- Email: [email protected]
Phentotypic, gentotypic antimicrobial resistance and pathogenicity of Salmonella enterica serovars Typimurium and Enteriditis in poultry and poultry products
Accepted Manuscript Phentotypic, gentotypic antimicrobial resistance and pathogenicity of salmonella enterica serovars Typimurium and enteriditis in poultry and poultry products Sher Bahadar Khan, Mumtaz Ali Khan, Irshad Ahmad, Tayyab ur Rehman, Shahid Ullah, Rahim Dad, Asad Sultan, Atta Muhammad Memon PII:
S0882-4010(18)30782-4
DOI:
https://doi.org/10.1016/j.micpath.2019.01.046
Reference:
YMPAT 3388
To appear in:
Microbial Pathogenesis
Received Date: 2 May 2018 Revised Date:
22 January 2019
Accepted Date: 30 January 2019
Please cite this article as: Khan SB, Khan MA, Ahmad I, ur Rehman T, Ullah S, Dad R, Sultan A, Memon AM, Phentotypic, gentotypic antimicrobial resistance and pathogenicity of salmonella enterica serovars Typimurium and enteriditis in poultry and poultry products, Microbial Pathogenesis (2019), doi: https:// doi.org/10.1016/j.micpath.2019.01.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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PHENTOTYPIC,
ANTIMICROBIAL
RESISTANCE
AND
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PATHOGENICITY OF SALMONELLA ENTERICA SEROVARS TYPIMURIUM AND
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ENTERIDITIS IN POULTRY AND POULTRY PRODUCTS
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Sher Bahadar Khan1 , Mumtaz Ali Khan2,3, Irshad Ahmad4, Tayyab ur Rehman4, Shahid
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Ullah5, Rahim Dad6, Asad Sultan7, Atta Muhammad Memon8
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Department of Animal Health, The University of Agriculture, Peshawar, Pakistan
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Civil Veterinary Hospital, SherGarh, Livestock and Dairy Development, Khyber
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Pakhtunkhwa, Pakistan
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University of Veterinary and Animal Sciences, Lahore, Pakistan
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Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan
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College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen
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518060, Guangdong, China
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Tandojam
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Department of Poultry Sciences, The University of Agriculture, Peshawar, Pakistan
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Livestock and Fisheries Department, Government of Sindh, Pakistan
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Corresponding author:
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Sher Bahadar Khan, Department of Animal Health, The University of Agriculture,
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Peshawar, Pakistan
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Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University,
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Email: [email protected]
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Abstract:
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For detection and isolation of Salmonella enterica, 650 meat and tissue samples were processed
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using Rappaport-Vassiliadis Enrichment broth and Salmonella Chromogenic agar followed by
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confirmation through specific antisera and polymerase chain reaction (PCR) targeting their
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Specific Serovar Genomic Regions (SSGRS). Isolates were tested for 15 antibiotics (CRO, AMX,
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GEN, STR, TET, CHL, CLR, LVX, OFX, GAT, CIP, SXT, AMP, LIN and AZM) according to
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the disc diffusion method and antimicrobial resistant genes (tet(A), tet(B), tet(C), strA/strB,
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aadA, aac(3)IV), aadB, sul1, sul2 and sul3, blaCMY-2, blaTEM and blaSHV) using PCR. The
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overall prevalence of Salmonella enterica was 12%, being higher in markets (15%) as compared
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to poultry farms(37.2%). The MPN of all positive meat and tissue samples was found 3.6
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MPN/gram (0.17-18). A total of 234 isolates were obtained, serovar Typimurium (139) and
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Enteridits (95) were the most prevalent. Antimicrobial resistance patterns were different in
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different serovars according to origin of Salmonella isolates. The overall isolates were highly
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resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%, 174/234),
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TET (64.5%, 151/234) and STR (64.5%, 151/234). Overall, the most common ARG was
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blaTEM (76%, 178/234 ), followed by blaSHV (71.7 %, 168/234), tet(A)(64%, 151/234) and
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tet(B) (64%, 150/234), while the least ARG was aadB ( 7.2%, 17/234). Both Typimurium and
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Enteridits were tested in the Balb/C mice for pathogenicity. Both Typimurium and Enteridits were found
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to cause successful colonization, 100% morbidity but Enteriditis were found to cause 33% mortality.
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Key words: Antibiotic resistance; antibiotic resistant genes; Salmonella; Serovar; Poultry;
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Poultry products; pathogenecity
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Introduction
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Globally, Salmonella is one of the leading causative agent of food-borne bacterial zoonosis
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(Daust and Maurer, 2007, Miranda et al., 2009). Salmonella has been estimated as a causative
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agent in about 70-80% of food borne bacterial outbreaks (Wang et al., 2007). It is estimated that
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Salmonella species are causing 1.4 million food-borne illness, 15,000 hospitalizations and 400
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deaths annually in the United States (Votesche et al., 2004, CDC, 2007). Salmonella genus
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consists of two species, Salmonella enterica and Salmonella bongori. More than 2500
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serovars of genus Salmonella have been reported (Popoff, 2001, Herikstad et al., 2002). A wide
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variety of warm blood animals is hosted by Salmonella enterica subsp. 1, which consists of
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almost 1500 serovars (Brenner et al., 2000, Grimont et al., 2000). A small proportion of
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Salmonella serovars are host specific, such as serovar Typhi, Cholerasuis, Dublin and
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Gallinarium are specific to human, swine, cattle and chicken, respectively (Kingsleyand and
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Baumler, 2000 and Rabsch et al., 2002, Wray et al., 2000). These serovars pose a potential threat
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to human health as food borne pathogens in contaminated meat and other food products
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(Bangtrakulnonth et al., 2004, Gebreyes et al., 2005, Foley and Lynne, 2008). An increase in the
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antimicrobial resistance in bacterial pathogens including Salmonella is a worldwide public health
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concern which is mainly associated with frequent use of antibiotics in production animals
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(Arslan et al., 2010, Alvarez-Fermandez et al., 2012, Chen et al., 2004). And then their
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subsequent transmission to human through animal food and food prodcuts (Foley and Lynne,
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2008 and EFSA, 2013). There are a variety of mechanisms through which these pathogens get
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resistance and among them ARGs is the most important (Aarestrup et al., 2003). This study is
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therefore carried out to find out the prevailing situation about Salmonella enterica subspecies
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enterica, its quantification, different serovars distribution, antibiotic resistance and antibiotic
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resistance genes in poultry and poultry products in north west of Pakistan.
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Materials and methods
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Sample collection
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A total of 650 samples were used in the study. From the poultry production system we collected
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250 cloacal swabs from 4 weeks old clinically healthy poultry birds from different herds of five
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intensive poultry farms (50 sample/farm). From the supply chain, 400 samples were purchased
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from different markets (100 each meat, liver, intestine and kidney) in both summer and winter.
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Samples were collected in sterile plastic bags and subjected for further process immediately upon
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arrival to the laboratory.
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Processing of samples
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Upon arrival to the laboratory, cloacal swabs were immediately washed with 0.5 ml PBS, and 0.1
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ml of them was inoculated into 9 ml Brain heart infusion broth (BHIB; BD Difco, USA) at 37°C
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for 24 hours. Out of 100 g meat and tissue samples purchased from the different markets, 50 g
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was cut into small pieces and mixed with 450 ml BHIB (BD Difco, USA) in homogenizer for
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two min. One ml of homogenized sample was mixed with 9 ml of BHIB and incubate at 37°C for
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24 h for further bacterial isolation. Sample homogenate was used for detection, MPN-PCR and
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isolation of Salmonella enterica subsp.I as described below.
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Serotyping using specific antisera
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Salmonellae are flagellated bacillia possessing three major antigens, H or flageller antigen, O or
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somatic antigen and Vi or capsular antigen. Specific antisera were used for the serotyping of
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Salmonella serovars.
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MPN series for enumeration of Salmonella
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Salmonella enterica subsp. I in sample homogenate was enumerated using a three-tube MPN as
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follows. MPN is an indirect means to calculate the probable number of certain number of
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microbes in food or water, particularly when the concentration of the microbe is relative low. For
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each sample homogenate, a tenfold serial dilution series was prepared using Butterfield's
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buffered phophate diluent (BBPD). Then 1ml of each original homogenate was transferred to 9
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ml BHIB in each of the three MPN tubes. Inoculated tubes and uninoculated control tube were
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incubated at 37°C for 24 h. After incubation, 1 ml of each MPN tube was processed for total
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DNA extraction using E.Z.Nce.A bacterial DNA kit (Omega Bio-Tek, USA).
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PCR targeting Salmonella specific invA gene
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The total DNA was first tested for Salmonella specific invA gene using the specific primers as
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described by Akiba et al., 2011. The details of the primers are given in Table 1. PCR
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amplifications were performed using 2 × Es Taq PCR master mix providing a concentration of 3
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mM MgCl2, Taq DNA polymerase, 2 × Es Taq PCR buffer and 400 µM dNTP mix (CWBIO,
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China), 200 ng template DNA and 10 µM of each primer in a total reaction volume of 25 µl with
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cycling condition including an initial incubation at 94 °C for 2 min, followed by 35 cycles at
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98°C for 10 s, 60°C for 30 s and 68°C for 30 s. PCR products were run on 2% agarose E-gels
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pre-stained with Gel red (Invitrogen A/S, Taastrup, Denmark) and visualized by UV-lighting.
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Bacterial isolation and DNA extraction
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The positive samples were processed for bacterial isolation. From the original homogenate stored
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at -20 °C, 0.1ml was transferred to 10 ml of Rappaport-Vassiliadis Enrichment both (RV, Oxoid,
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UK) and incubated at 37°C for 24 h. Then a loop full from Rappaport-Vassiliadis Enrichment
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broth into Salmonella Chromogenic agar plate (Hopebio, Qingdao Hope Biotechnology, China)
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was inoculated followed by incubation at 37°C for 24 h. Typical purple color colonies (three
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colony/sample) were selected for further analysis. Salmonella was confirmed using standard
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bacteriological biochemical tests using an API 20E system (bioMerieux, France). Genomic DNA
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was extracted from the Salmonella isolates using E.Z.Nce.A bacterial DNA kit (Omega Bio-Tek,
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USA). The genomic DNA was tested for specific invA gene and seven serovars targeting their
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three specific serovar genomic regions (SSGRS) for each serovar using the specific primers as
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described by Akiba et al., 2011. The details of the SSGRS and primers are given in Table 1. PCR
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amplifications were performed using 2 × Es Taq PCR master mix and PCR conditions as
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described above.
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Antimicrobial susceptibility testing (AST)
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The antimicrobial susceptibility of the Salmonella isolates were determined for 15 antimicrobials
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according to the disc diffusion method using Muller-Hinton agar (MHA, Qingdao hopebio
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technology Co., China). Interpretation of the results followed the recommendations of the
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Clinical and Laboratory Standards Institute (CLSI, 2016), the European Committee on
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Antimicrobial Susceptibility Testing (EUCAST) (Galni et al., 2008). AST was performed for the
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following antimicrobial agents (antimicrobial abbreviations and breakpoints are shown in
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parenthesis): Ceftriaxone (CRO, 30 µg), Amoxicillin (AMX, 20 µg), Gentamycin (GEN, 10 µg),
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Strptomycin (STR, 10 µg), Tetracycline (TET, 30 µg), Chloramphenicol (CHL, 30 µg),
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Clarithromycin (CLR, 15 µg), Levofloxacin (LVX, 5 µg), Ofloxacin (OFX, 5 µg), Gatifloxacin
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(GAT, 5ug), Ciprofloxacin (CIP, 5ug), Suphamethoxazole+Trimethpram (SXT, 25 µg),
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Ampicillin (AMP, 10 µg), Lincomycin(LIN, 2 µg) and Azithromycin (AZM, 15 µg). Isolates
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showing resistance to at least three antimicrobial agents belonging to different antimicrobial
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classes were considered multidrug resistance (MDR) strains.
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Detection of antibiotic resistance genes (ARGs)
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A set of multiplex PCRs was used for identifying major resistance genes following the procedure
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described by Kozak et al. (2009). The major genes conferring resistance for tetracycline [tet(A),
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tet(B), tet(C)], streptomycin (strA/strB, aadA and (aac(3)IV), gentamycin (aac(3)IV, aadB),
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sulfonamides (sul1, sul2 and sul3), and b-lactamases (blaCMY-2, blaTEM, blaSHV) were
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targeted. Primers and multiplex PCRs conditions used for detection of antibiotic resistance genes
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are given in the Table 2.
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Pathogenicity testing of Salmonella serovars using Balb/C mice
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For pathogenicity testing of Salmonella Typimurium and Enteriditis strains isolated from poultry
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and poultry products, 5-6 weeks old Balb/c female mice were used. Streptomycin treated mouse
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model was used in this study. In this model, mice are provided with streptomycin sulphate in
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their drinking water as a means to reduce their normal facultative flora so as to decrease bacterial
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competition for the infecting O157:H7 strain. Both salmonella Typimurium and Enteridits were
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tested. A group of 6 mice were used for each strain. The mice were provided with streptomycin
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sulphate 5 g/L in drinking water and were kept off fed for 12 hours. After 12 hours, mice were
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infected with 109 CFU of both Typimurium and Enteridits in 20% sucrose solution intragastrically
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(I/G) (Zhihong Ren et al., 2009). Colonization, morbidity including typical signs and symptoms,
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and mortality were noted.
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Histopathology
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The intestine and kidney were removed surgically from the dead mice/or and immediately after
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the mice were killed. Both the intestine and kidney were examined for pathological changes. The
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tissues were fixed in 10% formalin and were submitted to histopathology Lab. for further
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histopathological process.
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Results
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Prevalence and isolation of Salmonella enterica subsp.1 serovars
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Among the 650 samples, 78 samples (12%) were found positive for Salmonella using specific
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antisera and PCR targeting invA gene as shown in Table 3. Out of 250 cloacal swabs from five
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different farms, 18 (7.2%) swabs were found positive. Out of 400 tissue samples from different
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markets, 60 (15%) were found positive. The highest prevalence of Salmonella was found in
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intestine samples in winter (30/50, 60%) followed by meat in winter (15/50, 30%), meat in
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summer (10/50, 20%) and liver in winter (5/50, 10%). The MPN of all positive meat and tissue
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samples was found 3.6MPN/gram (0.17-18).
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Out of 650 samples processed for Salmonella isolation, 78 were found positive. A total of 234
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isolates(3 colony/sample) were obtained which were confirmed through specific antisera. PCR
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for specific invA gene and SSGRS were conducted as shown in Table 3. Out of 234 isolates, the
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most prevalent serovar was Typimurium (139) followed by Enteriditis (95). Except from meat
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samples in summer, it was observed that Typimurium can be easily isolated from all tissue
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samples.
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Antibiotic resistance in Salmonella serovars
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Out of 234 isolates, the most prevalent serovars was Typimurium (139) followed by Enteriditis
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(95). Antibiotic resistance was found different in both serovars (Figure 1). The overall isolates
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were highly resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%,
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174/234), TET (64.5%, 151/234) and STR (64.5%, 151/234). Resistance for GAT (8.9%, 21/234)
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and CRO (8.5%, 20/234) was the lowest in all the isolates. Typimurium was highly resistant for
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AMX (94.9%, 132/139) followed by LIN (93.5%, 130/139), AMP (92%, 128/139), TET and
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STR (84.1%, 117/139, 83.4, 116/139), but less resistant for CRO (6.4%, 9/139) and GAT (5.7%,
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8/139). Enteriditis was highly resistant for LIN (92.6%, 88/95) followed by AMX (74.6%,
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55/95), AMP (48.4%, 46/95), STR and SXT (37.8%, 36/95), but less resistant for GAT (13.6%,
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13/95) and CRO (11.5%, 11/95). Interestingly all the serovars possess highest resistance for LIN
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in contrast to other antibiotics. Multiple antibiotic resistance was found in different serovars.
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Prevalence of ARGs in Salmonella isolates along the PSCP
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The prevalence of ARGs in Salmonella isolates varied in different serovars which is
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consistent with the phenotypic data. Overall, the most common AR G was blaTEM (76%,
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178/234 ), followed by blaSHV (71.7 %, 168/234), tet(A)(64%, 151/234) and tet(B) (64%,
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150/234), while the least ARG was aadB ( 7.2%, 17/234) (Figure 2).
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Typimurium was found to possess higher blaTEM (94.9%, 132/139) followed by blaSHV (92%,
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128/139), tet(A) (84.1%, 117/139), tet(B) (84%,116/139) and strA/strB (82.7%, 115/139) as
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compared to other ARGs. While Enteriditis was found higher in blaTEM (48.4%, 46/95)
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followed by blaSHV (42.1%, 40/95), strA/B (37.8%, 36/95) tet(A) and tet(B) (35.7%, 34/95), as
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compared to other ARGs. Interestingly blaTEM was found higher in all serovars as compared to
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other ARGs (Figure 2).
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Pathogenicity testing
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Both Typimurium and Enteridits were tested in the Balb/C mice for pathogenicity. Both Typimurium and
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Enteridits were found to cause successful colonization, 100% morbidity but Enteriditis were found to
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cause 33% mortality. Typical clinical signs and symptoms as in appetence, diarrhea, hunched posture,
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ruffled feathers, tremor and lethargy were observed in the infected mice. Mortality in mice started 3 days
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post administration of serovars. Extensive necropsy (liver, heart, intestine, caecum, large intestine, large
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intestine and kidney) of the dead and infected mice revealed that small intestine were found to
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demonstrate the pathology of any kind. Histopathology of the intestine revealed changes in intestinal
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villi, epithelial integrity, and local visible accumulation of inflammatory cells in the lamina
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propria, mostly lymphocytes, mucosa and sub mucosa structure disappeared, visible necrosis of
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intestinal epithelial cells and cell debris shed full of the intestine as shown in Figure 3 and 4.
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Intestinal and kidney's sections of controlled group revealed no histopathological changes.
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Discussion
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The overall prevalence of Salmonella was 12%, being higher in markets as compared to
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poultry farms. Previous studies have shown different results of salmonella prevalence in
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different tissue samples such as 0-17.5% in tonsil swabs (Piras et al., 2011,) 12.2-16.4% in
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intestine samples (Hernandez et al., 2013), 9.3-12.5% in liver (Swanenburg et al., 2001) and
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Virira et al., 2011) and 27- 63% in kidneys (Arguello et al., 2012). China have also reported
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different results such as 20% in northen region （Yan et al., 2010）, 31% in Shanxi (Yang et al.,
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2010) and 26% in Hebei province (Jiang et al., 2006), while a Canadian study have reported 2%
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prevalence (Aslam et al., 2012). All these studies indicate that Salmonella is widely distributed
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in retail meat all over the world which increase salmonellosis. The inconsistency of our results
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with the previous studies may be due to several factors such as difference in country and origin,
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sampling season, slaughter house sanitation and isolation methods. Our MPN results are in close
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agreement with the previous studies (Bornadis et al., 2003).We compare the specificity of PCR
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method targeting SSGRS of different serovars as derscribed by Akiba et al., 2011 with specific
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antisera method.
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Here we report that serovar Typimurium and Enteirdits which is specially adopted to
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human and poultry, respectively have been isolated from cloacal swabs and tissue samples of
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poultry. Previous studies have shown that Typimurium, Enteriditis, Shurba, Djuju and Derby
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are the most common serovars worldwide (Bolton et al., 2013 and Yang et al., 2010). Serovar
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dublin from cattle was reported to have high resistance for Enrofloxacin followed by Ceftiofur
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(21.82% ) and SXT (20.9%) (Berge et al., 2008), while isolates from human was found to have
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resistance for streptomycin, sulphamethoxazole, tetracyclin and trimethoprim (43%) (Fashae et
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al., 2010). Similarly isolates from bulk tanks and milk filters have resistance for different
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antibiotics which is not consistent with the present study (Van Kessel et al., 2013). On the other
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hand, Typimurium isolates from pork have shown highest resistance for tetracycline,
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sulphamethxazole (90.9%)
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trimethoprim (72.7%) (Prendergast et al., 2009). Our results of antimicrobial resistance are not
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in consistent with the previous studies as antibiotic resistance may vary from region to region,
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followed
by streptomycin,
choramphenicol (81.1%)
and
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source of isolation, different antibiotics and other factors (Pan et al., 2010). Analyzing antibiotic resistance genes in Salmonella isolates was one of the aspect of this
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study. The b-lactamase genes are among the antibiotic resistance genes important from human
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health perspective. In this study blaTEM was the most common genes found in isolates resistant
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to AMP, AMX and CRO while the tet (A) and tet (B) were found to be more associated with
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tetracyclin resistance which is in consistent with the previous studies (Chuanchen et al., 2009
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and Aslam et al., 2012 ). Similarly serovars resistant to SXT were associated with sul1, sul2 and
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sul3 genes (Chuanchun et al., 2009), while those resistant to STR were found to be more
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associated with strA/strB gene as compared to aadA and aac(3)IV which is also in consistent
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with the previous studies (Aslam et al., 2012).
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The high prevalence of blaTEM genes followed by blaSHV, tet(A), tet(B) in Salmonella isolates
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may be due to common use of AMP, AMX, STR and TET during animal production for
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controlling bacterial infections and promotion of growth (Aslam et al., 2012). Presence of these
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genes on genetic mobile elements such as transposons and plasmids can facilitate their transfer
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(Schwarz et al., 2006). Our results of ARGs prevalence in different serovars associated with
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different antibiotics are not in consistent with the previous studies as different studies have
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reported different ARGs prevalence associated with different antibiotics (Cui et al., 2009, Li et
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al., 2007, 2013 ).
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Conflict of interest
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All the authors declare no conflict of interest
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Acknowledgment
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The study was supported by Department of Animal Health, The University of Agriculture,
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Peshawar, Livestock and Dairy development Department Khyber Pakhtunkhwa, Veterinary
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Research Institute, Peshawar, Pakistan.
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antimicrobial resistance of Salmonella in retail foods in
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northern China.
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Logsdon, Joan Mecsas, and Simin Nikbin Meydani, 2009. Effect of age on
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Table 1. Target genomic regions and primer sequences of the multiplex PCR assay
TSR2
TSR3
CSR1
Primer
Sequence
STM2896
InvA
invAF
5'-AAACCTAAAACCAGCAAAGG
605
invAR
5'-TGTACCGTGGCATGTCTGAG
TMP1F
5'-ATGCGGGTATGACAAACCCT
TMP1R
5'-TTAGCCCCATTTGGACCTTT
TMP2F
5'-CAGACCAGGTAAGTTTCTGG
TMP2R
5'-CGCATATTTGGTGCAGAAAT
TMP3F
5'-TTTACCTCAATGGCGGAACC
TMP3R
5-CCCAAAAGCTGGGTTAGCAA
CMP1F
5'-AGATATGCGGCTTGCTGACT
STM0292
STM2235
STM4493
SCH_0971
putativeRHS-family protein
Putative phage protein
Putative cytoplasmic protein
hypothetical protein
CMP1R
CSR2
SCH_2145
hypothetical protein
SCH_4349
hypothetical protein
ISR1
3113085– 114152
–d
ISR2
1067175– 068152
4621947– 622627
HSR1
1067175– 068152
2541120– 541716
–d
–d
AC C
HSR2
–d
EP
ISR3
–d
HSR3
ESR1
ESR2
ESR3
4675756– 676622
SEN0910
SEN1006
SEN2420
–d
hypothetical phage protein (pseudogene)
hypothetical protein
putative exported protein
94
196
303
100
5'-AGCGTGGCTCGAAACAGTAT
CMP2F
5'-GGCGAAAGAGCTTAACGTGA
CMP2R
5'-TTACCATCGGGACCAAATGT
CMP3F
5'-TCGGGATCGTCCTCTATACG
CMP3R
5'-CCCAAAAGCTGGGTTAGCAA
IMP1F
5'-GGTCATTGTCGGAAACCTGC
IMP1R
5'-ACATTCCCCCTTCCACTGCC
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CSR3
RI PT
TSR1
Product
SC
invAc
Representative geneb
PCR product size (bp)
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Region
a
IMP2F
5'-CGCGAAGAAGTGCATAAACC
IMP2R
5'-CGCGAAGAAGTGCATAAACC
IMP3F
5'-ACCTACTACTATCCCTGATG
IMP3R
5'-GCGAATTTTGCTACTTGAAG
HMP1F
5'-ACTTCATGCGCAGACTTGCC
HMP1R
5'-CAAGAATCGGGAAATTTTGA
HMP2F
5'-TACCGCCCCATTTGATATCT
HMP2R
5'-CCCTTGCGCAACATAAAACT
HMP3F
5'-GGATCTCAACAAAATGAGGT
HMP3R
5'ATGCATCACTGCATCTTCGT
EMP1F
5'-AATACAGCCTCAACCAGCTA
EMP1R
5'-ATTGGTTCACCCGTTGCAAT
EMP2F
5'-AGATAAGCCCTCCCTGCTTA
EMP2R
5'-CCCTCCTTTCACTGCAAGTC
EMP3F
5'-CAAAAGCGACAAATAATCTG
198
305
95
198
304
105
199
303
101
203
299
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conserved hypothetical protein
DSR2
SeD_A1104
conserved hypothetical protein
DSR3
SeD_A2276
lysozyme
GSR1
SG0266
conserved hypothetical protein
GSR2
SG3181
conserved hypothetical protein
hypothetical protein
5'-ATCGGTGCTGGGTAATTTTG
DMP1R
5'-AGGAACGAGAGAAACTGCTT
DMP2F
5'-ACGCGAAATCTGATGGTCTT
DMP2R
5'-GCCCACCAGTTGTGAAAGGC
DMP3F
5'-ATCACCCTCGCAAACTTGTC
DMP3R
5'-TCGGGCAATCAGGTCGCCGA
GMP1F
5'-CCGCACAACACATCAGAAAG
GMP1R
5'-AGCTGCCAGAGGTTACGCTG
GMP2F
5'-CTCCTGATCATGGCGCTACT
GMP2R
5'-GTGAGGATTTTGTCGTAGCA
GMP3F
5'-GCTAGGGGTTTCCTCCACTC
GMP3R
5'-CGATTTTGGAGCGGATAACC
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GSR3
SG1033
DMP1F
105
203
RI PT
SeD_A1226
5'-TTTCTCCGCCTGTTTTCGTT
SC
DSR1
EMP3R
296
97
206
301
a TSR, Typhimurium-specific (genomic) region; CSR, Choleraesuis-specific (genomic) region; ISR, Infantisspecific (genomic) region; HSR, Hadar-specific (genomic) region; ESR, Enteritidis-specific (genomic) region;
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DSR, Dublin-specific (genomic) region; GSR, Gallinarum-specific (genomic) region.
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Table 2. Primers and conditions used for antibiotic resistance genes Gene
Primer
Sequence
Final Conc of primer (M)
Annealing temp(°C)
Product size (bp)
1
sul1
sul1-Fb
CGGCGTGGGCTACCTGAACG
0.2
66
433
sul1-Bb
GCCGATCGCGTGAAGTTCCG
0.2
sulII-Lc
CGGCATCGTCAACATAACCT
0.3
sulII-Rc
TGTGCGGATGAAGTCAGCTC
0.3
sul3-GKa-Fd
CAACGGAAGTGGGCGTTGTGGA
0.2
sul3-GKa-Rd
GCTGCACCAATTCGCTGAACG
0.2
TetA-Lc
GGCGGTCTTCTTCATCATGC
0.1
TetA-Rc
CGGCAGGCAGAGCAAGTAGA
TetBGKF2m
CGCCCAGTGCTGTTGTTGTC
TetBGKR2m
CGCGTTGAGAAGCTGAGGTG
0.2
TetC-Lc
GCTGTAGGCATAGGCTTGGT
0.5
TetC-Rc
GCCGGAAGCGAGAAGAATCA
0.5
4Fe
GTGGATGGCGGCCTGAAGCC
0.1
4Re
AATGCCCAGTCGGCAGCG
0.1
strA-Ff
ATGGTGGACCCTAAAACTCT
0.4
2
2
3
3
tet (A)
tet (B)
tet (C)
aadA
strA/strB
strB-Rf 3
aac(3)IV
4
aadB
TGCTGGTCCACAGCTCCTTC
0.2
CGGATGCAGGAAGATCAA
0.2
GAGGAGTTGGACTATGGATT
0.2
CTTCATCGGCATAGTAAAAG
0.2
GKTEMFd
TTAACTGGCGAACTACTTAC
0.2
GKTEMRd
GTCTATTTCGTTCATCCATA
0.2
SHV-Fj
AGGATTGACTGCCTTTTTG
0.4
SHV-Rj
ATTTGCTGATTTCGCTCG
0.4
CMYFd
GACAGCCTCTTTCTCCACA
0.2
CMYRd
GGACACGAAGGCTACGTA
0.2
aadB-Li
5
blaSHV
AC C
5
blaTM
EP
aadB-Ri
5
blaCMY2
0.2
0.4
aac4-Rg
721
66
244
63
502
0.1
CGTCTAGGATCGAGACAAAG
aac4-Lg
66
SC
2
Sul3
M AN U
1
Sul2
TE D
1
RI PT
PCR
63
173
63
888
63
525
63
893
63
653
55
208
55
247
55
393
55
1000
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Table 3. Prevalence and isolation of salmonella enterica serovars in poultry Positive No. of Samples
Typimurium Enteriditis
Cloacal swabs
250
18 (7.2%)
34
20
IntestineS
50
-
-
-
IntestineW
50
30 (60%)
63
Liver S
50
-
-
Liver W
50
5 (10%)
9
Kidney S
50
-
-
KidneyW
50
-
-
MeatS
50
10 (20%)
5
MeatW
50
15 (30%)
28
17
Total
650
78 (12%)
139
95
EP AC C
27
-
SC
6
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summer, Wwinter, - negative
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S
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Type of sample
-
-
25
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SC
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Figure 1. Antibiotic resistance in Salmonella isolates.
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Figure 2. Antibiotic resistant genes in Salmonella isolates.
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Figure 3. Histopathological changes in the intestine.
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Figure 4. Histopathological changes in the intestine.
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The overall prevalence of Salmonella enterica was 12%, being higher in markets (15%) as compared to poultry farms(37.2%). The MPN of all positive meat and tissue samples was found 3.6 MPN/gram (0.17-18). Typimurium (139) and Enteridits (95) were the most prevalent. The overall isolates were highly resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%, 174/234), TET (64.5%, 151/234) and STR (64.5%, 151/234). Overall, the most common AR G was blaTEM (76%, 178/234 ), followed by blaSHV (71.7 %, 168/234), tet(A)(64%, 151/234) and tet(B) (64%, 150/234), while the least ARG was aadB ( 7.2%, 17/234).
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S0882-4010(18)30782-4
DOI:
https://doi.org/10.1016/j.micpath.2019.01.046
Reference:
YMPAT 3388
To appear in:
Microbial Pathogenesis
Received Date: 2 May 2018 Revised Date:
22 January 2019
Accepted Date: 30 January 2019
Please cite this article as: Khan SB, Khan MA, Ahmad I, ur Rehman T, Ullah S, Dad R, Sultan A, Memon AM, Phentotypic, gentotypic antimicrobial resistance and pathogenicity of salmonella enterica serovars Typimurium and enteriditis in poultry and poultry products, Microbial Pathogenesis (2019), doi: https:// doi.org/10.1016/j.micpath.2019.01.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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PHENTOTYPIC,
ANTIMICROBIAL
RESISTANCE
AND
2
PATHOGENICITY OF SALMONELLA ENTERICA SEROVARS TYPIMURIUM AND
3
ENTERIDITIS IN POULTRY AND POULTRY PRODUCTS
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Sher Bahadar Khan1 , Mumtaz Ali Khan2,3, Irshad Ahmad4, Tayyab ur Rehman4, Shahid
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Ullah5, Rahim Dad6, Asad Sultan7, Atta Muhammad Memon8
6
1
Department of Animal Health, The University of Agriculture, Peshawar, Pakistan
7
2
Civil Veterinary Hospital, SherGarh, Livestock and Dairy Development, Khyber
8
Pakhtunkhwa, Pakistan
9
3
University of Veterinary and Animal Sciences, Lahore, Pakistan
10
4
Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan
11
5
College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen
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518060, Guangdong, China
13
6
14
Tandojam
15
7
Department of Poultry Sciences, The University of Agriculture, Peshawar, Pakistan
16
8
Livestock and Fisheries Department, Government of Sindh, Pakistan
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Corresponding author:
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Sher Bahadar Khan, Department of Animal Health, The University of Agriculture,
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Peshawar, Pakistan
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Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University,
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Email: [email protected]
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Abstract:
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For detection and isolation of Salmonella enterica, 650 meat and tissue samples were processed
29
using Rappaport-Vassiliadis Enrichment broth and Salmonella Chromogenic agar followed by
30
confirmation through specific antisera and polymerase chain reaction (PCR) targeting their
31
Specific Serovar Genomic Regions (SSGRS). Isolates were tested for 15 antibiotics (CRO, AMX,
32
GEN, STR, TET, CHL, CLR, LVX, OFX, GAT, CIP, SXT, AMP, LIN and AZM) according to
33
the disc diffusion method and antimicrobial resistant genes (tet(A), tet(B), tet(C), strA/strB,
34
aadA, aac(3)IV), aadB, sul1, sul2 and sul3, blaCMY-2, blaTEM and blaSHV) using PCR. The
35
overall prevalence of Salmonella enterica was 12%, being higher in markets (15%) as compared
36
to poultry farms(37.2%). The MPN of all positive meat and tissue samples was found 3.6
37
MPN/gram (0.17-18). A total of 234 isolates were obtained, serovar Typimurium (139) and
38
Enteridits (95) were the most prevalent. Antimicrobial resistance patterns were different in
39
different serovars according to origin of Salmonella isolates. The overall isolates were highly
40
resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%, 174/234),
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TET (64.5%, 151/234) and STR (64.5%, 151/234). Overall, the most common ARG was
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blaTEM (76%, 178/234 ), followed by blaSHV (71.7 %, 168/234), tet(A)(64%, 151/234) and
43
tet(B) (64%, 150/234), while the least ARG was aadB ( 7.2%, 17/234). Both Typimurium and
44
Enteridits were tested in the Balb/C mice for pathogenicity. Both Typimurium and Enteridits were found
45
to cause successful colonization, 100% morbidity but Enteriditis were found to cause 33% mortality.
46
Key words: Antibiotic resistance; antibiotic resistant genes; Salmonella; Serovar; Poultry;
47
Poultry products; pathogenecity
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Introduction
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Globally, Salmonella is one of the leading causative agent of food-borne bacterial zoonosis
51
(Daust and Maurer, 2007, Miranda et al., 2009). Salmonella has been estimated as a causative
52
agent in about 70-80% of food borne bacterial outbreaks (Wang et al., 2007). It is estimated that
53
Salmonella species are causing 1.4 million food-borne illness, 15,000 hospitalizations and 400
54
deaths annually in the United States (Votesche et al., 2004, CDC, 2007). Salmonella genus
55
consists of two species, Salmonella enterica and Salmonella bongori. More than 2500
56
serovars of genus Salmonella have been reported (Popoff, 2001, Herikstad et al., 2002). A wide
57
variety of warm blood animals is hosted by Salmonella enterica subsp. 1, which consists of
58
almost 1500 serovars (Brenner et al., 2000, Grimont et al., 2000). A small proportion of
59
Salmonella serovars are host specific, such as serovar Typhi, Cholerasuis, Dublin and
60
Gallinarium are specific to human, swine, cattle and chicken, respectively (Kingsleyand and
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Baumler, 2000 and Rabsch et al., 2002, Wray et al., 2000). These serovars pose a potential threat
62
to human health as food borne pathogens in contaminated meat and other food products
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(Bangtrakulnonth et al., 2004, Gebreyes et al., 2005, Foley and Lynne, 2008). An increase in the
64
antimicrobial resistance in bacterial pathogens including Salmonella is a worldwide public health
65
concern which is mainly associated with frequent use of antibiotics in production animals
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(Arslan et al., 2010, Alvarez-Fermandez et al., 2012, Chen et al., 2004). And then their
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subsequent transmission to human through animal food and food prodcuts (Foley and Lynne,
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2008 and EFSA, 2013). There are a variety of mechanisms through which these pathogens get
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resistance and among them ARGs is the most important (Aarestrup et al., 2003). This study is
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therefore carried out to find out the prevailing situation about Salmonella enterica subspecies
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enterica, its quantification, different serovars distribution, antibiotic resistance and antibiotic
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resistance genes in poultry and poultry products in north west of Pakistan.
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Materials and methods
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Sample collection
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A total of 650 samples were used in the study. From the poultry production system we collected
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250 cloacal swabs from 4 weeks old clinically healthy poultry birds from different herds of five
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intensive poultry farms (50 sample/farm). From the supply chain, 400 samples were purchased
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from different markets (100 each meat, liver, intestine and kidney) in both summer and winter.
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Samples were collected in sterile plastic bags and subjected for further process immediately upon
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arrival to the laboratory.
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Processing of samples
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Upon arrival to the laboratory, cloacal swabs were immediately washed with 0.5 ml PBS, and 0.1
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ml of them was inoculated into 9 ml Brain heart infusion broth (BHIB; BD Difco, USA) at 37°C
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for 24 hours. Out of 100 g meat and tissue samples purchased from the different markets, 50 g
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was cut into small pieces and mixed with 450 ml BHIB (BD Difco, USA) in homogenizer for
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two min. One ml of homogenized sample was mixed with 9 ml of BHIB and incubate at 37°C for
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24 h for further bacterial isolation. Sample homogenate was used for detection, MPN-PCR and
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isolation of Salmonella enterica subsp.I as described below.
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Serotyping using specific antisera
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Salmonellae are flagellated bacillia possessing three major antigens, H or flageller antigen, O or
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somatic antigen and Vi or capsular antigen. Specific antisera were used for the serotyping of
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Salmonella serovars.
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MPN series for enumeration of Salmonella
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Salmonella enterica subsp. I in sample homogenate was enumerated using a three-tube MPN as
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follows. MPN is an indirect means to calculate the probable number of certain number of
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microbes in food or water, particularly when the concentration of the microbe is relative low. For
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each sample homogenate, a tenfold serial dilution series was prepared using Butterfield's
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buffered phophate diluent (BBPD). Then 1ml of each original homogenate was transferred to 9
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ml BHIB in each of the three MPN tubes. Inoculated tubes and uninoculated control tube were
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incubated at 37°C for 24 h. After incubation, 1 ml of each MPN tube was processed for total
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DNA extraction using E.Z.Nce.A bacterial DNA kit (Omega Bio-Tek, USA).
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PCR targeting Salmonella specific invA gene
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The total DNA was first tested for Salmonella specific invA gene using the specific primers as
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described by Akiba et al., 2011. The details of the primers are given in Table 1. PCR
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amplifications were performed using 2 × Es Taq PCR master mix providing a concentration of 3
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mM MgCl2, Taq DNA polymerase, 2 × Es Taq PCR buffer and 400 µM dNTP mix (CWBIO,
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China), 200 ng template DNA and 10 µM of each primer in a total reaction volume of 25 µl with
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cycling condition including an initial incubation at 94 °C for 2 min, followed by 35 cycles at
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98°C for 10 s, 60°C for 30 s and 68°C for 30 s. PCR products were run on 2% agarose E-gels
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pre-stained with Gel red (Invitrogen A/S, Taastrup, Denmark) and visualized by UV-lighting.
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Bacterial isolation and DNA extraction
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The positive samples were processed for bacterial isolation. From the original homogenate stored
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at -20 °C, 0.1ml was transferred to 10 ml of Rappaport-Vassiliadis Enrichment both (RV, Oxoid,
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UK) and incubated at 37°C for 24 h. Then a loop full from Rappaport-Vassiliadis Enrichment
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broth into Salmonella Chromogenic agar plate (Hopebio, Qingdao Hope Biotechnology, China)
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was inoculated followed by incubation at 37°C for 24 h. Typical purple color colonies (three
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colony/sample) were selected for further analysis. Salmonella was confirmed using standard
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bacteriological biochemical tests using an API 20E system (bioMerieux, France). Genomic DNA
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was extracted from the Salmonella isolates using E.Z.Nce.A bacterial DNA kit (Omega Bio-Tek,
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USA). The genomic DNA was tested for specific invA gene and seven serovars targeting their
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three specific serovar genomic regions (SSGRS) for each serovar using the specific primers as
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described by Akiba et al., 2011. The details of the SSGRS and primers are given in Table 1. PCR
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amplifications were performed using 2 × Es Taq PCR master mix and PCR conditions as
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described above.
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Antimicrobial susceptibility testing (AST)
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The antimicrobial susceptibility of the Salmonella isolates were determined for 15 antimicrobials
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according to the disc diffusion method using Muller-Hinton agar (MHA, Qingdao hopebio
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technology Co., China). Interpretation of the results followed the recommendations of the
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Clinical and Laboratory Standards Institute (CLSI, 2016), the European Committee on
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Antimicrobial Susceptibility Testing (EUCAST) (Galni et al., 2008). AST was performed for the
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following antimicrobial agents (antimicrobial abbreviations and breakpoints are shown in
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parenthesis): Ceftriaxone (CRO, 30 µg), Amoxicillin (AMX, 20 µg), Gentamycin (GEN, 10 µg),
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Strptomycin (STR, 10 µg), Tetracycline (TET, 30 µg), Chloramphenicol (CHL, 30 µg),
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Clarithromycin (CLR, 15 µg), Levofloxacin (LVX, 5 µg), Ofloxacin (OFX, 5 µg), Gatifloxacin
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(GAT, 5ug), Ciprofloxacin (CIP, 5ug), Suphamethoxazole+Trimethpram (SXT, 25 µg),
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Ampicillin (AMP, 10 µg), Lincomycin(LIN, 2 µg) and Azithromycin (AZM, 15 µg). Isolates
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showing resistance to at least three antimicrobial agents belonging to different antimicrobial
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classes were considered multidrug resistance (MDR) strains.
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Detection of antibiotic resistance genes (ARGs)
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A set of multiplex PCRs was used for identifying major resistance genes following the procedure
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described by Kozak et al. (2009). The major genes conferring resistance for tetracycline [tet(A),
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tet(B), tet(C)], streptomycin (strA/strB, aadA and (aac(3)IV), gentamycin (aac(3)IV, aadB),
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sulfonamides (sul1, sul2 and sul3), and b-lactamases (blaCMY-2, blaTEM, blaSHV) were
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targeted. Primers and multiplex PCRs conditions used for detection of antibiotic resistance genes
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are given in the Table 2.
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Pathogenicity testing of Salmonella serovars using Balb/C mice
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For pathogenicity testing of Salmonella Typimurium and Enteriditis strains isolated from poultry
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and poultry products, 5-6 weeks old Balb/c female mice were used. Streptomycin treated mouse
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model was used in this study. In this model, mice are provided with streptomycin sulphate in
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their drinking water as a means to reduce their normal facultative flora so as to decrease bacterial
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competition for the infecting O157:H7 strain. Both salmonella Typimurium and Enteridits were
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tested. A group of 6 mice were used for each strain. The mice were provided with streptomycin
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sulphate 5 g/L in drinking water and were kept off fed for 12 hours. After 12 hours, mice were
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infected with 109 CFU of both Typimurium and Enteridits in 20% sucrose solution intragastrically
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(I/G) (Zhihong Ren et al., 2009). Colonization, morbidity including typical signs and symptoms,
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and mortality were noted.
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Histopathology
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The intestine and kidney were removed surgically from the dead mice/or and immediately after
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the mice were killed. Both the intestine and kidney were examined for pathological changes. The
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tissues were fixed in 10% formalin and were submitted to histopathology Lab. for further
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histopathological process.
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Results
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Prevalence and isolation of Salmonella enterica subsp.1 serovars
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Among the 650 samples, 78 samples (12%) were found positive for Salmonella using specific
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antisera and PCR targeting invA gene as shown in Table 3. Out of 250 cloacal swabs from five
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different farms, 18 (7.2%) swabs were found positive. Out of 400 tissue samples from different
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markets, 60 (15%) were found positive. The highest prevalence of Salmonella was found in
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intestine samples in winter (30/50, 60%) followed by meat in winter (15/50, 30%), meat in
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summer (10/50, 20%) and liver in winter (5/50, 10%). The MPN of all positive meat and tissue
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samples was found 3.6MPN/gram (0.17-18).
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Out of 650 samples processed for Salmonella isolation, 78 were found positive. A total of 234
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isolates(3 colony/sample) were obtained which were confirmed through specific antisera. PCR
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for specific invA gene and SSGRS were conducted as shown in Table 3. Out of 234 isolates, the
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most prevalent serovar was Typimurium (139) followed by Enteriditis (95). Except from meat
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samples in summer, it was observed that Typimurium can be easily isolated from all tissue
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samples.
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Antibiotic resistance in Salmonella serovars
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Out of 234 isolates, the most prevalent serovars was Typimurium (139) followed by Enteriditis
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(95). Antibiotic resistance was found different in both serovars (Figure 1). The overall isolates
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were highly resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%,
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174/234), TET (64.5%, 151/234) and STR (64.5%, 151/234). Resistance for GAT (8.9%, 21/234)
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and CRO (8.5%, 20/234) was the lowest in all the isolates. Typimurium was highly resistant for
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AMX (94.9%, 132/139) followed by LIN (93.5%, 130/139), AMP (92%, 128/139), TET and
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STR (84.1%, 117/139, 83.4, 116/139), but less resistant for CRO (6.4%, 9/139) and GAT (5.7%,
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8/139). Enteriditis was highly resistant for LIN (92.6%, 88/95) followed by AMX (74.6%,
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55/95), AMP (48.4%, 46/95), STR and SXT (37.8%, 36/95), but less resistant for GAT (13.6%,
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13/95) and CRO (11.5%, 11/95). Interestingly all the serovars possess highest resistance for LIN
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in contrast to other antibiotics. Multiple antibiotic resistance was found in different serovars.
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Prevalence of ARGs in Salmonella isolates along the PSCP
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The prevalence of ARGs in Salmonella isolates varied in different serovars which is
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consistent with the phenotypic data. Overall, the most common AR G was blaTEM (76%,
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178/234 ), followed by blaSHV (71.7 %, 168/234), tet(A)(64%, 151/234) and tet(B) (64%,
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150/234), while the least ARG was aadB ( 7.2%, 17/234) (Figure 2).
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Typimurium was found to possess higher blaTEM (94.9%, 132/139) followed by blaSHV (92%,
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128/139), tet(A) (84.1%, 117/139), tet(B) (84%,116/139) and strA/strB (82.7%, 115/139) as
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compared to other ARGs. While Enteriditis was found higher in blaTEM (48.4%, 46/95)
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followed by blaSHV (42.1%, 40/95), strA/B (37.8%, 36/95) tet(A) and tet(B) (35.7%, 34/95), as
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compared to other ARGs. Interestingly blaTEM was found higher in all serovars as compared to
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other ARGs (Figure 2).
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Pathogenicity testing
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Both Typimurium and Enteridits were tested in the Balb/C mice for pathogenicity. Both Typimurium and
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Enteridits were found to cause successful colonization, 100% morbidity but Enteriditis were found to
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cause 33% mortality. Typical clinical signs and symptoms as in appetence, diarrhea, hunched posture,
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ruffled feathers, tremor and lethargy were observed in the infected mice. Mortality in mice started 3 days
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post administration of serovars. Extensive necropsy (liver, heart, intestine, caecum, large intestine, large
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intestine and kidney) of the dead and infected mice revealed that small intestine were found to
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demonstrate the pathology of any kind. Histopathology of the intestine revealed changes in intestinal
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villi, epithelial integrity, and local visible accumulation of inflammatory cells in the lamina
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propria, mostly lymphocytes, mucosa and sub mucosa structure disappeared, visible necrosis of
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intestinal epithelial cells and cell debris shed full of the intestine as shown in Figure 3 and 4.
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Intestinal and kidney's sections of controlled group revealed no histopathological changes.
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Discussion
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The overall prevalence of Salmonella was 12%, being higher in markets as compared to
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poultry farms. Previous studies have shown different results of salmonella prevalence in
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different tissue samples such as 0-17.5% in tonsil swabs (Piras et al., 2011,) 12.2-16.4% in
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intestine samples (Hernandez et al., 2013), 9.3-12.5% in liver (Swanenburg et al., 2001) and
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Virira et al., 2011) and 27- 63% in kidneys (Arguello et al., 2012). China have also reported
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different results such as 20% in northen region （Yan et al., 2010）, 31% in Shanxi (Yang et al.,
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2010) and 26% in Hebei province (Jiang et al., 2006), while a Canadian study have reported 2%
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prevalence (Aslam et al., 2012). All these studies indicate that Salmonella is widely distributed
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in retail meat all over the world which increase salmonellosis. The inconsistency of our results
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with the previous studies may be due to several factors such as difference in country and origin,
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sampling season, slaughter house sanitation and isolation methods. Our MPN results are in close
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agreement with the previous studies (Bornadis et al., 2003).We compare the specificity of PCR
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method targeting SSGRS of different serovars as derscribed by Akiba et al., 2011 with specific
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antisera method.
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Here we report that serovar Typimurium and Enteirdits which is specially adopted to
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human and poultry, respectively have been isolated from cloacal swabs and tissue samples of
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poultry. Previous studies have shown that Typimurium, Enteriditis, Shurba, Djuju and Derby
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are the most common serovars worldwide (Bolton et al., 2013 and Yang et al., 2010). Serovar
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dublin from cattle was reported to have high resistance for Enrofloxacin followed by Ceftiofur
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(21.82% ) and SXT (20.9%) (Berge et al., 2008), while isolates from human was found to have
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resistance for streptomycin, sulphamethoxazole, tetracyclin and trimethoprim (43%) (Fashae et
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al., 2010). Similarly isolates from bulk tanks and milk filters have resistance for different
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antibiotics which is not consistent with the present study (Van Kessel et al., 2013). On the other
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hand, Typimurium isolates from pork have shown highest resistance for tetracycline,
238
sulphamethxazole (90.9%)
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trimethoprim (72.7%) (Prendergast et al., 2009). Our results of antimicrobial resistance are not
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in consistent with the previous studies as antibiotic resistance may vary from region to region,
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followed
by streptomycin,
choramphenicol (81.1%)
and
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source of isolation, different antibiotics and other factors (Pan et al., 2010). Analyzing antibiotic resistance genes in Salmonella isolates was one of the aspect of this
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study. The b-lactamase genes are among the antibiotic resistance genes important from human
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health perspective. In this study blaTEM was the most common genes found in isolates resistant
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to AMP, AMX and CRO while the tet (A) and tet (B) were found to be more associated with
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tetracyclin resistance which is in consistent with the previous studies (Chuanchen et al., 2009
247
and Aslam et al., 2012 ). Similarly serovars resistant to SXT were associated with sul1, sul2 and
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sul3 genes (Chuanchun et al., 2009), while those resistant to STR were found to be more
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associated with strA/strB gene as compared to aadA and aac(3)IV which is also in consistent
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with the previous studies (Aslam et al., 2012).
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The high prevalence of blaTEM genes followed by blaSHV, tet(A), tet(B) in Salmonella isolates
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may be due to common use of AMP, AMX, STR and TET during animal production for
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controlling bacterial infections and promotion of growth (Aslam et al., 2012). Presence of these
254
genes on genetic mobile elements such as transposons and plasmids can facilitate their transfer
255
(Schwarz et al., 2006). Our results of ARGs prevalence in different serovars associated with
256
different antibiotics are not in consistent with the previous studies as different studies have
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reported different ARGs prevalence associated with different antibiotics (Cui et al., 2009, Li et
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al., 2007, 2013 ).
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Conflict of interest
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All the authors declare no conflict of interest
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Acknowledgment
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The study was supported by Department of Animal Health, The University of Agriculture,
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Peshawar, Livestock and Dairy development Department Khyber Pakhtunkhwa, Veterinary
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Research Institute, Peshawar, Pakistan.
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388
34. Popoff, M.Y., 2001. Antigenic Formula of the Salmonella Serovars, Eighth edition.
390 391 392 393 394
EP
WHO Colloborating centre for Reference on Salmonella, Paris, France.
AC C
389
TE D
382
35. Prendergast, D.M., Duggan, S.J., Gonzales-Barron, U., Fanning, S., Butler, F., Cormican, M., Duffy, G., 2009. Prevalence, numbers and characteristics of Salmonella spp. on Irish retail pork. International Journal of Food Microbiology, 131:233-239. 36. Rabsch, W., Andrews, H.L., Kinsley, R.A., Prager, R., Tschape, H., Adams, L.G.,
ACCEPTED MANUSCRIPT
395
Baumler, A. J., 2002. Salmonella enterica Typimurium and its host-adapted variants.
396
Infect. Immun. 70, 2249-2255. 37. Schwarz, S., Cloeckaert, A., Roberts, M.C., 2006. Mechanisms and spread of
398
bacterial resistance to antimicrobial agents. In: Aarestrup F.M. (Ed.), Antimicrobial
399
Resistance in Bacteria of Animal Origin. ASM Press, Washington, D. C, pp.73e98.
RI PT
397
38. Swanenburg, M., Berend, B.R., Urlings, H.A.P., Snijders, J.M.A., van Knapen, F.,
401
2001. Epideiological investigation into the sources of Salmonella contamination of
402
pork. Berliner und Munchenrer Tierarztliche Wochenschrift 114, 356-359.
SC
400
39. Van Kessel, J.S., Sonnier, J., Zhao, S., Karns, J.S., 2013. Antimicrobial resistance of
404
Salmonella enterica isolates from bulk tank milk and milk filters in the United
405
States. Journal of Food Protection, 76(1): 18-25.
M AN U
403
40. Vieira, M.J., Oliveira, M., Fontes, M.C., Themudo, P., Semedo-Lemsaddek, T., 2011.
407
Salmonella Sp. in edible offal (liver and tongue) from pigs slaughtered for
408
consumption. Proceedings of safe pork 2011, pp.202-205.
TE D
406
41. Voetsch, A.C., Van Gilder, T. J., Angulo, F. J., et al., 2004. "FoodNet estimate of
410
the burden of illness caused by nontyphoidal Salmonella infections in the united
411
States," Clinical infections and Disease, Vol.38, suppllement 3, pp.127-134.
413 414 415 416 417
42. Wang, J., Zheng R., Wang, J., 2007. Risk assessment of Salmonella in animal
AC C
412
EP
409
derived food. Chinese journal of animal quartine 24 (4), 23-25.
43. Wray, C., Davies, R.H., 2000. Salmonella infection in Cattle. In: Wray, C., Wray, A. (Eds.), Salmonella in Domestic Animals. CABI Publishing, Wallingford, UK, pp. 169-190. 44. Yan, H., Li, L., Alam, M. J.,Shinoda, S., Miyoshi, S., Shi, L., 2010. Prevalence and
ACCEPTED MANUSCRIPT
418
antimicrobial resistance of Salmonella in retail foods in
419
International Journal of Food Microbiology, 143:230-234.
northern China.
45. Yang, B., Qu, D., Zhang, X., Shen, J., Cui, S., Shi, Y., Xi, M., Sheng, M., Zhi, S.,
421
Meng, J., 2010. Prevalence and characterization of Salmonella serovars in retail
422
meats of marketplace in Shaanxi, China. International Journal of Food Microbiology,
423
141:63-72.
RI PT
420
46. Zhihong Ren, Raina Gay, Adam Thomas, Munkyong Pae, Dayong Wu, Lauren
425
Logsdon, Joan Mecsas, and Simin Nikbin Meydani, 2009. Effect of age on
426
susceptibility to Salmonella Typhimurium infection in C57BL/6 mice. J Med
427
Microbiol. 2009 Dec; 58(Pt 12): 1559–1567.
M AN U
SC
424
428
432
433
434
435
436
437
438
EP
431
AC C
430
TE D
429
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
439
ACCEPTED MANUSCRIPT
Table 1. Target genomic regions and primer sequences of the multiplex PCR assay
TSR2
TSR3
CSR1
Primer
Sequence
STM2896
InvA
invAF
5'-AAACCTAAAACCAGCAAAGG
605
invAR
5'-TGTACCGTGGCATGTCTGAG
TMP1F
5'-ATGCGGGTATGACAAACCCT
TMP1R
5'-TTAGCCCCATTTGGACCTTT
TMP2F
5'-CAGACCAGGTAAGTTTCTGG
TMP2R
5'-CGCATATTTGGTGCAGAAAT
TMP3F
5'-TTTACCTCAATGGCGGAACC
TMP3R
5-CCCAAAAGCTGGGTTAGCAA
CMP1F
5'-AGATATGCGGCTTGCTGACT
STM0292
STM2235
STM4493
SCH_0971
putativeRHS-family protein
Putative phage protein
Putative cytoplasmic protein
hypothetical protein
CMP1R
CSR2
SCH_2145
hypothetical protein
SCH_4349
hypothetical protein
ISR1
3113085– 114152
–d
ISR2
1067175– 068152
4621947– 622627
HSR1
1067175– 068152
2541120– 541716
–d
–d
AC C
HSR2
–d
EP
ISR3
–d
HSR3
ESR1
ESR2
ESR3
4675756– 676622
SEN0910
SEN1006
SEN2420
–d
hypothetical phage protein (pseudogene)
hypothetical protein
putative exported protein
94
196
303
100
5'-AGCGTGGCTCGAAACAGTAT
CMP2F
5'-GGCGAAAGAGCTTAACGTGA
CMP2R
5'-TTACCATCGGGACCAAATGT
CMP3F
5'-TCGGGATCGTCCTCTATACG
CMP3R
5'-CCCAAAAGCTGGGTTAGCAA
IMP1F
5'-GGTCATTGTCGGAAACCTGC
IMP1R
5'-ACATTCCCCCTTCCACTGCC
TE D
CSR3
RI PT
TSR1
Product
SC
invAc
Representative geneb
PCR product size (bp)
M AN U
Region
a
IMP2F
5'-CGCGAAGAAGTGCATAAACC
IMP2R
5'-CGCGAAGAAGTGCATAAACC
IMP3F
5'-ACCTACTACTATCCCTGATG
IMP3R
5'-GCGAATTTTGCTACTTGAAG
HMP1F
5'-ACTTCATGCGCAGACTTGCC
HMP1R
5'-CAAGAATCGGGAAATTTTGA
HMP2F
5'-TACCGCCCCATTTGATATCT
HMP2R
5'-CCCTTGCGCAACATAAAACT
HMP3F
5'-GGATCTCAACAAAATGAGGT
HMP3R
5'ATGCATCACTGCATCTTCGT
EMP1F
5'-AATACAGCCTCAACCAGCTA
EMP1R
5'-ATTGGTTCACCCGTTGCAAT
EMP2F
5'-AGATAAGCCCTCCCTGCTTA
EMP2R
5'-CCCTCCTTTCACTGCAAGTC
EMP3F
5'-CAAAAGCGACAAATAATCTG
198
305
95
198
304
105
199
303
101
203
299
ACCEPTED MANUSCRIPT
conserved hypothetical protein
DSR2
SeD_A1104
conserved hypothetical protein
DSR3
SeD_A2276
lysozyme
GSR1
SG0266
conserved hypothetical protein
GSR2
SG3181
conserved hypothetical protein
hypothetical protein
5'-ATCGGTGCTGGGTAATTTTG
DMP1R
5'-AGGAACGAGAGAAACTGCTT
DMP2F
5'-ACGCGAAATCTGATGGTCTT
DMP2R
5'-GCCCACCAGTTGTGAAAGGC
DMP3F
5'-ATCACCCTCGCAAACTTGTC
DMP3R
5'-TCGGGCAATCAGGTCGCCGA
GMP1F
5'-CCGCACAACACATCAGAAAG
GMP1R
5'-AGCTGCCAGAGGTTACGCTG
GMP2F
5'-CTCCTGATCATGGCGCTACT
GMP2R
5'-GTGAGGATTTTGTCGTAGCA
GMP3F
5'-GCTAGGGGTTTCCTCCACTC
GMP3R
5'-CGATTTTGGAGCGGATAACC
M AN U
GSR3
SG1033
DMP1F
105
203
RI PT
SeD_A1226
5'-TTTCTCCGCCTGTTTTCGTT
SC
DSR1
EMP3R
296
97
206
301
a TSR, Typhimurium-specific (genomic) region; CSR, Choleraesuis-specific (genomic) region; ISR, Infantisspecific (genomic) region; HSR, Hadar-specific (genomic) region; ESR, Enteritidis-specific (genomic) region;
AC C
EP
TE D
DSR, Dublin-specific (genomic) region; GSR, Gallinarum-specific (genomic) region.
ACCEPTED MANUSCRIPT
Table 2. Primers and conditions used for antibiotic resistance genes Gene
Primer
Sequence
Final Conc of primer (M)
Annealing temp(°C)
Product size (bp)
1
sul1
sul1-Fb
CGGCGTGGGCTACCTGAACG
0.2
66
433
sul1-Bb
GCCGATCGCGTGAAGTTCCG
0.2
sulII-Lc
CGGCATCGTCAACATAACCT
0.3
sulII-Rc
TGTGCGGATGAAGTCAGCTC
0.3
sul3-GKa-Fd
CAACGGAAGTGGGCGTTGTGGA
0.2
sul3-GKa-Rd
GCTGCACCAATTCGCTGAACG
0.2
TetA-Lc
GGCGGTCTTCTTCATCATGC
0.1
TetA-Rc
CGGCAGGCAGAGCAAGTAGA
TetBGKF2m
CGCCCAGTGCTGTTGTTGTC
TetBGKR2m
CGCGTTGAGAAGCTGAGGTG
0.2
TetC-Lc
GCTGTAGGCATAGGCTTGGT
0.5
TetC-Rc
GCCGGAAGCGAGAAGAATCA
0.5
4Fe
GTGGATGGCGGCCTGAAGCC
0.1
4Re
AATGCCCAGTCGGCAGCG
0.1
strA-Ff
ATGGTGGACCCTAAAACTCT
0.4
2
2
3
3
tet (A)
tet (B)
tet (C)
aadA
strA/strB
strB-Rf 3
aac(3)IV
4
aadB
TGCTGGTCCACAGCTCCTTC
0.2
CGGATGCAGGAAGATCAA
0.2
GAGGAGTTGGACTATGGATT
0.2
CTTCATCGGCATAGTAAAAG
0.2
GKTEMFd
TTAACTGGCGAACTACTTAC
0.2
GKTEMRd
GTCTATTTCGTTCATCCATA
0.2
SHV-Fj
AGGATTGACTGCCTTTTTG
0.4
SHV-Rj
ATTTGCTGATTTCGCTCG
0.4
CMYFd
GACAGCCTCTTTCTCCACA
0.2
CMYRd
GGACACGAAGGCTACGTA
0.2
aadB-Li
5
blaSHV
AC C
5
blaTM
EP
aadB-Ri
5
blaCMY2
0.2
0.4
aac4-Rg
721
66
244
63
502
0.1
CGTCTAGGATCGAGACAAAG
aac4-Lg
66
SC
2
Sul3
M AN U
1
Sul2
TE D
1
RI PT
PCR
63
173
63
888
63
525
63
893
63
653
55
208
55
247
55
393
55
1000
ACCEPTED MANUSCRIPT
Table 3. Prevalence and isolation of salmonella enterica serovars in poultry Positive No. of Samples
Typimurium Enteriditis
Cloacal swabs
250
18 (7.2%)
34
20
IntestineS
50
-
-
-
IntestineW
50
30 (60%)
63
Liver S
50
-
-
Liver W
50
5 (10%)
9
Kidney S
50
-
-
KidneyW
50
-
-
MeatS
50
10 (20%)
5
MeatW
50
15 (30%)
28
17
Total
650
78 (12%)
139
95
EP AC C
27
-
SC
6
M AN U
summer, Wwinter, - negative
TE D
S
RI PT
Type of sample
-
-
25
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 1. Antibiotic resistance in Salmonella isolates.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 2. Antibiotic resistant genes in Salmonella isolates.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
Figure 3. Histopathological changes in the intestine.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
Figure 4. Histopathological changes in the intestine.
ACCEPTED MANUSCRIPT
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SC M AN U TE D
•
EP
•
The overall prevalence of Salmonella enterica was 12%, being higher in markets (15%) as compared to poultry farms(37.2%). The MPN of all positive meat and tissue samples was found 3.6 MPN/gram (0.17-18). Typimurium (139) and Enteridits (95) were the most prevalent. The overall isolates were highly resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%, 174/234), TET (64.5%, 151/234) and STR (64.5%, 151/234). Overall, the most common AR G was blaTEM (76%, 178/234 ), followed by blaSHV (71.7 %, 168/234), tet(A)(64%, 151/234) and tet(B) (64%, 150/234), while the least ARG was aadB ( 7.2%, 17/234).
AC C
•