Isolation of medically significant Vibrio species from riverine sources in ...

Isolation of medically significant Vibrio species from riverine sources in South East Queensland David G. Myatt* and G.H.G. Davis Department of Microbiology, University of Queensland, St Lucia, Brisbane 4067, Australia (*Reprint address)

Abstract Vibrio and V/Mo-like bacteria were isolated from water, sediments, plants and faeces from eight riverine sites in South East Queensland, Australia, using filtrations, enrichments and selective growth on thiosulphate-citrate-bile salts-sucrose (TCBS) and Simidu agars. Isolates (402 in toto) were classified by numerical analysis of phenotypic characteristics and comparison with fifteen reference cultures. Of the isolates 41 (10.2%) were identified as Vibrio cholerae, 33 (8.2%) as V. fluvialis and 118 (29.4%) as motile Aeromonas species. Other isolates resembled V. parahaemolyticus, V. algino/yticus, V. vulnificus and V. hollisae. No isolates were positively identified as V. damsela or Plesiomonas shigelloides. Vibrio species tolerant of low salt levels and aeromonads were widely distributed in riverine locations but more halophilic species were restricted to more saline areas. Simidu agar failed to select for vibrios as effectively as TCBS agar. Enrichment for 18 h produced more isolates than for 6 h.

Introduction Eleven Vibrio species are acknowledged human pathogens responsible for intestinal and extra-intestinal infections (Morris and Black, 1985; Brayton et al, 1986). Fresh water courses in South East Queensland were shown to carry natural and persistent populations of V. cholerae by Rogers et al. (1980), and sporadic cholera cases in Queensland coastal areas in the last decade vindicate this finding (Bourke et al., 1986). The ubiquity of vibrios in marine, brackish and fresh waters has been well documented in America and Europe (Colwell, 1984) but not in Australia. The occurrence of potentially pathogenic vibrios in aquatic niches other than water itself is of interest in view of the reported associations of V. parahaemolyticus with chitinous microfauna (Kaneko and Colwell, 1975a) and sediment (Kaneko and Colwell, 1975b), and of V. cholerae associated with crustaceans, zooplankton and water hyacinth (Colwell et al.., 1985; Nalin et al., 1979; Spira et al., 1981). Diversification of sampling sites and methods for detection of environmental vibrios has been recommended (Spira, 1984). In the interests of assessing the usefulness of sampling a variety of niches in the aquatic environment and several isolation regimes we have surveyed the distribution of Vibrio and Vibrio-like bacteria in water courses in the Brisbane region of South East Queensland. Materials and methods Bacteria

Table 1 lists the fifteen bacterial reference strains used in this work.

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Published and © 1989by The Faculty Press 88 Regent Street, Cambridge, Great Britain

Culture media

Alkaline tryptone water (ATW), containing 10 g/1 tryptone (Oxoid, L42) and 10 g/1 NaCl at pH 8.6, was used for selective enrichment of all samples. Thiosulphate citrate bile salts sucrose (TCBS) agar (Eiken, Tokyo, Japan), and Simidu agar (Simidu and Tsukamoto, 1980) were employed for the selective isolation of Vibrio species. Isolates were stored on brain heart infusion agar (BHIA) prepared by the addition of 10 g/1 bacteriological agar (Oxoid, Lll) to brain heart infusion broth (Gibco), and sterilized at 121°C for 15 min. Tryptone yeast extract Lab-Lemco broth (TYLB) contained (g/1) tryptone (Oxoid, L42), 5; Lab-Lemco powder (Oxoid, L29), 5; yeast extract (Difco), 3; and NaCl, 20, at pH 7.0. Solid medium (TYLA) contained agar (Oxoid, Lll) 10 g/1 in TYLB. Saline base agar contained 20 g/1 NaCl and 10 g/1 agar (Oxoid, Lll). Sample sites

Eight sites within 50 km of Brisbane in South East Queensland, Australia, were chosen as follows: Lake Manchester (storage reservoir); University Lake (urban/recreation); Oxley Creek (urban drainage); Brisbane River A (urban) and B (rural/recreation); Logan River A and B (rural); and Albert River (rural). Sampling methods

All sites were sampled once, and water temperature, pH and salinity were measured before sampling. Samples were collected and processed as follows: (a) a 1 litre sample was collected ca 10 cm below the surface, transported to the laboratory, ca 1 g of sterile Kieselguhr (Ajax) added and filtered through a 47 mm 0.45 pm nitrocellulose filter (Sartorius SMI 13). For heavily particulate samples more than one filter was used. The filter(s) was rinsed with 40 ml of sterile 0.01 M phosphate buffered saline (PBS), (Smibert and Krieg, 1981) and transferred aseptically to 100 ml of sterile ATW. (b) The gauze filter method of Spira and Ahmed (1981) was carried out at the site by pouring 10 litres of water through ca 10 m of 10 cm wide cotton gauze bandage (sterile), transferring the gauze to 100 ml of ten-times normal strength ATW and making up the volume to 1 litre with site water, (c) Submerged mud, gravel and sand samples were collected in sterile disposable specimen jars, (d) Plants growing in or on the water were collected in plastic bags; and (e) bird faeces from waterside sites were collected when available. Samples were held at ambient temperature during transport, and enrichment began within 6 h of collection. In the laboratory, ca 20 g portions of samples (c), (d) and (e) were placed in 100 ml volumes of ATW. Enrichment cultures

A two phase system was used to minimize environmental stressing of the bacteria. After 6 h at 25°C ATW cultures were sampled from the surface by loop onto TCBS and Simidu agar. After a further 12 h at 37°C the surface subcultures to both solid media were repeated. 112 Microbios

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Selective culture and maintenance

TCBS cultures were incubated aerobically at 37°C for 30 to 40 h. Simidu agar cultures were incubated anaerobically (BBL Gaspak) at 25°C for 72 h (Simidu and Tsukamoto, 1980). Representatives of colony types appearing on the solid media were subcultured to TYLB (2 ml), incubated at 37°C for 24 h, 16-streak inoculated onto TYLA and observed for colony purity after 24—48 h at 37°C. The process was repeated if necessary to obtain pure cultures. Isolates were maintained on BHIA slopes at room temperature (23°C) in the dark. Strain characterization

Phenotypic characters (43 in toto) were recorded for 402 isolates and fifteen reference strains (Table 1). Strains were characterized in batches of 48 (always including UQM2773) and one complete batch was replicated to verify reproducibility of the system. Unless otherwise stated incubations were at 37°C, sterilization was at 121 °C for 15 min, and biochemical tests were inoculated with one drop of 18—24 h TYL broth culture. Colony and cellular characters were recorded from TYLA cultures. Broth growth characters were recorded from TYLB cultures. The following methods were used: amino acid decarboxylation was tested by the method of Moller (Cruickshank et al., 1975), modified by the addition of 1% (w/v) NaCl (West and Colwell, 1984) and recorded after 1, 2, 3 and 4 d. Arginine dihydrolase, using Thornley's method (Cowan and Steel, 1974) modified by reducing the final pH to 6.8 (West and Colwell, 1984), was determined after 3 d. Acetoin production (Voges-Proskauer test) from glucose (1% w/v) added to TYLB (2 ml volumes sterilized at 121°C for 1 min) was tested after 3 d by Barritt's method (Cowan and Steel, 1974). ONPG hydrolysis was tested according to Cowan and Steel (1974) using TYLB as base medium after 1, 2 and 3 d. Acid production from lactose, sucrose and arabinose was tested using 1% (w/v) sugars and 0.004% (w/v) bromocresol purple in TYLB (pH 7.2) at 1, 2, 3 and 6 d. Oxidation/fermentation of glucose was determined using TYLB (pH7.2) containing 0.004% (w/v) bromocresol purple, 0.5% (w/v) agar and 1% (w/v) glucose, inoculated while molten at 45°C, and results were recorded at 1 and 2 d for oxidation (yellow top), fermentation (yellow throughout), gas production (bubbles or fractured agar) and alkaline reversion (purple top, yellow butt). Aeromonas hydrophila medium described by Kaper et al. (1979), modified by the addition of 1% (w/v) NaCl, was stabbed after surface inoculation and examined at 1 and 2 d for motility, hydrogen sulphide production and indole production from tryptophan. Cytochrome c oxidase was tested with oxidase test strips (Cowan and Steel, 1974). Gelatin hydrolysis was determined on TYLA supplemented with gelatin (Oxoid L8,1.5% w/v) after 3 d by flooding with saturated ammonium sulphate solution. Corn oil hydrolysis was assessed by the method of Berry (1932), modified to use double layer plates with a layer of saline base agar under a layer of corn oil agar [TYLA containing 5% (v/v) corn oil] after 6 d by occurrence of a bright blue precipitate under growth, 15 min after addition of saturated aqueous copper sulphate. 113 Isolation of environmental vibrios

Growth without NaCl was assessed using CLED agar (Oxoid, CM301) after 2 d. Growth with 10% NaCl was recorded after 1, 2 and 3 d on TYLA containing 10% (w/v) NaCl. Growth with L-alanine as sole carbon source was assessed using the medium of Lee et al. (1981) after 2 d. Nitrate reduction was determined by method 1 of Cowan and Steel (1974) with TYLB as the base medium after 3 d. Haemolysis was recorded after 24 h using double layer 9% (v/v) horse blood agar with TYLA as base. Novobiocin sensitivity was determined using 5 /*g novobiocin discs (Oxoid) on the primary inoculum of TYLA streak plates. Sensitivity to 0/129 was tested using 10 jig and 150 /*g 0/129 discs prepared with 2,4-diamino-6,7-diisopropyl pteridine phosphate (Sigma). Numerical analysis

For each operational taxonomic unit (OTU), 43 binary characters were coded. Duplicated results for UQM2773 and OTUs in the duplicate batch were shown to be reproducible and included only once in the numerical analysis. Similarity coefficients (S) were calculated using the modified simple matching coefficient of Sokal and Michener (1958) and Skerman (1967). Cluster analysis was performed using the complete linkage modification of the single linkage algorithm of Sneath (1957) with the complete linkage B program of Szabo (1970) on a DEC PDP-10 computer. Subsequent sub-cluster analyses were performed by computer or manually.

Results Isolate characterization

Oxidation/fermentation test results were all fermentative or negative. Results for Thornley's arginine dihydrolase correlated exactly with those for Moller's arginine decarboxylase so the latter were omitted from numerical analysis. Numerical analysis

Initial computer analysis yielded seven groups at the 55% level of similarity with relatively large and homogeneous phenons around the reference strains of V. choleras, V. fluvialis (and V. furnissii), V. parahaemolyticus and A. hydrophila (Figure 1). Nine phenons failed to include any named reference strains. Closer scrutiny of the initial dendrogram and strain data resulted in segregation of minor atypical clusters from the major phenons. For example two peripheral OTUs in group 1 were unlike the other 46 OTUs (non-motile, indole negative, ornithine and lysine negative). The remaining V. cholerae strains differed in lactose and sucrose fermentation and acetoin production but conformed otherwise to the current description of V. cholerae. Similarly in group 2, six OTUs (phenon F3) which fused with the V. fluvialis cluster at a similarity of less than 70% were all negative for oxidase and growth on TCBS but reduced nitrate to nitrite and grew on CLED medium. These are not V. fluvialis or V. furnissii strains. The reference strains of V. fluvialis

114 Microbios

D. C. Myatt and G. H. G. Davis

100

Percentage similarity 80

60

Group

40

V. cholerae (46)

H Gram — rod Gram + rod

V. fluvialis (35) 2H

Phenon F3 (61 V. metschnikovii (1) Gram + rod

V. parahaemolyticus (69) 3H Phenon A (5) V. vu/nificus (6) V. damsela (21 V. a/gino/yticus (5)

Phenon E (66)

6

V. hollisae (12) Phenon C (7) Phenon D (14)

5

Phenon B (7)

4

Motile aeromonads (118)

7H

Phenon F (6) Phenon G (6) Phenon H (2) Plesiomonas shigel/oides (1)

Figure 1 Simplified dendrogram showing clustering of 402 potential vibrio isolates and fifteen reference strains. The number of strains per phenon is shown in parentheses, and the reference strains fell in the respective named phenons.

115 Isolation of environmental vibrios

and V. furnissii clustered together (at 90% S) and linked with the other 33 OTUs of the V. fluvialis phenon at 80% similarity. The reference strain of V. metschnikovii (UQM211) exhibited typical biochemical reactions, except for failure to utilize alanine, grow on TCBS agar and on CLED medium, i.e. in the absence of salt. One strain associated with UQM211 exhibited less than 72% S. The V. parahaemolytkus phenon in group 3 contained 69 OTUs and four sub-phenons were evident. One contained coccobacillary strains, and another contained OTUs tolerant of a wide range of salinities, i.e. growing on CLED medium and with 10% salt. Of the 69 OTUs in this phenon, 18% were sucrose positive (Table 2). Group 3 also contained OTUs clustered with the reference strain of V. alginolyticm. Their characters were not typical of V. alginolyticus but conformed to those of Vibrio, and similarly, the five OTUs which clustered with the type strain of V. vulnificus were not typical.

Table 1 Bacterial reference strains Reference number*

UQM2730 UQM2732 UQM2441 UQM2442

UQM2773 UQM2776 UQM2774 UQM2775 UQM2770 UQM2778 UQM211 UQM2852 UQM2853 UQM2768 UQM1134

Species

Supplier"

Source**

Vibrio cholerae non-01 V. cholerae non-01 V. cholerae non-01 V, cholerae 01 V. cholerae 01

AGAL AGAL QSHD QSHD RRC RRC RRC RRC RRC RRC ATCC CPHL CPHL RRC NCIB

Local isolate Local isolate Local isolate

V. parahaemolyticus V. flu via/is V, furnissii V. alginolyticus V. vulnificus V. metschnikovii V. ho/lisae V. damse/a Aeromonas hydrophila Plesiomonas shigelloides

Local isolate ATCC14035*** ATCC 17802*** NCTC11327*** NCTC11328 ATCC17749*** ATCC27562*** Not known ATCC33565 ATCC33537 ATCC7966*** CDC-RH798***

Designation in the University of Queensland Microbiology Culture Collection. AGAL, Australian Government Analytical Laboratories, Sydney, Australia; RRC, Professor R. R. Colwell, Department of Microbiology, University of Maryland, Baltimore, USA; ATCC, American Type Culture Collection, Rockville, USA; QSHD, Queensland State Health Department, Brisbane, Australia; CPHL, Commonwealth Public Health Laboratory, Sydney, Australia; NCIB, National Collection of Industrial Bacteria, Aberdeen, Scotland; NCTC, National Collection of Type Cultures, London, England; CDC, Centers for Disease Control, Atlanta, USA. Type culture

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Table 2

Character

Summarized characters of eight identified phenons Phenon*: Vc Vf

Number of OTUs Colony diameter < 1 mm Colony diameter > 2 mm Colony margin entire Colony elevation convex Colony elevation flat Colony mucoid Colony shiny Colony texture friable Colony adherent or sticky Colony not pigmented Gram-negative cells Coccobacilliform cells Bacilliform cells Pellicle/ring formed in broth Sediment deposited in broth Uniform turbidity in broth Cytochrome c oxidase activity Gelatin hydrolysis Corn oil lipolysis Haemolysis (horse blood) Nitrate reduction to nitrite Nitrate reduction beyond nitrite Acetoin production ONPG reaction Arginine dihydrolase Ornithine decarboxylase Lysine decarboxylase Acid production from lactose Acid production from sucrose Acid production from arabinose Glucose fermentation Gas production from glucose Alkaline reversion in 0/F test Hydrogen sulphide production Indole production from tryptophan Motility in semi-solid agar Growth on CLED agar (no salt) Growth on TCBS agar (bile) Growth with 10% NaCI Alanine as sole C source Sensitivity to novobiocin 5 /jg Sensitivity to 0/129 10 /tg Sensitivity to 0/1 29 1 50 jig

46 2" 20 98 98 2 0 98 2 0 100 100 2 98 76 80 100 100 100 0 98 100 0 43 100 0 100 100 37 67 0 100 0 26 0 78 98 100 98 11 22 100 100 100

Vp

0 84 99 97 3 6 100 0 4 100 100 30 68 54 100 100 99 97 0 50 100 1 0 94 1 100 100 14 19 74 100 0 1 0 91 100 25 99 39 99 100 100 100

3 51 100 100 0 0 100 0 0 100 100 3 97 11 94 100 100 100 89 94 100 0 0 100 100 0 0 11 100 100 100 11 3 0 3 97 100 97 23 100 41 97 100

69

35

A

Vv

Va

0 80 60 20 80 0 80 0 0 100 100 0 100 20 80 100 100 100 20 80 100 0 80 0 60 20 100 0 60 20 100 0 80 0 60 100 40 100 100 100 100 100 100

0 67 100 100 0 0 100 0 0 100 100 0 100 33 83 100 100 100 0 50 100 0 0 33 83 0 0 17 O 0 100 0 0 0 17 33 0 100 0 100 100 83 100

0 80 100 100 0 40 100 0 0 100 100 40 60 0 100 100 100 100 0 75 100 0 0 0 100 0 0 0 O 0 100 0 60 0 40 60 80 100 20 80 100 80 100

5

6

5

Vh

12 0 33 100 100 0 17 100 17 8 100 100 0 100 48 100 100 100 0 0 17 100 0 0 67 0 0 0 8 92 100 100 0 0 8 17 75 58 0 92 92 92 50 100

MA

118 0 2 100 100 0 0 100 3 0 100 100 0 100 23 96 100 97 97 3 97 100 1 28 100 100 1 96 4 51 14

100 53 31 0 88 100 99 92 1 10 72 0 9

Phenons shown in Figure 1. Vc, V. cholerae; Vf, V. fluvialis; Vp, V. parahaemolyticus; A, phenon A; Vv, V. vulnificus; Va, V. alginolyticus; Vh V. hollisae; MA, motile Aeromonas species. Percent strains positive.


Table 3 Association of vibrios with eight sample sites Sampling site**: Phenon*

LKM ULK

BCC ARC LRM LRP

BRC

OCC

Aeromonas species V. cholerae V. fluvialis V. parahaemolyticus V. alginolyticus V. vu/nificus V. hollisae

Temperature (°C) Salinity (ppm)

22

24

22

22

25

23

23

47

65

90

100

200

300

1000

3000

pH

5.5

6.9

7.6

6.5

6.0

6.9

5.6

6.6

Refer to Figure 1 and Table 2 LKM Lake Manchester; ULK, University Lake; BCC, Brisbane River (rural); ARC, Albert River; LRM Logan River A; LRP, Logan River B; BRC, Brisbane River (urban); OCC, Oxley Creek.

Table 4

Vibrios isolated from various samples

Phenon*

Sample type**: MF GF

Sediment

Plant

Faeces

V. cholerae V. fluvialis V. parahaemolyticus V. alginolyticus V. vulnificus V. hollisae Aeromonas species

1

Refer to Figure 1 and Table 2. Sediment samples included mud, rocks and sand. MF, membrane filtered 1 litre water sample. GF, gauze filtered 10 litre water sample.

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Five OTUs (phenon A) formed a cluster in group 3 without including any named reference strain; however they displayed characters typical of Vibrio species (Table 2). Phenon B (group 4) comprised seven glucose non-fermenting organisms. The phenon formed around the V. hollisae reference strain (twelve OTUs) in group 5 was the only Vibrio phenon associated with the Aeromonas and other non-Vibrio clusters (group 7). Phenons C, D and E (groups 5 and 6) consisted of oxidase negative glucose-fermenting OTUs. The Aeromonas phenon in group 7 contained five sub-clusters, with the type strain (UQM2768) in a sub-cluster showing negative results for acetoin and gas from glucose tests. Phenons F and G contained glucose non-fermenters and oxidase-negative OTUs, respectively, and two OTUs (phenon H) did not resemble the reference strain of P. shigelloides. On the basis of these considerations a summary of the characters of the identifiable phenons was made (Table 2). Environmental distribution

The sample sites from which the OTUs in seven named phenons were isolated are summarized in Table 3. The physicochemical data recorded for each site are also included in Table 3. Two sites, BRC and OCC, are tidal and relatively contaminated by urban drainage (BRC) and sewage effluent (OCC). The associations of major phenons with the samples collected are shown in Table 4. Isolation methods The comparative efficacy of ATW enrichment for 6 and 18 h and selective isolation on TCBS and Simidu agars is shown in Table 5. Table 5 Effects of enrichment time and selective media on isolation of environmental vibrios

Phenon*

Enrichment time in TCBS medium: 6h 18h

5

V. parahaemolyticus

4

V. fluvialis

3*»

V. cholerae

2

V. vulnificus

0

V. alginolyticus

0

V. hollisae

34

TOTAL

20

Aeromonas species

10 5 10 0 0 0 22 47

Enrichment time in Simidu medium: 6h 18h

1 0 4 2 0 6 2 15

5 5 9 1 3 0 18 41

* Refer to Figure 1 and Table 2. ** Number of samples yielding OTUs in this phenon.


Discussion Isolate identification

The characters used for strain differentiation and clustering were chosen either for their known value in differentiating medically significant Vibrio species or because they were simple to perform. All characters used showed some degree of strain discrimination. However, the number of characters employed (43) is at the lower limit of the range considered desirable for numerical analysis and it is recognized that insufficient distinguishing characters were employed to effectively differentiate some non-pathogenic vibrios from those of interest. Nevertheless, the method produced a clustering pattern for the 402 OTUs which associated many of them with type and reference strains often species. V. cholerae (46 OTUs), V. fluvialis (35 OTUs), V. parahaemolyticus (69 OTUs) together with another 18 OTUs with characters typical of the genus Vibrio in group 3 (Figure 1), comprised 91% of the 185 OTUs that clustered together as vibrios. Amongst the V. cholerae isolates, sub-clusters contained strains which did not ferment sucrose. Desmarchelier and Reichelt (1984) noted that it is not valid to call such strains V. mimicus although it is noteworthy that one sub-phenon was also acetoin negative. Negative corn-oil lipolysis did not confirm the V. mimicus-like nature of these isolates (Farmer et al., 1985). V. harveyi also shares many traits with V. cholerae. The 33 strains which clustered with the V. fluvialis and V. furnissii reference strains were predominantly anaerogenic and therefore mostly V. fluvialis. This phenon was very homogeneous. Six OTUs associated with the V. fluvialis cluster exhibited characters more typical of V. metschnikovii, and it is probable that they are enterobacterial isolates since all were isolated on Simidu agar. The V. parahaemolyticus phenon was less homogeneous and exhibited atypical results for ONPG and variable sucrose utilization. Growth at 10% NaCl was also variable and this may indicate that some isolates were actually V. alginolyticus. Similarly, OTUs associated with the V. alginolyticus reference strain tolerated 10% NaCl although decarboxylase results indicated that they might be other halophilic Vibrio species. Some isolates clustering with V. vulnificus exhibited atypical results for ONPG, and H2S and indole production, and the single isolate associated with V. damsela was not typical of that species. Phenon A (Figure 1) contains five strains which clustered with the halophilic vibrios in group 3. Negative amino acid decarboxylation and sugar fermentation results indicate that they may be V. splendidus type II strains (West and Colwell, 1984). Baumann et al. (1984) suggest that differentiation of V. parahaemolyticus, V. alginolyticus, V. harveyi, V. campbelli and V. vulnificus requires testing growth on cellobiose, gluconate, ethanol, L-serine, L-leucine, L-glutamate and putrescine. Figure 1 also shows the generalized grouping of species tolerant of low salt levels (groups 1 and 2) apart from more halophilic species (group 3). These two major divisions fuse at an S value of 46%. Eleven OTUs clustered with the reference culture of V. hollisae, but showed atypical characters (motility, sucrose fermentation, indole production). These 120 Microbios


were linked with the Aeromonas cluster and several unidentified groups (phenons C to H). All 87 OTUs in clusters C, D and E were oxidase negative, but fermented glucose, reduced nitrate, cleaved ONPG, were motile and failed to hydrolyse gelatin. These traits are typical of the family Enterobacteriaceae, although some genera may be excluded by the results of gelatin hydrolysis and ONPG tests. Of the strains in clusters C, D and E, 82% were isolated on Simidu agar, and of these 69% were from 6 h enrichment, indicating that the majority of these isolates were not selected by the typical vibrio isolation regime of ATW enrichment and TCBS agar selection. The cluster of 118 motile aeromonads contained sub-phenons discernible by gas and acetoin production from glucose. Two sub-phenons (36 OTUs) resembled A. hydrophila, 56 anaerogenic OTUs in two clusters resembled A. caviae and 26 aerogenic but acetoin negative OTUs resembled A. sobria (Popoff, 1984). The association of aeromonads with enterobacterial isolates is consistent with the recent description of the new family Aeromonadaceae, which recognizes the close phylogenetic relationship of these species to the Enterobacteriaceae rather than the Vibrionaceae (Colwell et al., 1986). Only oxidase positive and glucose non-fermenting strains, and oxidase negative and glucose fermenting strains constituted the three small phenons (fourteen OTUs) associated with P. shigelloides. Isolation techniques

The gauze filtration method of Spira and Ahmed (1981) yielded fewer Vibrio isolates in our hands than the conventional membrane filtration method, but both appear useful for water studies. Gauze filtration is not complicated by suspended paniculate matter which makes membrane filtration very difficult. In general more samples yielded vibrios after 18 h enrichment (Table 5). Enrichment for 6 h promoted isolation of strains similar to V. alginolyticus and V. hollisae. These organisms were isolated only using Simidu agar. V. hollisae is known to be inhibited on TCBS agar (Hickman et al., 1982), and the Eiken brand of TCBS was shown by West et al. (1982) to be more inhibitory than other brands. TCBS agar was not apparently selective against motile aeromonads. When used in conjunction with enrichment, the relative number of vibrio isolates to aeromonads increased with longer enrichment. In contrast, results for Simidu agar showed a parallel increase in isolation frequencies for vibrios and aeromonads with longer enrichment. There was no apparent correlation between the physicochemical characteristics of each sampling location with the exception of salinity. The two sites showing markedly higher salinity levels yielded a greater diversity of vibrios than less saline sites. The least saline sites, the University Lake, Lake Manchester and the rural Brisbane River site, yielded only isolates of species tolerant of low salt levels, V. choleras, V. fluvialis and aeromonads. More halophilic species, resembling V. parahaemolyticus, V. vulnificus and V. alginolyticus, were isolated only from sites with salinities greater than 100 ppm. Aeromonas species were widely distributed (at all sites) and tolerated a wide salinity range.


We conclude that the diversified sampling and isolation methods which were used yielded isolates of Vibrio species of known medical interest. The potentially diarrhoeagenic species V. cholerae, V. parahaemolyticus and V. fluvialis predominated. Motile Aeromonas species were also commonly isolated. However, the isolation regimes employed did not effectively select against non-pathogenic species, hence subsequent identification must be based on a fairly large array of differential phenotypic properties. Regimes incorporating isolation on Simidu agar were also shown to be less effective than TCBS agar in reducing the rate of isolation of enterobacteria-like organisms. Acknowledgements The authors would like to thank Lindsay Sly for valued advice. This work was funded by the Mayne Bequest Fund at the University of Queensland.

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Accepted 21 June 1989
