Low prevalence of antibiotic-resistant gram-negative bacteria isolated from rural south-western Ugandan groundwater

The objective of this study was to determine antibiotic resistance patterns and specific resistance genes in Gram-negative enteric bacteria recovered from 42 different drinking water sources servicing 2 rural villages in south-western Uganda. These water sites were prone to contamination by both human and cattle activity. Of the 52 isolates examined, 26 carried antibiotic resistance genes with 25 being ampicillin resistant, 21 carrying the blaTEM gene, and no isolate carrying genes coding for extended-spectrum β−lactamases. Twelve isolates were tetracycline resistant and these bacteria carried between 1 and 3 different tet genes, with the tet(A) gene the most common. Six isolates carried the macrolide resistance mef(A) and/ or the macrolide-lincosamide-streptogramin B resistance erm(B) genes. Four isolates carried the sul1 gene, and 4 isolates carried the sul1 and int1 genes indicating the presence of Class 1 integrons. The Ugandan isolates in this study had lower than expected carriage rates of antibiotic and multi-drug resistance genes, carriage of Class 1 integrons and lacked genes coding for extended-spectrum β−lactamases as compared to antibiotic resistance carriage in clinical African isolates.


Introduction
reported on the isolation and biochemical characterisation of water-borne Gram-negative bacteria isolated from boreholes, ponds and valley water tanks used by 2 rural villages, from Nyabushozi County in the Mbarara District of south-western Uganda.Both humans and animals used the water sources and the E. coli counts confirmed that 38 of the 42 water sites did not meet international drinking water standards.The inhabitants of these villages were settled and seminomadic Bahima pastoralists, Bairu agriculturalists and a few internal migrants.The villages had undergone dramatic water resource changes as a result of land privatisation and the creation of a national park in 1989.The villagers were 10 to 15 km from the nearest local private medical clinic, which charged money for its services and 40 to 50 km from the nearest hospital.These communities lacked the infrastructure required for basic services, such as health care and a municipal drinking water supply.As a result, these people had little interaction with western medicine and relied on traditional herbal treatments for human and livestock diseases (authors' unpublished observations).
The currently published literature suggests that levels of antibiotic-resistant bacteria are high and continue to rise in Africa (Okeke et al., 2007).There are no data on antimicrobial susceptibilities of water bacteria from Uganda.Therefore it was of interest to characterise the level of antibiotic resistance and corresponding resistance genes in the Ugandan bac-teria previously isolated from 42 of the 47 Ugandan water sites tested which serviced the 2 villages.In this study, the antibiotic resistance phenotypes and genotypes of 52 randomly selected Gram-negative enteric bacteria from Nyabushozi County in the Mbarara District of south-western Uganda were characterised.

Bacterial isolates
Previously, water-borne Gram-negative bacteria were recovered from 1 mℓ water samples taken from 47 water sites within 2 villages in Nyabushozi County in the Mbarara District of south-western Uganda.The water samples were plated onto EC 3M TM Petrifilm TM according to the manufacturer's instructions and incubated at 37 o C for 24 h.Forty-two of the 47 water sites were positive for enteric bacteria.Random isolates were taken from the Petrifilms and biochemically identified and then representative isolates were verified by sequencing the variable region of their 16S rRNA gene as previously reported (Pearson et al., 2008).Many of the 16S rRNA gene sequences obtained allowed identification to the genus but not species level.From the initial study, 52 genetically distinct archived bacteria were available for further study.These included: 2 Citrobacter spp., 20 E. coli, 7 Enterobacter spp., 5 Klebsiella spp., 3 Morganella morganii, 2 Proteus spp., 3 Providencia rettgeri, 4 Pseudomonas spp., 4 Salmonella spp., and 2 Serratia odorifera.Because E. coli 0157:H7 had previously been reported in the area (Majalija et al., 2008), we cultured the 20 E. coli isolates on Sorbitol-MacConkey medium-SMAC (Remel, Inc., Lenexa, KS, USA; March and Ratnam, 1986).No growth was observed indicating that they were not E. coli 0157:H7.Two laboratory strains, E. coli HB101 and E. coli DH5α were used as recipients in conjugation experiments.

Detection of antibiotic resistance genes
Polymerase chain reaction (PCR) assays were used for the detection of tetracycline resistance genes [tet(A), tet(B), tet(C), tet(D), tet(E), tet(G) and tet(M)]; macrolide or combinations of the intI1 integrase for Class 1 integron and sul1 gene coding for sulphonamide resistance were performed as previously described (Miranda et al., 2003;Soge et al., 2006a).Positive and negative controls were used for each PCR assay.The PCR products were verified by DNA-DNA hybridisation with internal probes as previously described (Soge et al., 2006a).The ampicillin resistance genes bla TEM and bla SHV were identified as previously described (Soge et al., 2006b).The PCR primers and probes are listed in Table 1.

Plasmids isolation
Plasmid DNA was extracted using a modified alkaline lysis procedure and electrophoresed through 0.7% agarose gel with the E. coli V517 (58 kb); R1 (100 kb) and R478 (274.5 kb) used as markers for plasmid size estimation as previously described (Soge et al., 2006a).
Transconjugants were confirmed biochemically and their antibiotic resistance genes verified by PCR assays followed by DNA-DNA hybridisation of the PCR products using radio-labelled internal probes as previously described (Soge et al., 2006a).

Antibiotic susceptibility and antibiotic resistance genes
Of the 52 Gram-negative isolates examined, 26 were susceptible to all antibiotics and 26 isolates were resistant to > 1 of the antibiotics examined, with 14 isolates exhibiting multidrug  2).Two isolates did not carry either the bla TEM , and bla SHV or bla CTX-M genes.
Twelve isolates were tetracycline resistant (Tc r ) with 7 isolates carrying a single tet gene.Three isolates carried the tet(A) gene and 1 isolate each with the tet(B), tet(D), tet(G) or the tet(M) gene.The remaining 5 isolates carried multiple tet genes and included 3 isolates with the tet(A), tet(C), and tet(G) genes, 1 isolate with both the tet(A) and tet(M) genes, and 1 isolate with both the tet(C) and tet(G) genes.Eleven isolates had erythromycin MIC >128 mg/ℓ and 5 of these isolates carried macrolide [mef(A)] and/or macrolide-lincosamide-streptogramin B [erm(B)] resistance genes, while all 11 isolates were negative for mph(A), mph(B), mph(C), mph(D), ere(A) and ere(B).Eight isolates carried the sul1 gene and 4 of these isolates also carried the int1 gene suggestive of an integron.One isolate was chloramphenicol resistant though the resistance gene was not determined.
All transconjugants carried the bla TEM conferring resistance to Ap r , the sul1 gene but differed in the tet genes that were transferred to the transconjugants.E. coli 302, E. coli 304 donors transferred tet(A), tet(C) and tet(G) genes, while E. coli 387 donor transferred only the tet(A) gene.

Discussion
In the current study, 26 out of 52 water-borne Ugandan Gramnegative bacteria were antibiotic resistant and 14 were multidrug resistant.None of the isolates carried extended-spectrum β-lactamases, and 4 isolates carried genes consistent with a Class 1 integron.Ap r was the most common resistance phenotype among the Ugandan isolates.In the E. coli isolates, Ap r was associated with conjugative plasmids [58, 65, 75 kb], while the other Gram-negative isolates appeared to have chromosomally mediated Ap r genes, which we were unable to conjugally transfer in the study.None of the antibiotic-resistant isolates were resistant to cefotaxime and ceftazidime, while clinical human African enteric bacteria are usually resistant to cephalosporins and often carry multiple β-lactamases encoded by bla CTX-M , bla CMY , and bla VIM type genes in addition to bla SHV and bla TEM-1 (Frank et al., 2006;Gray et al., 2006;Ktari et al., 2006;Soge et al., 2006b).Two isolates from the current Ugandan study, Proteus 421 and Enterobacter 307, were Ap r but did not carry either bla SHV , bla TEM-1 , bla CTX-M genes.Similarly, 2 Citrobacter spp.283 and 104 had erythromycin MICs > 128 mg/ℓ but did not carry any of the 8 common MLS genes examined.One SXT r E. coli did not carry the sul1 gene while all 12 Tc r isolates carried previously characterised tet genes.
Three of the 8 isolates used as donors were able to conjugally transfer antibiotic resistance to the recipients.The resulting transconjugants carried a single plasmid, which carried 1-3 tet genes, sul1 and bla TEM-1 genes.The bla SHV-1 was not associated with these conjugative plasmids and was not transferred in the experiments.In contrast, the remaining 5 donor isolates did not generate transconjugants.
In one Nigerian study, small differences in antibiotic susceptibility between the clinical bacteria and bacteria isolated from soil, industrial effluent, food and drinking water were found (Lateef et al., 2005), suggesting the possibility that clinical and water-borne bacteria may have the same level of antibiotic resistance.Little work on the level of antibiotic carriage in Ugandan Gram-negative bacteria is available; however, in a 1998 paper, the authors reported that 92% of the endemic Shigella isolated in Mbarara, Uganda were resistant to cotrimoxazole and 58% were resistant to ampicillin (Legros et al., 1998).Previous studies on water enteric bacteria from other African countries also showed higher rates of antibiotic resistance than those found in this study.For instance, two separate Nigerian studies have found high levels of antibiotic-resistant (93 to 94%) water-borne enteric bacteria, isolated from communal well water, and from the lower Niger Delta River (Ibiebele et al., 1989;Ogan et al., 1993).Lin et al. (2004) characterised 113 enteric bacteria, including E. coli, Klebsiella sp., C. freundii, Enterobacter spp., S. marcesens, isolated from the Mhlathuze River in South Africa and found that 94.7 % of these bacteria were resistant to > 1 antibiotic and 75.2% of the isolates were multidrug resistant.The low level of Class 1 integrons found in the Ugandan isolates differs from recent studies of Class 1 integrons where 12% of the E. coli isolated from a remote community of Guarani Indians in Bolivia, and 40% of 100 multi-drug resistant Gram-negative bacteria, from the River Torsa, India, carried Class 1 integrons (Pallecchi et al., 2007;Mukherjee and Chakraborty, 2006).Unregulated use of antibiotics in agriculture, animal husbandry, and medical therapy has been a major influence in Africa and is often cited as a major reason why there is high prevalence of multidrug resistant bacteria (Okeke et al., 2007).Why the Ugandan water bacteria in this study differ from other studies from remote areas in Africa is intriguing.However, this study does illustrate that the level of antibiotic resistance found in water-borne Gram-negative bacteria can vary among Gram-negative bacteria isolated from various remote parts of the world where exposure to western medicine and antibiotics is minimal.