Phenotypic detection of Carbapenamase among Klebsiella pneumoniae isolated from clinical samples using modified Hodged test

Carbapenemases are microbial enzymes that confer resistance to virtually all available beta-lactam antibiotics and the most frequent carbapenemases are the Klebsiella pneumoniae Carbapenamase (KPC). Detection of carbapenemases is a significant infection control strategy as the enzymes are often associated with extensive antimicrobial resistance, therapeutic failures and mortality associated with infectious diseases. A total of 400 clinical samples were collected from different groups of patients in Abubakar Tafawa Balewa University Teaching Hospital, Bauchi, Nigeria and 118 K. pneumoniae were isolated using standard microbiological techniques. The isolates were subjected to antibiotic susceptibility testing by Kirby-Bauer disc diffusion method, then screened for Carbapenamase production using modified Hodge test. The results indicated that the isolates were resistant to Ampicillin (61.9%), Ceftriaxone (50.8%) and Ceftazidime (50.8%), then Ciprofloxacin (54.2%), but predominantly sensitive to Imipenem (66.9%), Eterpenem (60.2%) and Meropenem (65.3%). It was found that 38 (32.2%) of the isolates phenotypically shows the presence of Carbapenamase, with highest frequency of (40.7%) among patients, mainly adult females with cases of Urinary Tract Infections (UTIs) and the least from wound (11.8%).This study revealed that the isolates produced other betalactamases than KPC or variants of Carbapenamase that cannot be detected by modified Hodge test, thus shows low resistance to carbapenems. Therefore further studies is needed to genotypically confirm the presence of KPC in these isolates.


Introduction
Klebsiella is a Gram-negative bacterium that can cause different types of infections, including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis. Klebsiella bacteria are normally found in the human intestines, as commensals. Klebsiella infections commonly occur among sick patients who are receiving treatment for other conditions, whose care requires devices like ventilators or intravenous catheters, and patients who are on prolonged antimicrobial therapy [1] Klebsiella pneumoniae have increasingly developed antimicrobial resistance to some antibiotics like carbapenems due to production of an enzyme known as Carbapenamase, thus referred to as KPC-producing bacteria. Infections caused by KPC-producing bacteria can be difficult to treat because fewer antibiotics are effective against them [2]. In such cases, a microbiology laboratory must run tests to determine which antibiotics will treat the infection. Klebsiella pneumoniae carbapenemases (KPCs) producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide [2,3]. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams [3].
Carbapenamase is an enzymes that hydrolyse a group of antibiotic called carbapenems which was a drug of choice for the treatment of multidrug resistance bacteria especially Extended spectrum beta lactamase producers by binding to the penicillin binding proteins (PBAs), inhibiting the synthesis of bacterial cell wall [4]. Among the beta-lactamases, the carbapenemases are the most crucial, because of their ability to hydrolyze all beta-lactam antibiotics, including the carbapenems. The major health concern is with transmissible carbapenemases, which can be acquired unpredictably by important nosocomial pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa [5].
Carbapenemases belong to molecular classes A, B, and D. The class A enzymes (Bush group 2f) are inhibited to various degrees by Clavulanate and usually hydrolyze penicillins or cephalosporins more efficiently than carbapenems [6]. For this reason, some, such as KPC enzymes, lack potent Carbapenamase activity and may be considered ESBLs that also hydrolyze carbapenems [7]. There are three classes of Carbapenamase usually identified, class A of the KPC type, class B of the New Delhi Metallo-βeta-lactamase (NDM-1), IMP and Verona Integron-encoded metallo-βeta-lactamase (VIM type), and class D of the OXA-type [5,6].
Detection of Carbapenamase-producing bacteria can be difficult, as their presence does not always produce a resistant phenotype on conventional disc diffusion or automated susceptibility testing methods. These enzymes are often associated with laboratory reports of false susceptibility to carbapenems which can be potentially fatal [8]. However, most clinical laboratories do not attempt to detect carbapenemases on routine basis, due to the lack of availability of guidelines and procedures or lack of knowledge and expertise. The routine susceptibility tests may be unreliable and special tests are therefore required to detect the resistance mechanisms involved [8]. Considering the infection control implications and the high morbidity and mortality associated with infections caused by K. pneumoniae, it is essential that susceptibility methods identify carbapenem resistance rapidly and reliably [9,10].
Modified hodged test is very crucial for the detection of Carbapenamase producing bacteria, as automated method such as VITEK-2 is not reliable because Carbapenamase-producers have MIC below the clinical carbapenems break point [11,12]. The test has been widely used in clinical setting and it's been recommended as phenotypic techniques for identification of Carbapenamase activity by CLSI guideline [5,13]. Carbapenamase-producing Klebsiella pneumoniae isolates are important causative agents of nosocomial infections associated with significant mortality rates [14,15]. Rapid detection and typing of these bacteria is critical especially for surveillance purposes, to prevent outbreaks and optimize antibiotic therapy. This study therefore aimed to determine the effectiveness of modified Hodged test as one of the most sensitive phenotypic test for detection of KPC producers among Klebsiella pneumoniae.

Study Design
This is a hospital based, descriptive and cross-sectional study involving both in and out-patients attended Abubakar Tafawa Balewa University Teaching Hospital Bauchi from June to December, 2016.

Sample collection
Total of four hundred (400) clinical samples were collected from Abubakar Tafawa Balewa University Teaching Hospital, the samples includes urine, sputum, wound swab and High vaginal swab.

Culture and Identification of K. pneumoniae
The samples were inoculated on CLED, MacConkey and Blood agar media (Oxoid, UK). The plates were incubated at 37 0 C for 24 hours. Preliminary identification of K. pneumoniae was carried out based on morphological features, Gram reaction, microscopy and biochemical tests according to Microbiological standards [16,17].

Antimicrobial susceptibility testing
Susceptibility of all isolates to the groups antimicrobial agents such as fluoroquinolones, third generation cephalosporins and carbapenems were determined by the disk diffusion method on Mueller-Hinton agar using antibiotic disks from Oxoid Ltd. (Basingstoke, UK) according to the recommendations and interpretation of CLSI (2017). The antibiotics used were Ampicillin (10µg), Amikacin (30µg), Ciprofloxacin (5µg), Eterpenem (10µg), Imipenem (10µg), and Meropenem (10µg). Strains of K. pneumoniae ATCC 1705 and ATCC 1706 were used as positive and negative control respectively). Isolates that shows susceptible or intermediate to disk diffusion (i.e. 16mm-21mm) were selected for Carbapenamase detection.

Modified Hodge test
A loopful of 1:10 dilution of Escherichia coli (ATCC 25922) was used as carbapenems susceptible organism recommended by CLSI (2014). The dilution was streaked on Mueller Hinton agar plate and a 10µg of Meropenem antibiotic disk was placed at the centre of the plate, the test organism (K. pneumoniae) and control organisms (K. pneumoniae ATCC 1705 and 1706) were streaked in straight line from the edged of the disk to the edge of the plate. The plates were then incubated at 35 O C for 16-24hours [17]. The results were interpreted as positive modified hodged test by clover leaf-like indentation of the E. coli 25922 susceptible strain growing along the test organism.

Statistical analysis
The data obtained were analyzed with SPSS version 20.0 using Chi-square at 95% to test degree of association.

Results
In this study, a total of 118 K. pneumoniae isolates were recovered from 400 clinical, specimens out of which 38 (32.2%) were Carbapenamase-producers ( Table 1). Most of the isolates were from female patients (55.1%) within 41 to 50 years age (22.9%), but highest Carbapenamase-producers (34.2%) was found in 31 to 40 age group.

Discussion
Carbapenems resistance can be mediated by production of carbapenemases or by the combination of outer membrane porin expression disruption and production of various β-lactamases [18]. Carbapenemases may confer resistance to virtually all available beta-lactam antibiotics. The most frequent carbapenemases are the Klebsiella pneumoniae Carbapenamase (KPC) enzymes. KPC-producing bacteria are found in many countries [19,20].While the KPC enzyme is still found most commonly in association with K. pneumoniae, the blaKPC gene has now been identified in numerous other members of the Enterobacteriaceae [10]. Carbapenamase producers in their study in Portugal,13(28%) were susceptible at lower frequency to imipenem and 10 (22%) to Meropenem.
The high antimicrobial sensitivity of K. pneumoniae to carbapenems in our study is an indication that the isolates might be harbouring other beta-lactamases than KPC. It can be another variants of Carbapenamase present, which cannot be detected by our study, hence the reason for low MHT positive. The low resistance to carbapenems in this study could be due to the fact that, our hospitals does not have a regular prescription pattern for carbapenems probably due to their high costs and are not usually the first line drugs used in the treatment of infectious disease in this area [24]. Other factors which contribute to carbapenem resistance is that the enzymes have social and microbiological epidemiology which embraces patient to patient transmission of KPC [27], which is low in our study area. However, these methods also inhibit AmpC activity and are, therefore, not specific to KPC [30]. The performance of automated systems for the detection of KPC-producing K. pneumoniae has been also evaluated [14].Matrix-assisted laser desorption-ionization time-of-flight-mass spectrometry (MALDI-TOP MS) is a promising, rapid and economical method for the detection of Carbapenamase-producing K. pneumoniae strains that could be successfully introduced into the routine diagnostic microbiology laboratories [14,15]. approving Carbapenamase production, but should accompany with PCR method for the bla-KPCgene, which has the additional advantage for confirming the specific KPC presenting the bacterial strains

Conclusion
The present study found that MHT might not be an appropriate phenotypic method for confirming Carbapenamase production, considering the low positive test results and high sensitivity of the isolates to common carbapenems drugs. Other standard methods, including molecular assays should therefore be employed to ascertain the presence of Carbapenamase in Klebsiella pneumoniae.

Disclosure of ethical approval
Ethical approval was obtained from the ATBU teaching hospital research and ethics committee, with verbal informed consent also sought from all patients, prior to specimen and data collection.