Benzoic acid and Salmonella: The latest knowledge

 

 

By Dr Christophe Paulus, Dr Matthias Wiemann and Dr Rolando Valientes, DSM Nutritional Products

The antimicrobial effects of organic acids on food-borne pathogenic bacteria have been well known since antiquity. Organic acids are still used as food and feed preservatives in a large number of applications ranging from canned foods to maize silage. In addition, the ban on antibiotics in feed within the European Union and elsewhere has put the spotlight on organic acids as most effective growth promoters.

 

The antimicrobial properties of organic acids first of all result from their ability to create an acidic, and thus unfavourable, environment within which most pathogenic bacteria cannot live and thrive. In this respect the more acidic the environment, or the stronger the acid, the greater the antibacterial effect. This is described as a ‘bacteriostatic effect’ because the bacteria are not directly killed but instead their functions are inhibited resulting in slower growth of their colonies.

 

But there are also organic acids that readily destroy bacteria and they do so through penetrating bacteria cells and disrupting vital functions such as DNA replication and expression. To be able to penetrate the outer bacterial membrane organic acids must be in their non-dissociated form, which depends on their chemical structure and on the pH of the surrounding environment. Once inside the bacterial cytoplasm they dissociate and the resultant acidity leads to detoxification reactions requiring energy from the bacterial cells leading to disruption of multiplication and leading to bacterial death, an action referred to as bacteriolytic.

 

Salmonella is one of the most frequent food-borne diseases and pork is quite often a source of Salmonella for humans in many countries. In live pigs Salmonella can be present as a sub-clinical infection, causing mild diarrhoea and depressed growth performance. In severe clinical outbreaks Salmonella can lead to acute septicaemia and death. Nevertheless, the most prevalent form of salmonellosis is that of low level sub-clinical infection.

 

Humans can be infected by being in close proximity to living infected pigs. More likely for the general population, however, is infection through eating contaminated pork that has been inadequately cooked. The presence of Salmonella in edible pork products results from animal infection and from poor hygiene in the slaughterhouse. Although efforts are continuously made to improve slaughterhouse conditions it is only recently that serious thought has been given to reducing animal infection levels, at least in a coordinated manner.In fact recent regulations in the European Union require specific targets to be set in the reduction of Salmonella infection levels in live pigs within the next few years. The ultimate goal is to reduce the risk of human infection by food-borne Salmonella to manageable levels, especially as recent evidence suggests an increasing resistance of current Salmonella strains to antibiotics used in human medicine. It is thus imperative in terms of general population health that effective measures are taken at farm level.

 

Practical experience in animal production, plus existing knowledge from the human food-chain industry, has meant that the first efforts in countering Salmonella in pig farms have concentrated around identifying the organic acids most effective against this particular bacteria. For this, two essential steps are required. The first features in vitro (laboratory) microbiological screening of organic acids using strains of Salmonella collected from infected pigs. The second takes the in vivo approach using actual animals to verify the results of the in vitro study under practical conditions. This is indeed a laborious procedure and one which can only be undertaken by the best equipped and staffed research centers if commercially-acceptable results are to be achieved.

 

The microbiology team at the DSM’s CRNA (Research Center for Animal Nutrition) has recently conducted an extensive in vitro study with ten organic acids screened under a variety of conditions. For this study three Salmonella serovars were collected from the intestinal contents of infected pigs.

 

These – typhimurium, derby, and enteritidis – represent the salmonellosis most likely to be found in pigs worldwide. The test suspension broth contained 105 CFU (colony forming units), a number adequate for causing infection in young pigs. Two indexes were used to assess the effectiveness of each organic acid against Salmonella. The first was the Minimum Inhibitory Concentration (MIC) representing the minimum concentration of each organic acid required to stop the growth of Salmonella (bacteriolytic effect). The second was the Growth Retardation (GR) index, representing the percentage level of reduction in Salmonella growth over a defined time period (bacteriostatic effect). The combined analysis revealed the most effective acid for combating Salmonella.

 

Results indicated that at high concentrations (above 25 mmol/l) all organic acids were equally capable of stopping Salmonella growth. However when concentration was reduced the most effective acids proved to be sorbic, benzoic, and citric. Indeed, MIC50 (50% inhibition of growth) with these acids was achieved at about half, or less than half, the concentration required for all other acids (Table 1). These results were more or less comparable among all three serovars.

 

 

Growth retardation results revealed that benzoic acid, followed by citric and then sorbic acid, retarded growth by 32, 17, and 16% more than all the other organic acids, which were practically ineffective in this respect. Again, similar results were obtained with all three serovars tested.

 

Under neutralised conditions, such as those when stomach contents reach the small intestine, only sorbic (6%) and benzoic acid (13%) retained antimicrobial activity.

 

From all these indications based on in vitro studies, our team selected benzoic acid as the best candidate against Salmonella infection. This was then verified in the next phase with in vivoexperiments.

 

As this is a rather recent development in animal nutrition, results from field (i.e. practical) studies are still coming in. Although the use of benzoic acid in piglet diets is not new, its use against Salmonella has been only recently evaluated. From these efforts, two challenge trials are presented here.

 

The first trial involved 96 pigs at about 42 kg liveweight. All animals were infected with 109 CFU of Salmonella and growth performance was monitored during the following 90 days. As expected, growth was depressed in all infected animals.

 

Those receiving antibiotics specific against Salmonella had the least overall growth depression with daily liveweight gain (DLWG) averaging 848 g. The only other group that resisted growth depression as well as the group receiving antibiotics was that receiving benzoic acid at 5 kg/tonne of feed with 830 g average DLWG. Other treatments, including a yeast additive and a plant extract, were virtually ineffective, growth depression being quite severe in such cases. Interestingly, the least growth depression in the period immediately following the infection was observed in the group receiving benzoic acid and not in the group receiving antibiotics, as might have been expected. This led to the second trial in which 120 pigs (18 kg liveweight), previously naturally infected by Salmonella and successfully treated with antibiotics, were observed to note the effects of benzoic acid during the recuperation period.

 

The experimental design included two levels of benzoic acid (zero and 5 kg/tonne) and three different antibiotic treatments (see Table 2). After 42 days pigs were tested for any presence of Salmonella. In fact, its presence was verified and attributed to a carry-over effect from the previous infection incidence. Results indicated that the use of benzoic acid or antibiotics reduced mortality compared to control. There were no interactive effects in any trait studied in this trial. The benzoic acid treatment resulted in the lowest mortality rate (1.2% versus 6.1% in the negative control). During the first 28 days, benzoic acid or tylosin also resulted in the highest growth performance.

 

At the end of the trial, pigs receiving the therapeutic antibiotic or benzoic acid were 7 and 4 kg heavier than control pigs respectively. Although benzoic acid did not replace the therapeutic properties of an antibiotic (in this case tylosin) for unspecific respiratory diseases, it obviously helped in the control of the disease giving excellent results, better than the ones where virginiamycin was used as antibiotic growth promoter.

 

Similar results have been observed under other more commercial conditions: the monitoring of 41 farms by E. Sieverding and K. Bode showed that feed supplementation with 0.5% benzoic acid during the fattening period significantly reduced detected Salmonella. Both the detection of Salmonella in anal swabs from pigs and the serological detection rates in meat juice at slaughter decreased (Figure 1).

 

 

It is evident that organic acids offer many advantages in the fight against food-borne pathogens eliminating, asthey do, the risk of increasing resistance to antibiotics. To this end, and in the case of Salmonella, it has been clearly demonstrated both in vitro and in vivo that VevoVitall, an ultra-pure grade of benzoic acid, is the most potent organic acid and one offering results very close to those supported by antibiotics specific for Salmonella. Thus this benzoic acid, already known for its growth-promoting properties in piglets, can be an effective tool in preserving animal growth performance, profitability and most importantly, public health.