2013 iMedPub Journals THE INTERNATIONAL ARABIC JOURNAL Vol. 3 No. 3:5 Our Site: http://www.imedpub.com/ OF ANTIMICROBIAL AGENTS doi: 10.3823/737 Evaluation of Rula Al-Dawodi1,3, Rawan Liddawi1,3, Raed Ghneim1,3, Randa Kattan1,3, Issa Siryani1,3, Afaf Abu-Diab1, Riyad Meropenem, Ghneim1, Madeleine Zoughbi1,3, Abed-El-Razeq Issa1,3, Randa Al Qass1,3, Sultan Turkuman1, Hiyam Marzouqa1, and Imipenem and Musa Hindiyeh1,2,3* Ertapenem 1 Caritas Baby Hospital, 3 Palestinian Forum for Medical * Correspondence: Bethlehem, Palestine; Research (PFMR), Ramallah, Impregnated 2 Bethlehem University, Palestine Bethlehem, Palestine; [email protected] MacConkey * Musa Y Hindiyeh, Caritas Baby Hospital Bethlehem Agar Plates for Palestine. the Detection of Carbapenem Abstract Resistant Background: Rapid detection of carbapenem resistant bacteria, in particular, members of the Enterobacteriaceae family (CRE), is of utmost importance for the Enterobacteriaceae management of infected or colonized patients. Methods: Three carbapenems; meropenem, imipenem and ertapenem, with two different concentrations (0.5 mg/ml and 1.0 mg/ml), were impregnated in Mac- Conkey agar. The carbapenem impregnated MacConkey agar plates; ([Mac-Mem], [Mac-Imp] and [Mac-Ert]), were then evaluated for the detection of carbapenem resistant Gram-negative bacteria in particular the blaKPC producing Enterobacteria- ceae. The Limit of Detection (LOD) of the plates was determined in triplicate after serial logarithmic dilution of the bacterial strains in saline. This was followed by inoculating the plates and counting the colonies that grew after 24 hours of incu- bation. The specificity and the shelf-life of the plates were determined by testing the plates with six Extended Spectrum β-lactamases (ESBL) producing members of the Enterobacteriaceae family and one genus with the blaAmpC phenotype. Finally, the 0.5 mg/ml Mac-Mem plates were further challenged by incorporating them in the routine active surveillance program for the detection of carbapenem resistant bacteria. Results: Of the three carbapenems impregnated plates, Mac-Ert plates gave the lowest LOD; however, its specificity was poor as it failed to inhibit the growth of some of the ESBL producing strains. The Mac-Imp plate LOD and specificity were both unsatisfactory for several strains of the bacterial isolates tested. On the other hand the 0.5 mg/ml Mac-Mem plates showed low LOD for the blaKPC producing bacteria and had excellent specificity as it prevented the growth of all the ESBL and blaAmpC positive isolates tested. Not only that, but the shelf-life of the 0.5mg/ ml Mac-Mem plate was long, up to one month at 4oC. Conclusions: The high specificity and the good selectivity, in addition to the long shelf-life allowed the 0.5µg/ml Mac-Mem agar to be used as a cost effective selec- tive medium for the isolation of carbapenem resistant Gram-negative bacteria, in particular the blaKPC producing members of the Enterobacteriaceae family. This article is available from: Key words: blaKPC, blaOXA-48, Meropenem Agar (Mac-Mem), Carbapenemase, www.iajaa.org Palestine © Under License of Creative Commons Attribution 3.0 License 1 2013 iMedPub Journals THE INTERNATIONAL ARABIC JOURNAL Vol. 3 No. 3:5 Our Site: http://www.imedpub.com/ OF ANTIMICROBIAL AGENTS doi: 10.3823/737 Introduction and utilized for the rapid detection of blaKPC producing bac- teria. Chromogenic agar, CHROMagarTM KPC (CHROMagar, Rapid detection of carbapenem resistant bacteria, in par- France), has been evaluated for the detection of blaKPC posi- ticular, members of the Enterobacteriaceae family (CRE) is of tive bacteria. However, this agar base medium still missed utmost importance for the management of infected or colo- certain strains of blaKPC positive Enterobacteriaceae (1, 13, TM nized patients (1). Carbapenems are widely used to treat se- 14). Similar performance between the CHROMagar KPC vere bacterial infections that are often hard to manage. Sever- and MacConkey Agar impregnated with 2 µg/ml imipenem al carbapenem resistance mechanisms have been implicated was noted by Adler et al. (15). More recently, the Drigalski in how bacterial strains become resistant to carbapenems. agar-based culture medium was reported to have excellent Based on the molecular properties of the β–lactamases, four sensitivity and specificity for the detection of bacteria carry- classes of carbapenemases have been identified (2). These ing the blaKPC resistance mechanism (14). include the molecular classes (A, C, and D) β-lactamases with the amino acid serine at their active site, and the molecular In this study, we report the evaluation of cost effective MacCo- class B where the β-lactamases are all metalloenzymes with nkey Agar plates impregnated with one of the carbapenems an active-site zinc (2). Class A enzymes, an Ambler molecu- (meropenem, imipenem and ertapenem) for the detection of CRE’s. In addition, the specificity, analytical sensitivity and lar class enzymes (ex: blaKPC), have been the most common detected carbapenemases with worldwide distribution (1). the plates’ shelf-life were also evaluated. The concentrations of the antibiotics evaluated were 1.0 µg/mL and 0.5 µg/mL. For any health care facility, it is important to understand the These concentrations were chosen after careful evaluation level of CRE spreading in its different medical departments. It of the carbapenems lowest minimal inhibitory concentration has been recommended by the American Centers of Disease (MIC) interpretation guidelines published by the Clinical and Control and Prevention (CDC), that in areas which are not Laboratory Standards Institute (CLSI) (17). endemic for CRE, the acute care facilities should review all the microbiology records for the preceding 6-12 months to determine if CRE’s have been isolated at the institution (3). Materials and Methods Upon the identification of CRE cases, surveillance cultures from high risk areas should be performed to identify any Reagents unrecognized cases. Meropenem trihydrate (Catalogue # M2574) and imipenem CRE’s active surveillance cultures (perianal/rectal swabs), tak- monohydrate (Catalogue # I0160) were supplied by Sig- en from patients upon admission are of great importance for ma-Aldrich, USA. Ertapenem sodium, Invanz (Catalogue # the identification of unrecognized CRE-colonized patients, in 7290004997960) was supplied by Merck Sharp and Dohme order to take immediate and appropriate isolation precau- Corp, USA. All reagents were stored and diluted according tions (4, 5). Studies have shown that in areas where CRE’s to the manufacturer’s guidelines. are endemic, up to 5.4% of hospitalized patients can be car- riers of CRE’s, of which only one-third had a positive clinical Bacterial Strains culture (4). Moreover, surveillance swabs have been reported to considerably lower the cost of patient hospitalization (6). Carbapenem resistant Enterobacteriaceae members (N=9), Active surveillance cultures taken from patients coming from Extended Spectrum β–Lactamases (ESBL) producing Entero- highly CRE endemic medical settings upon admission have bacteriaceae strains (N= 6), blaAmpC β-lactamase (N=1) and been used as part of a comprehensive strategy to combat the multi-drug resistant Acinetobacter baumannii (N=1), all iso- spread of CRE’s. Studies have shown a 4.7-fold reduction in lated from patients’ samples at Caritas Baby Hospital (CBH) the incidence of CRE infection following the activation of the laboratory, were used to evaluate the performance of the active CRE surveillance program (7, 8). carbapenems impregnated MacConkey Agar. The carbap- enem resistant isolates included: 3 Klebsiella pneumoniae Several highly sensitive Polymerase Chain Reaction (PCR) as- blaKPC positive; 2 Enterobacter species blaKPC positive; 2 E. says and Real-Time PCR assays have been developed and coli blaKPC positive; 1 Citrobacter species blaKPC positive and 1 Klebsiella pneumoniae bla positive. The ESBL positive validated for the rapid detection and identification of blaKPC OXA-48 positive bacteria (9-12). However, one of the disadvantages of isolates were (K. pneumoniae, Salmonella species, Shigella these assays was the need for specialized equipment and well sonnei, E. coli , Proteus species and Citrobacter species); while trained personnel. Consequently, screening agar plates, that the blaAmpC positive bacteria was an Enterobacter species. have incorporated one of the commonly used carbapenems Moreover, 14 Klebsiella pneumoniae blaOXA-48 positive and (ertapenem, meropenem or imipenem), have been developed 2 E. coli blaOXA-48 positive were used to evaluate the analyti- 2 © Under License of Creative Commons Attribution 3.0 License 2013 iMedPub Journals THE INTERNATIONAL ARABIC JOURNAL Vol. 3 No. 3:5 Our Site: http://www.imedpub.com/ OF ANTIMICROBIAL AGENTS doi: 10.3823/737 cal sensitivity of the 0.5 µg/ml Mac-Mem agar plates for the Determination of the specificity and Shelf-Life of detection of low level carbapenemase producing blaOXA-48 the different Carbapenems Impregnated Plates Enterobacteriaceae. MacConkey agar plates (Mac-Mem, Mac-Imp, and Mac-Ert) All bacterial strains were identified according to the Clinical impregnated with the two different concentrations (0.5 µg/ Microbiology Procedures Handbook, and their antimicrobial ml and 1.0 µg/ml) of the three carbapenems, were prepared susceptibility testing was performed in duplicate according and stored at 4-8oC. The plates were inoculated with colonies to the Clinical and Laboratory Standard Institute