increased mortality and morbidity due to antibiotic treatment failure make antimicrobial resistance ( amr ) one of the 21st century 's major global public health challenges . overuse and misuse of antibiotics are considered major reasons for the emergence of resistant bacteria in many low - income countries [ 2 , 3 ] . antibiotic resistance has been documented for enteric bacteria from various water sources and these water sources could facilitate dissemination of resistant bacteria to a wider community of people and animals . this is particularly true for low - income countries like tanzania where water sources are frequently shared between animals and people [ 5 , 6 ] . for example , a report from kenya reported a high prevalence of antibiotic resistance e. coli from water and fish in lake victoria [ ampicillin ( 64% ) , tetracycline ( 76% ) , and cotrimoxazole ( 80% ) ]   where untreated water is consumed routinely . tanzanian hospitals have reported a high proportion ( 80%90% ) of clinical e. coli isolates that are resistant to antibiotics such as ampicillin , cotrimoxazole , tetracycline , gentamicin , and amoxicillin / clavulanic acid . these bacteria infect people within a healthcare system where , in most cases , there are no laboratory diagnostics to guide antibiotic treatment [ 810 ] . another study reported a high number of antibiotic resistant e. coli , possessing resistance to cephalosporins , from free - range buffalo , zebra , and wildebeest . these animals were located in mixed grazing areas with potential contact with people and livestock . contaminated water was suspected as the source of resistant bacteria found in these wild animals . contaminated water most likely plays a role in the dissemination of antibiotic resistant bacteria and the probability of transmission likely increases when people and animals use that water . antimicrobial resistance ( amr ) genes are transferred to other bacteria , sometimes at the species level , by horizontal gene transfer ( transduction , transformation , and conjugation ) . plasmid - mediated horizontal transfer of multidrug resistance between different bacteria is a major concern because this contributes to the evolution and emergence of antibiotic resistant bacteria in the environment [ 12 , 13 ] . for the last decade , polymerase chain reaction - based replicon typing ( pbrt ) has been used to identify major plasmid types found in enterobacteriaceae , including incompatibility ( inc ) groups ( hi2 , hi1 , i1- , x , l / m , n , fia , fib , fic , w , y , p , a / c , t , k , and b / o ) [ 14 , 15 ] . plasmids belonging to group incf frequently harbor blactx - m-15 that is often associated with blatem-1 , blaoxa-1 , and aac(6)-ib - cr resistance genes . replicon groups inca / c and l1 are frequently associated with enterobacteriaceae and harbor multiple resistance genes including resistance for extended - spectrum cephalosporins and carbapenems [ 1719 ] . in northern tanzania , surface water such as rivers and ponds is often shared between animals and people on daily basis . consequently , these water sources become polluted with human and animal excreta and might harbor antibiotic resistant enteric bacteria . consumption of water containing these bacteria is likely to increase the risk that antibiotic resistant and pathogenic bacteria will be transmitted . nevertheless , to date , no studies have been conducted in tanzania to determine if drinking water represents a risk factor for transmission of antibiotic resistant bacteria to people and animals . the objective of this study was to characterize the replicon types of plasmids that harbor drug resistant traits , their conjugation efficiencies , and the complement of antibiotic resistance genes for a panel of multidrug resistant e. coli isolates that were obtained from drinking water sources in northern tanzania . each source was visited twice and one sample was collected from each source per visit ( in tanzania , march is the rainy season and august is during dry season ) . sample locations included the kilimanjaro region ( moshi municipal , moshi rural , and hai districts ) , the arusha region ( arusha city , arumeru , longido , and monduli districts ) , and the manyara region ( simanjiro and babati districts ) . a convenience approach was used to select sampling sites with appropriate permission from local authorities . water samples were collected in 500  ml sterile bottles and were transported in cooler boxes with ice packs to the laboratory for processing within 6  h of collection . out of 500  ml , 100  ml water samples were analyzed using a standard membrane filtration technique with minor modifications . following filtration , each filter membrane was placed on a chromogenic selective agar plate ( hicrome e. coli agar , himedia laboratories prt . ltd . , the agar plates were initially incubated at 37c for 4  h , followed by incubation for 1622  h at 44c . plates produced 1200 cfu of which individual colonies were subcultured to ensure purity for further characterization . the identity of e. coli isolates was confirmed using a pcr genotyping test that detects the presence of the uida gene . antibiotic break point assays were used to determine the resistance profile of each e. coli isolate against a panel of important antibiotics . macconkey ( mac ) ( thermo oxoid remel ) agar plates with each antibiotic at their clsi recommended minimum inhibitory concentrations   ( table 1 ) were used to perform the break point assays . e. coli strains k-12 ( negative control ; susceptible to all antibiotics tested ) and h4h e. coli ( positive control ; resistant to all antibiotics tested ) were used as reference strains for antibiotic susceptibility testing . a set of 31 e. coli isolates that were susceptible to nalidixic acid and resistant to more than 2 antibiotics tested were chosen for this study . filter - mating experiments were performed to determine the conjugation rates of plasmids with the nalidixic acid susceptible mdr ( wild - type ) e. coli isolates as donors and a plasmid - free recipient strain ( e. coli k-12 , nalidixic acid resistant , nal ) as recipient as described earlier [ 23 , 24 ] with minor modifications . briefly , single colonies of e. coli k-12 and potential donor strains were grown separately overnight in lb medium ( luria - bertani medium ; difco lb broth lennox , sparks , md , usa ) at 37c . equal quantities ( 10  l ) of overnight cultures of donor and recipient strains were added on top of a nitrocellulose ( ~1  cm ) membrane overlaid on lb agar with no antibiotics . after 24  h of incubation at 37c , cells from the membrane were suspended in 500  l of sterile phosphate - buffered saline ( pbs , ph 7.0 ) and spread onto lb agar plates containing 32  g / ml nalidixic acid ( sigma - aldrich ) and another antibiotic to which the donor cells were resistant ( table 2 ) . the conjugation efficiency of plasmid was calculated by dividing the number of transconjugants by the number of donor cells . transconjugants were screened for their donor 's antibiotic resistance phenotypes and presence of tet(a ) , tet(b ) , blatem-1 , blashv , and blactx - m genes [ 25 , 26 ] . ctx - m grouping ( group 1 , group 2 , and group 9 ) was further evaluated for all ctx - m positive isolates . all ctx - m e. coli isolates were positive for ctx - m group 1 and the pcr products were subsequently sequenced by functional bioscience ( madison , wi ) . sequencher ( ver 5.0 ) software was used to process sequence traces , and the final sequences were analyzed with clc genomic workbench 7.0.2 ( clc bio aarhus , denmark ) and compared with the reported sequences from genbank ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . pcr - based replicon typing was used with genomic dna of the transconjugants using the methods described by johnson et al . . briefly , pellets from 1  ml of overnight culture were resuspended with 200  l of nanopure water and placed in a heating block at 100c for 10  min . the lysed suspension supernatant was transferred to a new vial and stored at 80c until ready for testing against 18 different sets of primers   that were grouped into three multiplex primer panels . the following pcr conditions were used : 5  min at 94c , 30 cycles of 30  s at 94c , 30  s at 60c , and 90  s at 72c , and a final extension of 5  min at 72c . the amplified pcr products were visualized using 1.5% tris - acetate - edta agarose gel containing 0.2  g / ml ethidium bromide alongside a 1  kb ladder ( gene ruler 1 kb , life technologies ) . thirty - one mdr isolates were selected and used as donors to test if the resistance determinants were transferrable to recipient e. coli isolates by conjugation . of these , antibiotic resistance traits were successfully transferred by 25 isolates with conjugation efficiencies ranging from 10 to 10 ( table 2 ) . penta - resistant  phenotype that included resistance to ampicillin , streptomycin , sulfamethoxazole , tetracycline , and trimethoprim . pcr testing of transconjugants showed that tet(a ) was most commonly associated with conjugative plasmids ( 33% ) followed by blatem-1 ( 24% ) , tet(b ) ( 17% ) , blactx - m ( 8% ) , and blashv-1 ( 0% ) . a total of 11 replicon types were detected among the 31 mdr isolates ( table 3 ) . incf replicon types ( incf ia , ib , ic , and iia ) were predominant ( 49% ) and were mainly associated with e. coli isolates that were resistant to ampicillin , streptomycin , sulfonamide , tetracycline , and trimethoprim . replicon types incx , incw , incl / m , incy , inchi1 , inct , and inc k were not detected . replicon types n , h12 , fib , and fia were associated with blactx - m-15 and resistance to ampicillin , ceftazidime , streptomycin , sulfamethoxazole , tetracycline , and trimethoprim . after conjugation , one recipient was positive for four different replicons ( i1 , fib , fia , and k / b ) . plasmid - mediated horizontal transfer of multidrug resistance traits plays a key role in the dissemination of antimicrobial resistance around the world . our study shows that e. coli isolated from tanzanian water sources harbor multiple plasmids belonging to major plasmid replicon types such as incf , a / c , l1 , and n. most of these plasmids were associated with transfer of antibiotic resistance traits via conjugation with rates that varied between 10 and 10 using filter - mating assays . moreover , these plasmids harbor multiple antibiotic resistance genes that are associated with plasmid replicon types such as incf , a / c , n , l1 , h12 , and b / o . studies show that plasmid - mediated horizontal gene transfer occurs within and between e. coli and pseudomonas isolated from sewage and lake water . given the presence of resistant e. coli in biologically contaminated water from tanzania , it is likely that their presence contributes to the long - term persistence of resistance traits in people and animals who share these water resources . among the 11 replicon types found , incf group plasmids were detected more frequently than other tested groups . this is in accordance with previous studies where incf plasmids were found predominantly in e. coli from clinical samples ( rectal samples , gastric aspirate samples , and vaginal sample ) [ 28 , 29 ] and in e. coli from people ( feces and uti patients ) and poultry ( fecal swab ) . incfib was the most frequently detected ( 16% ) replicon type , similar to what has been reported for e. coli isolates collected from fecal samples of healthy people and cattle in nigeria . the overall proportion of incf - positive e. coli was 49% , which is lower than that observed in germany ( 71% )   but higher than that observed in e. coli isolates from fecal samples of healthy people and food animals in switzerland ( 45% ) . narrow  host range although they are well adapted to e. coli and are frequently associated with the presence of tet(a ) , blatem-1 , and blactx - m [ 31 , 32 ] . in this study , plasmid type incf was associated with tet(a ) , blatem-1 , and blactx - m-15 . the ctx - m-15 -lactamases are disseminated worldwide and are usually located in the conjugative plasmids . detection of this trait in e. coli isolated from water sources is a public health concern because ctx - m-15 -lactamases are commonly associated with urinary tract infections . another study from a hospital in tanzania found ctx - m-15 in klebsiella pneumoniae that can be associated with neonatal sepsis . in this study , detection of ctx - m-15 in e. coli from water suggests a possible contamination of human , livestock , and wild animal excreta and thus consumption of this untreated water is clearly a potential risk for transmission back to people . the inci1 plasmid type was the second most prevalent replicon type ( 13% ) and these plasmids also harbor multiple resistance genes . e. coli can reportedly maintain inci1 plasmids without antibiotic selection pressure and with little or no apparent fitness cost to the host bacterium . importantly , it is also a conjugative plasmid commonly detected in e. coli recovered from humans and animals with the conjugation efficiencies ranging between 10 and 10 [ 15 , 16 , 28 , 34 ] . the inci1 plasmid carrying blactx - m-15 and blatem-1 has been associated with the recent 2011 outbreak of e. coli o104 in germany . in addition , bacteria carrying lncl1 plasmids were responsible for community and hospital acquired infections [ 38 , 39 ] . inca / c plasmids are typically larger ( ~150  kb ) than others with lower conjugation efficiencies [ 24 , 40 , 41 ] . about 10% of e. coli isolates from our water samples harbored inca / c plasmids and these were associated with resistance to ampicillin , streptomycin , sulfonamide , tetracycline , and trimethoprim . importantly , inca / c plasmids can harbor a large number of antimicrobial resistance genes and the broad - host spectrum coupled with an ability to spread via conjugation transfer within bacteria communities means that they can transfer an arsenal of resistance traits to pathogens of people and animals [ 40 , 42 , 43 ] . isolation of e. coli with inca / c from environmental water samples that are consumed by humans and animals on daily basis is a major public health concern . the replicon typing methods have some pitfalls including the obvious inability to detect unknown replicons . for example , 14 isolates in the current study transferred their resistance phenotypes to the recipients ' cells but no plasmid replicon was detected . detection of these public health important conjugative plasmids and antibiotic resistant traits in tanzanian water suggests the possible pollution of these water sources from human , livestock , and wild animal wastes and also shows the potential of these water sources in the maintenance and transmission of these resistance traits between environment , animals , and people . therefore , appropriate intervention strategies should be identified and implemented to reduce the water pollution .
the aim of this study was to identify the replicon types of plasmids , conjugation efficiencies , and the complement of antibiotic resistance genes for a panel of multidrug resistant e. coli isolates from surface waters in northern tanzania . 
 standard membrane filtration was used to isolate and uida pcr was used to confirm the identity of strains as e. coli . 
 antibiotic susceptibility was determined by breakpoint assay and plasmid conjugation was determined by filter - mating experiments . 
 pcr and sequencing were used to identify resistance genes and pcr - based replicon typing was used to determine plasmid types . 
 filter mating experiments indicated conjugation efficiencies ranged from 101 to 107 . 
 over 80% of the donor cells successfully passed their resistance traits and eleven different replicon types were detected ( inci1 , fic , p , fiia , a / c , fib , fia , h12 , k / b b / o , and n ) . incf plasmids were most commonly detected ( 49% of isolates ) , followed by types inci1 and inca / c . 
 detection of these public health - relevant conjugative plasmids and antibiotic resistant traits in tanzanian water suggests the possible pollution of these water sources from human , livestock , and wild animal wastes and also shows the potential of these water sources in the maintenance and transmission of these resistance traits between environments , animals , and people .