Research Article

 

Multidrug Resistance Pattern of Enterobacter Spp. Isolated from Acute Respiratory Tract Infected Camels (Camelus dromedarius)

 

Sandeep Kumar Sharma1*, Rahul Yadav2, Prerna Nathawat3

1Department of Veterinary Microbiology & Biotechnology, Post Graduate Institute of Veterinary Education and Research (PGIVER)- Jaipur, Rajasthan University of Veterinary and Animal Sciences, Bikaner, India; 2Department of Veterinary Microbiology & Biotechnology; 3Centre for Studies on Wildlife Management and Health, College of Veterinary & Animal Science- Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner, India.

Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.

Received | December 27, 2014; Revised | January 16, 2015; Accepted | January 20, 2015; Published | January 26, 2015

*Correspondence | Sandeep Kumar Sharma, Rajasthan University of Veterinary and Animals Sciences, Bikaner, India; Email: drsharmask01@hotmail.com

Citation | Sharma SK, Yadav R, Nathawat P (2015). Multidrug resistance pattern of Enterobacter spp. isolated from acute respiratory tract infected camels (Camelus dromedarius). Adv. Anim. Vet. Sci. 3(2): 128-132.

DOI | http://dx.doi.org/10.14737/journal.aavs/2015/3.2.128.132

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2015 Sharma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

 

Introduction

 

Camel (Camelus dromedarius) is a state animal of Rajasthan and has significant role in economy of poor farmers through various ways such as camel milk, wool, riding and draft. Camel has also important role in agricultural utilities along with especial adaptations for Rajasthan desert but in certain harsh condition like sudden environment changes make it susceptible for opportunistic infections. Pulmonary diseases are among the emerging problems of camels that are causing considerable loss in production and death (Njage et al., 2012; Al-Juboori et al., 2013). Genus Enterobacter is more specifically a nosocomial opportunistic pathogen and is sought out to be one of the many key causes for extra intestinal infections next to Escherichia coli (Osterblad et al., 1999). In the 1970s, Enterobacter was first noted as a common cause of nosocomial infections in immuno-compromised hosts with respiratory, urinary, and gastrointestinal tracts infections (Wilberger et al., 2012). Thus it was observed that Enterobacter commonly associated with respiratory, gastrointestinal and urinary tract infections in addition to wound, bloodstream, and central nervous system infections (Bordeanu et al., 2012).

 

The physical examination of Enterobacter respiratory tract infections may include high fever, tachycardia, hypoxemia and cyanosis. Infected animal with pulmonary consolidation may present with crackling sounds, dullness to percussion, tubular breath sounds and egophony. Pleural effusion may manifest as dullness to percussion and decreased breath sounds (Edward and Ewing, 1972; Gohary et al., 2012). Since during the last decade Enterobacter has emerged as an important hospital pathogen responsible for nosocomial respiratory tract infections with exhibiting high resistance to broad-spectrum antibiotics and the emergence of extended-spectrum cephalosporin-resistant strains has been also documented (Thiolas et al., 2005). In addition to diseases causing, genus Enterobacter is also capable to acquire antibiotic resistance in short time with various mechanism such as Beta-lactamase & Extended-Spectrum Beta-lactamase (ESBL) production, multidrug efflux system, low outer membrane permeability, mutations in chromosomal genes and additional acquired resistance genes via plasmids, transposons and phage makes this organism highly resistant (Harbottle et al., 2006). Though multidrug resistance pattern of Enterobacter among human patients explored well but in animals especially camels it has not studied very much. Thus the present study was carried out to characterize genus Enterobacter from respiratory tract infection in camels on the basis of their biochemical properties and to determine multidrug resistance pattern of organism for prevention of further morbidity and mortality of animals because of resistant opportunistic pathogens.

 

 

Material and method

 

Isolation and identification

A total of 46 samples of deep nasal discharge from acute respiratory tract infected camels were collected aseptically with sterile absorbent swabs soaked in nutrient broth. All samples has been collected from clinical complex of college of veterinary and animal science, Bikaner (Rajasthan) India on the basis of clinical symptoms of acute respiratory tract infection without discrimination of age, sex and breed of camels. The samples were inoculated on nutrient agar plates and then processed for isolation and identification of Enterobacter spp. (Cowan and Steel 1974; Quinn et al., 1994). Out of the 46 samples 16 suspected isolates were proceeded on the basis of phenotypic and biochemical properties such as cultural characteristics, motility, lactose fermentation, IMViC pattern, H2S production in TSI agar, lysine decarboxylase and urease activity.

 

Antibiotic sensitivity test

To determine the antibiogram of the isolates against various 25 antibiotics (Table 1) the method of Bauer et al. (1966) was followed. The isolates were inoculated in sterile 5 ml nutrient broth tubes and incubated for 18 hour at 37oC. The opacity was adjusted to 0.5 McFarland opacity standards (Quinn et al., 1994) and inoculums were well spread over the agar surface with the help of sterilized swab. Plates were allowed to dry for 10 minute at 37oC and then antibiotic discs (Hi Media, Mumbai) were carefully placed on the surface with enough space around each disc for diffusion of the antibiotic. Plates were incubated for 24 hour at 37oC and the diameter of zone of inhibition of growth around each disc was measured in millimeters. After inhibition zone measurement, result interpretation was made with standard chart provided by disc manufacturer (Hi Media, Mumbai).

 

 

Results

 

In the present investigation 16 Enterobacter spp. were isolated from 46 nasal samples from acute respiratory tract infected camels on the basis of their phenotypic and biochemical properties. All the isolates showed pink lactose fermenting, non- metallic sheen, mucoid colonies on respective culture media with typical IMViC pattern (- - + +) and not produced H2S gas in Triple Sugar Iron (TSI) agar. Typically all isolates were motile, urease positive and negative for lysine decarboxylation. In antibiogram study, all isolates were 100% sensitive to gentamicin and imipenem while completely resistant to ampicillin, bacitracin, erythromycin, clindamycin, rifampicin, vancomycin and oxacillin. All Enterobacters showed multidrug resistance pattern with minimum resistance to fifteen antibiotics. The studied isolates were sensitive with following decreasing percentage such as cefepime & ciprofloxacin (93.75%), norfloxacin & cefotaxime (87.50%), ceftazidime & co-trimoxazole (81.25%) and 56.25% isolates were resistant to tetracycline, 62.50% to cephalothin, 68.75% to ampicillin/sulbactum, 87.50% to nalidixic acid and 93.75% isolates were resistant to penicillin. All other antibiotics showed variable efficacy as described in table 1.

 

 

Table 1: Antibiogram of Enterobacter spp. obtained from acute respiratory tract infected camels

S. No.	Antibiogram disc	Conc. (mcg/disc)	Percent (Number of isolates)
			Sensitive	Intermediate	Resistant
1	Gentamicin (G)	120	100 (16)	-	-
2	Imipenem (I)	10	100 (16)	-	-
3	Cefepime (Cpm)	30	93.75 (15)	-	6.25 (1)
4	Ciprofloxacin (Cf)	5	93.75 (15)	6.25 (1)	-
5	Norfloxacin (Nx)	10	87.50 (14)	6.25 (1)	6.25 (1)
6	Cefotaxime (Ce)	30	87.50 (14)	-	12.50 (2)
7	Ceftazidime (Ca)	30	81.25 (13)	12.50 (2)	6.25 (1)
8	Co-trimoxazole (Co)	23.75/1.25	81.25 (13)	6.25 (1)	12.50 (2)
9	Colistin (Cl)	10	68.75 (11)	6.25 (1)	25 (4)
10	Chloramphenicol (C)	30	62.50 (10)	-	37.50 (6)
11	Kanamycin (K)	30	56.25 (9)	-	43.75 (7)
12	Trimethoprim (Tr)	5	56.25 (9)	18.75 (3)	25 (4)
13	Tetracycline (T)	30	31.25 (5)	12.50 (2)	56.25 (9)
14	Cephalothin (Ch)	30	25 (4)	12.50 (2)	62.50 (10)
15	Ampicillin/Sulbactum (A/s)	10/10	18.75 (3)	12.50 (2)	68.75 (11)
16	Nalidixic Acid (Na)	30	12.50 (2)	-	87.50 (14)
17	Cloxacillin (Cx)	10	6.25 (1)	56.25 (9)	37.50 (6)
18	Penicillin (P)	10 unit	6.25 (1)	-	93.75 (15)
19	Ampicillin (A)	10	-	-	100 (16)
20	Bacitracin (B)	10 Units	-	-	100 (16)
21	Erythromycin (E)	15	-	-	100 (16)
22	Clindamycin (Cd)	2	-	-	100 (16)
23	Rifampicin (R)	5	-	-	100 (16)
24	Vancomycin(Va)	30	-	-	100 (16)
25	Oxacillin(Ox)	1	-	-	100 (16)

 

 

Discussion

In the present study, Enterobacter spp. showed typical biochemical and cultural phenotypic characteristic as described in literature (Edward and Ewing, 1972; Cowan and Steel, 1975). Since chromosomal DNA not so rapidly change in comparison to plasmid and most of phenotypic and cultural properties govern by chromosomal DNA so there may possibilities that Enterobacter existing long without any phenotypic variations (Holmes and Jobling, 1996). On the basis of several easily performed biochemical tests, Zabransky et al. (1969) and Iversen et al. (2006) has also characterize Enterobacter spp. obtained from clinical cases. Similar to present study they found biochemical characterization is a reproducible technique for epidemiological surveillance up to genus identification but for precise species differentiation further genotypes (DNA cluster groups based on partial 16SrDNA sequence analysis) characterization is required.

 

For antibiotic susceptibility pattern, present study also found similar observation without resistance to cefotaxime, aztreonam, imipenem, gentamicin, nalidixic acid and ciprofloxacin (Osterblad et al., 1999) and in the study of Magnet et al. (2013), Enterobacter spp. were resistant to most of antibiotics, but were moderately sensitive (50%) to ciprofloxacin, tetracycline and doxycycline.

Observations in present study had accordance with the Al-Juboori et al. (2013), who revealed that the Enterobacter isolates from clinical and subclinical mastitis from camel milk showed moderate sensitivity to carbenicillin, streptomycin, sulphamethoxazole and gentamicin while less sensitive or even resistive towards ampicillin, colistin, penicillin G and tetracycline. Greenup and Blazevic, (1971) found slight variations that all the 28 strains of Enterobacter were sensitive towards gentamicin, chloramphenicol and nalidixic acid followed by sulfisoxazole, kanamycin, tetracycline, streptomycin and all were resistant to ampicillin. The Enterobacter was resistant to ampicillin (81.3%), chloramphenicol (75.0%), ciprofloxacin (6.3%), enrofloxacin (18.8%), neomycin (37.5%), norfloxacin (25.0%), streptomycin (56.3%) and tetracycline (75.0%) in the study of antimicrobial susceptibility of Enterobacter aerogenes from free-range chickens (Ojo et al., 2012) and the study by Nyenje et al. (2012) found that Enterobacter cloacae isolates registered 100% susceptibility to ciprofloxacin and various percentages of susceptibility was reported to chloramphenicol and gentamicin (91%) each, nalidixic acid (97%) and streptomycin (94%). In support of present study, these all variable patterns of resistance may prove that Enterobacter not only having variable mechanisms of antibiotic resistance but also has variable multidrug resistance patterns with different source of samples. It may understand that acquired antibiotic resistance may result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms and these mechanisms most commonly governed by mobile genetic elements such as plasmids, transposons and integrons (Harbottle et al., 2006). The mobile genetic elements are most variable genetic material with organisms, environments and cross transmission conditions thus not only earlier studies but also present observed variable pattern of multidrug resistance among Enterobacter and these genetic component may also explain variation of antibiotic resistance with different source of samples, geographic regions and host animals (Ojo et al., 2012; Al-Juboori et al., 2013).

 

Although, less information is available regarding Enterobacter antibiotic resistance patterns in veterinary medicine; however, emergence of resistance to beta-lactam agents indicates indiscriminate and excessive use of these antibiotics in food producing animals and for therapeutic management to prevent various infections among animal population (Reisbig and Hanson 2004). According to veterinarians practicing in study area, tetracycline, norfloxacin and cephalosporin are more commonly prescribed antibiotics in comparison to gentamicin and yet imipenem is not in veterinary practice thus the usages of these antibiotics positively correlate with increased resistance among Enterobacter strains in this investigation. Wilberger et al. (2012) also found similar positive correlation of increased use of antibiotic and their resistance for enrofloxacin and gentamicin in the USA during study of antibiotic resistance among Enterobacter spp. isolated from infection in animals.

McEwen and Fedorka-Cray (2002) has also concluded that excessive use of antibiotics induces the selection of resistant strains by producing hydrolytic enzymes and decreasing the active drug concentration via the alteration of permeability in outer membranes and enhances persistence and dissemination of antibiotic resistance not only in hospitals but also in food chains and ecosystems. In the presence of above facts, present study may conclude that antibiotic resistance not only govern by various inherited and acquired mechanisms but also by indiscriminate use of antibiotic thus the present study suggest prudent and wise use of antibiotics and further molecular studies to find exact mechanism of antibiotic resistance along with genetic characterization of Enterobacter strains to curb mortality and morbidity due to resistant infections.

 

Acknowledgments

 

We acknowledge the support and facilities provide by Head of department of veterinary microbiology and biotechnology, Dean of college of veterinary and animal science, Bikaner and Dean of Post Graduate Institute of Veterinary Education and Research, Jaipur for this study.

 

References

 

Al-juboori AA, Kamat NK, Sindhu JI (2013).Prevalence of some mastitis causes in dromedary camels in Abu Dhabi, United Arab Emirates. Iraqi J. Vet. Sci. 27(1): 9-13.

Bauer AW, Kirby WM, Sherris JC, Turck M (1966). Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45(4): 493-496.

Bordeanu AD, Krupaci FA, Kiss T, Spinu M (2012). Study of seasonal dynamics in respiratory microbial flora in extensively raised goats. Vet. Med. (8)4: 38-45.

Cowan ST, Steel KJ (1975). In: Cowan and Steel’s Mannual for the identification of medical bacteria. Cambridge University Press, Cambridge.

Edward RR, Ewing WH (1972). Identification of Enterobacteriaceae. 3rd Ed. Mennepolis, Gurgess Publ. Co.

Gohary AHEL, Osman AO, Safwat ELS (2012). Some bacteriological studies on Enterobacter cloacae And Other Enterobacteria In genital tract of small ruminant. J. Egypt. Vet. Med. Assoc. 72(4): 651-663.

Greenup P, Blazevic DJ (1971). Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens. Appl. Microbiol. 22(3): 309-314.

Harbottle H, Thakur S, Zhao S, White DG (2006). Genetics of antimicrobial resistance. Anim. Biotechnol. 17(2): 111-124. http://dx.doi.org/10.1080/10495390600957092

Holmes RK, Jobling MG (1996). Genetics. In: Medical Microbiology, 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston.

Iversen C, Waddington M, Farmer JJIII, Forsythe S (2006). The biochemical differentiation of Enterobacter sakazakii genotypes. BMC Microbiol. 6: 94. http://dx.doi.org/10.1186/1471-2180-6-94

Magnet MDMH, Arongozeb MD, Khan GM, Ahmed Z (2013). Isolation and identification of different bacteria from different types of burn wound infections and study their antimicrobial sensitivity pattern. Int J. Res. Appl., Nat. Soc. Sci.1(3): 125-132.

McEwen SA, Fedorka-Cray PJ (2002). Antimicrobial use and resistance in animals. CID. 34(3): S93–S106. http://dx.doi.org/10.1086/340246

Njage PMK, Dolci S, Jans C, Wangoh J, Lacroix C, Meile L (2012). Ampicillin resistance and extended spectrum β-lactamases in Enterobacteriaceae isolated from raw and spontaneously fermented camel milk. African J. Microbiol. Res. 6(7): 1446-1452.

Nyenje ME, Tanih NF, Green E, Ndip RN (2012). Current Status of Antibiogram of Listeria ivanovii and Enterobacter cloacae isolated from ready-to-eat foods in Alice, South Africa. Int. J. Environ. Res. Pub. Health. 9(9): 3101-3114. http://dx.doi.org/10.3390/ijerph9093101

Ojo OE, Ogunyinka OOG, Agbaje M, Okuboye JO, Kehinde OO, Oyekunle MA (2012). Antibiogram of Enterobacteriaceae isolated from free-range chickens isolated from free-range chickens in Abeokuta, Nigeria. Vet. Arhiv. 82(6): 577-589.

Osterblad M, Pensala O, Peterzens M, Heleniusc H, Huovinen P (1999). Antimicrobial susceptibility of enterobacteriaceae isolated from vegetables. J. Antimicrob. Chemo. 43(4): 503-509. http://dx.doi.org/10.1093/jac/43.4.503

Quinn PJ, Carter ME, Markey BK, Carter GR (1994). Clinical Veterinary Microbiology. Wolfe Publishing, Mosby-Year Book Europe Ltd. Lynton House. Pp. 7-12.

Reisbig MD, Hanson ND (2004). Promoter sequences necessary for high-level expression of the plasmid associated ampC b-lactamase gene bla MIR-1. Antimicrob. Agents Chemother. 48(11): 4177-4182. http://dx.doi.org/10.1128/AAC.48.11.4177-4182.2004

Thiolas A, Bollet C, Scola BL, Raoult D, Pages JM (2005). Successive Emergence of Enterobacter aerogenes Strains Resistant to Imipenem and Colistin in a Patient. Antimicrob. Agents Chemother. 49(4): 1354-1358. http://dx.doi.org/10.1128/AAC.49.4.1354-1358.2005

Wilberger MS, Anthony KE, Rose S, McClain M, Bermudez LE (2012). Beta-Lactam Antibiotic Resistance among Enterobacter spp. Isolated from Infection in Animals. Adv. Microbiol. 2: 129-137. http://dx.doi.org/10.4236/aim.2012.22018

Zabransky RJ, Hall JW, Day FE, Needham GM (1969). Klebsiella, Enterobacter, and Serratia: Biochemical Differentiation and Susceptibility to Ampicillin and Three Cephalosporin Derivatives. App. Microbiol. 18(2): 198-203.