Abstract
Five brands of commercially sold milk samples were routinely evaluated for the presence of microorganisms and screened for anti-microbial substances using both the standard agar-well diffusion method and by a qualitative rapid antibiotic residue test kit for a period of 15 months. Bacillus spp. was recovered in higher frequencies in all the milk samples and milk was contaminated with coliforms, S. aureus, and S. agalactae. Anti-microbial activity were present in all brands of milk samples against the standard test strain of S. aureus using the standard agar-well diffusion method but were absent during the cooler months of the year. When milk samples with positive anti-microbial activity were subjected to heat treatment in order to denature milk proteins, the anti-microbial activity still persisted suggesting that these anti-microbial substances were not protein in nature. The rapid antibiotic residue detection method revealed the presence of antibiotics of the beta-lactam group. The presence of anti-microbial substances and widespread microbial contamination in the milk samples tested in Oman underlined the necessity to employ stricter enforcement of quality control procedures during the production of milk and milk products.
Keywords:
Introduction
Microbial resistance to antibiotics is a global issue and part of this concern is due to the inappropriate use of these agents in human and veterinary medicine. Intensive animal production involves administering livestock with antibiotics to promote growth and treat infections. In addition, high milk production is generally associated with a higher incidence of clinical and sub-clinical mastitisCitation1 and subsequently an increased use of anti-microbial therapeutic regimes.
There is a close association between the rate of resistance development and the quantities of anti-microbial agents used.Citation2 The inappropriate use of antibiotics can result in the selection and transmission of antibiotic-resistant bacteria. These bacteria move through the food chain and environment by a variety of routes, and their presence ultimately has consequences for humans, like the establishment of antibiotic resistant bacterial populations and development of allergic reactions. The increasing awareness concerning the connection between the overuse of anti-microbial drugs in both human medicine and the agriculture industry and the emergence and spread of antibiotic resistant bacteria has forced the dairy industry to put forth a number of protective measures and regulations in the use of antibiotics in their products.Citation3
Due to the potential hazardous effects of antibiotics for human health, some measures or studies must be undertaken to detect the presence of anti-microbial substances as well as the presence of pathogenic microorganisms in milk.Citation4 The objectives of this study were to identify the presence of microorganisms and anti-microbial substances in five brands of fresh pasteurized milk sold in Oman for a period of 15 months.
Materials and Methods
A total of 259 pasteurized fresh milk samples from five different milk producers representing both imported (IA and IB) and locally produced (LA, LB, and LC) milk were examined weekly for microorganisms and anti-microbial substances within three days of their production dates. These milk samples were purchased randomly from supermarkets, transported on ice-cooled box at 4–8°C to the laboratory for analysis.
The total aerobic plate count was done using the standard plate count (SPC, Oxoid, UK). A standardized test portion of 0.5 mL of one-tenth decimal dilutions of milk samples were inoculated by the standard surface spread method and incubated at 37.0 ± 0.5°C for 48 h to enumerate the mesophilic microorganisms and another plate at 37.0 ± 0.5°C for 3–5 days to detect the presence of pigmented bacteria, which are part of the thermoduric group. Results were expressed as a number of colony forming units (CFU) in a given volume of the sample.Citation5 To determine the total coliform (TC) and fecal coliform (FC) counts, a one in ten dilution of milk samples was plated on Cystine Lactose Electrolyte Deficient agarCitation6 and the colony counts expressed as CFU mL−1. Coliform like colonies underwent an oxidase test. Oxidase negative organisms were identified by the API 20 E multi-test identification systems for the members of the family Enterobacteriaceae. Milk samples were also plated on 5% sheep Blood Agar (BA) at 37.0 ± 0.5°C under aerobic condition to identify Staphylococcus aureus. Colonies were Gram stained and tested for catalase reaction and biochemical identification was performed by API STAPH multi-test identification system. Direct culturing on BACitation7 under microaerophillic incubation atmosphere at 37.0 ± 0.5°C for 24–48 h was performed for the detection of Streptococcus agalactiae. Citation8 For the isolation of thermoduric microorganisms, typical colonies of Bacillus from the SPC agar were sub-cultured on BA. Colonies with α-hemolysis were Gram stained for typical Gram-positive spore-former rods. Noncoliform colonies were sub-cultured onto Pseudomonas agar for the detection of typical pyocyanin pigment and the oxidase/catalase test and Gram stain were also done.Citation6
The milk samples were tested for anti-microbial activity against three frank and opportunistic pathogens.Citation9 Standard control strains included the following: Escherichia coli NCTC 10418 (Oxford university collections, UK) as a representative of coliform organisms, Pseudomonas aeruginosa N.Citation9 CTC 10662 representing a Gram-negative oxidase positive aerobic bacilli, and S. aureus NCTC 6571, a Gram-positive aerobic cocci. The inoculums size was standardized to a 0.5 McFarland standard, which gave a density equivalent to 108 CFU mL−1.Citation10 The rotary plating method was used to inoculate the control strains to the standard antibiotic sensitivity-testing medium using the diagnostic sensitivity test agar. Under aseptic conditions, 8 mm diameter wells were cut from the agar using a sterile cork-borer, and 100 µL of milk samples were delivered into the wells.Citation11 After overnight incubation at 37°C, plates were examined for any zones of growth inhibition. The zones of inhibition were measured using calipers and the diameter was recorded in mm. Standardized quantities of 5.0 mL of milk samples which showed positive anti-microbial activity were heat-treated at 100°C for 15 min to denature natural milk proteins such as lactoferrin, lactoperoxidase, and lysozyme.Citation12 They were then allowed to cool to room temperature and the above procedure of anti-microbial screening test was repeated.
Anti-microbial residues in milk were qualitatively assayed using rapid milk antibiotic residue test cartridges (Snap®, IDEXX, USA). A test portion of 450 µL of milk sample was incubated at 45 ± 5°C and placed in test cartridges for 2 and 5 min. The 2-min test was to detect tetracycline group, which includes chlortetracycline, oxytetracycline and tetracycline, and the 5-min test was to detect beta-lactam group that includes penicillin G, ampicillin, ceftiofur, and cephapirin. These tests were done only on milk samples that showed positive results to the standard agar-well diffusion method against control strains of S. aureus from April to June 2002.
Finally, six milk samples with and without heat treatment were tested (May, 2003) using the standard agar-well diffusion method against control strains of S. aureus and Methecillin Resistance S. aureus (MRSA) to detect the presence of anti-microbial activity. In addition, the rapid snap antibiotic screening test was also done to detect the antibiotics of the beta-lactam group.
Data were analyzed using the Statistical Analysis System.Citation13 Nonparametric Chi-square goodness of fit test was used to analyze the frequency of occurrence of anti-microbial activity by milk samples.Citation14 Two-way analysis of variance (months of milk collection and five brands of milk samples) in a completely randomized design was usedCitation15 to compare the effect of treatment on SPC, TC, FC, and total S. aureus counts in these milk samples. Duncan's multiple range means comparison test was used to examine the differences between treatment means. In addition, correlation analysis was used to determine the relationship between all variables in this study.
Results
Isolation of Microorganisms
Bacillus spp. (Table ) were recovered with the highest frequencies in all milk samples. Other microorganisms were also detected at lower frequencies. The coliform Citrobacter was absent in LB and LC milk samples and Enterobacter spp. was absent in IA and LC milk samples. Escherchia spp. was present in all three local milk products but was absent in the imported milk products. However, Klebsiella spp. was present in all milk samples with the highest frequency in LA milk. Pigmented bacteria and Pseudomonas spp. were found in all milk samples with varying frequencies.
Table 1 Frequency of isolation of major microbial genera from five brands of milk samples
There was no effect of months of milk collection on SPC, TC, FC, and S. aureus but all five brands of milk were found to contain these bacteria. The mean SPC count from LA milk samples was higher (P < 0.05) than all other milk samples (Table ).
Table 2 Mean microbial counts in milk samples from five producers
There was a positive (P < 0.05) correlation between SPC and the presence of TC and FC. The highest mean TC bacilli were also from LA milk samples and this was similar (P > 0.05) to LB milk samples. However, the TC of LB milk samples were similar (P > 0.05) to LC and IA milk samples. The IB milk samples had the lowest (P < 0.05) TC counts and this was similar to IA and LC milk samples. The highest mean FC count was again found in LA milk samples and this was similar (P > 0.05) to LB and LC milk samples. However, mean FC from LB and LC milk samples was similar (P > 0.05) to IA and IB milk samples. Once again the mean S. aureus count from LA milk samples was higher (P < 0.05) than from all other milk samples tested. S. agalactiae was isolated only in three batches in LA and two batches in LB milk samples (data not shown). Overall, the LA milk samples were more heavily contaminated with bacteria than all other milk samples.
Screening for the Anti-microbial Activity in Milk Samples
The five brands of milk samples had varying degrees of anti-microbial activity during different months of milk collection only against the standard test strain of S. aureus using the standard agar-well diffusion method (Table ). The frequency of occurrence of anti-microbial activity was lower (P < 0.05) in IB milk samples compared to other milk samples. There was no inhibition of S. aureus growth (P < 0.05) in all samples tested from October 2001 until March 2002, which coincides with cooler months with peak milk production in the Middle Eastern region.
Table 3 Percentage of milk samples with anti-microbial activity against S. aureus collected from April 2001 to June 2002 using the standard agar-well diffusion method
The mean zones of S. aureus growth inhibition was similar (P > 0.05) by IA, LA, LB, and LC milk samples and these were higher (P < 0.05) than IB milk samples (Table ). The anti-microbial activity was present only in one IB milk sample (April 2002) out of all the 55 milk samples that were analyzed with an inhibition zone of only 10 mm. When the pasteurized milk samples that were found positive for anti-microbial activity were further subjected to heat treatment (100°C for 15 min) in order to denature milk proteins and the anti-microbial activity still persisted in these milk samples. In addition, the zones of S. aureus growth inhibition were negatively correlated (P = 0.066) with the presence of this microorganism in these milk samples.
Table 4 Mean zones of inhibition against S. aureus by milk samples collected from April 2001 to June 2002 using the standard agar-well diffusion method and the presence (+) of S. aureus in these milk samples
The rapid snap antibiotic screening test revealed the presence of anti-microbial residues of the beta-lactam group, which is active against Gram-positive bacteria (Table ) for the three tested months from April to June 2002, but the antibiotics of the tetracycline group was absent in all these milk samples. The results from the rapid snap antibiotic screening test (Table ) were in general agreement with the standard agar-well diffusion method (Tables and ) for the presence of anti-microbial activity in these milk samples during the similar period tested. However, 20% of the IB milk samples collected in April 2002 inhibited the growth of S. aureus (Table ) but were negative to the rapid antibiotic screening test (Table ).
Table 5 Percentage of occurrence of antibiotic residues of the beta-lactam group in milk samples tested from April to June 2002 by the Snap rapid antibiotic screening test
To verify the absence of any anti-microbial activity in milk samples collected during the cooler months of the year, two batches of milk samples from all five milk producers were again tested on two occasions (November and December of 2002) of the following year by both the methods and results were again negative.
There were 10 cases of mastitis in LC dairy cows from October 2001 until March 2002 and antibiotic infusions of the beta-lactam group were used to treat these cows. However, the pasteurized milk samples sold from the LC dairy during these cooler months did not cause any inhibition of S. aureus growth (Table ).
In the final experiment, four of the six milk samples (both pasteurized and heat-treated) were positive for the presence of anti-microbial activity against S. aureus and these milk samples were also positive for the rapid snap antibiotic screening test for the beta-lactam group. However, these milk samples did not inhibit the growth of MRSA strain.
Discussion
Microbial Quality of Milk Samples
Pasteurized milk is defined as milk which has been suddenly cooled after all its particles undergo a process of heating to a certain temperature for a fixed time (72°C for 15 s) in such a manner to ensure that all pathogenic organisms and most of other microorganisms are destroyed with a minimum effect on its properties.Citation16 The quality and shelf life of pasteurized milk is dependent on the quality of the raw milk and other ingredients used and on an effective cleaning and sanitation program. Quality defects in pasteurized milk products are most often the result of microbial contamination, growth, and spoilage. In this regard, microbiological analyses can lend more insight into potential quality defects in dairy products.
In this study, Gram-negative and Gram-positive bacterial strains from five milk producers were identified (Table ). The majority of the Gram-positive isolates belonged to the genus Bacillus. Microbial contamination of pasteurized milk can occur from different sources. Because of this, determining the cause of bacterial contamination is not always straightforward. Though there is often one source of bacteria that cause high counts, high counts of bacteria can also result from a combination of factors like dirty milking equipment, inefficient pasteurization, contamination from the environments, and poor packaging.Citation17 Citation18 The Bacillus are spore-forming bacteria and their spores are able to survive even after pasteurization.Citation19 Citation20 Certain strains of psycrotrophic spores can grow during cold storageCitation21 and can be found in milk samples in such large numbers that the milk will be unsuitable for consumption. Bacillus cereus is the most important with respect to the limitation of the shelf life of pasteurized milk.Citation22 Citation23 A study in Addis Ababa showed that of the total bacterial counts in pasteurized milk, psychrophilic, thermoduric, and thermophilic organisms made up 53, 39.5, and 7.5% respectively and the isolates mostly belonged to the genera Bacillus, Streptococcus, Lactobacillus, Arthrobacter, Alcaligenes, Aeromonas, and Pseudomonas.Citation24 The degree of cleanliness of the milking system and pasteurization efficiency probably influence the total milk bacteria count as much, if not more than any other factor.Citation25 Also, E. coli was detected in 12% of the samples of raw milk studied and in 5% in those of the pasteurized milk but they found no E. coli in samples taken directly from the pasteurizing equipment.Citation26 The secondary contamination with E. coli resulted from the vessels and equipment in the course of the milk processing.
Of the Pseudomonas counts recovered in all milk samples in this study, the highest isolation frequency was in LB and LA milk samples. Pseudomonas is an environmental contaminant commonly originating in water sources. Excessive environmental bacteria have adverse effects on milk flavor and shelf life and milk handlers routinely test for overall bacteria levels (SPC). Water pipes may become contaminated with Pseudomonas and use of water from these pipe lines to clean milking equipment may lead to high Pseudomonas counts. In comparison with these findings, 18 strains of P. aeruginosa were isolated during a period of one year from raw milk and two P. aeruginosa strains were isolated from pasteurized milk.Citation27 The strains were devitalized when exposed to pasteurization (72°C for 20 s). At cold store temperatures (4°C), P. aeruginosa strain cells propagated on average by two folds, inhibitory effects of low temperatures were recorded only with one strain. In another study, pasteurized milk was found to be spoiled essentially by P. fragi, P. lundensis, and P. fluorescens biovar III suggesting that industrial recontamination of pasteurized milk are directed against Pseudomonas. Citation28
Several methodologies are used to evaluate the bacterial content of milk and the most common procedure is the SPC. This test is used throughout the dairy industry and is required by FDACitation29 and state regulatory agencies. In this study, there was no seasonal effect on SPC in all milk samples that were tested (data not shown). On the average, all the milk samples tested had SPC below (Table ) the recommended limit of 100,000 CFU mL−1.Citation30 In our study, all the milk samples were tested within 4 days after pasteurization for SPS. If the initial raw milk quality was very good, the keeping quality of all pasteurized milk was greater than 22 days and in some cases, the milk still had acceptable SPC even after 42 days of storage.Citation31
Milk should be free from FC, S. aureus and S. agalactiae, and the TC should not exceed 10 CFU mL−1.Citation30 However, they were present in all the milk that was tested in Oman. The coliform organisms of the raw milk varied depending on the season.Citation32 The regimes of pasteurization killed 100% of the coliform organisms that were present in the raw milk. After the thermal treatment, the milk was additionally contaminated with coliforms by the containers and the equipment and depends on the conditions of washing and disinfections. Most commonly encountered were the coliforms of the genera Citrobacter, Enterobacter, Klebsiella, and Escherichia. The milk samples commercialized without due authorization were found to contain mesophilic microorganisms and TC (68%) above the maximum limits for pasteurized milk and they also found that 18% of the samples tested were contaminated by FC.Citation33
Staphylococcus aureus counts were highest (P < 0.05) in LA milk samples compared to the other four sources of milk (Table ). In 1996, 50% of the milk samples tested were contaminated by coagulase-positive Staphylococcus and only 8.3% of the samples were found to comply with the required legal standards.Citation33 These results are suggestive of unsatisfactory hygienic and sanitary conditions of the raw milk and impose a great health risk to the consumers, especially when the product is taken without being boiled. In this study, S. agalactiae was negative for all 15 months in IA, IB, and LC milk samples, whereas it was positive three times in LA milk samples and twice in LB milk samples (data not shown). There is evidence that a mastitis bacterium may have caused an increase in the milk bacteria count. This seems to hold true more for Streptococcus spp. than for S. aureus, which appears to be shed into the milk in lower numbers.Citation18 S. agalactiae and S. aureus are not thought to grow significantly on soiled milking equipment or under conditions of marginal or poor cooling. Their presence in milk is considered strong evidence that they originated from infected cows.Citation34 Citation35
Screening for the Anti-Microbial Activity
There is increasing pressure from the consumer to ensure that all milk supplied is free from residues of antibiotics. There is no reason for any dairy farmer to have antibiotic residues in their milk, if the guidelines are strictly followed and they milk the cows in a way to avoid the accidental transfer of contaminated milk into the bulk tanks and then to the processing plant.Citation36 However, pasteurization does not make drug residues go away. The potential for an increased use of animal drugs to control mastitis is offset by the current animal drug residue-testing program employed by the dairy industry.Citation37 Any positive tankers are disposed of before the milk enters the processing facility. Lastly, producers are responsible for supplying milk from nondiseased animals.
In this study, all tested pasteurized milk samples were found to exhibit anti-microbial activity with varying intensities and frequencies only against the standard test strain of S. aureus by using the standard agar-well diffusion method (Table ). The standard test strain of S. aureus was used to represent the Gram-positive cocci, which cause the most common types of chronic mastitis in dairy cows. Anti-microbial agents are being used routinely to treat infections in livestock. In the dairy industry, a variety of antibiotics are being used to treat mastitis. Recent studies showed the presence of anti-microbial residues in pasteurized milk sold in EU and US markets.Citation38 Citation39
The results from the rapid snap antibiotic screening test (Table ) is in agreement with the results from standard agar-well diffusion method against S. aureus (Table ) for all milk samples tested during the same period of three months. These results confirm that the inhibition zones seen in the standard agar-well diffusion method were in fact caused by antibiotics of the beta-lactam group. There is widespread concern about the presence of anti-microbial drugs in milk. The presence of drug residues in milk may have public health implications. Similarly, beta-lactam group of antibiotics was found in milk samples in Barbados and Jamaica.Citation40 The presence of these antibiotic residues reinforces the necessity to screen the bulk tank milk prior to processing for human consumption.
In the present study, the anti-microbial activity persisted in heat-treated milk samples. This strongly suggest that the anti-microbial substances were not proteins. A survey on the incidence of inhibitory activity in market milk supplied in Pretoria identified penicillin in the fresh milk samples and the samples of pasteurized milk.Citation41 Some of the pasteurized milk contained inhibitory activity equivalent to more than 1.0 IU penicillin mL−1 and in some of the fresh milk samples, this figure exceeded 5.0 IU penicillin mL−1. Milk residues from drugs in the beta-lactam family, (e.g., penicillin), have been dramatically reduced from about 13% before 1962, to less than 5% in 2000 in Canada.Citation42
Surprisingly, we found no evidence of anti-microbial activity in any of the milk samples tested from October 2001 until March 2002 (Tables and ). It was unclear whether the incidence of S. aureus or any other causative agents of mastitis are seasonal in nature. A higher incidence of mastitis would be expected during the period of higher milk production during cooler months of the year. It is possible however that any anti-microbial activity might have been diluted out to below detection limits.
It is not surprising that S. aureus was present in 10 batches when there was no zone of inhibition against the control strain of using the standard agar-well diffusion method (Table ) during the cooler months with higher milk production. However, on the other hand 7 batches were positive for S. aureus as well as inhibited the growth of S. aureus using the standard agar-well diffusion method. It is likely that the S. aureus found in these milk samples could be resistant to the beta-lactam group.Citation43
A number of milk proteins including lactoferrin, lactoperoxidase, lactoferricin, and lysozyme display anti-microbial activity. However, the mechanisms by which these three milk proteins exhibit anti-microbial activity are quite different. Combined with antibiotics they can increase the effectiveness of preventing mastitis.Citation43 When the therapeutic potential of bovine lactoferrin or lactoferrin in combination with penicillin G against S. aureus was evaluated, the combination of lactoferrin with penicillin increased the inhibitory activity of penicillin and reduced the growth rate in S. aureus strains.Citation44 S. aureus were grown in the presence of a combination of penicillin and lactoferrin changed the protein profile of the bacteria, including the disappearance of several protein bands due to the presence of lactoferrin. These finding suggests that bovine lactoferrin or lactoferrin in combination with beta-lactam antibiotics can increase the antibacterial activity of these antibiotics against S. aureus resistant to other antibiotics.
The records of mastitis treatment at LC herd revealed that anti-microbial activity of the beta-lactam group was present in milk samples sold even after three months of last mastitis treatment in March 2002 (Tables and ). The recommended maximum withdrawal period of milk after treatment is only three days. However, the results indicate that the presence of this anti-microbial activity some months after treatment is suggestive that these substances may still be present in the milking machines and milk storage tanks even after thorough cleaning. It is also possible that the feed may be contaminated with molds that can synthesize these substances and subsequently passed in the milk or even that the rumen micro-flora might have synthesized these anti-microbial substances.
The convincing evidence for presence of antibiotics of the beta-lactam group came from the final experiment were four of the six milk samples (both pasteurized and heat-treated milk) inhibited the growth of the control strains of S. aureus and was negative for MRSA. The milk samples that inhibited the control strains of S. aureus showed positive results to the rapid snap antibiotic screening test for the beta-lactam group. The fact that the presence of these anti-microbial substances in milk even several months after the treatment of mastitis in LC herd and the possible role that the feed may be contaminated with penicillin mold needs to be further investigated. The pasteurized milk sold in Oman was found to be contaminated with a variety of different micro-organisms. In addition, antibiotic residues of beta-lactam group were commonly encounted. It is imperative that stricter quality control measures be imposed in Oman to assure that consumers are provided with truly wholesome milk and milk products.
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