Letters in Applied Microbiology 1995, 21, 207-209

Detection of coliphages and enteroviruses in sewage and aerosol from an activated sludge wastewater treatment plant

A. Carducci, S. Arrighi and A. Ruschi Department of Experimental, Infective and Public Biomedicine, University of Pisa, Pisa, Italy

RG/47: received and accepted 10 April 1995

A. CARDUCCI, s. ARRIGHI AND A. RUSCHI. 1995. Coliphages and enteroviruses were monitored over 12 months in sewage and air adjacent to an activated sludge plant. Both showed temporal variation bu t the mean count of phages in enterovirus-positive samples was not significantly different from that in enterovirus-negative samples. Hence coliphages are not necessarily a good indicator of enteroviruses in sewage and aerosols.

INTRODUCTION

Aerosols generated from sewage treatment plants may contain pathogenic micro-organisms. The evaluation of pathogenic viruses in aerosols is difficult because of their low concentration. Nevertheless, enteroviruses have been isolated from sewage generated aerosols (Teltsch and Katz- nelson 1978; Moore et al. 1979; Spendlove and Fannin 1982; Fannin et al. 1985). The difficulty in detecting viruses in environmental samples has led to coliphages being suggested as possible indicators of viral pollution (IAWPRC 1991). The aim of this report was to evaluate this suggestion by monitoring coliphages and enteroviruses in sewage and air at an activated sludge treatment plant in a densely populated suburb of Pisa, Italy.

MATERIALS AND METHODS

Samples were taken twice-monthly, from May 1992 to April 1993, from sewage at the inflow to the aeration tanks and from the air at their border. Air samples for coliphages were taken at 2 m, 20 m and 30 m from the tank and for enteroviruses at 2 m only. Sewage (100 ml) was collected in sterile bottles.

Aerosol was taken using the Surface Air System (SAS, International Pool Bioanalysis Italiana S.p.A, Milan), an impactor sampler with contact agar plates (Rodac) (Borioli et al. 1988); these plates contain a medium appropriate to the microbiological examination to be performed. For the detection of both coliphages and enteroviruses the sample volume was 0-9 m3 of air.

To detect coliphages in sewage, 1 ml of fresh nutrient broth culture of Escherichia coli K 12 (bacteriophage host

Correspondence to : Dr Annalaura Carducci, Dipartimento di Biomedicina Spcrimmiale, Infit iva e Pubblica, Universita degli studi di Pisa, Via S. Zrno 35, 56100 Pisa, Italy.

0 1995 The Society for Applied Bacteriology

cells), grown for 1 6 1 8 h at 37"C, was seeded with 0.1 ml of sewage. Seven millilitres of molten dilute agar (Plate Count Agar 7 g 1 - with sodium and calcium salts) were mixed with the culture (Jofre 1991). The mixture was then overlaid on the surface of a solid nutrient agar plate and incubated at 37°C for 12-14 h. The phage plaques were then counted and confirmed.

T o detect coliphages in aerosol, an agar layer method was used (Bolton et al. 1976): 5 ml of melted diluted agar (10 g 1 - with sodium and calcium salts) were inoculated with 0.5 ml of broth Escherichia coli K12 culture and over- laid onto the surface of a solid nutrient agar plate. Rodac plates, prepared as described, were used with SAS for air sampling and were then incubated at 37°C for 12-14 h. The phage plaques were counted and confirmed.

T o confirm the presence of coliphages, each suspected phage plaque was removed and inoculated into 10 ml of broth Escherichia coli K12 culture. After 16-18 h incu- bation at 37"C, the culture was centrifuged for 30 min at 2100 g and the supernatant fluid was collected and heated at 56°C for 30 min to inactivate bacteria. This material was then inoculated into an Escherichia coli K12 agar mixture overlaying solid agar plates as described above. Plaques were read after incubation at 37°C for 6 8 h.

For enterovirus detection in sewage, 10 ml of sample were mixed with 1 ml of chloroform, shaken for 30 min and centrifuged at 1200 g for 20 min. The supernatant fluid, aired for 3 h to eliminate chloroform, was inoculated onto monolayers of Buffalo Green Monkey kidney cells (BGM) (Dahling and Wright 1986) and Human Laringeal Carcin- oma cells (Hep-2) (Schmidt 1989). These cells, incubated at 37"C, were observed microscopically daily for 2 weeks or until a cytopathic effect appeared ; samples both positive and negative for cytopathic effect were passed an additional two times onto monolayers of BGM and Hep-2 cells to confirm negativity or positivity. The presence of entero- viruses was confirmed by indirect immunofluorescence,

208 A. CARDUCCI ET A L .

In T o detect enterovirus in aerosol, Rodac plates with Trypticase Soy Agar (TSA) were used (Hierholzer 1990). After sampling, the agar surface was sliced off into 8-10 ml of beef extract (3%, p H 9) and was vigorously mixed and centrifuged at 800 g for 30 min at 4°C. The supernatant fluid was neutralized with 1 mol 1 -' HCI and processed as described above for sewage samples. The recovery effi- ciency of elution from the agar surface was evaluated by spreading 0.1 ml of 1.13 x lo7 pfu ml-' viral suspension onto a Rodac plate with TSA, sampling pure air, slicing off the agar surface, and processing as described above. T o cal- culate recovery efficiency of virus elution, virus concentra- tion (pfu m1-I) found in the eluate was compared with the one of initial viral suspension. This evaluation was made on three repeated trials, each of two plates. The recovery effi- ciency of virus elution ranged from 19 to 68%, with an average of 40.3%.

2oo May Jun Jul Aug Sap Oct Nov Dec Jan Feb Mar Apr '' 6 ) Month

Flg. 1 Monthly mean values of coliphage concentration and number of positive samples for enteroviruses in sewage, May 1992-April 1993; two samples were taken per month. ., Coliphages; iJ, enteroviruses

using a human serum anti-enterovirus (Behringwerke AG, Marburg, Germany) and a fluorescein-conjugated goat RESULTS AND DISCUSSION

affinity-purified antibody to human IgG (Organon Teknika International, Turnhout, Belgium).

1 A r .. n- 12

0

Month Fig. 2 Monthly mean values of coliphage concentration and number of positive samples for enteroviruses in aerosol, at a distance of 2 and 20 m from the aeration tank, May 1992-April 1993; two samples were taken per month. ., Coliphages (2 m); A, coliphages (20 m); IJ, enteroviruses (2 m)

Coliphages in sewage showed seasonal variation with highest monthly mean values in summer (600-700 pfu m1-I) but falling to about 300 pfu ml-' in November (Fig. 1). Enteroviruses were recovered from 58% of sewage samples, but all the positive samples were taken from June to January. A similar seasonal pattern was found in aerosols (Fig. 2) : the number of coliphages decreased with distance from the tank. At 2 m, 8-13 pfu m-3 were observed in summer months and 0-3 pfu m P 3 in winter; at 20 m, 1-4 pfu m P 3 were found, but only in the summer months and October. No coliphages were found at 30 m. Positive aerosol samples for enteroviruses (25% of samples) were found only from August to November.

The temporal patterns for coliphages and enteroviruses appeared to be out of phase. Thus statistical analysis showed no significant difference between the mean count of phage in enterovirus-positive samples and that in negative samples. This was the case for both sewage and aerosol samples (Table 1).

Twenty-five per cent of the aerosol samples examined were enterovirus-positive. This suggests a possible risk of

Table 1 Results of statistical comparison between concentration of coliphages in enterovirus-positive samples and that in enterovirus-negative samples

Phage concentration

Enterovirus-positive Enterovirus-negative samples samples

Mean n S.D. Mean n S.D. P*

Sewage 534 14 193 423 10 151 > 0.05 Aerosol 3.5 6 4.7 5.5 18 6.8 > 0.05

~~ ~

* Student r-test. Phage concentrations are pfu ml-' for sewage and pfu m - 3 for aerosol; n, number of samples; s.D., standard deviation.

0 1995 The Society for Applied Bacteriology, Letters in Applied Microbiology 21, 207-209

COLIPHAGES AND ENTEROVIRUSES IN AEROSOL 209

airborne viral infection at least for people employed in wastewater plants. Because aerosols were sampled close to the tank one cannot discuss the possible risk to the general population of the area.

Earlier studies have demonstrated that the aerosol density of enteroviruses, around wastewater treatment plants, is very low when compared with faecal indicator bacteria and coliphages, and that their detection requires sampling methods with high sensitivity. I t has been sug- gested that coliphages (Fannin et al. 1977) and also faecal streptococci (Crawford and Jones 1979) are potential indi- cators of viral aerosol contamination. The results presented here demonstrated the presence of coliphages in aerosol and their spread up to 20 m, but no relation was found with the presence of enteroviruses. Therefore, the usefulness of phages as enterovirus indicators in aerosol was not con- firmed.

REFERENCES

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Moore, B.E., Sagik, B.P. and Sorber, C.A. (1979) Procedure for the recovery of airborne human enteric viruses during spray irrigation of treated wastewater. Applied and Environmental Microbiology 38, 688-693.

Schmidt, N.J. (1989) Cell culture procedures for diagnostic virology. In Diagnostic Procedures for Viral, Rickettsia1 and Chlamydia1 Infections eds Schmidt, N.J. and Emmons, R.W. pp. 51-100. Washington, DC: American Public Health Associ- ation.

Spendlove, J.C. and Fannin, K.F. (1982) Methods of character- ization of virus aerosol. In Methods in Environmental Virology ed. Gerba, C.P. and Goyal, S.M. pp. 261-329. New York: Marcel Dekker.

Teltsch, B. and Katzenelson, E., (1978) Airborne enteric bacteria and viruses from spray irrigation with wastewater. Applied and Environmental Microbiology 35, 29@-295.

0 1995 The Society for Applied Bacteriology, Letters in Applied Microbiology 21. 207-209