Drug susceptibility test of Escherichia coli isolates from healthy yak of Qinghai

T. Yun,¹ L. Chengping¹ and X. Luzhong²

1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P.R. China
2. Faculty of Agricultural and Animal Science of Qinghai University, Xining 810003, Qinghai, P.R. China

Summary

There is very limited information on the susceptibility of Escherichia (E) coli to antibiotics in healthy yak. Disk diffusion susceptibility test, an international standard test, was applied in the drug susceptibility test of 68 strains of E. coli isolates from healthy yak in Qinghai to 12 kinds of antibiotics. The results showed that, among the 12 antibiotics, the inhibitory effects of Norfloxacin and Amikacin were the greatest and all the E. coli strains were susceptible. The effects of Ciprofloxacin, Chloramphenicol, Ampicillin, Gentamicin, Trimethoprim/ Sulfamethoxazole, Kanamycin, Tetracycline and Streptomycin were medium and 52.9–98.5% strains were susceptible to these antibiotics. None of the strains was susceptible to Penicillin and Rifampin. This study suggests that acquired drug resistance is not an actual problem in E. coli strains in herds of healthy yak in Qinghai, where antibiotics are rarely used.

Keywords: Drug susceptibility test, E. coli, yak

Introduction

More than 90% of the world's yak population is found in China; Qinghai-Tibetan Plateau is the main region of yak rearing (Editing Committee 1989). Yak production is still principally low-input and extensive. Antibiotics are rarely administrated. There is limited information on the susceptibility to antibiotics of E. coli in healthy yak. This paper reports results of susceptibility test of 68 strains of E. coli isolates in healthy yak from Qinghai to 12 kinds of antibiotics.

Materials and methods

Strains and detection of virulence factors

Sixty-eight strains of E. coli were isolated from slaughtered healthy yak in Xining City of Qinghai Province in 1998. E. coli of ATCC 25922 (Institute of Microbiology of Academia Sinica) was used as control to quantify the susceptibility. Haemolytic test (Gottschalk et al. 1995) and skim milk plate test (Quinn et al. 1994) were applied to identify virulence factors of haemolysins and extracellular proteases of the E. coli strains. E. coli SP12 and XZ19 from chickens were used as positive controls.

Antimicrobial disks

Twelve kinds of antimicrobial disks containing Norfloxacin (NOR), Amikacin (AKN), Ciprofloxacin (CIP), Chloramphenicol (CMP), Ampicillin (AMP), Gentamicin (GEN), Trimethoprim/Sulfamethoxazole (SXT), Kanamycin (KAN), Tetracycline (TET), Streptomycin (STR), Penicillin (P-G) and Rifampin (RIF), were purchased from the Shanghai Yihua Medicine Technology Limited Corporation.

Experimental methods

MaConkey culture medium (No. 981217, Jiangsu Health and Epidemic Prevention Station) was used to culture the bacteria. M.H. culture medium (No. 000224, Shanghai Reagents Research Center of Diarrhea Diseases Control of China) was applied according to the K-B method (Carter 1986; Cao 1992). After inoculation for 18 hours, the size of the zones of inhibition was measured to identify the isolates' susceptibility to antibiotics. The zones of inhibition were recorded as susceptible (S), intermediate (I) and resistant (R) in relation to the standards of National Committee for Clinical Laboratory Standards (NCCLS) in USA. The inhibition standard of P-G was from that of Cao (1992).

Results

Detection of virulence factors

Haemolytic test and skim milk plate test both had negative results in 68 strains of E. coli, while the control strains SP12 and XZ19 were positive.

Susceptibility test of strains for quality control

The quality control of the susceptibility test of ATCC 25922 to the 12 kinds of antimicrobial disks conformed to the inhibition zones of NCCLS for all items and P-G's susceptibility test was also identical to that of Cao (1992).

Susceptibility test of isolates

The inhibition zones of 68 strains of E. coli demonstrated that only one strain was resistant to the antibiotics CMP, AMP, GEN and KAN, two were resistant to SXT, five to STR and eight to TET. No strains were resistant to NOR, AKN and CIP (Annex I).

Among the 68 strains studied, 23 (33.8%) were susceptible to 10 antibiotics, 20 (29.4%) were susceptible to 9 antibiotics, 17 (25.0%) were susceptible to 8 antibiotics, and 7 (10.3%) were susceptible to 7 antibiotics. No strains were sensitive to five or six antibiotics. Only one strain (1.5%) was susceptible to four antibiotics. None was susceptible to one to three antibiotics (Figure 1).

Figure 1. The susceptibility spectrum of 68 strains of E. coli to 10 kinds of antibiotics.

Among the 12 antibiotics, resistance was highest to P-G (77.9% of the E. coli strains) and RIF (97.1%). No strains were resistant to NOR, AKN and CIP (Table 1).

Table 1. Resistance percent (R%) and the intermediate percent (I%) of 68 strains of E. coli to 12 kinds of antibiotics.

	Antibiotics
	NOR	AKN	CIP	CMP	AMP	GEN	SXT	KAN	TET	STR	P-G	RIF
R%	0	0	0	1.5	1.5	1.5	2.9	1.5	11.8	7.4	77.9	97.1
I%	0	0	2.9	0	0	1.5	0	26.5	19.1	39.7	22.1	2.9

All the strains were susceptible to NOR and AKN. 52.9–98.5% of the strains had medium susceptibility to CIP, CMP, AMP, GEN, SXT, KAN, TET and STR. The susceptibility of P-G and RIF was the lowest: No strains were susceptible ( Table 2).

Table 2. The susceptibility percent (S%) of 68 strains of E. Coli to 12 kinds of antibiotics.

	Antibiotics
	NOR	AKN	CIP	CMP	AMP	GEN	SXT	KAN	TET	STR	P-G	RIF
S%	100	100	97.1	98.5	98.5	97.0	97.1	72.0	69.1	52.9	0	0

Discussion

Yak inhabits the high mountain pasture on the Qinghai-Tibetan Plateau, where, to date, there is no use of antibiotics. In this study, the natural susceptibility to antibiotics of E. coli strains from healthy yak was evaluated.

The standards for drug susceptibility test issued by NCCLS and approved by World Health Organization (WHO) were adopted in the test, and the control was E. coli ATCC 25922. The drug susceptibility of 68 strains of E. coli isolates from Qinghai was tested. Therefore, the results of the experiment should be reliable.

E. coli develops drug resistance easily, either natural or acquired resistance. Our test demonstrated that among the 68 E. coli strains studied none was resistant to NOR, AKN and CIP, and only 1.5–11.8% of strains had the medium resistance to CMP and six other antibiotics. Almost 100% strains were resistant to P-G and RIF. The invalidity of P-G to Gram-negative bacillus is well known and RIF is mainly applied to tuberculosis. These results suggested that acquired drug resistance was not a problem in E. coli strains in Qinghai yak.

Drug diffusion susceptibility test was only an initial susceptibility test. The determination of minimum inhibitory concentration (MIC), the analyses of plasmids spectrum and the detection of resistant plasmids are subjects of future studies.

References

Carter G.R. 1986. Essentials of veterinary bacteriology and mycology (third edition). Lea & Febiger, New York, USA. 91 pp.

Editing Committee. 1989. China yakology. Sichuan Science and Technology Press, Chengdu, Sichuan, P.R. China. 5 pp. [in Chinese].

Gottschalk M.G., Lacouture S. and Dubreuil J.D. 1995. Characterization of Streptococcus suis capsular type 2 haemolysin. Microbiology 141:189–195.

Quinn P.J., Carter M.G., Markey B.K. and Carter G.R. 1994. Clinical veterinary microbiology. Wolf publishing, London, UK. 662 pp.

Shuze C. 1992. Techniques of veterinary microbiology and immunology. Beijing Agricultural University Press, Beijing, P.R. China. pp. 49–54. [in Chinese].

Annex I. Results of inhibitory diameter for 68 strains of E. Coli isolates (mm).