2006 Poultry Science Association, Inc.
Isolation, Characterization, and Antimicrobial Drug Resistance Pattern of Escherichia coli Isolated from Japanese Quail and their Environment P. Roy,* V. Purushothaman,* A. Koteeswaran,* and A. S. Dhillon†1 *Central University Laboratory, Centre for Animal Health Studies, Tamilnadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai-600051, India; and †Avian Health and Food Safety Laboratory, Washington State University, 7613 Pioneer Way E, Puyallup 98371
Primary
Audience: Veterinarians, Poultry Microbiologists
Veterinarians,
Poultry
Scientists,
SUMMARY Escherichia coli isolates were cultured from diseased Japanese quail and their environment. Of 31 E. coli isolates, 11 were cultured from heart blood of dead Japanese quail and 20 were from dead-in-shell embryos, fluff samples, and footbath and drinking water samples. All E. coli isolates were moderately positive on the Congo red binder test and 14 out of 31 isolates produced hemolysis on sheep blood agar. Twenty-seven isolates were grouped under serogroups O4, O9, O38, O42, and O88, whereas 4 isolates could not be typed. Of the E. coli isolates cultured from Japanese quail infected with colibacillosis, 54.5% belonged to serogroup O9 and the same serotype was predominant in the hatchery environment. All the E. coli isolates showed high resistance to multiple drugs with 100% resistance observed against ampicillin/cloxacillin, chloramphenicol, tetracycline, and cotrimoxazole. The highest sensitivity was observed against nitrofurantoin. This study shows that hatchery hygiene should be improved to control colibacillosis and reduce production losses. At the same time, indiscriminate use of antibiotics should be avoided as it increases the risk of development of drug-resistant strains of bacteria. Key words: Escherichia coli, drug resistance, Japanese Quail 2006 J. Appl. Poult. Res. 15:442–446
DESCRIPTION OF PROBLEM Escherichia coli is a part of the common microbial flora of the intestine of poultry and most isolates are nonpathogenic. About 10 to 15% of intestinal coliforms are pathogenic serotypes [1]. Pathogenic E. coli are also present in the poultry environment. Escherichia coli causes a variety of lesions in poultry, including yolk 1
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sac infection, omphalitis, cellulitis, swollen head syndrome, coligranuloma, and colibacillosis [2]. Colibacillosis is an economically important disease, which is prevalent throughout the world [3]. Several reports are available about the involvement and serotypes of E. coli and the presence of disease in poultry [4, 5, 6]. Involvement of Newcastle disease also causes early chick mortality and the disease is widely preva-
ROY ET AL.: ESCHERICHIA COLI IN JAPANESE QUAIL lent in the Tamil Nadu state of India. A differential diagnosis is essential. Colibacillosis can be controlled with antibiotic therapy but a significant increase in drug-resistant strains of E. coli has complicated the problem in the poultry industry [7, 8]. Japanese quail are reported to be resistant to many diseases. The present paper describes the characterization and drug resistance pattern of isolates of E. coli obtained from Japanese quail and their environment.
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Dead Japanese quail were necropsied and swabs were collected aseptically for the isolation of bacteria. Eleven heart blood swab samples were collected aseptically from the dead Japanese quail. Proventriculus, lung, spleen, liver, and brain tissues were collected aseptically from the dead quail for virus isolation. Fifteen swab samples were collected from DSE, and 2 fluff samples and 1 footbath sample were collected from the hatchery. Two drinking water samples were collected from the farm for bacterial isolation. The unhatched eggs or DSE were swabbed with alcohol and aseptically opened to collect samples using sterile swabs.
key agar were tentatively identified as E. coli. Further confirmation was done by indole, methyl red, Voges-Proskauer, and citrate use tests [9]. The E. coli isolates were selected based on colony morphology and biochemical tests. Antimicrobial Drug Sensitivity Test. In vitro susceptibility of the isolates against antimicrobial agents was determined by the standard disk diffusion procedure [10]. The following antibiotic discs [11] were used: gentamicin (G; 30 g), ciprofloxacin (CF; 30 g), cotrimoxazole (Co; 25 g), chloramphenicol (C; 30 g), tetracycline (T; 30 g), ampicillin/cloxacillin (AX; 10 g), and nitrofurantoin (NF; 300 g). The E. coli isolates were inoculated in tryptic soy broth and incubated at 37°C for 5 h. The broth was diluted in normal saline solution to a density of 0.5 McFarland turbidity standard. Cotton swabs were used for streaking the diluted broth onto Mueller-Hinton agar plates. After air drying, antibiotic discs were placed 30 mm apart and 10 mm away from the edge of the plate. Plates were incubated at 37°C for 18 to 20 h. The zone of inhibition and resistance was measured, recorded, and interpreted according to the recommendation of the disc manufacturer. Serogrouping. Serogrouping of the E. coli isolates was done at the National Salmonella and Escherichia Center [12]. Congo Red Binding. To evaluate the Congo red binding, bacteria were grown at 37°C for 24 h on tryptic soy agar supplemented with 0.02% Congo red [13] and 0.15% bile salt [14]. Positive colonies appeared red, whereas negative colonies were pale. Based on the intensity of red color, the binding was scored as +, ++, and +++. Hemolysis. Overnight bacterial cultures were streaked on sheep blood agar and incubated at 37°C for 24 h. The appearance of a zone of erythrocyte lysis around or under bacterial colonies indicated hemolysis.
Bacteriology
Virology
Fluff samples were rinsed in a minimum volume of PBS and incubated at 37°C for 30 min. A loop full of rinse PBS was inoculated onto MacConkey agar and sheep blood agar. All the swab samples and water samples were also cultured on MacConkey agar and sheep blood agar and incubated overnight at 37°C. Bacterial colonies that were lactose positive on MacCon-
Twenty percent (wt/vol) tissue homogenates were prepared in PBS and clear supernatants were tested for hemagglutination with 1% chicken erythrocytes as described previously [15]. Supernatants were filtered through a 0.22m filter and filtrates were inoculated into embryonated chicken eggs for Newcastle disease virus (NDV) isolation as described [15]. Three
MATERIALS AND METHODS History In a commercial hatchery of Japanese quail, hatchability, dead-in-shell embryos (DSE), and 1-d-old Japanese quail chick mortality were 70, 2, and 6%, respectively. Japanese quail were also raised on a farm adjacent to the commercial hatchery. Sporadic mortality in 1- to 5-wk-old Japanese quail flocks of 30,000 birds for a period of 10 d ranged from 0.01 to 0.5%. An investigation was undertaken to find out the cause of increased mortality at the affected farm and the decrease in hatchability. Necropsy
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Table 1. Virulence characteristics and serogroups of Escherichia coli isolates from Japanese quail and their environment
Description
E. coli isolates (n)
Fluff Footbath Drinking water Dead-in-shell embryos
2 1 2 15
Dead Japanese quail
11
No. of E. coli
Congo red positive (n)
Serotyped
Untyped
O9 (2) O9 (1) O9 (1) O9 (8); O42 (2); O38 (2); O4 (2); O88 (1) O88 (1), O9 (6), O42 (1)
— — 1 —
++ ++ ++ ++
3
++ (11)
blind passages were made before a sample was declared negative for NDV.
RESULTS AND DISCUSSION Most of the birds had fibrinous perihepatitis. Livers were discolored and pale; kidneys and duodenum were congested; lungs were congested, frothy, and on occasion, consolidated. About 5% of the hearts had epicardial congestion. Pure colonies of bacteria were isolated in MacConkey agar plates from all the samples. All isolates were identified as E. coli based on morphological and biochemical characteristics. From 31 E. coli isolated, 27 isolates were serogrouped and 4 isolates could not be serotyped with available antisera. The 27 serotyped isolates belonged to O4, O9, O38, O42, and O88 (Table 1). All of the 31 E. coli isolates had Congo red binding scores of ++. Fourteen E. coli isolates caused hemolysis in sheep blood agar plates (Table 1). All of the isolated E. coli showed resistance to 2 or more antibiotics and a pattern of multiple drug resistance was observed. The highest rates of resistance were against AX, C, T, and Co (100% each), followed by CF (67.7%), G (61.3%), and NF (51.3%). Thirty-one E. coli isolates elicited 7 different patterns of antibiotic resistance to the agents used in this study (Table 2). The most common resistance pattern was AX/G/CF/C/NF/T/Co (38.7%) and the least common resistance pattern was AX/C/NF/T/Co (3.2%; Table 2). Samples were found negative for hemagglutination activity and NDV could not be isolated in embryonated chicken eggs using 3 egg passages, indicating that NDV was not a factor in the birds studied.
(2) (1) (2) (15)
Hemolysis positive (n; serotype) 2 1 1 4
(O9) (O9) (O9) (O9); 1 (O38); 1 (O88)
3 (O9); 1 (O42)
Japanese quail farming has recently gained popularity where birds are raised for commercial meat production. The Japanese quail are said to be resistant to many diseases; however, cellulitis caused by E. coli has been reported recently [16]. In the present study, necropsy lesions of dead birds were from colibacillosis confirmed by isolation of pure cultures of E. coli. The serotype O9 was predominant and present at a level of 54.5% (Table 1). The samples collected from hatchery also contained serotype O9 of E. coli in the fluff and footbath water samples (Table 1). Out of 15 E. coli isolates from DSE, 53.3% of E. coli belonged to serotype O9 (Table 1). All E. coli isolates in the present study were positive for Congo red binder and the majority of E. coli O9 serotypes were hemolytic (Table 1). Congo red binding has been used as a potential virulence marker [17], which indicated that the isolates were pathogenic. However, a clear distinction between pathogenic and nonpathogenic E. coli could not be established based on hemolytic activity [18]. However, involvement of O9 and O88 serotypes of E. coli has been reported to cause cellulitis and other colibacillosis lesions in the broiler chickens [19]. In the present study, the O9 serotype of E. coli was isolated from the fluff, footbath, DSE, and dead quail samples. The O88 serotypes of E. coli were isolated from DSE, not from colibacillosis cases. Besides the known pathogenic E. coli serotypes O9 and O88, other serotypes identified in this study included O4, O38, and O42. Pathogenicity in this case was indicated by a positive Congo red marker test. The E. coli were found to be responsible for high early chick mortality and reduced hatchability. This study indicates that hatchery and
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Table 2. Resistance pattern of Escherichia coli isolates1 Antimicrobial agent2 Resistance pattern ID
AX
G
CF
C
NF
T
Co
1 2 3 4 5 6 7 Resistant E. coli isolates (n)
R R R R R R R 31
R S R S S R S 19
R R R S R S S 21
R R R R R R R 31
R R S R S S S 16
R R R R R R R 31
R R R R R R R 31
E. coli Isolates (n) 12 3 3 1 3 4 5
R = resistant; S = sensitive. AX = ampicillin/cloxacillin; G = gentamicin; CF = ciprofloxacin; C = chloramphenicol; NF = nitrofurantoin; T = tetracycline; and Co = cotrimazole.
1 2
farm hygiene improvement is needed to control E. coli infection and drug resistance. Newcastle disease has been previously reported in Japanese quail [20]. Newcastle disease virus is endemic in Tamil Nadu state (India); hence, an attempt was made to rule out its possible involvement. Samples were found negative for NDV by the hemagglutination test and by virus isolation in embryonated hen’s eggs. Present E. coli isolates showed high resistance to multiple drugs. All E. coli isolates were found resistant to AX, C, T, and Co. In earlier studies, 94% resistance to T and 100% resistance to tetracycline-ampicillin were reported in E. coli isolates of chicken origin [7, 21]. Antibiotics are used as feed additives to improve feed efficiency and weight gain [22]. Many antibiotics are also used in feed and water to control disease. Indiscriminate use of antibiotics has provided selective pressure for the emergence of drugresistant strains of bacteria associated with poultry products [8, 22, 23]. The United States Food and Drug Administration emphasizes the spread of drug resistance in the enterobacteriaceae family from antibiotic-fed animals to human beings
[21]. Transmission of the R-plasmid from E. coli of poultry to human occurs very commonly [24]. Earlier studies revealed that use of fluoroquinolones in poultry was not appropriate due to the cross-resistance with fluoroquinolones used to treat human enteric infections. High resistance to chlortetracycline and oxytetracycline is of major concern because of the use of the same antibiotics in human medicine and poultry or in other food animals and the emergence of drug-resistant human pathogens [25, 26]. All of the 31 E. coli in the present study were resistant to AX, C, T, and Co and these drugs are also used in human medicine. Colibacillosis is mainly a fecal- and waterborne-transmitted ailment. Effective sanitizers such as chlorine need to be used to make the water free of E. coli. Hatchery environments should also be made free of bacteria with appropriate fumigation techniques, good sanitation, and strict biosecurity measures. Hatcheries and farms should not be located in same place because there is high possibility of microbial transmission from farm to hatchery or vice versa.
CONCLUSIONS AND APPLICATIONS 1. Escherichia coli were isolated from diseased Japanese quail, dead-in-shell embryos, fluff samples, and footbath and drinking water samples from a hatchery and the isolates were serogrouped as O4, O9, O38, O42, O88, and 4 untypeable isolates. 2. Escherichia coli isolates cultured from Japanese quail infected with colibacillosis were predominantly in serogroup O9 (54.5%) and the same serotype was also predominant in the hatchery environment. 3. The isolates were resistant to many antibiotics and caused mortality in Japanese quail. 4. Hatchery hygiene should be improved to control colibacillosis and reduce production loss.
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5. Indiscriminate use of antibiotics should be avoided because it may lead to the development of drug-resistant strains of bacteria.
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