Prevalence and antibiotic resistance of Salmonella spp. in South Punjab-Pakistan.

Present study aimed at investigating the magnitude of the prevalence and antibiotic resistance among four Salmonella spp. i.e., S. typhi, S. paratyphi A, S. paratyphi B and S. typhimurium. Raw milk and environment samples were collected from the five districts of southern part of the province of Punjab in Pakistan i.e., Multan, Bahawalpur, Lodhran, Dera Ghazi Khan and Muzaffargarh. Extent of antibiotic resistance was also determined and classified as resistant, intermediate and susceptible. District-wise prevalence data on Salmonella spp. in milk and environmental samples indicated higher S. typhi, S. paratyphi B and S. typhimurium count in Bahawalpur, D.G. Khan and Muzaffargarh districts, respectively. Amongst 13 tested antibiotics, chloramphenicol and ofloxacin were found to be the most susceptible against Salmonella spp. Increased emergence of antibacterial resistance was noted with respect to the type of antibiotics among Salmonella spp. isolates. The study suggests serious interventions to be practiced by the farmers and raw milk vendors in animal husbandry and milk marketing, respectively to curb the burden of Salmonella spp. prevalence in milk. Further, active engagement of animal health division and enforcement agencies to ensure sagacious use of antibiotics at farm level may also help in containment of antimicrobial resistance in Salmonella spp.

Introduction

Raw milk is considered as a primary source of essential nutrients by a variety of farming families and workers. Traditionally, milk processing is discouraged in some cultures and raw milk is preferred for consumption despite the fact that raw milk is reported to be the best breeding site for pathogenic microbial strains. Hence milk safety turns up as a challenge for consumers given the risk of animal udder infection and poor sanitary condition in milking area [1].

Salmonella has been considered as the major foodborne pathogen leading to an upsurge in enteric infection cases. Three groups of Salmonella serotypes have been considered responsible for causing distinctive clinical syndromes including typhoid fever, enteritis and bacteremia [2]. Likewise, infections by non-typhoid Salmonella serovars have been shown to result in acute gastroenteritis with extra intestinal localized infections that may eventually affect some organs [3]. Reportedly, 99% Salmonella infections in humans are associated with strains in the O-antigen serogroups such as serogroups A, B, C1, C2, D and E of S. enterica subspecies entericae [4]. Mechanistically, the onset of disease proceeds with intestinal phase once the food contaminated with typhoid and Salmonella enteritis is ingested [5].

Recently, emergence of multidrug-resistant (MDR) S. enterica serovars, including resistance to quinolone group (fluoroquinolones) and the third generation antibiotics (cephalosporin) has led to serious public health issues throughout the world. Emergence of antimicrobial resistance was reported among Salmonella spp. during conventional farming indicating 10% of the isolates being resistant against commonly used treatment regime i.e., cephalosporin and fluoroquinolones against salmonellosis [6]. Sufficient evidence is available to support the emergence of antibiotic resistance among Salmonella strains which is directly associated with intensive use of antibiotics to treat Salmonella infections and incorporation of growth promoters in animal feed [7, 8]. The wider distribution of MDR Salmonella spp. in foods have been reported by various researchers worldwide [9-11]. A substantial body of literature confirmed emergence of antibiotic resistance in Salmonella spp. isolates from milk and milk products as a major public health issue worldwide [12, 13].

S. enterica serovars typhi and paratyphi (A, B and C) have been reportedly developing resistance against a range of antibiotics thereby distressing 21 million people worldwide. Morbidity and mortality rate associated with these microbial infections had been much higher on account of infections by Salmonella spp. For example more than 14 million cases of enteric fever are reported annually resulting in 135,000 deaths. Prevalence rate of S. typhi and S. paratyphi infections in South Asian regions were reported higher indicating excessive use of antibiotic to exacerbate emergence of multidrug resistance in these strains [14, 15]. MDR Salmonella serotypes have become widespread in developed economies including USA. Treatment cost of infections from antibiotic resistant bacteria amounts to 4–5 billion US dollars annually. In addition to substantial financial losses caused in disease management, antibiotic resistant pathogens have been hampering international trade owing to threats of cross borders proliferation of infectious diseases [16].

Available evidences suggest increased prevalence of Salmonella spp. in foods especially raw milk and milk products leading to a surge in the onset of infections among humans and the farm animals. Resultantly, the injudicious and indiscreet use of antibiotics to treat such infections has been engendering heightened multidrug resistance among bacterial strains. Besides that, no epidemiological surveillance, monitoring and control of pathogenic microbes and associated microbiological infections is in place. The objective of the present study was to scale the prevalence of Salmonella spp. at farms in Southern part of the Punjab province and to ascertain the extent of development of antibiotic resistance in Salmonella spp. isolated from raw milk and farm environment. District–wise data on prevalence rates of Salmonella spp. and emergence of drug resistance would serve as baseline information for key stakeholders on potential risk factors for milk microbiological safety in milk producing zones of South Punjab. The data would further help in designing effective strategies and plans to mitigate microbiological food safety issues and corresponding disease burden in the region.

Materials and methods

Chemicals and reagents

All chemicals and reagents were of analytical grade unless otherwise mentioned and procured from Oxoid, Ltd., Hampshire, UK through the local supplier. Xylose Lactose Tergitol 4 agar and antimicrobial diffusion disks i.e., HardyDiskTM were purchased from Hardy Diagnostics, Santa Maria, CA.

Sampling plan and sampling

A cross-sectional study was designed to find out the prevalence of Salmonella spp. in raw milk and environmental samples collected from five major districts of South Punjab (Fig 1). A total of 3000 samples of raw milk and environment samples including farm manure, farm soil, animal feed, animal bedding, potable water, milk container, milking parlor, personnel and animals’ teat were collected in three visits from twenty tehsils / towns of five districts. Detection and isolation of Salmonella spp. were carried out to estimate the extent of the prevalence of Salmonella spp. i.e. S. typhi, S. paratyphi A, S. paratyphi B and S. typhimurium. Sampling was performed in three visits of each sampling site during September 2014 to August 2015 to draw raw milk (15 samples; five on each visit) and 135 environment samples. Sampling was carried from the following sites presented here in a format {town; (vendor; coordinates; type of sample)} Shershah (Sattar dairy farm; 30°08'21.9"North, 71°26'44.9"East; Milk and environment), Qadirpur (Al-noor livestock; 30°16'25.0"North, 71°37'57.8"East; Environment), Makhdom Rasheed (Makhdoom dairy farm; 30°05'48.1"North, 71°38'24.8"East; Milk and environment), Laar (Shabab dairy; 30°02'28.3"North, 71°29'07.3"East; Milk and environment), Shujabad (Bismillah dairy farm; 29°51'53.2"North, 71°14'31.5"East; Milk and environment), Jalalpur pirwala (Abdullah Rehman dairy farm; 29°36'45.2"North, 71°09'08.0"East; Milk and environment), Bahawalpur (Lodhi organic dairy farm; 29°25'51.6"North, 71°39'41.1"East; Milk and environment), Yazman road (Jattala dairy and cattle farm; 29°07'49.7"North, 71°46'36.0"East; Environment), Khairpur tamewali road (Muhammad dilshad cattle farm; 29°36'46.5"North, 72°00'55.0"East; Environment), Ahmadpur east (Al-fallah cattle farm; 29°10'11.3"North, 71°15'29.8"East; Milk and environment), Hasilpur (Shahdin dairies; 29°43'40.5"North, 72°32'09.0"East; Milk and environment), D.G. Khan (Ashiq mirani cattle farm; 30°04'51.4"North, 70°46'47.4"East; Milk and environment), Taunsa shareef (Jarwar cow farm; 30°39'22.5"North, 70°36'20.1"East; Milk and environment), Lodhran (Maqbool dairy farm; 29°35'08.3"North, 71°48'18.0"East; Milk and environment), Dunyapur (Ch.Saeed saqib dairy farm; 29°48'10.0"North, 71°44'45.5"East; Milk and environment), Kahror paka road (Baloch cow farm; 29°40'31.0"North, 71°54'30.1"East; Environment), Muzaffargarh (Fazal farm Ltd.; 30°09'15.0"North, 71°13'20.3"East; Environment), Alipur (Abdullah Rehman dairy farm; 29°23'22.4"North, 70°54'12.7"East; Milk and environment), Kot adu (Hafiz ramazan dairy farm; 30°24'00.1"North, 70°54'50.7"East; Milk and environment) and Jatoi (Fahd jameel dairy farm; 29°32'24.8"North, 70°47'09.2"East; Milk and environment). Raw milk (approx. 50 ml in sterilized airtight glass containers), environmental samples including water, soil, manure, feed and bedding (100 g/ 100ml in sterilized zip lock bags) and surface swabs of milk containers, hands and animal’s teats were collected, tightly sealed, kept in ice box and immediately shipped to the laboratory for analyses.

Fig 1

Map showing five major districts (shaded) of South Punjab–Pakistan covered for surveillance and antibacterial resistance in Salmonella isolates from raw milk and environment.

Detection, isolation and confirmation of Salmonella spp.

Procedure from ISO 6579:2002 standard (Microbiology of food and animal feeding stuffs) guidelines were followed for detection, isolation and confirmation of Salmonella. Thoroughly mixed raw milk and environmental samples were transferred aseptically into 225 ml sterile peptone water and incubated for a period of 24 hrs at 37°C. One milliliter of the primary enrichment was further transferred to Rappaport–Vassiliadis soya broth (9 ml) and another 1 ml to 9 ml of tetrathionate broth. Selective enrichments i.e., Rappaport–Vassiliadis soya broth and tetrathionate broth were incubated for 24 hrs at 42°C and 43°C, respectively. Rappaport–Vassiliadis and tetrathionate broth cultures were streaked onto bismuth sulfite agar plates and xylose lysine deoxycholate agar plates, respectively and incubated for a period of 24 hrs at 35°C. Confirmation test of Salmonella strains by culturing on xylose lactose tergitol– 4 agar. Morphological confirmation and identification of Salmonella strains was performed by biochemical analysis using triple sugar iron (TSI), lysine iron, Methyl Red Voges-Proskauer (MR-VP) and urease production reaction tests.

Determination of bacterial antibiotic resistance

Salmonella spp. positive raw milk and environmental samples were further tested for determination of antibiotic resistance using HardyDisk™ antimicrobial sensitivity testing. Isolates from the frozen stocks were grown onto tryptic soya agar overnight at 37°C. Culture colonies were transferred to tryptic soya broth and concentration was spectrophotometrically adjusted to an absorbance of 0.125 at 550 nm. Known concentration cultures were thus transferred to Mueller Hilton Agar by swabbing. Hardy disks loaded with known potencies antimicrobials including ciprofloxacin (5 μg), ampicillin (30 μg), gentamycin (10 μg), co-trimoxazole (25 μg), amoxicillin (30 μg), ofloxacin (10 μg), ceftazidime (30 μg), cefuroxime (30 μg), cefepime (30 μg), imipenem (10 μg), ceftazidime (30 μg), moxalactam (10 μg), chloramphenicol (30 μg) and oxytetracycline (30 μg) were incubated for 18 hrs at 37°C. The selection of tested antibiotics was made, based on the present therapeutic use of these antibiotics to treat Salmonella infections in humans and farm animals. Zones of inhibition were measured with meter ruler after 18 hrs.

Isolates were declared resistant, intermediate and susceptible against the tested antibiotics according to the Clinical & Laboratory Standard Institute (CLSI) guidelines. All chemicals and bacterial culture media were of analytical- reagent grade if otherwise noted.

Statistical analysis

The data for prevalence of Salmonella spp. so obtained were subjected to statistical analysis and positive and negative samples were taken to calculate percentage prevalence of different Salmonella spp. in raw milk and environmental samples. Significance between prevalence of Salmonella spp. in districts or different type of samples was computed by using Chi-square analysis. A p- value ≤0.05 was considered statistically significant.

Results

Prevalence of Salmonella spp.

Statistically significant association in district wise prevalence of Salmonella spp. i.e., S. typhi (p = 0.03) and S. paratyphi B (p = 0.000) was observed (Fig 2). While there were insignificant differences in S. paratyphi B and S. typhimurium prevalence among selected districts. Collectively, highest rate of prevalence of Salmonella spp. was observed in D. G. Khan i.e., 32% followed by Muzaffargarh (31%) and Bahawalpur (28%) while the lowest rate of Salmonella spp. prevalence i.e., 20% was witnessed from the milk and environmental samples collected from the towns of Lodhran district (Fig 2). Highest average prevalence percentage of S. typhi (11.9%) and S. paratyphi B (7.3%) was recorded in Bahawalpur and D.G. Khan districts, respectively.

Fig 2

Prevalence percentage of Salmonella spp. in milk and environmental samples of dairy farms in Southern part of the Punjab.

Salmonella spp. contamination was recorded in all samples sources while highest load was monitored in environmental samples (Table 1). The data analyzed to determine variability in prevalence of S. typhi among raw milk and environmental samples reported highest positive samples from farm manure i.e., 16% followed by bedding (14%), milk container (11%) and raw milk (11%) (Table 1). Identical trend was observed for prevalence of S. typhimurium wherein average positive samples proportion from farm manure and bedding were 16% and 16.7%, respectively. Nearly 23% of the milk samples were tested positive for salmonella spp. while extent of prevalence of S. typhi was highest i.e., 11% followed by S. typhimurium (8%), S. paratyphi A (2%) and S. paratyphi B (2.3%).

Table 1

Prevalence (%) of Salmonella spp. in raw milk and environmental samples.

Sample sources	Prevalence
	S. typhi	S. paratyphi A	S. paratyphi B	S. typhimurium
	n (%)	n (%)	n (%)	n (%)
Feed	17 (6)	11 (3.7)	8 (2.7)	25 (8.3)
Manure	47 (16)	16 (5.3)	19 (6.3)	50 (16.7)
Bedding	41(14)	15 (5)	22 (7.3)	48 (16)
Cattle teat	26 (9)	9 (3)	10 (3.3)	34 (11.3)
Milk container	33 (11)	8 (2.7)	17 (5.7)	33 (11)
Milking parlor	27 (9)	9 (3)	14 (4.7)	40 (13.3)
Personnel hand	24 (8)	7 (2.3)	11 (3.7)	28 (9.3)
Potable water	16 (5)	7 (2.3)	11 (3.7)	25 (8.3)
Raw milk	32 (11)	6 (2)	7 (2.3)	24 (8)
Shed soil	16 (5)	4 (1.3)	5 (1.7)	16 (5.3)
χ2; (p–value)	39.55; (0.000)	14.76; (0.10)	22.91; (0.006)	37.54; (0.00)

Data presented in Table 2 depict % prevalence of S. typhi (8.33%), S. paratyphi A (2.78%), S. paratyphi B (3.67%) and S. typhimurium (10.89%) isolated from 233 positive sample screened from 900 raw milk and environmental samples. Comparably, S. typhimurium remained to be the most frequent Salmonella serovar, however variability in prevalence rate was non-significant (p>0.05). All six towns significantly differed (p<0.05) for prevalence rate with highest prevalence of S. typhimurium (16.67%) in milk and environmental samples of dairy farms of Shuja Abad. This site indicated overall highest prevalence (39.33%) of Salmonella spp. with higher number (n = 59) of positive samples followed by Band Bosan town with 29.33% (n = 43) and Sher Shah with 26.67% (n = 40). Relative to these sites, Shah Rukn Alam was identified as microbiologically safe area with 14.67% (n = 22) prevalence of Salmonella spp. (Table 2).

Table 2

Prevalence (%) of Salmonella spp. isolated from raw milk and environment samples in dairy farms from district Multan.

Towns	TS	PS	S. typhi	S. paratyphi A	S. paratyphi B	S. typhimurium	Total Prevalence
			n (%)	n (%)	n (%)	n (%)
							(%)
BB	150	43	12 (8.0)	3 (2.0)	8 (5.33)	20 (13.33)	29.33
SRA	150	22	7 (4.67)	4 (2.67)	2 (1.33)	9 (6.0)	14.67
MPS	150	31	15 (10.0)	4 (2.67)	3 (2.0)	9 (6.0)	20.67
SS	150	40	11 (7.33)	5 (3.33)	7 (4.67)	17 (11.33)	26.67
SAB	150	59	19 (12.67)	6 (4.0)	7 (4.67)	25 (16.67)	39.33
JPPW	150	38	11(7.33)	3 (2.0)	6 (4.0)	18 (12.0)	25.33
Total	900	233	75 (8.33)	25 (2.78)	33 (3.67)	98 (10.89)	25.89

Town; BB: Band Bosan, SRA: Shah Rukne Alam, MPS; Musa Pak Shaheed, SS: Sher Shah, SAB: Shuja Abad, JPPW: Jalal Pur Pir Wala.

TS; Total number of samples, PS; Total number of positive sample, n = Number of positive samples of respective spp; χ2(df = 5,α = 0.05) = 13.3256 at p value = 0.03815 for towns; χ2(df = 3,α = 0.05) = 6.9249 at p value = 0.0743 for Salmonella spp.

Among five experimental sites in district Bahawalpur, Ahmad Pur East was shown to elicit the highest prevalence (%) for all four Salmonella spp. with S. typhi being more visible (Table 3). S. typhi also turned up as the most prevalent Salmonella spp. i.e. 11.8% (n = 89) in Bahawalpur district as a whole, followed by S. typhimurium i.e. 10.0% (n = 75). Notwithstanding, prevalence (%) of S. paratyphi A & B marked a non-significant difference (p>0.05) and they appeared to be the least prevalent Salmonella spp. i.e. 3.2% (n = 24) and 2.8% (n = 21) respectively in the region. When it came to the town level prevalence rate, Hasil Pur seemed to have been microbiologically the least tainted site in district Bahawalpur. A total of 209 samples (27.87%) were found positive for four strains of Salmonella isolated from raw milk and environment (Table 3).

Table 3

Prevalence (%) of Salmonella spp. isolated from raw milk and environment samples in dairy farms from district Bahawalpur.

Towns	TS	PS	S. typhi	S. paratyphi A	S. paratyphi B	S. typhimurium	Total Prevalence
			n (%)
				n (%)	n (%)	n (%)	(%)
BWP	150	37	17 (11.33)	5 (3.33)	4 (2.67)	11 (7.3)	24.67
APE	150	56	24 (16.0)	7 (4.67)	8 (5.33)	17 (11.33)	37.33
HPR	150	29	12 (8.0)	2 (1.33)	3 (2.0)	12 (8.0)	19.33
YZN	150	41	13 (8.67)	6 (4.0)	3 (2.0)	19 (12.67)	27.33
KPT	150	46	23 (15.33)	4 (2.67)	3 (2.0)	16 (10.67)	30.67
Total	750	209	89 (11.8%)	24 (3.2%)	21 (2.8%)	75 (10.0%)	27.8

Towns; BWP: Bahawalpur, APE: Ahmad Pur East, HP: Hasil Pur, YZN: Yazman, KPT: Khairpur Tamewali.

TS; Total number of samples, PS; Total number of positive sample, n = Number of positive samples of respective spp.; χ2(df = 4,α = 0.05) = 6.488 at p value = 0.1655 for towns; χ2(df = 3,α = 0.05) = 9.2245 at p value = 0.0265 for Salmonella spp.

Lodhran is relatively a smaller district of South Punjab and is located on northern side of the River Sutlej with its three towns viz Lodhran, Dunya Pur, Kehror Pakka. A total of 209 (20.22%) samples from raw milk and environment appeared as positive for Salmonella spp. (Table 4). Considering the extent of Salmonella spp. contamination in different towns, comparative results for prevalence (%) of Salmonella spp. revealed that the environment of sites from district Dunya Pur, was the most polluted with S. typhi (11.33%) and S. typhimurium (9.33%) both being more prevalent as compared to other Salmonella spp. (Table 4).

Table 4

Prevalence (%) of Salmonella spp. isolated from raw milk and environment samples in dairy farms from district Lodhran.

Towns	TS	PS	S. typhi	S. paratyphi A	S. paratyphi B	S. typhimurium	Total Prevalence
			n (%)	n (%)	n (%)	n (%)	(%)
LDN	150	28	11 (7.33)	1 (0.67)	4 (2.67)	12 (8.00)	18.67
DPR	150	40	17 (11.33)	5 (3.33)	4 (2.67)	14 (9.33)	26.67
KPA	150	23	07 (4.67)	3 (2.00)	2 (1.33)	11 (7.33)	15.33
Total	450	209	35 (7.78)	09 (2.00)	10 (2.22)	37 (8.22)	20.22

Towns; LDN: Lodhran, DPR: Dunya Pur, KPA: Kehror Pakka.

TS; Total number of samples, PS; Total number of positive sample, n = Number of positive samples of respective spp; χ2(df = 4,α = 0.05) = 3.3584 at p value = 0.1865 for towns; χ2(df = 3,α = 0.05) = 6.8869 at p value = 0.0756 for Salmonella spp.

S. typhi was found to be the most prevalent (11.0%) strain followed by S. typhimurium (10.33%) from D.G. Khan and Taunsa Sharif (Table 5). Town wise total percentage of Salmonella spp. in D.G. Khan town was 27.33% (n = 41) whereas Taunsa Sharif showed higher rate i.e. 37.33% (n = 56) with an overall percentage of 32.33% in the whole district. A total of 97 (32.3%) out of 300 raw milk and environment samples were tested positive for Salmonella spp. in D.G. Khan district. Differences among Salmonella spp. were non-significant (p>0.05) with regard to prevalence in towns of D.G. Khan district as shown in Table 5.

Table 5

Prevalence (%) of Salmonella spp. isolated from raw milk and environment samples in dairy farms from district D.G. Khan.

Towns	TS	PS	S. typhi	S. paratyphi A	S. paratyphi B	S. typhimurium	Total Prevalence
			n (%)	n (%)	n (%)	n (%)	(%)
DGK	150	41	12 (8.00)	05 (3.33)	07 (4.67)	17 (11.33)	27.33
TSA	150	56	21 (14.00)	06 (4.00)	15 (10.00)	14 (9.33)	37.33
Total	300	97	33 (11.00)	11 (3.67)	22 (7.33)	31 (10.33)	32.33

Towns; DGK: D.G. Khan, TSA: Taunsa Sharif.

TS; Total number of samples, PS; Total number of positive sample, n = Number of positive samples of respective spp; χ2(df = 1,α = 0.05) = 1.5466 at p value = 0.2137 for towns; χ2(df = 3,α = 0.05) = 4.1571 at p value = 0.2450 for Salmonella spp.

Muzaffargarh is one the known districts in D.G. Khan division of Punjab in Pakistan. Muzaffargarh city is located on the banks of the Chenab River. Out of 600 samples screened for Salmonella, 31.33% (n = 188) samples were found positive with the most prevalent Salmonella spp. S. typhimurium 13.67% (n = 82) followed by S. typhi 7.83% (n = 47), S. paratyphi B 6.0% (n = 36) while S. paratyphi A accounted for 3.83% (n = 23) being the least prevalent Salmonella spp. (Table 6). Amongst all experimental sites, Ali Pur was observed to be highly infected with 40.67% (n = 61) prevalence of Salmonella spp. followed by Kot Addu 32.67% (n = 49), Jatoi 28.67% (n = 43) and Muzaffargarh 23.33% (n = 35). Kot Addu and Ali Pur showed high prevalence rate of S. typhi and S. typhimurium with 10.0% (n = 15) and 18.0% (n = 27), respectively. Similar prevalence rate of S. paratyphi A 3.3% (n = 5) was observed in Muzaffargarh and Kot Addu areas whereas least occurrence (2.7%) of S. paratyphi B was recorded in Muzaffargarh town. The results presented in Table 6 showed differences among Salmonella spp. as non-significant (p>0.05).

Table 6

Prevalence (%) of Salmonella spp. isolated from raw milk and environment samples in dairy farms from district Muzaffargarh.

Towns	TS	PS	S. typhi	S. paratyphi A	S. paratyphi B	S. typhimurium	Total Prevalence
			n (%)	n (%)	n (%)	n (%)	(%)
MZG	150	35	10 (6.67)	5 (3.33)	4 (2.67)	16 (10.67)	23.33
KAU	150	49	15 (10.00)	5 (3.33)	7 (4.67)	22 (14.67)	32.67
APR	150	61	12 (8.00)	7 (4.67)	15 (10.0)	27 (18.00)	40.67
JTI	150	43	10 (6.67)	6 (4.00)	10 (6.67)	17 (11.33)	28.67
Total	600	188	47 (7.83)	23 (3.83)	36 (6.00)	82 (13.67)	31.33

Towns; MGR: Muzaffargarh, KAU: Kot Addu, JTI: Jatoi, APR: Ali Pur.

TS; Total number of samples, PS; Total number of positive sample, n = Number of positive samples of respective spp χ2(df = 3,α = 0.05) = 5.1095 at p value = 0.1639 for towns; χ2(df = 3,α = 0.05) = 6.8247 at p value = 0.0777 for Salmonella spp.

Antimicrobial resistance in Salmonella spp.

Data presented in Table 7 revealed the extent of resistance of Salmonella spp. against an array of antibiotics. S typhi emerged as a highly resistant Salmonella strain against OTC (70.11%) followed by AMP (38.79%), TMP (33.45%), CPl (29.54%) and AMX (28.11%) whilst same strain had shown to be the least resistant against OFL (0.00%), MOX (0.00%) and CPE (1.07%) suggesting these antibiotics to be employed against S. typhi infections. Four antibiotics viz OFL, CXM, IMP and MOX were noted to be remarkably effective against S. paratyphi A infection whereas this strain was identified to be highly resistant against OTC (25.84%) and TMP (47.19%). S. paratyphi A had also shown increased tendency towards switching over from sensitivity zone to intermediate level resistance against OTC (24.72%), TMP (21.35%), CXM (19.10%), and MOX (14.61%) suggesting a more cautious use of these antimicrobials against S. paratyphi A infection. Our results indicated that S. paratyphi A had not yet acquired multidrug resistance against these antibiotics, therefore these drugs could be equally applied as treatment options against illness caused by this microorganism. S. paratyphi B has almost manifested similar patterns for antibiotic resistance against the tested antibiotics as that of S. paratyphi A however, the microbe depicted increased sensitivity against GEN (94.21%) and CZA (87.60%) over S. paratyphi A (Table 7). Our results further demonstrated that S. paratyphi B was shown to make a rapid transition from its extant sensitivity to developing resistances against MOX (33.88%) and TMP (25.62%). Comparing S. typhimurium with rest of the three Salmonella spp. tested for development of antibiotic resistance against 13 antibiotics as mentioned in materials & method section, this strain had exhibited nearly a similar response with being least resistant against OFL, MOX and IMP in addition to CZA (Table 7).

Table 7

Antimicrobial susceptibility pattern of Salmonella isolates from raw milk and environment samples in dairy farms from South Punjab- Pakistan.

Antibiotic (μg)	S. typhi n (%)			S. paratyphi A n (%)			S. paratyphi B n (%)			S. typhimurium n (%)
	Sen	Int	Res	Sen	Int	Res	Sen	Int	Res	Sen	Int	Res
GEN (10)	228 (81.14)	34 (12.10)	19 (6.76)	78 (87.64)	7 (7.87)	4 (4.49)	114 (94.21)	7 (5.79)	0 (0.00)	267 (81.40)	28 (8.54)	33 (10.06)
CPL (30)	152 (54.09)	46 (16.37)	83 (29.54)	64 (71.91)	19 (21.35)	6 (6.74)	84 (69.42)	22 (18.18)	15 (12.40)	224 (68.29)	73 (22.26)	31 (9.45)
AMP (10)	123 (43.77)	49 (17.44)	109 (38.79)	62 (69.66)	8 (8.99)	19 (21.35)	95 (78.51)	15 (12.40)	11 (9.09)	175 (53.35)	66 (20.12)	87 (26.52)
OTC (30)	52 (18.51)	32 (11.39)	197 (70.11)	44 (49.44)	22 (24.72)	23 (25.84)	63 (52.07)	26 (21.49)	32 (26.45)	129 (39.33)	86 (26.22)	113 (34.45)
CIP (05)	239 (85.05)	11 (3.91)	31 (11.03)	78 (87.64)	2 (2.25)	9 (10.11)	114 (94.21)	5 (4.13)	2 (1.65)	221 (67.38)	77 (23.48)	30 (9.15)
OFL (05)	254 (90.39)	27 (9.60)	0 (0.00)	84 (94.38)	5 (5.62)	0 (0.00)	103 (85.12)	18 (14.88)	0 (0.00)	302 (92.07)	26 (7.93)	0 (0.00)
AMX(30)	168 (59.79)	34 (12.10)	79 (28.11)	57 (64.04)	19 (21.35)	13 (14.61)	82 (67.77)	16 (13.22)	23 (19.01)	262 (79.88)	41 (12.50)	25 (7.62)
CXM (30)	246 (87.54)	23 (8.19)	12 (4.27)	72 (80.90)	17 (19.10)	0 (0.00)	101 (83.47)	13 (10.74)	7 (5.79)	224 (68.29)	88 (26.83)	16 (4.88)
CZA (30)	208 (74.02)	39 (13.88)	34 (12.10)	70 (78.65)	14 (15.73)	5 (5.62)	106 (87.60)	15 (12.40)	0 (0.00)	297 (90.55)	26 (7.93)	0 (0.00)
CPE (30)	252 (89.68)	26 (9.25)	3 (1.07)	70 (78.65)	14 (15.73)	5 (5.62)	93 (76.86)	24 (19.38)	4 (3.31)	273 (83.23)	46 (14.02)	9 (2.74)
IMP (10)	240 (85.41)	28 (9.96)	13 (4.63)	87 (97.75)	2 (2.25)	0 (0.00)	109 (90.08)	12 (9.92)	0 (0.00)	312 (95.12)	16 (4.88	0 (0.00)
TMP (25)	137 (48.75)	50 (17.79)	94 (33.45)	28 (31.46)	19 (21.35)	42 (47.19)	75 (61.98)	31 (25.62)	15 (15.40)	204 (62.20)	51 (15.55)	73 (22.26)
MOX (10)	250 (88.97)	31 (11.03)	0 (0.00)	76 (85.39)	13 (14.61)	0 (0.00)	80 (66.12)	41 (33.88)	0 (0.00)	309 (94.21)	19 (5.79)	0 (0.00)

GEN; Gentamicin, CPL; Chloramphenicol, AMP; Ampicillin, OTC; Oxytetracycline, CIP; Ciprofloxacin, OFL; Ofloxacin, AMX; Amoxicillin, CXM; Cefuroxime, CZA; Ceftazidime, CPE; Cefepime, IMP; Imipenem, TMP; Trimethoprim, MOX; Moxalactam.

Sen; Sensitive, Int; Intermediate, Res; Resistant.

Numbers in parenthesis indicate percentage prevalence; Antibiotic resistance tested through chi squre χ2(df = 12,α = 0.05) = 278.07 at p value = 0.0000 for S.typhi; χ2(df = 12,α = 0.05) = 162.39 at p value = 0.0000 for S. paratyphi A; χ2(df = 12,α = 0.05) = 139.39 at p value = 0.0000 for S. paratyphi B; χ2(df = 12,α = 0.05) = 134.60 at p value = 0.0000 for S. typhimurium.

Discussion

The area under study has been one of the most distressed regions of Pakistan in terms of health care system and provision of medical facilities. Poverty remains to be a challenge which results in increased disease burden. Present study reflected higher rate of prevalence of Salmonella spp. in different districts of Southern Punjab indicating heighted incidences of salmonellosis. For example, overall prevalence of Salmonella spp. in all towns of Multan district was noted to be 25.89% (Table 2) and similar findings were also presented by Rahman et al. [17] who reported 21.89% Salmonella spp. in different samples. Other studies demonstrated the prevalence levels of Salmonella spp. to be ranging from 7.61% to 11.9% attributing the same to a variety of factors important being hygiene, sanitation and training of the food handling staff [18, 19]. Variability and significant differences in temperature at experimental sites in the present study could be a key determinant for difference in level of prevalence of Salmonella spp.

Our data revealed prevalence of S. typhi isolated from raw milk and environmental samples in district Bahawalpur to be to the tune of 11.9% (Table 3). Similar results were presented by Addis et al. [20] who reported Salmonella at 10.76% (n = 21/195) either from milk or feces samples. Similarly, 35.71% milk samples were found to be positive for S. typhi in Bangladesh [21]. Apart from Southern Punjab, more reports are available to signify the overwhelming effects of S. enteritidis among a number of population groups. Akin to other districts, S. typhi and S. typhimurium indicated the similar trend for prevalence irrespective of the sampling sites and sample type in district Lodhran which is a proxy of overall environment at dairy farms in the area (Table 4). Explanation to this opinion was better reflected from data presented in Table 1 suggesting overall hygiene of dairy farm including milking parlor environment, manure and inputs like bedding and feed as not merely the significant carriers of Salmonella spp. but also serve as potential milk contaminants.

Current study further revealed 32.3% Salmonella spp. samples being positive in district D.G. Khan. The results of present study are in agreement with the finding of Pangloli et al. [22] who isolated 40–92% Salmonella spp. from animal and environment samples. High prevalence of Salmonella spp. was ascribed to the poor hygienic condition of dairy farms, seasonal variation and improper personnel cleanliness. Our results further confirmed prevalence of S. typhi (11.0%) in D.G. Khan being less than extent of prevalence reported by Soomro et al. [23] who identified high prevalence of Salmonella enteritidis from chicken meat samples. The low prevalence of Salmonella spp. in this area was of S. paratyphi A with a prevalence rate of 3.67%. Almost identical results were obtained by other researchers who isolated Salmonella spp. from Kariesh cheese samples [24]. Increased prevalence of Salmonella spp. was also reported by Ghada et al. [25] and Wallaa, [26] who observed isolated Salmonella spp. from milk and cheese at 10% and 4% respectively.

Comparing the town wise prevalence of Salmonella spp. in Muzaffargarh, Ali pur was shown to indicate higher positive samples of Salmonella spp. (Table 5). Prevalence rate of Salmonella spp. however might not be attributed to any specific determinant and no relationship with respect to prevalence rate and region was established except the reasons described above i.e., farm hygiene and training of the farm staff. Data are not scant to indicate that the prevalence of Salmonella spp. at farms is not farm type specific e.g., beef cattle farm or dairy farms. These researchers were of the view that variation in prevalence might be a result of location of the farms and the focus on pathogen isolation from fecal or other animal-based samples [27-30]

Our results have further substantiated that the difference in prevalence of Salmonella spp. and the sources statistically differed with variability in region and source type. Overall results of this study demonstrated that no raw milk and environmental sample from selected sites might be considered up to the defined standards with respect to microbiological safety of the food, and control and monitoring of the dairy farms. Murinda et al. [29] reported 2.2% of bulk tank milk samples contaminated with Salmonella spp. attributing the presence of Salmonella spp. in tanks to be the result of cross-contamination from milking environmental sites instead of animal sites. A few recent studies with small sample size indicated Salmonella spp. to be present in raw farm bulk milk at 12% [31]. Results from a similar recent study from Ghana explicated reduced prevalence of Salmonella enterica in cow milk i.e., 7.3% [32].

A perusal of earlier studies to contemplate and compare the extent of prevalence of Salmonella spp. in South Asian regions portrayed that the prevalence rate in dairy and dairy products was more or less the same. Findings from Singh et al. [33]and Pant et al. [34] substantiated a kind of similar prevalence rate in India. Kaushik et al. [35] observed similar prevalence rate of Salmonella spp. in market milk samples in Patna, Bihar. Bangladesh as a region in subcontinent was not an exception for higher Salmonella spp. prevalence where the presence of S. typhi was found to be 35.17% in vendor’s milk. More studies confirmed these results showing Salmonella spp. prevalence to the tune of 9.5% and 4.2% [21, 36, 37]. This variation justified high prevalence of Salmonella spp. in various South Asian regions especially those located in subcontinent i.e. Pakistan and India because cultural, atmospheric and social conditions were quite the same therefore we might have witnessed the prevalence level being reported from these areas to be more or less similar.

Looking into the scale of emergence of antibiotic resistance among Salmonella spp. and efficacy of the 13 antibiotics tested in this study, we suggest OFL and MOX to be the most promising drugs of this time to treat Salmonella spp. infections. While most of the other antibiotics were shown to be in a transitional phase and are consistently losing their effectiveness against emerging and re-emerging microbes.

Researchers have recently ascribed the presence of antibiotic residues and antibiotic resistance bacteria in the animals’ manure to be the underlying cause of increased spread of antibiotic resistance. Besides, they reported a rise in antibiotic susceptibility among dairy manure isolates of bacterial pathogens with 15% of tested bacteria to be resistant against some antibiotics [38]

Most of the bacterial strains have been undergoing genetic modification for evolving resistance on account of indiscriminate and injudicious use of antibiotics for treating animal and human infections. Results of the present study demonstrate similar tendencies as all five experimental sites were shown to have been contaminated with Salmonella spp. A similar study depicted the same picture suggesting Salmonella spp. isolates from lactating cows, individuals handling them and the environment to be resistant to at least one of the tested antibiotics with 100% to ampicillin. Ciprofloxacin and amoxicillin appeared to be relatively effective as isolates were sensitive to these drugs [39]. More recently, researchers confirmed Salmonella enterica isolates from milk to be increasingly resistant to erythromycin (86.0%). Investigators further recorded susceptibility pattern as ciprofloxacin (100.0%), chloramphenicol (91.0%), ceftriaxone (91.0%), tetracycline (86.0%) and ampicillin (86.0%) attributing the increased emergence of resistance to imprudent and indiscreet exploitation of antimicrobials to treat animals against infectious diseases in dairy farms in Ghana and Uruguay [32, 40]. Lately, Sobur et al. [41] delineated an upsurge in resistance among Salmonella spp. against several antibiotics including oxytetracycline, tetracycline, erythromycin, azithromycin, and ertapenem. Researcher corroborated that Salmonella spp. were widely distributed in dairy farms and their environment and this scenario called for one health approach to override the growing health risks. They suggested judicious and wise use of antibiotics among dairy cattle for their treatment against salmonellosis.

Conclusion

Our study validated increased prevalence of Salmonella spp. in raw milk and environmental samples collected from the dairy farms of the Southern part of Punjab, which is well known for livestock production in Pakistan. Primarily, higher prevalence of Salmonella spp. in these regions badly contaminate the farm environment and farm produce leading to the onset of more frequent infections among farm animals and humans. Milk-borne pathogenesis and emergence of antibiotic resistance have been globally recognized as issues of public health significance and myriad containment strategies are underway. However, absence of new antimicrobials with increased efficacy has come out as a serious issue that warrants grave attention of the global health professionals. Available treatment options remain to be the conventional antibiotics being injudiciously used for treating Salmonellosis, leaving the microbes more resistant against them. Apparently, appropriate documentation and surveillance of bacterial infections and outbreaks badly lack in this region resulting in greater health risks and increased disease burden. The study concludes on precise, pragmatic and comprehensive strategies and initiatives have to be brought forward at farm level for preventing Salmonella spp. infections and the containment of multi drug resistance.

(XLSX)

Click here for additional data file.

(XLSX)

Click here for additional data file.