Sample Collection

Different sample from irrigation ditch water, rhizosphere, roots and from phyllo sphere were collected from different region of Faisalabad. Three sample from each of farm was randomly collected. For collection of roots and rhizosphere, a sharp sterilized blade in 70% Ethanol was used. With the help of blade, roots from different plants were cut and collected in sterilized falcon tube. Approximately 2 to 3 roots per plant and each length of each root was around 9 – 11 cm was collected. Falcon tube was shaken for 3 to 4 min until the release of soil from roots. Then using the sterilized forceps remove the roots from the falcon tube and put into another 50ml falcon tube labelled by root sample, and previous tube was properly labelled as rhizosphere sample. Time and date were also mentioned on falcon tube. Samples were transported at 4°C and processed within 24 hours. For collection of water sample from different source sterilized falcon tube was used.  Using gloved hands 30ml water sample was taken in 50ml of falcon tube from irrigation ditch. Properly labelled the falcon tube and transported the water sample to lab for further processing. Then leaves sample were collected using sterilized scissor in 70% of ethanol. Approximately 2 to 4 leaves per plant were collected and length of each leave was around 8 to 12 cm. Leaves were placed in properly labelled 50ml falcon tube. All the samples were safely transported to laboratory and screened for Salmonella.

Isolation of Salmonella by culturing Water and soil samples

Isolation of Salmonella was carried out by filtering the water by using syringe filter. Water samples were diluted 10-folds and then pre-enriched by mixing the 10 mL of water sample in 100 mL of nutrient broth. Similarly, soil samples were also diluted by making 10-fold dilution then pre-enriched by putting 10g of sample in 100ml of nutrient broth. Both the samples were incubated at 37°C for 24 hours. Then Salmonella Shigella (SS) agar was used for further isolation and purification. 1ml aliquot of each pre-enriched suspension was transferred to Salmonella shigella (SS) agar plate, incubated at 37°C for 24 to 36 hours. Colorless with black centers Salmonella colonies appeared, that were further confirmed as Gram negative by Gram staining.

Leaves and Root Samples

Collected leaves and root sample were dipped in 30 ml sterile phosphate buffered saline, vortexed for 2-3 min, and then 2 ml aliquot of each suspension was transferred to 18 ml of nutrient broth, incubated at 37°C for 24 hours. Then 1ml of aliquot of each suspension plated onto the Salmonella shigella agar plate and incubated at 37°C for 24 to 36 hours. Colorless with black centers Salmonella colonies appeared, that were picked and confirmed as Gram negative by Gram staining.

Biochemical Characterization

Isolated organisms which showed Salmonella growth characteristics on SS media, were further confirmed by performing the following biochemical tests named as, Catalase, Oxidase, Triple Sugar Iron Test (TSI), Methyl Red/Voges-Proskauer (MR-VP) and Indole Test.

Testing of susceptibility of the isolates to antimicrobial agents

Susceptibility of pure isolates were checked to eleven (11) commonly used antimicrobial agents with the following disk contents; sulfamethoxazole /trimethoprim (25 µg), ampicillin (10 µg), levofloxacin (5 µg), Tobramycin (10 µg), Amikacin (30 µg), Tetracycline (10 µg) and Chloramphenicol (30 µg), gentamycin (30 µg), cefoxitin (30 µg), Cefepime (30 µg), Cefuroxime (30 µg) by the disk diffusion method [17] and based on recommendations of Clinical and Laboratory Standards Institute guidelines (CLSI, 2010). Mueller–Hinton agar (Oxoid) was made and poured into plates, spread the culture onto the agar by using the sterile wire loop and allow to dry for five minutes. Antimicrobial discs were placed on the agar plates with antibiotic disc dispenser.  Plates were incubated at 37 ℃ for 24 hours.

Figures & Tables

Contamination of Salmonella spp. in maize comprises negative effects on consumer and also has devastating financial impact on the maize industry. Thus, to improve public health, there is much need of research to check from where and how the contamination is being occur in crops including Zea mays L. This study was conducted to evaluate the presence of Salmonella in the Zea mays L farms located near the poultry farms, and to check the antimicrobial susceptibility of recovered isolates. Our study showed that 11.25% of sample were positive to Salmonella spp. from different sources. From irrigation water source 25% sample were positive for Salmonella spp. it means water was contaminated and could serve as a carrier of Salmonella sp. to fields. Our study co related with study that, Irrigation water used in the fields contain lower than the 40% isolation rate [18]. It was reported worldwide that occurrence of Salmonella in various water bodies is ranging from 3 to 100% with frequency of positive samples [19].

The major source of human pathogens, such as Salmonella, is the poultry drainage or chicken litter, as poultry waste has the potential to pollute surface or ground water. In this study water showed the major reservoir of Salmonella with high density, it means poultry farm near the Zea mays L farms can impact on the Salmonella contamination of irrigated water. Furthermore, samples were collected from the Zea mays L farms located near the poultry farms area, so we presume that Salmonella contamination in ditch water was due to poultry drainage. It is anticipated that Irrigated water serve as reservoir to transmitted Salmonella to rhizosphere, roots, phyllo sphere of maize in field. As in this study, crop was irrigated with ditch water, not by any other method like spray irrigation, so after high Salmonella contamination in water, rhizosphere have 12.5% of Salmonella contamination found in this study. It is also reported in several studies that Salmonella can persist in farm environment for long period of time [20]. Several studies reported that there is improper waste management in Pakistan [21]. It is also indicated that country producing more organic waste, then the risk is also increased because the S. typhimurium acts as a potentially farm pathogen.

It is known that Salmonella is able to colonize fruit and vegetables and also leaf surfaces and it has been shown fruit contamination is due to early leaf colonization.  S. Typhimurium have ability to survive in the rhizosphere, in the phyllo sphere and in the soil for at least 26- 28 days after the irrigation. In this study the isolation rate of Salmonella from phyllo sphere (leaves) is 2.50% and a previous study reported 1.5 % of confirmed Salmonella spp. from leaves [22]. Unexpectedly very low percentage of Salmonella confirmed on leaves surfaces. Many of factors can change the prevalence of pathogen on leaves surfaces. It was hypothesized that survival of Salmonella on plant surfaces could influenced by variations in environmental conditions (such as temperature, sunlight etc.) [22]. Level of pathogen could higher on plant surface when applied spray irrigation of water [22] but in this study we select those farms where water is applied through ditch water. From root sample more isolation of Salmonella 7.40% is confirmed then phyllo sphere because we hypothesized that irrigated water directly contacts with rhizosphere and root surface in contrast with phyllo sphere.

Several studies revealed that Salmonella shows multi drug resistance pattern [23]. Only 2 isolates in this study shows multidrug resistance. Multi drug resistance shown by the Salmonella in this study, suggests that there could be improper antibiotic improper usage or misused in that environment from where these isolates originated [24], or it is also possible that resistance to multiple antibiotics is due to isolates may have acquired the genes. Hence, from vegetables or fruit multidrug resistance strains of Salmonella conformation is a major concern for public health and food quality. One isolate from rhizosphere and one from irrigated water was particularly observed to be resistant to multidrug resistance. This shows a great public health concern as certain cases of salmonellosis require antibiotic therapy for treatment. In many studies it is reports that MDR Salmonella isolates have been suggested to be more virulent than non MDR Salmonella isolates [25]. This study requires great attention after detection of these multiple drug resistant Salmonella strains. Our findings indicate that these isolates recovered in this study have the potential to develop resistance for commonly used antimicrobial drugs and shows considerable health hazards to consumers, hence control measures should be adopted otherwise it can be life threatening condition. From our result it is confirmed that Salmonella was present in rhizosphere, root, and plant surface and able to colonize on the plant surface for prolong period of time after irrigation with contaminated water.

From all of above findings it is conclude that poultry waste mixed with the irrigation water through irrigations ditches and reach to the crops. As no any other significant source for the Salmonella reported. In this study we found Salmonella as the MDR pathogen so intake of food with such contamination is very lethal for human being. There must be a check in the disposal of poultry and dairy waste. It must be dumped to away from the agriculture farms to avoid contamination with irrigation water. In Faisalabad there is no such mechanism of poultry and dairy waste disposal, improper disposal leads to farm to fork contamination as reported in this study. So this study is very significant for the awareness among common man and for higher authorities to take significant action to avoid in future circumstances.

Author Contributions

Conceived and designed the experiments: Arslan Ali, Saima Muzammil, Nimra Amjad.
Performed the experiments: Fatima javed, Arslan Ali, Nimra Amjad, Muhammad Zeeshan Ahmad, Muhammad Umar
Analyzed the data: Zain Ul Abbas, Fatima javed
Contributed materials/analysis/tools: Saima Muzammil, Sadaf Oranab, Maira Sajid
Wrote the paper: Arslan Ali.

Conflict of Interest

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