008 Afr. J. Environ. Sci. Technol.give
no indication of the sources of the microbial pollution.Despite efforts to minimize fecal input
into coastal water-ways and beaches, the problem persists, partly due to an
inability to reliably identify non point sources. These sour-ces
may include inefficient sewage treatment plants, lea-king septic
systems, agricultural runoff, or wildlife (Stritt-holt et al., 1998).Identification of the source of the bacterial
contami-nation is an essential first step in seeking to control
fecalcontamination of water. In particular, it is important
to det-ermine whether the source of fecal contamination is of
human, livestock, or wildlife origin, as microorganisms ofhuman
origin are regarded as having greater potential tocause disease
in humans (Puech et al., 2001). Bacterialsource tracking (BST) (also called microbial source trac-king, fecal source tracking or fecal typing) is new metho-dology that is being developed to determine the sourcesof fecal bacteria from environmental samples (e.g. fromhuman, livestock, or wildlife origins). According to Par-veen et al. (1997), bacterial source tracking is the deter-mination of the animal origin of fecal bacteria in naturalwaters which result from point or non point pollution.There are two types of BST methods, phenotypic andgenotypic (Scott,
2002; Simpson et al., 2002). Phenotypicmethods are based on characteristics expressed by fecalbacteria and genotypic methods are based on DNA seq-uences. Three primary genetic techniques are availablefor BST. Ribotyping characterizes a small, specific por-tion of the bacteria’s DNA sequence (Samadpour et al.,2005; Scott et al., 2003); pulse-field gel electrophoresis(PFGE) is similar to ribotyping but typically is performedon the entire genome of the bacteria (Lu et al., 2004).The polymerase chain reaction (PCR), amplifies selected(nonribosomal) DNA sequences in the bacteria’s genome(Makino et al., 1999). Phenotypic techniques generallyinvolve either a nutrient utilization technique where dif-ferent nutrient sources are used to produce a metabolicprofile of microorganisms (Garland and Mills, 1991) orantibiotic resistance analysis, where resistance patternsfrom a suite of different concentrations and types of anti-biotics are measured (Hagedorn et al., 1999; Wiggins,1996). According to some researchers (Hagedorn, 2004;Martellini et al., 2005; Meays et al., 2004), several met-hods are currently available for bacterial source tracking,however all of the BST methods are still being developedand/or evaluated to varying degrees. The ultimate goal ofbacteria source tracking (BST) is to identify the source ofindicator bacteria isolated from surface waters. Fecal coli-forms,
E. coli
and the bacteria of the genus
Enterococcus
are used extensively in the US and throughout the worldas indicator organisms to signal fecal contamination inwater (Garland and Mills, 1991). The objectives of thisstudywere: (1) to design a methodology to determine nut-rient utilization profiles for
E. coli
isolates as a phenol-typic fingerprinting methodology, and (2) to identify thepossible sources of the fecal coliform bacteria
E. coli
inSilver Lake, Delaware County, Iowa using the nutrient uti-lization technique.
MATERIALS AND METHODSSample collection and Isolation of E. coli
The
E. coli
isolates used in this study were obtained from fecalsamples collected from Silver Lake and its Watershed, Delaware,Iowa. Out of a total number of 300
E. coli
isolates, 123 isolatescame from geese, 97 isolates came from cattle, 41 isolates camefrom lake water, 22 isolates came from hogs, 11 isolates came frombirds, and 6 isolates came from soil near the outdoor restroomfacilities. The fecal samples were made into slurry by putting 1 g offecal material into sterile phosphate buffer solution (PBS). Theslurry was then streaked onto MacConkey agar and incubated at35°C for 24 h. All isolates exhibiting the characteristics of fecal coli-forms were then further analyzed to confirm them as
E. coli
by gro-wing them in EC broth with 4-methylumbelliferyl-
-D-glucuronide(MUG) (Fisher Scientific, Chicago, IL). After 24 h of incubation at37ºC, test tubes were removed from the incubator and were obser-ved using a long-wave ultraviolet (UV) light (365 nm). Most strainsof
E.coli
produce
-glucuronidase which hydrolyzes MUG to thefluorogenic compound, 4-methyl-umbelliferone. Therefore any isola-tes fluorescing under the UV light were confirmed as
E. coli
. Thesepositive samples were transferred to Tryptic Soy Agar (TSA) (FisherScentific, Chicago, IL) slants, properly labeled, grown at 35°C, andstored at 4°C, until needed for analysis.
BIOLOG GN2 microplate™ preparation
The method used in the study is modified from that developed byBiolog, Inc., (Hayward, CA.). The BIOLOG GN2 Microplates wereused and consist of 96 wells, with 95 containing pre-selected nut-rient sources, tetrazolium violet and a blank well (A1) with no subs-trate. The tetrazolium violet is a redox dye that serves as an indica-tor of the utilization of the nutrient. Prior to analysis, pure cultures of
E. coli
isolates were grown on Trypticase Soy Agar (TSA) (DIFCOLaboratories) at appropriate temperature 37ºC for up to 24 h. Thecells must be freshly grown, since many strains lose viability andmetabolic vigor in stationary phase. The innoculum was prepared in20 mm diameter test tubes with sterile saline. A colony was chosenfrom the TSA plate using a sterile wire loop, and suspended in a0.40% saline (0.4 g of sodium chloride in 100 mls of water) andmixed to obtain a uniform solution using a squeeze bulb pipette.Three drops of a 5 mM concentration of sodium thioglycolate (Sig-ma, Chicago) (7.6 g of thioglycolate in 100 ml of saline) were addedto keep the cells from using their own biofilm or cell walls as a car-bon source, which would give a false positive reaction. The thio-glycolate is an anticapsule agent, and partially or completely inhibitsthe purple color in the A-1 well and other negative wells that canform when bacteria metabolize their polysaccharide capsule as acarbon source. The concentration of bacteria was determined byplacing the tube containing the bacteria in a spectronic 20 spec-trophotometer and adjusting the transmittance to 59 - 61%. Thissolution was then used to inoculate 96-well BIOLOG Microplates™containing water blank well and 95 different dried carbon sources.All the wells started out colorless when inoculated, and the micro-plates were incubated at 37ºC for 24 h. After incubation, the wellsare examined for the formation of purple color similar to the BIO-LOG procedure. The appearance of a purple color indicates theutilization of the carbon source in a particular well. Because the cellcan utilize the carbon source, it respires and after oxygen is deple-ted, the cells reduce the alternate electron acceptor, redox dyetetrazolium, to form the purple color. The absorbance of each wellin the plates were then read at 590 nm for color, using a SPECTRA
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