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A Study on the Prevalence of Gram-Negative Bacteria in Bulk Tank Milk1
A Study on the Prevalence of Gram-Negative Bacteria in Bulk Tank Milk1 B. M. JAYARAO2 and L. WANG Minnesota-South Dakota Dairy Food Research Center Department of Dairy Science South Dakota State University Brookings, 57007-0647
ABSTRACT
INTRODUCTION
Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota were examined for coliforms and noncoliform bacteria. Coliforms were detected in 62.3% of bulk tank milk samples. Counts ranged from 0 to 4.7 log10 cfu/ml. The mean count was 3.4 log10 cfu/ml. Gram-negative noncoliform bacteria were observed in 76.3% of bulk tank milk. Counts ranged from 0 to 6.2 log10 cfu/ml. The mean count was 4.8 log10 cfu/ml. A total of 234 isolates from bulk tank milk were examined to species level; 205 isolates belonged to 28 species. Coliforms and gram-negative noncoliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively. Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from bulk tank milk in this study. These organisms have not been reported previously in bulk tank milk. A total of 116 isolates of Pseudomonas spp. were isolated from raw milk; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to their species level. Pseudomonas was the most predominant genus. Pseudomonas fluorescens was the most predominant species isolated from bulk tank milk and accounted for 29.9% of all isolates examined. The results of the study suggest that counts of coliforms and noncoliform bacteria in bulk tank milk vary considerably. The isolates represent a wide variety of Gramnegative bacterial species. Examination of bulk tank milk for coliforms and noncoliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality. (Key words: coliforms, noncoliforms, bulk tank milk)
Bulk tank milk (BTM) on the farm can become contaminated with gram-negative bacteria present on teats, the teat ends, teat canal, udder surfaces, mastitic udders, and contaminated water used to clean the milking systems and those that are resident in the milking system (1, 19). Gram-negative bacteria of concern in BTM include organisms that are pathogenic to humans and animals and those that lower the quality of milk. Gram-negative bacteria from the genera Acinetobacter, Aeromonas, Campylobacter, Citrobacter, Enterobacter, Escherichia, Flavobacterium, Klebsiella, Moraxella, Pseudomonas, Salmonella, Serratia, Yersinia, and Xanthobacter have been isolated from raw milk (9, 14, 15, 16). Gram-negative bacteria of public health significance such as Campylobacter, enterohemorrhagic strains of Escherichia coli, Salmonella, and Yersinia have been shown to be present in a small percentage of BTM surveyed and have been incriminated in several outbreaks of milkborne disease outbreaks following consumption of raw milk (14, 15). Gram-negative organisms associated with lowering of milk quality can be placed into two groups, coliforms and noncoliforms. Coliforms include Citrobacter spp., Enterobacter spp., Escherichia coli, and Klebsiella spp. Legal limits for coliform counts, unlike for pasteurized milk, have not been established for BTM. However, it is generally accepted that counts >1000 cfu/ml of raw milk indicate milk produced under unhygienic conditions (2). Gram-negative noncoliform bacteria in BTM have been shown to be a large, heterogeneous group of species belonging to the genera Acinetobacter, Aeromonas, Flavobacterium, Moraxella, Pasteurella, Pseudomonas, and Xanthobacter. Bacteria belonging to these genera, Pseudomonas in particular, have been shown on several occasions to be responsible for defects in raw milk, pasteurized milk, and milk products (16). The objective of this study was to determine prevalence and distribution of coliforms and noncoliforms in BTM on dairy farms.
Abbreviation key: BTM = bulk tank milk.
Received March 17, 1999. Accepted August 3, 1999. 1 Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 2112 of the Journal Series. 2 Current address: Department of Veterinary Science, Pennsylvania State University, PA 16802. 1999 J Dairy Sci 82:2620–2624
MATERIALS AND METHODS Bulk Tank Milk Samples Bulk tank milk from 131 dairy producers was examined. All of the BTM samples were collected during
2620
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GRAM-NEGATIVE BACTERIA IN BULK TANK MILK
March 1997. Two BTM samples were collected in sterile snap cap milk collection vials (Capital Vials, Fultonville, NY) from each dairy producer. Milk samples were collected in the morning, 1 to 2 h after milking. The milk in the bulk tank was inclusive of that day’s morning and previous day’s evening milk. The milk in the bulk tank was agitated for at least 5 min and collected with a sterile dipper. The interval between collecting the first and second samples was 7 d. Samples were brought to the laboratory on ice and processed within 1 to 2 h of receipt. Bacteriological Analysis of Bulk Tank Milk The milk samples were mixed thoroughly by gently inverting the milk vial 20 to 25 times. One milliliter of milk was transferred to a sterile tube containing 9 ml of quarter-strength Ringer’s solution (Oxoid, Unipath Ltd., England), which has been recommended as a diluent by the International Dairy Federation/International Standard Organization (8). The 10-fold diluted sample was vortexed at high speed for 10 to 15 s and 50 ml was plated with a spiroplater (Autoplate 4000, Spiral Biotech, Bethesda, MD) on MacConkey’s agar No. 3 (Oxoid) for determination of coliform counts and noncoliform counts. Plates were incubated at 32°C for 48 h. The coliform and noncoliform counts were determined by Autoplate 4000 user guide (Spiral Biotech). Bacterial counts from the first and second bulk tank milk samples were averaged and expressed as log10 cfu/ ml of bulk tank milk. Based on colony morphology and lactose fermentation on MacConkey’s agar No. 3 (Oxoid), isolates were selected for bacterial identification. Gram stain, oxidation-fermentation test, triple-sugar iron agar fermentation test, oxidase test, methyl red test, production of indole, and acetyl methyl carbinol and utilization of citrate were as described by Harley and Prescott (4). Coliforms and noncoliforms were identified to species level with API 20 E or API 20 NE bacterial rapid identification kits (BioMe´ rieux, Hazelwood, MO). Isolates that had excellent to very good identification profiles were considered as correctly identified species. All isolates yielding acceptable (≥90 ± 0.25% identification) profiles based on the API 20 E identification code book values (BioMe´ rieux) were retested to confirm API 20 E identification profiles. RESULTS AND DISCUSSION A total of 130 BTM samples were examined for coliforms. Coliform counts for one farm were accidentally not recorded. Coliforms were detected in 62.3% of BTM samples. Counts ranged from 0 to 4.7 log10 cfu/ml, with
TABLE 1. Coliform and noncoliform counts in bulk tank milk. Coliforms1,2 (n = 130)
Counts3
Noncoliforms1,2 (n = 131)
Percent bulk tank 1. Distribution of counts <0 0<1 1<2 2<3 3<4 4<5 >5 2. Range 3. Mean 4. Median
37.7 6.2 29.2 21.5 4.6 0.8 ... 0 to 4.7 3.4 2.3
23.7 6.1 15.2 32.1 12.2 9.9 0.8 log10 cfu/ml 0 to 6.2 4.8 3.1
1 Count represents the average of two samples from the same bulk tank. 2 Figures in parentheses indicate the number of bulk tanks examined. 3 Counts expressed as log10 cfu/ml.
a mean of 3.4 log10 cfu/ml of BTM (Table 1). The mean coliform counts observed in this study are higher than those reported by Hogan et al. (5), which were reported to be 2.0 log10 cfu/ml. Unlike pasteurized milk and milk products, where coliform count standards have been established, there are no regulatory standards for coliform counts in raw milk. About 5.4% of the samples in this study had counts greater than 3.0 log10 cfu/ml. Bray and Shearer (2) have suggested guidelines to interpret coliform counts in BTM. Based on these guidelines, a count greater than 3.0 log10 cfu/ml suggests problems associated with cleaning of the milking system or improper disinfection of teats before milking (2). A total of 131 BTM samples were examined for noncoliforms. Noncoliform counts were observed in 76.3% of BTM samples and counts ranged from 0 to 6.2 log10 cfu/ml, with a mean of 4.8 log10 cfu/ml (Table 1). The noncoliform counts mean was higher than that reported by Hogan et al. (5) of 2.0 log10 cfu/ml. There are no regulatory standards or recommended guidelines for evaluating noncoliform counts in raw milk. About 0.8% of BTM had counts greater than 105 cfu/ml, which suggests poor-quality milk, as counts of noncoliform >105 cfu/ml were reported in raw milk with defects (18, 19). Noncoliforms are widely distributed in the environment such as in soil, grass, hay, water, and feces (6, 10, 18, 19). These organisms could have gained access into BTM through untreated water used to clean the milking system or improper udder preparation before milking (18, 19). A total of 234 isolates of gram-negative bacteria from BTM were examined; 201 belonged to 28 species. Three coliforms and 30 noncoliform isolates could not be idenJournal of Dairy Science Vol. 82, No. 12, 1999
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TABLE 2. Total number of gram-negative bacterial genera isolated from bulk tank milk. No. Coliforms 1. 2. 3. 4. Noncoliforms 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Bacterial genera Citrobacter spp. Enterobacter spp. Escherichia spp. Klebsiella spp. Acinetobacter spp. Aeromonas spp. Agrobacterium spp. Bordetella spp. Comamonas spp. Hafnia spp. Listonella spp. Moraxella spp. Ochrobactrum spp. Oligella spp. Pasteurella spp. Pseudomonas spp. Xanthomonas spp. Yersinia spp. Serratia spp.
Total (n = 234)1 77 7 25 17 28 157 2 2 1 6 3 11 3 1 1 1 2 116 3 1 4
(32.9)2 (3.0) (10.7) (7.3) (12.0) (67.1) (0.9) (0.9) (0.4) (2.6) (1.3) (4.7) (1.3) (0.4) (0.4) (0.4) (0.9) (49.6) (1.3) (0.4) (1.7)
Percent of total isolates 100.0 9.1 32.5 22.1 36.4 100.0 1.3 1.3 0.6 3.8 1.9 7.0 1.9 0.6 0.6 0.6 1.3 73.9 1.9 0.6 2.5
1
Total number of bacteria isolated and examined. Figures in parenthesis indicate percentage of total number of isolates. 2
tified to species level. (Tables 2 and 3). Of the 234 isolates, coliforms accounted for about 32.9% of the isolates; Citrobacter spp., Enterobacter spp., E. coli, and Klebsiella spp. accounted for 3.0, 10.7, 7.3, and 12.0% of the 234 isolates, respectively (Table 3). The presence of coliforms in raw foods is generally regarded as direct contamination of foods with fecal material. This assumption might not be true in some instances with raw milk, as the resident flora of the milking system might be partly made up of coliforms that may gain access into BTM during the milking. Another mode of the entry of coliforms, particularly E. coli into BTM has been suggested by Jayarao et al. (9), who studied the prevalence of E. coli subtypes in feces, quarter milk, teat liners, and BTM by a PCR-based DNA fingerprinting technique. Their results showed that the E. coli subtypes present in feces were also observed in quarter milk, teat liner surfaces, and BTM. They suggested that the probable route of transmission of fecal origin subtypes of E. coli could be through “temporary habitation” of the streak canal between milkings; during milking E. coli would gain access to BTM. Coliform bacteria can also gain access directly into bulk tank milk when cows with subclinical coliform mastitis are milked and have been shown to increase the coliform counts of bulk tank milk (1). Several studies have shown that pathogenic E. coli comprise a very small percentage of the total E. coli present in raw milk (12). With regard to this study, the potential risk of BTM contamination Journal of Dairy Science Vol. 82, No. 12, 1999
with pathogenic strains of E. coli is probably low; only 7.3% of BTM were found to be contaminated with E. coli. Noncoliforms accounted for 67.1% of total isolates and belonged to 15 genera (Table 2). Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from BTM in this study (Table 3). None of these organisms has been reported previously to occur in BTM. These organisms are naturally distributed in soil and plants (Agrobacterium radiobacter and Comamonas testosteroni), human genitourethral tract (Oligella urethralis), marine environment and fresh water (Listonella damsela), mammalian epithelial cilia of respiratory tracts (Bordetella spp.), and human clinical specimens (Ochrobactrum anthropi) (6, 10). The mode of entry of these organisms in BTM is not clear. A total of 116 isolates of Pseudomonas spp. were isolated from BTM; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to species level (Tables 2 and 3). The nine Pseudomonas spp. have been shown to occur in BTM (9, 16). Pseudomonas spp. accounted for 49.6% of the total isolates (n = 234) and 73.9% of noncoliforms, respectively (Table 2). Pseudomonas was the most predominant genus, and P. fluorescens was the most predominant species isolated from BTM in this study. Pseudomonas fluorescens and P. putida accounted for 47.2 and 18.9% of all noncoliforms, respectively (Table 3). Ternstro¨ m et al. (17) showed that P. fluorescens accounted for 55.6% of the all bacterial isolates in raw milk. Gennarl and Dragotto (3) observed that P. fluorescens was present in 84% of the raw milk samples examined. The prevalence of P. fluorescens can be explained from the following reports. P. fluorescens is a psychrotrophic organism that has a short-generation time at refrigeration temperatures. Ingraham and Stokes (7) showed that P. fluorescens generation time was 30.2 h at 0 to 2°C, 6.7 to 7.2 h at 4 to 6°C, and 1.4 h at 20°C, respectively. The ability to grow rapidly at low temperatures puts P. fluorescens at an advantage over other gram-negative bacteria. In addition, P. fluorescens also has the ability to produce adhesive exopolysaccharides, which could facilitate the formation of biofilm (13). Once a biofilm is formed, P. fluorescens could become a potential bacterial reservoir and resist the effects of chemicals and sanitizers if the milking system remains unclean for long periods (11). Pseudomonas fluorescens could contaminate BTM when released from a biofilm. The results of the study suggest that numbers of coliforms and noncoliforms in bulk tank milk vary considerably and include a wide variety of gram-negative bacterial species. The study also reveals that a bulk
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GRAM-NEGATIVE BACTERIA IN BULK TANK MILK TABLE 3. Species of gram-negative bacteria isolated from bulk tank milk. No. Coliforms 1. 2. 3. 4. 5. 6. 7. Noncoliforms 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Total (n = 201)2
Bacterial species1 Citrobacter freundii Enterobacter agglomerans Enterobacter cloacae Enterobacter sakazakii Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Acinetobacter lwoffii Aeromonas salmonicida ssp. salmonicida Aeromonas schubertii Agrobacterium radiobacter Comamonas testosteroni Hafnia alvei Listonella damsela Moraxella lacunata Ochrobactrum anthropi Oligella urethralis Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas diminuta Pseudomonas fluorescens Pseudomonas mendocina Pseudomonas mesophilia Pseudomonas pickettii Pseudomonas putida Pseudomonas stutzeri Xanthomonas maltophilia Yersinia enterocolitica
74 6 9 14 1 17 7 20 127 2 1 1 1 3 11 3 1 1 1 4 2 1 60 2 1 1 24 3 3 1
(36.8)3 (3.0) (4.5) (7.0) (0.5) (8.5) (3.0) (10.0) (63.2) (1.0) (0.5) (0.5) (0.5) (1.5) (5.5) (1.5) (0.5) (0.5) (0.5) (2.0) (1.0) (0.5) (29.9) (1.0) (0.5) (0.5) (11.9) (1.5) (1.5) (0.5)
Percent of total isolates 100.0 8.1 12.2 18.9 1.4 23.0 9.5 27.0 100.0 1.6 0.8 0.8 0.8 2.4 8.7 2.4 0.8 0.8 0.8 3.1 1.6 0.8 47.2 1.6 0.8 0.8 18.9 2.4 2.4 0.8
1 A total of 33 isolates belonging to the genus Citrobacter (1), Bordetella (6), Enterobacter (1), Klebsiella (1), Pasteurella (2), Pseudomonas (18), and Serratia (4) could not be identified to species level. 2 Total number of bacterial isolates. 3 Figures in parentheses indicate percent.
tank milk can be examined not only to determine milk quality and the presence of foodborne pathogens but perhaps can also be used to detect pathogens of animal health significance such as Bordetella spp., Moraxella spp., and Pasteurella spp, which have been well documented as important agents of the respiratory diseases in calves and adult cows. The findings of the study clearly show the importance of examining bulk tank milk for coliforms and noncoliforms, in particular, enumeration of Pseudomonas organisms, as this could reveal potential problems associated with bacterial counts and milk quality. ACKNOWLEDGMENTS The authors thank the Minnesota-South Dakota Dairy Foods Research Center (Brookings, SD) for funding the project. The authors acknowledge the support of D. R. Henning, Alfred Chair, for use of laboratory facilities.
REFERENCES 1 Bramley, A. J., and C. H. McKinnon. 1990. The microbiology of raw milk. Pages 163–208 in Dairy Microbiology. 2nd ed. R. K. Robinson, ed. Vol. 1. Elsevier Appl. Sci., New York, NY. 2 Bray, D. R., and J. K. Shearer. 1996. Trouble-Shooting a Mastitis Problem Herd. Circular 1162, Department of Dairy and Poultry Science, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. 3 Gennarl, M., and F. Dragotto. 1992. A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water. J. Appl. Bacteriol. 72:281–288. 4 Harley, J. P., and L. M. Prescott. 1993. Laboratory Exercises in Microbiology. 2nd ed. Wm. C. Brown Publishers, Dubuque, IA. 5 Hogan, J. S., K. H. Hoblet, K. L. Smith, D. A. Todhunter, P. S. Schoenberger, W. D. Hueston, D. E. Pritchard, G. L. Bowman, L. E. Heider, B. L. Brockett, and H. R. Courad. 1988. Bacterial and somatic cell counts in bulk tank milk from nine well managed herds. J. Food Prot. 51:930–934. 6 Holt, J. G., N. R. Krieg, P.H.A. Sneath, J. T. Staley, and S. T. Williams. 1994. Bergey’s Manual of Determinative Bacteriology. 9th ed. Williams & Wilkins, Baltimore. 7 Ingraham J. L., and J. L. Stokes. 1959. Psychrotrophic bacteria. Bacteriol. Rev. 23:97–108. 8 Janus, J. A. 1980. Investigations into the bactericidal effect of diluents applied for the enumeration of Enterobacteriaceae in Journal of Dairy Science Vol. 82, No. 12, 1999
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food products. Report of Group E 50. The Netherlands: International Dairy Federation; 1980. ISO/TC 34/SC 9. Jayarao, B. M., L. Wang, L. An, R. Bangalore, D. R. Henning, and G. Stegeman. 1997. A study on the prevalence of Gramnegative bacteria in a dairy herd. J. Dairy Sci. 80(Suppl. 1):172 (Abstr.). Krieg, N. R., and J. G. Holt. 1984. Bergey’s Manual of Systematic Bacteriology. Vol. 1. Williams and Wilkins, Baltimore. Mosteller, T. M., and J. R. Bishop. 1993. Sanitizer efficacy against attached bacteria in a milk biofilm. J. Food Prot. 56:34–41. Padhye, N. V., and M. P. Doyle. 1991. Rapid procedure for detecting enterohemorrhagic Escherichia coli 0157:H 7 in food. Appl. Environ. Microbiol. 57:2693–2698. Read, R. R., and J. W. Costerton. 1987. Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens. Can. J. Microbiol. 33:1080–1090. Rohrbach, B. W., F. A. Draughon, P. M. Davidson, and S. P. Oliver. 1992. Prevalence of Listeria monocytogenes, Campylobacter je-
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juni, Yersinia enterocolitica and Salmonella in bulk tank milk: risk factors and risk of human exposure. J. Food. Prot. 52:93–97. Steele, M. L., W. B. McNab, C. Poppe, M. W. Griffiths, S. Chen, S. A. Degrandis, L. C. Fruhner, C. A. Larkin, J. A. Lynch, and J. A. Odermeru. 1997. Survey of Ontario bulk tank milk for foodborne pathogens. J. Food Prot. 60:1341–1346. Suhren, G. 1989. Producer microorganism. Pages 3–27 in Enzymes of Psychrotrophs in Raw Foods. R. C. McKellar, ed. CRC Press Inc., Boca Raton, FL. Ternstro¨ m, A., A. M. Lindberg, and G. Molin. 1993. Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus. J. Appl. Bacteriol. 75:25–34. Thomas, S. B. 1966. Sources, incidence and significance of psychrotrophic bacteria in milk. Milchwissenschaft 21:270–275. Thomas, S. B., and B. F. Thomas. 1973. Psychrotrophic bacteria in refrigerated bulk-collected milk. Part I. Dairy Ind. 38:11–15.
ABSTRACT
INTRODUCTION
Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota were examined for coliforms and noncoliform bacteria. Coliforms were detected in 62.3% of bulk tank milk samples. Counts ranged from 0 to 4.7 log10 cfu/ml. The mean count was 3.4 log10 cfu/ml. Gram-negative noncoliform bacteria were observed in 76.3% of bulk tank milk. Counts ranged from 0 to 6.2 log10 cfu/ml. The mean count was 4.8 log10 cfu/ml. A total of 234 isolates from bulk tank milk were examined to species level; 205 isolates belonged to 28 species. Coliforms and gram-negative noncoliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively. Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from bulk tank milk in this study. These organisms have not been reported previously in bulk tank milk. A total of 116 isolates of Pseudomonas spp. were isolated from raw milk; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to their species level. Pseudomonas was the most predominant genus. Pseudomonas fluorescens was the most predominant species isolated from bulk tank milk and accounted for 29.9% of all isolates examined. The results of the study suggest that counts of coliforms and noncoliform bacteria in bulk tank milk vary considerably. The isolates represent a wide variety of Gramnegative bacterial species. Examination of bulk tank milk for coliforms and noncoliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality. (Key words: coliforms, noncoliforms, bulk tank milk)
Bulk tank milk (BTM) on the farm can become contaminated with gram-negative bacteria present on teats, the teat ends, teat canal, udder surfaces, mastitic udders, and contaminated water used to clean the milking systems and those that are resident in the milking system (1, 19). Gram-negative bacteria of concern in BTM include organisms that are pathogenic to humans and animals and those that lower the quality of milk. Gram-negative bacteria from the genera Acinetobacter, Aeromonas, Campylobacter, Citrobacter, Enterobacter, Escherichia, Flavobacterium, Klebsiella, Moraxella, Pseudomonas, Salmonella, Serratia, Yersinia, and Xanthobacter have been isolated from raw milk (9, 14, 15, 16). Gram-negative bacteria of public health significance such as Campylobacter, enterohemorrhagic strains of Escherichia coli, Salmonella, and Yersinia have been shown to be present in a small percentage of BTM surveyed and have been incriminated in several outbreaks of milkborne disease outbreaks following consumption of raw milk (14, 15). Gram-negative organisms associated with lowering of milk quality can be placed into two groups, coliforms and noncoliforms. Coliforms include Citrobacter spp., Enterobacter spp., Escherichia coli, and Klebsiella spp. Legal limits for coliform counts, unlike for pasteurized milk, have not been established for BTM. However, it is generally accepted that counts >1000 cfu/ml of raw milk indicate milk produced under unhygienic conditions (2). Gram-negative noncoliform bacteria in BTM have been shown to be a large, heterogeneous group of species belonging to the genera Acinetobacter, Aeromonas, Flavobacterium, Moraxella, Pasteurella, Pseudomonas, and Xanthobacter. Bacteria belonging to these genera, Pseudomonas in particular, have been shown on several occasions to be responsible for defects in raw milk, pasteurized milk, and milk products (16). The objective of this study was to determine prevalence and distribution of coliforms and noncoliforms in BTM on dairy farms.
Abbreviation key: BTM = bulk tank milk.
Received March 17, 1999. Accepted August 3, 1999. 1 Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 2112 of the Journal Series. 2 Current address: Department of Veterinary Science, Pennsylvania State University, PA 16802. 1999 J Dairy Sci 82:2620–2624
MATERIALS AND METHODS Bulk Tank Milk Samples Bulk tank milk from 131 dairy producers was examined. All of the BTM samples were collected during
2620
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GRAM-NEGATIVE BACTERIA IN BULK TANK MILK
March 1997. Two BTM samples were collected in sterile snap cap milk collection vials (Capital Vials, Fultonville, NY) from each dairy producer. Milk samples were collected in the morning, 1 to 2 h after milking. The milk in the bulk tank was inclusive of that day’s morning and previous day’s evening milk. The milk in the bulk tank was agitated for at least 5 min and collected with a sterile dipper. The interval between collecting the first and second samples was 7 d. Samples were brought to the laboratory on ice and processed within 1 to 2 h of receipt. Bacteriological Analysis of Bulk Tank Milk The milk samples were mixed thoroughly by gently inverting the milk vial 20 to 25 times. One milliliter of milk was transferred to a sterile tube containing 9 ml of quarter-strength Ringer’s solution (Oxoid, Unipath Ltd., England), which has been recommended as a diluent by the International Dairy Federation/International Standard Organization (8). The 10-fold diluted sample was vortexed at high speed for 10 to 15 s and 50 ml was plated with a spiroplater (Autoplate 4000, Spiral Biotech, Bethesda, MD) on MacConkey’s agar No. 3 (Oxoid) for determination of coliform counts and noncoliform counts. Plates were incubated at 32°C for 48 h. The coliform and noncoliform counts were determined by Autoplate 4000 user guide (Spiral Biotech). Bacterial counts from the first and second bulk tank milk samples were averaged and expressed as log10 cfu/ ml of bulk tank milk. Based on colony morphology and lactose fermentation on MacConkey’s agar No. 3 (Oxoid), isolates were selected for bacterial identification. Gram stain, oxidation-fermentation test, triple-sugar iron agar fermentation test, oxidase test, methyl red test, production of indole, and acetyl methyl carbinol and utilization of citrate were as described by Harley and Prescott (4). Coliforms and noncoliforms were identified to species level with API 20 E or API 20 NE bacterial rapid identification kits (BioMe´ rieux, Hazelwood, MO). Isolates that had excellent to very good identification profiles were considered as correctly identified species. All isolates yielding acceptable (≥90 ± 0.25% identification) profiles based on the API 20 E identification code book values (BioMe´ rieux) were retested to confirm API 20 E identification profiles. RESULTS AND DISCUSSION A total of 130 BTM samples were examined for coliforms. Coliform counts for one farm were accidentally not recorded. Coliforms were detected in 62.3% of BTM samples. Counts ranged from 0 to 4.7 log10 cfu/ml, with
TABLE 1. Coliform and noncoliform counts in bulk tank milk. Coliforms1,2 (n = 130)
Counts3
Noncoliforms1,2 (n = 131)
Percent bulk tank 1. Distribution of counts <0 0<1 1<2 2<3 3<4 4<5 >5 2. Range 3. Mean 4. Median
37.7 6.2 29.2 21.5 4.6 0.8 ... 0 to 4.7 3.4 2.3
23.7 6.1 15.2 32.1 12.2 9.9 0.8 log10 cfu/ml 0 to 6.2 4.8 3.1
1 Count represents the average of two samples from the same bulk tank. 2 Figures in parentheses indicate the number of bulk tanks examined. 3 Counts expressed as log10 cfu/ml.
a mean of 3.4 log10 cfu/ml of BTM (Table 1). The mean coliform counts observed in this study are higher than those reported by Hogan et al. (5), which were reported to be 2.0 log10 cfu/ml. Unlike pasteurized milk and milk products, where coliform count standards have been established, there are no regulatory standards for coliform counts in raw milk. About 5.4% of the samples in this study had counts greater than 3.0 log10 cfu/ml. Bray and Shearer (2) have suggested guidelines to interpret coliform counts in BTM. Based on these guidelines, a count greater than 3.0 log10 cfu/ml suggests problems associated with cleaning of the milking system or improper disinfection of teats before milking (2). A total of 131 BTM samples were examined for noncoliforms. Noncoliform counts were observed in 76.3% of BTM samples and counts ranged from 0 to 6.2 log10 cfu/ml, with a mean of 4.8 log10 cfu/ml (Table 1). The noncoliform counts mean was higher than that reported by Hogan et al. (5) of 2.0 log10 cfu/ml. There are no regulatory standards or recommended guidelines for evaluating noncoliform counts in raw milk. About 0.8% of BTM had counts greater than 105 cfu/ml, which suggests poor-quality milk, as counts of noncoliform >105 cfu/ml were reported in raw milk with defects (18, 19). Noncoliforms are widely distributed in the environment such as in soil, grass, hay, water, and feces (6, 10, 18, 19). These organisms could have gained access into BTM through untreated water used to clean the milking system or improper udder preparation before milking (18, 19). A total of 234 isolates of gram-negative bacteria from BTM were examined; 201 belonged to 28 species. Three coliforms and 30 noncoliform isolates could not be idenJournal of Dairy Science Vol. 82, No. 12, 1999
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TABLE 2. Total number of gram-negative bacterial genera isolated from bulk tank milk. No. Coliforms 1. 2. 3. 4. Noncoliforms 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Bacterial genera Citrobacter spp. Enterobacter spp. Escherichia spp. Klebsiella spp. Acinetobacter spp. Aeromonas spp. Agrobacterium spp. Bordetella spp. Comamonas spp. Hafnia spp. Listonella spp. Moraxella spp. Ochrobactrum spp. Oligella spp. Pasteurella spp. Pseudomonas spp. Xanthomonas spp. Yersinia spp. Serratia spp.
Total (n = 234)1 77 7 25 17 28 157 2 2 1 6 3 11 3 1 1 1 2 116 3 1 4
(32.9)2 (3.0) (10.7) (7.3) (12.0) (67.1) (0.9) (0.9) (0.4) (2.6) (1.3) (4.7) (1.3) (0.4) (0.4) (0.4) (0.9) (49.6) (1.3) (0.4) (1.7)
Percent of total isolates 100.0 9.1 32.5 22.1 36.4 100.0 1.3 1.3 0.6 3.8 1.9 7.0 1.9 0.6 0.6 0.6 1.3 73.9 1.9 0.6 2.5
1
Total number of bacteria isolated and examined. Figures in parenthesis indicate percentage of total number of isolates. 2
tified to species level. (Tables 2 and 3). Of the 234 isolates, coliforms accounted for about 32.9% of the isolates; Citrobacter spp., Enterobacter spp., E. coli, and Klebsiella spp. accounted for 3.0, 10.7, 7.3, and 12.0% of the 234 isolates, respectively (Table 3). The presence of coliforms in raw foods is generally regarded as direct contamination of foods with fecal material. This assumption might not be true in some instances with raw milk, as the resident flora of the milking system might be partly made up of coliforms that may gain access into BTM during the milking. Another mode of the entry of coliforms, particularly E. coli into BTM has been suggested by Jayarao et al. (9), who studied the prevalence of E. coli subtypes in feces, quarter milk, teat liners, and BTM by a PCR-based DNA fingerprinting technique. Their results showed that the E. coli subtypes present in feces were also observed in quarter milk, teat liner surfaces, and BTM. They suggested that the probable route of transmission of fecal origin subtypes of E. coli could be through “temporary habitation” of the streak canal between milkings; during milking E. coli would gain access to BTM. Coliform bacteria can also gain access directly into bulk tank milk when cows with subclinical coliform mastitis are milked and have been shown to increase the coliform counts of bulk tank milk (1). Several studies have shown that pathogenic E. coli comprise a very small percentage of the total E. coli present in raw milk (12). With regard to this study, the potential risk of BTM contamination Journal of Dairy Science Vol. 82, No. 12, 1999
with pathogenic strains of E. coli is probably low; only 7.3% of BTM were found to be contaminated with E. coli. Noncoliforms accounted for 67.1% of total isolates and belonged to 15 genera (Table 2). Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from BTM in this study (Table 3). None of these organisms has been reported previously to occur in BTM. These organisms are naturally distributed in soil and plants (Agrobacterium radiobacter and Comamonas testosteroni), human genitourethral tract (Oligella urethralis), marine environment and fresh water (Listonella damsela), mammalian epithelial cilia of respiratory tracts (Bordetella spp.), and human clinical specimens (Ochrobactrum anthropi) (6, 10). The mode of entry of these organisms in BTM is not clear. A total of 116 isolates of Pseudomonas spp. were isolated from BTM; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to species level (Tables 2 and 3). The nine Pseudomonas spp. have been shown to occur in BTM (9, 16). Pseudomonas spp. accounted for 49.6% of the total isolates (n = 234) and 73.9% of noncoliforms, respectively (Table 2). Pseudomonas was the most predominant genus, and P. fluorescens was the most predominant species isolated from BTM in this study. Pseudomonas fluorescens and P. putida accounted for 47.2 and 18.9% of all noncoliforms, respectively (Table 3). Ternstro¨ m et al. (17) showed that P. fluorescens accounted for 55.6% of the all bacterial isolates in raw milk. Gennarl and Dragotto (3) observed that P. fluorescens was present in 84% of the raw milk samples examined. The prevalence of P. fluorescens can be explained from the following reports. P. fluorescens is a psychrotrophic organism that has a short-generation time at refrigeration temperatures. Ingraham and Stokes (7) showed that P. fluorescens generation time was 30.2 h at 0 to 2°C, 6.7 to 7.2 h at 4 to 6°C, and 1.4 h at 20°C, respectively. The ability to grow rapidly at low temperatures puts P. fluorescens at an advantage over other gram-negative bacteria. In addition, P. fluorescens also has the ability to produce adhesive exopolysaccharides, which could facilitate the formation of biofilm (13). Once a biofilm is formed, P. fluorescens could become a potential bacterial reservoir and resist the effects of chemicals and sanitizers if the milking system remains unclean for long periods (11). Pseudomonas fluorescens could contaminate BTM when released from a biofilm. The results of the study suggest that numbers of coliforms and noncoliforms in bulk tank milk vary considerably and include a wide variety of gram-negative bacterial species. The study also reveals that a bulk
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GRAM-NEGATIVE BACTERIA IN BULK TANK MILK TABLE 3. Species of gram-negative bacteria isolated from bulk tank milk. No. Coliforms 1. 2. 3. 4. 5. 6. 7. Noncoliforms 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Total (n = 201)2
Bacterial species1 Citrobacter freundii Enterobacter agglomerans Enterobacter cloacae Enterobacter sakazakii Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Acinetobacter lwoffii Aeromonas salmonicida ssp. salmonicida Aeromonas schubertii Agrobacterium radiobacter Comamonas testosteroni Hafnia alvei Listonella damsela Moraxella lacunata Ochrobactrum anthropi Oligella urethralis Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas diminuta Pseudomonas fluorescens Pseudomonas mendocina Pseudomonas mesophilia Pseudomonas pickettii Pseudomonas putida Pseudomonas stutzeri Xanthomonas maltophilia Yersinia enterocolitica
74 6 9 14 1 17 7 20 127 2 1 1 1 3 11 3 1 1 1 4 2 1 60 2 1 1 24 3 3 1
(36.8)3 (3.0) (4.5) (7.0) (0.5) (8.5) (3.0) (10.0) (63.2) (1.0) (0.5) (0.5) (0.5) (1.5) (5.5) (1.5) (0.5) (0.5) (0.5) (2.0) (1.0) (0.5) (29.9) (1.0) (0.5) (0.5) (11.9) (1.5) (1.5) (0.5)
Percent of total isolates 100.0 8.1 12.2 18.9 1.4 23.0 9.5 27.0 100.0 1.6 0.8 0.8 0.8 2.4 8.7 2.4 0.8 0.8 0.8 3.1 1.6 0.8 47.2 1.6 0.8 0.8 18.9 2.4 2.4 0.8
1 A total of 33 isolates belonging to the genus Citrobacter (1), Bordetella (6), Enterobacter (1), Klebsiella (1), Pasteurella (2), Pseudomonas (18), and Serratia (4) could not be identified to species level. 2 Total number of bacterial isolates. 3 Figures in parentheses indicate percent.
tank milk can be examined not only to determine milk quality and the presence of foodborne pathogens but perhaps can also be used to detect pathogens of animal health significance such as Bordetella spp., Moraxella spp., and Pasteurella spp, which have been well documented as important agents of the respiratory diseases in calves and adult cows. The findings of the study clearly show the importance of examining bulk tank milk for coliforms and noncoliforms, in particular, enumeration of Pseudomonas organisms, as this could reveal potential problems associated with bacterial counts and milk quality. ACKNOWLEDGMENTS The authors thank the Minnesota-South Dakota Dairy Foods Research Center (Brookings, SD) for funding the project. The authors acknowledge the support of D. R. Henning, Alfred Chair, for use of laboratory facilities.
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