Antimicrobial Susceptibilities of Campylobacter jejuni Isolates from Poultry from Alberta, Canada

Abstract

One hundred four isolates of Campylobacter jejuni from poultry in Alberta, Canada, collected during 2001 were tested for resistance to 10 antimicrobial agents using agar dilution. This study provides a baseline of resistance profiles and the mechanisms of resistance observed in C. jejuni in poultry from Alberta, Canada.

Campylobacter jejuni is a major food- and waterborne pathogen associated with gastroenteritis worldwide (6). Human C. jejuni infection occurs primarily through consumption of or contact with raw or undercooked poultry (5, 6, 21). The increased incidence of antimicrobial resistance in C. jejuni over the past 2 decades (6, 7, 11) is likely due to the agricultural use of antimicrobials for prophylaxis and growth promotion (10, 24).

Baseline levels of antimicrobial resistance in C. jejuni from environmental sources would be useful in developing policies regulating the use of antimicrobials in food production and human therapies. This study reports the susceptibility profiles of 104 C. jejuni isolates obtained from peptone washes of retail broilers from Alberta, Canada (Agri-Food Laboratories, Alberta Agriculture), to 10 antimicrobial agents in 2001 and the mechanisms of resistance present in resistant isolates.

Etest and disk diffusion methods were used to screen isolates for resistance to tetracycline, erythromycin, chloramphenicol, nalidixic acid, ciprofloxacin, kanamycin, imipenem, doxycycline, gentamicin, and amoxicillin-clavulanic acid (8). Any discrepancies in results were investigated by agar dilution. At the time this study was completed, breakpoint criteria for C. jejuni were not available, nor was there a designated C. jejuni control strain (17). Representative bacterial cutoffs were used as outlined by the Clinical and Laboratory Standards Institute (19). Control strains included several C. jejuni isolates for which MICs are well established in our laboratory (25).

The gyrA gene in ciprofloxacin-resistant isolates (28) and a fragment of the tet(O) gene in tetracycline-resistant isolates (11) were amplified by PCR. Plasmid and chromosomal DNAs were used as templates for tet(O) gene PCR, as the gene has been found to integrate into the chromosome (11, 22). The entire tet(O) gene (1.92 kbp) was analyzed as described by Gibreel et al. (11) from C. jejuni strains whose tetracycline MICs fell between 128 and 512 μg/ml.

Tetracycline resistance was observed in 69% (72/104) of the isolates, with MICs of 64 to 128 μg/ml for 49 isolates and 256 to 512 μg/ml for 6 isolates. Doxycyline resistance was observed in 64 poultry isolates, with MICs of 128 μg/ml for 7 isolates and 256 μg/ml for 1 isolate.

Approximately 11% (11/104) of the isolates were resistant to nalidixic acid, with MICs of 128 to 256 μg/ml for 7 isolates. Ciprofloxacin resistance was observed in 8% of the isolates (8/11 nalidixic acid-resistant isolates), with MICs of 32 μg/ml for 4 isolates.

All isolates were susceptible to amoxicillin-clavulanic acid, chloramphenicol, gentamicin, imipemen, kanamycin, and erythromycin. Multidrug resistance was observed in nine isolates, with six isolates being resistant to tetracycline, doxycycline, and both fluoroquinolones. One isolate was resistant to the tetracyclines and nalidixic acid. Two isolates were resistant to tetracycline and both fluoroquinolones (Table 1).

TABLE 1.

MICs of multidrug-resistant isolates of C. jejuni, as determined by agar dilution

Isolate	MIC (μg/ml)
	Tetracycline	Doxycycline	Nalidixic acid	Ciprofloxacin
CJ608	32	16	128	0.125
CJ713	32	16	256	16
CJ714	32	16	256	16
CJ559	64	64	64	32
CJ601	64	0.25	128	32
CJ604	64	32	256	32
CJ717	128	16	256	4
CJ449	256	16	16	32
CJ412	256	0.016	64	32

Seven ciprofloxacin-resistant isolates contained a C-to-T transition at nucleotide position 256 of the gyrA gene (codon 86; Thr→Ile), which is known to mediate high levels of quinolone resistance in Campylobacter (28). One isolate did not possess the mutation but was resistant to 4 μg/ml of ciprofloxacin.

The tet(O) gene was carried on a plasmid in 67 of 72 tetracycline-resistant isolates and on the chromosome in 5 isolates. In comparison with the original tet(O) gene sequence (16) from an isolate with a lower MIC (64 μg/ml), the tet(O) sequences of 21 high-level tetracycline-resistant poultry isolates revealed seven base changes in three isolates with MICs of 128 μg/ml, 256 μg/ml, and 512 μg/ml, as reported previously (11) (Table 2). Another isolate (MIC, 128 μg/ml) had six of the seven base changes, with the T910C transition absent. A C704T base change, which results in a change from alanine to valine, was present in 12 of 15 tetracycline-resistant isolates (MIC, 128 μg/ml). The various MICs for the strains with these base changes suggest that the substitutions may not be solely responsible for conferring tetracycline resistance at 512 μg/ml but may have some effect on the expression of tet(O) or the function of tetracycline resistance determinants in isolates resistant to tetracycline at 128 to 256 μg/ml.

TABLE 2.

Base pair and subsequent amino acid changes detected in sequenced tet(O) genes of 21 tetracycline-resistant C. jejuni isolates

Isolate	Tetracycline MIC (μg/ml)	Presence of nucleotide and amino acid changes
		A884G (Y→C)	T910C (S→P)	A993G (I→M)	A1036C (I→L)	T1063C (S→P)	T1111G (C→G)	A1784G (Y→C)	C704T (A→V)
CJ655	512	+	+	+	+	+	+	+	−
CJ625	256	+	+	+	+	+	+	+	−
CJ758	128	+	+	+	+	+	+	+	−
CJ617	128	+	−	+	+	+	+	+	−
CJ755	128	−	−	−	−	−	−	−	+
CJ431	128	−	−	−	−	−	−	−	+
CJ432	128	−	−	−	−	−	−	−	+
CJ468	128	−	−	−	−	−	−	−	+
CJ494	128	−	−	−	−	−	−	−	+
CJ548	128	−	−	−	−	−	−	−	+
CJ570	128	−	−	−	−	−	−	−	+
CJ574	128	−	−	−	−	−	−	−	+
CJ666	128	−	−	−	−	−	−	−	+
CJ669	128	−	−	−	−	−	−	−	+
CJ717	128	−	−	−	−	−	−	−	+
CJ724	128	−	−	−	−	−	−	−	+
CJ730	128	−	−	−	−	−	−	−	−
CJ412	256	−	−	−	−	−	−	−	−
CJ449	256	−	−	−	−	−	−	−	−
CJ638	256	−	−	−	−	−	−	−	−
CJ762	256	−	−	−	−	−	−	−	−

Antimicrobial resistance in C. jejuni is increasing globally (2, 4, 10, 13, 23, 26) and is reflected in clinical isolates from Canada (7, 11). Fluoroquinolone-resistant C. jejuni appeared following the introduction of enrofloxacin and sarafloxacin in the 1990s to treat poultry for Escherichia coli infections (1, 9, 10, 14, 18, 27). Ciprofloxacin-resistant C. jejuni has been suggested to be more fit and preferentially selected in the guts of chicks without any antibiotic selective pressure (15). The appearance of ciprofloxacin-resistant isolates is of concern, as this antibiotic is widely used for the empirical treatment of gastroenteritis and is recommended for the treatment of infections caused by macrolide-resistant Campylobacter (6).

The use of tetracyclines such as doxycycline in agriculture is likely responsible for the large number of tetracycline-resistant C. jejuni isolates. One study suggested that the tet(O) gene may be rapidly and spontaneously transferred in vivo without antimicrobial selection pressure between C. jejuni strains in the digestive tracts of chickens (3). Surveillance of tetracycline resistance is important due to the potential for plasmid-mediated transfer of the tet(O) gene (25) as well as genes encoding resistance to other antimicrobials (20) for other potential pathogens.

While multidrug resistance in C. jejuni isolates from poultry (9/104) is not common, the report of multidrug-resistant conjugative plasmids within C. jejuni (20) combined with the minimal regulation of antimicrobial usage (12) may result in the appearance of more such isolates.

Although poultry is suspected to be the primary source of C.jejuni infections for humans, other environmental sources such as water, wildlife, and cattle may also be important. The high incidence of plasmid-mediated tetracycline resistance and the emergence of ciprofloxacin resistance in C. jejuni poultry isolates support the need for antimicrobial resistance surveillance in order to develop policies regulating the use of antimicrobials in food production and human therapies.