Nathalie Tijet

Evaluation of the Carba NP Test for Rapid Detection of Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

ABSTRACT The Carba NP test was evaluated against a panel of 244 carbapenemase- and non-carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates. We confirmed the 100% specificity and positive predictive value of the test, but the sensitivity and negative predictive value were 72.5% and 69.2%, respectively, and increased to 80% and 77.3%, respectively, using a more concentrated bacterial extract. False-negative results were associated with mucoid strains or linked to enzymes with low carbapenemase activity, particularly OXA-48-like, which has emerged globally in enterobacteria.

Molecular Analysis of Antimicrobial Resistance Mechanisms in Neisseria gonorrhoeae Isolates from Ontario, Canada

ABSTRACT Surveillance of gonococcal antimicrobial resistance and the molecular characterization of the mechanisms underlying these resistance phenotypes are essential in order to establish correct empirical therapies, as well as to describe the emergence of new mechanisms in local bacterial populations. To address these goals, 149 isolates were collected over a 1-month period (October-November 2008) at the Ontario Public Health Laboratory, Toronto, Canada, and susceptibility profiles (8 antibiotics) were examined. Mutations in previously identified targets or the presence of some enzymes related to resistance (r), nonsusceptibility (ns) (resistant plus intermediate categories), or reduced susceptibility (rs) to the antibiotics tested were also studied. A significant proportion of nonsusceptibility to penicillin (PEN) (89.2%), tetracycline (TET) (72.3%), ciprofloxacin (CIP) (29%), and macrolides (erythromycin [ERY] and azithromycin; 22.3%) was found in these strains. Multidrug resistance was observed in 18.8% of the collection. Although all the strains were susceptible to spectinomycin and extended-spectrum cephalosporins (ESC) (ceftriaxone and cefixime), 9.4% of them displayed reduced susceptibility to extended-spectrum cephalosporins. PBP 2 mosaic structures were found in all of these ESCrs isolates. Alterations in the mtrR promoter, MtrR repressor (TETr, PENns, ESCrs, and ERYns), porin PIB (TETr and PENns), and ribosomal protein S10 (TETr) and double mutations in gyrA and parC quinolone resistance-determining regions (QRDRs) (CIPr) were associated with and presumably responsible for the resistance phenotypes observed. This is the first description of ESCrs in Canada. The detection of this phenotype indicates a change in the epidemiology of this resistance and highlights the importance of continued surveillance to preserve the last antimicrobial options available.

New Delhi Metallo-β-Lactamase, Ontario, Canada

To the Editor: The New Delhi metallo-β-lactamase (NDM-1) was first characterized in 2009 from Klebsiella pneumoniae and Escherichia coli isolated from a patient in Sweden who had received medical care in New Delhi, India (1). Further studies have shown broad dissemination of this β-lactamase gene (blaNDM-1) in India, Pakistan, Bangladesh, and the United Kingdom (2). Additional isolates have been detected in other countries, and many of the patients with NDM-1–producing Enterobacteriaceae reported receiving medical care in the Indian subcontinent (1–7). We describe detection and characterization of an NDM-1–producing K. pneumoniae isolated in Ontario, Canada. In August 2010, a urinary tract infection was diagnosed in a 36-year-old woman in a hospital in Brampton, Ontario. An E. coli strain sensitive to multiple antibacterial drugs (including carbapenems) was isolated from a midstream urine sample; the patient was successfully treated with ciprofloxacin. One week after treatment, when the patient did not have a fever or other clinical signs, a urine culture was repeated, and a carbapenem-resistant K. pneumoniae isolate (GN529) was recovered. Travel history indicated that the patient had recently returned from India, where in mid-July she had had a miscarriage and had been hospitalized in Mumbai for 2 days. At that time, no antimicrobial drug treatment was prescribed. Susceptibility profiles of K. pneumoniae GN529 and its E. coli transconjugant were obtained by using Etest (bioMerieux, Marcy l’Etoile, France) and the agar dilution method based on the Clinical and Laboratory Standards Institute guidelines (8). Multilocus sequence typing (MLST) of isolate GN529 was performed as described (9). The Pasteur Institute online database (www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) was used to assign the allelic numbers and sequence type (ST). To screen for the most commonly known β-lactamase genes in enterobacteria, we performed multiplex PCRs (10). Primers were designed (NDM-F, 5′-AATGGAATTGCCCAATATTATGC-3′; NDM-R, 5′-CGAAAGTCAGGCTGTGTTG C-3′) for the specific detection of blaNDM-1 and included in 1 of the multiplex PCRs (multiplex V). Primers NDM-F and NDM-R2 (5′-TCAGCGCAGCTTGTCGGC-3′) were used to amplify and sequence the entire blaNDM-1 gene. The samples were screened for the presence of six 16S methylase genes (armA, rmtA–D, and npmA) by PCR. E. coli J53 transconjugants were selected on Luria-Bertani plates containing sodium azide and meropenem (100 µg/mL and 1 µg/mL, respectively). The plasmid harboring blaNDM-1 was identified by Southern blot analysis by using a specific digoxigenin-labeled blaNDM-1 probe (Roche Diagnostics, Indianapolis, IN, USA). K. pneumoniae GN529 was highly resistant to all β-lactams, aminoglycosides, quinolones, tetracycline, nitrofurantoin, and co-trimoxazole. MICs of 0.5 µg/mL for colistin (European Committee on Antimicrobial Susceptibility Testing colistin breakpoint for Enterobacteriaceae: susceptibility <2 μg/mL) and 1 μg/mL for tigecycline (European Committee on Antimicrobial Susceptibility Testing and US Food and Drug Administration tigecycline breakpoint for Enterobacteriaceae: susceptibility <1 and <2 μg/mL, respectively) were also obtained (Table). Table Antibacterial drug susceptibility profiles and resistance genes of Klebsiella pneumoniae GN529 clinical isolate and its Escherichia coli transconjugant, Ontario, Canada, 2010* Considering the travel history of the patient and the high level resistance to all β-lactams, molecular screening of β-lactamases in strain GN529 was initiated to identify possible carbapenemases (e.g., blaNDM-1) in that isolate. Five β-lactamases genes (blaNDM, blaSHV, blaTEM, group 1 blaCTX-M , and blaOXA) and one 16S rRNA methylase (armA) were detected. By using primers for amplification of complete genes, we obtained sequences of blaNDM-1, 2 extended-spectrum β-lactamases (blaCTX-M-15 and blaSHV-12), 3 broad-spectrum β-lactamases (blaSHV-11, blaTEM-1 and blaOXA-1), and methyltransferase armA. No AmpC β-lactamases were linked to this isolate. Southern blotting identified a plasmid of ≈150 kb harboring blaNDM-1 (data not shown). A transconjugant E. coli positive for blaNDM-1 (E. coli J529, Table) was resistant to all β-lactams and aminoglycosides tested. In addition, blaSHV-12 and armA were detected in strain J529 (Table), indicating the potential for the horizontal spread of these resistance genes. K. pneumoniae GN529 was typed by MLST as ST147, the same type as a clinical NDM-1–producing strain isolated in Australia (6) but distinct from ST14 and ST16 strains described (1,7). There are insufficient MLST data to confirm polyclonal dissemination of NDM-1, but previous pulsed-field gel electrophoresis results support that hypothesis (2). K. pneumoniae GN529 was isolated from a patient who had recently received emergency medical care in India, suggesting importation of this clinical strain. In the United Kingdom, where Enterobacteriaceae containing blaNDM-1 are increasingly common, carriage of these organisms has been closely linked to receipt of medical care in the Indian subcontinent (2). Similar association as a risk factor was observed in other regions, including blaNDM-1-positive clinical strains isolated in North America, Australia, and Africa (3–6,10). The NDM-1–producing enterobacteria described in this study previously had low MICs only for colistin and tigecycline (1,2,5,6). However, an NDM-1 isolate resistant to these antimicrobial drugs has also been described (2). Early detection and implementation of infection control interventions is essential for preventing the spread of multidrug-resistant organisms such as these. It may be prudent to consider medical exposure in the Indian subcontinent as a risk factor for possible infection, colonization, or both with multidrug-resistant, NDM-1–producing Enterobacteriaceae.

New Endemic Legionella pneumophila Serogroup I Clones, Ontario, Canada

Identifying geographic distribution can improve surveillance and clinical testing procedures.

Detection of carbapenemase activity in Enterobacteriaceae: comparison of the carbapenem inactivation method versus the Carba NP test

Sir, Carbapenem resistance mediated by plasmid-encoded carbapenemases has emerged worldwide and become a major concern. The detection of the activity of carbapenemases has a strong impact on hospital infection control, because the detection of their presence can initiate measures to avoid potential outbreaks and lateral spread of the resistance. Although molecular detection by PCR is considered the gold standard for carbapenemase gene identification, some limitations are clearly recognized. Between them, falsenegative results (the presence of a carbapenemase gene not tested in the PCR reaction, or mutations affecting annealing of primers) or the detection of inactive genes (i.e. no carbapenemase expression) can delay infection-control measures or, oppositely, initiate them when they are not required. A fast and accurate phenotypic method, the Carba NP test (CNPt), was developed; it detects carbapenemase activity with very high sensitivity and specificity and lower costs compared with those of PCR. This method is now being recommended in the CLSI guidelines for carbapenemase activity detection. However, recent studies have shown that this test has lower sensitivity particularly against isolates expressing b-lactamases with low carbapenemase activity, such as OXA-48like, or expressing mucoid colonies. Another new test, the carbapenem inactivation method (CIM), has shown very promising results based on its sensitivity, specificity, low cost and easy interpretation. Briefly, the CIM consists of two steps: (i) incubation of a meropenem disc with the isolate to be tested; and (ii) incubation of this meropenem disc with the Escherichia coli ATCC strain. After this second incubation step, the presence of carbapenemase activity can be easily detected: the absence of an inhibition zone indicates enzymatic hydrolysis of meropenem during the first incubation step, whereas a ‘clear inhibition zone’ appears when the tested isolate does not express carbapenemase activity. The goal of this study was to compare the performance of the CIM and the CNPt against a panel of well-characterized enterobacteria. A total of 182 Enterobacteriaceae were tested (Table 1). Of these, 82 were negative controls with variable carbapenem susceptibilities, which were determined by Etest and their results interpreted using the CLSI guidelines. These 82 isolates had PCR results that were negative for carbapenemase genes, and none displayed carbapenemase activity by phenotypic tests (modified Hodge test, CNPt and KPC/MBL Confirm Kit, Rosco Diagnostica). We also included 100 carbapenemase-producing isolates (including KPC, NDM, VIM, IMP, OXA-48-like, NMC/IMI and SME producers), all confirmed by PCR results and sequence analysis (Table 1). Both the CNPt and the CIM were performed in triplicate for each isolate as described previously. All non-carbapenemase producers were negative by both phenotypic methods (Table 1). The fact that some of these negative controls had high carbapenem MICs (≥32 mg/L) suggests the presence of other mechanisms of resistance, such as AmpC and/or ESBL production plus impermeability. These results confirm the specificity and a positive predictive value of 100% for both methods. For carbapenemase producers, the CNPt had a few false-negative results, most of them related to OXA-48-like producers (two OXA-48, two OXA-181, two OXA-232 and one OXA-244), but also with two NDM-1 producers (one of which showed mucoid colonies) and one KPC producer (mucoid). Both methods failed to detect a swarmingproducing Proteus mirabilis positive for IMP-27. However, the carbapenemase activities of this enzyme as well as the NDM-1 from the mucoid Providencia rettgeri were identified by both methods in transformant or transconjugant E. coli strains, as described previously. These results gave a sensitivity (90.1%) and negative predictive value (88.2%) for the CNPt that were similar to those previously reported. However, considerably higher sensitivity (98.8%) and negative predictive value (99%) were obtained by the CIM. The CIM proves to be an accurate method for detection of carbapenemase activity. In our hands, its main disadvantage was that, in practice, it required an overnight incubation of the plates to obtain results (in contrast to the 8 h incubation period claimed in the original report) versus a maximum incubation of 2 h using the CNPt. On the other hand, the CIM had multiple advantages: (i) it is an easier-to-perform test; (ii) only water and a 10 mg meropenem susceptibility-testing disc per isolate were necessary to perform the test; (iii) four isolates plus positive and negative controls can be tested on the same Mueller–Hinton agar plate (standard size) inoculated with E. coli ATCC 25922 (these three first points highlight the low costs of the test); and (iv) the interpretation of results is easy (no inhibition zones versus ≥20 mm for negative carbapenemase activity; in our tests, with the exception of one false-negative result, we did not detect a carbapenemase producer exhibiting meropenem inhibition zones .6 mm). In summary, both methods proved to be very efficient in the detection of carbapenemase activity, with pros and cons for their implementation in microbiological laboratories. Although the CNPt showed an excellent specificity and a short turnaround time for carbapenemase detection (from 10 min to 2 h), it was unable to detect around 10% of the carbapenemase (mainly OXA-48-like) producers. The CIM, on the other hand, showed excellent specificity and sensitivity and low costs (no specialized reagents, equipment

Klebsiella pneumoniae Carbapenemase, Canada

To the Editor: Carbapenems are used to treat life-threatening infections caused by extremely drug-resistant gram-negative pathogens; these drugs represent the last line of defense in the antimicrobial drug armamentarium against serious or invasive infection (1). The rapid global spread of Klebsiella pneumoniae that produces K. pneumoniae carbapenemase (KPC), especially in the northeastern United States (e.g., New York state), is of major concern (2,3). KPC β-lactamases belong to the family of serine carbapenemases and are usually found in K. pneumoniae and Escherichia coli. KPC hydrolyzes β-lactam agents, thereby reducing their action. KPC activity has been reported, albeit less frequently, in other family Enterobacteriaceae (K. oxytoca, Enterobacter spp., Salmonella spp., Citrobacter freundii, and Serratia spp.) as well as in Pseudomonas aeruginosa (1). The blaKPC genes have been identified on conjugative plasmids and pose an infection control problem because plasmids could theoretically be transmitted from one species to another (4). The few therapeutic options for treating infections caused by organisms containing these β-lactamases are aminoglycosides, glycylcyclines, polymyxins, or combinations (1). A major concern is that routine susceptibility testing methods based on existing breakpoints can falsely identify KPC producers as susceptible to carbapenems. Such results pose the potential risk for increased illness and death, longer hospital stays, and nosocomial spread of infection. In 2008, the Public Health Laboratory in Toronto received clinical isolates of K. pneumoniae from urine and sputum of 1 patient. The hospital laboratory had forwarded the isolates to the Public Health Laboratory because they were possible KPC producers. The patient was a 73-year-old man with a history of emphysema and hypertension, seen at a tertiary care hospital in the Toronto area, 80 miles from the New York state border, for a laparoscopic right radical nephrectomy because of hypernephroma. He had no risk factors for acquisition of KPC producers, e.g., travel to the United States or prior carbapenem exposure. Susceptibility testing of K. pneumoniae was performed by the agar dilution method, using breakpoints set by the Clinical and Laboratory Standards Institute (5,6). The sputum isolate (7315) was susceptible to meropenem (MIC 4 μg/mL), and the urine isolate (7184) was intermediately susceptible (MIC 8 μg/mL). The K. pneumoniae isolates were screened for extended-spectrum β-lactamases (ESBLs) and AmpC production according to Ontario guidelines (7). Briefly, to screen for ESBL enzymatic activity, a double-disk diffusion method was used: a clavulanic acid–containing disk was placed adjacent to a disk containing one of several cephalosporins such as ceftazidime and cefotaxime. Enhanced killing of the organism in the area between the drug with and without clavulanate indicates ESBL. Cefoxitin resistance (zone <17 mm) indicates AmpC-like β-lactamase activity. In addition, testing for ESBL/AmpC was performed according to Clinical and Laboratory Standards Institute guidelines (6). When the screening result for ESBL or AmpC is positive, the clinical laboratory issues a warning that no β-lactam except carbapenems can effectively treat this infection. The Table summarizes results of initial susceptibility testing and supplementary laboratory testing for KPC. Table Results of initial susceptibility and supplementary testing for Klebsiella pneumoniae carbapenemase in urine and sputum samples from 73-year-old man, Canada* The initial result was consistent with a possible AmpC/ESBL producer for the sputum and urine isolates (6,7). However, because the patient responded poorly to empiric vancomycin and imipenem therapy and because of the elevated MIC to meropenem for isolate 7184, further laboratory testing was conducted to rule out the possibility of carbapenemase activity. The modified Hodge test is a phenotypic test proposed to confirm the presence of carbapenemase activity such as KPC in K. pneumoniae and E. coli (8). Universal primers for blaKPC family, Uni-KPC-F (5′-ATGTCACTGTATCGCCGTCT-3′) and -R (5′-TTACTGCCCGTTGACGCCC-3′), were used for the entire 882-bp coding sequence. Amplicons were bidirectionally sequenced by using the BigDye Terminators method and a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and primers Uni-KPC-F and -R. Multiple nucleotide and protein sequence alignments were performed with the ClustalW2 software (www.ebi.ac.uk/Tools/clustalw2/index.html). To aid the clinician, an Etest method was used to measure the MIC of this KPC-producing K. pneumoniae isolate to colistin (0.5 μg/mL) and tigecycline (2.0 μg/mL). However, before this information could be used, the patient had died of respiratory failure, presumably caused by K. pneumoniae. Infection control measures and laboratory screening were undertaken in the hospital to limit transmission to other patients. This report shows that KPC-producing organisms such as K. pneumoniae may pose a major risk for clinical disease and a challenge for infection control if they were to spread to other hospitals in Canada. Current testing algorithms focus on ESBL- and AmpC-producing gram-negative bacteria, which may not detect KPC-producer strains. We suggest that reference laboratories validate a screening method coupled with confirmatory phenotypic assay for carbapenemase activity for suspected organisms, especially K. pneumoniae and E. coli. Our in-house validation studies confirm that use of the ertapenem disk followed by the modified Hodge test to confirm carbapenemase activity may be effective (D.R. Pillai et al., unpub. data). Public health officials should be aware that this report further expands the international distribution of KPC-producing K. pneumoniae.

rmtD2, a New Allele of a 16S rRNA Methylase Gene, Has Been Present in Enterobacteriaceae Isolates from Argentina for More than a Decade

ABSTRACT The first allele of a 16S rRNA methyltransferase gene, rmtD2, conferring very high resistance to all clinically available aminoglycosides, was detected in 7/1,064 enterobacteria collected in 2007. rmtD2 was located on a conjugative plasmid in a Tn2670-like element inside a structure similar to that of rmtD1 but probably having an independent assembly. rmtD2 has been found since 1996 to 1998 mainly in Enterobacter and Citrobacter isolates, suggesting a possible reservoir in these genera. This presumption deserves monitoring by continuous surveillance.

Characterization of Multiple NDM-1-Producing Enterobacteriaceae Isolates from the Same Patient

ABSTRACT A male patient was admitted to a community hospital in Ontario, Canada, with an infected sacral ulcer after returning from India, where he was hospitalized. Carbapenem-resistant Escherichia coli (isolated from blood cultures), Enterobacter cloacae, and Providencia stuartii (from urine samples), all positive for blaNDM-1, were recovered. Comparative NDM-1 plasmid analysis suggests both lateral plasmid transfer and independent acquisition of the blaNDM-1 gene in these clinical isolates.

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.

Reply to “Further Proofs of Concept for the Carba NP Test”

We acknowledge the [letter by Dortet et al.][1] ([1][2]) in response to our evaluation of the Carba NP test ([2][3]), performed in two independent Canadian laboratories (the National Microbiology Laboratory, the national reference lab, and Public Health Ontario, a provincial reference lab). In