Antimicrobial Drug Resistance Patterns among Cattle- and Human-Associated Salmonella Strains

Antimicrobial Drug Resistance Patterns among Cattle- andHuman-Associated Salmonella Strains

Y. SOYER,1* J. RICHARDS,2 K. HOELZER,2 L. D. WARNICK,3 E. FORTES,2 P. MCDONOUGH,3 N. B. DUMAS,4

Y. T. GROHN,3 AND M. WIEDMANN2

1Department of Food Engineering, Middle East Technical University, 06800 Ankara, Turkey; 2Department of Food Science and 3Department of Population

Medicine and Diagnostic Sciences, Cornell University, Ithaca, New York 14853, USA; and 4Wadsworth Center, New York State Department of Health,

Albany, New York, USA

MS 13-018: Received 15 January 2013/Accepted 14 June 2013

ABSTRACT

During the year 2004, 178 human and 158 bovine clinical Salmonella isolates were collected across New York State to

better understand the transmission dynamics and genetic determinants of antimicrobial resistance among human and bovine hosts.

Serotyping, sequence typing, and pulsed-field gel electrophoresis typing results have been reported previously. Here we tested all

isolates for phenotypic susceptibility to 15 antimicrobial drugs that are part of the National Antimicrobial Monitoring System

bovine susceptibility panel. PCR was performed on a representative subset of unique isolates (n ~ 53) to screen for the presence

of 21 known antimicrobial resistance genes (i.e., ampC, blaTEM-1, blaCMY-2, blaPSE-1, cat1, cat2, cmlA, flo, aadA1, aadA2,aacC2, strA, strB, aphA1-IAB, dhrfI, dhrfXII, sulI, sulII, tetA, tetB, and tetG); selected fluoroquinolone- and nalidixic acid–

resistant (n ~ 3) and –sensitive (n ~ 6) isolates were also tested for known resistance-conferring mutations in gyrA and parC.

Genes responsible for antimicrobial resistance were shared among isolates of human and bovine origin. However, bovine isolates

were significantly more likely than human isolates to be multidrug resistant (P , 0.0001; Fisher’s exact test). Our analyses

showed perfect categorical agreement between phenotypic and genotypic resistance for beta-lactam and chloramphenicol. Our

data confirm that resistance profiles of amoxicillin–clavulanic acid, chloramphenicol, kanamycin, and tetracycline were strongly

associated with the presence of blaCMY or ampC, flo, aphA1-IAB, and tetA, respectively. Our findings provide evidence for the

clinical value of genotypic resistance typing if incorporating multiple known genes that can confer a phenotypic resistance

profile.

Salmonella is an important human and animal pathogen

worldwide, which can be transmitted to humans through

contaminated food or water or through direct contact with

infected hosts (32). Salmonella spp. have been estimated to

be responsible for as many as 11% of all foodborne illnesses

with known etiology in the United States (50); infections

with nontyphoidal Salmonella cause an estimated 1.0

million human cases and 450 deaths in the United States

annually (50).There are currently over 2,500 recognized Salmonella

serotypes (27). Genotypic approaches have been widely

used to further differentiate among Salmonella subtypes,

including multilocus sequence typing (1, 57, 63), pulsed-

field gel electrophoresis (PFGE) (22, 47, 48, 52, 64),multilocus variable number of tandem repeat analysis (15,18, 37), and DNA microarrays (20, 24, 51). Among these

subtyping methods, PFGE is the most widely used

molecular subtyping method for Salmonella (6). PFGE

results can easily be shared electronically among laborato-

ries, allowing participating members of the U.S. and

International PulseNet systems to rapidly compare isolates,

thus greatly facilitating outbreak investigations (58, 59).The emergence and spread of multidrug-resistant

(MDR) Salmonella strains have become a public health

concern worldwide (11, 14, 33, 44, 54, 65). The

antimicrobial resistance profiles of the most prevalent

Salmonella serotypes, such as Newport and Typhimurium,

have been characterized in great detail (26, 30). In the

United States, the National Antimicrobial Resistance

Monitoring System (NARMS) has been monitoring antimi-

crobial resistance among enteric bacteria since 1997 (13).Isolates are collected each year from human and animal

clinical cases, animals, and retail food samples to monitor

trends in antimicrobial resistance (13). In the NARMS

system, all isolates are tested for sensitivity to a panel of 15

antimicrobial drugs by using the Sensititre system, and

MICs are interpreted according to the standards of the

Clinical Laboratory Standards Institute, formerly National

Committee for Clinical Laboratory Standards (13, 16).Phenotypic methods are commonly used for determin-

ing antimicrobial resistance profiles of Salmonella isolates

(5). Antimicrobial susceptibility is commonly measured via

dilution or diffusion techniques (4); results obtained with* Author for correspondence. Tel: z90 312 210 5633; Fax: z90 312 210

2767; E-mail: [email protected].

1676

Journal of Food Protection, Vol. 76, No. 10, 2013, Pages 1676–1688doi:10.4315/0362-028X.JFP-13-018Copyright G, International Association for Food Protection

either method can be expressed as ‘‘susceptible,’’ ‘‘inter-

mediate,’’ or ‘‘resistant’’ by using established breakpoints

such as those defined by the Clinical Laboratory Standards

Institute (16). However, some drawbacks can potentially

limit the usefulness of such approaches (5, 53). For

example, the accuracy of serial dilution tests, used

commonly by diagnostic laboratories, is dependent on a

number of factors, such as the quality of microwell plates,

bacterial level, and volume of inocula (53). Critical factors

in the agar diffusion method are the agar type and depth,

antimicrobial content of the disks, the level of inocula, and

incubation time (5). In addition, both methods are

performed in an artificial environment for organisms, which

might cause false results (53). For example, for some

antimicrobial drugs such as first- and second-generation

cephalosporins, cephamycins, and aminoglycosides, in vitro

susceptibility may not necessarily be predictive of clinical

efficacy (13). Besides the potential clinical implications

associated with these drawbacks, these factors can compli-

cate comparisons of resistance rates among countries

because no common international standards that would

define antimicrobial drug dosages for resistance breakpoints

exist (53). Moreover, because phenotypic methods depend

on bacterial growth inhibition, several days are generally

required before results can be obtained (36, 40).However, especially in clinical settings, expeditious

determination of resistance profiles is critical to permit

adequate treatment of patients. In-house genotypic screen-

ing of isolates for the absence or presence of genes

responsible for antimicrobial resistance may potentially

offer an attractive alternative to phenotypic screening in

external laboratories and may provide more rapid, reliable,

and cost-effective antimicrobial sensitivity results (36).Here we describe the distribution of multidrug

resistance among 336 Salmonella isolates from human and

cattle, representing 51 different serotypes. We also inves-

tigate the genetic determinants of antimicrobial resistance

among both human and bovine isolates, which have

previously been characterized by multilocus sequence

typing and PFGE. In addition, we evaluate the reliability

of genotypic testing methods for predicting phenotypic

resistance profiles.

MATERIALS AND METHODS

Salmonella isolates. Except for five human clinical isolates,

all isolates incorporated in this study have previously been

described (1, 55). Briefly, a total of 336 Salmonella isolates,

including 178 of human origin and 158 of bovine origin, were

obtained from clinical salmonellosis cases that occurred between

January and December 2004. Human isolates were obtained from

the New York State Department of Health (NYSDOH) and

represented a convenient sample of human clinical Salmonellaisolates submitted to NYSDOH in 2004. Briefly, in every month,

10 to 20 human isolates were selected by NYSDOH and submitted

to Cornell University; these isolates represent a subset of all human

clinical isolates received by the NYSDOH in a given month.

Isolates were typically selected to represent the first five isolate

submissions that were serotyped each week. All bovine clinical

Salmonella isolates were obtained from the Animal Health

Diagnostic Center at Cornell University. The bovine clinical

Salmonella isolates were obtained either as a result of a routine

veterinary submission or as part of a prospective study on the

burden of clinical bovine salmonellosis (1). Bovine isolates

originated from 64 farms in New York State and 8 farms in

Vermont. Of the initial 158 bovine isolates, 61 were excluded from

most statistical analyses (see ‘‘Results’’), as they represented

multiple isolates from the same farm with the same phenotypic and

genotypic characteristics. Specifically, for any given combination

of serotype, sequence type (ST), PFGE type, and resistance profile,

only one isolate was selected from each farm to ascertain

representativeness. The remaining 97 unique bovine isolates were

used for further analysis.

Serotyping of human and bovine isolates was performed by

the NYSDOH and the National Veterinary Services Laboratory

(U.S. Department of Agriculture—Animal and Plant Health

Inspection Service—Veterinary Services, Ames, IA), respectively,

and has previously been described (1). Isolates also have

previously been assigned STs based on a three-gene (manB, fimA,and mdh) multilocus sequence typing, as also detailed by Alcaine

et al., 2006 (1). PFGE subtyping data generated by using restriction

endonuclease XbaI and the standard Centers for Disease Control

and Prevention PFGE protocol (47) have also been previously

reported for all isolates (55) (see Suppl. Table 1; all supplemental

material is available at http://foodscience.cornell.edu/cals/foodsci/

research/labs/wiedmann/links/soyer-et-al-2013.cfm).

Antimicrobial susceptibility testing. Resistance of all

isolates to antimicrobial drugs included in the standard NARMS

panel (61) was evaluated with the Sensititre system (Trek

Diagnostic Systems Ltd., Cleveland, OH) as previously described

(2). The antimicrobial agents tested included amikacin (Amk;

64 mg/ml), amoxicillin–clavulanic acid (Amc; 32 mg/ml), ampicil-

lin (Amp; 32 mg/ml), cefoxitin (Fox; 32 mg/ml), ceftiofur (Cef; 8 mg/

ml), ceftriaxone (Cro; 64 mg/ml), chloramphenicol (Chl; 32 mg/ml),

ciprofloxacin (Cip; 4 mg/ml), gentamicin (Gen; 16 mg/ml),

kanamycin (Kan; 64 mg/ml), nalidixic acid (Nal; 32 mg/ml),

streptomycin (Str; 64 mg/ml), sulfisoxazole (Suf; 512 mg/ml),

tetracycline (Tet; 16 mg/ml), and trimethoprim-sulfamethoxazole

(sulfisoxazole with trimethoprim) (Sxt; 4/76 mg/ml).

Isolate selection, PCR, and DNA isolation. PCR assays

were performed to test a representative subset of isolates (see

Suppl. Table 2) for the presence of 21 known antimicrobial

resistance genes (Suppl. Table 3). A total of 53 Salmonella isolates

were selected for these analyses, including 30 human and 23

bovine isolates. Isolates were selected to represent each unique

combination of phenotypic antimicrobial resistance profile,

serotype, ST, and host species. Three pansusceptible human

isolates, belonging to serotypes Newport, Abony, and Typhimur-

ium, and two pansusceptible bovine isolates of serotypes Agona

and Infantis were also included in the final isolate set (see Suppl.

Table 2).

Purified Salmonella DNA was isolated for PCR with the

QIAamp DNA minikit (Qiagen Inc., Valencia, CA) according to

the manufacturer’s instructions; DNA concentrations were stan-

dardized to 12.5 ng/ml in sterile distilled water. PCR primers used

for detection of the 21 Salmonella antimicrobial resistances genes

(i.e., ampC, blaTEM-1, blaPSE-1, tetA, tetB, tetG, dhrfI, dhrfXII, sulI,sulII, cat1, cat2, cmlA, aacC2, flo, aadA1, aadA2, blaCMY-2, strA,strB, and aphA1-IAB) have previously been reported by Chen et al.

(2004), Casin et al. (2003), Randall et al. (2004), and Orman et al.

(2002) (12, 14, 41, 46) (see Suppl. Table 3). The thermocycling

reaction conditions included an initial denaturation at 95uC for

10 min, followed by 32 to 35 cycles at 95uC for 30 s, annealing at

J. Food Prot., Vol. 76, No. 10 ANTIMICROBIAL RESISTANCE IN CATTLE- AND HUMAN-ASSOCIATED SALMONELLA 1677

the appropriate temperature for the specific primer (see Suppl.

Table 3) for 1 min, and 72uC for 1 min, followed by a final

extension step of 72uC for 7 min. The only exception to these

reaction conditions was for the PCR for amplification of cat1,which utilized an initial 20 cycles with a touchdown protocol,

where the annealing temperature was decreased by 0.5uC per cycle

from 55 to 45uC, followed by 20 cycles with an annealing

temperature of 45uC.

parC and gyrA sequencing. PCR-amplified gene segments

were further analyzed by DNA sequencing to test selected isolates

for resistance-conferring mutations in gyrA and parC. Specifically,

all fluoroquinolone- and nalidixic acid–resistant Salmonellaisolates (n ~ 3) as well as six randomly selected isolates sensitive

to these antimicrobial drugs were tested. Previously described gyrA(STGYRA1 and STGYRA12) and parC (SYPARC1 and

STPARC2) PCR primers were used under the reported PCR

conditions (25). PCR products were purified by using ExoSAP

(New England Biolabs, Inc.) and sequenced with the respective

PCR primers.

Statistical analyses. Associations between isolate source

(i.e., human or cattle) and resistance type (resistant to more than

one antimicrobial drug, i.e., MDR, or not; pansusceptible, or

sensitive to one or more antimicrobial drugs) or resistance gene

presence or absence were evaluated by Fisher’s exact test. For

extension of Fisher’s exact test to more than two categories, we

used the %RUN_FISHERS macro (SAS Global Forum 2010). All

statistical analyses were performed with SAS 9.1 or 9.2 (SAS

Institute Inc., Cary, NC).

Evaluation of results from molecular detection ofantimicrobial-resistant profiles. Resistance profiles were

grouped into five main antimicrobial groups (beta-lactam,

chloramphenicol, aminoglycoside, sulfisoxazole with and without

trimethoprim, and tetracycline antimicrobials) to facilitate com-

parisons between phenotypic results based on the NARMS panel

(i.e., the reference system or ‘‘gold standard’’) and results for

genotypic resistance profiling (i.e., the test system under

evaluation); for this purpose, isolates with phenotypic resistance

to one or more drugs in a drug class were classified as resistant.

TABLE 1. List of farms yielding identical clinical Salmonella isolates at multiple sample submission dates and description of isolates

Farm

ID

No. of farm visits yielding

Salmonella-positive samples ST/PFGE type/serotype/antibiotic-resistant pattern (no. of isolates)a

510 20 ST11/121/Newport/AmcAmpCefFoxChlKanStrSufTet (15); ST11/122/Newport/

AmcAmpCefFoxChlKanStrSufTet (1); ST11/121/Newport/AmcAmpCefFoxChlStrSufTet (2);

ST11/121/Newport/AmcAmpCefFoxKanStrSufTet (2); ST11/121/Newport/

AmcAmpCefFoxCroChlKanStrSufTet (1); ST6/94/4,5,12:i:-/AmcAmpCefFoxChlStrSufTet (1)

261 22 ST6/89/4,5,12:i:-/sensitive (5); ST6/91/4,5,12:i:-/sensitive (1); ST6/95/4,5,12:i:-/sensitive (1); ST6/

94/4,5,12:i:-/AmcAmpCefFoxChlStrSufTet (10); ST6/90/4,5,12:i:-/

AmcAmpCefFoxChlStrSufTet (1); ST6/94/Typhimurium/AmcAmpCefFoxChlStrSufTet (1);

ST17/Kentucky/sensitive (5)

223 15 ST60/107/Infantis/sensitive (13); ST60/108/Infantis/sensitive (1); ST60/109/Infantis/sensitive (1)

329 5 ST9/119/Montevideo/sensitive (1); ST44/7/Muenster/sensitive (3); ST62/157/Thompson/sensitive (1)

186 4 ST75/44/Adelaide/sensitive (1); ST8/104/Typhimurium/AmcAmpCefFoxChlKanStrSufTet (2)b;

ST8/104/Typhimurium/AmcAmpCefFoxCroChlKanStrSufTet (2)b

524 5 ST6/90/4,5,12:i:-/AmcAmpCefFoxChlStrSufTet (1); ST11/127/Newport/

AmcAmpCefFoxChlStrSufTet (3); ST11/126/Newport/AmcAmpCefFoxChlStrSufTet (1)

152 4 ST11/126/Newport/AmcAmpCefFoxChlStrSufTet (4)

490 4 ST11/126/Newport/AmcAmpCefFoxChlStrSufTet (1); ST11/127/Newport/

AmcAmpCefFoxChlStrSufTet (1); ST11/127/Newport/AmcAmpCefFoxChlKanStrSufTet (1);

ST11/129/Newport/AmcAmpCefFoxChlKanStrSufTet (1)

488 4 ST11/126/Bardo/AmcAmpCefFoxChlStrSufTet (1); ST11/126/Newport/

AmcAmpCefFoxChlStrSufTet (3)

163 3 ST60/114/Infantis/sensitive (1); ST11/126/Newport/AmcAmpCefFoxChlStrSufTet (2)

259 3 ST44/4/Muenster/AmcAmpCefFoxChlStrSufTet (1); ST44/2/Muenster/

AmcAmpCefFoxChlStrSufTet (1); ST44/6/Muenster/sensitive (1)

584 3 ST2/165/Agona/AmcAmpCefFoxCroChlKanStrSufTet (1); ST2/166/Agona/

AmcAmpCefFoxChlKanStrSufTet (1); ST6/64/Typhimurium/AmpChlStrSufTet (1)

764 2 ST6/60/Typhimurium/AmpChlStrSufTet (2)

415 2 ST6/70/Typhimurium/sensitive (1); ST9/119/Montevideo/sensitive (1)

97 2 ST8/104/ Typhimurium /AmpKanStrSufTet (1)c; ST8/104/ Typhimurium /AmpKanStrSuf (1)c

125 2 ST6/79/Typhimurium/sensitive (2)

105 2 ST11/121/Newport/AmcAmpCefFoxChlKanStrSufTet (1); ST8/102/Typhimurium/

AmpKanStrSufTetSxt (1)

208 2 ST6/66/Typhimurium/sensitive (2)

303 2 ST11/126/Newport/AmcAmpCefFoxChlStrSufTet (2)

320 2 ST11/126/Newport/sensitive (1); ST11/126/Newport/AmcAmpCefFoxCroChlStrSufTet (1)

a Sequence, PFGE, and serotype data were obtained from Alcaine et al., 2006 (1), and Soyer et al., 2010 (55).b Serotyping identified one isolate with serotype Typhimurium var. 5 (previously known as Typhimurium Copenhagen) among these two

Typhimurium isolates from farm 186.c Serotyping identified both isolates from farm 97 as serotype Typhimurium var. 5 (previously known as Typhimurium Copenhagen).

1678 SOYER ET AL. J. Food Prot., Vol. 76, No. 10

Genotypically, the presence of ampC, blaTEM-1, blaCMY-2, and

blaPSE-1 was classified as beta-lactam resistance; that of cat1, cat2,cmlA, and flo as chloramphenicol resistance; that of aadA1, aadA2,aacC2, strA, strB, and aphA1-IAB as aminoglycoside resistance;

that of dhrfI, dhrfXII, sulI, and sulII as resistance to sulfisoxazole

with or without trimethoprim; and that of tetA, tetB, and tetG as

tetracycline resistance. Isolates with at least one of the genes

associated with resistance to a main antimicrobial group were

classified as resistant. Similarly, isolates with phenotypic resistance

to at least one of the antimicrobial drugs in a main antimicrobial

group were classified as resistant to that particular antimicrobial

drug group. Isolates classified as intermediate based on phenotypic

characterization were assigned to the ‘‘nonresistant’’ category. For

example, isolate FSL S5-383 was phenotypically resistant to

ampicillin and carried gene blaTEM-1, conferring resistance to beta-

lactams; this isolate was thus classified as resistant by both

phenotypic and genotypic typing. To evaluate misclassification

between the phenotypic reference method (i.e., the gold standard)

and the genotypic test method, very major and major error rates

were calculated as described previously (56) according to the

following formulas.

Very major error rate (false susceptible result):

no: of isolates

genotypically susceptible

but phenotypically

resistant

0BBB@

1CCCA|

100

total no: of isolates

phenotypically resistant

Major error rate (false resistant result):

no: of isolates

genotypically resistant

but phenotypically

susceptible

0BBB@

1CCCA|

100

total no: of isolates

phenotypically resistant

Percent categorical agreement:

no: of tests with categorical agreement

total no: of tests

� �|100

For each antimicrobial group, the very major error rate describes

the fraction of truly resistant (i.e., based on phenotype, according

to the NARMS panel) isolates that have been incorrectly classified

as susceptible by genotyping, while the major error rate describes

the fraction of truly susceptible (i.e., based on phenotype,

according to the NARMS panel) isolates that have been incorrectly

classified as resistant by genotyping. Categorical agreement

describes the number of correctly classified tests.

RESULTS

Temporal dynamics of antimicrobial-resistantSalmonella on dairy farms. The 158 bovine Salmonellaisolates included 113 isolates that were obtained over

multiple sampling visits to 20 farms (Table 1). Initial

analyses were thus carried out to characterize antimicrobial

resistance patterns for isolates belonging to a given subtype

that were isolated repeatedly from the same farm. On 10

farms, multiple isolates with the same Salmonella serotype

and ST, but different antimicrobial resistance profiles, were

obtained. For example, on farm 320, one SalmonellaNewport ST11 isolate was sensitive to all antimicrobial

drugs tested, while the other isolate with the same subtype

was resistant to nine antimicrobial drugs (Table 1).

Similarly, on farm 510, MDR Salmonella Newport ST11

was isolated on 18 sampling dates over a 4-month sampling

period (August to December), with 20 of the 21 isolates

representing closely related phenotypic profiles with

TABLE 2. Salmonella serotypes that include drug-resistant isolatesa

Serotype

No. of

STs

No. of

PFGE

types

No. of human isolates No. of bovine isolates

Pansusceptible

Resistant to $1

antimicrobial

drug

Resistance profile

(no. of isolates)b Pansusceptible

Resistant to $1

antimicrobial

drug

Resistance profile

(no. of isolates)

Typhimurium 5 32 19 11 2 Ab (2); 5 Ab

(6); 6 Ab (3)

11 12 5 Ab (5); 6 Ab (2);

8 Ab (2); 9 Ab

(1); 10 Ab (2)

Newport 6 22 11 7 4 Ab (1); 7 Ab

(1); 8 Ab (3);

9 Ab (2)

2 32 8 Ab (21); 9 Ab

(10); 10 Ab (1)

Enteritidis 2 8 21 5 1 Ab (5) 0 0 NA

4,5,12:i:2 2 9 7 3 3 Ab (2), 4 Ab (1) 3 4 8 Ab (4)

Heidelberg 3 4 8 2 3 Ab (1), 9 Ab (1) 0 0 NA

Montevideo 5 6 5 1 2 Ab (1) 3 0 NA

Agona 2 7 0 3 2 Ab (1); 3 Ab (2) 2 3 9 Ab (1); 10 Ab (2)

Muenster 1 6 1 0 NA 5 2 8 Ab (2)

Infantis 1 7 2 1 10 Ab (1) 4 0 NA

Muenchen 2 3 2 1 2 Ab (1) 0 0 NA

Schwarzengrund 1 2 1 2 6 Ab (2) 0 0 NA

4,12:r:2 1 2 0 2 1 Ab (2) 0 0 NA

Bardo 1 2 0 0 NA 0 2 8 Ab (2)

Hadar 1 1 0 2 1 Ab (1); 2 Ab (1) 0 0 NA

Stanley 2 2 1 1 4 Ab (1) 0 0 NA

Rough o:i:1,2 1 1 0 0 NA 0 1 5 Ab (1)

a Details on STs and PFGE types are given in Suppl. Tables 2 and 4.b Resistance profiles are defined here as resistance to different numbers of antibiotics, e.g., ‘‘2 Ab’’ indicates resistance to two antibiotics.

NA, not available.


resistance to eight or nine antimicrobial drugs and one

isolate phenotypically susceptible to all tested drugs. On

farm 524, four MDR Salmonella Newport ST11 isolates

with identical resistance profiles were isolated between July

and September. Overall, MDR Salmonella Newport ST11

was repeatedly isolated on seven farms.

Among the 18 Salmonella 4,5,12:i:2 ST 6 isolates from

farm 261, 7 isolates were sensitive to all antimicrobial drugs

tested, while 11 isolates were resistant to eight antimicrobial

drugs (Table 1). Notably, sensitive isolates were overrepre-

sented at the beginning of sampling (among eight isolates

obtained between 15 June and 10 September, only one was

MDR), while on later sampling dates (15 September through 8

December) only resistant isolates were obtained; the frequen-

cy of MDR isolates in these two time periods was significantly

different (P ~ 0.0011, Fisher’s exact test). PFGE analysis

(55) showed that both resistant and sensitive isolates obtained

before 10 September differed from those obtained after 15

September. Sensitive isolates with ST6 represented PFGE

patterns 89, 91, and 95, which are very similar to PFGE

patterns 90 and 94, which were found among the MDR

isolates; there is only one band difference between patterns 89

and 90 and between patterns 95 and 94, while there is a three-

band difference between patterns 91 and 90 (Soyer et al., 2010

(55); detailed information can be found in Fig. 1B). Since it

cannot be excluded that acquisition of a genetic element

carrying multiple resistance genes has led to the change in

PFGE patterns, it is difficult to determine whether our

findings indicate (i) clonal replacement of a pansusceptible

ST6 strain by an MDR ST 6 strain or (ii) acquisition of an

MDR phenotype by pansusceptible Salmonella strain

4,5,12:i:2 ST 6 with concurrent PFGE type conversion.

The observation that multiple Salmonella isolates

obtained from the same farm at different sampling times

showed the same serotypes, STs, PFGE types, and

antimicrobial resistance profile provides strong evidence

for reisolation of persistent subtypes on given farms. Thus,

only one isolate representing each unique serotype–ST–

PFGE type–resistance profile combination for a given farm

was included in the summary statistics and additional

TABLE 3. PFGE types that were found two or more times and included isolates with phenotypic resistance to at least one antimicrobial drug

PFGE

type no. ST Serotype

Total no.

of isolates

Antimicrobial-resistant profiles found in isolates of indicated origin (no. of isolates)a

Human Bovine

14 4 Schwarzengrund 2 Amp/Cip/Nal/Suf/Tet/Sxt (2) NA

32 36 Enteritidis 6 Nal (5) NA

57 3 Heidelberg 7 Amp/Gen/Suf (1) NA

60 6 Typhimurium 8 Amp/Chl/Kan/Str/Suf/Tet (1);

Amp/Chl/Str/Suf/Tet (3);

Amp/Chl/Str/Suf/Tet/Sxt (1)

Amp/Chl/Str/Suf/Tet (2); Amp/Chl/Kan/

Str/Suf/Tet (1)

76 6 Typhimurium 4 Kan/Tet (1) NA

89 6 Typhimurium,

4,5,12:i:2

8 Gen/Suf/Tet (1); Amc/Amp/

Cef/Fox (1)

NA

90 6 4,5,12:i:2 2 NA Amc/Amp/Cef/Fox/Chl/Str/Suf/Tet (2)

94 6 Typhimurium,

4,5,12:i:2

3 NA Amc/Amp/Cef/Fox/Chl/Str/Suf/Tet (3)

104 8 Typhimurium 5 NA Amp/Kan/Str/Suf (1); Amp/Kan/Str/Suf/

Tet (1); Amc/Amp/Cef/Fox/Cro/Chl/

Kan/Str/Suf/Tet (2); Amc/Amp/Cef/

Fox/Chl/Kan /Str/Suf/Tet (1)

121 11 Newport 13 NA Amc/Amp/Cef/Fox/Chl/Kan/Str/Suf/Tet

(4); Amc/Amp/Cef/Fox/Chl/Str/Suf/


Kan/Str/Suf/Tet (1); Amc/Amp/Cef/

Fox/Kan/Str/Suf/Tet (1)

125 41 Hadar 2 Tet (1); Str/Tet (1) NA

126 11 Newport 14 Amp/Cef/Fox/Chl/Str/Suf/Tet (1);

Amc/Amp/Cef/Fox/Chl/Str/

Suf/Tet (1)

Amc/Amp/Cef/Fox/Chl/Kan/Str/Suf/Tet

(1); Amc/Amp/Cef/Fox/Chl/Str/Suf/


Str/Suf/Tet (2)

127 11 Newport 5 NA Amc/Amp/Cef/Fox/Chl/Str/Suf/Tet (4);

Amc/Amp/Cef/Fox/Chl/Kan/Str/Suf/

Tet (1)

132 11 Newport 2 NA Amc/Amp/Cef/Fox/Chl/Str/Suf/Tet (2)

166 2 Agona 2 NA Amc/Amp/Cef/Fox/Chl/Kan/Str/Suf/Tet

(1); Amc/Amp/Cef/Fox/Chl/IKan/Str/

Suf/Tet/Sxt (1)

168 11 Newport 3 Amc/Amp/Cef/Fox/Cro/Chl/

Str/Suf/Tet (2)

Amc/Amp/Cef/Fox/Chl/Str/Suf/Tet (1)

a NA, not applicable; this indicates that no isolates with antimicrobial resistance were found in the category.


analyses reported below to avoid overrepresentation of a

subtype due to resampling. For example, while 19

Salmonella Newport ST11 isolates with the same resistance

profile were obtained from farm 510 (Table 1), only one of

the isolates with this subtype combination from farm 510

was included in the statistical analyses. This yielded a total

of 97 bovine isolates for inclusion in the analyses reported

below, including in Figures 1 and 2 and Tables 2 and 3.

Drug resistance and multidrug resistance amonghuman and bovine isolates. Among the 275 human and

bovine Salmonella isolates analyzed, 35.6% (n ~ 98) were

phenotypically resistant to at least one of the antimicrobial

drugs tested. When defining multidrug resistance as

resistance to more than one antimicrobial drug, 32.7% (n~ 90) of all isolates could be classified as MDR. Among

the 178 human Salmonella isolates, 23% (n ~ 41) were

phenotypically resistant to one or more antimicrobial drugs,

with a range in resistances from 1 to 10 antimicrobials;

18.5% (n ~ 33) of human isolates could be classified as

MDR. Among the 97 bovine isolates, 58.8% (n ~ 57) were

phenotypically resistant to one or more antimicrobial drug,

with a range in resistances from 4 to 10 antimicrobial drugs

(Fig. 1); a total of 58.8% of bovine isolates could thus also

be classified as MDR. Most MDR Salmonella isolates from

cattle (31.9%; n ~ 31) were resistant to eight antimicrobial

drugs (Fig. 1). Overall, bovine isolates were significantly

more likely than human isolates to be MDR (P , 0.0001;

Fisher’s exact test) or resistant to at least one antimicrobial

drug (P , 0.0001; Fisher’s exact test).

Among the bovine isolates resistant to at least one

antimicrobial drug, 100% showed resistance to streptomy-

cin and sulfisoxazole (Fig. 2). Additionally, these bovine

isolates were commonly resistant to ampicillin (98%),

tetracycline (98%), chloramphenicol (89%), amoxicillin–

clavulanic acid (85%), cefoxitin (84%), and ceftiofur

(84%). Among the human isolates resistant to at least one

antimicrobial drug, resistance to tetracycline (80%) or

sulfisoxazole (70%) was most common (Fig. 2).

Among the 51 serotypes represented in our isolate set, a

total of 16 serotypes included at least one antimicrobial-resistant

isolate (Table 2). Among bovine isolates, antimicrobial-

resistant isolates predominantly grouped into Salmonellaserotypes Newport (32 isolates), Typhimurium (12 isolates),

and 4,5,12:i:2 (4 isolates) (Table 2). Among human isolates,

antimicrobial-resistant isolates predominantly grouped into

Salmonella serovars Typhimurium (11 isolates), Newport (7

isolates), and Enteritidis (5 isolates) (Table 2). Only four

serotypes (Newport, Typhimurium, 4,5,12:i:2, and Agona)

included antimicrobial-resistant isolates from both humans

and cattle (Table 2). Nine serotypes included antimicrobial-

resistant isolates collected only from human cases; two of

those serotypes (Montevideo and Infantis) were also found

among pansusceptible bovine isolates (Table 2).

Presence of antimicrobial resistance genes in thegenomes of antimicrobial-resistant isolates. Among 53

representative isolates, including 48 isolates with resistance

to at least one antimicrobial drug, we found resistance genes

that corresponded to the observed phenotype in all but one

instance. Notably, one human isolate (FSL S5-666) of

serotype 4,5,12:i:2 showed phenotypic resistance to

sulfisoxazole, but PCR failed to detect known sulfisoxazole

resistance genes (i.e., dhrfI, dhrfXII, sulI, and sulII). None of

the five phenotypically pansusceptible isolates contained

FIGURE 1. Prevalence of resistance to one or more antimicrobialdrugs among human and bovine isolates.

FIGURE 2. Prevalence of resistance todifferent antimicrobial drugs among humanand bovine isolates.


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any of the 21 antimicrobial resistance genes investigated in

this study. However, in the genomes of five human isolates

of serotypes Montevideo, Schwarzengrund, Stanley, Hadar,

and Muenchen (FSL S5-403, FSL S5-456, FSL S5-464,

FSL S5-543, and FSL S5-636) phenotypically susceptible to

aminoglycosides (i.e., amikacin, gentamicin, kanamycin,

and streptomycin), we detected genes encoding aminogly-

coside resistance, including (i) aadA1 in one Salmonellaserotype Montevideo (FSL S5-403); (ii) aadA2 in two

isolates representing serotypes Schwarzengrund and Stanley

(FSL S5-456 and FSL S5-464); and (iii) both strA and strBin two isolates of serotype Hadar and Muenchen (FSL S5-

543 and FSL S5-636). Similarly, in the genomes of two

human isolates of serotypes Typhimurium and 4,5,12:i:2

(FSL S5-501 and FSL S5-635), genes encoding resistance to

folate inhibitor pathway antimicrobial (sulI) and tetracycline

antimicrobial (tetB) drugs were present in the absence of

phenotypic resistance to the associated drugs (i.e., sulfisox-

azole with and without trimethoprim, and tetracycline).

Positive PCR results for ampC and blaCMY-2 showed perfect

correlations with phenotypic results, consistent with the fact

that the ampC PCR primers amplify blaCMY-2 (43), which is

a specific ampC allelic variant, and indicating that no ampCgenes other than blaCMY-2 were present among the isolates

tested.

Overall, we observed strong associations between gene

presence and associated antimicrobial resistance profiles.

For example, we found significant associations between (i)

individually, cefoxitin and ceftiofur resistance and the

presence of blaCMY/ampC; (ii) chloramphenicol resistance

and the presence of flo; (iii) kanamycin resistance and the

presence of aphA1-IAB; (iv) ‘‘sulfisoxazole with trimetho-

prim resistance’’ and the presence of dhfrXII; and (v)

tetracycline resistance and the presence of tetA, regardless of

the origin of the isolates (Table 4). We failed to detect a

statistically significant association between phenotypic

resistance to amoxicillin and clavulanic acid and the

presence of blaTEM1, potentially indicating that blaCMY-2/ampC might be the dominant gene encoding amoxicillin and

clavulanic acid resistance profile among bovine isolates.

Among the human isolates only, we failed to detect

statistically significant associations for ceftriaxone resis-

tance and blaCMY-2/ampC, as well as for kanamycin and

aadA1 or aadA2 and for streptomycin and aadA2 or aphA1-IAB, likely due to the very small number of positive isolates.

Similarly, among the bovine isolates only, we failed to

detect statistically significant associations between resis-

tance to amoxicillin and clavulanic acid and the presence of

blaTEM1, as well as between ‘‘sulfisoxazole with trimetho-

prim’’ and sulI, again likely due to the very small number of

positive isolates. Surprisingly, among the bovine isolates we

also failed to detect significant associations between

(i) ampicillin resistance and the presence of blaTEM1 or

blaCMY-2/ampC, (ii) streptomycin resistance and any of the

streptomycin resistance genes, or (iii) sulfisoxazole resis-

tance and sulI or sulII, despite relatively large sample sizes,

potentially indicating the presence of additional resistance

genes among bovine Salmonella isolates (Table 4).

Human ciprofloxacin-resistant Salmonella Schwar-zengrund isolates carry gyrA and parC mutations thathave previously been linked to fluoroquinolone resis-tance. While no bovine isolates were resistant to nalidixic

acid or ciprofloxacin, two human isolates (representing

serotypes Enteritidis and Stanley) were resistant to nalidixic

acid and one human isolate (serotype Schwarzengrund) was

resistant to both nalidixic acid and ciprofloxacin (Suppl.

Table 4). Sequencing of parC and gyrA regions where

mutations have been shown to confer resistance to nalidixic

acid and/or ciprofloxacin showed that the SalmonellaSchwarzengrund isolate carried two gyrA and two parCmutations that had previously been linked to high-level

resistance to fluoroquinolones in human MDR Schwarzen-

grund isolates from Taiwan (7).

NARMS panel results and genotyping results showperfect categorical agreement for beta-lactam andchloramphenicol resistance. Our analyses showed perfect

categorical agreement between phenotypic and genotypic

resistance for beta-lactam and chloramphenicol. In addition,

we found .90% categorical agreement between phenotypic

and genotypic methods for sulfisoxazole with and without

trimethoprim (96.2%) and tetracycline (98.1%). For both

drug classes, as well as aminoglycosides, we found 100%

categorical agreement among the bovine isolates but a very

major error rate of 28.6% among the human isolates. The

sulfisoxazole with and without trimethoprim class showed a

major error rate of 5%. Major error rates for aminoglyco-

sides, sulfisoxazole with and without trimethoprim, and

tetracycline were 28.6, 10, and 14%, respectively. Overall,

the categorical agreement exceeded 90% for the five drug

classes evaluated, except for the aminoglycoside class

(83.3%).

While many genes responsible for drug resistanceare common among bovine and human isolates, thepresence of some resistance genes is associated withisolate source. Three previously described resistance genes

(cat2, cmlA, and aacC2) were not found among any of the

53 isolates tested (Table 5). Notably, dhfr1 and tetB were

found only among human isolates and cat1 was found only

among bovine isolates; these resistance genes were overall

rare among the isolates tested, and none of these three genes

was significantly associated with isolate source (i.e., cattle

or human). We determined overall significant differences in

gene presence between human and bovine isolates for

ampicillin (P ~ 0.0144), sulfisoxazole (P ~ 0.040),

kanamycin (P ~ 0.048), and streptomycin (P , 0.001)

resistance.

MDR isolates of STs 6 and 11 are the most prevalent

among both human and bovine sample sets. MDR

Salmonella represented 14 different STs, including 11 and

5 different STs with MDR isolates found among human and

bovine isolates, respectively. ST6 and ST11 were the two

most prevalent STs among MDR isolates (n ~ 90),

representing 26.7 and 45.5% of all human and bovine

MDR isolates, respectively. ST6 includes serotypes Typhi-

murium, 4,5,12:i:2, and 4,12:i:2 (4,12:i:2 was found once


among bovine isolates) and comprised 19.7% (n ~ 35) of

all 178 human isolates; 40.4% (n ~ 14) of human ST6

isolates were MDR. Among the 97 bovine isolates, 27.7%

(n ~ 24) were ST6; 41.6% (n ~ 10) of these ST6 isolates

were MDR. ST11 includes serotypes Newport and Bardo.

Among human isolates, 5.1% (n ~ 9) were ST11; 78%

(n ~ 7) of these ST11 isolates were MDR. ST11 bovine

isolates comprised 37.1% (n ~ 36) of all bovine isolates;

94% (n ~ 34) of these ST11 isolates were MDR. These

data indicate that two specific clonal groups (i.e., sequence

types), which both include multiple serotypes, represent the

majority of human and bovine MDR.

DISCUSSION

While a number of studies have evaluated antimicrobial

resistance profiles among Salmonella isolates from either

human or animal hosts (14, 17, 33) or among isolates from

both hosts representing the most common serotypes (26,31), limited comparative studies on genotypic and pheno-

typic resistance profiles among diverse Salmonella sero-

types from human and bovine hosts have been reported to

date. In this study, 336 Salmonella isolates from human and

bovine clinical cases representing 51 different serotypes

were evaluated for both genotypic and phenotypic resistance

profiles; the resulting findings provide a number of

important new insights into the diversity and ecology of

human- and bovine-associated antimicrobial drug–resistant

Salmonella strains, as discussed below. These data are

important, as they will help evaluate the importance of

bovine hosts as sources of MDR Salmonella that could be

transmitted to humans, e.g., through food.

Persistence of MDR Salmonella appears to berelatively common on some dairy farms. Among 20

farms for which Salmonella isolates from multiple sample

visits were characterized, 12 farms showed indication for

reisolation of MDR Salmonella strains with identical or

closely related subtypes (based on serotype, multilocus

sequence typing, and PFGE data), including reisolation of

MDR Salmonella Newport ST 11 isolates on seven farms.

These findings are consistent with another report showing

persistence of MDR Salmonella Newport on two farms in

the state of Washington (19). While our data provide

convincing evidence for persistence of MDR Salmonella on

some farms, our data also illustrate that even with use of

multiple subtyping techniques it can be challenging to

TABLE 5. Distribution of antimicrobial resistance genes among 48 representative human and bovine isolates resistant to one or moreantimicrobial drugsa

Gene

No. (%) of isolates with indicated gene

P valuebHuman (n ~ 27) Bovine (n ~ 21) All (n ~ 48)

Beta-lactam resistance genes

blaTEM-1 5 (18.5) 8 (38.1) 13 (27.1)

blaPSE-1 3 (11.1) 2 (9.5) 5 (10.4)

blaCMY-2 8 (29.6) 15 (71.4) 23 (47.9) 0.004

ampC 8 (29.6) 15 (71.4) 23 (47.9) 0.004

Chloramphenicol resistance genes

cat1 0 2 (9.5) 2 (4.2)

cat2 0 0 0

flo 10 (37.0) 17 (81.0) 27 (56.3) 0.0023

cmlA 0 0 0

Aminoglycoside resistance genes

aadA1 7 (25.9) 8 (38.1) 15 (31.3)

aadA2 6 (22.2) 10 (47.6) 16 (33.3)

strA 11 (40.7) 15 (71.4) 26 (54.2) 0.0343

strB 11 (40.7) 14 (66.7) 25 (52.1)

aphA1-IAB 2 (7.4) 13 (61.9) 15 (31.3) ,0.0001

aacC2 0 0 0

Sulfisoxazole/SMZTMP resistance genes

dhfrI 3 (11.1) 0 3 (6.3)

dhfrXII 2 (7.4) 1 (4.8) 3 (6.3)

sulI 12 (44.4) 11 (52.4) 23 (47.9)

sulII 11 (40.7) 15 (71.4) 26 (54.2) 0.0343

Tetracycline resistance genes

tetA 18 (66.7) 18 (85.7) 36 (75)

tetB 3 (11.1) 0 3 (6.3)

tetG 3 (11.1) 2 (9.5) 5 (10.4)

a Isolates were tested for the presence of antimicrobial resistance genes and resistance-conferring genes.b P values (calculated with Fisher’s exact test) that are ,0.05 are given.


differentiate Salmonella persistence and in particular to

differentiate acquisition of resistance genes by a persistent

strain from clonal replacement by a closely related strain.

For example, we identified one instance where we initially

isolated pansusceptible Salmonella strain 4,5,12:i:2 ST 6

over multiple visits to a farm, followed by repeated isolation

of MDR Salmonella strain 4,5,12:i:2 ST 6 over multiple

subsequent visits to the same farm. Even with PFGE

characterization, we could not definitively determine

whether this represents an instance of resistance gene

acquisition or clonal replacement. Further in-depth genetic

characterization of isolates, e.g., through genomic micro-

arrays or whole-genome sequencing, would be necessary to

fully explore this issue.

Common serotypes show considerable phenotypicand genotypic resistance profile diversity. Among the

isolates characterized here, we identified 29 distinct

phenotypic resistance profiles as well as 32 resistance gene

profiles (based on gene presence or absence). SalmonellaTyphimurium showed a total of 12 distinct phenotypic

resistance profiles as well as 13 resistance gene profiles,

indicating considerable resistance gene diversity and a

considerable number of independent gene acquisition events

in this serotype. Consistent with this observation, MDR

Salmonella Typhimurium represented two sequences types,

ST6 and ST8, with ST6 isolated from both humans and

cattle, while ST8 strains were obtained only from cattle. One

group of Typhimurium ST6 PFGE pattern 60 isolates was

characterized by the presence of a set of five resistance

genes (blaPSE-1, flo, aadA2, sulI, and tetG), which had

previously been shown to be located in a class I integron

within Salmonella genomic island 1; this integron has also

been associated with MDR Salmonella Typhimurium

DT104 (34). While flo, aadA2, and sulI were found in a

number of isolates representing different Salmonellaserotypes, both blaPSE-1 and tetG were found only in five

Salmonella Typhimurium ST6 isolates, indicating that the

specific blaPSE-1-flo-aadA2-sulI-tetG class I integron is

limited in its distribution, even though others have reported

its presence in a number of serotypes including serotypes

Agona and Newport (62). All four MDR SalmonellaTyphimurium isolates that were resistant to eight or more

antimicrobial drugs were obtained from animals and

represented ST8 and PFGE pattern 104, which was isolated

only from cattle and has not been found in humans in our

studies (1, 55). These MDR ST8 PFGE pattern 104 isolates

showed the presence of antimicrobial resistance genes

(blaTEM-1, aadA1, strA, strB, sulI, and sulII) previously

described on a chromosomally located type 2 integron in

Salmonella Typhimurium DT193 as well as additional

resistance genes (e.g., tetA, aadA1, and aphAI-IAB) that had

not been mapped to this integron. Two bovine Typhimurium

as well as one rough o:i:2 isolates resistant to four and six

antimicrobial drugs were characterized by the presence of

blaTEM-1, aadA2, aphAI-IAB, sulI, and tetA; the common

co-occurrence of blaTEM-1 and tetA is consistent with the

occurrence of these two genes on a resistance plasmid as

reported by Pasquali et al. (42). Notably, the Salmonella

Schwarzengrund isolate resistant to ciprofloxacin and five

other antimicrobial drugs contained a core set of similar

drug resistance genes (blaTEM-1, aadA2, suI, and tetA),

potentially indicating the presence of a resistance gene

cluster (or clusters) similar to that found in some

Typhimurium isolates.

All four MDR 4,5,12:i:2 isolates characterized here

showed phenotypic resistance profiles and resistance gene

profiles distinct from those of all MDR SalmonellaTyphimurium isolates characterized, even though Salmo-nella 4,5,12:i:2 is closely related to and appears to have

evolved from Salmonella Typhimurium (24). This suggests

independent acquisition of MDR characteristics in

4,5,12,i:2 rather than emergence from an MDR SalmonellaTyphimurium. The three human 4,5,12,i:2 isolates resistant

to three antimicrobial drugs showed three distinct and

unique resistance gene patterns, even though tetB, strA, and

strB (which were found in one isolate that also contained

blaTEM-1 and sulII) had previously been described in a

Salmonella Typhimurium mobile element (39). Notably, in

our isolate set, a bovine 4,5,12:i:2 isolate was resistant to

eight antimicrobial drugs and showed an ACSSuT pheno-

type (i.e., resistance to ampicillin, chloramphenicol, strep-

tomycin, sulfonamides, and tetracyclines) with additional

resistance to ceftiofur and cefoxitin. While this strain thus,

except for ceftiofur and cefoxitin resistance, resembles the

phenotypic resistance profiles previously described for

MDR 4,5,12:i:2 strains isolated in Spain (28), the bovine

MDR 4,5,12:i:2 strain described here carried resistance

genes distinct from those found on a plasmid carried by the

Spanish MDR isolates (i.e., sulI, cmlA, tetA, aadA, and

blaTEM) (28), consistent with previous findings indicating

that Spanish and North American 4,5,12:i:2 strains may

have emerged independently.

All 32 bovine MDR Salmonella Newport isolates

represented ST11, and MDR isolates with this ST also

represented 50% of human Salmonella Newport isolates,

suggesting potential transmission of this MDR Salmonellasubtype between cattle and humans; this is also supported

by other studies (8, 29). As Salmonella Newport ST11 is the

most common bovine-associated ST, it is also tempting to

speculate that cattle may represent a reservoir for these

MDR strains (1, 30); further studies with larger isolate

collections representing different source populations will be

important, though, to identify the reservoirs of the different

Salmonella Newport phylogenetic lineages that have been

identified (49). While MDR Salmonella Typhimurium and

closely related serotypes (e.g., 4,5,12,i:2) represented a

diverse population as discussed above, MDR Newport

strains were found to be more homogeneous, possibly

suggesting evolution from a single ancestor, followed by

clonal dispersion, at least in cattle in New York. This is

supported by the fact that all 35 MDR Salmonella Newport

isolates represented the same ST and by the observation that

all isolates characterized by antimicrobial resistance gene

PCR showed the same core set of six resistance genes

(blaCMY-2, flo, strA, strB, sulII, and tetA; only one isolate

lacked strB, and another lacked blaCMY-2); interestingly, this

resistance gene profile is distinct from the previously


described SG1 resistance gene island in Salmonella New-

port isolates collected in France between 2000 and 2002

(21). As Poppe et al. reported that blaCMY-2, flo, strA, strB,sulII, and tetA resistance genes were located on 80- to

90-MDa plasmids that were self-transmissible at a high

frequency (45), we propose that the MDR SalmonellaNewport strains here may have acquired all or parts of this

plasmid. A total of four MDR Salmonella Newport isolates

(three bovine and one human) in our study carry additional

resistance genes (i.e., aphAI-IAB, cat1, and aadA1),

suggesting acquisition of additional resistance genes, e.g.,

through insertion of gene cassettes, such as a type 1

integron, that contain the aadA1 gene, as previously

reported for two Salmonella Newport isolates (60).

Genotyping can reliably predict phenotypic resis-tance to amoxicillin–clavulanic acid, cefoxitin, ceftiofur,chloramphenicol, and kanamycin, but among bovineisolates phenotypic resistance to ampicillin, streptomy-cin, and sulfisoxazole appears to be encoded byadditional resistance genes. Consistent with previous

studies (14, 23, 43), we found excellent agreement between

(i) presence of blaCMY-2 ampC and resistance to amoxicil-

lin–clavulanic acid, cefoxitin, and ceftiofur, (ii) presence of

flo and resistance to chloramphenicol, and (iii) presence of

aphAI-IAB and resistance to kanamycin. Notably, in our

study chloramphenicol resistance was linked to the presence

of flo, while this resistance was linked to the presence of

cat1 and/or cat2 in isolates obtained from retail meats in

China (14) and pigs in Kenya (33). Consistent with our

results, a study in Wisconsin recently showed that 45 cattle

isolates, resistant to chloramphenicol, carried floR but not

cat1 or cat2 (38). While the cat and flo genes are highly

mobile, cat genes are typically plasmid-borne genes and flogenes have been found in plasmids and genomic islands (3).This may complicate genotypic analysis for chloramphen-

icol resistance, since one gene might not be the only

resource of the resistant profile. In contrast to the findings

of our study, Chen et al., 2004 (14) showed a strong

association between the presence of the aacC2 gene and

antimicrobial resistance profiles of gentamicin and kanamy-

cin, while we did not detect the aacC2 gene in our isolates

among either gentamicin- or kanamycin-resistant isolates.

Especially in clinical laboratories, rapid determination

of sensitivity profiles is of paramount importance. We found

that categorical agreement between genotyping results and

phenotyping results generated by using the NARMS panel

for beta-lactam, chloramphenicol, sulfisoxazole with and

without trimethoprim, and tetracycline exceeded 90%,

indicating excellent agreement. However, in our analysis

the strength of association differed among resistance genes

and Salmonella host species. Therefore, instead of using one

gene to predict the phenotypic antimicrobial-resistant

profiles, we suggest to use groups of genes to predict

antimicrobial-resistant groups, since multiple genes can

generate equivalent resistance phenotypes. However, our

findings are based on a limited number of isolates from two

host species collected from the same geographical region

during a 1-year time span. Validation of our study results

with a larger isolate set including isolates from different host

sources and geographical regions is therefore clearly

needed. Also, our study was necessarily limited to a

relatively small number of genes and based on known

resistance genes among Salmonella. Our study excluded

resistance genes that are found in closely related organisms

that could easily transfer genes to Salmonella, such as

commensal Escherichia coli (43). Classification of isolates

as susceptible based on the absence of known antimicrobial

resistant genes must be interpreted carefully, because the

antimicrobial resistance mechanisms in the organisms might

be affected by more than one gene or set of genes or there

might be new genes acquired by Salmonella isolates. Use of

highly parallel screening approaches for larger sets of

resistance genes (e.g., based on arrays (9, 10, 35)) will likely

address some of these issues.

Our data support that while MDR Salmonella strains that

cause human clinical disease can originate from a variety of

sources, cattle likely represent an important reservoir of MDR

Salmonella subtypes that are associated with human disease.

While use of gene-based screening approaches for resistance

genes, in addition to phenotypic assays, has considerable

potential to further our understanding of MDR Salmonellaemergence and diversity as well as for diagnostic applications,

whole-genome sequencing approaches are likely to become

increasingly used for characterization of MDR isolates and

will further improve our understanding of MDR strain

diversity across different serotypes and host sources.

ACKNOWLEDGMENTS

Support for this project was provided by the National Institute of

Allergy and Infectious Diseases, National Institutes of Health, Department

of Health and Human Services, under contract numbers N01-AI-30054-

ZC-006-07 and N01-A1-30055. K.H. was supported by Morris Animal

Foundation Fellowship Training Grant D08FE-403.

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Antimicrobial Drug Resistance Patterns among Cattle- and Human-Associated Salmonella Strains

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