Detection of Coliform Bacteria in Raw Milk Samples Collected from Industrial Cities of Pakistan

Antibiotics and heavy metals-resistant bacteria in livestock environments can result in economic losses and raise public health and environmental problems. There is a crisis in the world's access to and pipeline for antibiotics. Objective: To screen raw milk samples collected from three different industrial cities Gujranwala, Lahore, and Sheikhupura, situated in the province of Punjab, Pakistan. Methods: In this regard, a total of 26 samples were having 84 coliform strains. Separated coliform colonies were processed for Gram's staining, catalase, indole production, and Simmon's citrate and motility tests. Results: Metal resistance of bacterial strains was also checked and 39.5% and 45.23% of bacteria were found to be resistant to ZnCl2 1% and 0.5%. 69.045% and 77.38% bacteria were found to be resistant to CuSo4 salt solution1% and 0.5%. 17.85% and 27% bacteria were found to be resistant to Na2CrO4 salt solution1% and 0.5% respectively. 80% of bacteria were found to be resistant to Cefuroxime, 26.19% to Cephradine, 84.52% to Aztroeonam 41.67% to Erythromycin, 91.667% to Trimethoprim 89.28% to Lincomycins. Conclusions: The raw milk samples were not only contaminated with coliforms but the bacteria were also resistant to heavy metals and certain antibiotics which might be considered indicative of industrial and anthropogenic pollution. Cephradine, 84.52% to Aztroeonam 41.67% to Erythromycin, 91.667% to Trimethoprim 89.28% to Lincomycins. Conclusions: The raw milk

Coliforms are also lactose fermenting bacteria including several genera like Escherichia, Citrobacter, Enterobacter, and Klebsiella.They are considered the indicator of fecal contamination.They normally live in the intestines of humans and animals.Their presence in milk is of severe health concern.In Pakistan, milking practices are very poor and unhygienic [3].To increase the reproductive potential heavy metals are extensively used as a therapeutic agent in farms [4].Heavy metals represent major contaminants with severe health and environmental problems [5,6].Due to their presence in nature and persistence, heavy metals are marked as hazardous to ecosystems and human beings

I N T R O D U C T I O N
S i n c e t h e s t a r t o f 2 0 t h -c e n t u r y, a g r i c u l t u r e mechanization, industrialization, and urbanization have been causing an increase in metals and antibiotics resistance among the bacteria.Major milk-producing countries are the United States, Russia, India, and Pakistan is ranked as fourth.Pakistan has 45 million tons of annual milk production.The rural area population's (30-40%) income source is solely the rearing of livestock.In Pakistan, there are 34 million cattle and 31 million buffaloes.Buffaloes contribute to 62% of milk and are major milk producers [1].Black gold buffaloes in Pakistan are a major milk source as their milk is consumed by all age groups [2].
[7].The presence of heavy metals in numerous media is of great concern.Irrigation of agricultural land with sewage and industrial wastewater is the basic cause of the development of heavy metal resistance [8].Food items grown in contaminated soil or with wastewater are reservoirs of heavy metals and transfer agents of heavy metals to humans and animal bodies [9].Animals c o n s u m i n g c o n t a m i n a te d fo d d e r p r o d u c e m i l k contaminated with heavy metals [10].Heavy metal accumulations in dairy animals result in their excretion in milk [11].Milk consumption of cattle and buffalo which had been feeding at polluted places leads to several human health problems [12,13].The livestock industry uses antibiotics and heavy metals as the most common supplements [14].Antibiotic resistance has become one of the biggest issues in the world in treating bacterial infections [15].During livestock production, use of antimicrobial agents causes the development of antimicrobial resistance which is a serious health concern for the public.By increasing the antibiotics in the environment, antibiotic resistance is more pronounced in the environment [16,17].Usage of drugs in animal food is a vital tool for animal welfare and health.Despite various health bene ts of antimicrobial drug usage, the production of antimicrobial-resistant bacteria is also an adverse condition [18].There is a positive correlation between increases in antibiotic resistance among disease-causing bacteria with the application of antibiotics to farming animals [19].Further evolution and increase in antibiotic resistance in pathogenic microbes enhance the severe health issues for animals and humans [20].When food containing the antimicrobials is given to animals outside the regimen dose label and durations then antimicrobial resistant microbes are produced [21].The use of antibiotics in growth promoters and medicine causes an increase in antibiotic-resistant bacteria [22].Bacteria showing antibiotic resistance have been found in milk, vegetables, cheese, meat, and fruits [23].Coliforms having antibiotic resistance are pathogenic bacteria that cause water pollution and environmental and public health issues in developing countries [24,25].Genes for antibiotic resistance are localized in mobile genetic elements and are transferred by bacteria in the food to the bacteria living in the human body by a process of horizontal gene transfer.Strains of E. coli are naturally more abundant in the gastrointestinal tract [26)].From the previous 50 years, E. coli has increased in multidrug resistance by 56.4%.This spread might be due to bacteria belonging to different ecosystems because resistance genes are localized on mobile vectors [26].The present study focused on the raw milk quality in terms of antibiotics and heavy metals resistance bacterial content of three cities.

Sample Collection
Twenty-six samples were collected, from three cities Gujranwala, Lahore, and Sheikhupura of Punjab Province, Pakistan.Raw milk samples were collected in autoclaved sterile bottles from local shops and livestock farms, from 6 November 2017 to 25 May 2018.they were transported to Microbiology Lab, Zoology Department, University of Punjab, Lahore, Pakistan, and stored at 40C till further use.Processing of Samples for Bacterial Colony-Forming Units In 99 ml of distilled autoclaved water, 1 ml of raw milk was mixed.From this dilution 1ml was, mixed with 99 ml of distilled water and so on [27].From diluted milk 0.1 ml was spread on EMB agar with the help of a spreader and incubated for 24 hours at 370C ( 27).Colony-Forming Unit (CFU) has then been enumerated for each category of the bacterial colonies.One separated colony was picked up with the help of a sterile loop and streaked on Nutrient agar, using the quadrate streaking method.The plates were incubated routinely and a well-separated colony from nutrient agar was picked up and again streaked on EMB agar to get pure culture.After getting pure culture characterization of bacterial colonies i.e., con gurations, margins, elevation, color, and size were determined and noted [28].A total of 78 bacterial isolates were processed.Biochemical Analysis Differential Grams staining test, motility test, catalase test, indole test, and Simmons citrate agar production test were performed [29], using pure cultured bacteria.In bacterial strain identi cation, "S" represents Shiekhupura; "L" represents Lahore and "G" represents Gujranwala as well as in milk sample labeling.

Metal Sensitivity Test
To check metal susceptibility Cu, Cr, and Zn salts solutions like (CuSo4), ( Na2CrO4) and (ZnCl2) with 1% and 0.5% concentrations were used, and discs from lter paper of equal size were cut, inoculated with 6 µl of each salt solution and these disc were autoclaved for 20 minutes at 1210C temperature and 15 psi pressure, these inoculated autoclaved disc were placed on nutrient agar plates aseptically and incubated for 24 hours at 370C temperature and metal sensitivity results were recorded [30].

R E S U L T S
In the table 1, rst column represents the number of samples.In "G1a" G represents Gujranwala "1" represents the sample number and "A" represents strain type.Similarly, all other strains are represented in the same way, bacteria like Salmonella or Shigella spp, Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli.All bacteria were found grams negative in staining Table 1:

Metals and Antibiotics Resistance of Coliforms
When bacterial isolates were grown in the presence of 1% and 0.5% (CuSo4), (Na2CrO4), and (ZnCl2) salts of zinc (Zn), Copper (Cu), and Chromium (Cr).39.5% and 45.23% bacteria were found to be resistant to ZnCl2 salt solution1% and 0.5% concentrations.69.045% and 77.38% bacteria were found to be resistant to CuSo4 salt solution1% and 0.5% concentrations.17.85% and 27% bacteria were found to be resistant to Na2CrO4 salt solution1% and 0.5% concentrations respectively.Antibiotic susceptibility of coliform isolates was also tested against nine different antibiotics with three modes of action i.e.-cell wall inhibitors, proteins inhibitors, and nucleic acid synthesis inhibitors.79.76% bacteria were found to resistant for Cefuroxime, 26.19% bacteria were found to resistant for Cephradine, 84.52% bacteria were found to resistant for Aztroeonam 41.67% bacteria were found to resistant for Erythromycin,91.667%bacteria were found to resistant for Trimethoprim 89.28% bacteria were found to resistant for Lincomycins as shown in gure 3(a) and 3 (b).

Table 2: Results of Kruskal-Wallis Test of milk samples collected from different cities
Figure 1 shows the prevalence of different species identi ed in different cities.Following the signi cance Post-Hoc test applied, test results are shown in gure 2 which shows that milk samples from Gujranwala and Sheikhupura were signi cantly different from each other while the comparison of Sheikhupura -Lahore and Gujranwala-Lahore showed to be non-signi cant-respectively at p< 0.05 ( gure 2).

Viable Bacterial Count
The CFU data corresponding to the cities of Sheikhupura, Lahore, and Gujranwala were statistically analyzed using the Kruskal-Wallis test a non-parametric, alternative to one ANOVA.Results showed signi cance at p<0.05 (table 2).The bacterial count was high in Sheikhupura which was collected from local dairy shops.Lahore and Gujranwala showed signi cantly lower viable bacterial counts as they were collected directly from cow udder and milkmen's buckets, respectively.Milk samples of Lahore city were taken directly from animals.In the case of Sheikhupura city milk samples S1-S12 were taken from local shops.Samples G1 to G5 all were taken from milkman buckets, whereas samples G6 to G9 and L1-L5 were taken directly from animals.

D I S C U S S I O N
The milk of a healthy cow is free from any contamination when secreted into the alveoli of the udder.Fresh milk contains a very low microbial load ranging few to 1000 CFU/ml [32,33].Microbial contamination might result during milking storage, handling, refrigeration, and transportation increasing microbes by 100 or even 1000 folds.In the present research, the total bacteria count taken directly from the udder during milking (L1, L2, G8) was 4 relatively low ranging at 10 CFU/ml.An increase in bacterial CFU/ml was observed from milk during its transportation from the milk farm to milk containers as (G1 to G5) samples were taken from buckets.Coliforms are indicators of fecal contamination.Their presence in large numbers predicts the very poor hygienic condition of the study area, even milk taken directly from the farm was not acceptable for consumption.So, dairy farm owners should be educated to render fresh milk suitable for human consumption.Many coliforms were resistant to four antibiotics and copper showing environmental pollution and its in uence on our diet.As coliforms are mostly fecal in origin, their presence in milk is a health hazard emphasizing to improve milk handling.

Figure 1 :
Figure 1: Presumptive Identi cations of the Bacterial Isolates from Milk Samples of Gujranwala, Lahore and Sheikhupura.

Figure 2 :
Figure 2: CFU\ml Median values of milk collected from Gujranwala, Lahore and Sheikhupura, letter showing signi cance at p<0.05.

Figure 3 :
Figure 3: Mean sensitivity and resistance of coliforms against different metals (a) and antibiotics (b).S=Sensitive R=Resistant; results are the mean of three replicates.

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O N C L U S I O N S DOI: https://doi.org/10.54393/fbt.v4i01.90Aslam A et al., Coliform Bacteria in Raw Milk Murdoch et al., and Bagré et  al.,whose test results showed most coliforms were resistant to different antibiotics [50-52].Other coliform strains did not show such results.Most bacterial strains were sensitive to NOR and TOB.Antibiotic resistance of bacterial isolates of milk is agreed with literature ndings [53, 54].Coliforms are gram's negative rods agreed with the [55].Coliform mostly were non-motile and least motile.Some coliform strain has catalase enzymes that were in support of scienti c ndings [56, 57].Bacteria having catalase enzyme produced bubbles on adding a few drops of H O .Tryptophanase enzyme is present in most 2 2coliforms and present research indole positive test results are in favor of previously reported results[29, 58, 59].Escherichia coli can be identi ed with Eosin Methylene Blue (EMB) agar.The presence of green-metallic sheen in three strains of E. coli was observed in milk sampled from Sheikhupura whose results agreed with previous ndings[60, 61].The citrate utilization test on Simmons citrate agar and several strains gave citrate positive test by changing the green color of media into blue these results are compatible with previous research[54].