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Phenotypic Screening for ESBL, MBL and AmpC [beta]-Lactamases in Enterobacteriaceae Isolates from Equines.


Acquired resistance to [beta]-lactam antibiotics by ESBL (Extended spectrum [beta]-lactamases), AmpC [beta]-lactamases and MBL (Metallo-[beta]-lactamases) in Gram-negative pathogens such as Enterobacteriaceae is a growing problem worldwide and considerable threat to animal health care and public health (Kraker et al., 2013). The possible zoonotic spread of antimicrobial-resistant bacteria is possible. ESBL/AmpC-producing E. coli isolates are now being found in increasing numbers in food producing animals leading to the hypothesis that animals might become infection sources or even reservoirs, the natural persistent source of infection, contributing to the spread of these bacteria (Dierikx et al., 2012). After ESBLs and AmpC [beta]-lactamases, the emergence of novel Metallo [beta]-lactamases (MBLs) has contributed to an increased prevalence of carbapenem resistant Entero-bacteriaceae (CRE). An ESBL producer may harbour AmpC or Metallo-beta-lactamase or both (Yong et al., 2009 and Miriagou et al., 2010). The characteristics of ESBL, AmpC and MBLs are given in Table 1.

The extended spectrum [beta]-lactamases often remain undetected by the current isolation and susceptibility methods as ESBLs have different levels of activity against various cephalosporins. If an ESBL is detected, all penicillins, cephalosporins and Aztreonam should be reported as resistant, even if in-vitro test results indicate susceptibility (Dhara et al., 2012). The co-existence of different classes of [beta]-lactamases in a single bacterial isolate may pose diagnostic and treatment challenges. Molecular methods like polymerase chain reaction, sequencing and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) are the key tools for their detection but the facilities for them are not available in most of the laboratories, especially in developing countries.

Materials and Methods

The present study screened 32 Enterobacteriaceae isolates from various Equine samples and specimens from organised studs by antibiotic susceptibility testing (ABST) and isolated 10 multi drug resistant Enterobacteriaceae strains which were further screened for ESBL, Amp C [beta]-lactamases and

MBL using following phenotypic detection methods:-

i) Double disc synergy test (DDST)

ii) Modified double disc synergy test (MDDST)

iii) Modified Hodge test (MHT)

iv) MBL-ESBL-Amp C Co-existence Detection MIC strips

Antimicrobial Susceptibility Testing

Total 32 Enterobacteriaceae isolates (20 E. coli isolates, 12 Klebsiella pneumonia isolates) were tested for their antimicrobial susceptibilities by disc diffusion method according to CLSI 2017 guidelines and out of 32, 10 multidrug resistant isolates (06 E. coli isolates, 04 Klebsiella pneumonia isolates) were further screened by DDST, MDDST, MHT and strip method.

Testing for ESBL Production- DDST, MDDST

All strains which were resistant for Cefotaxime and Ceftriaxone were selected for checking the ESBL production by DDST and MDDST. The ESBL production was tested by double disc synergy test (DDST) using 30[micro]g Cefotaxime (3GC - 3rd generation cephalosporin) disc and Amoxicillin-Clavulanate disc (AMC 20/10 [micro]g) placed at 30 mm spacing on the lawn culture of test isolate on MHA plate. Extension of inhibition zone around the Cefotaxime disk towards AMC disc is indicative of ESBL production (Dhara et al., 2012) (Fig. 1). MDDST performed on MHA plate (with the lawn culture of the test isolate) placing AMC disk in the centre of the plate and discs of 3GCs-Cefotaxime, Ceftriaxone, Cefpodoxime and 4GC (4th generation Cephalosporin) - Cefepime were placed 15mm and 20mm apart respectively, centre to centre to that of the AMC disc. Any distortion or increase in the zone towards the AMC disc was considered as positive for ESBL production (Dhara et al., 2012). Escherichia coli 25922 was used as negative control for ESBL production.

Modified Hodge Test (MHT)

Performed according to CLSI, 2017 document (Fig. 3). Enhanced growth of the indicator organism is indicative of carbapenemase production. No enhanced growth is negative for carbapenemase production (CLSI, 2017). In the present study, both Imipenem and Ertapenem were used and isolates were retested twice.

MBL-ESBL-AmpC Co-existence Detection MIC strips

MBL-ESBL-AmpC Co-existence Detection strips, HIMEDIA (EM 134--MBL + ESBL detection strip and EM 135--MBL + AmpC detection strip) used in this study and composition was given in the Fig. 4(a). Isolates which were showing reduced susceptibility to Cefotaxime, Imipenem and Ertapenem in ABST, were selected for screening with both EM 134, EM 135 strips on lawn culture of test isolate on the same MHA plate (Fig. 4) and interpretation was done according to manufacturer's interpretation chart.


Results of the above methods are given in Table 2.


ESBLs constitute a serious threat to [beta]-lactam therapy. Due to difficulty in their detection by the current CLSI methods, many of these strains have been falsely reported to be susceptible to widely used broad-spectrum [beta]-lactams (Rubin et al, 2014). The ESBL producers are intrinsically resistant to all cephalosporins even if they exhibit an in vitro susceptibility as resistance can be transferred horizontally (Wieler et al., 2011). Cefepime, a 4GC is more reliable for detecting ESBLs in the presence of AmpC [beta]-lactamases, as this drug is stable to AmpC [beta]-lactamase. Thus, it will demonstrate the synergy which arises from inhibition of ESBL by Clavulanate in presence of AmpC enzyme. In our study, DDST identified only 3 ESBL producers and failed to identify ESBL producers carrying AmpC [beta]-lactamases. Dhara et al. (2012) demonstrated the superiority of MDDST over DDST for ESBL detection. So, 4GC-Cefepime has been recommended as an alternative cephalosporin for the ESBL detection in the presence of AmpC. In our study, we used both 3GCs and 4 GC in MDDST.

The Carbapenems including Imipenem, Ertapenem are broad spectrum antibiotics with high stability against most [beta]-lactamase enzymes and isolation of Carbapenem resistant strains from animals is a cause of concern (Rubin et al., 2014). The Modified Hodge test (MHT) has been widely used as a general phenotypic method for detection of carbapenemase production (CLSI, 2017) and the test allows for carbapenem inactivation by carbapenemase-producing bacteria. This test is sensitive for Class A and Class D enzymes. However, metallo-[beta]-lactamases (e.g. NDM-1, New Delhi Metallo [beta]-lactamase) have consistently shown high false negative rates (Yamada et al., 2016). In our study, 2 isolates having resistance to carbapenams were falsely detected as carbapenemases by MHT (later confirmed with strip method). Over production of ESBLs along with AmpC [beta]-lactamase combined with the porin loss may have provided resistant to carbapenems in them as described by Bradford et al., 1997. In our study, better MHT results obtained with an Ertapenem disk than with an Imipenem disk upon retesting. Hence MHT is more useful as a confirmatory test rather than as a screening tool.

Commercially available MBL-ESBL-AmpC Coexistence Detection MIC strips are superior compared to other phenotypic detection methods because of their composition (Clavulanic acid--a ESBL inhibitor, Cloxacillin--a AmpC inhibitor, EDTA - MBL inhibitor). The ESBLs are encoded by plasmids which also carry resistant genes for other antibiotics. A co-resistance to quinolones and aminoglycosides is common (Yong et al., 2009). We have isolated an MBL-ESBL-AmpC positive E. coli strain resistant to 13 various non beta lactum antibiotics (fluoroquinolones, tetracyclines and aminoglycosides) in the present study (Fig 5). An optimum identification of the ESBL producing isolates is essential to formulate policies for an empirical antimicrobial therapy, especially in high-risk units where the infections which are caused by these organisms are common. It also helps in monitoring the development of anti-microbial resistance and in implementation of proper hospital infection control measures.


The authors are thankful to the DGRVS, QMG branch, IHQ of MOD for providing the necessary facilities to carry out the work.


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S.B. RamaRaju Sagi (1), Rahul Sharma (2), Chhabil Singh (2) and Vikas Thakur (3)

Central Military Veterinary Laboratory (CMVL) Sardhana Road Meerut Cantt.

Meerut - 250001 (Uttar Pradesh)

(1.) Lab Officer and Corresponding author. E-mail:

(2.) Lab Officer

(3.) Commandant

42nd Annual Congress of ISVS held at Navsari

The 42nd Annual Congress of Indian Society for Veterinary Surgery (ISVS) and National Symposium on 'Advances in Diagnostic Imaging and Surgical Techniques in Farm and Companion Animals' was held at College of Veterinary Science and Animal Husbandry, Navsari Agricultural University (NAU), Navsari, Gujarat during 22nd-24th November, 2018. More than 300 delegates including faculties and post graduate scholars from various Veterinary colleges, scientists from National Institutes, practicing Veterinarians and Veterinary officers from State Animal Husbandry department participated in the conference.

More than 200 papers were presented in nine sessions by participating delegates in anaesthesiology, avian, equine, ruminant, small animal and wild animal surgery, orthopaedic and ophthalmology as well as radiology and imaging with worthy deliberations.

The general body of ISVS also deliberated on their common issues including the BSMA syllabus for post graduate studies. The general body agreed to host the 43rd Annual Congress of ISVS and National Symposium on 'Recent Advances on Amelioration of Anaesthetic and Surgical Stress in Farm and Companion Animals' on 14-16th November, 2019 at College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana.

Intas Animal Health actively participated during the conference and had an exhibit stall. The stall was visited by conference participants and eminent Surgeons from across the country.
Table 1: Characteristics of ESBL, Amp C and MBLs (As adopted from
Pitout, 2012)

S.   Enzymes            Amber (1980)    Spectrum         Inhibitors
No.                     classification  of resistance

1    ESBL               Class A         Penicillins,     Clavulanic
                                        Cephalosporins,  acid,
                                        Monobactams      Tazobactam,
2    Amp C              Class C         Penicillins,     Cloxacillin,
                                        Cephalosporins,  Boronic acid
3    Carbapenemases-    Class B         Penicillins,     EDTA and
     Metallo-                           Cephalosporins,  other metal
     [beta]-lactamases                  Cephamycins,     chelators
     (MBL)                              Carbapenems

Table 2: Results of various tests

S.  Isolates based on enzyme   DDST   MDDST   MHT     Co-existence
No  production                                      Detection MIC Kit

1.  ESBL producers               3      10     -           10
2   ESBL-AmpC producers          -       7     -            7
3   ESBL-AmpC-MBL Producers      -      -      -            2
4   Carbapenemases producers     -      -      5            3
5   Only MBL producers           -      -      1            1
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Title Annotation:Research Article
Author:Sagi, S.B. Ramaraju; Sharma, Rahul; Singh, Chhabil; Thakur, Vikas
Publication:Intas Polivet
Date:Jul 1, 2018
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