Sorbitol-fermenting Bifidobacteria are indicators of very recent human faecal pollution in streams and groundwater habitats in urban tropical lowlands

Douglas Mushi Department of Biological Sciences, Sokoine University, P.O. Box 3038, Morogoro, Tanzania Denis Byamukama Department of Biochemistry, Makerere University, P.O. Box 7062, Kampala, Uganda Amelia K. Kivaisi Department of Molecular Biology and Biotechnology, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania Robert L. Mach Andreas H. Farnleitner (corresponding author) Institute of Chemical Engineering, Research Area Applied Biochemistry and Gene Technology, Research Group Environmental Microbiology and Molecular Ecology, Vienna University of Technology, Getreidemarkt 9/166-5-2, A-1060, Vienna, Austria Tel.: +43 1 58801 17256 E-mail: a.farnleitner@aon.at Sorbitol-fermenting Bifidobacteria (SFB) proved to be an excellent indicator of very recent human faecal pollution (hours to days) in the investigated tropical stream and groundwater habitats. SFB were recovered from human faeces and sources potentially contaminated with human excreta. SFB were undetectable in animal faeces and environmental samples not contaminated with human faeces. Microcosm studies demonstrated a rapid die-off rate in groundwater (T90 value 0.6 days) and stream water (T90 value 0.9–1.7 days). Discrimination sensitivity analysis, including E. coli, faecal coliforms, total coliforms and Clostridium perfringens spores, revealed high ability of SFB to distinguish differing levels of faecal pollution especially for streams although high background levels of interfering bacteria can complicate its recovery on the used medium. Due to its faster die-off, as compared to many waterborne pathogens, SFB cannot replace microbiological standard parameters for routine water quality monitoring but it is highly recommendable as a specific and complementary tool when human faecal pollution has to be localized or verified. Because of its exclusive faecal origin and human specificity it seems also worthwhile to include SFB in future risk evaluation studies at tropical water resources in order to evaluate under which situations risks of infection may be indicated.


INTRODUCTION
Maintaining the microbial quality of water resources requires target-oriented management strategies. Microbial faecal source tracking can help to identify the perpetrators of environmental pollution, to establish best management practices and to prevent any further contamination (Jagals & Grabow 1996;Parveen et al. 1999;Scott et al. 2002;Nebra et al. 2003;Bonjoch et al. 2004;Reischer et al. 2008).

The traditional microbial indicators (faecal coliforms,
Escherichia coli, enterococci) do not allow pollution source differentiation, as they occur in animal as well as human faeces. Efforts to type strain libraries of traditional faecal indicators by genotypical or phenotypical methods are hindered by poor adaptation of these bacteria and laborious procedures (Orskov & Orskov 1981;Parveen et al. 1999;Scott et al. 2002;Nebra et al. 2003;Bonjoch et al. 2004).
In the tropics, these classical indicators are also suspected to originate from non-faecal sources (such as soil) and to proliferate in tropical aquatic habitats under favourable situations and thus can be detectable at levels which may not reflect the original extent of faecal contamination doi: 10.2166/wh.2010.116 (Carrillo et al. 1985;Rivera et al. 1988;Jimenez et al. 1989;Perez-Rosas & Hazen 1989;Wright 1989;Hazen & Toranzos 1990;Solo-Gabriele et al. 2000;Desmarais et al. 2002). As a result, microbial standard indicators may yield biased results. Alternative indicators are thus needed in order to complement or replace standard indicators under situations where its (single) use is no longer justified in tropical waters.
However, these approaches were developed and tested comprehensively in temperate regions where the biological, physicochemical and socio-economic characteristics differ greatly from those of tropical regions (Toranzos & McFeters 1997). Very few SFB studies exist in developed tropical countries (Carrillo et al. 1985;Toranzos & McFeters 1997) and none in most tropical developing countries despite the problem of frequent outbreaks of waterborne diseases resulting from human source of the type of enteric pathogens such as Salmonella enterica serovar Typhi, Shigella spp. or Hepatitis A virus (Parveen et al. 1999;Scott et al. 2002). Additionally, bacterial flora of faeces from people living in widely different circumstances in different parts of the world may have differences in the type, number and frequency of isolation of bacterial groups (Drasar 1974). This could limit their use as indicators of recent and human specific faecal contamination. Furthermore, most SFB studies have been on surface waters such as streams, rivers and reservoirs (Mara & Oragui 1983;Carrillo et al. 1985;Long et al. 2003); studies on the suitability of SFB in assessing human faecal contamination of groundwater have not been done so far.
The aim of this study was to determine the presence of SFB in faecal sources including human and animal faeces of different host groups and environmental samples and furthermore to evaluate its use as a human-specific faecal indicator to monitor the microbiological water quality of aquatic resources in tropical and developing countries such as eastern Africa. We hypothesized that SFB exist only in human-specific faecal sources or in habitats where human faecal pollution was happening very recently. Soil samples from different locations within the study area were also investigated for the presence of SFB in order to have information on whether they originate from or regrow in the soil. In addition, the detection of SFB, standard coliforms (E. coli, faecal coliforms, total coliforms) and alternative Clostridium perfringens spores were compared in presumptively differently polluted stream and groundwater sources using the established and so-called 'faecal pollution gradient approach' of Byamukama et al. (2005).
Furthermore, the survival rate for SFB in the stream and groundwater was evaluated by microcosm experiments to complement the information from the field study and to enable a better understanding of their ecology in tropical aquatic environments of eastern Africa.

Description of the study area and sites
Dar es Salaam City is located on the coastal area of Tanzania, between 6851 0 30 00 S and 6847 0 30 00 S and 39815 0 E and 39817 0 E with an area of about 135 km 2 ( Figure 1).
The city experiences both tropical and coastal climate with mean daily temperatures varying between 178 and 348C and average humidity of about 67 -96%. The annual rainfall averages between 1,000 and 1,400 mm, with the wettest period of the year being March to May. The evaporation rate is over 2,100 mm per annum. Being a coastal area, the city is characterized by sandy soils overlaying sandstone and limestone bedrock that allow fast percolation of the surface water, especially during rainy periods.
In this study, 15 sampling sites were selected from streams and groundwater sources located in two municipalities of Dar es Salaam City (Figure 1); these included nine (9) boreholes and six (6) stream sites (selected from 3 streams). The samples were collected twice a month at each site from May to July 2005, during the time of the year when a mixture of rainy and dry patterns is evident. Over the whole sampling period, six (6) samples were collected from each sampling site, reflecting a total of 90 water samples. The borehole risk evaluation questionnaire described in WHO (1997) was used to select a gradient of boreholes differing in the probability of faecal contamination. The questionnaire contained ten sanitary conditions from which the boreholes were assessed. To each borehole, the risk scores were computed and later used to classify the borehole to low, medium or high risk (WHO 1997).
Three motorized boreholes (BL1, BL2, BL3) had risk scores of between 0 and 2. The boreholes were from the periphery of the city characterized by a low human population density. Although the boreholes were not fenced, there were no observable sources of contamination during the sampling period and they were thus classified as low-risk boreholes. Three other motorized boreholes (BH1, BH2, BH3) had risk scores of between 5 and 6. The boreholes were located at the squatter area in the city and characterized by the presence of pit latrines and/or septic tanks at a distance of 3 to 5 m from the boreholes.
The boreholes were also located very close to the road and human residences; they were not fenced and stagnant waste water pools were observable in close proximity. These were classified as medium-risk boreholes. Open boreholes (OP1, OP2, OP3) had a risk score of between 8 and 10.
The boreholes were located in the squatter areas and close to either pit latrines or septic tanks (about 3 -5 m).
The boreholes were neither fenced nor protected from surface run-off and their walls had cracks, which allowed the inward and outward movement of water. Water for domestic use is drawn from these boreholes using a plastic or rubber pail attached to a rope, which is left lying on the ground in between uses, a process that poses further risk of contamination of the water. These boreholes were considered to be under the highest risk of the groundwater sources.
As for the stream sites, three streams each with two sites -upstream and downstream sampling points -were surveyed. The stream sites US1, US2 and US3 were located upstream in low-density population catchments which do not receive discharge of sewage effluent; these were categorized as low influenced sites while the corresponding stream sites DS1, DS2 and DS3 were located downstream in highly populated areas whereby effluent channels join the respective streams. These were categorized as high influenced sites.
Probes were calibrated at 258C before sampling and the calibration was verified upon returning from the field.
Membrane electrode method (4500-O G) was used to measure five days' biochemical oxygen demand (BOD 5 ).
Total suspended solids (TSS) were assessed using the gravimetric technique (2540D). For determination of nitrates plus nitrites ðNO 2 3 þ NO 2 2 Þ, a defined volume of sample water was filtered through premuffled glass fibre filters (GF/C; Whatman, Springfield Mill, England), and the filtrate was analysed using calorimetric method (4500-NO 3 2 F).

Sampling and analysis of SFB in soil, faeces and sewage
Single samples of faeces were obtained from 15 healthy adults, 10 dogs feeding on leftovers from human diet, 10 pigs feeding frequently on cereal products and occasionally on leftovers from human diet, 10 hens feeding on varieties of cereal products, 10 goats and 10 cattle feeding solely on different plant materials from the farm. Each single sample of faeces from the respective individual was obtained from its own site. Soil samples were obtained from ten sites selected at random in areas with and without settlements. A soil auger was used to obtain soil sample core of the top 10 cm soil layer from ten randomly selected spots within a 50 m radius of the sampled site (Byamukama et al. 2005). water samples was filtered and tested for SFB as described above while for total coliforms (TC), faecal coliforms (FC), Escherichia coli and Clostridium perfringens spores (CP) were tested according to Byamukama et al. (2005).  (Byamukama et al. 2005).

Survival of SFB and coliforms in water
Statistical significance of detected differences between compared groups was checked using the non-parametric Mann Whitney Test (c.f. Table 5).
First-order die-off kinetics was assumed to be a reasonable model for the microcosm die-off experiments; die-off coefficients were computed from the slope of the regression line obtained from the ln-transformed data set.
SFB concentrations from various potential pollution sources were compared after unit conversion from gram and ml to cubic centimetre following standard conversions assuming 1 ml equals 1 cm 3 , and 1 cm 3 < 1 g, respectively.

RESULTS
Sources of sorbitol-fermenting Bifidobacteria (SFB) in the tropical environment SFB could be isolated from human faeces, sewage and water polluted with sewage but not from any of the other animal faeces, tap water and soil samples ( Table 1).
The concentration of SFB was highest in human faeces with a median of log 10 11.4 cfu per 100 cm 3 . Observed median concentrations of SFB in sewage and water polluted with sewage were log 10 6.8 cfu per 100 cm 3 and log 10 3.5cfu per 100 cm 3 , respectively. As compared to the concentration range of SFB from human faeces (approx. log 10 3 of variation), the observed concentration range of SFB was far increased for sewage and polluted water samples -as related to the kind of sewage, stage of treatment and the extent/age of faecal pollution. The isolation of SFB on HBSA agar was paralleled by the growth of potentially interfering background bacteria (Table 1). Assuming a and between BOD 5 and TSS ( p , 0.05). Groundwater had significantly higher levels of chloride, hardness, electrical conductivity, NO 3 2 þ NO 2 2 and salinity compared to stream water ( p , 0.01). There were also observable significant differences ( p , 0.05) in physicochemical parameters between sites (i.e. selected water source types).
The noted differences in the physicochemical characteristics between water source types were reflected by cluster analysis based on coefficients of similarity measured by squared Euclidean Distance ( Figure 2). Generally, cluster analysis divided the sites into two large clusters (I and II) based on groundwater and stream physicochemical data.
Cluster I consisted of groundwater sites while cluster II contained stream sites. Site OP1, however, was exceptional; it clustered with stream water sites. Grouping was confirmed by principal component analysis (data not shown).
The analysis indicated that the investigated stream and groundwater habitats represent unique physicochemical habitats. It should be mentioned that the physicochemical characterization is a valuable basis facilitating the correct interpretation of results as recovered from the further microbiological studies.
Occurrence of SFB in the investigated water source types SFB were not detected in either BL or BH sites (Table 3).
However, SFB were detected in samples from OP sites with pit latrines and septic tanks in the vicinity, US sites receiving minor faecal material from low-density  settlements and DS sites that received sewage effluent from heavily populated areas. SFB levels ranged from not detectable to log 10 5.0 cfu, not detectable to log 10 5.5 cfu and log 10 4.7 cfu to log 10 6.8 cfu per 100 ml for OP, US and DS sites, respectively (

Survival of SFB and coliforms in surface and groundwater microcosms
The survival of SFB and coliforms (i.e. E. coli, FC, TC) in the stream and groundwater microcosms incubated at  5.8 4.7-6.8 23 * SFB were not detectable (nd) above the median detection limit of log 0.28 cfu/100 ml (range log 0.1-1.7 cfu/100 ml). † SFB were not detectable (nd) above the median detection limit of log 0.60 cfu/100 ml (range log 0.1-1.5 cfu/100 ml). Abbreviations: For BL, BH, OP, US and DS see Table 2 (Table 5).

DISCUSSION
Human faeces are the primary source of SFB in the considered Tanzanian tropical environment under the given method and detection limit ( Table 1). The concentrations of SFB determined in human faeces in this study (geometric mean log 9.4 cfu/cm 3 ) are in remarkable accordance with that of temperate regions (geometric mean log 9.8 cfu/cm 3 ) (Mara & Oragui 1983) and only slightly differ from those reported in other tropical regions (geometric mean log 8.1 cfu/cm 3 , Zimbabwe; geometric mean log 9.1 cfu/cm 3 , Nigeria) (Mara & Oragui 1985) using the HBSA method. The observed slight difference with data from the tropical study could be due to diet differences, as SFB require rigorous nutrients for their survival/growth (Sinton et al. 1998;Nebra et al. 2003). Dilution and physical processes (Rhodes & Kator 1999;Nebra et al. 2003) as well as injury due to the presence of oxygen or its derivatives (Mara & Oragui 1983) (Mara & Oragui 1983).
The densities of SFB reported for surface water types in this study are comparable to that reported by Resnick & Levin (1981) for river samples collected near the outfall and much higher than those reported in tropical rainforest watershed in Puerto Rico (Carrillo et al. 1985) and in freshwater streams and rivers in Zimbabwe and Nigeria (Mara & Oragui 1985). In general, the high values of SFB were observed at downstream sites (c.f. DS, Table 3) during the rainy month (March, 2005) (Table 5). It should be mentioned that also C. perfringens spores, closely followed by E. coli, were able to distinguish US vs. DS sites in a statistically significant way (Table 5). However, the median concentration differences in indicator levels between US and DS (c.f. Table 5) were 5-to 6-fold higher for SFB (log 10 BCRM 1.4) as compared to CP (log 10 BCRM 0.6) or E. coli (log 10 BCRM 0.7). Our observations are thus in agreement with previous studies that recommended the use of Bifidobacteria such as SFB for surface water monitoring after observing extended survival and regrowth of coliforms in the warm tropical climate (Evison & James 1975;Carrillo et al. 1985;Hazen & Toranzos 1990).
In contrast to the performed surface water comparisons (US vs. DS), almost all the investigated bacterial indicators showed good discrimination ability between category I comparisons (within groundwater source types) and category II comparisons (river vs. groundwater sources) (c.f. Table 5). Remarkably, in five out of seven comparisons, TC revealed the highest median differences (log 10 BCRM up to 5.6) in comparison with the rest of the studied setup. For example, immediate bacterial surface influence from river water to well water would be most sensitively detected by TC rather than by E. coli, C. perfringens spores or SFB.
However, it has to be emphasized that TC are unlikely linked to specific faecal pollution but rather to a general surface influence event in the investigated well habitats (i.e. associated with soil, sediment and sewage influence from the surface). (SFB to background bacteria) was evidence that background bacteria may overgrow or inhibit the growth of SFB.
Practically, they can complicate enumeration of the SFB colonies in the respective method by the existence of false positives, a scenario that could explain the 17% false positives obtained during the confirmation experiment.
Thus, there is a need to improve the existing method in order to make it more applicable and also cheaper for its routine use.
On the basis of our results, the main source of SFB in the studied urban tropical lowland environment is human faeces. SFB does not occur in soil uncontaminated with recent human faecal material nor does it regrow in river and groundwater habitats. As a result of the rapid die-off, SFB exhibit a specific value as an indicator of very recent human faecal pollution (diagnostic time frame hours to days) in aquatic habitats in this tropical region of eastern Africa. It is clear that this low persistence has to be considered when selecting an appropriate investigation design in order to avoid false negative results (e.g. in case faecal associated pathogens are available but not SFB due to rapid die-off).
However, according to the performed faecal discrimination analysis, SFB showed better indication values for different levels of faecal pollution for surface water (stream water) than coliform fractions or C. perfringens spores. Taken together, SFB can be considered an excellent parameter for situations when recent human faecal pollution has specifically to be detected in the tropical aquatic environment.
Because of its exclusive faecal origin and human specificity it seems also worthwhile to include SFB in future risk evaluation studies at tropical water resources in order to evaluate under which situations risks of infection may be indicated.