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College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi 712100, ChinaJoint Institute for Food Safety and Applied Nutrition, and Department of Nutrition and Food Science, University of Maryland, College Park 20742
The aim of this study was to investigate the existence and characteristics of Salmonella enterica in dried milk-related infant foods. Twenty-four (3.4%) of 705 samples, including 5 (2.0%) of 246 powdered infant formula, 18 (4.0%) of 445 infant rice cereal, and 1 (7.1%) of 14 other infant foods, were positive for Salmonella. Fifteen serotypes were identified in 40 Salmonella isolates; Salmonella Duesseldorf (15.0%) and Salmonella Indiana (15.0%) were more frequently detected than other serotypes. Resistance to chloramphenicol (82.5%) was most common, followed by tetracycline (57.5%), ceftiofur (52.5%), kanamycin (52.5%), streptomycin (50.0%), gentamycin (45.0%), nalidixic acid (35.0%), ceftriaxone (32.5%), ciprofloxacin (25.0%), amikacin (20.0%), and cefoxitin (15.0%). Twenty-eight (70.0%) isolates were resistant to ≥8 antimicrobials, with 5 (12.5%) being resistant to 14 antimicrobials. Amino acid substitutions in gyrase A (GyrA) were most frequently detected as Ser83Arg/Asp87Glu and in p53-associated Parkin-like cytoplasmic protein (ParC), they were all Ser80Arg; the quinolone resistance gene qnrS (47.5%) was commonly detected as well as aminoglycoside acetyltransferase [aac(6′)-Ib; 25.0%], qnrA (17.5%), and qnrB (15.0%) genes. Thirty distinct pulsed-field gel electrophoresis patterns were identified among 40 isolates; no identical pulsed-field gel electrophoresis pattern was detected among Salmonella isolates with the same serovar that was recovered in 2010 and 2012. Our results suggest that dried milk-related infant foods could be contaminated with Salmonella and highlight that the dangers to infant health should not be neglected.
Dried infant foods, including powdered infant formula (PIF), infant rice cereal (IRC), and other infant foods (OIF) are not sterile products and may be intrinsically contaminated with pathogens, such as Salmonella enterica and Cronobacter sakazakii, which can result in serious illness among infants (i.e., children aged <1 yr;
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2004. Enterobacter sakazakii and other microorganisms in powdered infant formula: Meeting report. Microbiological Risk Assessment Series 6. FAO, Rome, Italy.
). Although C. sakazakii has become an infant food safety issue due to the severity of disease it may cause in infants, concerns regarding S. enterica in milk-related infant foods have been somewhat overshadowed (
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2006. Enterobacter sakazakii and Salmonella in powdered infant formula: Meeting report. Microbiological Risk Assessment Series 10. FAO, Rome, Italy. Assessed Dec. 10, 2013. http://www.who.int/foodsafety/publications/micro/mra10/en/
Currently, the tolerance limits for Salmonella in milk and milk-related products in Canada, Cuba, Chile, European Union, the Netherlands, Australia, New Zealand, China, Singapore, and South Africa are all 0 cfu/25 g (mL) of sample, which means no Salmonella should be detected in milk, PIF, cheese, or yogurt (
ICMSF (International Commission on Microbiological Specifications for Foods) Potential application of risk assessment techniques to microbiological issues related to international trade in food and food products.
). Although efforts have been made to control Salmonella in different milk-related products, in the 20-yr period from 1985 to 2005, at least 6 outbreaks of Salmonella infection have occurred in the United Kingdom, the United States, Canada, Spain, France, and Korea, associated with consumption of PIF, involving approximate 287 infants (
Molecular fingerprinting defines a strain of Salmonella enterica serotype Anatum responsible for an international outbreak associated with formula-dried milk.
). The common characteristic of these outbreaks was the low level of Salmonella detected in the implicated PIF; thus, Salmonella may be ignored or missed during routine testing. The outbreaks likely represent only a tiny proportion of the actual number of Salmonella infections among infants that had been involved in PIF and OIF (
In this study, the prevalence and characterization of Salmonella isolates recovered from dried milk-related infant foods were investigated in Shaanxi Province (China) to provide meaningful data for better understanding of infant food safety.
Materials and Methods
Sample Collection and Salmonella Isolation
A total of 705 dried milk-related infant foods, including 445 IRC, 246 PIF, and 14 OIF samples covering 71 brands, were randomly collected from 51 supermarkets and 32 retail stores in 12 cities (Xi’an, Yangling, Baoji, Meixian, Qishan, Qianxian, Xingping, Fufeng, Liquan, Zhouzhi, Wugong, and Xianyang) in Shaanxi Province (China) in 2010 and 2012. Two to 5 representative supermarkets and retail stores were selected in each city for sampling. In each supermarket/store, specific brands of IRC and PIF, including imported and domestically produced ones, were sampled.
Salmonella was isolated according to the standard procedures described in the National Standards of the People’s Republic of China (GB 4789.4-2010). Briefly, 25-g food samples were placed in 225 mL of buffered peptone water (Beijing Land Bridge Technology Co. Ltd., Beijing, China) and incubated at 36 ± 1°C for 8 to 18 h. A 1-mL aliquot of preenriched culture was transferred to a 10-mL aliquot of tetrathionate (TT) broth (Beijing Land Bridge Technology Co. Ltd.) and selenite cystine (SC) broth (Beijing Land Bridge Technology Co. Ltd.). The TT was incubated at 42 ± 1°C and SC at 36 ± 1°C for 18 to 24 h. One loop of TT broth was streaked onto bismuth sulfite (BS; Beijing Land Bridge Technology Co. Ltd.) and SC broth onto Hektoen enteric (HE; Beijing Land Bridge Technology Co. Ltd.) agars. The BS plates were incubated at 36 ± 1°C for 40 to 48 h and HE plates for 18 to 24 h, and examined for the presence of Salmonella. The presumptive isolates on BS and HE plates were subcultured on Luria-Bertani agar plates (Beijing Land Bridge Technology Co. Ltd.) at 36 ± 1°C from 18 to 24 h, and 1 colony on Luria-Bertani plate was selected and confirmed with API20E identification kits (bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s instructions.
Serotyping
The O and H antigens of Salmonella isolates were characterized with hyperimmune sera (S&A Reagents Lab Ltd., Bangkok, Thailand; Statens Serum Institut, Copenhagen, Denmark). The serotype was identified using the slide agglutination method and assigned following the manufacturer’s instructions and the Kauffmann-White classification scheme.
Antimicrobial Susceptibility Testing
Antimicrobial MIC were determined by an agar dilution method using Mueller-Hinton agar (Beijing Land Bridge Technology Co. Ltd.) according to the guidelines recommended by the Clinical and Laboratory Standards Institute (
). The antibiotics amikacin, gentamycin, kanamycin, streptomycin (STR), ampicillin, amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin (CIP), nalidixic acid (NAL), and tetracycline were selected. Escherichia coli ATCC 25922, E. coli ATCC 35218, and Enterococcus faecalis ATCC 29212 were used as quality control organisms in MIC determinations. The breakpoints for interpretation of resistance and susceptibility were according to the interpretive standards of the Clinical and Laboratory Standard Institute (
Screening for qnr and Aminoglycoside Acetyltransferase Genes, and Quinolone Resistance-Determination Region Gene Amplification and Sequencing
Salmonella isolates being resistant to NAL but susceptible or low-level resistant to CIP were examined for the presence of quinolone resistance (qnr) genes, including qnrA, qnrB, and qnrS, and the aminoglycoside acetyltransferase [aac(6′)-Ib] gene. Isolates that showed both NAL and CIP resistance were examined for amino acid substitutions in quinolone resistance-determination regions (QRDR) of gyrase A (GyrA) and p53-associated Parkin-like cytoplasmic protein (ParC). All genes were amplified using MyCycler PCR (Bio-Rad Laboratories Inc., Hercules, CA) as described previously (
). The primers were synthesized by TaKaRa Biotechnology Co. Ltd. (Dalian, China) and are listed in Table 1. The PCR products were stained with ethidium bromide and visualized under UV light after gel electrophoresis in 1% agarose. For DNA sequence analysis, PCR products were purified with a kit (TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0; TaKaRa Biotechnology Co. Ltd.), and sent for sequencing at AuGCT biotechnology Co. Ltd. (Beijing, China). The DNA sequence data were analyzed and aligned using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).
Table 1Primers used for detection and sequencing of target genes
). Briefly, agarose-embedded DNA was digested with 50 U of the restriction enzyme XbaI (TaKaRa Biotechnology Co. Ltd.) for 1.5 to 2 h in a water bath at 37°C. The restriction fragments were separated by electrophoresis in 0.5 × TBE (Tris, borate, and EDTA) buffer at 14°C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad Laboratories Inc.) with pulse times of 2.16 to 63.8 s. Salmonella Braenderup H9812 was used as the control strain. The gels were stained with ethidium bromide and visualized with UV transillumination (Bio-Rad Laboratories Inc.). The PFGE results were analyzed using BioNumerics software (version 3.0; Applied Maths, Kortrijk, Belgium) manually; the extent of variability was determined by use of the Dice coefficient and clustering was based on the unweighted pair group average method.
Statistical Analysis
The software DPS (DPS 9.5; Institute of Insect Science, Zhejiang University, Hangzhou, China) was used to process the prevalence, serovar distribution, antimicrobial susceptibility, and multidrug resistance of Salmonella isolates. Significant (P < 0.05) variation of positive rates among various foods (IRC, PIF, and OIF), serotypes, and antimicrobial susceptibility were determined using the Pearson chi-squared test at the 5% (with α = 0.05) level.
Results
Prevalence of Salmonella enterica
Twenty-four (3.4%) of 705 dried milk-related infant food samples, including 5 (2.0%) of 246 PIF, 18 (4.0%) of 445 IRC, and 1 (7.1%) of 14 OIF, were positive for Salmonella (Table 2). A total of 40 Salmonella isolates of 15 serotypes were recovered. Among the isolates, Salmonella Duesseldorf (15.0%) and Salmonella Indiana (15.0%) were significantly (P < 0.05) more commonly detected than Salmonella Djugu (2.5%), Salmonella Mbandaka (2.5%), Salmonella Nola (2.5%), Salmonella Papuana (2.5%), Salmonella Poitiers (2.5%), Salmonella Remo (2.5%), and Salmonella Somone (2.5%). No significant difference (P > 0.05) was found among the prevalence of Salmonella Duesseldorf, Salmonella Indiana, and Salmonella Blockley, Salmonella Enteritidis, Salmonella Thompson, Salmonella Typhimurium, Salmonella Rissen, and Salmonella Kiambu (Table 3). Salmonella Duesseldorf was the dominant serovar in 2010; isolates with this serovar were mainly recovered from IRC, whereas Salmonella Enteritidis was the dominant serovar in 2012 and mainly recovered from PIF (Figure 1).
Table 2Prevalence of Salmonella in powdered infant formula (PIF), infant rice cereal (IRC), and other infant foods (OIF)
Antibiotic resistance was most frequently found to chloramphenicol (82.5%), the rate of which was significantly (P < 0.05) higher than those of tetracycline (57.5%), ceftiofur (52.5%), kanamycin (52.5%), STR (50.0%), gentamycin (45.0%), NAL (35.0%), ceftriaxone (32.5%), CIP (25.0%), amikacin (20.0%), and cefoxitin (15.0%). No significant (P > 0.05) difference was found among resistance rates of ampicillin (75.0%), amoxicillin/clavulanic acid (75.0%), and sulfamethoxazole/trimethoprim (65.0%; Table 4).
Table 4Antimicrobial resistance of Salmonella serovars derived from powdered infant formula (PIF) and infant rice cereal (IRC; n = 40)
All isolates could resist at least 1 of the testing antimicrobials. Isolates that coresisted 8 testing antimicrobials could be found significantly more often (P < 0.05) compared with those coresistant to 5, 10, and 13 antimicrobials. No significant (P > 0.05) difference was found among the prevalence rates of isolates that were coresistant to 1, 2, 5, 9, 10, 11, 12, 13, and 14 antimicrobials (Table 5).
Table 5Multidrug resistance of Salmonella serovars derived from powdered infant formula (PIF) and infant rice cereal (IRC; n = 40)
Screening for qnr and Aminoglycoside Acetyltransferase Genes, and QRDR Gene Amplification and Sequencing
Twenty-one (52.5%) Salmonella isolates were positive for at least one of the qnrA, qnrB, qnrS, and aac(6′)-Ib genes. Among these genes, qnrS (47.5%) was most frequently detected, followed by aac(6′)-Ib (25.0%), qnrA (17.5%), and qnrB (15.0%). Eleven isolates (27.5%) were detected to carry at least 2 of the qnrA, qnrB, qnrS, and aac(6′)-Ib genes, 8 (20.0%) carried at least 3 of the 4 genes, and 2 (5.0%) carried all 4 genes (Figure 1).
In 7 isolates, amino acid substitutions of QRDR in GyrA and ParC, and 1 of the qnrA, qnrB, qnrS, and aac(6′)-Ib genes could be simultaneously detected. Mutations of Ser83Arg/Asp87Glu in GyrA were the most frequently detected; all mutations in ParC were detected as Ser80Arg.
PFGE
Thirty distinct PFGE based genotype patterns were identified, 21 were found in isolates recovered in 2010 and 9 in 2012. No identical PFGE pattern was found among Salmonella isolates of the same serovar that were recovered in 2010 and 2012 (Figure 1). Six Salmonella Duesseldorf isolates recovered from IRC of different brands in 2010 exhibited similar but different PFGE patterns as well as 5 Salmonella Enteritidis isolates. One Salmonella Indiana isolate recovered from baby cake showed a similar PFGE pattern to 5 other isolates recovered from IRC and PIF in 2010 and 2012.
Discussion
Foodborne disease caused by nontyphoid Salmonella represents an important public health problem worldwide. Although data are not available in many countries, especially in developing countries, in the United States, the incidence of salmonellosis in infants was 121.6 laboratory-confirmed infections per 100,000 infants, which was approximately 8 times greater than the incidence in other age groups (
). Similarly high incidences of salmonellosis in infants have been reported as 181 cases per 100,000 infants in the United Kingdom and 92.8 cases per 100,000 infants in Israel (
). It is unclear if these Salmonella illnesses were associated with PIF and IRC; however, it is well known that PIF is the dominant food for the majority of infants, and IRC and OIF, such as baby cake, act as accessorial infant foods that are not treated by sterilization. As a result, Salmonella may be intrinsically colonized in these foods. Although the Food and Agriculture Organization of the United Nations and the World Health Organization (WHO) categorized Salmonella as a category A hazard, and the tolerance limit for Salmonella in milk and milk-related products in most countries is 0 cfu/25 g (mL) of food, Salmonella in infant food has been responsible for infections and illnesses among infants (
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2004. Enterobacter sakazakii and other microorganisms in powdered infant formula: Meeting report. Microbiological Risk Assessment Series 6. FAO, Rome, Italy.
). In addition to environmental contamination, PIF and IRC may become contaminated by Salmonella during preparation and handling in home and hospital settings, it is those preparations and handling that may introduce the hazards to PIF and IRC if hygienic practices are not followed (http://www.who.int/foodsafety/publications/micro/mra10/en/).
Compared with Salmonella serotypes among isolates recovered from retail chickens and meats, serotypes of isolates derived from milk-related infant foods were more diverse. These serotypes were unusual and not frequently detected; our results are consistent with the findings of the WHO (
; http://www.who.int/foodsafety/publications/micro/mra10/en/). According to the WHO reports, Salmonella of these rare serotypes associated with outbreaks could facilitate some outbreaks to be identified if the Salmonella surveillance network were well-established. Thus, if a serotype of Salmonella isolates that was recovered from milk-related infant foods during routine testing were identified the same as that in outbreaks, more attention should be paid to avoid illnesses among infants. Because we cannot share the PFGE data on Salmonella in the molecular subtyping network for foodborne bacterial disease surveillance (PulseNet) of China and the limited PFGE data we had, it is difficult for us to trace the source of Salmonella isolates recovered from PIF, IRC, and ORF.
Antimicrobial resistance in Salmonella has always been a significant public health concern worldwide (
). Our results indicate that all Salmonella isolates were resistant to at least 1 of the antimicrobial agents tested, and almost 70.0% of the isolates were resistant to at least 8 antimicrobial agents, with 12.5% resistant to more than 14 antimicrobials. Because antimicrobial susceptibility data on Salmonella isolates derived from PIF and IRC have seldomly been reported, the extent of antibiotic resistance of the Salmonella isolates in current study was not clear. A recent study indicated that 31 CIP-resistant clinical Salmonella isolates in China were also resistant to ≥8 other antimicrobial drugs, and 54% of human Salmonella isolates were multidrug resistant in Henan Province (
). On the other hand, qnrA, qnrB, qnrS, and aac(6′)-Ib associated with NAL and fluoroquinolones were detected frequently in Salmonella isolates recovered from PIF and IRC in the current study; as qnr genes are commonly carried by plasmids, which can be transmitted to other antibiotic-sensitive pathogens through conjugation, the presence of qnr genes in Salmonella isolates are potential threats to consumers, especially to infants (
International collaborative study on the occurrence of plasmid-mediated quinolone resistance in Salmonella enterica and Escherichia coli isolated from animals, humans, food and the environment in 13 European countries.
Salmonella isolates were detected at a low prevalence rate in retail PIF and IRC in China; 15 serotypes were detected among the 40 isolates that showed multiple PFGE genotypes and multidrug resistance. Plasmid-associated qnr and aac(6′)-Ib genes as well as amino acid substitution in GyrA and ParC were detected in quinolone- and fluoroquinolone-resistance isolates. Although it is not technologically feasible to reduce Salmonella contamination in sterile PIF and IRC, preventative measures such as hazard analysis critical control points (HACCP) should be taken to keep the levels of contamination of these products as low as possible.
Acknowledgments
This study was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology (Beijing, China; 2011BAD28B05) and the National Key Spark Program of China (2013GA85003).
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Two consecutive large outbreaks of Salmonella enterica serotype Agona infections in infants linked to the consumption of powdered infant formula.
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2004. Enterobacter sakazakii and other microorganisms in powdered infant formula: Meeting report. Microbiological Risk Assessment Series 6. FAO, Rome, Italy.
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2006. Enterobacter sakazakii and Salmonella in powdered infant formula: Meeting report. Microbiological Risk Assessment Series 10. FAO, Rome, Italy. Assessed Dec. 10, 2013. http://www.who.int/foodsafety/publications/micro/mra10/en/
Molecular fingerprinting defines a strain of Salmonella enterica serotype Anatum responsible for an international outbreak associated with formula-dried milk.
International collaborative study on the occurrence of plasmid-mediated quinolone resistance in Salmonella enterica and Escherichia coli isolated from animals, humans, food and the environment in 13 European countries.
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