comparisons were made using the NCBI-BLAST program.' RESULTS .... 6 (50). 2 (17). Senftenherg. I (33). 0. 0. 0. Typhimurium. (var. Copenhagen). Email 0 ...
MICROBIAL DRUG RESISTANCE Volume 10, Number 1. 2004 © Mary Ann Liehert, Inc.
Antimicrobial Resistance and Serotype Prevalence of Salmonella Isolated from Dairy Cattle in the Southwestern United States T.S. EDRINGTON.' C.L. SCHULTZ,' K.M. BISCHOFF.' T.R. CALLAWAY,' M.L. LOOPER,2 K.J. GENOVESE,' Y.S. JUNG.' J.L. McREYNOLDS,' R.C. ANDERSON,' and D.J. NISBET'
ABSTRACT Mature dairy cattle were sampled over a 2-year period (2001-2002) on six farms in New Mexico and Texas. Fecal samples (ii = 1560) were collected via rectal palpation and cultured for Salmonella, and one isolate from each positive sample was serotyped. Three isolates of each serotype, with the exception of Salmonella Newport (ii = 12), were examined for susceptibility to 17 antimicrobial agents. Twenty-two different serotypes were identified from a total of 393 Salmonella isolates. Montevideo was the predominant serotype (27%) followed by Mbandaka (15%), Senftenberg (11.4%), Newport (6.4%), Anatum (4.8%), and Give (4.8%). Salmonella Typhimurium and Dublin, two frequently reported serotypes, accounted for only I % of the observed serotypes in this study. Sixty-four percent of the serotypes were susceptible to all 17 antimicrobials, 14% were resistant to a single agent, and 22% were multiresistant (2-11 types of resistance). All isolates tested were susceptible to amikacin, apramycin, imipenem, ceftriaxone, nalidixic acid, and ciprofloxacin. The most frequent types of resistance' were to sulfamethoxazole, tetracycline, streptomycin, kanamycin, chloramphenicol, and ampicillin (ranging from 8.9 to 22.4%). Serotypes demonstrating multiple resistance included Dublin and Give (resistant to three or more antibiotics), Typhimurium (resistant to five antibiotics), and Newport (four and two isolates resistant to six and nine antibiotics, respectively). Class I integrons were present in only two Salmonella Dublin isolates and one Salmonella Newport isolate. The most prevalent resistance patterns observed in this study were toward antimicrobial agents commonly used in cattle, while all Salmonella isolates were susceptible to ceftriaxone and ciprofloxacin, antibiotics used in human medicine.
reported in 1963, after increased levels of resistance were observed in Salmonella Typliirnurium isolated from British feedlots. 6 Recentl y, an increasing number of Salmonella isolates in the United States have been reported with multiple resistance 3.12.1 7.!, Although information regarding the common Salmonella serotypes recovered from the feces of sick animals is well documented. 3 information regarding the serotype prevalence in apparently healthy animals is much less extensive. The most frequently reported clinical Salmonella serotype isolated from bovine sources in the United States in 2001 was Newport followed by Typhimurium (var. Copenhagen). Typhimurium, Dublin, and Montevideo. Hosvever, the most frequently observed nonclinical bovine isolates were Montevideo. l'y-
there are an estimated 1.3 million hucases of foodborne Salmonella infections and 553 deaths annually. 20 During the first 4 months of 2002. 47 people in five states were diagnosed with salmonellosis, of which 37% were hospitalized and I person later died. The causative agent was a multidrug-resistant Salmonella Newport strain with implications that the vehicle of transmission was undercooked ground beef. 12 The use of antihioics in agriculture for prophylaxis and growth promotion (which accounts for about 90% of the antibiotics used in agriculture)" has been implicated as a contributing source of resistant strains that can be transmitted to humans through the fiodchain. 6.23 Evidence of this was first N 1 H UNITED STATES,
'USDA-ARS-SPA. Food and Feed Safety Research Laboratory, College Station. TX. 77845. 2 USDAARSSPA. Date Bumpers Sn,alt Farm Research Center. Booneville, AR, 72927. 51
EDRINCTON ET AL. phimurium, Kentucky, Typhimurium (var. Copenhagen), and Anatum, with only a handful of Newport isolates reported. 3 The number of Newport isolates from all sources increased 59% from 1996 to 2001. The NAHM's Dairy '96 Study reported the most common Salmonella scrotypes isolated from dairy cows were Montevideo, Cerro, Kentucky. Menhaden, and Anatuni.29 Because scrotype prevalence continues to change and the prevalence of antimicrobial-resistant isolates appears to increase, the need for monitoring Salnonella in livestock continues. Dairy cattle serve as an important reservoir for Salmonella and have been implicated in cases of human salmonellosis. 326 This study reports the prevalence of Salmonella serotypes isolated from apparently healthy mature dairy cattle in the Southwestern United States and examines patterns of antimicrobial resistance of these isolates.
MATERIALS AND METHODS Sample collection, bacterial culture, and isolation Lactating and nonlactating Holstein dairy cattle (a = 1560 total samples) were sampled on six farms in New Mexico and Texas over a 2-year period (2001-2002). Five of the farms were sampled each year, whereas one farm was sampled only in 2002. Sixty lactating cows were sampled on each of the six farms at each sampling time and 60 mionlactating cows were sampled twice on one farm. Cows were restrained in self-locking stanchions and approximately 30 g of fecal material was collected via rectal palpation. Fecal samples were either shipped on ice or transported directly to the Food and Feed Safety Research Laboratory in College Station, TX for isolation of Salmonella as described below. Fecal material (10 g) was enriched in 90 ml of tetrathionate broth for 24 hr at 37°C. A portion (200 .d) of the enriched culture was transferred to S ml of Rapport- Vassilidis RIO broth and incubated an additional 24 hr at 42°C before spread plating on brilliant green agar supplemented with novobiocin (25 g/ml). Following incubation (24 hr. 37C), colonies exhibiting typical Salmonella morphology were confirmed biochemically using lysine and triple-sugar iron agars. Confirmation of Salmonella-positive samples was conducted using slide agglutination with SM-O antiserum poly A-I and V-I and group Cl factors. Salmonella isolates were stored as glycerol stocks (10%) in TSB at —80° C. One isolate from each positive sample was sent to the National Veterinary Services Laboratory in Ames, IA. for confirmatory serotyping. All media and agar were from Difco Laboratories (Detroit. MI). Reagents and antibiotics were obtained from Sigma Chemical Co. (St. Louis. MO).
Anti,nicrol,ial susceptibility determination Three isolates of each serotype. with the exception of Bredeny, Typhimurium, and Worthington (a = I each). Barranquilla and Oranienberg (mm = 2 each), and Newport (n = 12) were examined for antimicrobial susceptibility using the Scnsititre automated antimicrobial susceptibility system according to the manufacturer's directions (Trek Diagnostic Systems. Westlake, OH). Broth microdilution was used according to methods described by the National Committee for Clinical Lab-
oratory Standards (NCCLS) 22 to determine MICs for the following antimicrobials: amikacin, apramycin, gentamicin, kanamycin, streptomycin, imipenem, cephalothin, ceftiofur, ceftriaxone, ampicillin, amoxicillin/clavulanic acid, chloramphenicol. nalidixic acid. ciprofloxacin, sulfamethoxazole, trimethopri m/sulfamethoxazole, and tetracycline. Resistance breakpoints were determined using the NCCLS interpretive standards 22 unless unavailable, in which case breakpoints in the NARMS 2000 Annual Report 8 were used. Esclmerichia coIl ATCC 25922, E. coli ATCC 35218, and Enterococcusfaecalis ATCC 29212 were used as quality control organisms.
Detection of integron gene cassettes Integron gene cassettes were detected by polynierase chain reaction (PCR) using the 5-CS and 3-CS primer set described previously. 18 DNA was prepared by boiling a bacterial pellet at 95°C for 10 inin and the supernatant liquid (10 d) was used as the template in the PCR reactions. Each reaction (50 l) also contained: I X AmpliTaq Gold DNA polymerase reaction buffer (Applied Biosystems. Foster City, CA), 2.5 mM MgCl2, 1 mM dNTPs. 1 pmol/.d of each oligonucleotide primer (BioSynthesis, Lewisville. TX), and I unit of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City. CA). All reactions were first heated to 95°C for 10 min and then subjected to 30 cycles of PCR consisting of denaturation at 95°C (I mm), annealing at 55°C (1 mm) and extension at 72°C (I mi. PCR products were submitted for DNA sequencing at the DNA Core Facility in the Department of Veterinary Pathohiology, Texas A&M University (College Station. TX). Sequence comparisons were made using the NCBI-BLAST program.'
RESULTS Serotype prevalence A total of 393 Salmonella isolates were identified in this study and serotype prevalence is presented in Table I. Twentytwo different serotypes were identified and 13 isolates were untypeable. Salmonella Montevideo was the most prevalent serotype accounting for more than one-quarter of all the identified serotypes. The six most common serotypes (Montevideo. Mhandaka, Senftenberg, Newport. Anatum. and Give) accounted for 69% of all isolates.
0-Antigen grouping The prevalence of Salmonella isolates as classified by 0antigen grouping is presented in Table 2. The most prevalent 0-antigen group in this study was C 1 , accounting for almost one-half of the Salmonella isolates. Groups C2, E 1 . and E4 were common in this study accounting for over 38% of the groups identified. A total of II different groups were identified with the C and E groups representing nearly 85% of all the isolates.
Antibiotic resistance in Salmonella isolates The antimicrobial susceptibility profiles of 67 Salmonella isolates are presented in Table 3. Nineteen (28%) isolates were resistant to at least one antimicrobial. The most common resistances observed were to sul famnethoxazole, tetracycline. strep-
ANTIMICROBIAL RESISTANCE IN SALMONELLA
TABLE I. PREVAlENCE OF SALMONELLA SEROTYPES ISOLATED FROM MAILEE DAIRY CATTLE IN THE SOUTHWESTERN UNITED STATES OVER A TWO-YEAR PERIOD (2001-2002) SerotvpeNumber Percentage Agona Albany Anatum Barranquilla Bredeny Cerro Cubana Dublin Give Havana, atypical Kentucky Mhandaka Meleagridis Minnesota Montevideo Newport Oranienherg Scnftcnberg Soerenga Tennessee Typhimurium var. Copenhagen Worthington Untypeahle Total
3 6 19 3 I 14 9 3 19 3 18 59 15 4 106 25 3 45 15 8 I
0.76 1.5 4.8 0.76 0.25 3.6 2.3 0.76 4.8 0.76 4.6 15.0 3.8 1.0 27 6.4 0.76 11.5 3.8 2.0 0.25
tomycin, kanamycin, and trimethoprim/sulfamethoxazole. All of the isolates tested were susceptible to aniikacin, apramycin, imipenem, ceftriaxone, nalidixic acid, and ciprofloxacin. Each of the three isolates in 14 (86%) of the 22 different serotypes tested were susceptible to all of the antibiotics, including the two most prevalent serotypes Montevideo and Mhandaka. In only two cases (Typhimurium and Dublin) were all three isolates of the same serotype resistant to the same antibiotics. All isolates of the following serotypes were susceptible to all of the antibiotics tested: Agona. Anatum, Barranquilla, Bredeny. Havana, Kentucky, Mbandaka. Meleagridis, Minnesota, Montevideo, Oranienberg, Senftenberg, Soerenga, Tennessee, and Worthington. Multiple resistance was observed in 15 (22%) of the isolates. Five (7.5%) of the isolates were resistant to at least two antibiotics, II (16.4%) were resistant to at least five antibiotics. and two (3%) were resistant to nine antibiotics (Table 4). Five serolypes contained at least one isolate with multiple resistance: Albany, Dublin, Give, Newport, and Typhimurium. Two isolates from the serotype Albany were resistant to two antibiotics whereas all three isolates of Salmonella Give were resistant to at least two antibiotics and one isolate was resistant to five antibiotics. The one isolate of Salmonella Typhimurium variety Copenhagen was resistant to five antibiotics. Fifty percent (,z = 6) of the Newport isolates were resistant to six antibiotics and two isolates were resistant to nine antibiotics each (Table 4).
Integron gene cassettes The polymerase chain reaction was used to examine 67 Salinonella isolates for the presence of integron gene cassettes. Using primers specific for the 5' and 3' conserved regions that flank the variable region of the integron, a I .2-khp product was obtained from one Newport and two Dublin strains. Sequence analysis of these products indicated that both Salmonella Dublin cassettes were homologous to the aadA21 gene. 7 whereas the Newport cassette was homologous to aadA2. 27 These genes encode aminoglycoside adenyltransferases that confer resistance to aniinoglycoside antibiotics, including streptomycin.
DISCUSSION Nearly 400 Salmonella isolates collected from apparently normal, healthy, mature dairy cattle were serotyped and examined for susceptibility to antimicrobials of human and veterinary significance. To our knowledge, this is one of the few reports on Salmonella serotypes obtained from healthy dairy cattle. Salmonella Typhimurium and Dublin have been frequently reported in sick, diarrheic dairy cattle, 3.1. however there appears to be a difference in serotypes isolated from healthy animals. Wells and co-workers 3° reported the most common serotypes isolated from lactating cows and cull dairy cattle were Montevideo, Cerro, and Kentucky. In the current study, we also found a hi gh percentage of Salmonella Montevideo but only a few isolates of Cerro and Kentucky. The serotypes Mbandaka and Senftenberg were also frequently isolated in this research; however, to the author's knowledge, these two serotypes have not been reported elsewhere as significant serotypes in dairy cattle. Wells and co-workers 30 reported only 5% of the serotypes from lactating dairy cows were Mbandaka, while other research reported Mbandaka in dairy cattle due to the feeding of contaminated vegetable fat.' 5 The serotypes Mbandaka and Senftenherg were infrequent serotypes listed in CDC's report of nonclinical bovine isolates in 2001.
TAI3IJ 2. PREVALENCE or O-ANTIGEN GROUPS IN SALMONELLA ISOLATED FROM MATURE DAIRY CATrLL IN Till. SouTlIwI:sTERN UNITED SrAres OVER A 2-YEAR PERIOD (2001-2002) 0-antigen group Number Percentage B Cl C2 Dl El E4 G 1 K L N Untypeable Total
5 176 49 3 53 45 13 3 14 4 15 13 393
1.3 45 12.5 0.8 13.5 11.5 3.3 0.8 3.6 1.0 3.8 3.3
TABLE 3. ANTulicRoBi..\L RESISTANCE OF SAISIO,VELLA ISOLATES (BY SvRoFYPE) iROM DAIRY CATTLE
% Resistant Salmonellab Resistant breakpoint (p,'/inl) All, Cer Cub Dub Give N,, Sen Tvp Totalc
Gentamicin Kanamycin Streptomycin Cephalosporins Cephalothin Ceftiofur Cefoxitin Penici II ins Ampicillin A mox i ci II in/cl avulan ic acid Phenicols Chloramphenicol Sulfonamides and potentiated sulfonamides Sulfamethoxazole Trimethoprim/ Sulfarnethoxazole Tetracyclines Tetracycline
16 67 0 0 0 0 0 0 0 3.0 64 0 0 0 100 tOO 33 0 100 16.4 64 0 0 0 100 lOU 50 0 100 19.4 ^!32 0 0 0 0 0 17 0 0 3.0 0 0 0 0 0 17 0 0 3.0 ^!8 0 0 0 0 0 17 0 0 3.0 ^!32 0 0 C) 10) 0 17 0 100 8.95 ^32/16 0 0 0 0 0 17 0 0 3.0 ^!32 0 0 0 0 0 50 0 0 8.96
^!512 67 0 0 100 100 100 0 100 22.4 =^4/76 33 33 33 0 33 33 0 0 11.9 > 16 0 0 0 100 100 50 33 100 20.9
'MICs were determined by microdilution methods according to NCCLS standards. All isolates tested were susceptible to amikacin, apramycin. imipenern, ceftriaxone, nalidixic acid, and ciprofloxacin. 'Alb, Albany: Cer, Cerro; Cub. Cubana; Dub, Dublin; Np, Newport; Typ. Typhimurium variety Copenhagen. Three isolates per serotype examined except for Np = 12 isolates and Typ = 1 isolate. Percentage of all isolates (n = 67) resistant to each antibiotic.
Of the top live most prevalent serotypes identified in the cur- phimurium variety Copenhagen. Salmonella Dublin, another rent study (Montevideo, Mbandaka, Senftenberg. Newport, and frequently reported serotype in the past, accounted for less than Anatum) only two (Newport at #3 and Montevideo at #6) were 1% of the isolates in this research. Possible reasons for finding in the 20 most frequently reported Salmonella isolates from hu- only a few of these serotypes may include the geographic loman sources in 2001. 3 Salmonella l'yphimurium, was the most cation of the farms, or sampling and culture techniques. In the frequently reported human and nonhuman clinical isolate by the current research, we serotyped only one colony from each posCDC in 2001. Salmonella Typhimurium and Typhimurium "a- itive fecal sample that may have biased the results.. Previous reriety Copenhagen were also in the top four most isolated search reports that cattle shed multiple serotypes within a serotypes from clinical and nonclinical bovine isolates in 2001.' day' 30; however, because we serotyped almost 400 isolates. However, in the current study. we found only one isolate of Ty- we believe this would not have changed our results dramatiI
MULTIPLE RESISTANCE OF Sni..•iio,vi.ijt SEROTYPES ISOLA1ED FROM DAIRY CArri.E
Number resistant Number resistant Number resistant Number resistant to at least one to at least two to at least jive to at least nine antibiotic (%) antibiotics (%) antibiotics (%) antibiotics (%)
Albany Cerro Cubana Dublin Give Newport Senftenherg Typhimurium (var. Copenhagen)
3(100) 1 (33) 1(33)
2 (67) 0 0 3 (100)
0 0 0 3 (lOU) I (33) 6 (50) 0
0 0 0 0 0 2 (17) 0
ANTIMICROBIAL RESISTANCE IN SALMONELLA
cally. Others have shown evidence of geographic distribution differences for Salmonella Typhimurium DT 104, suggesting that epidemiology may differ among Salmonella strains. 2 Spatial and temporal clustering of Salmonella Typhimuriuni, Montevideo, and several other serotypes has been reported 21 in dairy cattle in California; however, these were all isolates obtained from diarrheic cattle, not healthy cattle as in the current study. Because we sampled in a relatively small region of the United States, this might explain the small number of Typhiniurium and Dublin isolates. A more probable explanation is that the prevalence of some Salmonella serotypes appears to change with time. While Salmonella Typhiniurium retained its spot as the number one isolate in 2001, there was a 26% drop in the percentage of isolations since 1996. Similarly. Enteritidis, another frequently reported isolate, dropped 41 010 from 1996 to 2001. However, the number of Salmonella Newport isolations increased 59% over this same time frame.3 Salmonella Newport accounted for over 6% of the total isolates in this study. Reports of Salmonella Newport dating back to 1962 suggested it is an important serotype in stressed aninials. 2 ' Significant losses of cattle in Manitoba due to Salmonella Newport were reported in 1969.25 More recently, Salmnond/a Newport gained attention in 1983 when a niultidrug resistant strain was isolated from several infected people that apparently acquired this Salmonella from ground beef originating from beef cattle.' 4 In 1985, Salmonella Newport was isolated from human patients in California with an unusual resistance pattern (including chloramphenicol) that was traced through hamburger to dairy farms. 26 Others have reported the prevalence on Salmonella Newport appeared to he increasing in California and that a chloramphenicol-resistant strain was the most common serotype identified in 1986.24 Most recently, numerous people in the Eastern United States were made ill and one later died as a result of a multidrug-resistant strain of Newport which was later implicated as coming from ground beef from dairy cattle 32 Multiple resistance to six antibiotics was observed in 50% of the Newport isolates, and two isolates were resistant to nine antibiotics. The most common resistance was to streptomycin, sulfamethoxazole, tetracycline, chloramphenicol, and kanamycin. Similar resistance patterns were reported in the isolates responsible for the Newport outbreak in the Eastern United States in 2002.32 and by others in dairy cattle isolates.24 lntegrons are genetic elements consisting of two conserved regions (the 5-CS and 3'-CS) that flank a variable region. In general, the 5' conserved region encodes a site-specific integrase and the 3' conserved region encodes the qacEM and soil genes, which confer resistance to quartcrnary ammonium compounds and sulfonamide, respectively. The variable region may contain one of more mobile antibiotic resistance gene cassettes.' Consequently, integrons potentially contribute to the dissemination and maintenance of multiple antimicrobial resistance genes. We detected integron gene cassettes in only three of 17 antimicrobial-resistant Salmonella isolates from dairy cattle (two Dublin and one Newport). DNA sequencing revealed that all three encoded an aminoglycoside adenyltransferase gene. Although integrons have been reported in a wide range of Salmonella serotypes,4 - " it is interesting to note that 10 of the 13 multidrug-resistant isolates did not possess gene cassettes, suggesting the presence of alternative genetic elements
that maintain multiple antimicrobial resistances. Our results are in agreement with a recent report on the prevalence of integrons among Salmonella isolated from swine in the United States, which suggested that integions are not widely disseminated among non-Salmonella Typhimuri um serotypes.3' The development of antimicrobial resistance in pathogenic bacteria species as a result of the use of antibiotics in animal production has been the subject of considerable debate and controversy. The prevalence of antimicrobial resistance in Saimnonella isolates in the current study was low (17 of 67 isolates), although multidrug resistance was common in these 17 isolates. However, it is important to note that the majority of these isolates were resistant to ampicillin, tetracycline, and sulfonamides, antibiotics commonly used in veterinary medicine. All isolates were susceptible to the Ilouroquinolones and all but two Salmonella Newport isolates were susceptible to the third-generation cephalosporins, the two classes of drugs indicated for treatment of salmonellosis in humans. The majority of the serotypes reported in this study, including the most prevalent (Montevideo and Mhandaka) were susceptible to all the antimicrobials examined. Results of this study provide an indication of the diversity of Salmonella species in dairy cows. These isolates were strictly from healthy cattle on the farm versus those from cull cattle markets or from cattle that were clinically ill. This may explain the differences in serotype prevalence and susceptibility patterns we observed versus much of the current literature. REFERENCES I. Altschul, S.F., T.L. Madden, A.A. SchälTer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. 2. Besser, 'I'.E., M. Goldoft, L.C. Pritchett, R. Khakhria, D.D. Hancock, D.H. Rice, J.M. Gay, W. Johnson, and C.C. Gay. 2000. Multiresistant Salmonella Typhimuriurn DT t 04 infections of humans and domestic animals in the Pacific Northwest of the United States. Epidemiot. Infect. 124:193-200.
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