were susceptible to ceftazidime/avibactam, amikacin and mecillinam, respectively. This study showed that there are non-carbapenem options (including orally administrable drugs) for the treatment of all of the situations of ESBL-Ec or ESBL-Kp infections, with ceftazidime/avibactam being the most efficient alternative.