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Prevalence and Antibiotic Resistance of Salmonella Spp. in South Punjab

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Prevalence and Antibiotic Resistance of Salmonella Spp. in South Punjab bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042705; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Research Article 2 Prevalence and antibiotic resistance of Salmonella spp. in South Punjab-Pakistan 3 Aftab Qamar1, Tariq Ismail1*, Saeed Akhtar1* 4 5 I Institute of Food Science and Nutrition 6 Bahauddin Zakariya University Multan 7 8 Running title: Prevalence and antibiotic resistance in Salmonella spp 9 10 Key words: Antibiotic Resistance, Salmonella, Raw milk, Pakistan 11 12 13 14 Correspondence: *Saeed Akhtar (Ph.D.) [email protected] 15 *Tariq Ismail (PhD.) [email protected] 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042705; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 32 Abstract 33 Present study aimed at investigating the magnitude of the prevalence and antibiotic resistance 34 among four Salmonella spp. i.e. S. typhi, S. paratyphi A, S. paratyphi B and S. typhimurium, 35 present in raw milk and environment samples from five districts of southern part of the Province 36 of Punjab in Pakistan i.e. Multan , Bahawalpur, Lodhran, Dera Ghazi Khan and Muzaffargarh. 37 A total of 3000 raw milk and environment samples were collected from these sites and analyzed 38 for detection and confirmation of Salmonella spp. Extent of antibiotic resistance was also 39 determined and classified as resistant, intermediate and susceptible. S. typhimurium was found to 40 be more prevalent in Multan region as compared to other districts. Increased emergence of 41 antibacterial resistance among Salmonella spp. from raw milk samples was noted. Amongst 13 42 tested antibiotics, chloramphenicol and ofloxacin were found to be the most susceptible against 43 Salmonella spp. Present study suggested serious interventions by relevant government agencies to 44 ensure best practices in animal husbandry at farm level and sagacious use of antibiotics for the 45 containment of antimicrobial resistance in Salmonella spp. 46 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042705; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 Introduction 48 Traditionally, raw milk has been considered as a source of nutrients by a variety of farming 49 families and workers which they believe, are destroyed during any kind of processing [1] however 50 raw milk and milk products are reported to be the best breeding sites for pathogenic microbial 51 strains hence safety of these products turns up as a challenge given the direct contact of raw milk 52 with contaminated surfaces such as air, soil, workers hygiene, feces, grass and excretion from the 53 udder of an infected animal and milking equipments in production and processing areas [1]. 54 Salmonella has been considered as the major foodborne pathogen leading to an upsurge in enteric 55 infection cases. Three groups of Salmonella serotypes have been considered responsible for 56 causing distinctive clinical syndromes including typhoid fever, enteritis and bacteremia [2]. 57 Likewise, infections by non-typhoid Salmonella serovars have been shown to result in acute 58 gastroenteritis with extra intestinal localized infections that may eventually affect some organs [3]. 59 Reportedly, 99% Salmonella infections in humans are associated with strains in the O-antigen 60 serogroups such as serogroups A, B, C1, C2, D and E of S. enterica subspecies entericae [4]. 61 Mechanistically, the onset of disease proceeds with intestinal phase once the food contaminated 62 with typhoid and Salmonella enteritis is ingested [5]. 63 Recently, emergence of multidrug-resistant (MDR) S. enterica serovars, including resistance to 64 quinolone group (fluoroquinolones) and the third generation antibiotics (cephalosporin) has led to 65 serious public health issues throughout the world. One study demonstrated emergence of 66 antimicrobial resistance among Salmonella during animal farming, indicating 10% of the isolates 67 being resistant against one or more tested antimicrobials commonly used to treat Salmonella 68 infections e.g. cephalosporin and fluoroquinolones[6]. Sufficient evidence is available to support 69 the emergence of antibiotic resistance among Salmonella strains which is directly associated with 70 intensive use of antibiotics to treat Salmonella infections and incorporation of growth promoters 71 in animal feed [7,8]. The wider distribution of multidrug resistant (MDR) Salmonella spp. in foods 72 have been reported by various researchers worldwide [9–11]. A substantial body of literature 73 confirmed the prevalence of antibiotic resistance among foodborne pathogens especially in milk 74 and milk products and such a resistance pattern also came out in Salmonella spp. in last few 75 decades as a major public health issue worldwide [12,13]. 76 S. enterica serovars typhi and paratyphi (A, B and C) have been reportedly developing resistance 77 against a range of antibiotics thereby distressing 21 million people worldwide. Morbidity and 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042705; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 78 mortality rate associated with these microbial infections had been much higher on account of 79 infections by Salmonella spp. For example more than 14 million cases of enteric fever are reported 80 annually resulting in 135,000 deaths. S. typhi and S. paratyphi infections have been shown to be 81 more prevalent in South Asian regions where excessive use of antibiotic has been exacerbating the 82 emergence of multidrug resistance in these strains [14,15]. Multi-drug resistance (MDR) 83 Salmonella serotypes have become widespread in developed economies as well including USA. 84 Treatment cost of infections from antibiotic resistant bacteria amounts to 4-5 billion US dollars 85 annually. In addition to substantial financial losses caused in disease management, antibiotic 86 resistant pathogens have been hampering international trade owing to threats of cross borders 87 proliferation of infectious diseases [16]. 88 Available evidence suggest increased prevalence of Salmonella spp. in foods especially raw milk 89 and milk products leading to a surge in the onset of infections among humans and the farm animals. 90 Resultantly, the injudicious and indiscreet use of antibiotics to treat such infections has been 91 engendering heightened multidrug resistance among bacterial strains. Besides that, no 92 epidemiological surveillance, monitoring and control of pathogenic microbes and associated 93 microbiological infections is in place. The objective of the present study was to scale the 94 prevalence of Salmonella spp. at farms in South Punjab and to ascertain the extent of development 95 of antibiotic resistance in Salmonella spp. isolated from raw milk and farm environment. 96 Aftermaths of this study would provide a baseline information for the policy makers, farm owners, 97 dairy industry and stake holders to initiate plans for mitigating microbiological food safety issues 98 and corresponding disease burden in the region. 99 Materials and Methods 100 A cross-sectional study was designed to find out the prevalence of Salmonella spp. in raw milk 101 and environmental samples collected from five major districts of South Punjab (Fig.1). A total of 102 3000 samples were collected from towns/tehsils of these districts and detection and isolation of 103 Salmonella spp. were carried out to estimate the extent of the prevalence of Salmonella spp. i.e. 104 S. typhi, S. paratyphi A, S. paratyphi B and S. typhimurium. The isolated Salmonella strains were 105 also tested against antibiotics i.e. Gentamicin (GEN), Chloramphenicol (CPL), Ampicillin (AMP), 106 Oxytetracycline (OTC), Ciprofloxacin (CIP), Ofloxacin (OFL), Amoxicillin (AMX), Cefuroxime 107 (CXM), Ceftazidime (CZA), Cefepime (CPE), Imipenem (IMP), Trimethoprim (TMP), 108 Moxalactam (MOX), to figure out the resistance pattern in the organisms. The selection of tested 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.15.042705; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 109 antibiotics was made, based on the present therapeutic use of these antibiotics to treat Salmonella 110 infections in humans and farm animals. 111 Please Insert Figure 1 Here 112 Figure-1 Map showing five major districts (shaded) of South Punjab – Pakistan covered for surveillance and 113 antibacterial resistance in Salmonella isolates from raw milk and environment; 114 115 A total of 15 raw milk and 135 environment samples (manure, soil, feed, bedding, potable water, 116 milk container, personnel and animals’ teat) were collected from dairy farms, bicycle milk vendors 117 and raw milk outlets from peri-urban areas of each targeted site. Raw milk (approx. 50 ml in 118 sterilized dry bottles) and environmental samples (almost 100 g in sterilized zip lock bags) were 119 collected, tightly sealed, kept in ice box and immediately shipped to laboratory for analyses. 120 Procedure from ISO 6579:2002 standard (Microbiology of food and animal feeding stuffs) 121 guidelines were followed for detection, isolation and confirmation of Salmonella.
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