Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands.

AIMS: The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique. METHODS AND RESULTS: Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts. CONCLUSIONS: The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types. SIGNIFICANCE AND IMPACT OF THE STUDY: Method standardization will improve the understanding of how sands affect surface water quality.

Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern California.

AIMS: To determine the levels and species distribution of enterococci in intertidal and marine sediments and coastal waters at two beaches frequently in violation of bacterial water standards. METHODS AND RESULTS: Faecal indicator bacteria were extracted from sediment and enumerated using membrane filtration. High levels of enterococci were detected in intertidal sediments in a seasonal river and near a storm drain outlet. Low levels were found in marine sediments at 10 m depths and in surf zone sand. Bacterial isolates presumptively identified as Enterococcus on mEI media were speciated. The predominant species found in both water and sediment included Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. A number of isolates (11-26%) from regulatory water samples presumptively identified as enterococci on mEI media were subsequently identified as species other than Enterococcus. At both study sites, the distribution of species present in water was comparable with those in sediments and the distribution of species was similar in water samples passing and exceeding bacterial indicator standards. CONCLUSIONS: High levels of Enterococcus in intertidal sediments indicate retention and possible regrowth in this environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Resuspension of enterococci that are persistent in sediments may cause beach water quality failures and calls into question the specificity of this indicator for determining recent faecal contamination.

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed.

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.