By Roberto G. Melano, David J Farrell, Donald E Low, and Samir N Patel
Public Health Ontario, Public Health Laboratory – Toronto, Toronto, ON Canada

Carbapenems, such as ertapenem, imipenem and meropenem, are the most potent class among ß-lactam antibiotics. They are highly active against most Gram-negative bacteria, including those that produce extended-spectrum ß-lactamases (ESBLs) or AmpC enzymes. Carbapenems are used as ‘agents of last resort’ for the treatment of serious infections caused by multidrug resistant Gram-negative bacteria. Consequently, carbapenem-resistant organisms represent a global public health concern.⁴

Carbapenem resistance in enterobacteria can be mediated via multiple different mechanisms, including (1) presence of ESBL plus impermeability^3,6,8,15, (2) hyperproduction of chromosomal/plasmid-mediated AmpC plus impermeability^6,11,15,16, or (3) production of carbapenem-hydrolyzing ß-lactamases, commonly called carbapenemases.¹⁰ With the emergence and subsequently spread of carbapenemase-producing Enterobacteriaceae (CPE) throughout the world, it has presented a great deal of concern among clinicians, infection control teams, laboratorians, and public health officials.

Carbapenemases can be grouped into different classes based on molecular group defined by Ambler class¹. Class A carbapenemases are serine ß-lactamases actively inhibited by clavulanic acid (e.g. Klebsiella pneumoniae carbapenemase [KPC]). Class B carbapenemases are metallo-enzymes not inhibited by any therapeutically useful inhibitor (e.g. clavulanic acid), but inhibited in vitro by EDTA or dipicolinic acid (e.g. New Delhi metallo-ß-lactamase [NDM]).^2,7  Finally, class D carbapenemases are also serine-ß-lactamases poorly inhibited by clavulanic acid (e.g. OXA-48). Most CPE developed and became endemic in specific geographic regions. For example, KPC-containing enterobacteria were first reported in North Carolina and subsequently spread throughout the eastern seaboard of the United States. These organisms are also found in Israel, Greece and China. Similarly, OXA-48-producing organisms are commonly found in Middle Eastern and Mediterranean countries. NDM is the most recently identified carbapenemase. NDM-producing organisms emerged in Indian subcontinent and subsequently spread throughout the world including Europe, Australia, and Americas. Increased global travel as well as medical tourism contributed to the introduction of these resistant organisms in areas where they were not commonly found. CPE organisms have the potential to spread rapidly in hospital settings. This situation highlights the need for surveillance and detection of CPE in Ontario in order to control spread in hospital settings. In December 2011, QMP–LS published their consensus recommendations for testing and reporting CPE. At the same time, Public Health Ontario (PHO) issued recommendations for screening methods for CPE. In addition, the Chief Medical Officer of Health requested that all confirmed CPE isolates be sent to the Public Health Ontario Laboratory (PHOL) for surveillance purposes. Here we report number and type of CPE confirmed by PHO from 2008 to 2011. This will serve as a baseline and will allow us to prospectively track incidence of CPE in Ontario.

Since the characterization of the first CPE in Ontario in 2008, the PHOL—the Reference laboratory for bacterial susceptibility testing in the province, has been improving phenotypic and molecular methods for the detection of CPE. Initially isolates that were non-susceptible to carbapenems were screened using the Modified Hodge Test (MHT). Organisms positive by the MHT method were subsequently tested for the presence of common carbapenemase genes using a multiplex PCR assay. However, it was noted that the MHT method was not reliable since it lacks specificity, particularly in detection of NDM-producing Enterobacteriaceae. In 2011, the MHT was replaced by the most reliable inhibitor screening test.¹² Also, a multiplex PCR assay specific for detection of the most common carbapenemase genes was implemented into routine clinical testing.¹³

Isolates received at PHOL for confirmation of carbapenemase resistance testing were subjected to the following algorithm: isolates with confirmed MICs >1 mg/L for imipenem or ertapenem, and/or >2 mg/L for meropenem were tested by phenotypic methods for carbapenemase activity detection (initially the MHT and, most recently, the inhibitor test). Subsequently, molecular characterization of the carbapenemase genes was performed in the positive isolates. Finally, molecular typing of E. coli and Klebsiella pneumoniae containing carbapenemase genes were performed by multilocus sequence typing (MLST) to determine whether emergence of these isolates are a result of clonal dissemination.

From January 2008 to December 2011, 73 Enterobacteriaceae isolates were confirmed by PHOL for the presence of carbapenemase genes. The carbapenemases found included KPC,⁹ NDM,¹³ the Verona integron-encoded metallo-ß-lactamase (VIM)¹⁴ and OXA-48. These isolates were referred from 18 laboratories across the province. Our data show that incidence of carbapenemase-producing strains has increased, particularly in the last 2 years (Figure 1). NDM-1-producing isolates became the most predominant CPE in Ontario comprising 49% of the cases in 2011, followed by KPC-2/3 (31%), OXA-48 (15.6%) and VIM-1 (4.4%).

Figure 1. Number of carbapenemase-producing Enterobacteriaceae referred to the Public Health Ontario Laboratories from 2008 to 2011 (N=73)

The 73 CPE were from 64 patients (38 male and 26 female), and were isolated from urine (31 patients), urine catheters (3 patients), intraperitoneal fluid (2 patients), skin or wound swab (5 patients), sputum (2 patients), blood (2 patients), bone (1 patient), or unknown specimens (3 patients). Twenty-four patients were positive for CPE isolated from rectal swabs (Figure 2). Most of these isolates were identified from hospitalized patients.

Figure 2. Carbapenemase-producing Enterobacteriaceae classified by site of isolation

K. pneumoniae was the predominant species (n=48) primarily as a result of clonal dissemination of bla_KPC and polyclonal spread of bla_NDM (Figure 3). The MLST data show that all KPC-producing K. pneumoniae (n=27) belonged to ST258 or closely related. Diversity of MLST-types was observed in NDM-producing K. pneumoniae (n=16) and E. coli (n=7) strains. Most of the CPE were isolated in the city of Toronto (TO, n=33) and the Greater Toronto area (GTA, n=30) (Figure 4).

Figure 3. Carbapenemase-producing Enterobacteriaceae classified by species

Figure 4. Carbapenemase-producing Enterobacteriaceae classified by geographic place of isolation. GTA: Greater Toronto Area, GHS: Greater Horseshoe valley, SW: Southwestern Ontario, TO: Toronto, Ott: Ottawa area

In conclusion, the incidence of CPE has increased in Ontario since 2008. The majority of these isolates were NDM- and KPC-producers. The total number of CPE is believed to be higher than what is reported here as submission of these isolates to PHOL is not mandatory and some laboratories have implemented molecular testing for confirmation of carbapenemase genes. All such laboratories are urged to submit confirmed isolates so that true incidence of CPE can be in tracked in Ontario.

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