ELSEVTER Journal of Microbiological Methods 23 (1995) 183-203

Evaluation of media for the enumeration of faecal streptococci from natural water samples

Lidia PO Catalao Dionisio”, Juan J. Borregob’*

‘V.C.T.R.A., University of Algarve, Campus de Gambelas, 8lXW-Faro, Portugal bDepartment of Microbiology, Faculty of Sciences, Vn)versity of Malaga,

Campus Vniversitario Teatinos, 20971 Malaga, Spain

Received 19 October 1994; revised 22 January 1995; accepted 15 February 1995

Abstract

The Most Probable Number technique using Azide dextrose broth and Ethyl violet azide dextrose broth, and the membrane filtration technique with the following agars: mEn- terococcus, KF, Pfizer selective enterococcus, Kanamycin aesculin azide, Bile-Aesculin, Thallous acetate (Barne’s), and Mitis-Salivarius were compared on the basis of their accuracy, specificity, selectivity, precision, and relative recovery efficiency characteristics. The results obtained indicate that none of the above mentioned procedures shows an optimal selectivity. The Most Probable Number technique was a good procedure on the basis of its high recovery efficiency and good selectivity. However, mEnterococcus agar using the membrane filtration technique showed the best performance characteristics of the enumeration media tested, because this method possessed the best recovery efficiency, precision and accuracy, and also a good specificity.

Keywords: Enumeration media; Faecal streptococci; Water

1. Introduction

Faecal streptococci have received widespread interest as indicators of faecal pollution of natural aquatic ecosystems, since (i) they show a high and close

* Corresponding author. borrego@CCUMA.UMA.ES

Tel: +34-52131893; fax: +34-52132000; E-mail:

0167-7012/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0167-7012(95)00014-3

184 i.. PO Cafatuo Dionisio. J.J. Borrego i 1. Microbiological Methods 23 (iYU5) 183-203

relationship with health hazards associated with bathing in marine and freshwater environments, mainly for gastrointestinal symptoms [ 1,2]; (ii) they are not as ubiquitous as coliforms [3]; (iii) they are always present in the faeces of warm-blooded animals [4]; (iv) they are unable to multiply in sewage-contaminated waters [5]; and (v) in seawater they live longer than coliforms [6,7].

The main problem with these universally used microorganisms is the lack of a standard methodology for their selective enumeration from natural waters. More than 70 media have been proposed for this purpose until now [8,9]. However, a standard methodology has not been adopted, since few studies have been conducted on their comparative recovery efficiencies from water samples.

Early attempts to quantify faecal streptococci relied on enrichment tube procedures associated with the use of the Most-Probable-Number method, Rothe Azide Dextrose medium followed by a confirmation in Ethyl Violet Azide broth being the procedure most widely accepted by researchers. The enumeration of faecal streptococci by a membrane filter procedure was first reported by Slanetz and Bartley [lo], and since then several media have been proposed, including Thallous Acetate agar [ll], KF agar [ 121, PSE agar [13], Kanamycin Aesculin Azide (KEA) agar [ 141, mSD [ 1.51, modified mE agar (Dufour, A.P., Abstract Ann. Meet. A.S.M., Miami Beach, 1980). Other media formulations and incubation procedures for faecal streptococci have been proposed for specific situations [16], increasing the membrane incubation period from 48 to 72 h to recover stressed faecal streptococci from chlorinated effluents. Rutkowski and Sjogren [17] developed a medium designated M2 to distinguish between human and animal pollution sources, and recently a rapid system for enumeration of faecal streptococci and enterococci from water samples using a miniaturized fluorogenic assay has been proposed [ 18,191.

An additional problem related to the selective enumeration of this group of microorganisms is its taxonomical and ecological heterogeneity. The group called faecal streptococci includes species of different sanitary significance and survival characteristics [20]; in addition, the proportion of the species of this group are not the same in animal and human faeces [17,19]. Therefore, a clearer definition of faecal streptococci is necessary in order to establish a specific standard meth- odology of enumeration. The taxonomy of this group has been subjected to extensive revision in recent years [21-231, comprising two genera, Enterococcus and Streptococcus, with the following species [24]: Enterococcus faecalis, E. faecium, E. durans, E. hirae, E. avium. E. gallinarum, E. cecorum, Streptococcus bovis, S. equines, S. alactolyticus, S. intestinalis, S. hyointestinalis and S. acidominumus.

The aim of the present study was to compare the currently used methods to recover and enumerate faecal streptococci from seawater samples. Recovery media were evaluated on the basis of their accuracy, specificity, selectivity, precision and relative recovery efficiency characteristics.

L. PO Calatao Dionisio, J.J. Borrego i J. Microbiological Methods 23 (1995) 183-203 185

2. Materials and methods

2.1. Microorganisms and growth conditions

The microorganisms used in this study are listed in Table 1. All the strains were grown in Tryptone Soya broth (TSB) (Oxoid) at 36°C for 18-24 h. Isolations were carried out on Tryptone Soya agar (TSA) (Oxoid).

2.2. Media

Tryptone Soya agar (TSA) and Plate Count agar (PCA) (Difco) were used as control media in the qualitative growth tests, and TSA was also employed for the isolation of colonies from recovery media. PCA was used as a reference medium in the selectivity tests.

The following selective media were used for the recovery of faecal streptococci from water samples: mEnterococcus agar (Difco), KF agar (Difco), Kanamycin Aesculin Azide agar (KEA) (Oxoid), Bile Aesculin Azide agar (PSE) (Difco),

Table 1 Qualitative growth of different microorganisms of faecal streptococci selective media

Strains Media

mEnterococcus KF PSE KEA Barne’s Rothe’s Litskv’s

Enterococcus faecalis ATCC 8213 E. faecalis ATCC 19433 E. faecium ATCC 10541 Streptococcus equinu.s CECT 973 S.A.S. S. mitis CECT 804 Staphylococcus aureus NCTC 8532 S. epidermidis NCTC 4276 Escherichia coli NCTC 9001 KlebsielIa aerogenes NCTC 418 K. pneumoniae ATCC 13883 Proteus vulgaris CECT 484 Salmonella typhimurium

ATCC 13311 Pseudomonas aeruginosa

ATCC 10145 Bacillus stearothermophilus

NCTC 10003 Candida albicans INSA 969

+++” +++ +++ +++ +++ +++ +++ +++ +++ +++ _c -

+++ +++ +++ +++ +++ +++ +++

+++ +++ +++ +

h

+++

+++

_ _ _ _

rc_ +

_

+++

+++

_

+++

_

+++

+++ +++ +++ +++ +++ ++d ++ +++ +++ ++ +++ +++

+++

+++

-

+++ +++ +++ +++ +++ +++ +++ +++ 2

+

+++

+++

+++

+++

+++

+++ +++ +++ +++ +++ -

-

4-c

+

-

ATCC. American Type Culture Collection; CENT, Coleccion Espaiiola de Cultivos Tipos; NCTC, National Collection of Type Cultures; INSA, Instituto National de Saude (Portugal). a Growth 24 h. b Reduced growth. ’ No growth. d Growth at 48 h. ’ Growth at 72 h.

IX6 L. PO Calatao Dionisia. J.J. Borrego I J. Microbiological Methods 23 (lYY5) 183%2U3

Bile Aesculin agar (BE) (Difco), Mitis-Salivarius agar (Oxoid), Thallous Acetate agar (Barne’s medium) (BioLife). Azide Dextrose broth (Rothe’s medium) (Difco) and Ethyl Violet Azide broth (Litsky’s medium) (Difco). All the media were prepared according to the instructions of the manufacturers.

2.3. Recovery methods

Water samples were collected in sterile glass bottles from polluted wells near

Faro (Portugal) and faecally polluted seawater from Ria Formosa (Faro, Portugal) over a l-year period (January, 1992 to January, 1993). The samples were taken from up to 30 cm below the water surface, stored in isothermic containers (about 4”C), and shipped to the laboratory, where they were processed

within 4 h of collection. The membrane filtration technique was used in conjunction with the solid

media mentioned above for the recovery of faecal streptococci from water samples. Three appropriate volumes of each sample were filtered using 0.45pm pore size membrane filters (HAWG 047, Millipore). Each sample was five-fold

replicated. Phosphate-buffered saline [25] was used as diluent and washing solution.

Typical colony appearance and the incubation procedures of the media tested were as follows: mEnterococcus agar, pink or dark red-brown colonies (36 + l”C,

48-72 h); KF agar, red or pink colonies (36 -+ l”C, 48 h); KEA, BE and PSE agars, translucent to whitish colonies surrounded by dark brown to black halos (36? 1°C. 24-48 h); Barne’s medium, white or pink colonies with red or

brown-red centers; and Mitis-Salivarius agar. brilliant blue colonies, sometimes with a ‘gum drop’ appearance. After the incubation period (36 ? l”C, 24-48 h), all the typical colonies grown on the filters were noted as presumptive counts.

The Most Probable Number (MPN) technique was also applied to recover faecal streptococci from the samples tested. A two-step method, using Rothe’s and Litsky’s media [25], as presumptive and confirmatory tests, respectively, was performed. Both broths were incubated at 36? 1°C for 48 h. The presence of growth with a red-brown deposit is considered as a positive result for faecal streptococci.

2.4. Verification

After incubation all the colonies grown in each medium were counted and recorded. Then, a selected number (about the square root of the count of typical and non-typical colonies) were isolated from each filter cultured with an optimal colony count range (between 20 and 100 colonies). The colonies isolated on TSA plates (incubation at 36 +- 1°C for 24-36 h) were examined by gram stain and tested for catalase reaction. The gram-positive cocci in chains and catalase- negative strains were subcultured on Brain Heart Infusion (BHI) agar (Difco) slants, and after incubation at 36°C for 24 h, were stored at 4°C. Stock cultures were also frozen in 10% glycerol and stored at -20°C.

L. PO Calatao Dionisio, J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203 187

The colonies selected as faecal streptococci- or enterococci-like were further screened applying the test systems specified by Facklam and coworkers [26,27]. To differentiate enterococci from other faecal streptococci, the tests proposed by Devriese et al. [28] were conducted, including growth in BHI broth supplemented with 6.5% NaCl, hydrolysis of aesculin, resistance to sodium azide and reduction of tryphenil tetrazolium chloride. The final confirmation of the isolates was accomplished with the API 20 STREP system (BioMerieux).

2.5. Efficiency tests

To compare the different selective media and procedures to enumerate faecal streptococci, the criteria proposed by Levin and Cabelli [29] and by El-Shaarawi and Pipes [30] were considered. These include the study of the qualitative growth, selectivity, specificity, recovery efficiency, precision and accuracy of the methods.

For the qualitative growth studies, the microorganisms were cultured in TSB and incubated at 36°C for 24-36 h. They were then diluted in phosphate-buffered saline with 1% of peptone (Difco) for inoculum adjustment. Each culture was streaked on the test media in petri dishes or in tubes for the broth media and incubated at 36 -+ 1°C for 24, 48 and 72 h. The experiment was replicated five times. PCA was used as a control medium.

The selectivity of the media was determined for each of the samples examined by comparing total (typical and non-typical) colony numbers grown on the different selective media plates (or MPN) with the total heterotroph density obtained on PCA spread plates.

Media specificities for faecal streptococci were determined from physiological and biochemical tests performed on both presumptive positive and negative colonies, grown from natural water samples on the media tested. The MPN method was not tested for this purpose. The above mentioned verification methodologies were applied for determining the specificity of all solid media tested.

To compare the different selective media used, seawater samples were analysed both by membrane filtration and MPN techniques. The relative recovery ef- ficiency of the media was evaluated, following the criteria pointed out by El-Shaarawi and Pipes [30]. The recovery efficiency of each medium was calculated with respect to the maximal count obtained for one medium and sample, applying the following equation:

Relative Recovery Percent (medium A) = [(count on medium A)l(maximum

cowt on any medium)] x 100.

For precision evaluation of the different media the D* Fisher index was used, applying the Eisenhart and Wilson [31] formula, expressed as:

D* = [N&i* - (Z,xi)*]lCxi

where xi is the count obtained in each plate of the same volume and sample, and

18X L. PO Calalao Dionisio. J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203

N is the number of replicated plates (5 for each volume assayed). The number of samples tested for the media was 13 for seawater and 8 for freshwater.

Cultures of four faecal streptococci strains (Enterococcus fuecium ATCC 10541, E. fuecalis ATCC 19433, Streptococcus equinus CECT 973 and S. mitis CECT 804) with cell densities varying between lo’-lo9 CFU per ml in TSB, were suspended in filtered seawater, saline solution (0.85%) and filtered freshwater. Appropriate volumes of cultures were used as inocula to obtain a concentration of ca. 10’ cells per ml and were stored at 4°C. Accuracy test of strain suspensions was carried out, in five replicates, at 0, 6, and 24 h by the spread plate method. The arithmetical mean for each experimental condition was calculated. Accuracy was determined by comparison of the strain recoveries obtained with each medium with those obtained with the reference media (PCA and TSA) by the following equation:

Z[(CFiJ for each mediumlmean CFU for reference media) x lOO]lnumber of

samples

2.6. Statistical analysis

The nonparametric test of Kendall concordance coefficient was used to compare the different selective media tested. This allows us to determine the order of the media on the basis of the efficiency characteristics studied (qualitative growth, selectivity, specificity, relative recovery, precision and accuracy). All statistical analyses were performed by using an Apple Macintosh IIsi computer and the StatView 512 Plus and Excel applications as software.

3. Results

A total of 15 strains and 7 different selective media were used in qualitative growth experiments repeated five times (Table 1). All the selective media achieved the growth of all faecal streptococci tested, except in the case of KEA agar in which a reduced growth of S. equinus was observed at the end of the tested period. Only mEnterococcus agar and KF agar were totally selective, since they allowed the optimal growth of all faecal streptococci strains at 24 h, and none of the other non-faecal streptococci strains could grow. On the other hand, PSE and KEA agars, which are routinely used for the recovery of enterococci and faecal streptococci, showed optimal growth of background microorganisms, such as Staphylococcus aureus, S. epidermidis. Pseudomonas aeruginosa and Candida albicans strains, and reduced growth of the strains of the genus Kfebsiella. Barnes agar and Azide Dextrose broth (Rothe’s medium) allowed the growth of almost all the strains tested. However, the confirmatory broth, Ethyl Violet Azide broth (Litsky’s medium), showed good selectivity (Table 1).

The selectivity of 8 different selective media was tested using 13 natural seawater samples collected during 1992 (Table 2). Selectivity measures the

L. PO Calatao Dionisio, J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203 189

Table 2 Selectivity of methods for enumerating faecal streptococci from natural seawater samples

Media Mean background reduction factor”

Spring (n=3)

Summer (n=l)

Fall (n = 7)

Winter (n =2)

Overall (n = 13)

mEnterococcus 106.4 78.3 243.8 113.8 179.4 KF 131.9 58.1 279.8 126.8 205.1 PSE 36.4 35.3 82.3 62.5 65.0 KEA 51.6 52.9 211.3 40.6 136.0 BE 4.1 1.5 7.3 2.8 5.4 Barne’s 47.4 29.5 197.7 51.4 127.6 Mitis-Salivarius 4.1 4.5 96.8 7.7 54.6 Rothe-Litsky 88.3 1.6 359.2 52.7 222.0

a Ratio between total colonies counted on control medium (PCA) and total colonies counted by each method tested. Values show the mean of eight samples for each test and sampling.

capability of one medium to inhibit or reduce the growth of the background microbiota present with the faecal streptococci in the samples studied. A 3-log reduction of the background organisms was not reached for any medium; the average reduction varied between 2.35 and 0.73 logs for Rothe-Litsky procedure and BE agar. The most selective media were Rothe-Litsky, KF and mEnterococ- cus, since their average reduction factors did not show a significant difference (p > 0.1) (2.35 versus 2.31 versus 2.25 logs). The reduction factors exerted by the media tested varied according to the microbial level of the samples. Thus, at levels higher than lo6 CFU per 100 ml, all the media showed higher selectivity factors, ranging between almost 3 logs (2.98) for Rothe-Litsky procedure and 1.32 logs for BE agar; at levels lower than lo6 CFU per 100 ml, only KF and mEnterococcus media showed 2-log reductions.

A medium is considered specific for faecal streptococci when 90% or more of the typical colonies grown are verified as belonging to that microbial group. Moreover, the percentage of faecal streptococci that grow as non-typical colonies must not be higher than 10%. The specificity of the different media tested by the membrane filtration technique was determined by examining several different types of seawater samples. More than 500 colonies, grown on the selective media, were isolated and identified following the methodology specified in Materials and Methods section. The highest percentages of typical colonies verified as faecal streptococci were found on KF and mEnterococcus agars (98.5 and 97.5%. respectively) (Table 3). On the other hand, none of the non-typical colonies were confirmed as faecal streptococci. These media were the only ones that fulfilled the specificity criteria. The other media tested presented a very low specificity, since the percentage of confirmation as faecal streptococci from typical colonies varied from 80.5% for KEA agar to 6.8% for BE agar. However, the false-negative percentages were acceptable for all media tested (lower than 10%). The verification, at genus level, of the typical colonies grown in the different media is specified in Table 4. All typical colonies (533) were screened by the API 20

IYU L. PO Calatao Dionisio. J-J. Borrego I J. Microbiological Methods 23 (1095) 1X3-203

Table 3

Comparative soecificitv of different selective enumeration media for faecal streotococci

Media No. of colonies examined Typical colonies Non-typical colonies

No. Percentage of No. Percentage of

examined confirmation examined confirmation

as faecal as faecal

streptococci streptococci

mEnterococcus x0 KF 68

PSE 79

KEA 69 BE 73

Barne’s 69

Mitis-Salivarius 95

Total s33

X0 Y7.5 0 0

66 98.5 2 (I

60 68.3 1Y I.3

41 80.5 28 1.4

44 6.8 2’1 1.4

68 67.6 1 0

77 13.0 1X 0

436 97

Table 4

Verification at genus level of the typical colonies grown in the different selective enumeration media tested

Media No. of colonies Percentage of verification of typical colonies as

examined

Enterococcus Streptococcus Aerococcusl Staphylococcus Gram- Micrococcus negatives

mEnterococcus 80 90.0 7.5 1.25 1.25 0

KF 66 95.5 3.0 0 1.5 0

PSE 60 53.3 15.0 0 3.3 28.3

KEA 41 73.2 14.6 0 0 12.2

BE 44 0 6.X 0 40.9 52.3

Barne’s 68 5S.Y 16.2 0 11.8 16.2

Mitis-Salivarius 77 7.x 6.5 1.3 24.7 59.7

STREP system, and the species identification profiles are given in Table 5. All the faecal streptococci media recovered mainly species belonging to Enterococcus genus, except BE agar, which only recovered Streprococcus species, none of which were faecal streptococci. From the results obtained, BE and Mitis- Salivarius agars were discarded from further comparative studies.

To compare the efficiency of detection and recovery of faecal streptococci, thirteen natural seawater samples, with different pollution levels were analysed in parallel by the multiple tube and membrane filtration procedures using the following media: Rothe-Litsky for MPN, and mEnterococcus, KF, PSE, KEA and Barne’s media for membrane filtration technique, respectively. All the media tested were equally efficient in the detection of faecal streptococci from water samples (100%). However, the recovery rates of the media demonstrated differences, considering both typical colonies and confirmed faecal streptococci numbers (Table 6). The highest average relative recovery percentage was achieved in mEnterococcus agar and membrane filtration procedure (84.7%),

L. PO Calatao Dionisio. J. J. Borrego J J. Microbiological Methods 23 (1995) 183-203 191

Table 5 Identification of the typical colonies grown on the selective faecal streptococci media

Microorganism Medium

mEnterococcus KF KEA PSE BE M-S Barnes

S.A.S. Enterococcus faecium E. faecalis E. durans E. gallinarum E. avium Sireptococcus spp. S. bovis S. lactisldiacetilus

Staphylococcus spp. S. hominis S. warneri S. aureus S. epidermidis s. xylosus S. lentus S. saprophyticus S. cohnii Micrococcus spp. Aerococcus viridans Vibrio parahaemolyticus V. fluvialis V. alginolyticus V. vulnifcus V. damsela Aeromonas hydrophila A. sobria Chromobacterium violaceum Acirtetobacter calcoaceticus Moraxella spp. Pseudomonas aeruginosa P. cepacia P. pausimovilis P. stutzeri P. testolalcaligenes P. pickettii P. putrefaciens P. putida Flavobacterium indologenes Achromobacter spp. A. xylosoxidans Pasteurella spp. Morganella morganii Citrobacter freundii Enterobactercloacae E. intermedium E. agglomerans E. sakazakii

34” 36 20 11 14 16 2 6 22 11 7 13 2 0 0 2 0 0 1 0 6 2 3 7 0 0 0 2 0 0 3 0 1 1

1

2

4 1 1

4

1 3

2

1 2 1

1

1 3

2

0

0

0 0

0

3 0 0 7 1

2 4 1 2 1

3

2

2 1

1

1

1

1 2

0 22 2 5 4 10 0 0 0 1 4 6 0 2 1 3 6 5 1 1 1 3 1 3 1 1 2 1 1 1

1 1

1 3 1

2 1 2

3

1 1 2

1 4 2 5 3 1 2 2

192 L. PO Calalao Dionisio. J.J. Borrego i J. Microbiological Methods 23 (1995) 183-203

Table 5 (continued)

Microorganisms Medium

mEnterococcus KF KEA PSE BE M-S Barne’s

Escherichia coli 2 2 E. adecarboxylata 2 Klebsiella oxyloca 6 1

K. pneumoniae spp. pneumoniae 2 5 K. pneumoniae spp. ozaenae I Yersinia infermedia 1

* Number of isolates

Table 6

Comparison of the efficiency of different media and procedures for recovery of faecal streptococci from seawater samples

Medm Sample no Average

1 1 3 4 5 6 7 x 9 IV 11 I? 13

mEnterococcus x4.4” x0.9 l(x) 75.1 73.‘l 93.3 1on 100 loo 81.4 72.4 91.0 43.0 84.7

KF 70.2 XI.8 51.7 1011 48.6 47.6 9x.5 6X.7 x3.2 R4.x 54.6 XI 1 40.4 70.1

PSE lol? 58.2 57.0 x4.(1 37.3 ?I. I 34.7 87.5 55 4 97 4 6X.3 17 I 42.7 59.0

KEA 71.Y 63.9 9.4 68.0 46.2 36.7 89.7 b8.7 80.8 78.7 41,s 49.u SO.3 65.Y

Barlle‘, 97.9 19.5 69.1 94 1 50.6 56.2 90.9 75.x 82.4 100 5x.4 58.0 74.6 71.3

Rothe-Litsky 60.1 lO(l 86.1 36.Y INI IOU 58.4 73.7 53.4 8b.5 IO0 loo loo 81.2

*Relative recovery efficiency percentage was obtained applying the following equation:

h Relative Recovery Percentage (Medium A) = ((Count on medium A)/(Maximum count on any medium)1 x 100

although no significant difference was observed by the MPN procedure using Rothe and Litsky broths (81.2%). On the other hand, a relative recovery rate of faecal streptococci as low as 59% was achieved by PSE agar in conjunction with membrane filtration technique.

The precision of the different media was graphically determined from disper- sion of Fisher index D’ values of the assay variability for mEnterococcus, KF, PSE. KEA, Barne’s and Mitis-Salivarius media. This index indicates if the existing variability is due to random distribution or influenced by other additional factors. In Fig. 1 the D* values for each medium calculated from the examination of 21 samples (13 of seawater and 8 of freshwater) are represented in five-fold replication, in comparison with the theoretical D’ values for the control limits for p equal to 0.5, 0.05, 0.025 and 0.005. The xi values were considered typical colonies grown on the different media. As it can be seen, there was a uniform distribution of the counts for mEnterococcus, KEA and Mitis-Salivarius agars, and no significant effect from plate to plate was detected. A good precision was also recorded for PSE medium, although two samples presented 0’ values higher than the confidence levels for p = 0.005. Barne’s and KF media showed the worst precision performances.

Accuracy tests of strain cultures (E. fuecium, E. faecalis, S. mitis and S. equinus) and their suspensions were carried out at 0, 6, and 24 h by the spread plate method using mEnterococcus, KF, PSE and KEA media. PCA and TSA were used as reference media. The results of the recovery of three test strains (S.

L. PO Calatao Dionisio, J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203 193

0.025 0.05

6 0.5 2

a 8 i; 5 i;

I ih-----o--------1-.- $--~__+~~~~---+----+__--_,_-

0 2 4 6 0 10 12 14 16 18 20 22

Number of samples

Fig. 1. Precision of different selective media as estimated from dispersion of DL Fisher values. Symbols: (0, mEnterococcus); (0, KF); (W, KEA); (A, PSE); (X Mitis salivarius); (+ Barne’s).

equinus died rapidly in the suspensions) are shown in Tables 7 and 8. From seawater suspensions all the media tested showed high recovery percentages, with an interval average recovery from 74.8% (for KF with S. &is) to 116.3% (for mEnterococcus with E. fuecuZis) (Table 7). On the other hand, from freshwater suspensions average recovery percentages varied from 66.7% (for PSE with S. mitis) to 111.7% (for KF with E. fuecium) (Table 8). Significant differences (p < 0.005) in the recovery of Enterococcus strains versus the Streptococcus strain were obtained by all the media from freshwater suspensions, and by all the media except KEA agar from seawater suspensions (data not shown). The ranking of the

Table I Relative recovery of. several pure cultures of faecal streptococci strains suspensions in filtered seawater on selective media

Media Relative recovery percentage of strain’

E. faecium ATCC 105431 E. faecaiis ATCC 8213 5. mi& CECT 804 stressed for(h) stressed for(h) stressed for(h)

0 6 24 Average 0 6 24 Average 0 6 24 Average

mEnterococcus 79.4b 96.2 92.1 89.2 115.6 96.2 137.2 116.3 76.6 81.1 81.0 79.6 KF 90.1 90.1 124.7 101.6 107.8 99.6 51.6 86.3 76.6 72.1 15.1 14.8 PSE 78.1 77.8 93.2 83.0 100.3 99.7 144.0 114.7 73.9 80.8 75.7 76.8 KEA 79.3 80.8 86.5 82.2 89.1 85.1 78.2 84.1 85.1 88.0 84.1 85.7

a Relative recovery percentage = (Mean number of colonies on selective media/mean number of colonies counted on reference media) X 100. b Average of three different experiments replicated live-fold.

194 L. t’o Calatao Dionisio. J.J. Borrego I J. Microbiological Methods 23 (1995) 1X3-20.3

Table 8

Relative recovery of several pure cultures of faecal streptococci strains suspensions in filtered freshwater on selective media

Media Relative recovery percentage of strain”

E. faecium ATCC 105431 E. faecalis ATCC 8213 S. miris CECT 804

stressed for(h) stressed for(h) stressed for(h)

0 6 24 Average 0 6 24 Average 0 6 24 Average

mEnterococcus 80.3h 84.2 105.5 90.0 80.1 68.7 140.3 96.4 82.2 61.2 19.4 14.3

KF 107.1 93.1 135.0 111.7 92.1 98.5 99.6 96.7 69.6 53.8 81.4 68.3

PSE 79.6 84.9 127.1 :::f 67.4 61.6 72.8 67.3 81.1 52.2 66.7 66.7

KEA 79.1 84.5 98.5 70.8 76.0 84.6 77.2 76.7 71.6 65.8 71.3

a Relative recovery percentage = (Mean number of colonies on selective media/mean number of colonies counted on

reference media) x 100.

h Average of three different experiments replicated five-fold.

media compared for the accuracy test for both types of suspensions was mEnterococcus agar (90.9%), KF agar (89.9%), PSE agar (84.3%), and KEA agar (81.3%).

4. Discussion

European Community directives [32,33] concerning the microbial quality of recreational waters, especially swimming waters, prescribe the microbial param- eters for determining the pollution level of these waters. One of them is faecal streptococci, the usefulness of which as faecal indicators has been repeatedly evaluated, and is generally accepted because of their inability to multiply in water under natural conditions [5,7,20]. However, the lack of standardization of the enumeration procedures is the main shortcoming for the universal use of this microbial group.

From the two procedures recommended by EEC for faecal streptococci quantification, the MPN technique and membrane filtration procedure have been compared in the present study on the basis of the following characteristics: qualitative growth, selectivity and recovery efficiency. The use of the MPN technique as recommended by WHO [34] using exclusively Rothe’s broth lacks selectivity for the faecal streptococci analysis, since this medium allows the growth of other Gram-positive and Gram-negative microorganisms. The MPN technique increases selectivity when the confirmatory test (using Litsky’s broth) is per- formed, although the number of false-positive results obtained is too high [35]. Furthermore, the MPN technique using Rothe’s and Litsky’s media presents a high recovery efficiency (81.2%) (Table 6). However, the MPN technique is time consuming (up to 4 days) and requires a high number of laboratory equipment. Membrane filtration procedure is easier, cheaper, shorter (l-2 days) [4], and allows the processing of a large volume of sample and the immediate biochemical testing of isolated colonies [36].

L. PO Calatao Dionisio, J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203 195

For these reasons, the present study was focused on the comparison of selective enumeration media for faecal streptococci in conjunction with the membrane filtration technique. From the results of this and other studies, it has become clear that an optimal medium for enumeration of faecal streptococci from water samples has not been established yet, due to the taxonomical and physiological heterogeneity of the microorganisms included in this group. Thus, the first aim of this study was to compare several commercial selective media designed for the faecal streptococci recovery, and on the basis of the results obtained, to propose the most adequate method to enumerate faecal streptococci from seawater samples.

The present study shows that only mEnterococcus and KF media were completely selective on the basis of the qualitative growth of pure laboratory strains (Table l), and that media widely used for the isolation and enumeration of faecal streptococci, such as Pzifer Selective Enterococcus (PSE) agar, Kanamycin-Aesculin-Azide (KEA) agar, and Thallous Acetate (Bame’s) agar allowed the growth of strains belonging to Staphylococcus, Pseudomonas and Candida genera, as well as different species of enterobacteria. Other authors have reported that these media should allow the growth of Staphylococcus, Proteus, Corynebacterium, Vibrio, Aeromonas, Listeria and Candida strains [8,9,13,37]. However, it must be considered that the qualitative growth of pure strains on different media cannot determine the specificity of a recovery medium, since the presence of different background organisms at different concentration levels in the natural samples can influence the performance characteristics of the media tested.

A medium is selective when its ability to reduce background bacteria exceeds 3 orders of magnitude. As can be seen in Table 2, the reduction factors obtained for the media tested were low, and none of them fulfilled the selectivity requirement pointed out by Levin and Cabelli [29]. The majority of the media formulated for the selective enumeration of faecal streptococci from aquatic environments base their selectivity on the incorporation in the medium composition of one or more inhibitory substances, such as sodium azide, ethyl violet, thallous acetate, Tween- 80, and antibiotics, such as nalidixic acid, kanamycin, to name a few. These inhibitors are used to suppress the growth of background microbiota, but can also inhibit the development of injured-faecal streptococci [38]. As reported by Levin et al. [15], we also found that seasonal variations may influence the selectivity results obtained by the same media (Table 2). In order to increase the selectivity of the conventional enumeration media several authors have increased the incubation temperature close to 44°C [20,39]. The results obtained in these published studies indicated that restrictive incubation temperatures (44°C) gener- ally reduce the number of typical colonies recovered, mainly due to the inability of some species of faecal streptococci to grow at this temperature, although the growth of marine vibrios that grow optimahy at 36°C is also reduced [39]. This author [39] also showed that a 42°C incubation temperature improved the KF agar performance, but no significant difference was found for mEnterococcus agar.

mEnterococcus and KF media showed a good specificity, since more than 90%

196 L. f'o Calatao Dionisio. J.J. Borrego I J. Microbiological Methods 2_? (199-5) l#_?-203

of the typical colonies, were confirmed as faecal streptococci from both media, and none of the non-typical colonies were recorded as faecal streptococci (Table 3). The verification at genus level of the typical colonies grown in the different selective enumeration media tested is given in Table 4. Confirmation rates of enterococci and faecal streptococci were similar for mEnterococcus and KF media, and none of them allowed the growth of Gram-negative microorganisms. The main background microbiota recovered by these media were Aerococcus/ Micrococcus, and Stuphylococcus. Similar results have been reported by several authors [4,12]. The species identified from the typical colonies grown on mEnterococcus or KF media were Enterococcus fuecium, E. duruns, and E. fuecalis. Similarly, Bayne et al. [40] and Yoshpe-Purer [9] showed that these species were the predominant components of the faecal streptococci group in the aquatic environment.

Different authors have reported that mEnterococcus agar should support the growth of lactobacilli [4] and non-faecal streptococci [lo]. Furthermore, there is now some doubt as to whether this medium recovers faecal streptococci or enterococci. Slanetz and Bartley [lo] originally designed this medium for the enumeration of enterococci, although actually this medium is recommended by the 17th and 18th editions of Standard Methods [25,41] for enumerating faecal streptococci from fresh and marine waters. On the other hand, it seems that faecal streptococci of animal faeces, such as Streptococcus bovis and S. equinus are poorly recovered on this medium [37]. However, Page1 and Hardy [42] reported that mEnterococcus was the only medium which showed ability to grow faecal streptococci species other than enterococci. These authors suggested the possibility of using the typical count on this medium to estimate the enterococcus population and the total number of colonies to provide a faecal streptococcal count.

Similarly, it has been reported that KF agar can isolate Pediococcus and Luctobucillus species [12], Staphylococcus uureus [43], Aerococcus, Micrococcus, Bacillus, Escherichiu coli and non-faecal streptococci, such as S. cremoris and S. sunguis [4,42], and Aerococcus, marine vibrios, Pasteurella, Aeromonus, Plesiomonus, Moruxellu, Agrobucterium. Pseudomonas and non-faecal Strep- tococcus species [9]. For this reason, several authors [5,9] considered this medium as inappropriate to recover faecal streptococci from seawater and shellfish samples. The role of KF agar to support the growth of S. bovis and S. equinus is in controversy, since some authors have reported the failure of this medium to recover faecal streptococci of an animal faecal origin [9,42,44], whereas others have isolated S. equinus and S. bovis from KF agar [8,37,45,46].

In the present paper, we could note that S. equinus grows optimally in mEnterococcus and KF agars in pure cultures growth experiments (Table 1); however, when S. equinus was inoculated to filtered seawater in the accuracy experiments, the inhibitory power of those media (mEnterococcus and KF) prevented the growth of this species. This finding can be explained by the fact of the low survival capability of this species outside the gastrointestinal tract [47] or in natural aquatic environments [6,48], and/or the low growth ability of their sublethally injured cells in selective media [49].

L. PO Calatao Dionisio, J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203 197

The specificity of PSE and KEA agars were low, since only 68.3% and 80.5%, respectively, of the typical colonies on the basis of the presence of dense black halos indicative of aesculin hydrolysis were confirmed as faecal streptococci (Table 3). Although aesculin hydrolysis has been proved to be a reliable means of separating group D from other streptococci [50,51], this test cannot be considered as an essential identification criterion [45], since a high number of microorganisms can hydrolyse aesculin, including Vibrio, Pseudomonas and Staphylococcus species [9]. These media presented a low capability of discrimination between typical and non-typical faecal streptococci colonies, and in addition allowed the growth of numerous pinpoint colonies [4,8]. The main background microbiota grown on these media were non-faecal streptococci, Staphylococcus, Vibrio, Chromobacterium, Acinetobacter, Pseudomonas, and members of the Enterobac- teriaceae family (Table 5). Similar results have been reported in several studies [8,9,13,37]. In addition, Volterra et al. [4] detected high percentages (up to 30%) of rod-shaped bacteria that were able to produce colonies with similar appearance to that of typical faecal streptococci (with a black halo on the reverse of the filter) in Mediterranean coastal samples. On the contrary, Brodsky and Schiemann [45] reported that PSE agar possessed a high selectivity for enterococci, since more than 90% of all colonies grown on membrane filters were confirmed as faecal streptococci and 86% were confirmed as enterococci. Furthermore, these authors reported an additional advantage of this medium on the basis of its shorter incubation period (24 h). Similarly to the results reported by Nowlan and Deibel [52], PSE allowed the growth of S. equines and S. bovis from both pure cultures and water samples. On the contrary, none of these species were able to grow on KEA agar (Tables 1 and 5). The use of the mSD medium and confirmation on aesculin iron agar proposed by Levin et al. [15] is also based on aesculin hydrolysis as a marker for faecal streptococci. This procedure has not been tested in the present study because of two shortcomings: (i) the necessity of a rapid method without a confirmation step, and (ii) the low specificity displayed by this procedure for Mediterranean samples [4,9].

The specificity of Barnes, BE and Mitis-Salivarius media are given in Tables 3-5. All these media possessed low specificity, since only 6.8%, 13% and 67.6% for BE, Mitis-Salivarius and Barne’s agars, respectively, of the typical colonies were confirmed as faecal streptococci (Table 3). The main background micro- organisms, at genus level, that grow as typical colonies were Gram-negatives, Staphylococcus, and AerococcuslMicrococcus (Table 4). None of the isolates on BE agar were Enterococcus species, and only three isolates were Streptococcus spp. The most frequently recovered species by this medium were Staphylococcus spp., S. epidermidis, and Vibrio parahaemolyticus. In addition, this medium also allowed the growth of Aeromonas, Moraxella, Pseudomonas and Flavobacterium (Table 5). The low specificity obtained by this medium is easily explained because it is used mainly as confirmative plating agar to demonstrate the aesculin hydrolysis of the isolates at 37°C [25].

The microbial species spectra isolated from Barnes medium was lower than from BE agar, including Staphylococcus, Vibrio, Pseudomonas, and enterobac- teria strains (Table 5). On the contrary, Mitis-Salivarius agar allowed the growth

198 L. PO Calatao Dionisio. J.J. Borrego I J. Microbiological Methods 23 (19%) 1X3-203

of a high number of Staphylococcus species, as well as enterobacteria strains (Table 5). Other background microbiota grown in this medium were non-faecal streptococci, Pseudomonas, Vibrio, Pasteurella, Achromobacier, and Micrococ- cus. On the other hand, this medium recovered low percentages of both enterococcal and faecal streptococcal species (7.8 and 6.5%, respectively). These results can be explained by the fact of the formulation of this medium, since it was designed to improve the isolation of ‘viridans’ streptococci [53]. Some authors include S. miiis and S. salivarius as faecal streptococcal species, because they were found in high concentrations in human faeces [.54]. However, it must be considered that these species are inhabitants of the nasopharyngeal tract and usually grouped with the ‘oral streptococci’ [24].

The relationship between the inhibitory and indicator substances contained in the media tested and their selectivity and specificity seems clear. The media with sodium azide and TIC were more selective than those with antibiotics and base their colonial differentiation power on the aesculin hydrolysis. Easterbrook and West [55] reported the same conclusions when they carried out a detailed comparative study for the isolation of group D streptococci from shellfish.

The comparison of the enumeration methods for faecal streptococci was conducted studying their relative recovery efficiency, where the maximum titre obtained for any method for the same sample was considered as reference value (100%). In Table 6, the relative recovery efficiencies of six enumeration methods are shown for 13 seawater samples. The highest average values of faecal streptococci recovery were obtained by mEnterococcus and membrane filtration (84.7%) and Rothe-Litsky broths with MPN technique (81.2%). The ranking of the methods tested was the following: mEnterococcus > MPN (Rothe-Litsky) > Barne’s > KF > KEA > PSE. However, if we apply the specificity rates of the media (Table 3) and considering the MPN (Rothe-Litsky) specificity rate obtained by Volterra et al. [4] (86.7%), the ranking is established as follows: mEnterococcus (82.6%) > MPN (Rothe-Litsky) (70.4%) > KF (69.0%) > KEA (53.0%) > Barne’s (48.2%) > PSE (40.3%). Similar results were obtained by Volterra et al [4], who found that MPN (Rothe-Litsky) and mEnterococcus agar were the methods that yielded the highest recoveries from Adriatic seawater samples, mSD and PSE media being the methods with lowest recoveries. On the contrary, for Tyrrenian seawater samples, the lowest recoveries were achieved by using the MPN (Rothe-Litsky) method. Levin et al. [15], comparing the recovery efficiency of mSD with those PSE and KF from water samples, reported that neither the PSE nor the KF recoveries approached those obtained with the mSD method, although those by the PSE and KF methods did approximate each other. Gauci [20] concluded that mEnterococcus provided higher faecal streptococci counts compared to those obtained on KF agar (mean average 88 versus 11) using Mediterranean seawater samples. On the contrary, Page1 and Hardy [42] analysing in parallel wastewater samples from a variety of sources using five media (PSE, mEnterococcus, KF. mSD and SB) found that the ranking of recovery efficiency was the following: mEnterococcus = KF > SB > PSE > mSD. These disagreements between the results obtained from the recovery efficiency of

L. PO Calatao Dionkio, J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203 199

the media have been pointed out previously by different authors [37,45], who reported significantly greater recoveries of faecal streptococci on KF agar compared with PSE agar. However, Dutka and Kwan [56] found no significant differences between counts on PSE and KF media for sewage and lagoon samples. These differences may be due to the sample origin or a function of a seasonal variation, such as water temperature or rainfall [15]. In another study in Mediterranean water samples, PSE and mEnterococcus yielded the highest counts of colonies, whilst the lowest recovery was detected by the MPN method and on membranes laid on KF agar [57].

The precision of a microbiological enumeration method has two error sources, experimental errors depending on the manipulation, and sampling errors. The first ones are easily resolved by standardization of the methods and carefully maintained manipulation [58]. The sampling error, however, may only be minimized by increasing the number of samples and the number of replicates [30]. The precision test of the enumeration methods calculated by the values of Fisher dispersion index (D2) is expressed in Fig. 1. Only three values of mEnterococcus exceeded the p = 0.5 limit. This indicates that the experimental variability of the counts is less than that expected for the random because of alterations in the preparation of the medium, the nature of the inhibitory agents or other causes from different sources [58]. The other media tested showed lower precision, ranging from PSE (77%) KF (62%) and KEA, Mitis-Salivarius and Barne’s with 61.5% of the samples were included within p = 0.5 limit. All these percentages are higher than those obtained by Levin et al. [15] for mSD agar, since only 39% of the samples were included into the p = 0.5 limit. In order to establish a media ranking of precision, the percentages of samples that exceeded 0.05, 0.025 and 0.005 limits were considered, obtaining the following precision ranking: mEnterococcus > PSE > KF > KEA > Mitis-Salivarius > Barne’s.

The relative recoveries of four media (mEnterococcus, KF, PSE and KEA) was evaluated in parallel using pure cultures of three reference strains under two experimental conditions (seawater and freshwater) (Tables 7 and 8). Average recoveries with the media tested varied depending on the type of samples and the strain tested. In seawater suspensions (Table 7), the most efficient medium to recover the stressed faecal streptococci was mEnterococcus (mean 95%), and the worst was KEA (84%), although all the media fulfilled the recovery requirement proposed by Levin and Cabelli [29], established at a minimum recovery of 75%. In the most stressful conditions, faecal streptococci recovery with the reference media (non-selective) was greater than with selective media. However, there were instances in which recovery with the selective media tested increased with time exposure to a stressful environment. These results suggest that injury to the cells occurs during the lag phase and that the percentage of injured cells is lower after a long exposure time to stressful conditions. The decreased percentage of injured cells could be explained by either the repair or the inactivation phenomena subsequent to the cell injury [38,59].

In freshwater suspensions (Table S), the most efficient medium was KF (mean 92.2%), whilst PSE showed the worst performance (77.1%). Again all the media

2(K) L. PO Calatao Dionisio. J.J. Borrego I J. Microbiological Methods 23 (1995) 183-203

tested possessed a recovery percentage higher than 75%. However, if we only consider the enterococcal strains tested, the efficiency ranking changes for both seawater and freshwater suspensions, yielding the following rankings: mEnterococcus > PSE > KF > KEA for seawater suspensions; and KF> mEnterococcus > KEA > PSE for freshwater suspensions. This change in the recovery capability may be due to the influence of the chemical composition of the stress suspension, since the recovery efficiency of the enumeration media from aquatic environments is affected by the source and nature of the sample [60]. The efficiency of the media tested to recover S. mitis from seawater and freshwater suspensions was very similar, ranging between 71.6 and 78.5%.

In choosing the best method for the enumeration of faecal streptococci from water samples, each of the performance categories tested was considered (Table 9). In a simple ranking scheme, the best overall performance was shown by mEnterococcus agar which possesses the best accuracy, precision and recovery efficiency; in addition, this medium is good on the basis of its selectivity and specificity. In conclusion, the membrane filtration method with mEnterococcus agar, and the MPN method using the Rothe-Litsky media are the best procedures for the enumeration of faecal streptococci from seawater samples.

Table 9

Performance characteristics of the selective media tested applying the concordance coefficient of

Kendall

Characteristics mEnterococcus KF PSE KEA Barnes’ BE Mitis- Rothe-

Salivarius Litskv

Qualitative growth

Faecal streptococci 3

Others 1.5 Mean 2.25

Selectivity (Mean) 3

Specificity

False-positive 2.5 False-negative 2 Mean 2.25

Recovery efficiency

Typical colonies I

Verified colonies 1

Mean 1 Precision

At 0.5 limit 1

At 0.005 limit 1

Mean 1 Accuracy

Seawater 1

Freshwater 2

Mean 1.5 Overall 11 Mean 1.83 Ranking 1

3 3 6 3 NT” NT 3

1.5 5 4 6 NT NT 3

2.25 4 5 4.5 NT NT 3

2 6 4 5 7 8 1

2.5 5

1 4

1.75 4.5

4 6

3 6

3.5 6

3 2

3 2

3 2

3 2

1 4

2 3

14.5 25.5

2.42 4.25

3 4

‘NT: Not tested.

6.5 2.5 2.5 6.5 NT

3 5 6 7 NT

4.75 3.75 4.25 6.75 NT

5 3 NT NT 2

4 5 NT NT 2

4.5 4 NT NT 2

5 5 5 NT NT

4 6 5 NT NT

4.5 5.5 5 NT NT

4 NT NT NT NT

3 NT NT NT NT

3.5 NT NT NT NT

26.25 22.75 16.25 14.75 6

4.37 4.55 5.42 7.37 2

5 6 7 8 2

L. PO Calatao Dionisio, J. J. Borrego I J. Microbiological Methods 23 (1995) 183-203 201

Acknowledgments

This study was partially supported by research grants No. 74-B and No. E-61 from the Acciones Integradas Hispano-Portuguesa, Ministerios de Educaci6n y Ciencias of the Spanish and Portuguese governments. We thank to Miss M.J. Navarrete for her assistance in the English revision of the manuscript.

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