Comparison of Three Phenotypic Methods and PCR for Rapid Detection of Carbapenemase ‎Production in Klebsiella spp. and Acinetobacter spp.

INTRODUCTION

The most common bacterial isolates identified from clinical specimens are Gram-negative bacilli belonging to the Enterobacteriaceae family. Entero-bacteriaceae are responsible for roughly 70% of urinary tract infections, 50% of septicemia cases, and a large percentage of intestinal infections [1]. Antibiotic resistance genes, such as extended-spectrum Beta-lactamases (ESBLs), AmpCs, and carbapenemases, can be acquired by these bacteria [2].

Multidrug resistance is spreading rapidly among a wide range of bacterial species, resulting in community-acquired and nosocomial illnesses [3]. Carbapenems, the last line of treatment, are routinely used to treat nosocomial infections, and rising resistance to this family of ß-lactams leaves us with fewer options.

Carbapenem resistance in Gram-negative bacteria has become a major public health issue. Production of Beta-lactamases, porin loss, Penicillin-binding protein change, and/or overexpression of efflux systems have all been proposed as mechanisms of carbapenem resistance [4]. In Gram-negative bacteria, carbapenemase synthesis is the most significant mechanism of carbapenem resistance. The appearance and dissemination of carbapenemases like K. pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), and Oxacillinase-48 (OXA-48) among Enterobacteriaceae isolated globally highlighted the recent decade [5].

For a variety of reasons, accurate detection of carbapenemase synthesis is critical. The most important goal is to direct infection control resources and treatments [6]. Colorimetric techniques for detecting carbapenemase production directly in bacterial isolates recovered from microbiological cultures (e.g. Carba NP and Blue Carba tests) are simple to use and reliable enough to be employed in normal laboratory work. They are based on the enzymatic hydrolysis of a carbapenem's Beta-lactam ring (usually imipenem), which results in the acidification of an indicator solution, which changes colour owing to pH change. Using colorimetric techniques for detection of carbapenemase production in Enterobacteriaceae (CPE) takes up to 2 hours [7].

The modified Hodge test (MHT) is a phenotypic test used to screen for carbapenemase producers microorganisms. This test is based on carbapenemase-producing microorganisms inactivating a carbapenem, allowing a sensitive indicator strain to grow toward a disc containing this antibiotic, along the streak of inoculum of the tested strain. The MHT has showed high sensitivity in detecting carbapenemase producers from classes A and D. Unfortunately, the MHT has a limited sensitivity for detecting NDM-producing isolates, with a sensitivity of less than 50% [8].The study aims to compare three phenotypic methods with PCR for rapid detection of carbapenemase in carbapenem-resistant gram negative bacteria (Klebsiella, and Acinetobacter).

MATERIALS‎ AND METHODS

This study was conducted in Central Microbiology Laboratory, Ain Shams University Hospitals. Various clinical samples were cultured on blood agar plate (Himedia, India) and MacConkey agar plate (Himedia, India).Isolates were identified using Vitek 2 compact system (BioMérieux, Marcy l’Etoile, France). Antimicrobial susceptibility testing was done for gram negative isolates using disk diffusion method to detect resistance to Imipenem or Meropenem [9]. Sixty gram negative Imipenem or Meropenem resistant isolates were collected. These isolates were screened phenotypically for carbapenemases production by chromID® CARBA SMART Agar (CARB/OXA) (BIOMERIEUX, France) [10], Modified Hodge test (MHT) [11] and Blue-Carba test [12]. These 50 isolates were preserved in glycerol tryptic soya broth in -80 C⁰ for testing by PCR for carbapenemases genes (bla_KPC, bla_IMP, bla_OXA-48, bla_VIM and bla_GES ) [13].

Phenotypic screening of carbapenemases

ChromID® CARBA SMART Agar (BIOMERIEUX, France) is made up of two chromogenic culture media dispensed onto a single Petri dish with two compartments (CARB/OXA). ChromID CARBA SMART Agar (CARB/OXA) is made out of a nutrient-dense foundation made up of several peptones. It comprises a cocktail of antibiotics that allows KPC and metallo-carbapenemase-type CPE to develop selectively in the CARB medium and OXA-48-type CPE in the OXA medium. It also includes three chromogenic substrates that may be used to identify the most often isolated CPEs. Escherichia coli produces spontaneous coloration (pink to burgundy) in strains producing ß-glucuronidase (ßGUR) and/or ß-galactosidase (ß-GAL), while Klebsiella, Enterobacter, Serratia, Citrobacter (KESC) produces spontaneous coloration (bluish-green to bluish-grey or purple) in strains producing ß-glucosidase (ß ) and Acinetobacter spp produces no  color [10]. Isolates were streaked on chromID® CARBA SMART Agar (CARB/OXA) (BIOMERIEUX, France) once on each side of the plate. The bacterial growth and color change of colonies were observed.

In brief, a lawn of 0.5 McFarland suspension of carbapenem susceptible strain E. coli ATCC 25922 was streaked over Mueller Hinton agar (MHA) plates (Himedia, India), and a meropenem antibiotic disc (10 ug) (Oxoid, UK) was inserted in the center of the MHA agar plate. The isolates were streaked in straight lines from the edge of the meropenem disc to the edge of the plate, together with a positive control (K. pneumoniae ATCC1705) and a negative control (K. pneumoniae ATCC1706). Plates were inoculated and allowed to dry at room temperature for 15 minutes before being incubated for 16–24 hours at 35 ± 2 ^C. When a clover leaf-like form was observed, the test was considered positive [11]. 

The Blue-Carba test (BCT) is a biochemical test for detecting carbapenemase synthesis in Gram-negative bacteria directly from bacterial culture in as short as two hours. It is based on bacterial colonies hydrolyzing imipenem in vitro (direct inoculation without previous lysis), which is identified by changes in pH values given by the indicator bromothymol blue [7].

The test solution consisted of an aqueous solution of bromothymol blue at 0.04% (El-Nasr pharmaceutical chemicals company, Egypt) adjusted to pH 6.0, 0.1 mmol/liter ZnSO4 (El-Nasr pharmaceutical chemicals company, Egypt), and 3 mg/ml of imipenem powder (Merck & Co, USA), with a final pH of 7.0. A negative-control solution (0.04% bromothymol blue solution, pH 7.0) was prepared to control the influence of bacterial components or products on the pH of the solution. In 96- well round bottom microtiter plate (CITOTEST, Haimen, China) a 100µL of imipenem solution were added in each well of the plate in addition to 50µL of Zn sulphate solution and 50µL of bromothymol blue solution. A loop (approximately5 µl) of a pure bacterial culture recovered from Mueller-Hinton agar (Himedia, India) was directly suspended in 200 µl of both test and negative-control solutions in the 96-well microtiter plate and incubated at 37°C. Carbapenemase activity was revealed when the test and negative-control solutions, respectively, were (i) yellow versus blue, (ii) yellow versus green, or (iii) green versus blue. Non-carbapenemase producers remained blue or green on both solutions. The test was performed in triplicate for all isolates, yielding reproducible results [12].

Genotypic detection of carbapenemases genes by real time PCR

Total DNA was extracted using Thermo Scientific Gene JET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific, Dreieich, Germany) according to manufacturer’s instructions. Amplification was performed using Maxima SYBER Green qPCR Master Mix (2x) (Thermo Scientific, Germany) and primers for amplification of (bla_KPC, bla_GES, bla_OXA-48, bla_IMP and bla_VIM) (table 1) [13] genes were used in a total volume of 25 µl. Each gene was run separately at a time. The amplification was carried out on Rotor-Gene Q (Qiagen, Germantown, USA) thermal cycler [14].

Statistical methods

The collected data were computerized and statistically analyzed using the Statistical Package for Social Sciences (SPSS 24 Inc. Chicago, IL, USA).  Chi-Square test was utilized to inspect the association between two qualitative variables (count >5).Fisher’s exact testwas implemented to evaluate the association between two qualitativevariables (count < 5). A p-value < 0.05 was considered statistically significant.

Supplementary table 1

RESULTS:

In the current  study, out of 60 gram negative Imipenem or Meropenem resistant isolates, there were 50 isolates carbapenemase producers (50/60, 83.3%). They included Klebsiella  spp. (28/50, 56%) and Acinetobacter spp. (22/50, 44%). The 50 isolates were genotypic tested by real time PCR for (bla_KPC, bla_IMP, bla_OXA-48, bla_VIM and bla_GES) genes with 48/50 (96%) isolates were positive to one or more of the tested genes coding for carbapenemase production, and 2/50 (4%) isolates were negative for these genes. All Klebsiella isolates 28/28 (100%) were positive for genes coding for carbapenemase by PCR, with predominance of bla_OXA-48 gene (table 2) while Acinetobacter isolates 20/22 (90.9%) were positive for genes coding for carbapenemase by PCR with predominance of bla_OXA-48 gene (table 2).

As regard ChromID® CARBA SMART Agar (CARB media), in the current study, all Klebsiella isolates grew on CARB media 28/28 (100%) and showed bluish green colonies. While 21/22 (95.5%) of Acinetobacter isolates grew on CARB media with no color and 1 isolate (2%) failed to grow on CARB media.

As regard ChromID® CARBA SMART Agar (OXA media), in the current study, among Klebsiella isolates 14/28 (50%) grew on CHROM OXA media with positive blue color, while other 14 isolates failed to grow. Concerning Acinetobacter isolates, 13/22 (59.1%) grew on CHROM OXA media with no color, while 9/22 (40.9%) failed to grow including the one failed to grew on CARB media.

In the current study, Comparing results of ChromID® CARBA SMART Agar and PCR showed 47/50 (94%) of positive isolates by chromogenic media were also positive by PCR, while only 2 positive isolates by chromogenic media were negative by PCR. Negative PCR isolates may carry another genes code for carbapenemases which are not included in our study and this may explain the cause of negative isolates by PCR which grew on positive ChromID® CARBA SMART Agar.  There was highly statistical significance between results of ChromID® CARBA SMART Agar and real time PCR (P value, 0.001) (table 3).  Diagnostic Performance of Chromogenic media were 100% sensitivity, specificity 0%, PPV 96%, NPV 0% for detection of carbapenemase production (Table, 6).

In the current study, ConcerningModified Hodge test, forty seven out of fifty isolates were positive by MHT, while 3 isolates were negative. All Klebsiella isolates 28/28 (100%) were positive, while 19/22 (86.4%) Acinetobacter were positive and 3/22 (13.6%) were negative. There was non- statistical significance between MHT and PCR (P value, 0.7) (table 4). Forty five isolates were positive by both MHT and PCR for carbapenemases, while three positive PCR isolates were negative by MHT and two isolates showed false positive results by MHT.  Diagnostic Performance of MHT were 93.8% sensitivity and specificity 0% for detection of carbapenemase production (table 6). 

In the current study, there were 48/50 (96%) isolates positive by blue carba test, while 2/50 (4%) were negative. Among the Klebsiella isolates 28/28 (100%) tested isolates were positive by blue carba test, while 20/22 (90.9%) Acinetobacter isolates were positive. There was highly-significant statistical association (P.‹0.001) between blue carba test and PCR as 48/48 (100%) positive by blue carba test were PCR positive and the 2/2 (100%) negative isolates by blue carba test were also PCR negative (table, 5). Diagnostic Performance of blue carba test was 100% sensitivity and specificity 100% for detection of carbapenemase production (table 6).

Table (3): Results of chromogenic media in relation to PCR.

Table (4): Result of MHT in relation to PCR.

Table (5): Results of blue carba test and PCR.

Table (6): Diagnostic Performance of all tests.

DISCUSSION