Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar Typhimurium DT104.

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37 degrees C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37 degrees C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25 degrees C than at 37 degrees C. Since the CP was shown to be produced at both 37 degrees C and 25 degrees C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.

Evaluation of chemical disinfectants for the elimination of Salmonella biofilms from poultry transport containers.

Containers used in transporting live poultry between production and processing units are a primary source of contamination for processed poultry products. Because disinfection of transport containers (TC) has been difficult to accomplish, it is probable that the choice of appropriate disinfectant and its application are partially or wholly responsible for the failure to adequately eliminate pathogens from TC. Therefore, 13 commercial disinfectants were selected and evaluated for their capacities to destroy Salmonella. The disinfectants were applied in various concentrations on prescribed areas (10 cm diameter circle) of galvanized steel surfaces (representative of TC material) that were artificially contaminated with Salmonella (10(8) cfu/mL) with a mixture of organic material. Likewise, coupons (1.9 cm2) made of the same metallic surfaces and covered with biofilms of Salmonella spp. were tested in the same manner for each disinfectant. Two of the disinfectants completely eliminated Salmonella on the artificially contaminated and biofilm-covered surfaces. These compounds produced logarithmic reductions in Salmonella populations as high as 7.18 within 2 min. One of the two effective disinfectants contained sodium hypochlorite and was effective at a concentration of 0.05% (vol/vol). The other disinfectant was an alkaline peroxide compound and was effective at a concentration of 1% (wt/vol). Evaluation of these two disinfectants under simulated conditions suggested that application under the prescribed regimen could result in effective elimination of Salmonella from TC within a limited period.

High-frequency recovery of quinupristin-dalfopristin-resistant Enterococcus faecium isolates from the poultry production environment.

The occurrence of resistance to the streptogramin quinupristin-dalfopristin in Enterococcus faecium isolates from chickens on the Eastern Seaboard, was evaluated. Quinupristin-dalfopristin resistance was found in 51 to 78% of E. faecium isolates from the food production environment. The high level of resistance in this organism suggests that this reservoir of resistance may compromise the therapeutic potential of quinupristin-dalfopristin.

Implications of multiple antimicrobial-resistant enterococci associated with the poultry environment.

Poultry are increasingly being associated with carriage of multiresistant organisms that may cause disease in humans. Among these organisms are the enterococci, not regarded as a common cause of hospital-acquired infections. The use of antimicrobials for growth promotion in poultry production envronments may facilitate the dissemination of resistance to Enterococcus spp. that have the potential to be clinically significant. To assess descriptively the degree of multiresistant enterococci in the poultry environment of the Delmarva (Delaware-Maryland-Virginia) East Coast region of the USA, litter samples from regional commercial poultry houses and transport container swabs from processing facilities were cultured for Enterococcus spp. Using a microtiter plate adaptation of a conventional biochemical screen, the predominant species identfied were E. faecalis (61.2%), E. faecium (18.6%) and E. gallinarum (2.4%). Resistance to the cephalosporin, macrolide and tetracycline classes of antimicrobials was uniform with broader resistance to penicillin and derivatives present in a majority of E. faecium isolates. High-level streptomycin resistance was evident in close to 30% of all isolates with a majority of E. faecalis variants possessing resistance. High-level gentamicin resistance was detected at a low frequency (2.6%) only within the E. faecalis group with resistance to low-level gentamicin levels present in a majority of both the E. faecalis group and subsets of E. faecium. No unexpected vancomycin resistance was detected. Of particular interest was resistance to the streptogramin quinupristin-dalfopristin (Q-D or Synercid), which was present in 70.4% of E. faecium and E. faecium variants.

Characterization of the contribution of water activity and moisture content to the population distribution of Salmonella spp. in commercial poultry houses.

Because Salmonella spp. can be spread from the production environment to the consumer, strategies are required to control Salmonella. One such intervention involves control of the distribution of the organism in poultry litter. In this regard, we have attempted to determine whether Salmonella spp. are uniformly distributed throughout the litter of commercial poultry houses, or if they are unevenly localized to "hot spots" associated with high surface litter water activity (Aw) and high total moisture levels. Of the 86 houses sampled, 48 (55.8%) were positive for Salmonella spp. using a combination of the drag swab and targeted litter culturing methods. Data are presented that show that elevated Aw and percentage moisture content values representative of targeted litter samples or whole-house moisture status are not statistically predictive of Salmonella contamination, using either drag swab or targeted litter culture results as indicators of contamination. This study suggests that favorable environmental conditions for the growth of Salmonella are unequally dispersed in poultry houses. Because there is not equivalent distribution of salmonellae in the surface litter, the drag swab technique is apparently necessary to adequately survey for Salmonella spp. contamination. The findings further suggest that the development of a methodology to detect areas within houses that possess risk factors favorable for Salmonella growth must also include elevated Aw and percentage moisture content levels. This capability may enable a grower to detect and intervene in these targeted areas with neutralizing procedures, agents, or other substances to provide significant reduction of Salmonella or other poultry-associated food-borne pathogens.

A colony lift immunoassay for the specific identification and quantification of Listeria monocytogenes.

A colony lift immunoassay (CLI) has been developed to detect Listeria monocytogenes after the organisms have been cultured on filter membranes or agar plates. Polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA), used in the CLI, were prewet with methanol and used to imprint colonies that were grown on the filter or agar plates. A positive control was applied to the edge of each membrane. The imprinted membranes were subsequently air dried, peroxidase neutralized, blocked, and reacted for 20 min with a 2-microg/ml unconjugated Mab EM-7G1 solution. The membranes were washed briefly and reacted for 30 min with a 1:2000 dilution of a commercially prepared peroxidase-labeled goat anti-mouse secondary antibody (Kirkegaard and Perry Laboratories (KPL), Gaithersburg, MD). After a second wash step, the membranes were exposed to a 3,3',5, 5'-tetramethylbenzidine membrane substrate (KPL), rinsed in deionized water, and allowed to dry. Colonies of L. monocytogenes were identified by a blue color reaction on the membrane, which could be used to reference the colonies either on the filter membranes or agar plates. The CLI was tested against a wide range of Listeria species as well as several non-Listeria species and was shown to have a high degree of sensitivity (96%) and specificity (90%). We have shown that it is useful as a simple and rapid method to detect and identify L. monocytogenes.

Development and evaluation of a 24-hour method for the detection and quantification of Listeria monocytogenes in meat products.

A 24-h filter monitor-based test, Listeria-SELeCT, has been developed to quantify Listeria monocytogenes organisms in meat samples with a sensitivity of < or = 1.0 CFU/g. The technique comprises a filter monitor-based system and a colony lift immunoassay to identify and enumerate the target organism. Meat homogenates were centrifuged and the eluate was filtered to trap and immobilize the microorganisms on the filter. Fraser broth was then added to the filter apparatus to allow the organisms to become established overnight and to inhibit contaminants, after which the filters were transferred onto Modified Oxford medium agar, a selective medium for L. monocytogenes. After 10 to 12 h, a colony lift immunoassay was used to confirm and enumerate suspect colonies on the filter. A correlation study between the Listeria-SELeCT method and the most probable number technique showed the Listeria-SELeCT to be considerably more accurate than the most probable number for quantitatively determining the number of viable organisms in meat samples. Because of ease and speed of testing, the Listeria-SELeCT system also provided major advantages over the most probable number technology.

Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 +/- 1 degree C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3',5,5'-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling.

Prevalence of Salmonella in broiler flocks: effect of litter water activity, house construction, and watering devices.

Litter samples from 24 flocks of broilers and four flocks of broiler breeders were evaluated for Salmonella contamination, water activity (Aw), and total moisture content (MC). The drag swab (DS) monitoring system was used to collect samples to detect Salmonella contamination. Simultaneously, representative samples of the uppermost surfaces of dry (loose) litter and wet (caked) litter were collected for Aw and MC analyses. On dry litter surfaces, high Aw values (0.90-0.95) were associated with flocks Salmonella-positive using DS; low Aw values (0.79-0.84) were associated with flocks Salmonella-negative by DS; and transition Aw values (0.85-0.89) were associated with flocks having an increased risk for the presence of Salmonella. The association of high Aw values with Salmonella risk was not observed for wet (caked) litter surfaces. Observations suggest that limiting Aw in the litter base of broiler houses may create a less favorable environment for the multiplication of Salmonella and thus a more hygienic environment for broiler production.

Evaluation of possible alternatives to double-strength skim milk used to saturate drag swabs for Salmonella detection.

The drag-swab Salmonella screening technique was evaluated using less expensive alternatives to double-strength skim milk (2 x SM) as a saturating medium for drag swabs. Ten pre-determined Salmonella-positive poultry houses were studied. In the first phase, Salmonella screening efficiency of drag swabs impregnated with 2 x SM and commercially available canned Carnation evaporated skim milk (CESM) were compared. Results showed CESM to be a less efficient alternative. In the second phase of the study, the Salmonella screening efficiency of drag swabs impregnated with 2% buffered peptone water (BPW), physiological saline (PS), and distilled water (DW) were evaluated along with an unimpregnated drag swab (dry drag swab) (DD) as possible alternatives to 2 x SM. The efficiency of Salmonella detection using various impregnation treatments were in the following order: 2 x SM > PS > BPW > DW > DD.

Correlation of water activity and other environmental conditions with repeated detection of Salmonella contamination on poultry farms.

Three flocks on 13 different broiler farms were monitored for Salmonella over three consecutive growout periods using the drag swab (DS) technique. One house was consistently negative for Salmonella contamination (7.7%); four houses were consistently positive (30.8%); and eight houses (61.5%) alternated between either a DS Salmonella-negative or -positive status. Simultaneously, numerous environmental parameters of the litter surface were measured, including water activity (Aw), ammonia, temperature, pH, moisture content (MC), ash content, and volatile solids. Analysis of these data as a corollary to either Salmonella-negative or -positive DS results revealed significant correlation coefficients for some of the parameters, especially Aw. The results suggest that there should be further exploration of remedial intervention based on control of some of the physical features of litter (e.g., controlling litter Aw and possibly MC and pH levels) in poultry houses.

The effect of exposure, storage times, and types of holding media on the drag-swab monitoring technique for Salmonella.

Four maintenance media were compared for the preservation of the sensitivity of drag swabs and to assess the survival of Salmonella spp. on drag swabs at reduced temperatures. The effects of Difco double-strength skim milk (2 x SM), 2% buffered peptone water (BPW), a modified liquid Cary-Blair transport medium (LCB), and lactose broth holding medium were compared, as were storage periods of various lengths. The results with enzyme-linked immunosorbent antigen capture assay and highly selective plating media detection systems showed that 2 x SM had the highest level of recovery of salmonellae after prolonged storage, both under refrigeration at 4 C for 3 days and under frozen conditions of -15 C for 2 weeks.