SEROTYPING AND ANTIMICROBIAL RESISTANCE OF SALMONELLA ISOLATES FROM FOOD MATRICES AND CLINICAL SPECIMENS FROM LEBANON

Salmonella (spp) is a genus of bacteria responsible for the most frequently reported cases of foodborne illnesses worldwide. In this study, serotyping was done by using highly specific manufactured antisera, to identify circulating Salmonella serovars isolated from food samples and clinical specimens. Antibiotic susceptibility testing was also performed to determine the level of resistance of these serotypes. This was done by using the disk diffusion method by testing 14 clinically important antibiotics on the Salmonella (spp) isolates. A total of 85 Salmonella (spp) strains, preidentified by biochemical testing, were collected over the course of 1 year. Serotyping of the isolates revealed the presence of 14 serotypes in the food samples with S. Infantis and S. Enteritidis, being the most common and 6 serotypes in the clinical samples with S. Enteritidis and S. Typhimurium being the most common. Antimicrobial susceptibility testing results showed resistance of isolates from both sample types to conventional antibiotics like Ampicillin and Piperacillin, and almost total resistance of the food isolates to a broad-spectrum antibiotic Imipenem, belonging to the Carbapenem class. Higher resistance was observed in food samples than in clinical ones. Moreover, 13 and 3 multidrug resistant serotypes of Salmonella (spp) were identified for the first time in Lebanon in the food and clinical samples respectively, with three of them were isolated from imported food samples.


INTRODUCTION
Foodborne bacterial diseases constitute a growing challenge to human health around the world, in both developing and industrialized countries (Sánchezvargas et al., 2011). Case reports of food intoxications and infections caused by these pathogens are increasing as a consequence of changes in the natural environment, human lifestyle and the haphazard use and prescription of antibiotics, causing bacterial resistance and outbreaks (Scallan et al., 2011). Several pathogens like Salmonella, Escherichia coli, Yersinia enterolitica, Staphylococcus aureus, Clostridium botulinum and many more, are causing foodborne outbreaks and becoming economic burdens on many societies, as they associate with new types of food vehicles and develop resistance to common antibiotics (Masterton, 2008;Pires et al., 2014). Of these pathogens, the genus Salmonella and specifically non-typhoidal Salmonella rank second after Norovirus in the top five pathogens contributing to domestically acquired foodborne illnesses published by the CDC (Scallan et al., 2011). The Salmonella genus encompasses many strains and serotypes that cause an array of diseases, ranging from mild gastroenteritis to life threatening major diseases like typhoid fever, caused by Salmonella Typhi and Paratyphi serotypes, especially in the poorer regions of the world and developing countries like Lebanon (Ryan et al., 2017). This genus contains more than 2,500 serotypes, at present, and has a complex nomenclature (Sloan et al., 2017). Hence, serotype identification is of great importance in order to monitor and contain outbreaks associated with salmonellosis cases, and link them with their respective contaminated food sources for the purpose of prioritizing effective food safety interventions. Continuous updates of the identification and tracking of new serovars is of utmost importance for the long term epidemiological surveillance of this ubiquitous pathogen found in the food chain, in order to establish control in the public health sector in Lebanon (Malaeb et al., 2016). Add to that, antibiotic resistance is actually reaching dangerously high levels in the world. New resistance mechanisms are emerging and spreading across the globe, compromising our ability to treat common infectious diseases. For an increasing number of infections, such as tuberculosis, sepsis, pneumonia, gonorrhea and foodborne diseases, treatment becomes more difficult, if not sometimes impossible, due to the loss of effectiveness of antibiotics (World Health Organization, fact sheet). The objective of our study was to identify, by serotyping, the circulating and predominant serovars of Salmonella isolated from clinical and food sources in Lebanon, and to establish the current antibiotic resistance patterns of the identified serotypes of Salmonella

Bacterial isolates Clinical isolates
Over the period of one year (October 2018 -October 2019), 16 clinical isolates already identified as Salmonella . with API 20E, were collected from different hospitals, pertaining from the stools of patients with salmonellosis.

Food isolates
Also during the period from October 2018 to October 2019, a total of 69 strains of Salmonella were collected from different food matrices and identified with API 20E. They were provided by the Lebanese Agricultural Research Institute (LARI) and the Chamber of Commerce, Industry & Agriculture at Tripoli & North Lebanon (CCIAT).

Serotyping
Serotyping was performed by following guides and protocols published by Bio-Rad. (n.d.-b). The detailed flowchart of Salmonella serotyping is represented in figure 1. The isolated Salmonella strains were streaked onto a solid Trypticase Soya Agar (TSA) (VWR International Eurolab, Barcelona, Spain), a non-selective agar medium, to obtain pure colonies. Cultures were incubated at 37°C for 18 hours.
Salmonella is a genus of bacteria responsible for the most frequently reported cases of foodborne illnesses worldwide. In this study, serotyping was done by using highly specific manufactured antisera, to identify circulating Salmonella serovars isolated from food samples and clinical specimens. Antibiotic susceptibility testing was also performed to determine the level of resistance of these serotypes. This was done by using the disk diffusion method by testing 14 clinically important antibiotics on the Salmonella isolates. A total of 85 Salmonella strains, preidentified by biochemical testing, were collected over the course of one year. Serotyping of the isolates revealed the presence of 14 serotypes in the food samples with S. Infantis and S. Enteritidis, being the most common and 6 serotypes in the clinical samples with S. Enteritidis and S. Typhimurium being the most common. Antimicrobial susceptibility testing results showed resistance of isolates from both sample types to conventional antibiotics like ampicillin and piperacillin, and almost total resistance of the food isolates to a broad-spectrum antibiotic imipenem, belonging to the carbapenem class. Higher resistance was observed in food samples than in clinical ones. Moreover, 13 and 3 multidrug resistant serotypes of Salmonella were identified for the first time in Lebanon in the food and clinical samples respectively, with three of them isolated from imported food samples.

Autoagglutination Test
The auto-agglutination test was performed by mixing pure colonies with one drop of physiological water/saline (0.85%). If auto-agglutination was observed, the strain was recorded as being unable to be serotyped.

Testing with Omni-O antiserum
All strains were tested with Salmonella Omni-O antiserum for the presumptive identification of O-agglutinable strains of Salmonella. If agglutination exists, the strain is O-agglutinable and can be further serotyped with specific antisera.

Testing for O Antigens
The tests for O Antigens (polyvalent O sera, monovalent O sera and Anti-Vi serum) and for H antigen phase I and phase II (Polyvalent H sera and monovalent H sera) were done according to the bacterial serotyping guide for Salmonella (Bio-Rad, FSD_14-0699.pdf).

Phase inversion -Sven Gard method
After identification of the first phase, the Sven Gard agar medium [supplied in tubes, 25 mL, Bio-Rad (Marnes-la-Coquette, France)] was melted in a water bath of 95°C. After Cooling to 44°C, one drop of SG sera, containing the agglutinin of the phase already determined, was added. Tubes were then mixed with circular motions, to ensure homogenization of the serum before pouring onto Petri-dishes. After solidification, 3 to 4 colonies from a pure culture of Salmonella were inoculated at the center of the plate and incubated, with the dish cover facing upwards, for 18 hours at 37°C. The H phase II antigen was then identified by slide agglutination, by collecting the culture from the periphery of the invasion zone.

Identification and the Antigenic formula
After the identification of the O, H-phase I and H-phase II antigens, they were placed in the antigenic formula to identify the serotype. This was done by referring to the White-Kauffman-Le Minor scheme (Grimont et al., 2007). The formula is as follows: O antigens in Arabic numeral, Vi (when present), H antigens phase I in lower case letters, H antigens phase II in Arabic numerals (when present). The serotyping steps described previously, are summarized in the flowchart represented in figure 1.

Antibiotic susceptibility testing
Antibiotic resistance testing was performed on the serotyped strains of Salmonella (spp). by using the disk diffusion method (Kirby Bauer method) according to the guidelines of the Clinical & Laboratory Standards Institute (CLSI) (Performance Standards for Antimicrobial Susceptibility Testing, 2017). The susceptibility testing was done using 14 antibiotics (total of seven classes) commonly used for the treatment of infections caused by Enterobacteriaceae (Table 1). Antibiotic disks were purchased from Sigma Aldrich (Darmstadt, Germany). These disks were placed on the surface of an agar plate previously inoculated with the bacterial strain and plates were incubated at 37 ˚C for 18 hours. After the incubation period, zones of inhibition were recorded using a ruler to the nearest millimeter and classified as resistant (R)

Serotyping
Serotyping of the clinical samples showed a high prevalence of S. Enteritidis (56.25%) being the most commonly isolated serovar, Followed by S. Typhimurium (12.50%) being the second most isolated one ( Figure 2). However, the most prevalent serotype in the food samples was S. Infantis (24.63%) followed by S. Enteritidis (18.84%) ( Figure 3). Of all the food origins, three food types are widely consumed by the Lebanese population as traditional cuisine: chicken Tawouk, raw meat and minced meat. S. Enteritidis was mostly isolated from chicken "Tawouk"; a traditional Middle Eastern food consisting of chicken cubes marinated in tomato paste and different spices, and minced meat. As for S. Infantis, this serovar was isolated from chicken dishes that can contain eggs in their breading, and ham in their filling; hence, source attribution for S. Infantis can be uncertain since this serotype can be isolated from chicken, eggs and swine. All the other serotypes in our study are mainly associated with their common reservoirs. The antigenic formula of the identified Salmonella serovars from clinical and food samples are represented in table 3.  (2017), who performed the serotyping of Salmonella spp. isolates, collected between 2011 and 2014 in Lebanon. Their study showed the prevalence of S. Enteritidis among both human and food samples, with S. Typhimurium being the second prevalent serotype in both types of samples. However, in our study, the first most prevalent serotype in the food samples was S. Infantis (24.63%) and S. Enteritidis (18.84%) had a lower prevalence, but still being the most commonly isolated serotype when added to the clinical serovars in term of percentages. The shift in prevalence from S. Typhimurium to S. Infantis is noted in many recent studies. The main reason can be explained by the emergence of new S. Infantis clones that have enhanced capabilities to colonize food animals, and spread rapidly (kalaba et al., 2017; Antunes et al., 2016).  al., 2015). Valuable information was deduced from the food sources of the isolates, poultry constituted the main food reservoir of the totality of food sources. This is in accordance with many studies that confirm poultry as the main reservoir responsible for Salmonella spp. outbreaks (Andino et al., 2015). Furthermore, the countries of origin of the foods also revealed that four imported food items found in this study contained three different serovars S. Senftenberg -India, S. Malmoe -India, and S. Muenchen -Brazil, with the two latter being uncommon serotypes.

Clinical strain
From the 16 clinical isolates, 13 were resistant to at least one antimicrobial agent from 14 tested antibiotic agents. All the isolates were susceptible to aztreonam (ATM). Almost total susceptibility was also observed for ciprofloxacin and ofloxacin (OFX) (93.75% each). In addition, 87.5 % of the isolates were susceptible to amoxicilinclavulanic acid (AMC) and 81.25% of the isolates in this study were susceptible to both trimethoprim -sulfamethoxazole (SXT) and gentamycin (CN). Moreover, 6.25% of the isolates were susceptible to piperacillin and 25 % were susceptible to ampicillin; both belonging to the penicillin antibiotic class. However, the use of piperacillin with a β lactamase inhibitor like tazobactam (TZP) increased the percentage of the susceptible isolates (75 %). Of particular note, none of the isolates was susceptible to imipenem (IMI) ( Table 4). The antimicrobial resistance profile of the clinical isolates is shown in table 5.
Results showed that three out of the 16 strains (18.75%) exhibited multidrug resistance profile (MDR), defined as resistance to at least three different families of antimicrobials. S. Lille displayed resistance to 5 antibiotic families and 7 antimicrobial agents, and was intermediate for a 6th antibiotic class also. S. chincol and the autoagglutinated strain displayed resistance to 6 and 5 antimicrobial agents respectively (Table 5).

Food strains
Results of antimicrobial susceptibility testing of Salmonella isolates from food are summarized in Table 6. Results showed that most of the isolates were susceptible to aztreonam (ATM) (95.31%), ceftriaxone (87.5%), cefamandole (MA) (85.94%), trimethoprim sulfamethoxazole (SXT) (85.94%) and amoxicillin clavulanic acid (AMC) (71.88%). Strains were commonly resistant to imipenem (IMI) (79.69 %) and showed resistance to ampicillin (50%) and piperacillin (25%). As for the clinical isolates, none of the food isolates was susceptible to imipenem ( Table 6). The table 7 show the antimicrobial resistance profile of food isolates where 58 among the 69 tested isolates, show a resistance to at least one family of the tested antibiotics. Among these isolates, 13 showed MDR profile (Table 7). MDR isolates were S. Senftenberg (n=2), S. Anatum (n=1), S. Newport (n=1), S. Muenster (n=1), S. Montevideo (n=1), S. Chincol (n=2), S. Infantis (n=1), Autoagglutinated (n= 1), S. Malmoe (n=1) and S. Muenchen (n=2) ( Table 7).   The increase of antimicrobial -resistant Salmonella strains is a global concern, it stems from the abusive use of antibiotics in humans and in farm animals for the mass production of food and animal byproducts (Kolár et al., 2001). Results of this study have shown that for both the clinical and food serovars, low susceptibility was observed for penicillins that include piperacillin and ampicillin, on the other hand, low resistance to almost none was observed for trimethoprim-sulfamethoxazole. Low susceptibility to ampicillin and low resistance to trimethoprim-sulfamethoxazole was also the case with the isolates of (Fadlallah et al., 2017). Moreover, other studies also indicate that conventional antibiotics; used as first-line treatment, like ampicillin are no longer an appropriate choice for the treatment of invasive salmonellosis (Su et al., 2004).
In addition, the results also indicate that none of the isolates were susceptible to imipenem in both sample types. This finding of carbapenem resistance is alarming, since this broad spectrum antibiotic is classified as a "critically important" and is often used as a "last resort agent", when patients with the infection become gravely ill or harbor resistant bacteria ( serotypes included the serotypes S. Muenchen, S. Malmoe and S. Senftenberg with the first two serotypes found in three imported food samples. According to the CDC, S. Muenchen is a commonly isolated MDR strain from South America (Brazil), and S. Senftenberg is considered a serotype that has higher resistance to heat up to 70 ˚C (Manas et al., 2003). It has a thermotolerance approximately 30 times more than S. Typhimurium, hence surviving high cooking temperatures and transmission to humans. By importing food, a variety of uncommon and MDR serovars of Salmonella are being introduced into the country, these strains have increased virulence and a high potential for survival and spreading to humans through the food chain, hence increasing the dissemination of more virulent serotypes of Salmonella in Lebanon and creating possible endemic outbreak situations. Infection with MDR Salmonella strains would result in an increased number of treatment failures, since the normally used antibiotics are no longer effective. Physicians later would have to use "last resort" antibiotics that are often more expensive and toxic ( . This can be an explanation to why the mean percent resistance in food samples was higher than the one for clinical samples in this study. These resistant bacteria are reaching humans by food consumption and causing major infections with a limited number of antibiotics to cure them. This is mostly observed in poultry production, which is one of the most consumed and increasing globally traded meat products, hence, it is crucial to restrict global usage of antibiotics in veterinary and farm settings to minimize the selection of resistant Salmonella (Hur et al., 2012; Kalaba et al., 2017). Antibiotic susceptibility testing is an important tool to keep track of antibiotic resistance levels in various microorganisms, in different countries and throughout the world. This surveillance tool enables us to notice that resistance to antibiotics has risen greatly and quickly over the years, it also helps to identify MDR pathogens capable of fast dissemination (