57 Journal of Food Protection, Vol. 77, No. 1, 2014, Pages 57–66 doi:10.4315/0362-028X.JFP-13-221

Quantification, Serovars, and Antibiotic Resistance of Salmonella Isolated from Retail Raw Chicken Meat in Vietnam YEN T. TA,1 TRUNG THANH NGUYEN,1 PHUONG BICH TO,1 DA XUAN PHAM,1 HAO THI HONG LE,1 GIANG NGUYEN THI,1 WALID Q. ALALI,2* ISABEL WALLS,3 AND MICHAEL P. DOYLE2 1National

Institute for Food Control, Ha Noi, the Socialist Republic of Vietnam; 2Center for Food Safety, University of Georgia, Griffin, Georgia 30223, USA; and 3U.S. Department of Agriculture, National Institute of Food and Agriculture, Washington, D.C. 20250, USA MS 13-221: Received 30 May 2013/Accepted 8 September 2013

ABSTRACT The objectives of this study were to quantify Salmonella counts on retail raw poultry meat in Vietnam and to phenotypically characterize (serovars and antibiotic resistance) the isolates. A total of 300 chicken carcasses were collected from two cities and two provinces in Vietnam. Salmonella counts on the samples were determined according to the most-probable-number (MPN) method of the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). A total of 457 isolates were serotyped and tested for antibiotic susceptibility. Overall, 48.7% of chicken samples were Salmonella positive with a count of 2.0 log MPN per carcass. There were no significant differences (P . 0.05) in log MPN per carcass by the study variables (market type, storage condition, and chicken production system). There was a significant difference (P , 0.05) in Salmonella-positive prevalence by chicken production system. Among the 22 Salmonella serovars identified, Albany was the most frequent (34.1%), followed by Agona (15.5%) and Dabou (8.8%). Resistance to at least one antibiotic was common (i.e., 73.3%), with high resistance to tetracycline (59.1%) and ampicillin (41.6%). Resistance to three antibiotics was the most frequently found multidrug resistance profile (17.7%, n ~ 81); the profile that was resistant to the highest number of drugs was resistant to nine antibiotics (0.7%, n ~ 3). Only Salmonella Albany posed phenotypic resistance to ceftriaxone (a drug of choice to treat severe cases of salmonellosis). The data revealed that, whereas Salmonella prevalence on raw poultry was high (48.7%), counts were low, which suggests that the exposure risk to Salmonella is low. However, improper storage of raw chicken meat and cross-contamination may increase Salmonella cell counts and pose a greater risk for infection. These data may be helpful in developing risk assessment models and preventing the transmission of foodborne Salmonella from poultry to humans in Vietnam.

One of the principal foodborne pathogens, Salmonella causes illness in humans and is frequently carried in the intestinal tract of food animals (5, 12, 15, 19, 32). An estimated 93 million cases of gastroenteritis, and 155,000 deaths, occur annually worldwide due to nontyphoid Salmonella infections (24). Many studies have reported cases of human salmonellosis associated with contaminated food such as raw meats, eggs, dairy products, and vegetables (15, 19, 31). Even though contaminated fresh vegetables and low-moisture food products are increasingly a concern for Salmonella transmission to humans, poultry and poultry products continue to be a significant source of human salmonellosis (4, 19, 29, 31, 32). Unlike most developed countries, Vietnam does not have a nationwide Salmonella surveillance program to monitor foodborne disease in the human population or pathogen levels on processed and retail meats. There are a few published studies with limited data on the prevalence and characterization of Salmonella isolates from live poultry and poultry meat in Vietnam (2, 23, 30, 37, 41, 42). * Author for correspondence. Tel: 770-467-6066; Fax: 770-229-3216; E-mail: [email protected].

However, there are no data on Salmonella cell counts on raw chicken meat in Vietnam. Since risk to human health is based on a dose-response relationship, low pathogen counts might pose a low risk even though the prevalence is high (33). Therefore, count data are helpful in assessing the risk of salmonellosis associated with exposure to poultry meat in Vietnam. Information on frequency of common and unique Salmonella serovars on raw poultry meat is also limited in Vietnam in terms of number of isolates serotyped or tested and their geographical distribution (2, 23, 37, 42). More than 2,500 Salmonella enterica serovars have been identified worldwide based on their antigenic formula (17). The distribution and frequency of Salmonella serovars may differ depending on the geographical world region. For instance, Salmonella serovars Enteritidis and Infantis are commonly reported in food animals in Europe, whereas Anatum and Rissen are more frequently detected in food animals in Asia (7, 12, 13). Data on antibiotic-resistant Salmonella are needed to assess the potential impact on public health of multidrugresistant isolates (those with resistance to three or more classes of antibiotics) from raw poultry meat. Antibiotic

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resistance development and transmission is a global problem (26, 27, 31, 35, 44). Single- and multidrug-resistant Salmonella have been isolated from food animals and can be transmitted to humans through direct contact with animals or indirectly through consumption of contaminated foods such as poultry products (3, 14, 18, 31, 44). Resistance to at least one antibiotic was found in 88.9% (n ~ 30) of Salmonella isolates from raw chicken meat in Vietnam, whereas 27.8% (n ~ 30) of the isolates were resistant to at least two antibiotics (41). The most frequent resistance was to tetracycline, ampicillin, chloramphenicol, streptomycin, nalidixic acid, trimethoprim, and sulphonamides (41). We determined the baseline Salmonella prevalence on raw poultry in five cities and seven provinces in Vietnam in 2011 (36). In the present study, our goals were to (i) quantify Salmonella counts on raw poultry at retail markets in two cities and two provinces with high, medium, and low Salmonella prevalence based on data from our previous study (36) and (ii) phenotypically characterize (serovars and antibiotic resistance) Salmonella isolates from both the previous and the current study to have a better geographical representation of Vietnam. MATERIALS AND METHODS Sample collection. Based on the Salmonella prevalence data from our previous study (36), 300 whole chicken carcasses were collected from two cities (i.e., Ha Noi and Ho Chi Minh) and two provinces (Phu Tho and Lam Dong) in Vietnam to determine Salmonella counts and to phenotypically characterize (serovar and antibiotic susceptibility) isolates. The geographical distribution of the sampling scheme in this study was based on the Salmonella prevalence data on retail chicken from the previous study (36) (i.e., target sampling). Therefore, the cities and provinces with historically high (Ha Noi city), medium (Ho Chi Minh city and Phu Tho province), and low (Lam Dong province) Salmonella prevalence were sampled again for the purpose of quantifying this pathogen. Of these, 270 samples were collected from wet markets and 30 samples from supermarkets. Most (,90%) of chicken meat in Vietnam is sold at wet markets (36). Wet markets (traditional markets) are open markets in which poultry, fish, pork, beef, fruit, and vegetables are all sold. Most of the chicken meat sold in wet markets is stored at ambient temperatures (15 to 35uC); however, most of the meat sold in Ho Chi Minh was at chilled temperatures (2 to 8uC). Supermarkets are large, nationally recognized chain stores that sell mostly chilled Vietnamese chickens. Within each city or province, districts and markets were selected randomly. The number of samples per city or province, and then district, was based on the relative population size of the selected cities, provinces, and districts of Vietnam, as described previously (36). Chicken carcasses at retail markets were stored at either ambient or chilled temperatures. Chicken meat was produced by nonintegrated local farmers (i.e., locally raised) and by integrated poultry companies (conventionally raised). Locally raised chickens were backyard broilers raised by nonintegrated small-volume producers. These birds were free-range, supplemented with nonmedicated feed, and were usually processed by butchers at the wet markets. Conventionally raised chickens were broilers raised by integrated large poultry companies, kept in enclosed houses during growout, supplemented with feed that may contain antibiotics, and processed at high-volume processing plants. Chicken samples collected in

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Lam Dong province and Ho Chi Minh city were analyzed at the local laboratories, whereas samples from both Ha Noi and Phu Tho were analyzed at the National Institute for Food Control (NIFC, Ha Noi, Vietnam). Salmonella isolates from Lam Dong province and Ho Chi Minh city were shipped to NIFC for further Salmonella phenotypic analyses. Salmonella quantification using the MPN method. The three-tube, three-dilution most-probable-number (MPN) technique was used to quantify Salmonella counts following the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) protocol (39). Each chicken sample was aseptically placed in a sterile bag, and 400 ml of buffered peptone water (BPW; Difco, BD, Sparks, MD; all broths and agars mentioned in this section were obtained from Difco, BD) was poured into the bag. The sample was rinsed with a rocking motion for 1 min. A series of three-tube, three-dilution tubes was set up as follows: three empty tubes, three tubes containing 9 ml of BPW, and three tubes with 9.9 ml of BPW. A 10-ml aliquot of the chicken rinse was added to each of the three empty tubes, a 1-ml aliquot to each of the 9-ml BPW tubes, and a 0.1-ml aliquot to each of the 9.9-ml BPW tubes. All nine tubes were held at 37uC for 20 to 24 h. Portions of 0.5 and 0.1 ml of these preenrichment solutions were transferred to 10 ml of tetrathionate-Hajna broth and 10 ml of Rappaport Vassiliadis broth, respectively; these were incubated at 42uC for 22 to 24 h. After incubation, one loopful of tetrathionateHajna and Rappaport Vassiliadis broth cultures were streaked onto xylose lysine Tergitol 4 agar and brilliant green sulfa agar plates and were incubated at 37uC for 20 to 24 h. Up to three typical presumptive Salmonella colonies were selected per plate, inoculated onto triple sugar iron and lysine iron agar slants, and incubated at 37uC for 22 to 24 h. Isolates with typical Salmonella colony biochemical reactions were confirmed by agglutination with Salmonella Poly-O antisera. Salmonella-positive BPW tubes were confirmed, and the MPN results per ml of rinsate were determined according to the USDA-FSIS MPN table (39). Salmonella Enteritidis ATCC 13076 and Escherichia coli ATCC 25922 were used as positive control and negative control reference strains for Salmonella number determinations, antibiotic susceptibility testing, and serotyping analyses. Serotyping. A total of 457 isolates were serotyped at the NIFC Laboratories, of which 347 isolates were from the banked collection obtained from the previous study (36) and 110 were from this study to provide a wider geographical distribution of isolate sources. Isolates were serotyped by slide agglutination following the protocol described in the Salmonella serotyping guide (9), and serovars were identified based on the somatic (O) and flagellar (H) antigen formula of the Kauffmann-White scheme (9, 17). The O and H antisera were manufactured by Bio-Rad (Marnes-la-Coquette, France) and Sifin (Berlin, Germany), respectively. Antibiotic susceptibility testing. All isolates (n ~ 457) were tested for antibiotic susceptibility using the disk agar diffusion method according to the laboratory protocol of the World Health Organization Global Foodborne Infection Network (45). Fourteen antibiotics representative of eight classes of drugs were used in this study (Table 1). All antibiotics were obtained from Difco, BD, except for ceftriaxone and cefepime, which were obtained from Nam Khoa (Ho Chi Minh, Vietnam). The antibiotic resistance results were interpreted as susceptible, intermediate, or resistant based on the standard breakpoints recommended by Clinical and Laboratory Standards Institute standard M100 (Table 1) (8). Intermediate MIC results were reclassified as susceptible.

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TABLE 1. Antibiotic disk diffusion zone diameter range and interpretation of results for antibiotics tested in this study Zone diam standard breakpoints (mm)a Abbreviation

Disk content (mg)

S

I

R

Aminoglycosides Gentamicin Kanamycin

GEN KAN

10 30

$15 $18

13–14 14–17

#12 #13

Cephalosporins (2nd generation) Cefuroxime

CXM

30

$23

15–22

#14

Cephalosporins (3rd and 4th generation) Cefixime Ceftriaxone Cefepime

CFM CRO FEP

5 30 30

$19 $23 $18

16–18 20–22 15–17

#15 #19 #14

Fluoroquinolones Ciprofloxacin Ofloxacin

CIP OFX

5 5

$21 $16

16–20 13–15

#15 #12

b-Lactamase combination Amoxicillin-clavulanate

AMC

20/10

$18

14–17

#13

Penicillins Ampicillin

AMP

10

$17

14–16

#13

Tetracyclines Tetracycline

TET

30

$15

12–14

#11

Phenicols Chloramphenicol

CHL

30

$18

13–17

#12

Folate pathway inhibitors Trimethoprim Sulfamethoxazole-trimethoprim

TMP SXT

5 23.75/1.25

$16 $16

11–15 11–15

#10 #10

Antibiotic

a

S, sensitive; I, intermediate; R, resistant.

Statistical analysis. The MPN data per ml were adjusted to the original rinse volume (400 ml) per carcass and then log transformed to approximate normality. Only MPN per carcass values that met or exceeded the limit of detection (i.e., 12 salmonellae per carcass) were used in the analysis. The relationship between the log MPN per carcass and the study variables (market type [wet market and supermarket], storage condition [ambient and chilled], and chicken production system [locally and conventionally raised]) was determined using a generalized linear model, with identity link function and adjustment for dependency within city using generalized estimated equations in STATA software version 10.1 (Stata Corp., College Station, TX). A difference was considered statistically significant at P , 0.05. The Salmonella prevalence data (presence of Salmonella at any count per total number of chicken carcasses tested) were cross-tabulated, with each of the study variables using a Fisher’s exact test or 2 | n likelihood ratio chisquare test, as appropriate, in STATA. Fisher’s exact test was used when the number of isolates in the contingency table was small (i.e., when the expected values in any of the cells of a contingency table were below 5). Otherwise, the chi-square test was used. Salmonella serovars were cross-tabulated by market type, storage condition, and chicken production system. The proportion of isolates resistant to each antibiotic was compared by market type, storage condition, production system, and serovar using either Fisher’s exact test or 2 | n likelihood ratio chi-square test, as appropriate, in STATA. Multidrug resistance (of 14 antibiotics) was compared by market type, storage condition, production system, and serovar using m | n likelihood ratio chi-square test.

For the purpose of data analysis, serovars that were not among the five most frequent serovars were collapsed into one category (‘‘other serovars’’).

RESULTS The overall Salmonella prevalence was 48.7% (n ~ 300) on chicken carcasses from retail markets for the two cities and two provinces included in this study. There were no significant differences in Salmonella prevalence (P . 0.05) by storage condition and market type. However, the prevalence on locally raised chickens (45.6%) was significantly (P , 0.05) lower than that of conventionally raised chickens (60.7%) (Table 2). The overall mean Salmonella count (log MPN per carcass) was 2.0 (95% confidence interval: 1.9 to 2.1). Sixty-six percent of retail chicken samples were contaminated, with Salmonella counts that ranged from 1.0 to 2.0 log MPN per carcass; 23% of these samples were contaminated with 2.1 to 3.0 log MPN per carcass and 11% with from 3.1 to 3.8 log MPN per carcass (Fig. 1). There were no significant differences (P . 0.05) in Salmonella counts by storage condition, market type, or chicken production system (Table 2). A total of 22 Salmonella serovars were identified among the 457 isolates; of these, the 5 most frequently identified were Albany, Agona, Dabou, Hadar, and Indiana (34.1, 15.5, 8.8, 6.8, and 6.1%, respectively) (Table 3).

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TABLE 2. Salmonella prevalence and mean log MPN values on chicken carcasses by study variables obtained from supermarkets and wet markets in Vietnam a Variable

Market type Supermarket (n ~ 30) Wet market (n ~ 270) Storage condition Chilled (n ~ 145) Ambient (n ~ 155) Chicken production system Locally raised (n ~ 239) Conventionally raised (n ~ 61) a

No. (%) of Salmonella-positive samples

13 (43.3) 133 (49.3)

A

69 (47.6) 77 (49.7)

A

109 (45.6) 37 (60.7)

A

A

A

B

Log MPN/carcass (95% CI)

1.6 (1.2–2.0) 2.0 (1.9–2.2)

A

2.0 (1.8–2.2) 2.0 (1.9–2.2)

A

2.1 (1.9–2.2) 1.8 (1.6–1.9)

A

A

A

A

n ~ 300 chickens. CI, confidence interval. Values in columns (percentages or log MPN per carcass) within each variable that are followed by the same letter are not significantly different (P . 0.05).

There was greater diversity in Salmonella serotypes in Ha Noi chicken samples compared with other cities and provinces (data not shown). The serovar distribution by market type, storage condition, and chicken production system is shown in Table 3. The top five serovars were, in general, more commonly identified in samples collected from wet markets than in those from supermarkets. The phenotypic resistance profiles of Salmonella isolates by market type, storage condition, and chicken production system are shown in Table 4, and by serovar in Table 5. All Salmonella isolates were susceptible to cefepime and amoxicillin-clavulanate. The highest proportion of single resistance was to tetracycline (59.1%), ampicillin (41.6%), chloramphenicol (37.4%), trimethoprim (34.6%), and sulfamethoxazole-trimethoprim (34.6%) (Table 4). The proportions of resistant Salmonella phenotypes to each antibiotic were not significantly different (P . 0.05) by the study variables, except for resistance to tetracycline by storage condition (ambient versus chilled) (P , 0.05) (Table 4). Salmonella serovar Albany was highly resistant to multiple antibiotics compared with other serovars (Table 5). Other serovars expressed resistance to tetracycline, FIGURE 1. Percentage bar chart illustrating the log most-probable-number distribution of Salmonella on broiler chicken carcasses at retail stores in Vietnam.

chloramphenicol, trimethoprim, and sulfamethoxazole-trimethoprim. The distribution of the overall phenotypic resistance to up to nine antibiotics among the Salmonella isolates is shown in Figure 2. The multidrug resistance phenotypes (up to nine) by serovars are shown in Table 6. Approximately 27% of the isolates were susceptible to all antibiotics, 56% were resistant to one to four drugs, and 18% were resistant to five to nine drugs. Overall, there was a significant (P , 0.05) difference among the proportions of multidrugresistant isolates by serovars (Table 6). Three serovars, Albany, Indiana, and Infantis (one of the serovars included in the ‘‘other serovars’’ group), had at least one isolate in the single and multidrug-resistant profiles (Table 6). DISCUSSION The overall Salmonella prevalence in retail fresh chicken obtained in the two provinces and two cities included in this study (i.e., 48.7%) was similar to the overall prevalence reported in our previous study (45.9%) (36). The prevalence of Salmonella on chicken carcasses in Vietnam was higher than that reported in several developing

a

156 71 40 31 28 25 24 14 11 10 9 8 8 7 4 3 2 2 1 1 1 1

(34.1) (15.5) (8.8) (6.8) (6.1) (5.5) (5.3) (3.1) (2.4) (2.2) (2.0) (1.8) (1.8) (1.5) (0.9) (0.7) (0.4) (0.4) (0.2) (0.2) (0.2) (0.2)

Subtotal (n ~ 457)

142 63 34 30 23 23 20 12 11 10 9 7 4 7 4 3 1 2 1 1 1 1

(34.7) (15.4) (8.3) (7.3) (5.6) (5.6) (4.9) (2.9) (2.7) (2.4) (2.2) (1.7) (1.0) (1.7) (1.0) (0.7) (0.2) (0.5) (0.2) (0.2) (0.2) (0.2)

Wet market (n ~ 409)

(29.2) (16.7) (12.5) (2.1) (10.4) (4.2) (8.3) (4.2) — — — 1 (2.1) 4 (8.3) — — — 1 (2.1) — — — — —

14 8 6 1 5 2 4 2

Supermarket (n ~ 48)

(28.3) (21.5) (4.30) (3.6) (3.9) (6.5) (7.2) (3.6) (3.9) (3.6) (3.2) (2.5) (1.4) (2.5) (1.4) (1.1) (0.4) — — 1 (0.4) 1 (0.4) 1 (0.4)

79 60 12 10 11 18 20 10 11 10 9 7 4 7 4 3 1

Ambient (n ~ 279)

1 2 1

1 4

77 11 28 21 17 7 4 4

(43.3) (6.2) (15.7) (11.8) (9.6) (3.9) (2.3) (2.3) — — — (0.6) (2.3) — — — (0.6) (1.2) (0.6) — — —

Chilled (n ~ 178)

Storage condition

Ambient storage conditions, 15 to 35uC; chilled storage conditions, 2 to 8uC. —, an isolate was not detected.

Albany Agona Dabou Hadar Indiana Typhimurium Rissen London Magherafelt Chester Enteritidis Infantis Muenster Stanley Bousso IV Derby Montevideo Newport Give Kunduchi Schleissheim Thompson

Serovar

Market type

No. (%) of isolates

(33.3) (13.7) (10.5) (7.8) (6.9) (4.6) (4.9) (2.9) (2.0) (1.6) (1.3) (2.3) (2.6) (2.0) (1.0) (0.7) (0.7) (0.7) — 1 (0.3) — 1 (0.3)

102 42 32 24 21 14 15 9 6 5 4 7 8 6 3 2 2 2

Local (n ~ 306)

1

1

1 1 1

54 29 8 7 7 11 9 5 5 5 5 1

(35.8) (19.2) (5.3) (4.6) (4.6) (7.3) (6.0) (3.3) (3.3) (3.3) (3.3) (0.7) — (0.7) (0.7) (0.7) — — (0.7) — (0.7) —

Conventional (n ~ 151)

Chicken production system

TABLE 3. Salmonella serovar distribution on raw poultry collected at two retail markets, two storage conditions, and two chicken production systems in Vietnam a

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b

a

(5.7) (3.1) (0.4) (0.4) (0.2) — 16 (3.5) 16 (3.5) — 190 (41.6) 270 (59.1) 171 (37.4) 158 (34.6) 158 (34.6)

26 14 2 2 1

Total (n ~ 457)

(5.4) (3.2) (0.5) (0.5) (0.2) — 14 (3.4) 14 (3.4) — 169 (41.3) 240 (58.7) 147 (35.9) 140 (34.2) 140 (34.2)

22 13 2 2 1

Wet market (n ~ 409)

4 (8.3) 1 (2.1) — — — — 2 (4.2) 2 (4.2) — 21 (43.8) 30 (62.5) 24 (50.0) 18 (37.5) 18 (37.5)

Supermarket (n ~ 48)

0.338 1.000 1.000 1.000 1.000 — 0.680 0.680 — 0.747 0.611 0.057 0.652 0.652

P valueb

(4.7) (3.2) (0.7) (0.7) (0.4) — 7 (2.5) 7 (2.5) — 115 (41.2) 152 (54.5) 107 (38.4) 98 (35.1) 98 (35.1)

13 9 2 2 1

Ambient (n ~ 279)

13 (7.3) 5 (2.8) — — — — 9 (5.1) 9 (5.1) — 75 (42.1) 118 (66.3) 64 (36.0) 60 (33.7) 60 (33.7)

Chilled (n ~ 178)

Storage condition

0.234 0.801 0.523 0.523 1.000 — 0.149 0.149 — 0.846 0.012 0.606 0.756 0.756

P value

(5.2) (3.3) (0.7) (0.7) (0.3) — 11 (3.6) 11 (3.6) — 125 (40.9) 185 (60.5) 113 (36.9) 100 (32.7) 100 (32.7)

16 10 2 2 1

Local (n ~ 306)

10 (6.6) 4 (2.7) — — — — 5 (3.3) 5 (3.3) — 65 (43.1) 85 (56.3) 58 (38.4) 58 (38.4) 58 (38.4)

Conventional (n ~ 151)

0.545 1.000 1.000 1.000 1.000 — 0.877 0.877 — 0.654 0.394 0.758 0.226 0.226

P value

Chicken production system

—, Salmonella isolate was susceptible to the antibiotic. P values are based on the Pearson chi-square or Fisher’s exact test, as appropriate. There were no significant differences of resistance phenotypes of Salmonella to each antibiotic by the study variables (market type, storage conditions, and chicken production system), except for resistance to tetracycline by storage condition (P , 0.05).

Gentamicin Kanamycin Cefuroxime Cefixime Ceftriaxone Cefepime Ciprofloxacin Ofloxacin Amoxicillin-clavulanate Ampicillin Tetracycline Chloramphenicol Trimethoprim Sulfamethoxazole-trimethoprim

Antibiotic

Market type

No. (%) of resistant isolatesa

TABLE 4. Antibiotic resistance profiles of Salmonella isolates on raw poultry collected at two types of retail markets, two storage conditions, and two chicken production systems in Vietnam

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,0.001 0.025 1.000 1.000 1.000 — ,0.001 ,0.001 — ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

c

b

a

6 25 4 6 6 16 28 17 20 20 79 97 74 67 67

2 2

—, Salmonella isolate was susceptible to the antibiotic. Serovars that were not among the five most common serovars were collapsed into one category (i.e., other serovars) and represented 28.7% of the total. P values are based on a likelihood ratio of chi-square or Fisher’s exact test of the differences in % of resistance by serovar.

61 73 51 42 42

3 3

(7.6) (3.8) (0.8) (0.8) — — (2.3) (2.3) — (46.6) (55.7) (38.9) (32.1) (32.1) 10 5 1 1

13 (46.4) 4 (14.3) — — — — 11 (39.3) 11 (39.3) — 20 (71.4) 24 (85.7) 22 (78.6) 16 (57.1) 16 (57.1) — 1 (3.2) — — — — — — — 8 (25.8) 23 (74.2) 3 (9.7) 7 (22.6) 7 (22.6) — — — — — — — — — (15.0) (62.5) (10.0) (15.0) (15.0) — — — — — — — — — (22.5) (39.4) (23.9) (28.2) (28.2) (1.9) (2.6) (0.6) (0.6) (0.6) — (1.3) (1.3) — (50.6) (62.2) (47.4) (43.0) (43.0) 3 4 1 1 1

(5.7) (3.1) (0.4) (0.4) (0.2) — 16 (3.5) 16 (3.5) — 190 (41.6) 270 (59.1) 171 (37.4) 158 (34.6) 158 (34.6) 26 14 2 2 1

Gentamicin Kanamycin Cefuroxime Cefixime Ceftriaxone Cefepime Ciprofloxacin Ofloxacin Amoxicillin-clavulanate Ampicillin Tetracycline Chloramphenicol Trimethoprim Sulfamethoxazole-trimethoprim

Hadar (n ~ 31) Dabou (n ~ 40) Agona (n ~ 71) Albany (n ~ 156) Total (n ~ 457) Antibiotic

No. (%) of resistant Salmonella serovarsa

TABLE 5. Antibiotic resistance profiles of the five most common Salmonella serovars on chicken meat in Vietnam

Indiana (n ~ 28)

Other serovars (n ~ 131)b

P valuec

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countries such as Colombia (27%; n ~ 1,003), Russia (31.5%; n ~ 698), and Thailand (18.7%; n ~ 75), as well as in developed countries such as the United States (5.2%; n ~ 3,275) and the United Kingdom (3.6%; n ~ 401) (1, 6, 10, 12, 40). Moreover, Salmonella prevalence in our study was also higher than that reported in other studies on chicken meat in Vietnam. Approximately 43% (n ~ 268) Salmonella prevalence was reported by Thai et al. (37) on chicken meat samples (25 g) collected from the Red River Delta region (Northern Vietnam), whereas Phan et al. (30) determined that 21% (n ~ 202) of chicken meat samples (25 g) in the Mekong River Delta region (Southern Vietnam) were Salmonella positive. In general, the limitation in the number of sampling locations, number of samples, sampling scheme, and Salmonella analytical method might have contributed to the differences in the findings among these studies (20). There are reports of several studies on the prevalence of Salmonella on raw poultry in Vietnam, but there are no reported data on Salmonella counts (2, 23, 37, 38, 41, 42). In this study, the mean Salmonella count on chicken meat collected from retail markets in Vietnam was 2.0 log MPN per carcass. This count is slightly higher than that reported on postchilled chickens at processing plants in the United States (mean log 1.75 MPN per carcass; n ~ 3,275) and higher than the Salmonella count on fresh retail chicken in The Netherlands, where 89% of fresh (nonfrozen chickens) had less than 1 log Salmonella per carcass (11, 40). As for the log MPN distribution (Fig. 1) compared with U.S. data, 66% of samples were contaminated with Salmonella counts that ranged from 1.0 to 2.0 log MPN per carcass in this study versus 3.7% of U.S. samples in the same range; 23% of our samples were contaminated with 2.1 to 3.0 log Salmonella MPN per carcass versus 1.2% of U.S. samples, and 11% of samples from 3.1 to 3.8 log MPN per carcass versus 0.27% of U.S. samples fell in that range (40). In Vietnam, most chickens are processed on-site at wet markets and sold immediately (the same day); thus, chickens have a short shelf life (time from slaughter to consumption). Foodborne salmonellosis risk to humans largely depends on the numbers of Salmonella cells on food (e.g., poultry meat); therefore, low counts of this pathogen may pose a lower risk even though the prevalence is high (i.e., 46%) (33). However, mishandling of raw poultry can increase the numbers of Salmonella cells on chicken meat and in the surrounding food preparation environment, thereby increasing the risk of foodborne illness. Salmonella Albany was the most common serovar (34.1%) isolated from chicken meat, followed by Agona (15.5%), Dabou (8.8%), Hadar (6.8%), and Indiana (6.1%) (Table 3). Salmonella Agona was the serovar most frequently (31%, n ~ 129) detected in a study by Luu et al. (23). Thai et al. (37) determined that Salmonella serovars Emek and Infantis were most commonly detected on chicken meat at 21.7 and 13.0% (n ~ 115), respectively; whereas Albany and Hadar were detected at lower percentages (i.e., 3.5 and 4.3%, respectively). Furthermore, Vo et al. (42) determined that Salmonella serovars Emek (38.8%) and Blockley (20.9%) (n ~ 67) were most

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FIGURE 2. Frequency bar chart illustrating the distribution of phenotypic antibiotic resistance to up to nine antibiotics among Salmonella isolates from retail chicken meat samples in Vietnam.

commonly detected in fecal samples from the lower intestinal tract of broilers obtained during processing, whereas serovars Albany and Hadar were detected at 4.5 and 9.0%, respectively. Among the total of 22 identified Salmonella serovars in this study, Albany, Agona, Hardar, Typhimurium, Rissen, London, Enteritidis, Infantis, Derby, Newport, and Stanley were also identified in previous studies (2, 23, 37, 41, 42). In addition, we identified several previously undetected Salmonella serovars on chicken meat in Vietnam, such as Dabou, Indiana, Magherafelt, Chester, Meunster, Bousso IV, Montevideo, Give, Kunduchi, Schleissheim, and Thompson (Table 3). These serovars were detected at low percentages in our study, except for Dabou and Indiana. Since the distribution of Salmonella serotypes is generally region dependent (16), it is notable that Salmonella serovars Enteritidis, Typhimurium, Indiana, Derby, Infantis, Agona, Thompson, Rissen, Hadar, Stanley, Newport, London, Schleissheim, and Albany were also identified in neighboring countries such as China, Cambodia, and Thailand (22, 28, 46).

More than five Salmonella serovars associated with human infections in Vietnam were detected on broiler carcasses by Vo et al. (42); of these, the two most frequent were Typhimurium and Enteritidis (37.5 and 12.5%, respectively) (42). Salmonella serovars Typhimurium and Enteritidis were identified in 5.5 and 2.0% of raw chicken meat samples in our study. This suggests that chicken may not be a major source of salmonellosis in Vietnam. Salmonella serovars Typhimurium and Enteritidis are also the most common cause of human salmonellosis in the United States and Europe (13, 19). Our findings revealed the presence of both single- and multidrug-resistant Salmonella phenotypes on chicken samples collected from retail markets in Vietnam. Van et al. (41) reported that proportions of Salmonella isolates in Vietnam with single-drug resistance to ampicillin, amoxicillin-clavulanate, tetracycline, kanamycin, gentamicin, ciprofloxacin, chloramphenicol, and trimethoprim were 22.2, 0.0, 38.9, 0.0, 5.6, 0.0, 11.1, and 5.6%, respectively. Antibiotic-resistant Salmonella percentages in our study were higher than those found by Van et al. (41). In 2010, Vo et al. (43) reported percentages of Salmonella with singledrug resistance to ampicillin, amoxicillin-clavulanate, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, tetracycline, and trimethoprim at 13.4, 3.0, 50.8, 0.0, 3.0, 20.9, 44.8, and 52.2%, respectively. The percentages of Salmonella isolates resistant to chloramphenicol, amoxicillin-clavulanate, kanamycin, and trimethoprim in our study were lower than those reported by Vo et al. (43), but percentages of Salmonella isolates resistant to ampicillin, tetracycline, ciprofloxacin, and gentamicin were higher. Among the antibiotics tested, the third-generation cephalosporins are drugs of choice to treat cases of invasive salmonellosis or in vulnerable patients (e.g., immunocompromised) with Salmonella infection. However, Salmonella resistance to these drugs is a worldwide public health concern. Salmonella resistance to third-generation cephalosporins has been associated with foodborne outbreaks of salmonellosis in developing countries in North Africa and in

TABLE 6. Multidrug resistance phenotypes of Salmonella isolates on raw chicken meat by top five common serovars in Vietnam No. (%) of resistant Salmonella serovarsa Multidrug resistance

0 1 2 3 4 5 6 7 8 9 a

b

Total (n ~ 457)

Albany (n ~ 156)

Agona (n ~ 71)

Dabou (n ~ 40)

Hadar (n ~ 31)

Indiana (n ~ 28)

122 91 48 81 35 55 10 4 8 3

27 34 14 30 21 24 3 1 1 1

31 10 10 13 3 4

13 15 4 8

5 11 7 8

2 3 1 2 3 5 3 2 6 1

(26.7) (19.9) (10.5) (17.7) (7.7) (12.0) (2.2) (0.9) (1.8) (0.7)

(17.3) (21.8) (9.0) (19.2) (13.5) (15.4) (1.9) (0.6) (0.6) (0.6)

(43.7) (14.1) (14.1) (18.3) (4.2) (5.6) — — — —

(32.5) (37.5) (10.0) (20.0) — — — — — —

(16.1) (35.5) (22.6) (25.8) — — — — — —

(7.1) (10.7) (3.6) (7.1) (10.7) (17.9) (10.7) (7.1) (21.4) (3.6)

Other serovars (n ~ 131)b

44 18 12 20 8 22 4 1 1 1

(33.6) (13.7) (9.2) (15.3) (6.1) (16.8) (3.1) (0.8) (0.8) (0.8)

Differences in multidrug-resistant Salmonella isolates were significant (P , 0.001) using the likelihood of the chi-square test. —, Salmonella isolate was susceptible to the antibiotics. Serovars that were not among the five most common serovars were collapsed into one category (i.e., other serovars) and represented 28.7% of the total.

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CHARACTERIZATION OF SALMONELLA ON RETAIL CHICKEN MEAT IN VIETNAM

India (25). In this study, only Salmonella Albany was resistant to ceftriaxone (0.2%), but not to cefepime (fourthgeneration cephalosporin) (Table 5). Salmonella isolates resistant to third-generation cephalosporins were not detected in other studies in Vietnam (41, 43). Multidrug resistance among Salmonella isolates in this study was most commonly to three antibiotics. Salmonella serovars Albany, Agona, Dabou, and Hadar most frequently expressed phenotypic resistance to three different antibiotics, whereas Salmonella Indiana most frequently expressed phenotypic resistance to eight drugs (Table 6). In China, Salmonella Indiana isolates (12.7%, n ~ 292) were reported to be resistant to more than nine antibiotics (46). In Cambodia, Salmonella serovar Enteritidis isolates (4.9%, n ~ 201) were resistant to six antibiotics (22). In our study, we determined that Salmonella serovars Albany, Indiana, and Infantis were resistant to nine antibiotics. This study presents findings on counts, serovar distribution, and antibiotic resistance profiles of Salmonella on raw poultry meat sampled over a wide geographical range in Vietnam. Since there is no foodborne surveillance and response system in Vietnam, there are no data on the attribution of poultry meat consumption outbreaks or cases of human salmonellosis. Poultry producers (both local and conventional) should follow good agricultural practices at the farm and during processing to reduce the prevalence and level of Salmonella on chicken carcasses. Although the most commonly identified Salmonella serovars in this study are not those most frequently detected in human salmonellosis in Vietnam (42), serovars such as Albany, Agona, and Hadar are known to cause human disease in different parts of the world (5, 13). Additional studies might help to pinpoint other food sources of Salmonella, e.g., produce. Due to international trade and complex global food production systems, Salmonella isolates can be disseminated among different agriculture production systems. It is interesting that Salmonella Rissen (the seventh most common serovar in this study, which has also been detected on raw chicken meat in Thailand and China but is not a common serovar in North America) was detected in a foodborne outbreak in the United States associated with white pepper imported from Thailand (21). Results of studies on antibiotic-resistant Salmonella from many countries have raised awareness about the potential for spread of antibiotic-resistant bacteria. It is important to monitor antibiotic-resistant Salmonella in developing countries where poor sanitation, inadequate health care systems, and abuse of antibiotics are common (26, 34, 35). This study provides preliminary data for a future Salmonella surveillance system in Vietnam, which should be established to identify, assess risk, and establish control practices to mitigate the occurrence of salmonellosis. ACKNOWLEDGMENTS This research was supported by the project ‘‘Data Collection for Salmonella in Raw Poultry in Vietnam’’ of the University of Georgia in collaboration with the World Health Organization Global Foodborne Infections Network. We thank the technical staff at the Local Department of Health in Vietnam.

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REFERENCES 1. Alali, W. Q., R. Gaydashov, E. Petrova, A. Panin, O. Tugarinov, A. Kulikovskii, D. Mamleeva, I. Walls, and M. P. Doyle. 2012. Prevalence of Salmonella on retail chicken meat in Russian Federation. J. Food Prot. 75:1469–1473. 2. Bao, V. N., R. Fries, K. H. Zessin, M. N. Kyule, R. Pinthong, and M. P. O. Baumann. 2006. Salmonella and Campylobacter in broiler carcasses in Vietnam. Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006. Available at: www.sciquest.org.nz. Accessed August 2006. 3. Butaye, P., G. B. Michael, S. Schwarz, T. J. Barrett, A. Brisabois, and D. G. White. 2006. The clonal spread of multidrug-resistant non-typhi Salmonella serotypes. Microbes Infect. 8:1891–1897. 4. Carrasco, E., A. Morales-Rueda, and R. M. Garcı´a-Gimeno. 2012. Cross-contamination and recontamination by Salmonella in foods: a review. Food Res. Int. 45:545–556. 5. Centers for Disease Control and Prevention. 2006. Salmonella surveillance: annual summary, 2006. Available at: http://www. cdc.gov/ncidod/dbmd/phlisdata/salmonella.htm. Accessed 8 April 2013. 6. Chaisatit, C., C. Tribuddharat, C. Pulsrikarn, and S. Dejsirilert. 2012. Molecular characterization of antibiotic-resistant bacteria in contaminated chicken meat sold at supermarkets in Bangkok, Thailand. Jpn. J. Infect. Dis. 65:527–534. 7. Chimalizeni, Y., K. Kawaza, and E. Molyneux. 2010. The epidemiology and management of non typhoidal salmonella infections. Adv. Exp. Med. Biol. 659:33–46. 8. Clinical and Laboratory Standards Institute (CLSI). 2011. Performance standards for antimicrobial susceptibility testing; twenty-first informational supplement. CLSI document M 100-S21. CLSI Publishing, Wayne, PA. 9. Danan, C., S. Fremy, F. Moury, M. L. Bohnert, and A. Brisabois. 2009. Determining the serotype of isolated Salmonella strains in the veterinary sector using the rapid slide agglutination test. AFSSA J. Ref. 2:13–18. French Food Safety Agency, Ploufragan, France. Available at: http://www.afssa.fr/euroreference/Documents/CR2-MethSeroSalmoEN.pdf. Accessed 13 March 2013. 10. Donado-Godoy, P., V. Clavijo, M. Leon, M. A. Tafur, S. Gonzales, M. Hume, W. Alali, I. Walls, D. M. Lo Fo Wong, and M. P. Doyle. 2012. Prevalence of Salmonella on retail broiler chicken meat carcasses in Colombia. J. Food Prot. 75:1134–1138. 11. Dufrenne, J., W. Ritmeester, E. Delfgou-van Asch, F. van Leusden, and R. de Jonge. 2001. Quantification of the contamination of chicken and chicken products in The Netherlands with Salmonella and Campylobacter. J. Food Prot. 64:538–541. 12. European Food Safety Authority. 2010. Scientific report of EFSA: analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J. 8:1503. 13. European Food Safety Authority. 2011. Scientific opinion on a quantitative estimation of the public health impact of setting a new target for the reduction of Salmonella in broilers. EFSA J. 9:2106. 14. Foley, S. L., and A. M. Lynne. 2008. Food animal-associated Salmonella challenges: pathogenicity and antimicrobial resistance. J. Anim. Sci. 86:173–187. 15. Food and Agriculture Organization of the United Nations and World Health Organization. 2002. Microbiological risk assessment series 2. Risk assessments of Salmonella in eggs and broiler chickens. Available at: www.fao.org/docrep/005/Y4392E/Y4392E00.HTM. Accessed 6 April 2013. 16. Galanis, E., D. M. Lo Fo Wong, M. E. Patrick, N. Binsztein, A. Cieslik, T. Chalermchikit, A. Aidara-Kane, A. Ellis, F. J. Angulo, and H. C. Wegener. 2006. Web-based surveillance and global Salmonella distribution, 2000–2002. Emerg. Infect. Dis. 12:381–388. 17. Grimont, P. A. D., and F.-X. Weill. 2007. Antigenic formulae of the Salmonella serovars, 9th ed. World Health Organization Collaborating Center for Reference and Research on Salmonella, Institut Pasteur, Paris.

66

TA ET AL.

18. Helmuth, R. 2000. Antibiotic resistance in Salmonella, p. 94. In C. Wray and A. Wray (ed.), Salmonella in domestic animals. CABI Publishing, Wallingford, Oxon, UK. 19. Hoelzer, K., A. I. M. Switt, and M. Wiedmann. 2011. Animal contact as a source of human non-typhoidal salmonellosis. Vet. Res. 42:1–27. 20. Jorgensen, F., R. Bailey, S. Williams, P. Henderson, D. R. Wareing, F. J. Bolton, J. A. Frost, L. Ward, and T. J. Humphrey. 2002. Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods. Int. J. Food Microbiol. 76:151–164. 21. Kennelly, P. 2010. Salmonella Rissen outbreak associated with white pepper consumption. Presentation at WAFDO (Western Association of Food and Drug Officials) 2010 Educational Conference. Available at: http://www.wafdo.org/events.html. Accessed 20 April 2010. 22. Lay, K. S., Y. Vuthy, P. Song, K. Phol, and J. L. Sarthou. 2010. Prevalence, numbers and antimicrobial susceptibilities of Salmonella serovars and Campylobacter spp. in retail poultry in Phnom Penh, Cambodia. J. Vet. Med. Sci. 73:325–329. 23. Luu, Q. H., R. Fries, P. Padungtod, T. H. Tran, M. N. Kyule, M. P. O. Baumann, and K. H. Zessin. 2006. Prevalence of Salmonella in retail chicken meat in Hanoi, Vietnam. Ann. N. Y. Acad. Sci. 1081:257– 261. 24. Majowicz, S. E., J. Musto, E. Scallan, F. J. Angulo, M. Kirk, S. J. O’Brien, T. F. Jones, A. Fazil, and R. M. Hoekstra. 2010. The global burden of nontyphoidal Salmonella gastroenteritis. Clin. Infect. Dis. 50:882–889. 25. Miriagou, V., P. T. Tassios, N. J. Legakis, and L. S. Tzouvelekis. 2004. Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella. Int. J. Antimicrob. Agents 23:547–555. 26. Okeke, I. N., K. P. Klugman, Z. A. Bhutta, A. G. Duse, P. Jenkins, T. F. O’Brien, A. Pablos-Mendez, and R. Laxminarayan. 2005. Antimicrobial resistance in developing countries. Part II: strategies for containment. Lancet Infect. Dis. 5:568–580. 27. Okeke, I. N., R. Laxminarayan, Z. A. Bhutta, A. G. Duse, P. Jenkins, T. F. O’Brien, A. Pablos-Mendez, and K. P. Klugman. 2005. Antimicrobial resistance in developing countries. Part I: recent trends and current status. Lancet Infect. Dis. 5:481–493. 28. Padungtod, P., and J. B. Kaneene. 2006. Salmonella in food animals and humans in northern Thailand. Int. J. Food Microbiol. 108: 346–354. 29. Painter, J. A., R. M. Hoekstra, T. Ayers, R. V. Tauxe, C. R. Braden, F. J. Angulo, and P. M. Griffin. 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998–2008. Emerg. Infect. Dis. 19: 407–415. 30. Phan, T. T., L. T. L. Khai, N. Ogasawara, N. T. Tam, A. T. Okatani, M. Akiba, and H. Hayashidani. 2005. Contamination of Salmonella in retail meats and shrimps in the Mekong Delta, Vietnam. J. Food Prot. 68:1077–1080. 31. Pui, C. F., W. C. Wong, L. C. Chai, R. Tunung, P. Jeyaletchumi, M. S. Noor Hidayah, A. Ubong, M. G. Farinazleen, Y. K. Cheah, and R. Son. 2011. Salmonella: a foodborne pathogen. Int. Food Res. J. 18: 465–473.

J. Food Prot., Vol. 77, No. 1

32. Sanchez-Vargas, F. M., M. A. Abu-El-Haija, and O. G. GomezDuarte. 2011. Salmonella infections: an update on epidemiology, management, and prevention. Travel Med. Infect. Dis. 9:263–277. 33. Singer, R. S., L. A. Cox, Jr., J. S. Dickson, H. S. Hurd, I. Phillips, and G. Y. Miller. 2007. Modeling the relationship between food animal health and human foodborne illness. Prev. Vet. Med. 79:186–203. 34. Sirinavin, S., and S. F. Dowell. 2004. Antimicrobial resistance in countries with limited resources: unique challenges and limited alternatives. Semin. Pediatr. Infect. Dis. 15:94–98. 35. Sosa, A. de J., D. K. Byarugaba, C. F. Ama´bile-Cuevas, P.-R. Hsueh, S. Kariuki, and I. N. Okeke. 2010. Antimicrobial resistance in developing countries. Springer, New York. 36. Ta, Y. T., T. T. Nguyen, P. B. To, D. X. Pham, H. T. H. Le, W. Q. Alali, I. Walls, D. M. Lo Fo Wong, and M. P. Doyle. 2012. Prevalence of Salmonella on chicken carcasses from retail markets in Vietnam. J. Food Prot. 75:1851–1854. 37. Thai, T. H., T. Hirai, N. T. Lan, and R. Yamaguchi. 2012. Antibiotic resistance profiles of Salmonella serovars isolated from retail pork and chicken meat in North Vietnam. Int. J. Food Microbiol. 156: 147–151. 38. Tran, T. H., T. T. Nguyen, Q. T. Hoang, T. T. Le, M. T. Lam, and T. H. Nguyen. 2006. Prevalence of Salmonella spp. in poultry in Vietnam. Ann. N. Y. Acad. Sci. 1081:266–268. 39. U.S. Department of Agriculture. 2008. Most probable number procedure and tables, MLG appendix 2.03. Available at: www.fsis. usda.gov/pdf/MLG_Appendix_2_03.pdf. Accessed 28 January 2008. 40. U.S. Department of Agriculture. 2008. The Nationwide Microbiological Baseline Data Collection Program: young chicken survey. Available at: www. Fsis.usda.gov/PDF/Baseline_Data_Young_ Chicken_2007–2008/pdf. Accessed 5 January 2013. 41. Van, T. T. H., G. Moutafis, T. Istivan, L. T. Tran, and P. J. Coloe. 2007. Detection of Salmonella spp. in retail raw food samples from Vietnam and characterization of their antibiotic resistance. Appl. Environ. Microbiol. 73:6885–6890. 42. Vo, A. T. T., E. van Duijkeren, A. C. Fluit, M. E. O. C. Heck, A. Verbruggen, H. M. E. Maas, and W. Gaastra. 2006. Distribution of Salmonella enterica serovars from humans, livestock and meat in Vietnam and the dominance of Salmonella Typhimurium phage type 90. Vet. Microbiol. 113:153–158. 43. Vo, A. T. T., E. van Duijkeren, W. Gaastra, and A. C. Fluit. 2010. Antimicrobial resistance, class 1 integrons, and genomic island 1 in Salmonella isolates from Vietnam. PLoS One 5:e9440. 44. World Health Organization. 2011. Tackling antibiotic resistance from a food safety perspective in Europe. ISBN 978 92 890 1422 9. Available at: www.euro.who.int/__data/assets/pdf/e94889.pdf. Accessed 3 October 2012. 45. World Health Organization Global Foodborne Infections Network. 2010. Laboratory protocol: ‘‘Susceptibility testing of Enterobacteriaceae using disk diffusion.’’ Available at: www. Antimicrobialresistance. dk. Accessed February 2010. 46. Yang, B., D. Qu, X. Zhang, J. Shen, S. Cui, Y. Shi, M. Xi, M. Sheng, S. Zhi, and J. Meng. 2010. Prevalence and characterization of Salmonella serovars in retail meats of marketplace in Shaanxi, China. Int. J. Food Microbiol. 141:63–72.