Use of the indirect immunofluorescence method for detectionand enumeration of Escherichia coli in seawater samples

G. Caruso, R. Zaccone and E. CrisafiIstituto Sperimentale Talassografico ± Consiglio Nazionale Ricerche, Messina, Italy

107/2000: received 28 March 2000 and accepted 13 June 2000

G . C A R U S O , R . Z A C C O N E A N D E . C R IS A F I . 2000. The determination of Escherichia coli in

marine waters through a rapid method, the microscopic indirect immuno¯uorescent

technique, is evaluated in comparison with the conventional count on m-FC agar medium.

The data obtained in seawater samples, collected monthly along the Messina coastline, show

good sensitivity of the analysis and agreement between the microscopic and culture

technique, with a detection limit of 102 cells 100 mlÿ1 for immuno¯uorescence.

INTRODUCTION

Recent proposals to modify the European Directive con-

cerning the suitability of seawaters for swimming have

been addressed with respect to research and quantitative

enumeration of Escherichia coli as the main indicator of fae-

cal pollution (Bonadonna et al. 1997). The choice of this

micro-organism is universally accepted by public health

surveillance services. Conventional methods prescribed for

its detection, however, are affected by certain limitations

(e.g. long incubation and response times, low accuracy)

which make their application to seawater monitoring inade-

quate (Volterra and Garizio 1997; Sartory and Watkins

1999). Rapid techniques developed in recent times include

speci®c enzymatic assays based on the evaluation of the

presence of b-glucuronidase using ¯uorogenic substrates,

such as 4-methylumbelliferyl-b-D-glucuronide (MUG test;

Caruso et al. 1998a), or immunological methods such as the

¯uorescent antibody technique (immuno¯uorescence) for

the speci®c detection of enteropathogenic serotypes

(Zaccone et al. 1995).

Within the general framework of the Consiglio

Nazionale Ricerche Strategic Project `Marine pollution

monitoring of the South of Italy', a comparative evaluation

of immuno¯uorescence and standard culture techniques

has been carried out in order to verify the sensitivity and

performance of the direct microscopic method.

MATERIALS AND METHODS

During the period April 1996 to March 1997, seawater

samples were drawn monthly from 15 stations located

along the Messina coastline, close to the outfall of urban

sewage. The samples were collected in Niskin bottles, kept

at 5 �C and treated within 2 h of sampling. A total of 229

heavily polluted samples, selected from among those col-

lected, were analysed at the same time by the conventional

culture method (FC) and by immuno¯uorescence (IF). For

culture analysis (FC), 100 ml subsamples were ®ltered in

duplicate and incubated on m-FC (Difco) agar medium

plates (APHA 1992) at 44�5 �C for 24 h, while a 100 ml

volume of the remaining sample was formalin-®xed (2%

®nal concentration) and then treated using the indirect

immuno¯uorescence (IF) staining procedures reported by

Crisa® et al. (1994) and Zaccone et al. (1995). A known

volume of water (at least 10 ml) was ®ltered through a

Nuclepore black membrane (0�22 mm pore diameter) and

washed three times with 5 ml phosphate-buffered saline

(PBS), pH 7�2. The ®lter was incubated at room tempera-

ture with 1 ml of a mixture of polyclonal E. coli antisera

(1:80) for 30 min. After washing with PBS, the ®lter was

then incubated with 1 ml of a ¯uorescein isothiocyanate

(FITC)-conjugated globulin antiglobulin (1:160) for 30

min. For the labelling, the immune sera Behring Serum

test Coli anti OK pool A, B and C, speci®c for entero-

pathogenic E. coli serotypes (pool A against E. coli O26,

O55, O111, O128; pool B against E. coli O86, O119, O125,

O126, O127; pool C against E. coli O114, O142 and O158)

and FITC-conjugate goat anti-rabbit IgG (Sigma), were

used.

Filters were observed with a Zeiss Axioplan epi¯uore-

scence microscope (®lter set BP 450±490, FT 510 and LP

520) and 30 microscopic ®elds were counted. Escherichiacoli cells appeared to be rod-shaped with a clear green out-

line.

Statistical evaluation of data was performed through cal-

culation of the mean value, standard deviation (S.D.) and

coef®cient of variation (C.V.� S.D./mean) (Kirchman et al.

Correspondence to: Dr Gabriella Caruso, Istituto Sperimentale Talassogra®co

± Consiglio Nazionale Ricerche, Spianata S. Raineri, 86±98122 Messina,

Italy.

Letters in Applied Microbiology 2000, 31, 274ÿ278

= 2000 The Society for Applied Microbiology

1982). The counts obtained with both techniques were also

evaluated for their conformity to assumption of homogeneity

through comparison of Fisher's dispersion index (D2) with

Chi-square (w2) critical values. Values of D2 lower than w2

indicated that there was homogeneity in bacterial counts

and therefore Poisson distribution was an appropriate

model to describe the data (El-Shaarawi et al. 1981).

Values which failed this assumption were discarded, and

the pair of data (184) which remained from the initial 229

was tested for internal variability through the calculation of

the coef®cient of variation among repeated measurements

(i.e. variability within 30 microscopic readings or within

two replicates on a plate) carried out within each sample.

The error rate (E%� IF-FC/FC) between the two methods

was also calculated.

RESULTS

The speci®city test of the sera used, which was performed

in order to exclude cross-reactions against other homolo-

gous and heterologous strains belonging to

Enterobacteriaceae and marine bacteria, showed no reaction

of the sera with autochthonous micro-organisms (Zaccone

et al. 1995).

The counts obtained by the IF technique, ranging from

a minimum of 1�3� 102 to a maximum of 1�1� 106 cells

100 mlÿ1, were comparable with those on m-FC agar,

which ranged from 5�2� 101 to 2�5� 106 cfu 100 mlÿ1.

Table 1 shows the monthly logarithmic mean, minimum

and maximum values of the bacterial densities.

The estimation of variability among replicates within

each sample showed lower C.V. values, and therefore

higher data reproducibility, for the FC than the IF method,

indicating that the latter method might be affected by a

higher variability due to the subjectivity of the microscopic

observation. The C.V. data obtained for plate counts

departed from the theoretical C.V. curve of the Poisson

distribution (C.V.� 1/p

x) more than microscopic counts

(Fig. 1).

Table 1 Monthly logarithmic mean, minimum and maximum values of bacterial densities obtained for the plate (FC) and

immuno¯uorescence (IF) methods

Months Samples Logarithmic mean Minimum Maximum

FC (cfu 100mlÿ1)

April 23 3�5 2�4 6�0May 11 3�9 3�2 5�1June 21 4�2 2�9 5�6July 13 4�1 2�7 5�5August 13 4�0 2�8 5�7September 19 3�8 2�4 5�4October 19 4�0 3�0 5�2November 19 3�4 2�8 5�9December 30 3�3 2�1 5�3January 19 3�5 2�6 5�4February 22 4�0 2�7 5�5March 20 3�9 3�0 5�6

IF (cells 100mlÿ1)

April 23 3�2 1�7 5�8May 11 3�5 2�5 5�2June 21 3�9 2�0 6�1July 13 3�7 2�0 4�9August 13 4�0 2�5 5�8September 19 3�5 1�7 6�1October 19 3�8 2�3 5�9November 19 3�4 2�1 5�4December 30 3�7 2�1 5�4January 19 3�5 2�2 6�4February 22 3�7 2�1 5�6March 20 3�7 2�1 6�4

275E . C OL I D E T E C T IO N B Y IM M U N O F LU O R E S C E N C E

= 2000 The Society for Applied Microbiology, Letters in Applied Microbiology, 31, 274ÿ278

The linear regression analysis of log10-transformed plate

counts vs IF counts (Fig. 2) showed a good correlation (R2

� 0�5841) between these two methods. The Student's t-values calculated for each sampling month (ranging from

0�17 to 0�91, P< 0�05) suggested that there was no signi®-

cant difference between the IF and FC data. The similar

course of the IF and FC counts is also evident in Fig. 3,

which reports the annual distribution of E. coli densities at

two representative sampling stations.

The error rate calculated between the two methods was

above 10% for samples with densities of faecal coliforms

ranging from 102 to 103 cfu 100 mlÿ1. This value decreased

to 0% for highly contaminated samples, with densities

higher than 104 cfu 100 mlÿ1 (Fig. 4).

DISCUSSION

Immuno¯uorescent methods have been successfully applied

for the speci®c detection of marine micro-organisms, i.e.,

belonging to Nitrosococcus (Zaccone et al. 1996) and

Synechococcus (Acosta Pomar et al. 1998). A new approach

based on the use of ¯uorescent probes (viable stains or

¯uorochrome-labelled antisera) together with ¯ow cytome-

try (FCM) seems to be very promising in marine microbial

ecology (Caruso et al. 1998b).

The method used here was designed to study the

amount and temporal course of E. coli densities discharged

into the sea. The data obtained in this investigation showed

no signi®cant difference between plate and microscopic

counts, despite some quantitative discrepancies of approxi-

mately two orders of magnitude which were probably due

to the inadequacy of FC medium for estimating viable but

non-culturable forms (as suggested also by Xu et al. 1982;

Roszack and Colwell 1987). On the basis of this observa-

tion, Brettar and Ho¯e (1992) suggested that immuno¯uore-

scent staining was a more sensitive technique than the plate

method for re¯ecting the total number of E. coli cells.

It should also be noted that the different approach of the

two methods, i.e., the ®rst directed towards the detection

of faecal coliforms and the second, towards E. coli, does

not affect the comparison between them, because E. colirepresents the main faecal coliform present in urban ef¯u-

ents.

Immuno¯uorescence data are also not signi®cantly dif-

ferent from enzymatic (MUG) values obtained on the same

coastal samples, as shown by the statistical analysis

(Student's t-values which ranged from 0�02 to 0�20, P<0�05) performed between these two methods (Caruso,

unpublished data). Furthermore, unlike the MUG assay,

the possibility of quantitative bias due to the detection of

Shigella, another enterobacterial genus phylogenetically

close to Escherichia (Brenner 1984), is avoided with IF.

This fact, in addition to other advantages such as the high

600

(a)

500

300

400

0

100

200

20·0Bacteria per field

c.v.

0·0 5·0 10·0 15·0

120

(b)

100

60

80

0

20

40

300200 250Bacteria per plate

c.v.

0 50 100 150

Fig. 1 Variability within each sample: coef®cient of variation

(C.V.) vs number of bacteria (a) per microscope ®eld (IF) or (b)

per plate (FC). (&), Theoretical C.V. curve (C.V.� 1/p

x) from

the Poisson distribution

Fig. 2 Linear regression analysis between immuno¯uorescence

(IF) and plate (FC) methods

276 G . C A R U S O E T A L .

= 2000 The Society for Applied Microbiology, Letters in Applied Microbiology, 31, 274ÿ278

sensitivity and speed (counts available within 2 h), further

encourages the use of this microscopic assay as a reliable

and effective tool for the speci®c quanti®cation of E. coli in

marine waters and therefore for the early warning of sew-

age pollution.

The application of this technique in environmental moni-

toring is, however, affected by a quantitative limit (102

cells 100 mlÿ1), which makes the use of immune sera advi-

sable for the sites characterized by high levels of contami-

nation, where the error rate decreases (Zaccone et al. 1995).

Depending on the quantity of bacteria and on the turbidity

of the sea water, the volume of ®ltered sample or the num-

ber of counted microscopic ®elds could be varied in order

to increase the accuracy of enumeration.

Furthermore, particular care has to be taken in the

choice of dilution of both antiserum and labelled immuno-

globulin to produce the maximum intensity of ¯uorescence.

Care also has to be taken in the choice of the test for anti-

serum speci®city in order to exclude the risk of cross-reac-

tivity due to non-speci®c bindings (Brayton and Colwell

1987).

Another possible limitation for the application of the

method relates to the availability and speci®c reactivity of

the immune sera. The commercially-available sera for E.coli, however, speci®cally recognize as `target' the surface

antigens of the enteropathogenic serotypes only, and there-

fore, IF does not allow quanti®cation of the global E. colipopulation because it is selective for a given serotype. The

production of new polyclonal antisera labelling a wider

number of serotypes, or the use of monoclonal antibodies

increasing the speci®city of the reaction, are objectives for

future research. Moreover, the development of new alter-

native methodologies (i.e., ¯uorescent in situ hybridization

with rRNA-targeted oligonucleotide probes) may make the

detection of E. coli in environmental samples more accu-

rate.

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(a)

0

1

2

3

4

5

F MJ

log

cfu

100

ml–1

log

n.ce

ll 10

0 m

l–1

A M J J A S N

6

0

1

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3

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6

(b)

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F MJ

log

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