Organic acids continue to receive considerable attention as feed additives for animal production. Most of the emphasis to date has focused on food safety aspects, particularly on lowering the incidence of foodborne pathogens in poultry and other livestock. Several organic acids are currently either being examined or are already being implemented in commercial settings. Among the several organic acids that have been studied extensively, is formic acid. Formic acid has been added to poultry diets as a means to limit Salmonella spp. and other foodborne pathogens both in the feed and potentially in the gastrointestinal tract once consumed. As more becomes known about the efficacy and impact formic acid has on both the host and foodborne pathogens, it is clear that the presence of formic acid can trigger certain pathways in Salmonella spp. This response may become more complex when formic acid enters the gastrointestinal tract and interacts not only with Salmonella spp. that has colonized the gastrointestinal tract but the indigenous microbial community as well. This review will cover current findings and prospects for further research on the poultry microbiome and feeds treated with formic acid.

Formic Acid as an Antimicrobial for Poultry Production: A Review.

Salmonella enterica is represented by >2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials S enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 001% of the sample Poultry is a major reservoir of Salmonella spp, including serovars that are frequently associated with human illness, as well as those that are not Numerous studies have examined the prevalence and diversity of Salmonella spp in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected In another sample, reads for the minority serovar comprised 0003% of the total number of Salmonella spacer reads The most abundant serovars identified were Salmonella enterica serovars Montevideo, Kentucky, Enteritidis, and Typhimurium CRISPR-SeroSeq also differentiated between multiple strains of some serovars This high resolution of serovar populations has the potential to be utilized as a powerful tool in the surveillance of Salmonella species IMPORTANCESalmonella enterica is the leading bacterial cause of foodborne illness in the United States and is represented by over 2,600 distinct serovars Some of these serovars are pathogenic in humans, while others are not Current surveillance for this pathogen is limited by the detection of only the most abundant serovars, due to the culture-based approaches that are used Thus, pathogenic serovars that are present in a minority remain undetected By exploiting serovar-specific differences in the CRISPR arrays of Salmonella spp, we have developed a high-throughput sequencing tool to be able to identify multiple serovars in a single sample and tested this in multiple poultry samples This novel approach allows differences in the dynamics of individual Salmonella serovars to be measured and can have a significant impact on understanding the ecology of this pathogen with respect to zoonotic risk and public health

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High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq.

Effect of Simulated Sanitizer Carryover on Recovery of Salmonella from Broiler Carcass Rinsates.

Neutralization of Bactericidal Activity Related to Antimicrobial Carryover in Broiler Carcass Rinse Samples.

Salmonella can resist various stresses and survive during food processing, storage, and distribution, resulting in potential health risks to consumers. Therefore, evaluation of bacterial survival profiles under various environmental stresses is necessary. In this study, the resistance profiles of five Salmonella isolates [serotypes with Agona, Infantis, Typhimurium, Enteritidis, and a standard strain (ATCC 13076, Enteritidis serotype)] to acidic, hyperosmotic, and oxidative stresses were examined, and the relative expressions of non-coding small RNAs were also evaluated, including CyaR, MicC, MicA, InvR, RybB, and DsrA, induced by specific stresses. The results indicated that although all tested strains displayed a certain resistance to stresses, there was great diversity in stress resistance among the strains. According to the reduction numbers of cells exposed to stress for 3 h, S. Enteritidis showed the highest resistance to acidic and hyperosmotic stresses, whereas ATCC 13076 showed the greatest resistance to oxidative stress, with less than 0.1 Log CFU/ml of cell reduction. Greater resistance of cells to acidic, hyperosmotic, and oxidative stresses was observed within 1 h, after 2 h, and from 1 to 2 h, respectively. The relative expression of sRNAs depended on the isolate for each stress; acidic exposure for the tested isolates induced high expression levels of DsrA, MicC, InvR, RybB, MicA, and CyaR. The sRNA RybB, associated with sigma E and outer membrane protein in bacteria, showed a fold change of greater than 7 in S. Enteritidis exposed to the tested stresses. CyaR and InvR involved in general stress responses and stress adaptation were also induced to show high expression levels of Salmonella exposed to hyperosmotic stress. Overall, these findings demonstrated that the behaviors of Salmonella under specific stresses varied according to strain and were likely not related to other profiles. The finding also provided insights into the survival of Salmonella subjected to short-term stresses and for controlling Salmonella in the food industry.

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Resistance Profiles of Salmonella Isolates Exposed to Stresses and the Expression of Small Non-coding RNAs.

SUMMARY Acid injury could prevent detection of Salmonella in feed and feed-type samples. A previous study showed that after incubation in commonly used pre-enrichment media, mixed feeds and feed ingredients reached a pH (4.0 to 5.0), capable of injuring or killing Salmonella. Approximately 105 colony forming units (CFU) of S. Enteritidis (SE), S. Heidelberg (SH), S. Kentucky (SK), or S. Typhimurium (ST) were individually placed into 50 mL of a citrate buffer at pH of 4, 4.5, or 5.0 for 6 or 24 h at 37°C. After holding at 37°C, each serotype was serially diluted in sterile physiological saline and plated onto xylose lysine tergitol 4 [for injury] and nutrient agar (NA) [for death]. These plates were enumerated and percent injury and/or death determined. The injury and death confirmed that pH 4.0 was detrimental to these Salmonella serotypes. After 24 h at pH 4.0, 60% or more of all 4 serotypes were killed with 100% of S. Kentucky killed. At pH 5.0, 65 to 75% of these organisms were uninjured, with death between 15 to 20%. Researchers testing feed/feed ingredients for Salmonella may not be aware of the acidic nature of the pre-enrichment step and the subsequent injury or death of any Salmonella present, whether healthy or stressed. Current protocols that are considered the “gold standard” may not be detecting Salmonella in samples containing fermentable substrates and extraneous microorganisms, which prevent the accurate detection of Salmonella. Injury or death of a few Salmonella cells could reduce the chance of recovery from any sample. More research is needed with additional Salmonella serotypes and strains to fully understand the challenges of isolating Salmonella from feed.

Injury and death of various Salmonella serotypes due to acidic conditions

In a recent study, the pH of commonly used Salmonella pre-enrichment media became acidic (pH 4.0 to 5.0) when feed or feed ingredients were incubated for 24 h. Acidic conditions have been reported ...

Impact of desiccation and heat exposure stress on Salmonella tolerance to acidic conditions.

The pH of Salmonella pre-enrichment media can become acidic (pH 4.0–5.0) when feeds/ingredients are incubated for 24 h. Salmonella in feed that have been stressed by heat and desiccation exhibit di...

Dry and heat stress affects H2S production of Salmonella on selective plating media.

The first step to detect Salmonella in feed and other dry contaminated samples is a pre-enrichment broth that can assist in the recovery of small numbers of stressed Salmonella cells. A previous study demonstrated that incubation of feed and feed ingredients in commonly used pre-enrichment media resulted in a low pH that injured or killed the Salmonella. The objective of this study was to determine which environmental samples and pre-enrichment and selective broths could interfere with the accuracy of Salmonella detection by allowing pH to drop. Samples were collected from commercial poultry operations. Triplicate 10-g subunits were dispensed into sterile 18-oz Whirl Pak bags and 90 mL of each of the media [lactose broth (LB), buffered peptone water (BPW), Universal Pre-enrichment (UP), minimal salts (M-9), tetrathionate broth (TT) and Rappaport-Vassiliadis broth (RV)] were added to the bags and incubated 24 h at 37°C (or 42°C for RV and TT). The pH was measured after incubation. With turkey litter, fluff, and eggshells, regardless of media, the pH never went below 5.7. Regardless of sample type, the lowest pH was 6.1, 6.2, and 6.4 for UP, M-9, and BPW, respectively. For the pre-enrichment medium commonly used in the US, (LB) the pH dropped to 4.7 to 4.9 with broiler litter and 4.2 for boot covers used in turkey or broiler houses. Many researchers testing these sample types are unaware of this potential for pH change during pre-enrichment and may be underestimating the presence of Salmonella.

The effect of environmental poultry samples on the pH of typical Salmonella pre-enrichment and enrichment media following incubation

Studies were conducted to investigate the recovery of Campylobacter from feed. The impact of feed moisture, water activity, pH, number of background microflora and the use of different antibiotic supplements in Campylobacter enrichment broth (CEB) on Campylobacter recovery were evaluated in five studies. Broiler starter feed was inoculated with 104 -105 cfu of Campylobacter/g and stored at 24 °C and 43% RH. Enrichment culture was conducted on the day of inoculation or 24 h post inoculation and every 48 h of storage thereafter for 14 d. Feed moisture, water activity, pH and level of background microflora were not correlated with Campylobacter recovery. The incubation of feed in CEB with no antibiotic supplement resulted in the number of background microflora increasing to 109 cfu/g and the pH of the media decreasing to pH 4-5 impacting recovery. Addition of certain antimicrobial supplements to CEB reduced background microflora growth and maintained a near neutral pH. Campylobacter was recovered up to 10 days post inoculation when using CEB containing antibiotic supplements compared to 1 day in CEB. These findings suggest that Campylobacter can be recovered from feed and the type of antimicrobial supplement utilized influences recovery by controlling extraneous microbial growth which occurs during enrichment.

K E Richardson

Papers

Injury and death of various Salmonella serotypes due to acidic conditions

Impact of desiccation and heat exposure stress on Salmonella tolerance to acidic conditions.

Dry and heat stress affects H2S production of Salmonella on selective plating media.

The effect of environmental poultry samples on the pH of typical Salmonella pre-enrichment and enrichment media following incubation

Recovery of Campylobacter from feed using different enrichment media.