Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Note: Comparison of media for the isolation of lactose-positive Salmonella

We studied the capacity of 10 selective media (Rambach agar, RB; salmonella-shigella agar, SS; SM-ID medium, SM; Hektoen enteric agar, HE; modified semisolid Rappaport-Vassiliadis agar, MSRV; bismuth sulphite agar, BS; MacConkey agar, MC; brilliant green agar, BG; novobiocin-brilliant green-glucose agar, NBG; and novobiocin-brilliant green-glycerol-lactose agar, NBGL), and the C₈-esterase test (MUCAP test, Biolife, Italy) to detect the growth of 14 strains of lactose-positive Salmonella (12 Salm. virchow and two Salm. montevideo ) and 16 Salm. arizonae. Suspensions of pure strain were plated on the aforementioned media and on Mueller-Hinton, used as a control, with inocula of 3 x 10² cfu ml^-1. The performance of BS was excellent, determining the 30 strains as typical Salmonella colonies (H₂S+). On NBG, 27 strains were detected. On MSRV, only some strains grew and only one produced swarming. On the other media, the two Salm. montevideo and the 12 Salm. virchow strains produced coliform colonies. Some of these latter were inhibited on BG and NBGL. The 16 Salm. arizonae strains produced typical colonies on all the media, except on RB, SM and MSRV. On NBGL, two strains did not produce H₂S. The C₈-esterase test was only successful with Salm. montevideo and Salm. virchow on NBG and RB (with a few exceptions on the latter). However, with Salm. arizonae the test was positive on SS, MC, HE, BG and NBG. In summary, BS was the best medium of those used (all the 30 strains were isolated), followed by NBG (27 isolates).     

Evaluation of Rambach agar for the differentiation of Salmonella species from other Enterobacteriaceae

Rambach agar (Merck) was evaluated for its reliability as a selective diagnostic medium for the differentiation of Salmonella species from other Enterobacteriaceae. Twenty-five Salmonella strains were cultured on each of three agar media, Rambach (RAM), xylose lysine desoxycholate (XLD) and bismuth sulphite (BSA). Typical, easily interpreted reactions and colony morphologies were achieved for 23 strains on RAM and BSA and 17 on XLD. Of 135 other Enterobacteriaceae cultured on RAM, 134 gave characteristics which differentiated them from Salmonella . One strain which looked like Salmonella was identified as Citrobacter freundii . Rambach agar has potential as a supplementary agar in testing foods for Salmonella , but as with other selective diagnostic agars, it has limitations.     

Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella

In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars. Biochemical confirmation of such colonies can be expensive and time-consuming. It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative). Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive. Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method. A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing. The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.     

Evaluation of different plating media used in the isolation of salmonellas from environmental samples

Different serotypes of salmonellas were compared for selectivity and efficiency of recovery using 11 plating media. No optimal growth was obtained after 24h incubation in any of the media, but after 48h, brilliant green, brilliant green-phenol red-lactose-sucrose, bismuth sulphite, xylose-lysine-deoxycholate and Hektoen enteric agars showed optimal recovery of all the salmonella serotypes.
Xylose-lysine-deoxycholate and brilliant green-phenol red-lactose-sucrose agars were the most selective media for all salmonella serotypes. Addition of 10 μg/ml of sodium novobiocin to the tryptic soy-xylose-lysine and tryptic soy-brilliant green agars significantly improved their selectivity but reduced or inhibited the growth of some salmonella serotypes, including Salmonella typhi. Xylose-lysine-deoxycholate agar gave the highest recovery percentage of stressed salmonellas with a double-agar layer technique. Good recovery was also obtained on brilliant green-phenol red-lactose-sucrose, tryptic soy-brilliant green, tryptic soy-brilliant green-novobiocin, tryptic, soy-xylose-lysine and tryptic soy-xylose-lysine-novobiocin agars. Salmonella-shigella agar was the least efficient medium for the recovery of salmonellas under stress-induced or non-stressed conditions.     

Evaluation of different plating media used in the isolation of salmonellas from environmental samples

Different serotypes of salmonellas were compared for selectivity and efficiency of recovery using 11 plating media. No optimal growth was obtained after 24 h incubation in any of the media, but after 48 h, brilliant green, brilliant green-phenol red-lactose-sucrose, bismuth sulphite, xylose-lysine-deoxycholate and Hektoen enteric agars showed optimal recovery of all the salmonella serotypes. Xylose-lysine-deoxycholate and brilliant green-phenol red-lactose-sucrose agars were the most selective media for all salmonella serotypes. Addition of 10 micrograms/ml of sodium novobiocin to the tryptic soy-xylose-lysine and tryptic soy-brilliant green agars significantly improved their selectivity but reduced or inhibited the growth of some salmonella serotypes, including Salmonella typhi. Xylose-lysine-deoxycholate agar gave the highest recovery percentage of stressed salmonellas with a double-agar layer technique. Good recovery was also obtained on brilliant green-phenol red-lactose-sucrose, tryptic soy-brilliant green, tryptic soy-brilliant green-novobiocin, tryptic, soy-xylose-lysine and tryptic soy-xylose-lysine-novobiocin agars. Salmonella-shigella agar was the least efficient medium for the recovery of salmonellas under stress-induced or non-stressed conditions.     

Agar underlay method for recovery of sublethally heat-injured bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0. 05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60 degrees C for 1.5 min in buffer or 80 degrees C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

Isolation of Salmonellae and Shigellae from an Artificial Mixture of Fecal Bacteria

Numerous selective media, available commercially, act by suppressing "normal" bacterial inhabitants of the intestine while permitting the growth of so-called pathogenic representatives of the family Enterobacteriaceae. This investigation attempts to evaluate the action of Salmonella-Shigella (SS) agar, xylose lysine desoxycholate (XLD) agar, and hektoen enteric (HE) agar. Salmonellae and shigellae, isolated from clinical material, were mixed in various ratios with escherichiae, Klebsiella-Enterobacter-Serratia group bacteria, and members of the tribe Proteeae, also of clinical origin. Several of the mixtures were plated in multiple dilutions on the three media. Stools in preservative were also used for evaluation of the media after the addition of definite numbers of the pathogenic bacteria. Results indicate that SS agar suppresses the shigellae along with the autochthonous members of Enterobacteriaceae. XLD and HE agars readily permit the recovery of shigellae as well as salmonellae. This recovery is not obscured by the higher yield of other species obtained with these media.     

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

Evaluation of Pfizer selective enterococcus and KF media for recovery of fecal streptococci from water by membrane filtration.

Pfizer selective enterococcus (PSE) and KF agars were compared for their recovery of fecal streptococci from sewage effluent on membrane filters. The results showed that PSE agar is highly selective for the enterococci. The tan color resulting from esculin hydrolysis, which was not always visible on the surfaces of the colonies, is not considered a necessary differential characteristic on PSE agar since more than 90% of all colonies recovered on membrane filters were confirmed as fecal streptococci and 86% were confirmed as enterococci. The detection of esculin hydrolysis on membrane filters was not improved by using the new Millipore type HC filter. KF agar recovered significantly greater numbers of organisms but was not as selective, with 83% of the typical colonies being confirmed as fecal streptococci and 54% as enterococci. An attempt to improve the selectivity of KF agar while retaining its inclusiveness by incubation at 45 C was not successful.     

Comparison of seven plating media for enumeration of Listeria spp.

The suitability of seven media for the enumeration of Listeria spp. was evaluated at 30 degrees C for 48 h. The media tested were (i) the original McBride Listeria agar formulation (with glycine); (ii) modified McBride agar containing glycine anhydride; (iii) LiCl-phenylethanol-moxalactam (LPM) agar; (iv) acriflavine-ceftazidime agar; (v) Rodriguez isolation agar (RISA); (vi) modified Vogel-Johnson (MVJ) agar; (vii) cyclohexanedione-nalidixic acid-phenylethanol agar; and tryptose agar as control. A total of 66 organisms were used including 11 Listeria monocytogenes strains and 5 other Listeria spp. For L. monocytogenes strains only, all media performed highly similarly. Of the other Listeria spp., only two grew on MVJ agar and three each grew on LPM and RISA. Only LPM agar inhibited the 50 non-listeriae, including five yeasts, while MVJ agar inhibited all but one yeast. The McBride Listeria agar formulation that contained glycine anhydride was less selective than the original. When pure cultures of 10 bacteria (including one L. monocytogenes strain) were combined and plated on four media, L. monocytogenes colonies were easiest to enumerate on MVJ agar, followed by LPM and RISA. These media ranked in the same order when plated with homogenates of various foods to which was added L. monocytogenes Scott A, but LPM agar was the best overall since Scott A was inhibited by MVJ. Upon microscopic examination of listerial colonies from the plating media, atypical cell morphology was noted with cells being about twofold in size on LPM, MVJ, and acriflavine-ceftazidime agars. Overall, LPM agar was the most suitable of the media tested even though it was inhibitory to Listeria grayi and Listeria murrayi.     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.     

IMMEDIATE DIFFERENTIATION OF SALMONELLA-RESEMBLING COLONIES ON BRILLIANT GREEN AGAR

A rapid biochemical system (OBIS) based on immediate enzymatic differentiation of Citrobacter, Proteus, Providencia, Hafnia and Morganella spp. from Salmonella on brilliant green agar was evaluated. A total of 96 field isolates from various Salmonella serotypes, 18 Citrobacter freundii and 25 isolates of other Enterobacteriaceae were tested. All Salmonella isolates were identified correctly by the kit, and none of the Enterobacteriaceae isolates were identified as Salmonella. The results indicate complete specificity for Salmonella colonies on brilliant green agar.     

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p < 0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p > 0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p < 0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P < 0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.     

Evaluation of enhanced haemolysis agar for detection of Listeria spp. and L. monocytogenes from production lines of fresh to cold-smoked fish.

Enhanced haemolysis agar (EHA) was compared to the two conventional Listeria isolation agars Oxford and PALCAM for its ability to detect Listeria spp. from production lines of fresh to cold-smoked fish. The ability of EHA for distinguishing L. monocytogenes colonies from other Listeria spp. was also evaluated.A total of 243 fish and environmental samples were analysed. Overall, 42 samples were found to contain Listeria spp. Only 34 samples were positive simultaneously by the three plating media. Two samples considered to be negative by the two conventional agars were found to be positive after isolation on EHA. All three selective agars were shown to be less effective in recovering Listeria spp. after primary enrichment in half-Fraser broth, compared to secondary enrichment in Fraser broth after 24 and 48 h.From 79 Listeria but presumptive negative L. monocytogenes colonies, EHA identified correctly 76 Listeria spp. and presented three false-negative results_three colonies further identified as L. monocytogenes but showing no noticeable haemolysis on EHA. Twenty-three of the thirty-three L. monocytogenes presumptive positive colonies, were confirmed positive and ten were identified as L. seeligeri.Despite its ability of distinguishing L. monocytogenes from the other Listeria spp., unless it is produced as a commercial medium, EHA cannot be an alternative to time-consuming classical identification because the preparation of this medium is both time and labour intensive.     

Comparison of methods for the isolation of Salmonella from imported frog legs.

Four methods of sample preparation were compared for their relative efficiency in recovering Salmonella from imported frog legs. No significant difference (P greater than 0.10) was observed in the efficiency of submersion, blending, and stomaching methods, but rinsing recovered significantly fewer (P less than 0.01) Salmonella-positive frog legs than the other three methods. No significant difference (P less than 0.25) was observed in the number of positive frog legs recovered by selenite cystine or tetrathionate broth. Salmonella-Shigella agar, when streaked from either of these broths, gave significantly fewer (P less than 0.01) Salmonella-positive frog legs than brilliant green, bismuth sulfite, xylose lysine deoxycholate, and Hektoen-enteric agars. Use selective agars, resulted in detection of an additional 11 and 6 Salmonella-positive frog legs, respectively. A variety of serotypes, mostly uncommon, was recovered. One Salmonella serotype (6,14,24:r,i:e,n,z15), possessing a heretofore unreported antigenic formula, was isolated.     

Comparison of methods for the isolation of Salmonella from imported frog legs.

Four methods of sample preparation were compared for their relative efficiency in recovering Salmonella from imported frog legs. No significant difference (P greater than 0.10) was observed in the efficiency of submersion, blending, and stomaching methods, but rinsing recovered significantly fewer (P less than 0.01) Salmonella-positive frog legs than the other three methods. No significant difference (P less than 0.25) was observed in the number of positive frog legs recovered by selenite cystine or tetrathionate broth. Salmonella-Shigella agar, when streaked from either of these broths, gave significantly fewer (P less than 0.01) Salmonella-positive frog legs than brilliant green, bismuth sulfite, xylose lysine deoxycholate, and Hektoen-enteric agars. Use selective agars, resulted in detection of an additional 11 and 6 Salmonella-positive frog legs, respectively. A variety of serotypes, mostly uncommon, was recovered. One Salmonella serotype (6,14,24:r,i:e,n,z15), possessing a heretofore unreported antigenic formula, was isolated.     

Detection of Salmonella by indicator agar media and PCR as affected by alfalfa seed homogenates and native bacteria

AIMS: To investigate and prevent the undesirable effect of native bacteria and alfalfa seed homogenates on detection of Salmonella in alfalfa seeds by indicator agar media and polymerase chain reaction (PCR). METHODS AND RESULTS: The relative sensitivity of five indicator agar media, including modified semisolid RV (MSRV), xylose-lysine-Tergitol 4 (XLT4), Hektoen enteric agar (HEA), brilliant green agar (BGA) and bismuth sulphite agar (BSA), for detection of Salmonella in the presence of a large number of native bacteria from alfalfa seeds was examined. The detection limit as measured by the ratio between the numbers of native bacteria and Salmonella was estimated to be 10(6) to 1 for MSRV and 10(3) to 1 for XLT4, HEA, BGA or BSA. Presence of alfalfa seed homogenates markedly reduced the sensitivity of Salmonella detection by PCR. The minimal number of Salmonella detectable by PCR was determined to be 1-10 and 100-1000 CFU in the absence and presence of seed homogenate, respectively. Application of anti-Salmonella immunomagnetic beads permitted detection of 2-5 CFU of heat-injured cells in 25 g of seeds within 24 h by PCR. CONCLUSIONS: The MSRV medium is more sensitive than other indicator agars for detecting a small number of motile Salmonella in samples containing a large number of native bacteria. Application of immunomagnetic beads eliminates the PCR-inhibitory activity of seed homogenates and improves the detection of Salmonella in inoculated seeds. SIGNIFICANCE AND IMPACT: The results generated from this study will aid the seed distributors, sprout growers and public health officials to identify and recall the Salmonella-contaminated seed lots to be used for sprout production.     

Lactose-Fermenting Salmonella from Dried Milk and Milk-Drying Plants

A study of 552 salmonella cultures revealed that 86 (15.6%) of the cultures fermented lactose. These had been isolated from dried milk products and milk-drying plants. Acid and gas were produced in lactose broth. Solid media containing lactose as the key ingredient for the differential reaction were not satisfactory for recognizing salmonella colonies. No problem was encountered in selecting salmonella colonies when bismuth sulfite agar was used.