Community-Level Physiological Profiling for Microbial Community Function in Broiler Ceca.

Poultry production is a major agricultural output worldwide. It is known that the gut health of broilers is essential for their growth and for providing wholesome products for human consumption. Previously, the microbial diversity of broiler ceca was studied at the genetic level. However, the functional diversity and metabolic activity of broiler cecal bacterial communities are not fully investigated. Recently, the EcoPlates™ from Biolog, Inc. have been used for characterizing bacterial communities from various environments. In this study, we applied these plates to physiologically profile cecal bacterial communities in broilers. The ceca were aseptically excised from 6-week-old broilers, and their contents were suspended in phosphate buffered saline. The cultures in the EcoPlates™ were incubated at 42 °C for 5 days in an OmniLog® system. Responses of the bacterial communities to the various chemicals as carbon sources were measured on formazan production. The results show sigmoidal growth curves with three phases in all 12 cecal samples. Cecal bacterial communities could not use 11 carbon substrates for carbon sources; instead, they used pyruvic acid methyl ester, glycogen, glucose-1-phosphate and N-acetyl-D-glucosamine most frequently. Each bacterial community metabolized various numbers of the substrates at different rates among broilers. In the future, modification of the culture conditions to mimic the gut environment is needed. More investigations on the effects of nutrients, Salmonella or Campylobacter on physiological functions of cecal bacterial communities will provide insights into the improvement of animal well-being, saving production expenditures for producers and providing safer poultry products for human consumption.

Growth of Campylobacter incubated aerobically in fumarate-pyruvate media or media supplemented with dairy, meat, or soy extracts and peptones.

The ability of Campylobacter to grow aerobically in media supplemented with fumarate-pyruvate or with dairy, meat, or soy extracts or peptones was examined. Optical densities (OD) of Campylobacter cultured in basal media, media supplemented with fumarate-pyruvate or with 1.0, 2.5, 5.0, or 7.5% beef extract was measured. Growth was also compared in media supplemented with other extracts or peptones. Finally, cfu/mL of Campylobacter recovered from basal media or media supplemented with fumarate-pyruvate, casamino acids, beef extract, soytone, or beef extract and soytone was determined. Results indicated that OD of cultures grown in media supplemented with fumarate-pyruvate or with 5.0 or 7.5% beef extract were higher than OD of isolates grown in basal media or media supplemented with lower concentrations of beef extract. Highest OD were produced by isolates grown in media supplemented with beef extract, peptone from meat, polypeptone, proteose peptone, or soytone. Also, more cfu/mL were recovered from media with fumarate-pyruvate, beef extract, soytone, or beef extract-soytone than from basal media or media with casamino acids. Findings indicate that media supplemented with organic acids, vitamins, and minerals and media supplemented with extracts or peptones containing these metabolites can support aerobic growth of Campylobacter.

Influence of in-package cold plasma treatment on microbiological shelf life and appearance of fresh chicken breast fillets.

The effect of in-package cold plasmas (CP) was studied on microbiological shelf life and surface lightness of fresh chicken fillets (pectoralis major). Chicken fillets were packaged in food trays in air or modified atmosphere (MA) gas (O2:CO2:N2 = 65:30:5) and stored at 4 °C after exposed to an in-package cold plasma (80 kV for 180 s) treatment. Populations of mesophiles, psychrophiles, and pseudomonas spp. were measured as indicators for microbiological shelf life and CIELAB L(∗) values as an indicator for raw meat appearance. Results show that regardless of microbial type, there were no significant differences in microbial counts between the control and CP treated chicken fillets packed in air. However, in the MA packages, microbial counts were consistently lower than the non-treated control during refrigerated storage. Regardless of CP treatment, the microbial counts on the samples packed in air were much higher than in MA. They were more than 6 logs cfu/g in air compared to fewer than 4 logs cfu/g in MA after 7 d storage and fewer than 6 logs cfu/g after 14 d storage. Regardless of CP treatment and gas composition in package, there were no significant differences in the surface L(∗) value between the fillets pre-treatment and those after storage at 4 °C. These results demonstrate that the effects of in-package CP treatments on microbiological shelf life of fresh chicken fillets depend upon headspace composition in packages. When fresh chicken fillets are packed in air, CP treatment has no effect on microbiological shelf life. MA packages with high O2 and CO2 significantly extend shelf life and CP treatment with MA can at least double shelf life of fresh chicken meat (more than 14 days). Regardless of headspace composition, in-package CP does not have negative effects on chicken meat appearance.

The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain.

The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n=45) and group B S. Heidelberg (n=11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.

Detection and Distribution of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Campylobacter jejuni Isolates from Chicken Livers.

Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide linked to the consumption of undercooked broiler livers. Application of bacteriophages during poultry production has been used as an alternative approach to reduce contamination of poultry meat by Campylobacter. To make this approach effective, understanding the presence of the bacteriophage sequences in the CRISPR spacers in C. jejuni is critical as they may confer bacterial resistance to bacteriophage treatment. Therefore, in this study, we explored the distribution of the CRISPR arrays from 178 C. jejuni isolated from chicken livers between January and July 2018. Genomic DNA of C. jejuni isolates was extracted, and CRISPR type 1 sequences were amplified by PCR. Amplicons were purified and sequenced by the Sanger dideoxy sequencing method. Direct repeats (DRs) and spacers of CRISPR sequences were identified using the CRISPRFinder program. Further, spacer sequences were submitted to the CRISPRTarget to identify potential homology to bacteriophage types. Even though CRISPR-Cas is reportedly not an active system in Campylobacter, a total of 155 (87%) C. jejuni isolates were found to harbor CRISPR sequences; one type of DR was identified in all 155 isolates. The CRISPR loci lengths ranged from 97 to 431 nucleotides. The numbers of spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates that could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases showed that most spacer sequences were homologous to Campylobacter bacteriophage DA10. The results of our study provide important information relative to the development of an effective bacteriophage treatment to mitigate Campylobacter during poultry production.

Filter sterilized carcass rinsate for recovery of Salmonella species with various concentrations of cetylpyridinium chloride.

Controlling Salmonella in poultry processing continues to be important to processors and consumers. Cetylpyridinium chloride (CPC) has proven to be effective in vitro in controlling Salmonella. This study evaluated the recovery of Salmonella after overnight storage in 4°C filter-sterilized carcass rinsate containing CPC from 0.44 to 909 ppm (µg/mL). Ten Salmonella serotypes (18 strains), of which 6 serotypes are commonly isolated from poultry products, were grown in Bacto-Tryptic Soy Broth overnight at 37°C. Serial dilutions of a CPC/propylene glycol solution were prepared in 24-well tissue culture plates containing filter-sterilized carcass rinsate. Approximately 107 cfu/mL of each Salmonella serotype was added to the appropriate wells. Inoculated plates were stored overnight at 4°C. After storage, triplicate plates of brilliant green agar with sulfapyridine (BGS) were surface inoculated with 10 µL of the contents for each well, streaked for isolation, and incubated at 37°C for 24 h. Three replications were conducted. The presence of typical colonies on BGS plates was recorded as growth and verified through biochemical and serological testing. Of the serotypes chosen, Salmonella Kentucky, Dublin, and Enteritidis were the least resistant to CPC with a median minimum inhibitory concentration (MIC) of 14.22 µg/mL (range from 3.55 to 56.88 µg/mL); S. Typhimurium demonstrated a median MIC of 114.00 µg/mL (range from 28.44 to 114.00 µg/mL). Residual CPC potentially remaining attached to a carcass or in the weep after processing could potentially alter which Salmonella serotype is recovered from a carcass rinse due to different growth patterns during regulatory testing, with a potential for more virulent strains not to be recovered.

Head-to-head comparison of CAMPYAIR aerobic culture medium versus standard microaerophilic culture for Campylobacter isolation from clinical samples.

Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%-99.7%) and 100% (95% CI 98%-100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%-99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79-1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.

Use of automated capillary immunoassays for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins.

The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In the present study, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations was also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. The chicken immunoglobin G (IgG) antibody response to the FliD protein from the immunized group was 1110- and 51,400-fold higher than that from the un-immunized chickens both two- and three-weeks post-vaccination, respectively. It was also observed that IgM antibody against the FliD protein from the immunized chickens was 1030-fold higher than that from the un-immunized chickens two weeks post-vaccination, but the IgM response declined to 120-fold between two groups from two weeks to three weeks after immunization. The IgM antibody response to the FimA protein from the immunized group was 1.84- and 1.12-fold higher than that from the un-immunized group, respectively, both two- and three-weeks post-vaccination, while the IgG antibody response from the immunized group was 8.07- and 27.6-fold higher than that from the un-immunized group, respectively, during the same period. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

CAMPYAIR, a New Selective, Differential Medium for Campylobacter spp. Isolation without the Need for Microaerobic Atmosphere.

Campylobacter spp. are considered the most frequent bacterial cause of acute gastroenteritis worldwide. Although the diarrhea produced by these bacteria is self-limiting, the pathogen has been associated with severe long-term sequelae following acute signs and symptoms of the illness. However, research on Campylobacter is hampered by costs and technical requirements for isolating and culturing the bacterium, especially in low and middle-income countries. Therefore, attempts have been made to simplify these culture methods and to reduce costs associated with conducting research on Campylobacter. Recently, a liquid medium which allows selective enrichment of Campylobacter using aerobic incubation has been described. However, a solid medium is also needed for the isolation of pure colonies, enumeration of bacterial populations, and other studies on the pathogen. Therefore, a new medium (CAMPYAIR) was developed, based on the formulation of the liquid medium. CAMPYAIR is a solid chromogenic medium that supports the growth of Campylobacter isolates within 48 h of incubation in aerobic atmospheres. Moreover, CAMPYAIR contains antibiotic supplements with an enhanced ability to recover Campylobacter from environmental samples that may also contain non-campylobacter bacteria. The addition of the indicator 2,3,5-triphenyltetrazolium (TTC) to the medium differentiates Campylobacter from other bacteria growing on the media. The findings from studies on CAMPYAIR suggest that the utilization of the new selective, differential medium could help to reduce the costs, equipment, and technical training required for Campylobacter isolation from clinical and environmental samples.

Prevalence and Characterization of Quinolone Resistance in Campylobacter spp. Isolates in Chicken Livers from Retail Stores in Georgia, USA.

ABSTRACT: Campylobacter is a bacterial pathogen that causes human foodborne illnesses worldwide, and outbreaks have been associated with consumption of undercooked chicken livers. The objectives of this study were to compare two PCR assays of 250 Campylobacter isolates for identification to species, to assess antibiotic resistance of the isolates, and to analyze genetic diversity of the quinolone resistance determining regions (QRDRs) of the isolates. A double-blind design was used to identify the species of Campylobacter; 181 (72%) of the isolates were identified as Campylobacter jejuni, and 69 (28%) isolates were identified as Campylobacter coli by both PCR assays. A total of 93 (37.2%) isolates were resistant to at least one antibiotic. Among 88 C. jejuni isolates, 33 (18%) were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), 25 (14%) were resistant to tetracycline (TET), and 18 (10%) were resistant to NAL and TET. Two C. jejuni isolates were resistant to four of the tested antibiotics, and one isolate was resistant to five antibiotics. Two C. coli isolates were resistant to TET, and two were resistant to NAL, CIP, and TET. The amino acid sequences of the QRDRs for the isolates had eight point mutations and could be classified into 12 groups. Thirty-eight C. jejuni isolates resistant to NAL and CIP had a point mutation at residue 86 (substitution from threonine to isoleucine). However, six isolates without this substitution were resistant to NAL and/or CIP. Ten isolates with a point mutation at residue 86 were susceptible to NAL and CIP. This observation suggests that in addition to the substitution at residue 86 other mechanisms may confer resistance to quinolones. Further studies are needed to elucidate mechanisms for quinolone resistance in Campylobacter. The Campylobacter spp. isolated from chicken livers in this study were resistant to quinolones and other classes of antibiotics.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Rapid Identification of Campylobacter Strains Cultured Under Aerobic Incubation Using Hyperspectral Microscope Imaging.

ABSTRACT: Campylobacter is an organism of concern for food safety and is one of the leading causes of foodborne bacterial gastroenteritis. This pathogen can be found in broiler chickens, and the level of allowable contamination of processed poultry is regulated by federal agency guidelines. Traditional methods for detecting and isolating this pathogen from broiler chicken carcasses require time, expensive reagents, and artificially generated microaerophilic atmospheres. An aerobic medium that simplifies the procedure and reduces the expense of culturing Campylobacter has been recently described, and Campylobacter can be grown in this medium in containers that are incubated aerobically. Hyperspectral microscopic imaging (HMI) has been proposed for early and rapid detection of pathogens at the cellular level. The objective of the present study was to utilize HMI to compare differences between Campylobacter cultures grown under artificially produced microaerobic atmospheres and cultures grown in aerobic medium. Hyperspectral microscopic images of three Campylobacter strains were collected cultures grown for 48 h microaerophilically and for 24 and 48 h aerobically, and a quadratic discriminant analysis was used to characterize the bacterial variability. Microaerobically cultured bacteria were detected with 98.7% accuracy, whereas detection accuracy of cultures grown in the novel medium was slightly reduced (-4.8 and -3.2% for 24 and 48 h, respectfully). The Mahalanobis distance multivariate metric was applied to quantify strain variability under all three treatment conditions. Across all strains and treatments, little cluster variation was present (4.22 to 4.42), indicating the consistency of the images collected from the three strains. The classification and spectral consistency was similar for cultures incubated in the aerobic medium for 24 h and cultures grown for 48 h under microaerobic conditions.

Bacterial Community Assessed by Utilization of Single Carbon Sources in Broiler Ground Meat after Treatment with an Antioxidant, Carnosine, and Cold Plasma.

ABSTRACT: Contaminated poultry meat is a major source of human foodborne illnesses. Many interventions have been developed to reduce and/or eliminate human foodborne pathogens in poultry products; however, treatments with cold plasma or carnosine or their combination have not been extensively investigated. In this study, the bacterial microflora of poultry meat samples after treatments with cold plasma and carnosine were characterized with EcoPlates in the OmniLog system. The plates were incubated at 25°C for 7 days in the OmniLog chamber, and bacterial growth was monitored by recording formazan production every 30 min at an optical density of 590 nm. The kinetics of lag, log, and stationary phases of bacterial growth followed the Gompertz sigmoidal model but with different inflection times and asymptotes at the log phase and the stationary phase, respectively. Results indicated that treatment of poultry meat samples with cold plasma technology and carnosine could inhibit growth of the bacteria in the treated meat samples. Of 31 chemicals tested, phenylethylamine, α-d-lactose, d,l-α-glycerol phosphate, 2-hydroxybenzoic acid, γ-hydroxybutyric acid, α-ketobutyric acid, and d-malic acid could not be metabolized by bacteria in the meat samples. Future research is required to determine whether these seven chemicals that inhibited growth of bacteria in these meat samples can be used as food preservatives for extending the shelf life of these products. Whether the bacterial flora can be an indicator of effectiveness for meat samples treated with cold plasma, carnosine, or both needs further study.

The effect of rosemary Extract and cold plasma treatments on bacterial community diversity in poultry ground meats.

To provide safer food, many technologies have been used to preserve food. One such technology is cold plasma, which can reduce viable bacterial counts in various food matrices. However, bacterial communities in food matrices before and after cold plasma treatment have not been investigated. In this communication, the EcoPlates™ were used to physiologically profile bacterial communities from poultry ground meat treated with rosemary, cold plasma or both. The cultures in the plates were incubated at 25 °C for seven days in an OmniLog® system. Responses of the bacterial communities to 31 chemicals were measured on formazan production. The results show that the three parameters of the Gompertz growth curves were observed in all samples, 2-hydroxybenzoic acid could not be used, while pyruvic acid methyl ester was used for a carbon source by the bacterial communities from all meat samples, each bacterial community metabolized different numbers of chemical compounds at different rates, and reduction of bacterial functional diversity was observed in the poultry meat samples treated with cold plasma and rosemary. In the future, investigations on whether the physiological profiling in bacterial communities be used as an indicator for effectiveness of cold plasma treatment of meat samples.

Bacterial flora of processed broiler chicken skin after successive washings in mixtures of potassium hydroxide and lauric acid.

Changes in the size of populations of different groups of bacteria composing the normal flora of processed broiler skin were examined after each of five consecutive washings in mixtures of potassium hydroxide (KOH) and lauric acid (LA). Portions of skin from commercially processed broiler carcasses were washed in distilled water (control) or in mixtures of 0.25% KOH-0.5% LA or 0.5% KOH-1% LA by using a stomacher laboratory blender to agitate the skin in the solutions. After each wash, skin was transferred to fresh solutions, and washing was repeated to provide samples washed one to five times in each solution. Bacteria in rinsates of the washed skin were enumerated on plate count (PC) agar, Staphylococcus (STA) agar, Levine eosin methylene blue (EMB) agar, lactic acid bacteria (LAB) agar, and Perfringens (PER) agar with TSC supplement. Selected isolates recovered on each medium were identified. Overall, no significant differences were observed in numbers of bacteria recovered on PC, STA, or EMB agars from skin after repeated washing in water, but there were significant reductions in the number of bacteria recovered on LAB and PER agars. Repeated washing of skin in 0.25% KOH-0.5% LA or 0.5% KOH-1% LA generally produced significant reductions in the number of bacteria recovered on all media. Furthermore, no bacteria were recovered on PER agar from skin washed five times in 0.25% KOH-0.5% LA. Likewise, no bacteria were recovered on EMB or LAB agars from skin washed three or more times in 0.5% KOH-1% LA or on PER agar from skin washed four or five times in this solution. Staphylococcus spp. were identified as the skin isolates with the highest degree of resistance to the bactericidal activity of KOH-LA. Findings indicate that although bacteria may be continually shed from poultry skin after repeated washings, bactericidal surfactants can be used to remove and kill several types of bacteria found on the surface of the skin of processed broilers.

Molecular Analysis, Biochemical Characterization, Antimicrobial Activity, and Immunological Analysis of Proteus mirabilis Isolated from Broilers.

Proteus mirabilis, a Gram-negative bacterium, is ubiquitous in the environment and is considered as the normal microflora in the human gastrointestinal tract. However, this bacterium is an opportunistic pathogen in humans, often causing urinary tract infections. Moreover, Proteus has been frequently isolated from food animals, including poultry. Whether this bacterium contributes to the foodborne illness in humans is unclear. In this report, P. mirabilis isolates recovered from broilers during housing in the units were characterized, their antimicrobial activity was assayed, and broiler immune response to the soluble proteins was determined. Cecal contents and fecal droppings were treated according to the standard protocol for isolation. Speciation based on biochemical reactions and the antimicrobial activity of the isolates were carried out using commercial kits. Immunoblot was assayed to determine immune status of broilers against P. mirabilis. A total of 10 isolates of P. mirabilis were selected for further characterization. These isolates could grow in pH 6.0 and 1% NaCl conditions. They were resistant to sodium lactate, troleandomycin, rifamycin SV, vancomycin, but sensitive to nalidixic acid, cefotaxime and novobiocin. Moreover, the CTX, ACC, CMY-1, BIC, NDM, VEB, qnrB and qnrD genes were detected by PCR amplification in all isolates. Sera from broilers harboring this bacterium reacted to the P. mirabilis soluble proteins, but not from litter- and age-matched P. mirabilis negative and SPF chickens, indicating that this bacterium infected chickens that could have humoral immune response against P. mirabilis. This study provides a rationale for further monitoring P. mirabilis during poultry production to determine whether this bacterium poses potential threats to public health.

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

Studies were conducted to examine the ability of three chemicals to neutralize residual antibacterial activity of commercial antimicrobial chemicals used in poultry processing. Chemical antimicrobial interventions used in poultry processing may have potential for carryover into whole poultry carcass buffered peptone water (BPW) rinses collected for monitoring Salmonella contamination. Such carryover may lead to false-negative results due to continuing bactericidal action of the antimicrobial chemicals in the rinse. To simulate testing procedures used to detect Salmonella contamination, studies were conducted by separately adding test neutralizers (highly refined soy lecithin, sodium thiosulfate, or sodium bicarbonate) to BPW and using these solutions as carcass rinses. Control samples consisted of BPW containing no additional neutralizing agents. One of four antimicrobial solutions (cetylpyridinium chloride, peroxyacetic acid, acidified sodium chlorite, and a pH 1 hydrochloric:citric acid mix) was then added to the rinses. The four antimicrobial solutions were prepared at maximum allowable concentrations and diluted with modified BPW rinses to volumes simulating maximum carryover. These solutions were then inoculated with a mixed culture of five nalidixic acid-resistant Salmonella serovars at 106 CFU/mL. The inoculated rinse was stored at 4°C for 24 h, and Salmonella was enumerated by direct plating on brilliant green sulfa agar supplemented with nalidixic acid. Results indicate that incorporation of optimal concentrations of three neutralizing agents into BPW neutralized the demonstrated carryover effects of each of the four antimicrobial solutions tested, allowing recovery of viable Salmonella at 106 CFU/mL (P > 0.05), equivalent to recovery from carcass rinses with no antimicrobial carryover. Incorporation of these neutralizers in BPW for Salmonella monitoring may reduce false-negative results and aid regulatory agencies in accurate reporting of Salmonella contamination of poultry.

Growth of Campylobacter in media supplemented with organic acids.

The growth of Campylobacter spp. in media supplemented with organic acids was examined. A Bioscreen C Microbiology Reader was used to measure growth of cultures incubated at 37 degrees C for 48 h in a tryptose-yeast extract basal broth medium and in basal broth supplemented with 10, 20, 30, 40, or 50 mM citric, fumaric, lactic, malic, or succinic acid. Growth of three of six isolates was significantly greater (P < or = 0.05) in media supplemented with 20 to 50 mM citric acid than in nonsupplemented media, growth of five of six isolates was significantly greater in media supplemented with 10 to 50 mM succinic acid than in nonsupplemented media, and growth of six of six isolates was significantly greater in media supplemented with 10 to 50 mM fumaric or malic acid or with 20 to 50 mM lactic acid than in nonsupplemented media. Isolates were also cultured in basal media supplemented with a mixture of 10, 20, 30, 40, or 50 mM fumaric, malic, lactic, and succinic acids. Results indicated that the growth of all Campylobacter isolates was significantly greater in media supplemented with mixtures containing each of these organic acids at 10 to 40 mM than in nonsupplemented media. These findings indicate that in vitro growth of Campylobacter spp. may be significantly enhancedin media supplemented with organic acids that support the growth of these bacteria.

Antimicrobial activity of potassium hydroxide and lauric acid against microorganisms associated with poultry processing.

The antimicrobial activity of solutions of potassium hydroxide (KOH) and mixtures of KOH and lauric acid against microorganisms associated with poultry processing was determined. In vitro tests were performed by enumerating viable microorganisms recovered from bacterial cultures suspended in peptone water (control) and in solutions of 0.1% KOH or mixtures of 0.1% KOH and 0.25 or 0.50% lauric acid. Additional studies were conducted to identify changes in the native microbial flora of poultry skin washed in distilled water, KOH, or KOH-lauric acid. Although results of in vitro studies indicated that significantly fewer bacteria (P < or = 0.05) were recovered from cultures suspended in KOH than from cultures suspended in peptone water, there were also significantly fewer bacteria recovered from cultures suspended in KOH-lauric acid than from cultures suspended in KOH. Results of experiments with broiler skin indicated that although rinsates of skin washed in 1.0% KOH solutions contained significantly fewer total aerobic bacteria and enterococci than did skin washed in water, significantly fewer of these microorganisms were generally recovered from rinsates of skin washed in mixtures of 1.0% KOH and 0.5, 1.0, 1.5, or 2.0% lauric acid than from skin washed in KOH alone. Washing of broiler skin in solutions of 0.25 to 1.00% KOH or mixtures containing these concentrations of KOH and two parts lauric acid (wt/vol) also significantly reduced the populations of bacteria and yeasts in the native flora of broiler skin. Enterococci, lactic acid bacteria, and staphylococci in the native flora of the skin had the highest level of resistance to the bactericidal activity of KOH-lauric acid. These findings indicate that the antimicrobial activity of KOH-lauric acid is significantly greater than that of KOH alone in vitro and on poultry skin. Thus, KOH-lauric acid may be useful for reducing the level of microbial contamination associated with poultry processing.

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

Numerous antimicrobial chemicals are currently utilized as processing aids with the aim of reducing pathogenic bacteria on processed poultry carcasses. Carryover of active sanitizer to a carcass rinse solution intended for recovery of viable pathogenic bacteria by regulatory agencies may cause false-negative results. This study was conducted to document the potential carryover effect of five sanitizing chemicals commonly used as poultry processing aids for broilers in a postchill dip. The effect of postdip drip time on the volume of sanitizer solution carryover was first determined by regression of data obtained from 10 carcasses. The five sanitizer solutions were diluted with buffered peptone water at 0-, 1-, and 5-min drip time equivalent volumes as determined by the regression analysis. These solutions were then spiked to 10(5) CFU/ml with a mixture of five nalidixic acid-resistant Salmonella enterica serovars, stored at 4°C for 24 h, and finally enumerated by plate count on brilliant green sulfa agar containing nalidixic acid. At the 0- and 1-min drip time equivalents, no Salmonella recovery was observed in three of the five sanitizers studied. At the 5-min drip time equivalent, one of these sanitizers still exhibited significant (P ≤ 0.05) bactericidal activity. These findings potentially indicate that the currently utilized protocol for the recovery of Salmonella bacteria from postchill sanitizer interventions may lead to false-negative results due to sanitizer carryover into the carcass rinsate.