==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 1756817610.5144/0256-4947.2007.195asm-3-195Brief ReportAcid resistance of Salmonella isolated from animals, food and wastewater in Tunisia Chatti Abdelwaheb Daghfous Douraid Landoulsi Ahmed From the Faculty of Sciences of Bizerte, Zarzouna, TunisiaCorrespondence and reprint requests: Abdelwaheb Chatti Laboratoire de Biochimie et Biologie Molécualire Faculté des Sciences de Bizerte, Zarzouna 7021 Tunisia, chattiabdel@yahoo.frMay-Jun 2007 27 3 195 198 01 3 2007 Copyright © 2007, Annals of Saudi Medicine2007This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. ==== Body Salmonella are important foodborne pathogens that infect a variety of hosts and cause a broad spectrum of diseases. During the past few decades, there has been an increase in salmonellosis worldwide. This can largely be attributed to an increase in transmission of broad host-range organisms to humans from infected food and water.1 However, it is well demonstrated that Salmonella can disseminate and survive in various environmental niches for long periods of time. They are pervasive in nature and may contaminate animals, vegetables, water and especially food during its production and distribution.2 The spread and dissemination of these enteric pathogens were accompanied by an important increase in resistance to antibiotics all over the world.3,4 Therefore, these bacteria are developing a cross-resistance to antibiotics often used in therapy or as additives.5–7 The occurrence of multiple-antibiotic-resistant Salmonella was reported in many developed and non-developed countries and continues to cause major health problems.8–12 Therefore, the use of antibiotics must be restricted to reduce the selection and spread of multi-resistant strains. This urgent need to replace antibiotics has prompted researchers to try to find other alternatives. Consequently, many compounds and chemicals, such as organic acids, were tested for their antimicrobial activities. Organic acids have long been used both for preserving foods and in livestock production for disease prevention or as growth-promoting feed additives.13–19 It has thus been demonstrated that organic acids have an antimicrobial activity which is pH dependent.13 Salmonella, as enteric pathogens, encounter a low pH value in the environment, especially during its transit in the host. These bacteria must face this barrier to replicate and proliferate in the hosts. The study of acid resistance in previous research showed that some strains of E. coli are able to survive at very low pH values, but stop growing at pH values less than 4.4.20–22 This study was undertaken to determine if ten isolates of Salmonella could grow on nutrient broth acidified by acids commonly present in foods or used as acidulants. It was also of interest to see whether acid adaptation enhanced the survival and growth of these strains upon subsequent exposure to a low pH environment. MATERIAL AND METHODS Strains of Salmonella used in this study are described in Table 1. These strains were isolated from foods, wastewater and animals. Bacteria were routinely incubated in nutrient broth (NB) (Pronadisa, Spain) at 37°C overnight with shaking. Salmonella isolates were assayed for susceptibility to eight different antibacterial agents by the disc diffusion method. All antibiotic discs used were supplied by Biomérieux (France). The respective quantities (μg/disc) of these compounds were as follows: ampicillin (AMP) (10); tetracycline (TET) (30); gentamicin (GEN) (10); nalidixic acid (NAL) (30); chloramphenicol (CHL) (30); amikacin (AMK) (30); neomycin (NEO) (30); streptomycin (STR) (10). The results were interpreted in accordance with the Clinical Laboratory and Standard Institute (CLSI). All isolates of Salmonella were tested for acid resistance. A fresh overnight culture was used to inoculate 50 mL (OD600=0.05) of the NB medium preadjusted with the appropriate acid to the desired pH, then incubated at 37°C with shaking. The optical density and pH of the medium were periodically measured. The pH of the samples were determined with a pH meter (HANNA Instruments pH 209) before and after inoculation and during the measurement time (pH 2.5, 3, 3.5, 4, 4.5, 5 and 7 were tested). For all of the acids, concentrations of both 1% and 10% were tested. The bacterial population was determined by plating on NB agar plates 0.1 mL of appropriately diluted cultures which were then incubated at 37°C for 24 hours. RESULTS In this study, eight antibiotics were tested: ampicillin, tetracycline, gentamicin, nalidixic acid, chloramphenicol, amikacin, neomycin and streptomycin. The findings obtained showed a greater resistance of all non-typhoidic Salmonella serotypes isolated from food, animals and water. Tetracycline was the only antibiotic that had no effect on any of the isolates tested. Ampicillin and streptomycin followed in lack of effect. On the other hand, amikacin, chloramphenicol, gentamicin and nalidixic acid were the most active against these isolates. Therefore, in the Salmonella population tested, Salmonella hadar (isolated from poultry) showed the highest rates of resistance (five resistant) and Salmonella newport the lowest (one resistant) (Table 1). The results showed that the antimicrobial activity of all acids increase as their concentrations increase. For periodic and acetic acids, a concentration of 1% was capable of reducing the number of colony-forming units by a factor of ten, whereas the same concentration of hydrochloric and citric acid reduced this number only by a factor of five. Figure 1 shows the growth kinetics of Salmonella hadar when incubated in an NB medium adjusted with the appropriate acid as an example of the responses of the other isolates tested. However, the results also showed that the minimum pH for growing of all Salmonella tested was dependant on the type of acid used. Therefore, the pH was 3 when using citric acid or hydrochloric acid as acidulants and 4.3 when using periodic or acetic acids. Lag time was similar as well for citric and hydrochloric acids. Moreover, acetic and periodic acids were significantly more inhibitory than both citric acid and hydrochloric acid towards all of the isolates tested. Figure 2 illustrates the growth kinetics of the ten isolates of Salmonella in an NB medium with citric acid added at a concentration of 10%. Therefore, except for Salmonella hadar which has a pH minimum for growth of about 3, all other isolates have a minimum of about 3.5. In addition, according to Figure 1, an increase in pH from 3.5 to 7 decreases the lag time of all Salmonella isolates. This decrease was serotype dependant; however, S. hadar showed the least lag time. For S. enteritidis, its lag time at pH 3.5 was different from all other isolates. DISCUSSION For years, antibiotics have been used in different fields such as medicine, agriculture and the food industry. However, the situation has changed in recent years due to the emergence of bacterial resistance and the decreased effectiveness of antibiotics.8,9,10,11,23 In Tunisia, few studies have been addressed the antibiotic resistance of Salmonella and its evolution. Our results show that the resistance profiles of all serotypes tested are very similar (Table 1). Tetracycline and ampicillin do not show any effect on the isolates. These antibiotics have been the most commonly used and therefore the very frequent occurrence of resistance among Salmonella is probably a consequence of this. Although this problem is well documented worldwide, the situation in Tunisia will become more dangerous in the near future.10,12,23 Abusive use of these molecules in fields such as agriculture, medicine and industry must be limited. Antibiotics must be used only when necessary, and the consumption of antibiotics for purposes other than the treatment of infectious diseases must be avoided as much as possible.24 Alternatives that are cheap, readily available and have few effects must be substituted. The majority of treatments used in Tunisia to eradicate pathogens from poultry contain chlorine and present a certain number of disadvantages. However, unlike chlorine, organic acids were recognized as safe for the environment and are generally used as preservatives in food. In this study, the efficiency of organic acids towards these pathogens was tested. Acetic and periodic acids showed great effectiveness on Salmonella tested at a concentration of 1% and pH 4. It is well known that organic acids have important antibacterial capacity and no negative effect on the environment.13 These molecules are thus generally used as food preserving additives. 12,14,15,17,18 At the same concentration and pH, citric acid and hydrochloric acid increase the doubling time of all serotypes tested without affecting their growth and survival. The killing effect of the acids used increased in the following way: citric acid < hydrochloric acid < acetic acid < periodic acid. The results showed that the situation would be clinically worse for the following reasons: First, acid tolerance is a contributing factor in the intracellular survival and virulence of Salmonella. Secondly, some isolates have also demonstrated high levels of resistance. In fact, Wain et al have suggested the existence of a relationship between virulence and drug resistance.25 Therefore, it should be stressed that pathogens have shown both high levels of drug resistance and also acid resistance, especially Salmonella hadar, which has the highest antibiotic and acid resistance. This link between acid and antibiotic resistance could have significant implications for the food industry and needs to be further investigated in foodborne pathogens in a real food-processing situation. The acid tolerance response provides cross-protection to heat, osmotic, and oxidative challenges.26 This work could help to assess the risk of stressed-adapted bacteria and alert public health professionals and inspection authorities. We are grateful to Mr. Ben Aissa Ridha (Institue Pasteur de Tunis) for providing strains of Salmonella. This work was supported by the Ministère de l’Enseignement Supérieur, Tunisie. Figure 1 Response of Salmonella hadar, incubated in an NB medium with acetic acid ( ), hydrochloric acid ( ), citric acid ( ) at the pH 3.5. All acids were tested at a concentration of 1%. For a control growth curve, Salmonella hadar was incubated in an NB medium pH 7 ( ) (OD = optical density). Figure 2 Salmonella isolates incubated in an NB medium adjusted by citric acid (1%) at range of pH 7 ( ), pH 4 ( ), pH3.5 ( ), pH 3 ( ) and pH 2.5 ( ). (OD = optical density). Table 1 Resistance profiles of Salmonella isolates Salmonella serovars Source Resistance profile S. enteritidis Poultry TET, STR S. mbandaka Meat TET, AMP, STR S. lindenburg Milk TET, AMP, STR S. nikolaifleet Wastewater TET, AMP, STR S. cerro Merguez* TET, STR S. montevideo Wastewater TET, AMP, NEO S. hadar Poultry TET, AMP, NEO, GEN, STR S. braenderup Milk TET, AMP, STR S. zanzibar Wastewater TET, NAL S. newport Wastewater TET * A Tunisian food prepared with meat. ==== Refs REFERENCES 1 Tauxe RV Emerging foodborne diseases: An evolving public health challenge Emerging Infect Dis 1997 3 425 34 9366593 2 Davies RH Wray C Seasonal variations in the isolation of Salmonella typhimurium, Salmonella enteritidis, Bacillus cereus and Clostridium perfringens from environmental samples J Vet Med Sci 1996 43 119 27 3 Moellering RC Antibiotic resistance: Lessons for the future Clin Infect Dis 1998 27 135 40 4 Cruchaga S Echeita A Aladuena A Garcia-Pena J Frias N Usera MA Antimicrobial resistance in salmonellae from humans, food and animals in spain in 1998 Antimicrob Agents Chemother 2001 47 315 21 5 David GW Shaohua Z Robert S Sherry A Sharon F Patrick FD Shawn MD David DW Jiangh OM The isolation of antibiotic-resistant Salmonella from retail ground meats N Engl J Med 2001 16 1147 54 6 Tellefson L Altekruse SF Potter ME Therapeutic antibiotics in animal feeds and antibiotic resistance Rev Sci Tech 1997 16 709 15 9501383 7 Witte W Medical consequences of antibiotic use in agriculture Science 1998 279 996 97 9490487 8 Levy SB Burke JP Wallace CK Antibiotic use and antibiotic resistance world-wide Rev Infect Dis 1987 9 231 16 9 Threlfall EJ Ward LR Skinner JA Rowe B Increase in multiple antibiotic resistance in non-typhoidal salmonellas from humans in England and Wales: a comparison of data for 1994 and 1996 Microb Drug Resist 1997 3 263 66 9270996 10 Beatriz G Sara S Santiago C Carmen M Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes Antimicrob Agents Chemother 2000 44 2166 69 10898692 11 Breuil J Brisabois A Casin I Armand-Lefèvre L Frémy S Collatz E Antibiotic resistance in Salmonellae isolated from humans and animals in France: comparative data from 1994 and 1997 Antimicrob Agents Chemother 2000 46 965 71 12 Asem A Shehabi Extra-intestinal infections with multiply drug-resistant Salmonella typhimurium in hosptialized patients in Jordan Eur J Clin Microbiol Infect Dis 1995 14 448 51 7556236 13 Cherrington CA Hinton M Media GC Chopra I Organic acids: Chemistry, Antibacterial activity and practical applications Adv Microb Physiol 1991 32 87 07 1882730 14 Risley CR Kornegay ET Lindemann MD Wood CM Eigel WN Effect of Feeding Organic Acids on Selected Intestinal Content Measurements at Varying Times Postweaning in Pigs J Anim Sci 1992 70 196 06 1582907 15 Gorden J Small PLC Acid resistance in enteric bacteria Infect Immun 1993 61 364 67 8418063 16 Lin J Lee IS Frey J Slonczewski JL Foster JW Comparative analysis of extreme acid suvival in Salmonella typhimirium, Shigella flexneri and Escherichia coli J Bacteriol 1995 177 4097 04 7608084 17 Russell JB Diez-Gonzalez F The Effects of Fermentation Acids on Bacterial Growth Ad Microb Physiol 1998 39 206 34 18 Partanen HK Mroz Z Organic acids for performance enhancement in pig diets Nutr Res Rev 1999 12 117 45 19087448 19 Foster JW Microbial reponses to acid stress Bacterial stress Response Storz G Hengge-Aronis R Washington DC American Society for Microbiology 2000 99 16 20 Small P Blankenhorn P Welty D Zinser E Slonczewski JL Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH J Bacteriol 1994 176 1729 37 8132468 21 Benjamin MM Datta AR Acid tolerance of enterohaemorrhagic Escherichia coli Appl Environ Microbiol 1995 61 1669 72 7747983 22 Pablo MM Alberto EP Marcelo ES Survival in acidic and alcoholic medium of Shiga toxin-producing Escherichia coli O157:H7 and non-O157:H7 isolated in Argentina BMC Microbiology 2003 3 17 12914672 23 Winokur PL Brueggemann DL Desalvo DL Hoffmann MD Apley MD Uhlenhopp EK Pfaller MA Doern GV Animal and Human Multidrug-Resistant Salmonella Isolates Expressing a Plasmid-Mediated CMY-2 AmpC (-Lactamase Antimicrob Agents Chemother 2000 2777 83 10991860 24 Martinez J Baquero F Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity and antibiotic resistance Clin Microbiol Rev 2002 15 647 79 12364374 25 Wain J Diep TS Anh HV Walsh AM Tuyet Hoa NT Parry CM White NJ Quantitation of bacteria in blood of typhoid fever patients and relationship between counts and clinical features, transmissibility, and antibiotic resistance J Clin Microbiol 1998 36 1683 87 9620400 26 Leyer GJ Johnson EA Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium Appl Environ Microbiol 1993 59 1942 47