We read with interest the article published by Muntean et al.1 about the comparison of two different techniques, i.e. the so-called rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) and the Carba NP test, for the identification of carbapenemase production among Enterobacterales hyperproducing an AmpC-type β-lactamase. Unfortunately, the data presented on the sensitivity and specificity of those techniques are biased by the selection of isolates that was made, being far from the epidemiologically representative picture. Eleven out of the 48 carbapenemase-producing Enterobacterales (CPE) strains (23%) included in the study were Proteus mirabilis producing either OXA-23 or OXA-58. As published by the authors themselves,2 those carbapenem-hydrolysing class D β-lactamases are identified mainly in Acinetobacter baumannii and very rarely in Enterobacterales. To provide a valid and useful comparison of the sensitivity and specificity of a technique, the nature of the CPE strains to be tested should mirror the...

The whole-genome sequencing analysis revealed a polyclonal dissemination of NDM-1 and NDM-9 variants in Escherichia coli (n=20) and Klebsiella pneumoniae (n = 2) in Tahiti since 2015 via interspecies transfer of three different bla(NDM)-carrying plasmids (IncR, IncHI2, and IncF) and patient-to-patient cross-transmission. It highlights the potential risk of importation of NDM producers in France, where French Polynesia is not considered stricto sensu a foreign country from which repatriated patients have to be screened.

The MAST® Carba PAcE test is a colorimetric test used to detect carbapenemase-producing Gram-negative bacilli from cultured colonies. The performances of this test were compared to β-CARBA™, Carba NP test and RAPIDEC® CARBA NP tests using a collection of 280 characterized isolates. Sensitivity and specificity of the MAST® Carba PAcE test were 79.8% (95%IC: 73.3%-85.1%) and 98.9% (95%IC: 92.9%-99.9%). The MAST® Carba PAcE sensitivity was the lowest mainly due to interpretation difficulties (particularly OXA-48-like).

OBJECTIVES:Detection of carbapenemase-producing Enterobacterales (CPEs) is sometimes difficult with AmpC-hyperproducing Enterobacterales (AHEs), as they may falsely be classified as CPEs. Here, we present a rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) that allows rapid and easy discrimination between AHEs and CPEs.METHODS:Enterobacterales (n = 249), including natural AmpC producers, AHEs, CPEs and non-carbapenemase-producing carbapenem-resistant control strains were evaluated, using Carba NP, rCIM and rCIM-A. The rCIM-A differs from the rCIM by the addition of cloxacillin (400 μg/mL) to the initial antibiotic incubation step.RESULTS:The rCIM-A yielded a sensitivity and specificity of 84.26% (95% CI: 76.00%-90.55%) and 99.29% (95% CI: 96.11%-99.98%), respectively, while those of the rCIM were 86.11% (95% CI: 78.13%-92.01%) and 80.85% (95% CI: 73.38%-86.99%), respectively; those of Carba NP were lower at 84.04% (95% CI: 75.05%-90.78%) and 91.37% (95% CI: 85.41%-95.46%), respectively, due to indeterminate results. The rCIM-A was capable of discriminating between AHEs and true CPEs, but still failed to identify OXA-23-producing Proteus mirabilis isolates and remained only partially reliable for identifying IMI-like producers and a few MBL (2 NDM-1, 1 LMB-1, 1 TMB-1 and 1 IMP-13) producers. One chromosomally encoded AmpC variant, MIR-10, gave repeatedly positive results using all three tests and was thus considered a false positive.CONCLUSIONS:Specificity for AHEs greatly improved with the rCIM-A without altering the test performance for the other resistance mechanisms. It may replace the rCIM as a cheap, easy, rapid and accurate CPE detection test.

A carbapenem-resistant Citrobacter sp. was recovered from routine screening of multidrug-resistant bacteria. This isolate coproduced OXA-48 and OXA-198. OXA-48 was carried by the prototypical IncL plasmid, whereas OXA-198 was carried by a peculiar IncHI-type plasmid. This carbapenemase gene was inserted within a class 1 integron located on a conjugative plasmid. This report describes the first occurrence of OXA-198 in Enterobacterales.

A novel real-time multiplex PCR assay, BD MAX Check-Points CPO, was evaluated to detect carbapenemase-producing organisms in clinical settings on the BD MAX system. A total of 175 well-characterized isolates (including 123 carbapenemase producers) and 128 rectal swab specimens (including 83 positives) of patients considered at high risk for carriage of carbapenemase producers were included. Bacterial suspensions were used to spike true-negative rectal swabs to mimic a clinical sample. Sample (50 mu L), containing either the spiked or the patient's sample, was processed. The BD MAX Check-Points CPO assay detected carbapenemases KPC, VIM/IMP, NDM, and OXA-48 like producers with a high sensitivity and specificity of 97.1% and 98.8%, respectively. Rare variants of the IMP type (IMP-11, IMP-13, and IMP-14) and one rare and distantly related OXA-48 variant (OXA-535) remained undetected. With patients' rectal swabs, sensitivity and specificity were 92.8% and 97.8%, respectively. Failure of detection was due to weak inoculum. The time to result was short: approximately. 2.5 hours for 12 samples (including extraction and PCR). The automated sample-in results-out platform is an efficient, quick, and easy-to-use tool for the detection of the main five carbapenemases. The lack of distinction between producers of VIM and IMP may be limiting in countries where these enzymes are widespread, as in Asia, but not in France, where IMP producers are extremely rare.

In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The bla(ACT-28) gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the bla(ACT-1) gene. Detection of carbapenemase activity was positive only for E. coli expressing the bla(ACT-28) gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests.

Objectives Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection. Methods The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories.

With the dissemination of carbapenemase-producing Enterobacteriaceae (CPE) strains worldwide, carbapenem-hydrolyzing enzymes are increasingly reported among isolates of Escherichia coli , the first hospital and community-acquired opportunistic pathogen. Here, we have performed an epidemiological survey of carbapenemase-producing E. coli (CP- Ec ) isolates received at the French National Reference Centre (F-NRC) in 2012 and 2013.

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.

Carbapenemase-producing Klebsiella pneumoniae (CPKp) are increasingly reported worldwide and represent a major health threat ([1][1]). Carbapenemases frequently encountered in CPKp belong to Ambler's class A (KPC enzymes), class B (NDM, VIM, and IMP enzymes), and class D (OXA-48-like enzymes) ([1][1

Carbapenemase-producing enterobacteriaceae (CPE) and non fermenters (CPNF; pseudomonadaceae and Acinetobacter sp.) have been increasingly reported worldwide. The most clinically relevant carbapenemases belong either to Ambler class A (KPC-type), Ambler class B, i-e metallo-b-lactamases (MBLs such as IMP-, VIM- and NDM-types) or Ambler Class D (OXA-48-like in enterobacteriaceae, Acinetobacter OXA group: OXA-23, OXA-40, OXA-58, OXA-143 and the intrinsic OXA-51-like enzymes, OXA-198 in P. aeruginosa). The Amplidiag® Carba-R + VRE, a qualitative multiplex nucleic acid-based in-vitro diagnostic test intended for the detection of carbapenemase-producing bacteria and vancomycin resistant enterococci, has been tested on a collection of 100 characterized Gram-negative isolates with reduced susceptibility to carbapenems, and on 200 isolates collected at the National Reference Center for Antibiotic Resistance from 20st January to 10th February 2016. The markers detected by this assay are blaKPC-like, blaNDM-like, blaVIM-like, blaIMP-like, blaOXA-48-like, Acinetobacter OXA genes including blaOXA-23, blaOXA-24/-40, blaOXA-58, and blaOXA-51 including upstream promoter ISAba1, and vanA and vanB. DNA was extracted using QiaAmp DNA extraction kits or by boiling extract. The Amplidiag® CarbaR + VRE was able to detect all KPC, NDM, VIM, IMP, OXA-48 and variants including OXA−162, −181, −204, −232, −244. Similarly, all A. baumannii producing OXA−23, OXA−24/−40, OXA−58 as well as over expression of the chromosomally-encoded OXA−51-like b-lactamase, due to the presence of ISAba1 upstream of the blaOXA-51 gene were detected. The most prevalent carbapenemases encountered in P. aeruginosa have also been detected. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES−5 in P. aeruginosa, GES−14 in A. baumannii), GIM−1, AIM−1, SPM−1, DIM−1 or OXA−198 in P. aeruginosa, or OXA−143-like in A. baumannii were not detected by the Amplidiag® CarbaR + VRE assay. The PCR worked equally well on purified DNA and on boiling extracted DNA from a colony. The biological performances of the Amplidiag® CarbaR + VRE on the prospective study of 200 CREs isolates collected at the NRC were of 100% and 96% sensitivity and specificity, respectively. Amplidiag® Carba-R's performances were high as it was able to detect the five major carbapenemases: NDM, VIM, IMP, KPC, and OXA−48, as well as OXA-type carbapenemases from Acinetobacter sp. It can provide a result directly on colonies growing on selective screening media within two hours. This test needs now to be evaluated on rectal swabs, since it is able to detect the most worrisome resistant traits in Gram negative bacteria.

ABSTRACT The emergence of colistin-resistant Klebsiella pneumoniae (CoR Kp ) is a public health concern, since this antibiotic has become the last line of treatment for infections caused by multidrug-resistant (MDR) Gram negatives. In this study, we have investigated the molecular basis of colistin resistance in 13 MDR K. pneumoniae strains isolated from 12 patients in a teaching hospital in Sousse, Tunisia. Whole-genome sequencing (WGS) was used to decipher the molecular mechanism of colistin resistance and to identify the resistome of these CoR Kp isolates. It revealed a genome of ca. 5.5 Mbp in size with a G+C content of 57%, corresponding to that commonly observed for K. pneumoniae . These isolates belonged to the 5 different sequence types (ST11, ST15, ST101, ST147, and ST392), and their resistome was composed of acquired β-lactamases, including extended-spectrum beta-lactamase and carbapenemase genes ( bla CTX-M-15 , bla OXA-204 , bla OXA-48 , and bla NDM-1 genes), aminoglycoside resistance genes [ aac ( 6 ′) Ib-cr , aph ( 3 ″)- Ib , aph ( 6 )- Id , and aac ( 3 )- IIa ], and fosfomycin ( fosA ), fluoroquinolone ( qnr -like), chloramphenicol, trimethoprim, and tetracycline resistance genes. All of the isolates were identified as having a mutated mgrB gene. Mapping reads with reference sequences of the most common genes involved in colistin resistance revealed several modifications in mgrB , pmr , and pho operons (deletions, insertions, and substitutions) likely affecting the function of these proteins. It is worth noting that among the 12 patients, 10 were treated with colistin before the isolation of CoR Kp . No plasmid encoding mcr-1 to mcr-5 genes was found in these isolates. This study corresponds to the first molecular characterization of a collection of CoR Kp strains in Tunisia and highlights that the small-transmembrane protein MgrB is a main mechanism for colistin resistance in K. pneumoniae .

OBJECTIVES Outbreaks of OXA-48-like carbapenemase producers are increasingly reported in many European countries and are often the result of difficulties in detection, especially for isolates with MICs of carbapenems that remain in the susceptibility range. METHODS An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect bla(OXA-48-like) genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants. RESULTS qPCR amplification using plasmid DNA was linear over 10 log dilutions (r(2) = 0.998 and slope = -3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other β-lactam resistance genes. The bla(OXA-48) qPCR assay was capable of detecting 10-50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 10(1) to 3 × 10(2) cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum β-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1-3 × 10(1) cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity. CONCLUSIONS The qPCR is likely to shorten the time for bla(OXA-48) detection from 48 to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.