“ Detection of various β-lactamases amongst gram negative bacilli from clinical isolates with special reference to Metallo β-Lactamases ”

“Detection of various β-lactamases amongst gram negative bacilli from clinical isolates with special reference to Metallo β-Lactamases” Dr. Sonal P. Chavan 1 , Dr. Sunanda N. Shrikhande 2 , *Dr.Ratna R.Prasad 3 Dr. Gajbhiye S R 4 .  1. Assistant Prof, Dept. of Microbiology, Indira Gandhi Govt. Med. College, Nagpur-440018, Maharashtra. 2. Prof. & HOD, Dept. of Microbiology, Indira Gandhi Govt. Med. College, Nagpur-440018, Maharashtra. 3. Junior ResidentII, Dept. of Microbiology, Indira Gandhi Govt. Med. College, Nagpur-440018, Maharashtra. 4. Assistant Prof, Dept. of Microbiology, Indira Gandhi Govt Medical College, Nagpur-440018, Maharashtra


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permits hydrolysis of all cephalosporins, penicillins and aztreonam. AmpC class β-lactamases are cephalosporinase that are poorly inhibited by clavulanic acid. They are differentiated from other ESBLs by their ability to hydrolyze cephamycins as well as other extended spectrum cephalosporins (Hyder et al., 2014). Metallo β-lactamases (MBLs) producing bacteria can hydrolyze a wide range of β-lactam antibiotics including penicillins, cephalosporins, carbapenems, cephamycins, but lack the ability to hydrolyze aztreonam. Moreover, their catalytic activities are generally not neutralized by commercially available β-lactamase inhibitors such as clavulanate, tazobactam, and sulbactam.
Laboratory detection of resistance to carbapenems has been reported to be difficult for many reasons; low expression of such resistance, degradation of the drug, the use of automated methods for identification and susceptibility testing, in addition to the lack of standardized methods of detection (Babey et al., 2009). The present study was carried out to detect the various β-lactamases in gram negative organisms with special reference to Metallo β-lactamases (MBLs) from a tertiary care centre.

Materials and Methods:-
The study was conducted over a period of six months from December 2014 to June 2015 at a tertiary care centre. A total of 569 gram negative bacteria (GNB) were isolated from various clinical specimens and identified according to standard guidelines (Konemann et al., 2006, Mackey et al., 2006. Antibiotic susceptibility testing (AST) was performed on Mueller-Hinton agar plates with commercially available discs (Hi media, Mumbai) by Modified Kirby-Bauer (Bauer et al., 1966) disc diffusion method and interpreted as per the CLSI 2015 guidelines. The following antibiotics were tested: Ampicillin (10 μg), Cefazolin (30 μg), Cephalothin (30 μg),Cefuroxime (30 μg) Cefotaxime (30 μg), Ceftazidime (30 μg), Cefepime (30 μg), Amoxycillin-clavulanate (20/10 μg), Piperacillintazobactum (100/10 μg), Gentamicin(10 μg), Amikacin (30 μg), Ciprofloxacin (5 μg), Levofloxacin (5 μg), Doxycycline (30 μg), Imipenem (10 μg), Meropenem (10 μg), Colistin (10 μg), Polimixin B(300 units). The detection methods of betalactamases were applied on the same day along with routine AST. Screening and Confirmatory test for ESBL detection:-Initial screening test for ESBL production was performed on Muller Hinton agar (MHA) by disk diffusion method using ceftazidime (30 µg) disk. The lawn culture of 0.5 McFarland inoculum of the test strain was exposed to a disk of ceftazidime. After incubation at 37 o C for 16-18 hours, the zone diameter ≤ 22 mm indicated ESBL production They were further confirmed by a double-disc synergy method. Muller Hinton agar plates were swabbed to form a lawn culture with 0.5 McFarland standard inoculum of the test strain. On the MHA plate, a disk of cefotaxime (30 μg) was placed 20 mm apart, centre to centre, from amoxyclav (20/10 μg) disk whereas piperacillin-tazobactam (100/10 μg) disk was placed 20 mm apart, centre to centre from cefepime (30 μg) disk. Plates were incubated at 37 o C overnight and were examined for enhancement of zone inhibition of cefotaxime and cefepime at the side facing amoxyclav and piperacillin-tazobactam disk respectively [ Figure 1]. Organisms that showed a clear extension of inhibition zone towards the disk of amoxyclav and piperacillin-tazobactam were considered ESBL positive (CLSI 2015, Khan et al., 2008) Screening and Confirmatory test for Amp C detection:-Screening for Amp C production was done by using cefoxitin disk (30 µg) on MHA. The lawn culture of 0.5 McFarland inoculum of the test strain was exposed to a disk of cefoxitin. After overnight incubation at 37 o C, the zone diameter ≤ 18 mm were suspected as AmpC producer (Jacoby et al., 2009). Further they were confirmed by Cefoxitin-cefotaxime disk antagonism for inducible AmpC detection. A lawn culture of 0.5 McFarland inoculum of the test strain was exposed to a disk of cefotaxime (30 µg) and cefoxitin (30 µg) placed at a distance of 1.5 cm from edge to edge [ Figure 2]. After overnight incubation, there was flattening of radius of zone of inhibition produced by cefotaxime on the side nearest the cefoxitin disk in case of AmpC β-lactamase (Miles et al., 2006).

Screening and Confirmatory test for Carbapenemase detection:-
Carbapenemase production was tested by performing both initial screening test and phenotypic confirmatory test. In initial screening test, lawn culture of 0.5 McFarland inoculum of the test strain was exposed to a disk of imipenem (10 μg). E. coli ATCC 25922 was used for quality control. After overnight incubation, the zone diameter around 16-21 mm indicated carbapenemase production (CLSI 2015). All these isolates were subjected to modified Hodge test (MHT) for confirmation of carbapenemase production. A 0.5 McFarland standard suspension of E. coli ATCC 25922 was prepared in a saline and it was further diluted in 1:10 in saline. The MHA plate was inoculated as per the routine disk diffusion procedure. The plate was allowed to dry 3 to 10 minutes. A single disk of meropenem (10 μg) was placed on the plate at the centre. Using a 10-μl loop, 3-5 colonies of test organism grown overnight on a blood agar plate were picked up. They were inoculated in a straight line (at least 20-25 mm in length) out from the edge of the disk [ Figure 3]. The plate was incubated at 37 0 C for 16-20 hours. Following incubation, the MHA plate was examined for enhanced growth around the test organism streak at the intersection of the streak and the zone of inhibition. The enhanced growth suggests positive test for Carbapenemase production (CLSI 2015). The carbapenemase producing strains were further subjected to three different phenotypic methods for detection of MBL production.

MBL production-Combined disk test (Disk potentiation test-DPT):-
It was used to detect metallo-β-lactamase (MBL) production (Picao et al., 2008). In this test, the lawn culture of 0.5 McFarland inoculum of the test strain was exposed to a disk of imipenem (10 µg) and imipenem-EDTA (10/750 µg). The difference of ≥ 7 mm in zones of inhibitions of two disks indicated MBL production. Since EDTA may have some bactericidal activity, a blank disc of EDTA was tested as control [ Figure 4]. DPT was performed with meropenem/ meropenem-EDTA and ceftazidime/ceftazidime-EDTA also (Pitout et al.2005).

Discussion:-
The newer β-lactamases like ESBL, AmpC and Carbapenemase has emerged as a cause of antibiotic resistance among the GNB worldwide in the recent years. Failure to detect these enzymes has contributed to their uncontrolled spread and sometimes to therapeutic failure (Doddaiah et al.2014). Study by Nagdeo et al. (Nagdeo et al.,2012) found ESBL production in 39.03 strains, AmpC in 9.29%, and MBL in 7.44%strains which is highly comparable to our study.
In the present study, ESBLs were found in 31.1% isolates which is comparable with Doddaiah et al.  (Haider et al. 2014), found pseudomonas to be the second commonest ESBL producer after E.coli. In our study also, Pseudomonas and Nonfermenters were found to be the remarkable ESBL producers. These figures reinforce the fact that ESBL production should be routinely screened in Gram-negative bacteria other than E. coli and Klebsiella. Piperacillin/tazobactam with cefepime was found to be more sensitive as compared to amoxyclav/clavulanic acid with cefotaxime. This finding is comparable with study done by Khan  The coexistence of different classes of β-lactamases in a single bacterial isolate may pose diagnostic and treatment challenges. The AmpC producing organisms can act as a hidden reservoir for the ESBLs (Oberoi et al., 2013). We detected coproduction of ESBL and AmpC in 31 isolates (5.5%) which is similar to Raffie et al., MHT identified 94.8% of strains as carbapenemase producer in our study. Bartolini et al., (Bartolini et al., 2014) found MHT to be 94% sensitive and 100% specific for identification of carbapenemase production. As per CLSI 2015 , MHT have some limitations like false positives in isolates producing ESBL or AmpC enzymes coupled with porin loss and false negative in NDM producing isolates. But MHT is a phenotypic test used to detect carbapenemase in isolates demonstrating elevated but susceptible carbapenem MICs and has demonstrated sensitivity and specificity exceeding 90% in identifying carbapenemase producing Enterobacteriaceae (CLSI 2015).
In present study, carbapenemase production was highest in Acinetobacter spp. (36.4%) followed by Klebsiella spp. pneumoniae. But in this study only enterobacteriaceae were included. Our study is comparable to Nagdeo et al., (Nagdeo et al.2012) who showed Nonfermenters to be the commonest followed by Pseudomonas and Enterobacteriaceae for carbapenemase production. Noyal et al., (Noyal et al., 2009) found carbapenemase production in 53% isolates of Acinetobacter spp. which is much higher than ours but 27.8% isolates of Pseudomonas spp. which is comparable.
The carbapenemase producing isolates were further subjected to three phenotypic methods for MBL detection. Over the past few years, MBL producing gram negative bacteria are being reported with increasing frequency from several parts of the world and have emerged as a most widespread and clinically significant carbapenem resistance mechanisms (Bora et al., 2014). The detection of MBL-producing isolates by PCR is expensive, requires specialized technicians and instruments, and, more importantly, is able to detect only previously described MBL-encoding genes. However, the detection of the MBL phenotype of resistance is of crucial importance for selecting the most appropriate therapy and applying infection control measures. For these reasons, an accurate and easy-to-perform phenotypic test is desirable and urgently necessary in hospitals with a high prevalence of MBL-producing isolates (Picao et al., 2008).
In our study, the rate of MBL production in gram negative bacilli was 14.7% (85/579). MBL production has been reported from various regions ranging from 7-65% (Deshmukh et al., 2011). Most of these studies reported MBL production in Pseudomonas aeruginosa and Acinetobacter baumannii. Very few studies have reported MBL production in Enterobacteriaceae. In our study, we reported 44 strains of MBL producing Enterobacteriacae. Of the 116 carbapenem -resistant strains, thirty one strains did not show MBL production. The likely reasons for carbapenem resistance in non MBL producers varied such as decreased cell membrane permeability, or activity of efflux pumps (Tellis et al., 2013).
In our study MEM (73.3%) and IMP (58.6%) were better than CAZ (44.8%) when combined with EDTA in DPT for detection of MBL. Picao et al., (Picao et al., 2008) reported similar findings. Kumar et al., (Kumar et al., 2012) and Manoharan et al., (Manoharan et al., 2010) found I/I-EDTA to be the better method for routine MBL detection than MEM/MEM-EDTA. Few other studies also reported I/I-EDTA as most sensitive method for detection of MBL production in gram negative bacilli (Pandya et al., 2011). Noyal et al., (Noyal et al., 2009) and a study from National Institute of Infectious Diseases, Tokyo, Japan (Arakawa et al., 2000) found CAZ/EDTA-CAZ combination to be better than MEM/MEM-EDTA combination. Our results were in contrary with the above mentioned studies. Our study is highly comparable with Sheikh et al. (Sheih et al., 2014) and Buchunde et al. (Buchunde et al., 2012). Both these studies (Sheih et al., 2014, Buchunde et al., 2012 have compared their results of DPT with the molecular methods. Overall, a combined disk diffusion testing with MEM and MEM-EDTA is easier, cost-effective and sensitive, and can be easily introduced on the same plate of Antibiotic susceptibility testing done routinely in the clinical laboratory.
Our study was limited by the fact that no molecular confirmation was done for above mentioned resistance mechanisms due to constraint of resources. But in a resource-poor developing country, these innovative disc placement methods are easy, affordable and reliable for detection of various beta-lactamases. Our study showed betalactamase production in a considerable number of gram negative bacilli. CLSI guidelines explain the method of ESBL detection in Enterobacteriaceae. But in our study we got variable number of ESBL producing Pseudomonas and Acinetobacter. Similarly guidelines for MBL detection are limited. Hence our study emphasises the importance of ESBL detection in isolates other than Enterobacteriaceae and MBL detection with CDT in gram negative bacilli.   (9) 1(11.1) 0(00) 0 1(11.1) Enterobacter species (5) 0(00) 0(00) 0 0(00) S.Typhi (2) 0(00) 0(00) 0 0(00) Nonfermenters (  failure and may result in increased mortality. Thus it is the need of the hour to standardise various cost effective and time saving methods to detect these resistance mechanisms with routine AST in a Clinical Microbiology laboratory.