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Applied and Environmental Microbiology, July 2005, p. 3524-3527, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3524-3527.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a Simple and Rapid Fluorogenic Procedure for Identification of Vibrionaceae Family Members

Gary P. Richards,^1* Michael A. Watson,¹ and Salina Parveen^2,

United States Department of Agriculture, Agricultural Research Service, Microbial Food Safety Research Unit, Delaware State University, Dover, Delaware,¹ Department of Agriculture and Natural Resources, Delaware State University, Dover, Delaware²

Received 3 November 2004/ Accepted 31 January 2005

Top Abstract Introduction Materials and Methods Results and Discussion References

 
We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The Vibrionaceae family contains a broad group of human and animal pathogens within the genera Vibrio, Aeromonas, Photobacterium, and Plesiomonas. Two other genera have been recommended to be placed in the Vibrionaceae family: Shewanella (9) and Listonella (8, 9). In screening for bacterial-virulence-enhancing enzymes, we recently identified and characterized a lysyl aminopeptidase (LysAP) activity associated with phosphoglucose isomerase (PGI) of Vibrio vulnificus (12). Subsequently, we demonstrated that PGI with LysAP activity (PGI-LysAP) hydrolyzed the amino-terminal lysyl residue from des-Arg¹⁰-kallidin, converting it to des-Arg⁹-bradykinin (11). Kinin metabolites are known to enhance virulence and mediate inflammatory reactions (10). Kinins and their metabolites influence a host of physiological functions, including inflammation, vasodilation, vascular permeability, and the contraction and relaxation of smooth muscle (2, 14). We also identified PGI-LysAP in chromatographically purified fractions from eight species of Vibrio (13). Weak LysAP activity, but no isomerase activity, was detected in purified preparations of Aeromonas hydrophila, Aeromonas veronii, and Plesiomonas shigelloides (13). No LysAP activity could be detected in non-Vibrionaceae pathogens (13).

Vibrio PGI-LysAP cleaves the amino-terminal lysyl residue from the synthetic substrate L-lysyl-7-amino-4-methylcoumarin, and enzyme activity can be measured spectrophotometrically (12). To date, the detection of PGI-LysAP has required substantial efforts to obtain and analyze the enzyme with chromatographic and spectrophotometric techniques (12). We sought to develop a rapid, simple, and inexpensive procedure to identify and enumerate members of the Vibrionaceae family without the need for sophisticated instrumentation. This paper reports on the development of a colony overlay procedure for peptidases (COPP) which may be used to identify and quantify a broad range of Vibrionaceae in food, environmental, and clinical samples based on the rapid and simple detection of PGI-LysAP.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains and culturing.
Sources for the bacterial cultures used in this study are as previously described (13), except for the Vibrio cholerae strains, which are listed in Table 1, and Proteus vulgaris and Pseudomonas aeruginosa, which were cultures 13315 and 10145, respectively, from the American Type Culture Collection, Manassas, VA. Non-Vibrio pathogens (Bacillus cereus, Enterobacter aerogenes, Escherichia coli O157:H7, Klebsiella pneumoniae, Listeria monocytogenes, Proteus mirabilis, P. aeruginosa, Salmonella enterica serovar Typhimurium, S. enterica serovar Enteritidis, Staphylococcus aureus, and Yersinia enterocolitica) were streaked onto plates of tryptic soy agar (TSA; Difco Laboratories, Detroit, MI). Vibrionaceae, including eight Vibrio spp., two Aeromonas spp., and Plesiomonas shigelloides, were streaked onto TSA containing 1% NaCl (TSA-N). All isolates were incubated at 37°C for 16 to 18 h.

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TABLE 1. Vibrio cholerae strains used in this study

Preparation of membranes containing synthetic substrate.
A stock solution of 20 mM L-lysyl-7-amino-4-trifluoromethylcoumarin (L-Lys-AFC) (catalog no. AFC-08; Enzyme Systems Products, Livermore, CA) was prepared in dimethyl sulfoxide and stored at –20°C. Cellulose acetate membranes (8 by 15 cm) (catalog no. 12200-78-150-K; Sartorius, Goettingen, Germany) were quickly (1 to 2 s) submerged, one at a time, into a solution containing 125 µl of L-Lys-AFC stock per 10 ml of distilled water, soaked for about 30 s, removed, and dried. Each membrane absorbed 2 ml of the solution. Small metal binder clips taped to the lower side of an overhead cabinet were convenient for hanging the membranes to dry. Once thoroughly dried, the membranes were placed in an opaque envelope and stored at room temperature. The shelf life of the prepared membranes was determined using the COPP procedure, as described below, by overlaying colonies of V. vulnificus MLT364 with membranes that had been prepared monthly for 6 months and then comparing the fluorescence intensities.

Colony overlay procedure for peptidases.
TSA and TSA-N plates were streaked or spread plated with the appropriate bacteria and incubated overnight at 37°C. Previously prepared membranes (1 month old) containing L-Lys-AFC were cut to a size appropriate for the area to be overlaid, labeled accordingly, and prewet for 5 s in 20 mM Tris-HCl, pH 9.5. While handling with forceps, excess buffer was dripped from the membranes for 3 to 5 s, and the membranes were carefully placed onto one or more colonies on the agar plates. Care was exercised to prevent bubbles from becoming trapped under the wet membranes. Plates were incubated for 10 min at 37°C without inversion. Each membrane was then removed with forceps and placed on a petri dish, colony side up. The dish was placed on a UV light box, and the membrane was viewed at 364 nm for fluorescent foci at the point of contact between the bacterial colony and the membrane. Membranes were photographed through a deep yellow filter no. 15 (Tiffen Manufacturing Corp., Hauppauge, NY) with a Polaroid camera (Polaroid Corp., Cambridge, MA) on a UV light box with Polaroid 667 film at an F stop setting of 4.5 and exposure for 1/8 to 1/30 s for a permanent record of the results. Results could also be recorded with a digital camera.

Dot blots.
For the comparison of fluorescence intensities among the Vibrionaceae and the non-Vibrio pathogens, templates were made with graph paper by marking a grid to represent columns and rows. The template was taped to the outside bottom of the TSA and TSA-N plates. Isolated colonies were stabbed on the plates according to markings on the template. Each plate was incubated overnight and subjected to the COPP.

Mixed cultures.
Sandy loam, raw sewage, oyster homogenates, and seawater were also subjected to the COPP assay. Sandy loam from a local (Dover, Delaware) flower garden and raw sewage from a municipal sewage treatment plant in Harrington, DE, were tested to determine the prevalence of fluorescence from organisms likely to be nonvibrios. The soil was diluted to a ratio of 1:5 with phosphate-buffered saline, vortexed for 15 s, and centrifuged for 5 min at 200 x g. Supernatant and 10-fold dilutions in phosphate-buffered saline were spread onto TSA plates (100 µl/plate), incubated overnight, and overlaid as described above. Oysters obtained from state-approved shellfish harvesting beds in New Jersey were collected and immediately transported to the laboratory, shucked under aseptic conditions, diluted to a ratio of 1:10 with 0.1% peptone, homogenized, and serially diluted in 0.1% peptone, and 100 µl of each dilution was spread plated onto TSA-N. One hundred microliters of seawater and 10-fold dilutions of seawater were also spread plated onto TSA-N. All plates were incubated overnight at 37°C and overlaid according to the COPP technique.

Bacterial identification.
Representative bacterial colonies from shellfish, seawater, and sewage were identified biochemically using API 20E and oxidase tests (BioMerieux Industries, Hazelwood, MO), according to the manufacturer's instructions, to determine the relative percentages of fluorescent and nonfluorescent isolates that were in the Vibrionaceae family. Colonies on the primary plates were dotted onto duplicate TSA-N plates and incubated overnight. The COPP assay was performed on one set of plates, and colonies producing fluorescent foci were picked from the duplicate plate for API 20E identification. Likewise, nonfluorescent colonies were picked from the duplicate plate and Gram stained, and gram-negative isolates were identified using API 20E. A total of 170 colonies were picked for identification from oysters and seawater cultures. For sewage, all eight isolates producing fluorescent foci were subjected to API 20E identifications.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The COPP procedure successfully discriminated between the Vibrionaceae and the non-Vibrionaceae. Pure cultures of clinical and environmental strains of the Vibrionaceae family dotted onto TSA-N plates all produced strong fluorescent foci on the membrane overlays (Fig. 1). Nine strains of V. cholerae, including serogroups O1, O139, O22, and O155 (Table 1), were also strongly positive. Fluorescent foci were not observed for the non-Vibrionaceae pathogens, except for a very weak and diffuse fluorescence occasionally associated with Enterobacter aerogenes and E. coli cultures that had been dotted onto plates (Fig. 1) and with K. pneumoniae and P. vulgaris cultures which were overlaid directly from a streaked culture (not shown). The diffuse fluorescence was easily discounted and was attributed to other lysyl aminopeptidases present in cell lysates, since activity was not present in chromatographic fractions from these species (13). The overlay of V. vulnificus with membranes that were prepared 0 to 6 months previously showed that membranes could be stored up to 2 months with only a slight loss of fluorescence intensity. Older membranes did not produce fluorescence.

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FIG. 1. Colony overlay procedure for peptidases showing PGI- LysAP activity in Vibrionaceae and non-Vibrio pathogens after bacterial isolates were stabbed onto a TSA-N plate and incubating for 24 h. Overlay was for 10 min, and the membrane was photographed while wet on a UV light box at an F stop setting of 4.5 for 1/30 s. Bacteria are as follows (left to right): (A) V. vulnificus strain MLT367, V. vulnificus strain MLT1003, V. cholerae O1, V. cholerae O139, V. parahaemolyticus (Kanagawa positive), V. parahaemolyticus (Kanagawa negative), Vibrio fluvialis; (B) Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus, Aeromonas hydrophila, Aeromonas veronii, Plesiomonas shigelloides, E. coli O157:H7; (C) Enterobacter aerogenes, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, Yersinia enterocolitica, Staphylococcus aureus, and Listeria monocytogenes. The last space in row C is blank.

The COPP assay was performed on oyster homogenates, seawater, sandy loam, and raw sewage. An overnight culture of diluted oyster homogenate on TSA-N (Fig. 2A) and the corresponding membrane overlay (Fig. 2B) show strongly fluorescent foci (Fig. 2B) associated with nearly all of the bacteria isolated from the oysters. Of 170 natural isolates from seawater and oyster cultures that were picked for biochemical identification over a 2-month period, 120 produced fluorescent foci. Only 93 of these could be identified by API 20E, and 87 (93.5%) were identified as Vibrionaceae, including Shewanella, whereas 6 (6.5%) were identified as Chryseobacterium (Flavobacterium) meningosepticum, a bacterium which is not presently classified as a Vibrionaceae family member but which causes cellulitis and tissue damage (1, 3), much like other Vibrionaceae (4, 5, 7, 15). The most prevalent Vibrionaceae family member detected was V. vulnificus, followed by Shewanella putrefaciens, Aeromonas spp., Photobacterium damselae, and V. parahaemolyticus. A lack of discrimination in some assays allowed a portion of the isolates to be identified only to the genus level. Among these, the API 20E listed several isolates as Vibrio spp. or Aeromonas spp. Only 13 of 50 nonfluorescent isolates could be identified by API 20E, and none were Vibrionaceae. They included Shigella spp., Pasteurella multocida, Morganella morganii, Pseudomonas spp., and Stenotrophomonas maltophila. Some of the isolates were gram positive and were not identified. The inability to identify some of the isolates by API 20E is commonly experienced for environmental samples, since API 20E was primarily designed for detecting human pathogens in clinical specimens. Bacterial colonies from garden soil (Fig. 2C) did not produce fluorescence when overlaid; however, three fungal colonies (Fig. 2C) produced strong fluorescence (Fig. 2D). Approximately 15% of the isolates obtained from raw municipal sewage produced fluorescent foci and were identified as A. hydrophila isolates. No other colonies producing fluorescent foci were detected in sewage. The quantification of Vibrionaceae in shellfish and seawater was accomplished with the COPP assay by the overlay of primary spread plates prepared with known amounts of sample. Obviously, quantification is not possible from the overlay of picked or streaked cultures.

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FIG. 2. TSA-N plate of oyster homogenate (A) and TSA plate of sandy loam supernatant (C) after incubation for 24 h. Plates were overlaid with membranes containing L-Lys-AFC for 10 min, and the resulting membranes were photographed (B and D) while wet on a UV light box at an F stop setting of 4.5 for 1/8 s. Panel C shows three fungal colonies from the TSA plate (arrows) that produced the fluorescent foci seen in panel D.

Several factors are critical to the success of the COPP technique. Cultures grown on TSA-N should be 24 h old and the cell density should be light to moderate, since older or overgrown cultures may acidify the medium, thus preventing PGI-LysAP from cleaving its substrate. Previously, we showed that the optimal pH for PGI-LysAP activity in V. vulnificus was 8.0, with a secondary peak activity at pH 9.5 (12). We also demonstrated that at pH 7.0, enzyme activity was only 20% of the activity obtained at pH 8.0 (12). If the COPP assay is modified to detect other peptidases using L-Lys-AFC or another fluorogenic substrate, it would be necessary to determine the optimal pH for the hydrolysis of that substrate. Membranes should be prewet in buffer approximating the optimal pH for the specific enzyme activity being sought.

The duration of the overlay should be carefully controlled. We chose a 10-min overlay period, which was sufficient to give clearly positive results for the Vibrionaceae and essentially negative results for the remaining bacteria. Longer overlays would allow other enzymes present in the bacteria to slowly cleave the substrate, giving various degrees of fluorescence. Once the substrate is cleaved, the fluorescence is nonreversible, leading to a permanent spot on the membrane. Any changes in the time course for the overlay must be determined empirically and should depend on the specificity of the reaction, the quantity of active enzyme, the presence of competing enzymes, the medium used, the pH of the culture, the pH optimum for the enzyme being sought, and the density and age of the culture.

The cellulose acetate membranes employed for the overlays can be readily adapted for use as thin strips of membrane, which can be placed on one or more bacterial colonies for 10 min to quickly detect the presence of a broad array of enzymes, depending on the substrate selected for use. Since the technology is inexpensive and does not require the use of any sophisticated instrumentation, the membranes and the COPP technique could become routinely used in many laboratories, especially if membrane strips were to be commercially marketed. The stability of the membranes at room temperature for up to 2 months provides an added advantage to this method. It should be noted, however, that cellulose acetate membranes must be used in this procedure, because they contain hydrophobic pockets to specifically bind the electronegative fluorine atoms of the amino-trifluoromethylcoumarin (6), thus allowing localization of both the reaction and any subsequently produced fluorescent product.

The only instrument required to perform the COPP assay is a long-wave UV light box capable of irradiating the membranes at 364 nm. Alternatively, an inexpensive "black light" may be used to view fluorescence. There is no need for sample preparation, and the materials used to screen for enzyme activity are inexpensive. Labor costs are substantially reduced by the simplicity and speed of the assay. The entire overlay procedure takes 20 min from overlay to viewing for fluorescence. Another advantage is the ability to test about a hundred colonies with the overlay of a single petri dish, thus allowing the screening for and enumeration of Vibrionaceae on a large scale.

Potential applications for this new technology include the use of COPP as follows: (i) to quantify the levels of Vibrionaceae present in food, water, and environmental samples as a potential mechanism to regulate vibrios or to gain a better understanding of their presence, seasonal distribution, and ecology; (ii) to rapidly screen food, water, and environmental samples for Vibrionaceae based on the simple presence or absence of fluorescence; (iii) to rapidly screen for Vibrionaceae in cultures from tissue, blood, and pus to provide preliminary evidence of Vibrionaceae presence or absence; and (iv) to identify the presence of any number of hydrolytic enzymes in bacteria after overlaying cultures with membranes containing various substrates. By selecting other fluorogenic substrates, the COPP assay would have expanded use for the detection of a host of bacterial, fungal, and yeast enzymes involved in proteolysis, housekeeping functions, and virulence. Since fish are highly susceptible to infection by Vibrionaceae, the COPP technique may be particularly valuable for monitoring water quality in the aquaculture industry.

 
We are grateful to the following individuals for providing bacterial cultures: Mark Tamplin and Jeffrey Call (U.S. Department of Agriculture, Wyndmoor, PA), Angelo DePaola and David Cook (U.S. Food and Drug Administration, Dauphin Island, AL), Ghada Khaled (Yale University, New Haven, CT), and M. Sirajul Islam (International Centre for Diarrhoeal Disease Research—Bangladesh, Dhaka, Bangladesh). We also thank David Bushek and Iris Burt (Haskin Shellfish Research Laboratory, Port Norris, NJ) for the collection and assay of oysters and for performing biochemical identifications of bacterial isolates.

 
* Corresponding author. Mailing address: USDA, ARS, Delaware State University, 1200 N. DuPont Hwy., James W. W. Baker Center, Dover, DE 19901. Phone: (302) 857-6419. Fax: (302) 857-6451. E-mail: grichard{at}desu.edu.

Present address: University of Maryland Eastern Shore, 2112 Center for Food Science and Technology, Princess Anne, MD 21853.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, July 2005, p. 3524-3527, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3524-3527.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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• RICHARDS, G. P., WATSON, M. A. (2006). A simple fluorogenic method to detect Vibrio cholerae and Aeromonas hydrophila in well water for areas impacted by catastrophic disasters.. Am J Trop Med Hyg 75: 516-521 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Richards, G. P. Articles by Parveen, S. Search for Related Content PubMed PubMed Citation Articles by Richards, G. P. Articles by Parveen, S. Agricola Articles by Richards, G. P. Articles by Parveen, S.