Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys

Abstract

In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F+) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1,410, E. coli organisms from <1 to 130,Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F+ phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks.

Human sewage identified as likely source of white pox disease of the threatened Caribbean elkhorn coral, Acropora palmata

“…Serratia marcescens (all strains) was consistently found in human sewage (100% of influent samples) and canals (77.2%) previously shown to be contaminated with human waste (Griffin et al, 1999). The bacterium was also recovered from seabird guano and from Key West beaches with a history of closures to public use due to elevated faecal indicator bacteria levels (Griffin et al, 1999;Nobles et al, 2000).…”

“…Serratia marcescens (all strains) was consistently found in human sewage (100% of influent samples) and canals (77.2%) previously shown to be contaminated with human waste (Griffin et al, 1999). The bacterium was also recovered from seabird guano and from Key West beaches with a history of closures to public use due to elevated faecal indicator bacteria levels (Griffin et al, 1999;Nobles et al, 2000). Encouragingly, our results indicate that the Key West wastewater treatment facility is effective at removing S. marcescens from wastewater; only one isolate of the bacterium was recovered from treated effluent during the entirety of our study (Table 3).…”

“…Our results suggest that human sewage is a direct source of the white pox pathogen in the Florida Keys. Contamination of nearshore waters in the Keys has been clearly demonstrated (Lapointe et al, 1990;Paul et al, 1995a,b;Griffin et al, 1999;Nobles et al, 2000;Lipp et al, 2002) and microbes found in human sewage can be detected in corals of the outer reef tract (Lipp et al, 2007). Currently the majority of the Florida Keys and much of the Caribbean utilize on-site waste disposal including septic systems, injection wells and illegal cesspools (Paul et al, 2000).…”

The simultaneous detection of both enteroviruses and adenoviruses in environmental water samples including tap water with an integrated cell culture-multiplex-nested PCR procedure

“…Among the viruses, enteroviruses and adenoviruses have been targeted by many researchers monitoring the viral contamination in water environments (Murrin and Slade 1997;Vantarakis and Papapetropoulou 1998;Chapron et al 2000;Fout et al 2003) for the following reasons: (i) the concentration of these viruses in water environments is higher than that of other viruses (Castignolles et al 1998;Vantarakis and Papapetropoulou 1998;Griffin et al 1999); (ii) the survivability of adenoviruses is high in external environments and during water treatment processes such as chlorination and UV irradiation (Enriquez et al 1995;Gerba et al 2002;Thompson et al 2003).…”

Feasibility of methods based on nucleic acid amplification techniques to fulfil the requirements for microbiological analysis of water quality

“…There may also be problems with false positives as a result of DNA cross-contamination if extreme care is not taken and confirmations are not performed (Limsawat and Ohgaki 1997;Lamothe et al 2003;Fey et al 2004). Low concentrations of DNA are also known to cause random fluctuations in PCR efficiency (Chandler et al 1997), which lead to tube-to-tube variability and thus to false negatives (Griffin et al 1999).…”