VETERINARSKI ARHIV 69 (3), 125-134, 1999

ISSN 1331-8055 Published in Croatia

Basing a selective method for isolating environmental
Vibrio cholerae on differences in the growth rate
of competing Vibrio metschnikovii

Vladimir Muic¹*, Mate Ljubicic¹, Ivan Vodopija²,
and Vladimir Mayer¹

¹Croatian National Institute of Public Health, Zagreb, Croatia

²Zagreb Public Health Institute, Zagreb, Croatia

* Contact address:
Dr. Vladimir Muic,
Croatian National Institute of Public Health, 10000 Zagreb, Rockefellerova 7, P.O. Box 684, Croatia;
Phone: 385 1 46 83 222

Muic, V., M. Ljubicic, I. Vodopija, V. Mayer: Basing a selective method for isolating environmental Vibrio cholerae on differences in the growth rate of competing Vibrio metschnikovii. Vet. arhiv 69, 125-134, 1999.

ABSTRACT

Some differential growth characteristics for Vibrio cholerae and Vibrio metschnikovii were looked at in a peptone water vibrio enrichment basic liquid medium by varying pH, ionic strength, as well as temperature and length of incubation. The purpose was to separate the two bacterial species, i.e. to establish which combination of the above cultivation conditions might enable a selective replication of V. cholerae (human and animal pathogen), while suppressing the growth of V. metschnikovii. The latter vibrio hinders visual identification of the V. cholerae of waste water and animal origin on a solid TCBS standard selective medium. A preliminary limited routine verification of potential methods, as applied to waste water samples, showed that a combination of cultivation conditions which involved an extension of incubation to 22 h at a heightened temperature of 41 °C might enable Vibrio cholerae to be simply and rapidly isolated, with visual inspection already providing its final identification and quantification.

Key words: selective isolation, Vibrio cholerae, Vibrio metschnikovii

Introduction

Methods for the quantitative demonstration of cholera vibrios are few, imperfect and laborious. They are particularly unreliable when used to analyse either urban sewage waters or seawater (JURAS et al., 1979; DePAOLA et al., 1987, 1988; NAIR et al., 1987). The result of all this is the creation of a lack of reliable information regarding the extent and size of surface water pollution with V. cholerae, often leading to a misinterpretation of the causative agent's sources, routes of spread and reservoirs. Another consequence is erroneous estimates of the epidemiological situation and roles of individual serovars (or pathovars) in human and animal pathology. Highly sensitive modern methods, which are based either on the replication of genetic material originating from live or dead vibrio cells, or on monoclonal antibodies, can not provide fully valid epidemiological arguments, unless accompanied by a confirmation of the presence of a specified live vibrio count (LOWENHAUPT et al., 1998; MUNRO et al., 1996; HASAN et al. 1994). While sporadic cases of animal disease caused by some cholera vibrios serovars are of very old date, there is a lack of more detailed information on this due to the fact that cholera vibrios are mainly the concern of microbiological laboratories for human medicine. As causative agents, in animal pathology one usually encounters - except for serovars O-1 and O-139 - those cholera vibrio serovars which are less important in human epidemiology (from O-2 to O-138, as well as from O-140, upwards).

All the above justifies an attempt to devise a simple V. cholerae isolation method which would - with or without the need for biochemical or serological confirmation - quantitatively determine the presence of V. cholerae in water, e.g. by using the multiple tube technique, or membrane filtration.

The purpose of this study was to discover a particular combination of temperature, length of incubation, pH and ionic strength for a culture medium to enable the rapid growth of V. cholerae and a slow or no growth of V. metschnikovii. There are earlier reports of such attempts (SINGLETON et al., 1982; DePAOLA et al., 1987, 1988; MUNRO et al., 1994, 1996). The regular presence of this latter vibrio in urban sewage waters impedes the identification of V. cholerae, because both organisms exhibit an equally abundant growth and morphology on the vibrio-selective TCBS plating medium.

Materials and methods

The used V. cholerae O-1 strains originated from laboratory collections. Two strains (D-3142-2, D-3134-2) were obtained from Dr. P. B. Bhattacharya, International Centre for Vibrios, Cholera Research Centre, Calcutta, India, whereas 20 non O-1 Vibrio cholerae strains were own isolates whose identification was confirmed by the Vibrio Reference Laboratory, PHL, Maidstone, England (Dr. A. L. Furniss). All 22 strains were of the eltor biotype.

The V. metschnikovii strains came from laboratory collections: one, V.35/78, from the Czech National Collection, the remaining 28 being own isolates that were identified and confirmed in the same way as the preceding strains.

Bacteriological media. Peptone water (PW) was prepared from a low NaCl content (1%) Bacto-peptone ("Difco") so that NaCl concentration in PW was only 0.01%. Three variants of PW were produced: PW 0, with no addition of NaCl; PW 0.5, with the addition of 0.5% NaCl, and PW 1.0 with the addition of 1% NaCl. With regard to pH, three variants were prepared by adding to them either HCl or NaOH, producing pH 6.0, pH 8.6 and pH 9.2, respectively. Standard, pre-calibrated, bacteriological test tubes were filled with 5 ml PW each, and sterilized at 121 °C for 20 min.

Inoculation of bacteriological media. Inoculation was performed with 0.5 mL of an overnight bacterial strain culture in a 0.5% NaCl PW, pH 8.6, with a count of 5 x 10⁶ CFU/mL.

Media incubation. This was carried out at 30 °C, 37 °C and 41 °C, the length of incubation being 8 or 22 h.

Turbidometric measurement of relative bacterial growth. To make this measurement a Coleman Model 54 (Perkin-Elmer Corp. Norwalk; Connecticut, USA) digital spectrophotometer was employed at a light wavelength of 700 nm. The mean optical density of spectrophotometric readings obtained from an unequal number (from 4 to 29) of bacterial suspensions tested was designated as t.

The values of quotient t found for V. cholerae and V. metschnikovii were considered to be the specificity index in vitro as determined by a given method for V. cholerae isolation. To represent the results graphically, of statistical methods we used histogram, scatterplot and the method of hierarchical agglomerative clustering, in addition to the determination of weighted pair group average and Euclidean distances.

Results

Results relate to differences between the growth rates of Vibrio cholerae and interfering Vibrio metchnikovii in five new combinations of nutrient medium and cultivating conditions. A part of the results described below pertains to a statistical estimate of the relatedness and unrelatedness of these new combinations (or potential routine laboratory isolation methods for Vibrio cholerae) compared with the widely used standard method. Only the last table presents a semi-quantitative assessment of preliminary results of comparative testing of the standard and two potential methods during a routine analysis of sewage for Vibrio cholerae.

Table 1 shows cultivation conditions for samples containing V. cholerae and V. metschnikovii with regard to the ionic strength and pH of the PW medium, as well as temperature and length of incubation.  It specifies the conditions in which the standard method is performed, as well as those for five of its versions.

Table 1. Standard method^a and five potential methods for selective isolation of Vibrio cholerae; use conditions in relation to temperature, ionic strength, pH and length of incubation.

Table 2 shows the sequence of six methods (one standard and five own versions of that standard) ranked by levels of mean optical density of V. cholerae growth (t_Vch ) in the PW medium under the conditions of the given method. The number of strains tested varied between 4 and 29.

Table 2. Growth rates of Vibrio cholerae and Vibrio metschnikovii (expressed as mean optical density, t) compared for the conditions of the standard^a and potential methods for the selective isolation of Vibrio cholerae in relation to Vibrio metschnikovii. The methods were ranked by the size of the optical density means for Vibrio cholerae^d

^a=standard method; ^b=the mean value of optical densities for the growth of Vibrio cholerae read off a spectrophotometer; ^c=the mean value of optical densities for the growth of Vibrio metchinkovii read off a spectrophotometer; ^d=the number of suspensions tested corresponds to the number of strains; ^e=with more strains tested, the results could have been positive

Table 3 shows the sequence of six methods ranked by specificity index for the V. cholerae isolation (in relation to V. metschnikovii) with pertinent values of optical density means for V. cholerae (t_V.ch.) and V. metschnikovii (t_V.m.).

Table 3. The standard^a method, as well as potential methods, for selective isolation of Vibrio cholerae with reference to Vibrio metschnikovii. Their ranking is by quotient t_V.ch/t_V.m size, i.e. by specificity index for the Vibrio cholerae species isolation from a culture mixed with Vibrio metschnikovii in vitro.

^a=standard method; ^b=mean value of optical densities for V. cholerae read from a spectrophotometer; ^c=mean value of optical densities for V. metschnikovii read from a spectrophotometer

The scatterplot diagram (Fig. 1) illustrates the relationship between the individual six methods, ranked by the height of the specificity index for the isolation of V. cholerae, and the same methods ranked by the height of optical density means of V. cholerae (t_V.ch.).

Fig. 1. This scatterplot shows the relationship between the six methods for selective isolation of V. cholerae, ranked by the size of specificity indexes^a, and the same methods ranked by the height of the mean optical density for the growth of V. cholerae, t_V.ch. (in vitro indicator of bacterial growth under the conditions of a given method).

Figure 2 is a graphic representation (based on a method of hierarchical agglomerative clustering) of the degree of similarity between the six methods (the standard one and five potentially so) by four conditions, i.e. ionic strength, pH, as well as temperature and length of incubation.

Fig. 2. Hierarchical agglomerative clustering of six methods (M1 to M6) for selective isolation of V. cholerae by four conditions: temperature (°C), NaCl (%), pH and length of incubation (h).

The relationship between mean optical densities of V. cholerae and V. metschnikovii (t_V.ch./t_V._m.)is represented graphically (Fig. 3) for respective methods. A semi-quantitative empirical estimate and a hypothetical estimate of the possibility of detecting either of the two bacterial species were superimposed on the graphic representation of the relationship between mean optical densities. The detection subsumes subsequent cultivation of the sample on a solid vibrio-selective TCBS medium.

Fig. 3. Mean optical density values for Vibrio cholerae (t_V.ch.) and Vibrio metschnikovii (t_V.m.) growths in liquid medium depending on the method (M1 to M6). Also shown is the empirically established threshold of detection on the solid selective TCBS medium (full line) and the assumed threshold of detection (dashed line) for V. cholerae, respectively V. metschnikovii. (^a=standard method)

Discussion

Some combinations of the six conditions in Table 1 suggested that V. cholerae might be grown selectively in a liquid medium, a precondition for devising a method for the quantitative determination of vibrios. Indicative of this is the height of specificity indexes in Table 2, which ranked the methods as M3, M2, M1, M4. Despite the obvious method unsuitability, the in vitro results of M5 do not mean that this combination of conditions would make the isolation of V. cholerae impossible. The conditions of M5 are the same as that of the standard method widely used until now. The latter, besides involving the pre-selection (not detrimental to V. metschnikovii but affecting other bacterial flora) of V. cholerae in a liquid medium and its sub-cultivation on the selective TCBS agar, uses visual checking of colony appearance and supplemental biochemical confirmation of the isolates.

Condition combinations in M3 and M2 prevent the growth of V. metschnikovii and could, theoretically, be considered ideal for and absolutely specific of the V. cholerae isolation. This indicates that biochemical confirmation of morphologically suspect colonies on TCBS agar could then become totally unnecessary.

According to own purely preliminary verification, in detecting V. cholerae in waste water samples potential methods M3 and M2 did not prove to be sufficiently effective. Although experimental sensitivities of these methods (t_V.ch.) were not the lowest, the growth of thermally stressed cells on the solid vibrio selective TCBS medium was unsatisfactory. Unlike these two, the standard method, M5, while less sensitive than M1, was highly non-specific (t_V.m.=1.27), and thus encumbered by the need for a vast number of biochemical tests to identify TCBS agar-grown colonies morphologically suspected of being V. cholerae.

As to the first four condition combinations in Table 1, M1 was tested most extensively practically on sewage water and faecal samples. Besides demonstrating satisfactory sensitivity, this method was highly specific in diagnosing V. cholerae. In practice, when used in combination with the vibrio-selective TCBS medium, its results even considerably exceeded those anticipated based on relatively high mean optical density growth value for V. metschnikovii oft_V.m.=0.42. Although more sensitive than in the theoretical model (t_V.ch.=0.72), the standard test M5 was totally non-specific in the demonstration of V. cholerae.

Figure 3 illustrates the difference between the theoretical model of the method and its practical effectiveness (which includes supplemental use of the solid vibrio-selective TCBS plating medium). A dendogram in Figure 2 suggests that the standard test M5 and the method M1 recommended by us have diametrically opposite properties. It is common for increased specificity (specificity index t_V.ch./t_V.m) to be accompanied by an undesirable reduction in the method's sensitivity (t_V.ch), as shown by the scatterplot diagram in Figure 1.

In conclusion, from preliminary investigations it could be said that verification findings on the growth of V. cholerae in peptone water liquid medium with 1% NaCl at a heightened temperature of 41 °C under an extended incubation of 22 h, suggest that a test for rapid qualitative and quantitative demonstration of V. cholerae in waste water may be devised. The test's high sensitivity, specificity, simplicity and rapidity (visual identification only suffices) would allow a reduction in, or complete dispensation of, the need for biochemical confirmation of isolates. Except for urban waste waters and other environmental samples, this method could equally effectively be applied to faecal samples, although the attendant V. metschnikovii flora is extremely rare in humans, but relatively common in domestic animals.

Acknowledgment
The authors are grateful to Dr. Eva Aldová of the National Epidemiological Institute, Prague, Czech Republic, for advice on vibrios. They also extend their thanks to Vilim Crlenjak, B.A., of the Croatian National Institute of Public Health for this translation.

References

DePAOLA, A., C. A. KAYSNER, R. M. McPHEARSON (1987): Elevated temperature method for recovery of V. cholerae from oysters (Crassostrea gigas). Appl. Environ. Microbiol. 53, 1181-1182.

DePAOLA, A., L. H. HOPKINS, R. M. McPHEARSON (1988): Evaluation of four methods for enumeration of Vibrio parahaemolyticus. Appl. Environ. Microbiol. 54, 617-618.

HASAN, J. A. K., A. HUQ, M. L. TAMPLIN, R. J. SIEBELING, R. R. COLWELL (1994): A novel kit for rapid detection for V. cholerae 01. J. Clin. Microbiol. 32, 249-252.

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LOWENHAUPT, E., A. HUQ, R. R. COLWELL, A. ADINGRA, P. R. EPSTEIN (1998): Rapid detection of V. cholerae O1 in West Africa. Lancet 351, 34.

MUNRO, P. M., BRAHIC, R. L. CLEMENT (1994): Seawater effects on various Vibrio species, Microbios 77, 191-198.

MUNRO, P. M., R. R. COLWELL (1996): Fate of V. cholerae O1 in seawater microcosmos. Water Res. 30, 47-50.

NAIR, G. B., S. MISRA, R. K. BHARDA, S. C. PAL (1987): Evaluation of the multitest medium for rapid presumptive identification of V. cholerae from environmental sources. Appl. Environ. Microbiol. 53, 1203-1204.

SINGLETON, F. L., R. W. ATWELL, M. S. JANGI, R. R. COLWELL (1982): Effects of temperature and salinity on V. cholerae growth. Appl. Environ. Microbiol. 44, 1047-1058.

Received: 3 February 1999
Accepted: 25 June 1999

Muic, V., M. Ljubicic, I. Vodopija, V. Mayer: Selektivno izdvajanje bakterije Vibrio cholerae iz okolisa na osnovi razlike u brzini rasta u odnosu na vrstu Vibrio metschnikovii. Vet. arhiv 69, 125-134, 1999.

SAZETAK

Iistrazivane su razlicite kombinacije uvjeta rasta i sastava peptonske vode, kao osnovnog tekuceg medija za razmnozavanje bakterija Vibrio cholerae i Vibrio metschnikovii obzirom na pH, ionsku jakost, te temperaturu i vrijeme inkubacije sa svrhom da se utvrde znacajke koje najvise pridonose razlikama u rastu spomenutih mikroorganizama. Svrha je bila odvojiti dvije navedene bakterijske vrste, tj. odrediti kombinaciju uvjeta kultivacije (obzirom na gore navedene pokazatelje) koja bi omogucila selektivno razmnozavanje V. cholerae (ljudskog i zivotinjskog patogena) uz supresiju rasta V. metschnikovii. Ovaj potonji vibrio pravi smetnje pri vizuelnoj identifikaciji V. cholerae iz okolisnih i zivotinjskih uzoraka za analizu - pri upotrebi standardnog krutog selektivnog TCBS medija za kultivaciju. Preliminarna provjera mogucih metoda (tj. novih kombinacija gore navedenih uvjeta kultivacije i svojstava medija za kultivaciju) u dnevnoj rutini (u nasem slucaju pri bakterioloskoj analizi otpadnih voda) je pokazala da bi kombinacija uvjeta za kultivaciju koja ukljucuje produzenje inkubacije na 22 sata pri povisenoj temperaturi od 41 °C mogla omoguciti brzu i jednostavnu izolaciju V. cholerae, pri cemu bi njegova definitivna identifikacija i kvantifikacija mogla biti obavljena vec i vizuelno.

Kljucne rijeci: selektivno izdvajanje, Vibrio cholerae, Vibrio metschnikovii