Evaluation of a Preharvest Bacteriophage Therapy for Control of Salmonella within Bovine Peripheral Lymph Nodes.

ABSTRACT: A series of proof-of-concept studies were developed to determine whether a commercial bacteriophage cocktail could be utilized for the mitigation of Salmonella in bovine peripheral lymph nodes (LNs). The first objective sought to determine whether exogenous phage could be isolated from the LNs following administration. If isolation were successful, the second objective was to determine whether the phage in the LNs could effectively reduce Salmonella. Salmonella Montevideo was inoculated intradermally at multiple sites and multiple times, followed by delivery of the phage cocktail subcutaneously in two injections around each of the right and left prescapular and subiliac LNs. At the conclusion of each study, animals were euthanized, and the popliteal, prescapular, and subiliac LNs were examined. The inoculated phage was successfully isolated from the LNs; transmission electron microscopy revealed phages in the LNs of the treated cattle, and these phages were identical to those in the cocktail. Levels of phage were higher (P < 0.01) in the prescapular and subiliac LNs in the phage-treated than in the control cattle. In subsequent studies, the protocols were modified to increase Salmonella and phage levels within the LNs. Compared with the first study, overall Salmonella levels were increased in the LNs, and phage treatment decreased (P < 0.01) Salmonella in the some of the LNs. Phage levels were numerically but not significantly increased (P = 0.12) in the treated cattle. The final study was modified, hypothesizing that a 48-h postmortem period before LN removal would facilitate phage-Salmonella interaction; however, no differences (P > 0.10) in Salmonella levels were found among treatments. Salmonella-specific phages administered to live cattle can translocate to the LNs; however, these phages had limited to no effect on Salmonella in these LNs under these experimental conditions.

Evaluation of the Efficacy of Three Direct Fed Microbial Cocktails To Reduce Fecal Shedding of Escherichia coli O157:H7 in Naturally Colonized Cattle and Fecal Shedding and Peripheral Lymph Node Carriage of Salmonella in Experimentally Infected Cattle.

Two experiments were conducted to evaluate the feeding of direct fed microbials (DFMs) on fecal shedding of Escherichia coli O157:H7 in naturally infected cattle (experiment I) and on Salmonella in the feces and peripheral lymph nodes (PLNs) of experimentally infected cattle (experiment II). Thirty cattle, 10 per treatment, were used in each experiment. Treatments in experiment I consisted of a control (lactose carrier only); DFM1, a 1:1 ratio of Enterococcus faecium and Lactobacillus animalis; and DFM2, a 1:1 ratio of Lactobacillus acidophilus and Pediococcus acidilactici. In Experiment II, DFM1 was replaced with DFM3, a 1:2 ratio of Lactobacillus reuteri and other Lactobacillus strains. Additives were mixed in water and applied as a top-dressing to each pen's daily ration for 50 days. Approximately half-way through each experiment, the DFM concentration was doubled for the remainder of the study. Fecal samples were collected throughout experiment I and cultured for E. coli O157:H7. Cattle in experiment II were inoculated intradermally with Salmonella Montevideo on days 32, 37, and 42 and then necropsied on days 49 and 50 (five cattle per treatment on each day). Innate immune function was assessed on days 29, 49, and 50. In experiment I, fecal concentration and prevalence of E. coli O157:H7 were not different (P > 0.10) nor was there an effect (P = 0.95) on the percentage of super shedders (cattle shedding ≥3.0 log CFU/g of feces). In experiment II, no treatment differences (P > 0.05) were observed for Salmonella in the PLNs except for the inguinal nodes, which had a significantly lower Salmonella prevalence in DFM-supplemented cattle than in the controls. Immune function, as measured by monocyte nitric oxide production and neutrophil oxidative burst, was decreased (P < 0.05) in the DFM treatment groups. Although results of this research indicate little to no effect of these DFMs on E. coli O157:H7 or Salmonella in cattle, an increase in the duration of administration to that similar to what is used for commercial cattle might elicit treatment differences.

Methylsulfonylmethane exhibits bacteriostatic inhibition of Escherichia coli, and Salmonella enterica Kinshasa, in vitro.

AIMS: To evaluate antibacterial properties of methylsulfonylmethane (MSM) on Escherichia coli (MDRE21) and Salmonella enterica serovar Kinshasa (SK132). METHODS AND RESULTS: Bacterial proliferation analysis was measured spectrophotometrically during log phase growth with 0, 3, 5, 7, 10, 12 and 16% MSM. To assess the mechanism of inhibition, cultures were grown overnight with 0-16% MSM and enumerated on unmedicated brain-heart infusion agar (BHIA) or BHIA with 0-16% MSM. The long-term viability studies were done to evaluate the impact of 10% MSM. Absorbance data indicated a dose-dependent inhibition from 0 to 16% MSM. There was no growth of MDRE21 or SK132 on BHIA in 10-16% MSM. Both strains enumerated on unmedicated BHIA from overnight cultures with 10-16% MSM were able to partially recover. CONCLUSIONS: Recovery after MSM removal may be indicative of a bacteriostatic mechanism of inhibition. The long-term viability studies illustrated that neither MDRE21 nor SK132 could be rescued from 10% MSM after 5 or 6 days respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Methylsulfonylmethane antibacterial activity may prove useful during pre or postharvest food safety as a disinfectant. The primary benefit being, its clinical safety to humans. Comparisons to other disinfectants would also need to be done to determine if MSM was superior to those already on the market and would be cost effective.

Inhibition of multidrug-resistant Staphylococci by sodium chlorate and select nitro- and medium chain fatty acid compounds.

AIMS: Determine the antimicrobial effects of 5 µmol ml-1 sodium chlorate, 9 µmol ml-1 nitroethane or 2-nitropropanol as well as lauric acid, myristic acid and the glycerol ester of lauric acid Lauricidin® , each at 5 mg ml-1 , against representative methicillin-resistant staphylococci, important mastitis- and opportunistic dermal-pathogens of humans and livestock. METHODS AND RESULTS: Three methicillin-resistant Staphylococcus aureus and two methicillin-resistant coagulase-negative staphylococci were cultured at 39°C in 5 µmol ml-1 nitrate-supplemented half-strength Brain Heart Infusion broth treated without or with the potential inhibitors. Results revealed that 2-nitropropanol was the most potent and persistent of all compounds tested, achieving 58-99% decreases in mean specific growth rates and maximum optical densities when compared with untreated controls. Growth inhibition did not persist by cultures treated solely with chlorate or nitroethane, with adaptation occurring by different mechanisms after 7 h. Adaptation did not occur in cultures co-treated with nitroethane and chlorate. The medium chain fatty acid compounds had modest effects on all the staphylococci tested except the coagulase-negative Staphylococcus epidermidis strain NKR1. CONCLUSIONS: The antimicrobial activity of nitrocompounds, chlorate and medium chain fatty acid compounds against different methicillin-resistant staphylococci varied in potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that differential antimicrobial activities exhibited by mechanistically dissimilar inhibitors against methicillin-resistant staphylococci may yield potential opportunities to combine the treatments to overcome their individual limitations and broaden their activity against other mastitis and dermal pathogens.

Interactions of organic acids with vancomycin-resistant Enterococcus faecium isolated from community wastewater in Texas.

AIMS: Investigate the interactions of organic acids (OAs), acetic, butyric, citric, formic, lactic and propionic acid against 50 Gram-positive vancomycin-resistant Enterococcus faecium (VRE) strains to determine whether pH, undissociated or dissociated acid forms correlate with bacterial inhibition. METHODS AND RESULTS: Concentrations of undissociated and dissociated OAs at the molar minimum inhibitory concentrations (MICM s) of the VRE were calculated using the Henderson-Hasselbalch equation. The pH at the MICM s of all VRE strains against acetic, butyric, formic and propionic acids was similar, 4·66 ± 0·07, but there was a 1·1 pH unit difference for all six OAs. Inhibition of VRE by all six OAs did not appear to be solely dependent on pH or on the undissociated OA species. The inhibition of VRE by all six dissociated acids was within Δ = 3·1 mmol l-1 . CONCLUSIONS: Vancomycin-resistant Enterococcus faecium inhibition correlated with the dissociated OA species. A small decrease in the concentration of the dissociated OAs from optimum may result in allowing VRE strains to escape disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: When an OA is used to disinfect VRE strains, the concentration of the dissociated OA should be carefully controlled. A concentration of at least 20 mmol l-1 dissociated OA should be maintained when disinfecting VRE.

The horizontal transfer of Salmonella between the lesser mealworm (Alphitobius diaperinus) and poultry manure.

There is need to determine the nature of enduring reservoirs of Salmonella contributing to perpetual contamination within poultry flocks. The dispersal of Salmonella between birds, litter and the lesser mealworm has been established, but the extent that these act as critical components in the epidemiology of Salmonella infection during broiler grow-out and flock rotation has not been delineated; in particular, the level of participation by the lesser mealworm beetles (LMB) as agents of retention and dispersal. This study defines this route of transmission and provides empirical data on bacterial loads that facilitate Salmonella transfer. Results showed differential Salmonella transfer dependent on bacterial concentration. At 103  cfu/ml, only a small, but not significant, amount of Salmonella was transferred, from the LMB to the manure and back to uninfected LMB; while from 105 to 107  cfu/ml, a significant acquisition and transfer occurred both internally and externally to the LMB over 4 and 24 hr exposures. These data will be used in correlation with facility management practices to develop intervention strategies to mitigate the establishment and spreading of reservoir Salmonella populations contributing to pre-harvest contamination of poultry flocks.

Transferability of antimicrobial resistance from multidrug-resistant Escherichia coli isolated from cattle in the USA to E. coli and Salmonella Newport recipients.

OBJECTIVES: This study aimed to evaluate conjugative transfer of cephalosporin resistance among 100 strains of multidrug-resistant Escherichia coli (MDRE) to Salmonella enterica serotype Newport and E. coli DH5α recipients. METHODS: Phenotypic and genotypic profiles were determined for MDRE as well as for Salmonella Newport (trSN) and E. coli DH5α (trDH) transconjugants. RESULTS: Of 95 MDRE donor isolates, 26 (27%) and 27 (28%) transferred resistance to trSN and trDH recipients, respectively. A total of 27 MDRE (27%) were confirmed as extended-spectrum ß-lactamase (ESBL)-producers based on the double-disk synergy assay and whole-genome sequencing (WGS). WGS was performed on 25 of the ESBL-producing isolates, showing that 2 isolates carried blaCTX-M-6, 22 possessed blaCTX-M-32 and 1 was negative for blaCTX-M genes. Fourteen of the ESBLs sequenced were qnrB19. Differential transfer of IncA/C and IncN from MDRE32 was observed between trSN32 and trDH32. IncN-positive trDH32 displayed an ESBL phenotype, whereas IncA/C-positive trSN32 displayed an AmpC phenotype. The rate of ESBL transfer to trSN and trDH recipients was 11% and 96%, respectively. CONCLUSIONS: Twenty-seven MDRE were phenotypically identified as ESBL-producers. WGS of 25 MDRE revealed that 2 and 22 isolates carried blaCTX-M-6 and blaCTX-M-32, respectively. One multidrug-resistant isolate exhibited conversion from an AmpC phenotype to an ESBL phenotype with the transfer of only the IncN plasmid. The rate of resistance transfer to Salmonella or E. coli recipients was nearly identical. However, the ESBL phenotype was transferred with significantly greater prevalence to E. coli compared with Salmonella Newport (96% and 11%, respectively).

The identification of fungi collected from the ceca of commercial poultry.

Under normal conditions, fungi are ignored unless a disease/syndrome clinical signs are reported. The scientific communities are largely unaware of the roles fungi play in normal production parameters. Numerous preharvest interventions have demonstrated that beneficial bacteria can play a role in improving productions parameters; however, most researchers have ignored the impact that fungi may have on production. The goal of the present study was to record fungi recovered from commercial broiler and layer houses during production. Over 3,000 cecal samples were isolated using conventional culture methodology and over 890 samples were further characterized using an automated repetitive sequence-based PCR (rep-PCR) methodology. Eighty-eight different fungal and yeast species were identified, including Aspergillus spp., Penicillium spp., and Sporidiobolus spp, and 18 unknown genera were separated using rep-PCR. The results from the present study will provide a normal fungi background genera under commercial conditions and will be a stepping stone for investigating the impact of fungi on the gastrointestinal tract and on the health of poultry.

Salmonella Persistence within the Peripheral Lymph Nodes of Cattle following Experimental Inoculation.

Utilizing a transdermal method of inoculation developed in our laboratory, the duration of infection of Salmonella in the peripheral lymph nodes of steers was examined. Thirty-six Holstein steers (mean body weight of 137 kg) were inoculated with Salmonella Montevideo (day 0) on each lower leg and both sides of the back and abdomen. Calves were euthanized beginning at 6 h and subsequently on each of days 1, 2, 4, 7, 9, 11, 14, and 21 postinoculation (four animals each time). The subiliac, popliteal, and superficial cervical (prescapular) lymph nodes were collected and cultured (quantitatively and qualitatively) for the challenge strain of Salmonella. The challenge strain was detected via direct culture within the lymph nodes at 6 h postinoculation and on each subsequent necropsy date. Salmonella levels in lymph node were 0.8 to 1.8 log CFU/g. Lymph nodes were generally positive after enrichment culture throughout the experiment. Salmonella elimination appeared to begin approximately 14 days postinoculation. However, elimination was not completed by day 21; therefore, a second experiment was conducted identical to the first except that the time from inoculation to necropsy was extended. Salmonella was recovered via direct culture on each of the necropsy days, and results in general were similar to those of experiment I, except that on days 20, 24, and 28 isolates from serogroups C2 and E1 were identified in addition to the inoculation strain C1 in multiple animals. The data from both experiments indicate that after a single inoculation event, Salmonella would be completely cleared by approximately 28 days. Further research with expanded times between inoculation and necropsy is required for verification.

In Vitro Effects of Thymol-ß-D-Glucopyranoside on Salmonella enterica Serovar Typhimurium and Escherichia coli K88.

Although thymol is bactericidal against many pathogens in vitro, its in vivo effectiveness against pathogens in the lower gastrointestinal tract is limited because of its rapid absorption in the proximal gut. Thymol-ß-D-glucopyranoside (ß-thymol), a conjugated form of thymol, can deliver thymol to the lower gastrointestinal tract and has shown antibacterial effects. In the present study, we examined the in vitro effects of ß-thymol on Salmonella enterica serovar Typhimurium (ST) and Escherichia coli K88 (K88). We inoculated one-half strength Mueller-Hinton broth with 5.8 ± 0.09 log CFU/ml novobiocin- and naladixic acid-resistant (NN) ST (NVSL 95-1776) and 5.1 ± 0.09 log CFU ml(-1) NN-resistant K88, with or without porcine feces (0.1% [wt/vol]) (fecal incubations). The resultant bacterial suspensions were distributed under N2 to triplicate sets of tubes to achieve initial concentrations of 0, 3, 6, and 12 mM for ST treatments and 0, 3, 12, and 30 mM for K88 treatments. Samples were incubated at 39°C and then plated onto NN-containing brilliant green agar and NN-containing MacConkey agar; ST and K88 CFU concentrations were determined via 10-fold dilutions, and viable cell counts were performed at 0, 6, and 24 h. No differences in ST CFU counts were observed in ß-thymol-treated tubes without the added porcine feces (i.e., pure culture) at 6 or 24 h. However, in tubes that contained fecal incubations, ST CFU counts were reduced (P < 0.05) from controls at 6 h in tubes treated with 6 and 12 mM ß-thymol, whereas in tubes treated with 3, 6, and 12 mM ß-thymol the CFU counts were reduced (P < 0.05) at 24 h. No differences were observed in K88 CFU counts in pure culture or in fecal incubations at 6 h, but K88 CFU counts were reduced (P < 0.05) in both pure and fecal incubations at 24 h. The results from this study demonstrate that ß-thymol, in the presence of fecal suspensions, has anti-Salmonella and anti-E. coli effects, suggesting a role of ß-glycoside-hydrolyzing microbes for the release of bactericidal thymol from ß-thymol.