Antimicrobial resistance in enteric pathogens isolated from Minnesota pigs from 1995 to 2004

Abstract

This study investigated the occurrence and antimicrobial resistance profiles of Escherichia coli and Salmonella sp. isolated from swine samples submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) in Saint Paul, Minnesota from 1995 to 2004. During this time period, a total of 5072 E. coli and 2793 Salmonella sp. was isolated. Most of these isolates were found to be resistant to the tetracycline and beta-lactam group of antibiotics. Resistance to spectinomycin was also frequently observed. An increasing trend in ampicillin resistance and a decreasing trend in apramycin resistance were seen in both pathogens, although ampicillin resistance was relatively higher in E. coli than in Salmonella. Aminoglycoside (amikacin) and quinolone (enrofloxacin) were the only antimicrobials to which minimum or no resistance was observed. The resistance of pig pathogens to several antibiotics indicates the need to routinely monitor the use of these antimicrobials and their associated resistance in pig populations.

Introduction

Increased antimicrobial resistance among pathogenic and commensal bacteria of animal origin is a growing concern in both veterinary and human medicine (1). Antimicrobial selection pressure during animal rearing is considered to be a factor in increased antimicrobial resistance among zoonotic pathogens. In the United States, 17 classes of antimicrobials including tetracyclines, macrolides, and beta lactam have been approved for promoting growth in animals (2). It is believed that some resistant bacteria found in humans and in food products originate from food animals (3,4). Studies have shown that retail meat products may be an important source of human exposure to antibiotic-resistant bacteria and that transfer of bacteria from food animals to humans may increase the distribution of antimicrobial resistance genes in human bacteria (5,6).

Escherichia coli and Salmonella enterica cause gastrointestinal infections and septicemia in pigs and the latter have been identified as common foodborne pathogens of humans (7). It is well known that E. coli can serve as a reservoir of antibiotic-resistance genes that can be transferred to bacterial pathogens of humans and animals (6,8). Escherichia coli and S. enterica in livestock are exposed to selective pressure because of widespread use of antimicrobials in food- producing animals (9). As a result, resistance to different antibiotics in these microorganisms is on the rise (10–12).

Since pigs are an important source of human food and since E. coli and Salmonella sp. are important pathogens of both humans and animals, this retrospective study was conducted to determine the prevalence of these pathogens and their antimicrobial resistance profiles in Minnesota pigs over a period of 10 y.

Materials and methods

Source of samples

Tissues from cases of septicemia and fecal samples from gastrointestinal illness in pigs are routinely submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) in Saint Paul, Minnesota for disease diagnosis. For isolation of E. coli, homogenates from these samples were inoculated on 5% sheep blood agar (SBA), followed by incubation at 37°C for 24 h. Suspect colonies were subjected to re-isolation on MacConkey Agar. For isolation of S. enterica, samples were enriched overnight in tetrathionate broth at 42°C and then plated on SBA followed by incubation at 37°C for 24 h. Suspect colonies were subjected to re-isolation on Brilliant Green Agar. The API-system (BioMérieux, Marcy l’Etoile, France) was used for confirmation of these isolates.

Antimicrobial susceptibility

The minimum inhibitory concentration (MIC) of spectinomycin, ampicillin, trimethoprim/sulphamethoxazole (TMP/SMX), gentamicin, ceftiofur, enrofloxacin, apramycin, amikacin, neomycin, oxytetracycline, chlortetracycline, and tetracycline was determined by broth microdilution technique using Sensititre AB1PF plates (Trek Diagnostic, Cleveland, Ohio, USA). Each isolate was classified as either resistant or susceptible to the given antimicrobial agent following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (13). For the purpose of this study, bacteria with intermediate resistance were considered to be resistant.

Results

Occurrence of E. coli and S. enterica

A total of 5072 E. coli and 2793 S. enterica were isolated from septicemic and diarrheal pigs during the 10-year study period. A consistent increase was seen in E. coli isolations over time; their numbers increased from 355 in 1995 to 955 in 2004 (Table I). The number of S. enterica isolates also doubled from 200 in 1995 to 406 in 2004 (Table II).

Table I.

Antimicrobial resistance in Escherchia coli isolated from pigs

Antibiotic class	Antibiotic	Range tested (mg/mL)	Percent of bacteria resistant to indicated antibiotic (number of isolates tested)
			1995 (355)	1996 (345)	1997 (324)	1998 (348)	1999 (386)	2000 (419)	2001 (509)	2002 (687)	2003 (744)	2004 (955)
Aminoglycoside	Amikacin	8 to 64	1	1	1	1	1	0	3	0	0	NT
	Gentamicin	1 to 16	37	37	36	32	29	34	36	38	32	48
	Apramycin	4.0 to 32	38	37	39	31	32	32	35	35	27	30
	Neomycin	4 to 32	51	54	46	48	48	49	54	55	51	66
Fluoroquinolone	Enrofloxacin	0.12 to 2	1	0	0	0	0	0	0	0	1	0
Cephalosporin	Ceftiofur	0.25 to 8	4	3	8	4	8	6	13	19	21	22
Sulfonamide	TMP/SMX	0.5:9.5 to 2:38	13	13	16	18	24	27	21	33	29	23
Beta-lactam	Ampicillin	0.25 to 16	41	37	41	40	53	48	55	59	62	99
Aminocyclitol	Spectinomycin	8 to 64	99	100	98	96	100	88	95	95	97	97
Tetracycline	Oxytetracycline	0.5 to 8	NT	NT	NT	NT	NT	95	97	96	98	97
	Chlortetracycline	0.5	NT	NT	NT	NT	NT	95	96	96	96	100
	Tetracycline	0.5	94	93	91	93	94	94	96	80	100	100

NT — Not tested.

Table II.

Antimicrobial resistance in Salmonella sp. isolated from pigs

Antibiotic class	Antibiotic	Range tested (mg/mL)	Percent of bacteria resistant to indicated antibiotic (number of isolates tested)
			1995 (200)	1996 (241)	1997 (294)	1998 (220)	1999 (216)	2000 (253)	2001 (386)	2002 (305)	2003 (272)	2004 (406)
Aminoglycoside	Amikacin	8 to 64	0	1	1	0	1	0	0	0	NT	NT
	Gentamicin	1 to 16	12	15	13	13	9	0	10	15	13	13
	Apramycin	4.0 to 32	11	15	13	13	9	0	10	15	13	13
	Neomycin	4 to 32	32	13	13	14	8	15	8	11	6	6
Fluoroquinolone	Enrofloxacin	0.12 to 2	0	0	0	0	0	1	0	0	0	0
Cephalosporin	Ceftiofur	0.25 to 8	5	7	8	3	3	9	2	8	11	11
Sulfonamide	TMP/SMX	0.5:9.5 to 2:38	3	4	3	7	6	15	6	8	13	7
Beta-lactam	Ampicillin	0.25 to 16	34	38	35	51	46	52	53	73	71	74
Aminocyclitol	Spectinomycin	8 to 64	NT	100	100	100	100	100	100	100	100	100
Tetracycline	Oxytetracycline	0.5 to 8	NT	NT	NT	NT	NT	20	86	94	88	89
	Chlortetracycline	0.5	NT	NT	NT	NT	NT	89	86	93	89	91
	Tetracycline	0.5	80	82	79	80	78	86	82	100	NT	NT

NT — Not tested.

Antimicrobial resistance in E. coli isolates

As shown in Table I, most E. coli isolates (more than 90%) were resistant to spectinomycin, oxytetracycline, chlortetracycline, and tetracycline. An increasing trend of resistance was seen against ceftiofur (4% in 1995 to 22% in 2004), TMP/SMX (13% in 1995 to 23% in 2004), ampicillin (41% in 1995 to 99%% in 2004), gentamicin (37% in 1995 to 48% in 2004), and neomycin (51% in 1995 to 66% in 2004). Resistance to apramycin (38% in 1995 to 30% in 2004), either remained stable or declined. The lowest frequency of resistance was seen against amikacin (0% to 3%) and enrofloxacin (0% to 1%). In 2004, more than 97% of E. coli isolates were found to be resistant to ampicillin, spectinomycin, oxytetracycline, chlortetracycline, and tetracycline.

Antimicrobial resistance in S. enterica isolates

Almost all S. enterica isolates were resistant to spectinomycin and resistance to the tetracycline group of antibiotics was also frequent as most of the isolates were resistant to tetracycline, oxytetracycline, and chlortetracycline (Table II). Lower resistance was observed against TMP/SMX (3% to 15%), ceftiofur (2% to 11%), gentamicin (0% to 15%), apramycin (0% to 15%), and the least resistance was observed against amikacin and enrofloxacin. An increasing trend of resistance was seen against ampicillin (34% in 1995 to 74% in 2004) and a decreasing trend against neomycin (32% in 1995 to 6% in 2004). Resistance to gentamicin and apramycin was stable over time.

Discussion

Since 1990, there has been a sustained increase in the occurrence of antibiotic resistance in foodborne enteric pathogens in both developed and developing countries (14,15). Foodborne enteric bacterial pathogens are reported to be more resistant to antibiotics than other pathogens (16). The surveillance of antimicrobial resistance in human and animal pathogens is important in order to develop strategies for preventing the spread of multidrug-resistant bacteria (17). Salmonellosis and colibacillosis are two of the most common enteric infections in both animals and humans (18). Monitoring antimicrobial resistance in Salmonella and E. coli may help to determine the choice of antimicrobials for combating these infections and to assess the risks associated with drug-resistant strains.

During the study period (1995 to 2004), a steady increase was observed in the number of E. coli and S. enterica isolates. This may be partly due to an increase in the number of diagnostic cases submitted to the MVDL for disease diagnosis. Frequent resistance to tetracycline in E. coli is in agreement with Kozak et al (19) who reported high tetracycline resistance in E. coli isolates from Canadian swine. Teshager et al (20) also reported high resistance to tetracyclines, sulphonamides, trimethoprim, and amoxicillin antibiotics in porcine E. coli isolates from Spain. These results are in contrast to those of Mayrhofer et al (16), however, who found only 33% of E. coli isolates were resistant to tetracycline.

The increased resistance against ampicillin found in our study is in contrast to the findings of Dunlop et al (21) who reported only 29% resistance in E. coli isolates from pigs in Canada. The low frequency of resistance in E. coli to quinolone antibiotics is in contrast to the findings of Mayrhofer et al (16) who found 42% of the isolates to be resistant to quinolones. As shown in Table I, in our study, E. coli isolates were found to be resistant to the aminoglycoside class of antibiotics such as gentamicin (37% to 29%), neomycin (51% to 48%), and apramycin (38% to 30%). Mathew et al (22) reported very high resistance to gentamicin (72% to 92%) in E. coli isolates from sows and pigs from 10 commercial farms. Resistance to this antibiotic was also low in E. coli isolates from pigs on 34 furrow-to-finish farms in Ontario, Canada (21). Resistance to neomycin was more stable over time (51% in 1995 to 55% up to 2002, except 66% in 2004), but was higher than 10% as reported for isolates from pigs in the Spanish study (20). The absence of resistance to quinolone (enrofloxacin) and aminoglycoside (amikacin) and low resistance to TMP/SMX and ceftiofur is encouraging from both the public and animal health perspective. Frequent resistance against tetracyclines, β-lactam (ampicillin), spectinomycin, and ampicillin, however, is a concern as these antibiotics are important for treating infections in humans and animals.

A higher frequency of resistance to tetracyclines, ampicillin, and spectinomycin was observed among S. enterica isolates in this study, which is in agreement with Huang et al (23) who reported a high prevalence of resistance to tetracycline (83.8%), ampicillin (55.8%), and spectinomycin (42.6%) among Salmonella isolates from diagnostic cases of swine. Farrington et al (24) also reported high tetracycline resistance among S. enterica isolates from slaughter pigs in the USA. Lynne et al (25) reported higher resistance to tetracycline (94.4%) and ampicillin (50.0%) antibiotics among S. enterica serotype Heidelberg isolates from swine.

Surveillance in 1996 and 2001 by the National Antimicrobial Resistance Monitoring (NARM) system showed an increasing trend of resistance to TMP/SMX and ampicillin antibiotics in the Salmonella isolates from the USA with some isolates being resistant to more than 5 antibiotics (11). In our study, however, low to medium resistance was seen against TMP/SMX (3% to 15%), gentamicin (0% to 15%), apramycin (0% to 15%), and ceftiofur (2% to 11%). These results are consistent with the findings of Huang et al (23) who also reported low levels of resistance to TMP/SMX, gentamicin, and ceftiofur in S. enterica isolates from clinically ill pigs. Rajic et al (12) found that only 8% of Salmonella isolates from Canadian swine were resistant to ampicillin, while Deckert et al (26) reported high resistance to ampicillin (40%) but low resistance to cephalothin, ceftiofur, gentamicin, and TMP/SMX. All Salmonella isolates in our study were highly susceptible to amikacin and enrofloxacin, although an increasing trend of resistance to quinolone has been reported from the USA (27). Our results on amikacin are similar to those of Sorensen et al (28) who found no amikacin resistance in Salmonella isolates from swine in a Canadian study.

Overall, the majority of E. coli and Salmonella isolates was resistant to tetracyclines, spectinomycin, and clindamycin. The increased frequency of tetracycline resistance could be due to widespread use of this class of antibiotics in swine production systems. Another reason for high levels of resistance in this study could be that all intermediate resistant isolates in this study were classified as being resistant. It should also be emphasized that the source of bacteria in this study was diseased pigs.

An increasing resistance trend was seen in both enteric pathogens to ceftiofur, TMP/SMX, and ampicillin. Although ceftiofur is not used to promote growth in swine, higher resistance to this antibiotic could be due to the use of other related cephalosporins, such as cefquinome (29), which is a fourth generation cephalosporin with pharmacological and antibacterial properties valuable in the treatment of coliform and other infections. Overall, E. coli isolates were more resistant than Salmonella isolates. The latter showed a decreasing resistance trend to apramycin and neomycin, while E. coli isolates showed a decreasing trend to gentamicin. The reason for this decrease is not clear at this point. It is possible, however, that the use of these particular antibiotics may have decreased in recent years.

Conclusions

The results of this study indicate a higher frequency of antimicrobial resistance in E. coli and S. enterica isolates from diseased pigs to tetracyclines, ampicillin, and spectinomycin. This indicates the need for continued surveillance studies so that appropriate strategies can be developed to combat the development of resistance in these and other pathogens.