Incidence of Multiple Antibiotic Resistant Organisms Isolated from Retail Milk Products in Hermosillo, Mexico

370

M. E. DIAZ DE AGUAYO*, A. B. LEON DUARTE, AND F. MONTES DE OCA CANASTILLO

Centro de Investigation en Alimentation Y Desarrollo, A.C. Apartado Postal 1735, Hermosillo, Sonora, Mexico Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/5/370/1664010/0362-028x-55_5_370.pdf by guest on 24 September 2021

(Received for publication February 6, 1990)

ABSTRACT Milk properly pasteurized will be free of organisms pathogenic to man. However, over the years, there have Retail milk samples (231) of pasteurized, whole milk powder been numerous foodborne illnesses attributed to the con (WMP), and ultrapasteurized (UP) were collected in Hermosillo, sumption of contaminated milk products. Upon investiga Sonora, Mexico, to determine the incidence of multiple antibiotic resistance. Pathogens such as Escherichia coli, Staphylococcus tion, this contamination has been shown to be a result of aureus, and Salmonella were determined as well as nonpathogens improper pasteurization or contamination after processing such as Pseudomonas, Klebsiella, Serratia, and Hafnia alvei. which may be a cross-contamination with raw milk, contact Drug resistance of each isolated culture was tested using the with insanitary equipment, or as a result of contamination Kirby-Bauer technique. The following antibiotic discs and con of the product by infected workers. centrations were used: amikacin 30 meg, ampicillin 10 meg, In 1985 a large outbreak of salmonellosis, consisting of carbenicillin 100 meg, cefamandole 30 meg, chloramphenicol 30 16,284 cases including two deaths, occurred in Chicago, meg, erythromycin 15 meg, gentamicin 10 meg, kanamycin 30 IL; in this case, pasteurized milk probably became contami meg, nitrofurantoin 300 meg, penicillin 10 U, polymixin B 300 U, tetracycline 30 meg, and tobramycin 10 meg. nated with raw milk due to the failure of a discharge in the Sixty-seven percent of the pasteurized milk samples con processing plant (77). In Massachusetts in 1983, 49 people tained fecal coliforms with multiple drug resistance. Four percent acquired listeriosis from pasteurized milk (77). contained Salmonella which was 100% resistant to penicillin and Studies revealed that Listeria is thermoresistant and showed some resistance to polymixin B and chloramphenicol. S. survives pasteurization, although a large inoculum would aureus which was drug resistant was present in 2.9% of the be necessary before survival of any organisms during pas samples. The WMP and UP samples were relatively pathogenic teurization would be expected. Postpasteurization storage free; however, nonenteric coliforms were detected showing mul of contaminated milk at refrigerator temperature (in es tiple drug resistance. E. coll isolated in 1 UP showed resistance to sence, cold enrichment) would permit selective growth of ampicillin, chloramphenicol, gentamycin, polymixin B, and tetra cycline. the remaining organism (77). In California in 1985, a A high incidence of drug-resistant organisms in milk prod Mexican-style cheese was the vehicle for listeriosis in ucts represents a public health hazard in that food poisoning which the mortality rate for the outbreak was approxi outbreaks would be difficult to treat and this pool of multidrug- mately 30% (14). In England, over 1000 school children resistant organisms in the food supply represents a reservoir for were affected in a widespread outbreak of staphylococcal transferable resistant genes. An active surveillance and monitoring intoxication in seven different schools due to spray-dried program should be mandated to minimize this risk for the con skim milk powder. Staphylococcus aureus was isolated sumer. from the milk (2). Of most recent concern is the presence of antibiotic- resistant pathogenic bacteria in our food supply. For more Cow's milk has long been considered a valuable hu than 45 years antibiotics have been successfully used for man food, but its nutrient composition also makes it a treating infectious diseases in man and animals. In addition suitable medium for bacterial growth, including organisms to their therapeutic uses, antibiotics are regularly included pathogenic to human. Raw milk was first implicated as a in livestock feeds for their growth promotive effects. Regu vehicle of human infectious disease of brucellosis which lations require proper conditions for use to assure food led to legislation requiring pasteurization in many coun safety (5). A major consequence of antibiotic usage world tries. In Mexico, regulations specify microbiological stan wide has been the emergence of resistant bacterial strains dards and pasteurization temperatures for milk for human (13). consumption. They are the same as in the United States and While resistance to single agents occurred early in the Canada (5). history of antibiotic use, resistance to multiple antibiotics

JOURNAL OF FOOD PROTECTION, VOL. 55, MAY 1992 ANTIBIOTIC RESISTANT ORGANISMS FROM MILK IN MEXICO 371 came later. This has complicated the treatment of even ceptible. An appropriate group of antibiotics for each organism common infectious agents. Work by Japanese investigators was chosen based upon the effectiveness of the antibiotic against showed that these multiple resistance determinants resided the organism (8). on transferable DNA elements, plasmids which can be RESULTS AND DISCUSSION transferred among bacteria of different species (4). Additionally, it has been suggested that there is a Of the total of samples analyzed, the incidence of relationship between antibiotic residues in bovine and microorganisms in retail milk products is shown in Table 1. chicken tissues and antibiotic-resistant bacteria isolated Fecal coliforms, Enterobacter sp., were detected in 67% of from the tissues. The samples were purchased at local the pasteurized milk samples. The presence of fecal colif markets in Hermosillo, Mexico (18). orms in processed foods indicates the food has been ex In Central America in 1968-1972, a bacillary dysentery posed to unsanitary conditions which may be from one or epidemic, the causative agent of which was resistant to all of the following: dirty equipment, poor employees antibiotics, occurred, causing hundreds of deaths (7,19). In hygiene, or direct contamination with fecal matter. Regula 1972 an epidemic occurred in Mexico, caused by Salmo tions for pasteurized milk in Mexico permit no more than nella typhi, a chloramphenicol-resistant bacterium, and again Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/5/370/1664010/0362-028x-55_5_370.pdf by guest on 24 September 2021 10 colonies per ml of coliforms, and the presence of fecal in India in 1976 (15). In Hermosillo, Mexico, 62% of the coliforms is generally interpreted as indicating a contami bacteria isolated from diarrhetic feces showed multiantibiotic nated product the sale of which is not permitted (5). Fifteen resistance (6). The U.S. Center for Disease Control found percent of the samples were positive for E. coli. that food animals were the source of 69% of resistant Enteropathogenic E. coli is known to cause diarrhea and Salmonella infections in humans and 46% of susceptible other intestinal disorders (10,16). Salmonella outbreaks (12). Holmberg et. al (12) reported on a 3-state outbreak of a foodborne illness from Salmo TABLE 1. Incidence of microorganisms in milk products in nella newport with particular ampicillin and tetracycline- Hermosillo, Mexico. resistant plasmid. Epidemiologic data implicated hamburger from cattle from a single farm in South Dakota. Product The objective of this study was to evaluate retail milk N = 170 N = 31 N == 30 products in Hermosillo, Mexico, to determine the incidence Organism P (%) WMP (%) UP (%) of antibiotic-resistant pathogens. E. coli 26 (15) ND 1 (3.3) MATERIAL AND METHODS Salmonella 7 (4) ND ND Collection of samples During the period of September 1987 to June of 1988, 170 S. paratyphi 5 (2.9) ND ND samples of pasteurized milk, including 66 samples of brand A, 56 of brand B, 51 of brand C , 31 of whole milk powder (WMP), and Arizona 1 (0.5) ND ND 30 samples of ultrapasteurized milk (UP), were collected from retail food markets in Hermosillo, Mexico. The pasteurized milk S. aureus 5 (2.9) ND 2 (6.6) samples were transported in ice to the laboratory and analyzed immediately. S. epidermidis 105 (61.7) 29 (93) 27 (90) Isolation of pathogens was performed (Salmonella sp. enteric Enterobacter sp. (67) coliforms, Staphylococcus sp.) according to techniques specified 115 2 (6.4) 3 (10) in Bacteriological Analytical Manual (9). For serotyping, cultures Pseudomona sp. 14 (8.2) 7 (22.5) 2 (6.6) of Escherichia coli and Salmonella were incubated in blood agar 8 at 37°C for 24 h. A drop of a suspension of 9 x 10 cells per ml H. alvei 30 (17.6) 3 (9.7) 2 (6.6) (estimated, using a turbidimetric method) of typical colonies in 0.85% saline solution was placed in a plate with antiserum. Poly Serratia 29 (17) 3 (9.7) 2 (6.6) A and Poly B antisera were used for E. coli and Salmonella "o". The antiserum (rabbit) kit was obtained from Fisher Diagnostic, Klebsiella 43 (25.3) ND 1 (3.3) Orangeburg, NY. P = Pasteurized. Antibiotic sensitivity testing WMP = Whole milk powdered. Each isolated culture was tested for its susceptibility/resis UP = Ultrapasteurized. tance to antimicrobial agents using the Kirby-Bauer technique (8). ND = Not detected. The following antibiotic discs and concentrations were used: amikacin 30 meg, ampicillin 10 meg, carbenicillin 100 meg, cefamandole 30 meg, chloramphenicol 30 meg, erythromycin 15 Four percent (9) of the pasteurized milk samples con meg, gentamicin 10 meg, kanamycin 30 meg, nitrofurantoin 300 tained Salmonella and 5 out of the 7 were positive for S. meg, penicillin 10 U, polymyxin B 300 U, tetracycline 30 meg, paratyphi and 1 Arizona. These organisms are known to tobramycin 10 meg (Becton Dickinson and Co., Cockeysville, MD). cause severe diarrhea, vomiting, and fever (10,16). Approximately five antimicrobial agent discs were placed on S. aureus was detected in 2.9% of the samples. S. each inoculated Muller-Hinton agar plate. Inoculated plates were aureus has been responsible for many foodborne intoxica incubated at 37°C for 24 h in the inverted position. Zones of tions (10,16). Staphylococcus epidermidis was detected in inhibition were interpreted as indicating the bacterium was sus 61.7% of the pasteurized milk samples, 93% of the WMP

JOURNAL OF FOOD PROTECTION, VOL. 55, MAY 1992 372 DE AGUAYO, DUARTE, DE OCA CANASTILLO

and 90% UP. No food poisoning situation has been re should be made with a complete history on the raw material ported for S. epidermidis as it is considered to be as to its origin and processing. nonpathogenic {3,10,16). The antibiotic resistance (%) of organisms isolated The WMP and UP samples were relatively free of from milk products in Hermosillo, Mexico, is presented in pathogens. Enterobacter sp. was 6.4% and 10% in WMP Table 2. A high level of multiresistance to the antibiotics of and UP, respectively. E. coli was positive for only 1 UP and 2 choice was detected for E. coli in pasteurized milk. Sixty- out of the 30 (6.6%) of the UP samples contained S. aureus. nine percent of the strains were polymixin B resistant. For Other enteric coliforms (Hafnia alvei, Serratia, Kleb Salmonella, 100% were penicillin resistant and 70% poly siella) and Pseudomonas were detected in WMP 13 out of myxin resistant. For S. aureus, 80% were resistant to 31 and 7 out of 30 UP samples. The presence of these organisms tetracycline and ampicillin, respectively. This relatively in these samples reflects unsanitary processing conditions. high level of multiantibiotic resistance for pathogenic or In the ultrapasteurization process, a temperature be ganisms represents a real public health hazard. This drug tween 130 and 150°C is maintained for 1 to 15 s. This resistance makes it very difficult to control infectious agents process should produce a practically sterile product (7). with drugs normally used in treating the disease. Because of the high incidence of organisms in this type of Enterobacter sp. in 67% of the pasteurized milk samples Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/5/370/1664010/0362-028x-55_5_370.pdf by guest on 24 September 2021 sample, it indicates a severe condition of inadequate pro showed a high level of multiantibiotic resistance, also some cessing or contamination after processing. Further investi antibiotic resistance was noted for the fecal coliforms gation in the plant processing of this product in Mexico isolated from the WMP and UP samples.

TABLE 2. Antibiotic resistance {%) of organisms isolated from milk products in Hermosillo, Mexico. Antibiotic Microorganisms PB Te AM TD CM GM NF EM EN AK MA CB E. coli @(26) P 69 53 23 23 15 7 (1)UP 100 100 100 0 100 100

Salmonella (7)P 70 13 100

Arizona (DP 100 0 100

S. aureus (5)P 80 80 0 20 80 (2)P 0 100 0 50 100

Enterobacter (114) P 72 42 82 9 44 (2) WMP 50 50 100 0 (3) UP 32 0 58 0 32

S. epidermidis (92) P 38 63 13 36 (28) WMP 0 54 12 12 18 52 (27) UP 0 59 8 0 11 59

H. alvei (19) P 25 0 12 (3) WMP 0 0 32 32

Serratia (17) P 41 0 82 17 82 (3) WMP 22 22 72 22 22 (2) UP 50 0 100 50 50

Pseudomona (14) P 0 21 0 23 42 21 (7) WMP 0 0 0 23 12 42 0 (2) UP 0 0 0 0 50 0 0

Klebsiella (43) P 18 11 4 11 25 (DUP 100 0 100 * PB=polymixin B, Te=tetracycline, AM=ampicillin, TO=tobramycin, CM=chloramphenicol, GM=gentamicin, NF=nitrofurantoin, EM=erythromycin, PN=penicillin, K=kanamycin, AK=amikacin, MA=cefamandole, CB=carbenicillin, @=these numbers mean the isolated bacterias from each product.

JOURNAL OF FOOD PROTECTION, VOL. 55, MAY 1992 ANTIBIOTIC RESISTANT ORGANISMS FROM MILK IN MEXICO 373

Of the 30 UP milk samples, 2 strains of S. aureus REFERENCES isolated showed resistance to penicillin, ampicillin, an eryth 1. Alais, C. 1980. Ciencia de la leche. Principios de tecnica lechera. Ed. romycin, and 1 of the strains also showed resistance to Cecsa. Mexico, pp.339-340. chloramphenicol. The E. coli strain isolated from UP milk 2. Anderson, P. H. R., and D. M. Stone. 1955. Staphylococcal food showed resistance to tetracyclines, polymyxin B, poisoning associated with spray dried milk. J. Hyg. Cambr. 53:387- chloramphenicol, ampicillin, and gentamycin. 397. 3. Arredondo, J. L. 1986. Antibioticos en diarreas. Bol. Med. Hosp. Inf. The S. epidermidis present in both WMP and UP Mex. 43:445-450 showed resistance to tetracyclines, ampicillin, tobramycin, 4. Datta, N. 1985. Plasmids as organisms, pp. 3-16 In Donald R. nitrofurans, erythromycin, and penicillin in very similar Helinski (ed.), Plasmid in bacteria. Plenum Press, New York. percentages with the highest level of resistance to ampicillin 5. Diario Oficial de la Federacion. 1988. Reglamento para el Control de la Leche. Tomo CDXII. Cap. IV: 29-36. for both products. In addition, some multidrug resistance 6. Diaz de Aguayo, M., E. Acedo y L.Quihui. 1989. Determination de was noted in all other nonpathogenic organisms isolated la Frecuencia bacteriana y su resistencia a Antibioticos en regardless their source. Enfermedades Gastrointestinales de la Infancia en Hermosillo Sonora. Boletin Clinico de la Asociacibn Medica del Hospital Infantil del The results of this study show the presence in milk Edo. de Sonora. 6:14-21.

products of potential food pathogens with multiple resis 7. Farrar, W. E. 1985. Antibiotic resistance in developing countries. J. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/5/370/1664010/0362-028x-55_5_370.pdf by guest on 24 September 2021 tance to drugs. Furthermore, many nonpathogenic drug- Infect. Dis. 152:1105-1107. resistant organisms in the food supply can serve as a 8. Finegold, S. M., and W. J. Martin. 1983. Diagnostico Microbiologico, reservoir of resistant genes that can be transferred to patho VI ed. 9. Food and Drug Administration. 1984. Bacteriological Analytical genic organisms (73). Manual. 6th ed. Division of Microbiology, Center for Food Safety and Applied Nutrition. U.S. Food and Drug Administration. Wash CONCLUSIONS ington, DC. 10. Frazier, W. C, and D.C. Westhoff. 1978. Food microbiology, 3rd ed. McGraw Hill, New York. pp. 419. Retail milk in Hermosillo, Mexico, was found to be 11. Fleming, D. W., S. L. Cochi, K. L. Mc Donald, J. Bronsdeen, P. S. contaminated with antibiotic-resistant pathogenic and Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, nonpathogenic bacteria. This represents a real public health and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407. hazard in a product that is widely consumed. The multidrug 12. Holmberg, S. D., J. G. Wells, and M. L.Cohen. 1984. Animal to man resistance of the pathogenic organisms would make it transmission of antimicrobial resistant Salmonella. Investigations of difficult to treat the infections if a food poisoning situation U.S. outbreaks, 1971-1983. Science 225:833-835. were to occur. Furthermore, the high level of nonpathogenic 13. Levy, S. B. 1982. Microbial resistance to antibiotics: An evolving and persistent problem. Lancet ii:83-88. organisms present with drug resistance can act as a reser 14. Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. voir of resistance genes. Salminen, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, A. This high incidence of drug resistance could be attrib Audurier, B. D. Plikaytis, S. L. Fannin, A. Kleks, and C. V. Broome. uted to the widespread use of antibiotics in treating ill 1988. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 319:823-828. nesses both in the human and animal population, since in 15. Parker, M. T. 1982. Antibiotic resistance in pathogenic bacteria. Mexico antibiotics can be purchased without prescription Who Chronicle. 36:19-26. and they are relatively inexpensive. Furthermore, samples 16. Pelczar, J. M., and E. C. S. Chan. 1981. Elementos de Microbiologia. are not routinely monitored for compliance with food regu McGraw-Hill Book Co., Mexico, pp. 673-679. 17. Vasvada, P. C. 1986. For safety's sake. Sci. Food and Agric. 4:2-9. lations. It is recommended that a program be established to 18. Vazquez-Moreno, L., M. C. Bermudez, A. Langure, I. Higuera prevent contaminated products from reaching the consumer. Ciapara, M. Diaz de Aguayo, and E. Flores. Antibiotic residues and The most contaminated product was fluid pasteurized drug resistant bacteria in beef and chicken tissues. J. Food Sci. milk that contained Salmonella, E. coli enteropathogenic, 55:632-634. 19. World Health Orgainzation. 1978. Surveillance for the prevention and S. aureus. It can be stated that processing procedures are and control of health hazards due to antibiotic resistanc Enterobac- inadequate or that the milk is contaminated after processing. teria. Tech. Rep. Series. Geneva No. 624.

JOURNAL OF FOOD PROTECTION, VOL. 55. MAY 1992