Molecular detection of biofilm, virulence and antimicrobial resistance associated genes of Salmonella serovars isolated from pig and chicken of Mizoram, India

Pork is currently considered as the most frequently consumed meat in the world followed by chicken, beef and mutton (USDA Foreign Agricultural Service, 2018). It is predicted that chicken will account for the largest share of meat consumption within 2025. Modernization of livestock farms and globalization of bird breeding trade also helps in transboundary spreading of food-borne bacteria such as Salmonella. Salmonellae are Gram-negative zoonotic bacteria and cause substantial morbidity in livestock and poultry and significant economic impact. The pigs and poultry can harbour several non-host adapted serovars of Salmonalla, which can be transmitted to human through food chain. A global estimate revealed 93.8 million cases of salmonellosis each year, of which 80 million cases are considered as food-borne infections.
               
Acquisition of antimicrobial resistance genes and biofilm production capacity makes the zoonotic transmission of Salmonella from pigs and poultry more complicated. Production of extended-spectrum β-lactamase (ESBL) enzymes is one of the major mechanisms instrumental in Enterobacteriaceae family for their resistance, which is detected against the penicillin, cephalosporins and monobactams (except cephamycins and carbapenems). Among the three classical ESBLs (i.e. TEM, SHV and CTX-M), CTX-M is observed as the most prevalent type throughout the world (Carattoli, 2013). Currently, multidrug resistant (MDR) non-typhoidal Salmonella (NTS) has raised major concern with an estimated 100,000 annual domestic cases and 40 deaths in United States alone (CDC, 2013). In Europe and North America, outbreaks of MDR Salmonella occurred in human due to consumption of pork or beef products (Mindlin et al., 2013; Laufer et al., 2015). In African countries, incidence of NTS infection especially with S. Typhimurium or S. Enteritidis has increased in recent times with the acquisition of MDR organisms (García et al., 2016). MDR Salmonella carrying bla_TEM-1 and bla_CMY-2 is already reported from diarrhoeic piglets of Mizoram, India (Kylla et al., 2018). Salmonellae are capable of forming biofilms in host and on a wide variety of contact surfaces, which contributes to survival in presence of stresses such as desiccation, extreme temperatures, antibiotics and antiseptics (Marin et al., 2009). Curli fimbriae and cellulose are the two main components found in Salmonella biofilms (Steenackers et al., 2012). Additionally, capsular polysaccharide, LPS and secreted protein BapA also contribute to biofilm formation (de Rezende et al., 2005). Further, the relative expression of the biofilm associated genes, such as csgD, csgB, adrA and bapA favors biofilm production by Salmonella (Pande et al., 2016). Moreover, pathogenecity of Salmonella mainly depends on presence of virulence genes, such as sefA (Clothier  et al. 1993), stn (Prager et al., 1995) and invA (Galan et al., 1992), which play significant role in bacterial adhesion and are frequently found in invasive strains of Salmonella.
       
There is paucity of scientific literatures regarding characterization of biofilm producing salmonellae from livestock and birds in India particularly the North-eastern states where consumption of pork and poultry meat is very high. In view of the above facts, the present study was carried out to characterize the salmonellae isolated from pigs and chicken of Mizoram in respect of their biofilm production capacity, antibiotic resistance properties including detection of possible linkage between biofilm producing genes, antibiotic resistance genes and virulence genes. 

Sampling
 
A total of 100 fecal specimens from pigs and 50 cloacal swabs from domestic chickens of Mizoram, India (Table 1) were collected during September 2016 to July 2017. Salmonellae were isolated and identified by primary culture in RV brothsmedium followed by sub-culturing on selective media like Hektoen Enteric agar (HiMedia, India), xylose lysine deoxycholate agar (HiMedia, India) and brilliant green agar (HiMedia, India) followed by microscopy and biochemical properties (Quinn et al., 1994). All the isolates were further identified by BD Phoenix™ automated bacterial identification system (Becton, Dickinson and Company, USA). The genotypic confirmation of the isolates were carried out by 16S rRNA genus specific PCR (Whyte et al., 2002). All the Salmonella isolates were serotyped at Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India. During the whole course of the study, all the isolates were maintained at -80°C in 25% glycerol. 
 

 
Biofilm formation assay
 
Microtiter plate biofilm assay was performed (Vestby et al., 2009) in sterile 96-well flat bottom polystyrene microtiter plates (Nunc, Denmark), filled with 180 μL of 1/20 diluted tryptic soya broth (TSB). The negative control wells (in triplicate) contained 200 μL of broth only. For each test isolate, 20 μL of overnight cultures in TSB broth was dispensed to the wells in triplicate followed by incubation aerobically at 37°C for 72 h. After incubation, the liquid contents of the plates were discarded and the wells were thoroughly washed thrice with PBS (pH 7.2). The adherent bacterial cells were stained with 200 μL of 0.5% (w/v) crystal violet stain per well for 10 min and washed thrice with 300 μL/well of sterile distilled water. The plates were air dried followed by addition of 250 μL of 33% glacial acetic acid in each well. The optical density (O.D.) of each well was measured at 590 nm using automated plate reader (BioRad, USA). Bacterial biofilms were classified based on the method of Stepanoviæet_al(2004).
 
Antimicrobial sensitivity pattern of Salmonella isolates
 
All the biofilm producing Salmonella isolates were subjected to in vitro antimicrobial sensitivity test by disc diffusion method against 11 commonly used antibiotics (gentamicin, ampicillin, amikacin, ciprofloxacin, norfloxacin, cefotaxime, ceftazidime, ceftriaxone, cefixime, cotrimoxazole and tetracycline) as per the guideline of CLSI (2014). The multiple antibiotic resistance (MAR) index was calculated as “a/b”, where “a” the number of antibiotics to which a particular isolate was resistant and “b” the total number of antibiotics tested (Krumperman, 1983).
 
PCR based screening of biofilm producing Salmonella isolates for biofilm associated genes, virulence genes and antibiotic resistance genes
 
All the phenotypically confirmed biofilm producing Salmonella isolates were subjected for detection of selected biofilm producing genes, viz. csgA (Akbari et al., 2015), csgD (Akbari et al., 2015) and adrA (Grantcharova et al., 2010); virulence genes, viz. invA (Rahn et al., 1992), stn (Murugkar et al., 2003) and sefA (Oliveira et al., 2003); antibiotic resistance genes  viz., bla_TEM (Weill et al., 2004) bla_SHV  (Weill et al., 2004) and bla_CTX-M (Monsteinet_al2007) by PCR using specific oligonucleotide primers and cycle conditions as described in Table 4. In brief, DNA lysate for PCR was prepared by standard boiling and snap chilling method. Amplification was carried out in a Master cycler™ (Eppendorf, Germany) with a reaction mixture of 25µl in a thin walled 0.2 ml PCR tube containing 4 µl of template DNA, 2.5 µl of 10X Taq DNA polymerase buffer [10 mmol l^-1 Tris HCl (pH 9.0), 50 mmol l^-1 KCL], 1.5 µl of 2.5 mmol l^-1 MgCl₂, 2.5 µl of dNTP mixture containing 2.5 mmol l^-1 of each dNTP, 1 µl (10 pmol l^-1) each of forward and reverse primer and 1.0 U of Taq DNA polymerase (Thermo Fisher Scientific, India). Final volume was made with nuclease free water. PCR products were analyzed by gel electrophoresis in 1.0% agarose gel (Amersham Biotech, UK) using 1X Tris-EDTA electrophoresis buffer and stained with ethidium bromide (5 mg ml^-1). A 100-bp and 50-bp DNA ladder (Thermo Fisher Scientific, India) were used as molecular weight marker. The DNA fragments were observed by ultraviolet transilluminator and photographed (Alpha Imager, Germany).
 

 
Statistical analysis
 
Occurrence of biofilm producing Salmonella isolates in pigs and chickens was compared by chi-square test using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA).

The present study was conducted to determine the biofilm producing ability, virulence and antimicrobial resistance of Salmonella isolates recovered from pig and poultry in Mizoram state of India.
 
Isolation of Salmonella
 
A total of 15 Salmonella were recovered and identified, of which 9 and 6 were from pigs and chicken, respectively (Table 1). Among the 15 isolates, 9 (60%) were of serovar Virchow, 5(33.33%) of serovar Typhimurium and remaining one was rough Salmonella strain. Most of the isolates from pig (7 out of 9) belonged to serovar Virchow, whereas serovar Typhimurium were mostly (4 out of 6) recovered from chicken (Table 1). Earlier, Murugkar et al., (2005) reported the recovery of 20.5% salmonellae from human, poultry and animals including pigs in North Eastern India. On contrary to this report, Sanjukta et al., (2016) could detect 7.03% salmonellae from animals of North Eastern India. Kylla et al., (2018) also reported 16.66% prevalence of Salmonella in pigs of Mizoram. Globally, the isolation rate of salmonellae remained in the range of 5.22% to 9% in animals (Sanjukta et al., 2016). Verma and Gupta (1996) also reported maximum prevalence of Salmonella Virchow from animal origin compared to other serotypes in India.
 
Biofilm formation assay
 
A total of 5 (55.55%) Salmonella isolates from pigs were identified as biofilm producer, of which 4 (80%) and 1 (20%) were recorded as weak and moderate biofilm producers, respectively (Table 1). Similarly, a total of 5 (83.33%) Salmonella isolates from chickens were found to be positive for biofilm production, of which 2 (40%), 2 (40%) and 1 (20%) isolates were recorded as weak, moderate and strong biofilm producer, respectively. No significant difference was detected among the number of biofilm producing strains isolated from pigs and chickens. However, S. Typhimurium was found be stronger biofilm producer than S. Virchow. All the 5 isolates of S. Virchow were weak biofilm producers whereas, in case of S. Typhimurium, 1, 3 and 1 isolates were recorded as strong, moderate and weak biofilm producers. Earlier, other workers also reported biofilm production by S. Typhimurium from poultry, poultry houses, raw chicken and chicken products (Nair et al., 2015; Ziech et al., 2016). Only one isolate of S. Typhimurium from pigs was moderate biofilm producer in the present study, which is also in agreement with earlier study (Piras et al., 2015). However, no report could be traced in literature on biofilm production by S. Virchow from pigs. The weak biofilm producing organisms may acquire new genetic traits, plasmids due to over usage of disinfectants, antibiotics or other external selective pressures, which can increase their pathogenic potential (Charlebois et al., 2014).
 
Antimicrobial resistance pattern of Salmonella isolates
 
As depicted in Table 2, majority of Salmonella isolates were resistant against ceftazidime (80%), ceftriaxone (80%), cefixime (70%), cefotaxime (70%), gentamicin (70%), cotrimoxazole (60%) and ampicillin (60%). Isolates of chicken origin were more resistant to commonly used antibiotics compared to pig origin. The multiple antibiotic resistance (MAR) index was ranging from 0.36 to 0.64. The highest MAR index value of 0.64 was found in two chicken S. Typhimurium isolates, which were resistant to 7 antibiotics. Observation from the present study indicated that seven Salmonella isolates were multidrug resistant (resistance to ³ 3 groups of antimicrobials) (Table 2, 3). Earlier, Sanjukta et al., (2016) from North Eastern India and El-Tayeb et al., (2017) from Saudi Arabia also reported salmonellae with high level of resistance against cephalosporin group of antibiotics. The widespread use of cephalosporin might be the primary reason for detection of MDR salmonellae in the food animals. Majority of the biofilm producing salmonellae were MDR type, which is also in corroboration with earlier findings (Barilli et al., 2018), where all the biofilm producing Salmonella isolates of pig origin was multidrug resistant. Ampicillin is no more the drug of choice in porcine or poultry medicine and that might be the major reason for detection of MDR Salmonella isolates susceptible to ampicillin (Sudhanthirakodi, 2016). The MAR index values of all the studied isolates were more than 0.2, which indicates high risk source of contamination, where antibiotics are often used (Paul et al., 1997). (Repetition).
 

 

 
Detection of biofilm associated genes, virulence genes and antibiotic resistance genes in Salmonella isolates
 
All the biofilm producing isolates were positive for adrA gene but not for csgA and csgD genes (Table 4). Similarly, all the isolates under the study were positive for at least two virulence genes (Table 4). All the biofilm producing isolates from pigs and chicken were positive for invA and stn genes and 3 (60%) isolates from chicken origin were positive for sefA gene. All the 5 biofilm producing isolates from pigs were positive for bla_TEM gene and one isolate was positive for bla_CTX-M gene. Among the 5 biofilm producing isolates from chicken, 1 (20%), 2(40%) and 2(40%) were positive for bla_TEM, bla_SHV and bla_CTX-M genes, respectively (Table 4).
 

       
Although, earlier studies revealed the role of both adrA and csgD genes in formation of biofilm by Salmonella isolates (S. Mbandaka and S. Oranienburg) on the egg shell (Pande et al., 2016) but formation of biofilm by Salmonella isolates are possible in absence of both the genes (Van Parys et al., 2010), which justified the detection of biofilm producing salmonellae carrying only adrA gene in the present study. In addition, majority of the biofilm producing Salmonella isolates from pig and chicken harboured bla_TEM gene, which is also a consistent finding compared with earlier reports (El-Sharkawy et al., 2017; Zhao et al., 2017). The bla_SHV is the least encountered gene in enteric bacteria of poultry (broilers) in India (Mahanti et al., 2018). In the present study, only 2 Salmonella   isolates from chicken were found to possess bla_SHV gene. The invA gene encoding protein is a putative inner membrane component of the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion system (TSST). It has been reported that invA is present only in Salmonella and therefore is used as a golden marker in genetic diagnosis of Salmonella (O’Regan et al., 2008). All the Salmonella isolates from pig and poultry under the present study were positive for invA and stn genes.  Detection of invA and stn gene in all the isolates also suggested their potential pathogenic role (Chaudhary et al., 2015: Kylla et al., 2018). The sefA gene is known to be specific to the poultry-associated Salmonella. Present study revealed that 60% Salmonella isolates from poultry were positive and all the Salmonella isolates from pigs were  negative for sefA gene, which is in corroboration with earlier study (El-Sharkawy et al., 2017). sef14 fimbriae consist of a repeating major subunit of the 14.3 kDa protein sefA, encoded by the sefA gene and are required for macrophage uptake and survival in intraperitoneal infections (Edwards et al., 2001).

The present study revealed the occurrence of biofilm producing Salmonella serovars in pigs and chickens in Mizoram, India. Majority of the isolates were capable of producing biofilm and also possessed genes encoding   virulence factors and resistance to commonly used antimicrobial agents. Moreover, the biofilm producing isolates were resistant to commonly used antibiotics, which indicate that antibiotic resistance is still increasing and evolving. Detection of such organisms from food animals and birds may be considered as major public health concern.

The authors are thankful to the Central Agricultural University-Imphal and  the Department of Biotechnology, GOI sponsored project on “Studies on antimicrobial and anti-biofilm activities of medicinal plant extracts from North Eastern India against pathogenic bacteria isolated from pigs, cattle and poultry of NER India and cattle, poultry and ducks of West Bengal” for providing all the support.