Differential susceptibility of aeromonads and coliforms to cefsulodin.

Cefsulodin was evaluated as a potential selective agent for aeromonads. Resistance of Aeromonas and coliform isolates was determined by using a standard disk diffusion technique. A total of 119 Aeromonas and 78 coliform strains were isolated. For 102 of 130 [corrected] Aeromonas isolates (environmental and reference strains), the MIC of cefsulodin was < 8 micrograms/ml. Results of MIC tests by the agar dilution method showed that a concentration of cefsulodin of 10 micrograms/ml or less inhibited the growth of 96% of isolates. In comparison, for 81 of 94 coliform isolates (environmental and reference strains), the MIC of cefsulodin was > 32 micrograms/ml. Because cefsulodin suppresses growth of Aeromonas and other oxidase-positive organisms, total coliform (TC) and Escherichia coli counts on Chromocult Coliform agar (CC agar) without cefsulodin and on CC agar with 10 mg of cefsulodin per liter (CC-CFS) were compared. Variance analysis of data from 14 sewage-polluted irrigation water specimens did not demonstrate any statistically significant difference in the enumeration of E. coli with CC and CC-CFS media. On average, the CC agar recovered 2.46 times as many TCs as CC-CFS. However, Aeromonas colonies made up an average of 58.6% of the TC counts on CC agar. Because no Aeromonas spp. were recovered on CC-CFS, background interference was eliminated and the counts that were obtained reflected more accurately the number of TCs. Results of this study suggest that cefsulodin may be a useful selective agent against Aeromonas spp. which should be included in coliform chromogenic media when high levels of accompanying flora are expected.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas. Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 micrograms/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas. The best temperature for the recovery of aquatic aeromonads was 28 degrees C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95.5% of 28 colonies tested with an Aeromonas-like morphology belonged to the genus Aeromonas. Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28 degrees C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas . Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 μg/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas . The best temperature for the recovery of aquatic aeromonads was 28°C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95·5% of 28 colonies tested with an Aeromonas -like morphology belonged to the genus Aeromonas . Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28°C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

The suitability of four media for enumerating Aeromonas spp. from environmental samples

Starch ampicillin agar (SA), dextrin fuchsin sulphite agar (DFS) and blood agar with 10 mg (BA-10) and 30 mg (BA-30) ampicillin/l, respectively, were evaluated for enumeration of Aeromonas spp. from environmental samples. Recovery from pure cultures was excellent on all media except for ampicillin-sensitive strains on the ampicillin-containing media. With natural samples, the ability to differentiate Aeromonas from the background microflora was best on SA agar where 85% presumptive Aeromonas colonies were confirmed, compared with 18% on DFS, 36% on BA-10 and 40% on BA-30. Prolonged incubation caused a decrease in the differentiating ability.     

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen? AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen? AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen? AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen? AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30°C under aerobic conditions, and specificity was high (i.e. confirmation rate usually <90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/1.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30 degrees C under aerobic conditions, and specificity was high (i.e. confirmation rate usually greater than 90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/l.     

Isolation and enumeration of Serratia entomophila—a bacterial pathogen of the New Zealand grass grub, Costelytra zealandica

Several agar media were tested for their use in a selective isolation and identification scheme for Serratia entomophila , a bacterium causing amber disease of the New Zealand grass grub, Costelytra zealandica (White). Soil dilutions were plated on caprylate thallous agar (CTA), selective for Serratia spp. Most strains of Ser. entomophila grew well on CTA; the mean efficiency of colony formation on CTA was 94 ± 3% of that on a non-selective medium. The identity of colonies growing on CTA was determined on the basis of their growth reactions on DNase-toluidine blue agar, adonitol agar and itaconate agar. Serratia entomophila could be distinguished from other Serratia spp. found in New Zealand soils, in particular Ser. proteamaculans , another causal agent of amber disease of grass grub. The identification scheme allowed the selective recovery of Ser. entomophila from field soils containing a diverse microflora.     

A comparison of different culture media for the membrane filter quantification of Aeromonas in water

A comparative assessment of culture media for the membrane filter enumeration of Aeromonas spp. in water was performed, testing the effects of different incubation conditions (aerobic and anaerobic), temperatures (30 and 37 degrees C) and times (24 and 48 h). Different water samples seeded with test suspensions of Aeromonas spp., fecal material or raw sewage were examined. Results indicate clearly that plates should be incubated aerobically at 30 degrees C for 24 h. If the bacterial contamination is likely to be low, the use of most sensitive culture media, such as SAA, mA, ADA or PADE Agar, is recommended. By contrast, samples with an expected high level of background microbial flora should be analysed through more selective media, such as MIX Agar. However, the low selectivity of all media tested and the high likelihood of false negatives based upon the macroscopic examination of colonies means that further research directed to the development of more efficient media is needed.     

Comparison of different media for the enumeration of Aeromonas sp. in freshwaters

I. KERSTERS, N. SMEYERS AND W. VERSTRAETE. 1996. Ampicillin-dextrin agar (ADA), m-Aeromonas agar (mA), starch glutamate ampicillin penicillin C-glucose agar (SGAP-10C), trehalose agar (TRE) and ampicillin bile salts inositol xylose agar (MIX) were evaluated for the isolation of Aeromonas sp. from aquatic environments. Recovery of pure cultures was excellent on all media, except for Aer. sobria on SGAP-10C and MIX agars. Recovery of Aeromonas sp. from freshwaters was comparable on ADA, mA, SGAP-10C and TRE. The most selective media were SGAP-10C and ADA, which yielded an average reduction factor of more than 2.9 log. The ability to differentiate Aeromonas sp. from the background microbiota present in freshwaters was best on ADA. The present findings indicate that ADA is the most adequate medium for the selective isolation of Aeromonas sp. from freshwaters.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25 degrees C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22 degrees C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22 degrees C and plating on CIN agar (48 h at 25 degrees C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

Relation between Aeromonas and faecal coliforms in fresh waters

A possible correlation between the presence of mesophilic aeromonads and the number of faecal coliforms present in three fresh-water habitats subject to differing levels of faecal pollution was investigated. Concentration of Aeromonas spp. between 10(2)-10(9) cfu/100 ml and faecal coliforms of between 9-10(7) cfu/100 ml were found in the waters. In water free from faecal pollution there was no correlation but in polluted waters there was a significant relationship between the numbers of aeromonads, faecal coliform and the concentration of organic matter measured by biological oxygen demand.     

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.     

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.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

A selective and differential medium for Vibrio harveyi.

A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V. harveyi. It is possible to differentiate V. harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium. This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp. and two strains of marine Flavobacterium spp. but to allow the growth of Photobacterium strains. Colonies displaying typical V. harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V. harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V. harveyi. This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V. harveyi from other marine and estuarine Vibrio species.     

A new cost-effective, selective and differential medium for the isolation of Cronobacter spp

The objective of the present study was to develop a new selective, differential and cost-effective medium (Kim and Rhee — KR-medium) for the isolation of Cronobacter spp. In this new medium, which contained salicin as a differential agent, Cronobacter spp. generated typical colonies with characteristic violet-colored centers surrounded by a transparent to opalescent border, and the growth of other microorganisms (40 strains) was inhibited or produced visually distinguishable colonies. Using healthy and heat- and desiccation-injured cells, the quantity of nutrients was adjusted to determine the optimal recovery rate, selectivity, differentiation and cost-effectiveness. Peptone and salicin concentrations were established as 10 and 8 g/L, respectively. The KR medium was then validated using salicin fermenting organisms, including Cronobacter spp. (52 strains), Enterobacter cloacae (50 strains) and Klebsiella pneumonia (10 strains) isolated from clinical and food specimens. All strains of Cronobacter spp. produced typical colonies and other salicin fermenting organisms were easily distinguishable from Cronobacter spp. with the exception of 2 E. cloacae strains. The verification of KR medium was carried out in powdered infant formula artificially inoculated with healthy, heat-injured, and desiccation-injured Cronobacter spp. and the expected typical colonies were appeared. The KR medium had a high specificity (98%) and sensitivity (100%), with no false-negative results. Moreover, we show that the cost of the KR medium is much lower than that of other selective and differential media. The use of the KR medium for the selective isolation of Cronobacter spp. in laboratories and food industry settings may therefore lessen the financial burden of Cronobacter spp. detection.