Abstract: Since last decade, there is increasing reports of presence of extended spectrum β-lactamases producing bacteria especially from enterobacteriacae family. The presence of Extended spectrum β-lactamases (ESBL) producing E. coli causes a serious public health threat to human beings. The present study reports the isolation of extended spectrum β-lactamases producing α hemolytic Escherichia coli in Yellow-wattled Lapwing (Vanellus malabaricus) chicks. Escherichia coli organisms were isolated from three chicks suffering from unusual clinical signs and died before rehabilitation in the month of August 2010. Further assessment of isolates revealed their ability to bind with Congo red dye on Tryptic soy agar media and α hemolytic nature on 5% chicken blood agar. As usual when drug sensitivity was performed it revealed their multi drug resistance pattern which on further examination with double disk method showed them to be extended spectrum β-lactamases producing Escherichia coli. The presence of enterohemorrhagic extended spectrum β-lactamases producing Escherichia coli in Yellow-wattled Lapwing is a matter of concern as it may be due to the transmission from human being as these Lapwings are residing in the close vicinity of university premises. Moreover, their nesting areas are also very near to the place where carcasses of dead animals were disposed off during that period of time. To the best or our knowledge, this appears to be the first report of pathogenic E. coli identified in Yellow-wattled Lapwing, implicating Yellow-wattled Lapwings as a new potential reservoir of these zoonotic pathogens.
INTRODUCTION
Since last few years the rising antimicrobial resistant strains of bacteria along with increase in emerging and re-emerging pathogens of man as well as animals have gained attention of the researchers all over the world (Verma et al., 2007, 2012; Garmyn et al., 2011; Kumar et al., 2011, 2012). Among them, extended-spectrum beta-lactamases producing E. coli (ESBL-E. coli) poses a major threat to man and animal, especially in the form of nosocomial infections (Guenther et al., 2011) and may cause bacteremia, wound infections, urinary tract infection, neonatal meningitis and gastrointestinal infections (Raina et al., 1999; Kumar et al., 2013). There are many reports that birds may act as reservoir of multi drug resistant (MDR) bacteria (Guenther et al., 2010) and play a crucial role in spread of antibiotic resistance (Garmyn et al., 2011). Mathur et al. (2002) reported 68% ESBL positivity rate in bacteria of enterobacteriacae family from India. Thus, global increasing trend in ESBL mediated resistance among bacteria particularly in E. coli has pulled the focus of researchers towards this public health issue. However, to the best of author’s knowledge, there is no report for these in Yellow-wattled Lapwing (Vanellus malabaricus). In this context, the present study describes the possible cause of death in three chicks of yellow-wattled bird (Vanellus malabaricus); presence of ESBL producing E. coli in Yellow-wattled Lapwing and chances of dissemination of antibiotic resistant bacteria in natural environment.
MATERIALS AND METHODS
Three chicks were found on roadside to Veterinary University (DUVASU) campus, Mathura appeared to suffering with certain illness showing clinical signs such as disability to walk, difficult respiration and inability to eat and drink on the grounds near to premises, where these birds stay throughout year and probably breed also. These chicks died within few hours before any rehabilitation was started, except the attempts of feeding and providing drinking water. Then post-mortem examinations were conducted to know the possible cause of death.
Sample collection: Died chicks were cleaned with 70% alcohol and then opened aseptically to collect the tissues in sterile container in phosphate buffered saline (pH 7.4) and heart blood. Tissues were collected from heart, liver, lung and kidney. The collected tissues were immediately shifted to laboratory for further isolation of microbial agents.
Microbiological culture and identification: Microbiological isolation was attempted from tissues (lungs, heart, liver and kidney) and heart blood samples of dead chicks. Samples were inoculated onto nutrient agar, MacConkey Lactose agar (MLA), Sabouraud’s Dextrose agar (SDA) and 5% chicken blood agar plates and incubated aerobically/microaerobically at 37°C for 24 h (Quinn et al., 2002). After 24 h incubation, the plates were observed for presence of bacterial and fungal colony. Lung tissues for mycoplasma isolation were homogenized in 1 mL PBS and then 100 μL of this solution was transferred into Pleuropneumonia like Organism broth (PPLO broth) enriched with horse serum and incubated at 37°C, 5% CO2 for 48 h. (Rosengarten et al., 1994) afterwards. Samples from PBS (pH 7.4) were triturated, filter sterilized (0.22 micron syringe filter) and inoculated on Maiden Durby Bovine Kidney (MDBK) cell lines and incubated at 36°C, 5% CO2 for 48h in CO2 incubator for virus isolation.
Lung samples for the isolation of mycoplasma were cultured and observed according to the method described by Ter Laak et al. (1992). Small pieces from altered lungs were homogenized in PBS and 10% cell suspension was cultured. The inoculated tubes were incubated at 37°C and plated on days 3 and 7. The plates were incubated at 37°C in 5% CO2 atmosphere and were examined under a stereomicroscope every 2 days. Blood samples collected aseptically were also inoculated in the similar pattern with the help of sterilized bacteriological loop.
Isolation and identification of E. coli: All the pink colonies suspected to be of E. coli were further re-streaked on Macconkey’s Lactose Agar (MLA) and after incubation at 37°C for 24-48 h pink colonies from each MLA plate were picked up and streaked on Eosin Methylene Blue (EMB) agar for observing the metallic sheen. Bacterial isolates suggestive of E. coli on MLA and EMB were studied on the basis of their cultural, morphological and motility characteristics. Then biochemical studies of E. coli isolates were carried out as per the method of Cruickshank et al. (1975).
Assessment of pathogenicity: All the isolates were assessed for their pathogenicity by Congo red binding and production of hemolysins. For this the colonies confirmed biochemical, cultural and morphological basis were grown at 37°C for 24 h on Tryptic soy agar supplemented with 0.02% Congo red and 0.15% bile salt and 5% sheep blood agar, respectively. Positive colonies for Congo red binding appeared red in color whereas haemolytic isolates produced a clear zone of haemolysis in surrounding to colonies.
Antibiogram: In vitro antibiotic susceptibility testing of all the isolates of E. coli recovered during study was conducted against antibiotic discs Streptomycin (10 μg), Chloramphenicol (30 μg), Amikacin (30 μg), Ampicillin (10 μg), Cephalexin (30 μg), Tetracycline (30 μg), Gentamicin (10 μg), ciprofloxacin (5 μg) and Norfloxacin (10 μg) using disk diffusion method (Bauer et al., 1966). The zone size around each antibiotic disc was elucidated as sensitive, intermediate or resistant according to NCCLS (2002).
ESBL determination by double disk method: The disk approximation method was used with the antimicrobial disks of cefpodoxime and clavulanic acid. A Mueller-Hinton agar plate was inoculated with a suspension made from an overnight blood agar culture of the isolates as recommended for a standard disk diffusion susceptibility test. Disks containing the standard cefpodoxime (10 μg) are placed 15 mm apart (edge to edge) and from an amoxicillin-clavulanic acid disk containing 10 μg of the latter compound as per the recommendation of (Coudron et al., 1997) to have greater sensitivity. Following incubation for 16-20 h at 35°C, any enhancement of the zone of inhibition between a beta-lactam disk and that containing the beta-lactamase inhibitor is indicative of the presence of an ESBL was observed with all the precautions (Moland and Thompson, 1994).
RESULTS AND DISCUSSION
The Yellow-wattled Lapwing, Vanellus malabaricus, is a commonly observed bird in Indian subcontinent though some reports also indicates their rare appearances in India. These belong to the family Charadriidae and found in dry plains of sub continent even in the close vicinity of human populations. They are ground birds and their nest is a mere collection of tiny pebbles within which their well camouflaged eggs are laid. These lapwings breed in the dry season with peak breeding in March to May ahead of the monsoons (Jayakar and Spurway, 1965). The incubation period was 27-30 days. The chicks are nidifugous, leaving the nest shortly after hatching and following their parents to forage for food. These Yellow-wattled Lapwings are a common feature on the grounds near the university premises, here in Mathura, India.
In the present investigation, the absence of growth in SDA, PPLO broth and further in PPLO agar, ruled out the possibilities of fungal pathogens and mycoplasma infections, while the growth on nutrient agar and MLA plates revealed bacterial growth. There were no changes in MDBK cell lines as well. The further confirmation on the basis of cultural, morphological and biochemical characters confirmed it as E. coli. On blood agar these isolate revealed a hazy wider zone of haemolysis and also produced red color colonies on tryptic soya agar enriched with Congo red dye. Antibiogram of these isolates revealed resistance against streptomycin, tetracycline, ciprofloxacin, norfloxacin, ampicillin and cephalexin. Intermediate sensitivity was observed with chloramphenicol whereas isolates were sensitive to amikacin and gentamicin. Moreover, double disk diffusion revealed the presence of ESBL producing character of these isolates.
Thus, ESBL producing E. coli might be the cause of death. The probable source of infection might be the feeding habit of bird like beetles, termites and other invertebrates picked from the ground. Moreover, the place where they have nested was also near to the place where the carcasses of dead animals were deposited at that time and from where the parents of these chicks might have received these pathogens which appeared to be the cause of their death.
Yellow-wattled Lapwings parents visit water and wet their breast feathers {"belly soaking"; they may stay for as much as 10 min to soak water (Maclean, 1974)} which is then be used to cool the eggs or chicks (Jayakar and Spurway, 1964), might be the source of infection due to presence of these pathogens in contaminated water. As Escherichia coli are an important pathogens group in community and hospital-acquired infections and it is unfortunate to see the increasing resistance against common among gram-negative bacteria and ultimately making empirical therapy decisions more difficult and confusing. Resistance patterns among gram-negative organisms not only include resistance to extended spectrum of cephalosporin and penicillin but also against the third generation of antibiotics (Motta et al., 2003). Murugan et al. (2011) have also reported difficulties in the treatment of food and water associated gastrointestinal diseases due to E. coli and this problem is compounded by the continued emergence of antibiotic resistance to a growing number of antibiotics i.e. carbenicillin, tetracycline, streptomycin (Walia et al., 2004), norfloxacin, amoxycillin, trimethoprim, nitrofurantoin (Goettsch et al., 2000), nalidixic acid, gentamicin, cefuroxime (Shehabi et al., 2004), ampicillin, ceftriaxone, ciprofloxacin, ceftazidime and cefotaxime (Patoli et al., 2010) etc. Increased resistance to antibiotics is a global problem and makes the treatment extremely difficult or virtually impossible in some instances (El-Astal, 2004). Similar to methicillin resistant staphylococcus extended spectrum beta Lactamases producing strains of Enterobacteriaceae have now a day among major emerging problems particularly in hospitalized as well as community based patients. Moreover, the infections due to ESBLs-producers mainly affecting a range of patients from uncomplicated urinary tract infections (UTI) to life threatening sepsis producing conditions (Bhattacharya, 2006). The status of ESBL producing E. coli is also alarming in India with the 68% ESBL positivity rate in Enterobacteriacae isolates (Mathur et al., 2002).
CONCLUSION
The level of increasing ESBL mediated resistance amongst E. coli isolates worldwide and particularly in developing countries is among major public health threats. Now the presence of these ESBL producing multi drug resistant α hemolytic E. coli in Yellow-wattled Lapwing has posed another challenge to public health.
ACKNOWLEDGMENTS
The authors are highly thankful to Manoj Kumar Gupta, Incharge, Department of Veterinary Microbiology and Immunology, Dean, College of Veterinary Sciences and Hon’ble Vice Chancellor, Uttar Pradesh Pandit Deen Dayal Upadhaya Pashu Chikitsa Vigyan Vishwavidhyalaya Evam GoAnusandhan Sansthan, Mathura, India for providing all the necessary facilities and supports to conduct this study.