BACKGROUND
since  <dig>  kenya has suffered many outbreaks of cholera.
from  <dig> to  <dig>  outbreaks were reported every year with an average case fatality rate of  <dig> %  <cit> .
for instance, the  <dig> cholera outbreaks started in kwale on the kenyan coastline and affected  <dig> districts in the coast province; kwale, mombasa and taita-taveta.
between  <dig> and  <dig>  more than  <dig>  notified cases of cholera were reported in kenya, representing 10% of all cholera cases reported from the african continent during this period  <cit> .
from  <dig> to  <dig>  cases ranging from  <dig> to  <dig>  were reported each year except for  <dig>  in which  <dig> cases were reported  <cit> .
more cases have been reported locally since  <dig>  <cit>  and the recent outbreak in  <dig> had a case fatality of up to  <dig> %  <cit> .
strains belonging to serotype ogawa were frequently isolated from cholera outbreaks in kenya in the 1970s through 1990s and many isolates from this period were known to exhibit resistance to a diverse range of antibiotics including chloramphenicol, tetracycline, ampicillin, streptomycin, sulfonamides and doxycycline  <cit> .
in the recent past however, serotype inaba has emerged as the main cause of epidemics in kenya and these isolates are frequently not susceptible to chloramphenicol, streptomycin, sulphonamides, sulfamethoxazole and trimethoprim .
a mobile genetic element  belonging to the sxt family of ices, was shown to confer this phenotype in the strains isolated during the 1998- <dig> period  <cit> .
it is however unknown if strains isolated prior to and after this period harbour this element.
the integrase gene of the sxt family of ices is highly related to the one found in the r <dig> element  <cit>  and is also closely related to the one found in conjugative transposons and bacteriophages  <cit> .
upon conjugation, sxt/r391-like ices integrate into the prfc, a gene found on the large v. cholerae chromosome  <cit> .
in the sxt-like elements, genes encoding antibiotic resistance are integrated into the rumb thus interrupting the rumab operon while in the r <dig>  this operon is not interrupted  <cit> .
an sxt element, sxtmo <dig>  was detected in v. cholerae from a o <dig> biotype strain from madras, india and is known to confer the chl-str-sul-trim phenotype  <cit> .
this element is related to icevchind <dig> found in o <dig> and el tor strains  <cit> .
burrus et al.
gave a detailed review of the ice biology and classification  <cit> .
we investigated  <dig> strains exhibiting the chl-str-sul-trim phenotype isolated from various parts of kenya from  <dig> through  <dig> for the presence of sxt/r391-like elements and for evidence of integration of the element into the host chromosome.
we also determined the diversity of rstr genes encoding the cholera ctx-prophage repressor from the  <dig> strains isolated from the same period.
although most sequences in the ctxΦ-prophage genomes are similar in the el tor and classical biotypes strains, the rstr specific to the biotype-specific prophages differ.
the el tor and classical biotype strains carry the ctxetΦ and the ctxclassΦ repressor types, respectively  <cit>  while the ctxcalcΦ and ctxenvΦ encode the calcutta and environmental rstr types, respectively  <cit> .
strains known as the matlab variants belonging to the el tor biotype but harbouring the ctxclassΦ prophage have been isolated in bangladesh  <cit> , india  <cit>  and mozambique  <cit> .
three classes of multiresistant  integrons  are known to harbour genes encoding resistance to antibiotics  <cit> .
integron class  <dig> is commonly found in v. cholerae and is referred to as a super integron .
although integrons are not capable of self-transposition, they are known to associate with insertion sequences , transposons, and/or conjugative plasmids which serve as vehicles for the intra- and interspecies transmission of genetic material  <cit> .
like the r <dig> ice elements, transposon tn <dig> and its relatives frequently harbour genes conferring resistance to mercury and antibiotics-containing integrons that integrate into the left arm adjacent to the tnpm gene  <cit> .
transposon tn <dig> is also known to associate with integron class  <dig> and is therefore an important mge  <cit> .
we therefore also analysed the  <dig> strains for the presence of integron classes  <dig>   <dig>   <dig> and  <dig>  conjugative plasmids, the tnpm gene of transposon tn <dig> and the transposase of tn <dig> transposon.
methods
sources of vibrio cholerae strains
strains that were included in this study were obtained from distinct outbreaks occurring in different parts of kenya between  <dig> and  <dig> as indicated in figure  <dig>  for consistency, a distinct outbreak was defined as a gap of at least  <dig> months between the last known cholera case and a report of a new case in the same location.
archived isolates were initially subcultured on thiosulphate citrate bile salts sucrose agar  and confirmation of strain identity was done by serology using polyvalent, anti-ogawa, and anti-inaba antisera .
haemolysis test was done by growing v. cholerae on 5% sheep blood nutrient agar plates incubated at 37°c overnight.
antimicrobial susceptibility testing
antimicrobial susceptibility tests were performed using commercial discs following manufacturer's instructions .
susceptibility to β-lactam antibiotics was tested using ampicillin  while susceptibility to cephalosporins was determined using cefixime , cefotaxime , cefepime  cefoxitin , cefuroxime , ceftriaxone , and ceftazidime .
ciprofloxacin , norfloxacin  and nalidixic acid  were used for testing susceptibility to the quinolones.
aztreonam , a monobactam antibiotic, was also included in the assay.
aminoglycosides used in susceptibility tests included kanamycin , amikacin , streptomycin , gentamicin , neomycin , and tobramycin .
tetracycline antibiotics included minocycline , doxycycline  and tetracycline .
other antibiotics included chloramphenicol , furazolidone , rifampicin  and nitrofurantoin .
sulphamethoxazole , trimethoprim  and sulphonamides  were also tested.
β-lactam and β-lactamase inhibitor combinations included augmentin  and a combination of piperacillin  and tazobactam .
e. coli atcc  <dig> was used as a control for bacterial growth and potency of antibiotic discs.
susceptibility tests were interpreted using the clinical and laboratory standards institute guidelines  <cit> .
pcr amplification
dna used as template for pcr reactions was prepared from overnight l-broth cultures incubated at 37°c.
bacterial cells were harvested by centrifugation and re-suspended in  <dig> ml  <dig> mm tris/hcl  containing  <dig> mm edta.
template dna was obtained by boiling for  <dig> min and separated by centrifugation at  <dig>  × g for  <dig> min and then stored at -20°c until analysed.
pcr was carried out in  <dig> μl reaction volumes containing  <dig> μl 10× concentrated pcr buffer ,  <dig> μl  each of primer,  <dig> μl dntp mix ,  <dig>  μl  taq dna polymerase,  <dig> μl of template dna and  <dig>  μl sterilized distilled water.
all pcr assays were performed using an automated thermal cycler .
pcr products were analysed by electrophoresis in  <dig> % agarose gels, stained with ethidium bromide, visualized under uv light and recorded with the aid of a gel documentation system
conjugation experiments and pcr screening for antibiotic resistance genes
the mating assays were carried using the rifampicin-resistant e. coli c <dig> strain as the recipient.
conjugations were carried out at 37°c for  <dig> hr without shaking.
transconjugants were selected on mueller-hinton agar plates  containing trimethoprim  and rifampicin  <dig> μg/ml.
in order to confirm that the antibiotic resistance gene markers were transferred during conjugation, the donor and transconjugants were analysed using pcr methods.
screening of the sulii gene encoding resistance to sulfamethoxazole, dfra <dig> encoding resistance to trimethoprim and strb encoding resistance to streptomycin was done as described previously by ramachandran et al.
<cit>  while detection of the flor conferring resistance to chloramphenicol and dfra- <dig> gene that also confers resistance to trimethoprim was done as described previously  <cit> .
genomic dna from v. cholerae o <dig> strains atcc  <dig>  co <dig> and vo <dig> were used as positive controls templates for the screening of sulii, dfr <dig>  strb and sxt respectively and that from o <dig> biotype el tor strains ko <dig> was used for the screening for the dfra <dig> gene.
detection of mobile genetic elements
all strains were further tested for the presence of the 3'-conserved sequence  of integron class  <dig> using the forward primer targeting the qaceΔ <dig> and the reverse primer of the suli <dig> gene encoding resistance to quaternary ammonium compounds  and sulphonamides, respectively.
the gene cassettes flanked by the 5'-cs and the 3'-cs were amplified using a combination of primers that target the 3'-cs and the 5'-cs of the integron class  <dig>  primers and pcr conditions for the detection of inti <dig> gene belonging to integron class  <dig> was done as described previously by rivera et al.
<cit>  while detection of the 3'-cs and the variable cassette region was done as described previously by dalsgaard et al.
<cit> .
detection of inti <dig> was performed as previously described by falbo et al.
<cit> .
screening for the integrase specific to integron class  <dig>  and integron class  <dig>  was performed as detailed previously by machado et al.
and shi et al.
respectively  <cit> .
we also conducted pcr experiments using the genomic dna isolated from donors and transconjugants to verify the transfer of the tn <dig> and the sxt/r391-like element.
detection of tn <dig> transposon was done using trpm-specific primers and pcr conditions published previously by villa et al.
<cit>  while detection of tn <dig> was done using pcr conditions and primers described previously by hansson et al.
<cit> .
the presence of the ice was detected using primers for amplification of a  <dig> bp fragment of the integrase gene specific for the sxt/r391-like element as described previously by bhanumathi et al.
<cit> .
integration of the ice into the chromosome was demonstrated by amplification of a pcr product of  <dig> bp corresponding to the right junction between the attp element of the ice and the prfc chromosomal gene of the bacteria.
primers and pcr conditions used are similar to those published before by pugliese et al.
<cit> .
strains from our culture collection known to harbour various genes of interest were used as appropriate positive controls in corresponding pcr experiments.
analysis of vibrio cholerae virulence genes
all strains were screened for the presence of genes encoding virulence determinants in v. cholerae including cholera toxin , zonula occludens toxin , accessory cholera enterotoxin , hemolysin , and nag-specific heat-stable toxin .
detection of the tcpa gene specific to the el tor and classical biotypes was done using a common forward primer and biotype-specific reverse primers.
similarly, two forward primers were used for the detection of the biotype-specific haemolysin gene .
pcr conditions and primers used for the detection of tcpa, ompu, tcpi, toxr and hyla genes were similar to those described previously by rivera et al.
<cit>  while detection of the ctxa gene was done using primers and conditions described before by fields et al.
<cit> .
genomic dna from v. cholerae o <dig> strain atcc  <dig> was used as a positive control in screening for ctxa, zot, ace, tcpa, ompu, tcpi, and toxr genes.
for detection of the four rstr gene alleles, a single reverse primer was used in combination with forward primers specific for each of the four rstr gene alleles as described previously by nusrin et al.
<cit> .
plasmid analysis
dna for plasmid analysis was extracted using the method of kado and liu  <cit>  with a few modifications  <cit> .
dna was resuspended in  <dig> μl of te buffer containing  <dig> mm tris, and  <dig> mm edta  and separated by electrophoresis on  <dig> % agarose gel for  <dig> hours at  <dig> volt/cm.
plasmids of known sizes isolated from e. coli v <dig> and 39r <dig> were used as controls.
pfge
selection of strains used for genotypic analysis was based on location and year of isolation and antibiotic resistance profiles.
pfge was performed using the pulsenet recommended procedure  <cit> .
the plugs containing agarose-embedded dna were digested with  <dig> units of sfii  or noti .
fragments from xba1-digested salmonella braenderup h <dig> were used as molecular size markers.
RESULTS
antibiotic susceptibility tests and conjugation tests
all the  <dig> strains showing resistance to the chl-strep-sul-trim combinations transferred this phenotype to e. coli c <dig> en bloc.
the frequency of transfer, expressed as number of transconjugants per recipients, ranged between  <dig>  × 10- <dig> and  <dig>  × 10- <dig> with an average of  <dig>  × 10- <dig>  pcr analysis of the donor strains and the e. coli c <dig> transconjugants amplified a  <dig> bp fragment of sulii gene encoding resistance to sulfamethoxazole, a  <dig> bp amplicon corresponding to the dfra <dig> gene encoding resistance to trimethoprim, a  <dig> bp fragment of strb encoding resistance to streptomycin, a  <dig> bp fragment of flor gene conferring resistance to chloramphenicol and a  <dig> bp fragment corresponding to the integrase gene of the sxt/r <dig> ice family, thus confirming co-transfer of resistance markers and this element.
the trpm gene of the transposon tn <dig> was not detected in the transconjugants indicating that this transposon had not been acquired via conjugation.
the dfra <dig> gene was not detected in any of the isolates analysed.
similarly, attempts to isolate plasmids in the donor strains and transconjugants were not successful.
these results are in agreement with those obtained by pugliese et al.
<cit>  who demonstrated the co-transfer of the sxt-like element with the genes encoding the chl-strep-sul-trim phenotype in o <dig> strains isolated locally in during the 1998- <dig>  these workers also found that some strains had an incc plasmid harbouring a gene conferring resistance to tetracycline and while other strains were resistant to ampicillin but we did not identify any strain in our collection bearing these resistance patterns.
v. cholerae o <dig> strains resistant to tetracycline have previously been reported in kenya  <cit>  and zambia  <cit>  in the 1990s, but those isolated from ethiopia  <cit>  and somalia  <cit>  in the same period were susceptible to this antibiotic.
furthermore, strains isolated from previous outbreaks in kenya were known to exhibit resistance to ampicillin  <cit> , doxycycline and streptomycin  <cit> .
none of the strains we studied were resistant to furazolidone as was the case with strains isolated from mozambican immigrants in south africa  <cit> .
similarly, none of these strains were resistant to ceftriaxone, cefotaxime, nalidixic acid, amikacin and gentamicin as has been the case with strains previously reported from ghana  <cit> .
all the strains we analysed also lacked the dfra <dig> gene encoding resistance to trimethoprim in o <dig> and non-o <dig> strains isolated in india  <cit> .
genetic and environmental factors that may be responsible for the apparent serotype shift from ogawa to inaba in recent outbreaks in kenya remain to be elucidated.
while strains that do not harbour the sxt/r391-like element and those bearing the incc plasmids were not available for analysis alongside those included in our study, it is apparent that the gradual emergence of a population of v. cholerae o <dig> strains bearing the sxt/r391-like element as a major cause of cholera outbreaks in kenya has occurred independent of antibiotic resistance acquisition.
it remains to be determined exactly when the sxt/r391-like ice emerged in pathogenic v. cholera strains in kenya because isolates obtained locally between  <dig> and  <dig> were known to exhibit resistance to antibiotics encountered in the chl-strep-sul-trim phenotype  <cit>  that has lately been associated to the presence of the sxt-type ices  <cit> .
although it is well established that cholera came to africa from asia in the 1970s, it is only suspected that the sxt-like elements have been present in african vibrio spp even before the emergence of the v. cholerae o <dig> from which the first sxt element, sxtmo <dig>  was identified  <cit> .
ice-like elements have been detected in o <dig> clinical strains isolated in  <dig> in angola and v. parahaemolyticus clinical strains from the same country isolated in  <dig> were also shown to contain sxt-related ices that do not mediate resistance to antibiotics  <cit> .
similarly, analysis of o <dig> el tor clinical isolates from algeria isolated in  <dig> suggests the presence of sxt-like ices mediating trimethoprim resistance  <cit> .
however, the isolates from the  <dig> outbreak in the goma refugee camp in zaire did not harbour this element  <cit> .
our study demonstrates that the o <dig> el tor strains bearing the sxt/r391-like ice were in circulation in kenya in the 1994- <dig> period and have continued to persist in recent outbreaks.
this may suggest that the  <dig> strains isolated from the two outbreaks in 1994- <dig> in kwale, a coastal town of kenya, are some of the oldest strains in the region known to harbour this integrating conjugative element in this part of the continent.
analysis for mobile genetic elements and vibrio cholerae pathogenicity island
all the  <dig> o <dig> strains were positive for all the v. cholerae pathogenic genes except for the nag-specific heat-stable toxin .
these strains were also positive for the inti <dig> integrase belonging to integron class  <dig>  asuper-integron believed to be important in shuffling the vibrio cholerae genome  <cit> .
it is worth noting that the st gene normally occurs as a cassette  within int <dig> region in some v. cholerae strains but not in others  <cit> .
besides the st gene, another pathogenicity determinant, mrha, is frequently detected in si region of o <dig> and non-o1strains  <cit> .
it is not clear whether the presence of the st gene improves biological fitness of the host but appears to be dispensable without compromising the ability to cause disease in pathogenic strains.
besides the sxt elements, other mobile genetic elements implicated in the spread of antibiotic resistance phenotype in v. cholerae from africa include conjugative plasmids belonging to class c  <cit> , integron class  <dig>  <cit> , and integron class  <dig>  <cit> .
although the isolates we studied carried the sxt element, they lacked the class  <dig>   <dig>  and  <dig> integrons and did not harbour any conjugative plasmids.
all the strains were negative for the transposase gene belonging to tn <dig> but were positive for the trpm gene associated with tn <dig>  the tn <dig> has frequently been detected in gram negative strains containing integron class  <dig>  <cit> .
on the other hand, tn <dig> and its relatives are major agents in the dissemination of mercury resistance and antibiotic resistance genes in gram negative bacteria but not all tn21-like transposons are associated with antibiotic resistance and there are variations in the diversity of antibiotic resistance genes detected in tn21-like transposons that harbour antibiotic resistance markers  <cit> .
pcr analysis of transconjugants did not detect the tn <dig> implying that this transposon was not co-transferred with the sxt/r391-like element during conjugation.
we were however not able to determine if this element confers mercury resistance to the strains we studied or if it is physically linked to any antibiotic resistance markers.
it is also not clear if this transposon has all the other genes responsible for transposition such as tnpa, tnpr, res, and inverted repeats or if it exists as a defective transposon in these strains.
however, the presence of the trpm gene suggests that although the strains carrying the sxt/r391-like elements lack multiple resistant integrons, this transposon is genetically ready to accept such elements because integrons are normally located adjacent to this gene  <cit> .
it has been suggested that tn21-like transposons which confer multiple antibiotic resistance descended from an ancestral mercury resistance transposon like tn <dig> by successive insertions of antibiotic resistances and/or insertion sequences  <cit> .
it is therefore important to further characterize tn <dig> in pathogenic v. cholerae strains.
all the  <dig> strains were positive for the ctxetΦ but negative for all the other ctxΦ phage repressor gene alleles and this contradicts with the study on o <dig> el tor strains isolates from mozambique  <cit>  and india  <cit>  which have been reported to harbour the ctxclassΦ repressor.
such el tor strains carrying the ctxclassΦ repressor are now designated as the matlab variants of v. cholerae  <cit> .
our finding on the diversity of the ctxetΦ repressor and the absence of the other rstr genes in all the strains further indicate the need for detailed studies on the genetic diversity of v. cholerae strains from different parts of the continent to gain insight into the evolutionary trends of v. cholerae species causing epidemics in africa.
pulsed field gel electrophoresis
analysis of macro-restricted dna using not <dig> demonstrated that all the sxt-carrying strains produced indistinguishable digest patterns regardless of the year and region of isolation .
similarly, restriction digest analysis using sfi <dig> showed that all strains were clonal .
the fact that all our strains showed identical pattern in antibiotic susceptibility patterns, pathogenicity genes, and the diversity of mobile genetic elements strongly suggest that this population of o <dig> strains that have caused outbreaks since  <dig> to as recent as  <dig> are clonally related.
the absence of the st gene   <cit>  and the absence of the classical biotype-specific tcpa and hyla genes in these strains further indicates that genetic exchanges between this population and other v. cholerae serotypes that might be in circulation in kenya have been highly restricted.
in a previous study by jiang et al.
<cit>  it was noted that a number of o <dig> strains from kenya failed to cluster with those isolated from other parts of the world when using amplified fragment length polymorphism  genotyping technique.
similarly, the study by pugliese et al.
<cit>  showed that strains that carried the sxt-element alone or in combination with an incc plasmid belonged to a unique rapd cluster iv.
in the same study  <cit> , strains without this ice were shown to belong to other cluster types shared by isolates from ethiopia and somali.
it is also interesting to note that none of the isolates from 1998- <dig> study shared a rapd cluster with strains isolated in india and bahrain isolated in  <dig> and  <dig>  such observations have led to a theory that some toxigenic v. cholerae strains circulating in different countries may not have originated from a single clone in asia as is popularly believed, but may have been derived locally from genetic exchange between the asian o <dig> strains and the o <dig> or non-o <dig> strains from local environments  <cit> .
CONCLUSIONS
we observed that antibiotic susceptibility and genomic content of the strains bearing the sxt/r391-like ice that have been in circulation in kenya between  <dig> and  <dig> has not changed significantly and there are indications that these strains have undergone minimum genotypic changes during this entire period.
in the absence of older isolates for molecular characterization, it is not possible to determine whether other clones of v. cholerae bearing this ice have existed in kenya and is also difficult to determine when the sxt/r391-bearing clone emerged as the major cause of outbreaks in kenya.
in order to understand the epidemiological trends of cholera outbreaks in the region, there is need for further studies to determine evolutionary trends among strains isolated from the african region and compare them with those from other parts of the world.
authors' contributions
jnk designed and coordinated the study, carried out molecular characterization studies and drafted the manuscript.
smk participated in the design and supervision of the study and revision of the manuscript.
bmg revised the manuscript and supervised the study.
ncw participated in manuscript revision.
sms provided strains from earlier outbreaks and revised the manuscript.
pb participated in the study design, supervision of molecular characterization studies in belgium and revision of manuscript.
all authors read and approved the final manuscript.
authors' information
jnk is a research scientist at the kenya medical research institute  and doctoral fellow at katholieke universiteit leuven, belgium.
he holds an msc  and msc  where he is currently pursuing a phd in bioscience engineering at the department of biosystems.
his work is supported by a scholarship from the vlaamse interuniversitaire raad , belgium.
smk, ncw and sms are scientists at kemri, kenya.
smk is a wellcome trust research fellow and an opinion leader in the field of antibiotic resistance in the east african region while ncw is the former director centre for microbiology research, kemri.
bmg is professor of immunology at the k.u.leuven  and the university of ghent , belgium while pb is a senior research scientist at the veterinary and agrochemical research centre  and an expert in the field of antibiotic resistance in belgium.
he is also a professor at the faculty of veterinary medicine at ugent.

 since kenya has suffered many outbreaks of cholera. from to outbreaks were reported every year with an average case fatality rate of %. for instance, the cholera outbreaks started in kwale on the kenyan coastline and affected districts in the coast province; kwale, mombasa and taita-taveta. between and more than notified cases of cholera were reported in kenya, representing 10% of all cholera cases reported from the african continent during this period. from to cases ranging from to were reported each year except for in which cases were reported. more cases have been reported locally since and the recent outbreak in had a case fatality of up to %. strains belonging to serotype ogawa were frequently isolated from cholera outbreaks in kenya in the 1970s through 1990s and many isolates from this period were known to exhibit resistance to a diverse range of antibiotics including chloramphenicol, tetracycline, ampicillin, streptomycin, sulfonamides and doxycycline. in the recent past however, serotype inaba has emerged as the main cause of epidemics in kenya and these isolates are frequently not susceptible to chloramphenicol, streptomycin, sulphonamides, sulfamethoxazole and trimethoprim. a mobile genetic element belonging to the sxt family of ices, was shown to confer this phenotype in the strains isolated during the 1998- period. it is however unknown if strains isolated prior to and after this period harbour this element. the integrase gene of the sxt family of ices is highly related to the one found in the r element and is also closely related to the one found in conjugative transposons and bacteriophages. upon conjugation, sxt/r391-like ices integrate into the prfc, a gene found on the large v. cholerae chromosome. in the sxt-like elements, genes encoding antibiotic resistance are integrated into the rumb thus interrupting the rumab operon while in the r this operon is not interrupted. an sxt element, sxtmo was detected in v. cholerae from a o biotype strain from madras, india and is known to confer the chl-str-sul-trim phenotype. this element is related to icevchind found in o and el tor strains. burrus et al. gave a detailed review of the ice biology and classification. we investigated strains exhibiting the chl-str-sul-trim phenotype isolated from various parts of kenya from through for the presence of sxt/r391-like elements and for evidence of integration of the element into the host chromosome. we also determined the diversity of rstr genes encoding the cholera ctx-prophage repressor from the strains isolated from the same period. although most sequences in the ctxΦ-prophage genomes are similar in the el tor and classical biotypes strains, the rstr specific to the biotype-specific prophages differ. the el tor and classical biotype strains carry the ctxetΦ and the ctxclassΦ repressor types, respectively while the ctxcalcΦ and ctxenvΦ encode the calcutta and environmental rstr types, respectively. strains known as the matlab variants belonging to the el tor biotype but harbouring the ctxclassΦ prophage have been isolated in bangladesh, india and mozambique. three classes of multiresistant integrons are known to harbour genes encoding resistance to antibiotics. integron class is commonly found in v. cholerae and is referred to as a super integron. although integrons are not capable of self-transposition, they are known to associate with insertion sequences, transposons, and/or conjugative plasmids which serve as vehicles for the intra- and interspecies transmission of genetic material. like the r ice elements, transposon tn and its relatives frequently harbour genes conferring resistance to mercury and antibiotics-containing integrons that integrate into the left arm adjacent to the tnpm gene. transposon tn is also known to associate with integron class and is therefore an important mge. we therefore also analysed the strains for the presence of integron classes and conjugative plasmids, the tnpm gene of transposon tn and the transposase of tn transposon. methods sources of vibrio cholerae strains strains that were included in this study were obtained from distinct outbreaks occurring in different parts of kenya between and as indicated in figure for consistency, a distinct outbreak was defined as a gap of at least months between the last known cholera case and a report of a new case in the same location. archived isolates were initially subcultured on thiosulphate citrate bile salts sucrose agar and confirmation of strain identity was done by serology using polyvalent, anti-ogawa, and anti-inaba antisera. haemolysis test was done by growing v. cholerae on 5% sheep blood nutrient agar plates incubated at 37°c overnight. antimicrobial susceptibility testing antimicrobial susceptibility tests were performed using commercial discs following manufacturer's instructions. susceptibility to β-lactam antibiotics was tested using ampicillin while susceptibility to cephalosporins was determined using cefixime, cefotaxime, cefepime cefoxitin, cefuroxime, ceftriaxone, and ceftazidime. ciprofloxacin, norfloxacin and nalidixic acid were used for testing susceptibility to the quinolones. aztreonam, a monobactam antibiotic, was also included in the assay. aminoglycosides used in susceptibility tests included kanamycin, amikacin, streptomycin, gentamicin, neomycin, and tobramycin. tetracycline antibiotics included minocycline, doxycycline and tetracycline. other antibiotics included chloramphenicol, furazolidone, rifampicin and nitrofurantoin. sulphamethoxazole, trimethoprim and sulphonamides were also tested. β-lactam and β-lactamase inhibitor combinations included augmentin and a combination of piperacillin and tazobactam. e. coli atcc was used as a control for bacterial growth and potency of antibiotic discs. susceptibility tests were interpreted using the clinical and laboratory standards institute guidelines. pcr amplification dna used as template for pcr reactions was prepared from overnight l-broth cultures incubated at 37°c. bacterial cells were harvested by centrifugation and re-suspended in ml mm tris/hcl containing mm edta. template dna was obtained by boiling for min and separated by centrifugation at × g for min and then stored at -20°c until analysed. pcr was carried out in μl reaction volumes containing μl 10× concentrated pcr buffer, μl each of primer, μl dntp mix, μl taq dna polymerase, μl of template dna and μl sterilized distilled water. all pcr assays were performed using an automated thermal cycler. pcr products were analysed by electrophoresis in % agarose gels, stained with ethidium bromide, visualized under uv light and recorded with the aid of a gel documentation system conjugation experiments and pcr screening for antibiotic resistance genes the mating assays were carried using the rifampicin-resistant e. coli c strain as the recipient. conjugations were carried out at 37°c for hr without shaking. transconjugants were selected on mueller-hinton agar plates containing trimethoprim and rifampicin μg/ml. in order to confirm that the antibiotic resistance gene markers were transferred during conjugation, the donor and transconjugants were analysed using pcr methods. screening of the sulii gene encoding resistance to sulfamethoxazole, dfra encoding resistance to trimethoprim and strb encoding resistance to streptomycin was done as described previously by ramachandran et al. while detection of the flor conferring resistance to chloramphenicol and dfra- gene that also confers resistance to trimethoprim was done as described previously. genomic dna from v. cholerae o strains atcc co and vo were used as positive controls templates for the screening of sulii, dfr strb and sxt respectively and that from o biotype el tor strains ko was used for the screening for the dfra gene. detection of mobile genetic elements all strains were further tested for the presence of the 3'-conserved sequence of integron class using the forward primer targeting the qaceΔ and the reverse primer of the suli gene encoding resistance to quaternary ammonium compounds and sulphonamides, respectively. the gene cassettes flanked by the 5'-cs and the 3'-cs were amplified using a combination of primers that target the 3'-cs and the 5'-cs of the integron class primers and pcr conditions for the detection of inti gene belonging to integron class was done as described previously by rivera et al. while detection of the 3'-cs and the variable cassette region was done as described previously by dalsgaard et al.. detection of inti was performed as previously described by falbo et al.. screening for the integrase specific to integron class and integron class was performed as detailed previously by machado et al. and shi et al. respectively. we also conducted pcr experiments using the genomic dna isolated from donors and transconjugants to verify the transfer of the tn and the sxt/r391-like element. detection of tn transposon was done using trpm-specific primers and pcr conditions published previously by villa et al. while detection of tn was done using pcr conditions and primers described previously by hansson et al.. the presence of the ice was detected using primers for amplification of a bp fragment of the integrase gene specific for the sxt/r391-like element as described previously by bhanumathi et al.. integration of the ice into the chromosome was demonstrated by amplification of a pcr product of bp corresponding to the right junction between the attp element of the ice and the prfc chromosomal gene of the bacteria. primers and pcr conditions used are similar to those published before by pugliese et al.. strains from our culture collection known to harbour various genes of interest were used as appropriate positive controls in corresponding pcr experiments. analysis of vibrio cholerae virulence genes all strains were screened for the presence of genes encoding virulence determinants in v. cholerae including cholera toxin, zonula occludens toxin, accessory cholera enterotoxin, hemolysin, and nag-specific heat-stable toxin. detection of the tcpa gene specific to the el tor and classical biotypes was done using a common forward primer and biotype-specific reverse primers. similarly, two forward primers were used for the detection of the biotype-specific haemolysin gene. pcr conditions and primers used for the detection of tcpa, ompu, tcpi, toxr and hyla genes were similar to those described previously by rivera et al. while detection of the ctxa gene was done using primers and conditions described before by fields et al.. genomic dna from v. cholerae o strain atcc was used as a positive control in screening for ctxa, zot, ace, tcpa, ompu, tcpi, and toxr genes. for detection of the four rstr gene alleles, a single reverse primer was used in combination with forward primers specific for each of the four rstr gene alleles as described previously by nusrin et al.. plasmid analysis dna for plasmid analysis was extracted using the method of kado and liu with a few modifications. dna was resuspended in μl of te buffer containing mm tris, and mm edta and separated by electrophoresis on % agarose gel for hours at volt/cm. plasmids of known sizes isolated from e. coli v and 39r were used as controls. pfge selection of strains used for genotypic analysis was based on location and year of isolation and antibiotic resistance profiles. pfge was performed using the pulsenet recommended procedure. the plugs containing agarose-embedded dna were digested with units of sfii or noti. fragments from xba1-digested salmonella braenderup h were used as molecular size markers.  antibiotic susceptibility tests and conjugation tests all the strains showing resistance to the chl-strep-sul-trim combinations transferred this phenotype to e. coli c en bloc. the frequency of transfer, expressed as number of transconjugants per recipients, ranged between × 10- and × 10- with an average of × 10- pcr analysis of the donor strains and the e. coli c transconjugants amplified a bp fragment of sulii gene encoding resistance to sulfamethoxazole, a bp amplicon corresponding to the dfra gene encoding resistance to trimethoprim, a bp fragment of strb encoding resistance to streptomycin, a bp fragment of flor gene conferring resistance to chloramphenicol and a bp fragment corresponding to the integrase gene of the sxt/r ice family, thus confirming co-transfer of resistance markers and this element. the trpm gene of the transposon tn was not detected in the transconjugants indicating that this transposon had not been acquired via conjugation. the dfra gene was not detected in any of the isolates analysed. similarly, attempts to isolate plasmids in the donor strains and transconjugants were not successful. these results are in agreement with those obtained by pugliese et al. who demonstrated the co-transfer of the sxt-like element with the genes encoding the chl-strep-sul-trim phenotype in o strains isolated locally in during the 1998- these workers also found that some strains had an incc plasmid harbouring a gene conferring resistance to tetracycline and while other strains were resistant to ampicillin but we did not identify any strain in our collection bearing these resistance patterns. v. cholerae o strains resistant to tetracycline have previously been reported in kenya and zambia in the 1990s, but those isolated from ethiopia and somalia in the same period were susceptible to this antibiotic. furthermore, strains isolated from previous outbreaks in kenya were known to exhibit resistance to ampicillin, doxycycline and streptomycin. none of the strains we studied were resistant to furazolidone as was the case with strains isolated from mozambican immigrants in south africa. similarly, none of these strains were resistant to ceftriaxone, cefotaxime, nalidixic acid, amikacin and gentamicin as has been the case with strains previously reported from ghana. all the strains we analysed also lacked the dfra gene encoding resistance to trimethoprim in o and non-o strains isolated in india. genetic and environmental factors that may be responsible for the apparent serotype shift from ogawa to inaba in recent outbreaks in kenya remain to be elucidated. while strains that do not harbour the sxt/r391-like element and those bearing the incc plasmids were not available for analysis alongside those included in our study, it is apparent that the gradual emergence of a population of v. cholerae o strains bearing the sxt/r391-like element as a major cause of cholera outbreaks in kenya has occurred independent of antibiotic resistance acquisition. it remains to be determined exactly when the sxt/r391-like ice emerged in pathogenic v. cholera strains in kenya because isolates obtained locally between and were known to exhibit resistance to antibiotics encountered in the chl-strep-sul-trim phenotype that has lately been associated to the presence of the sxt-type ices. although it is well established that cholera came to africa from asia in the 1970s, it is only suspected that the sxt-like elements have been present in african vibrio spp even before the emergence of the v. cholerae o from which the first sxt element, sxtmo was identified. ice-like elements have been detected in o clinical strains isolated in in angola and v. parahaemolyticus clinical strains from the same country isolated in were also shown to contain sxt-related ices that do not mediate resistance to antibiotics. similarly, analysis of o el tor clinical isolates from algeria isolated in suggests the presence of sxt-like ices mediating trimethoprim resistance. however, the isolates from the outbreak in the goma refugee camp in zaire did not harbour this element. our study demonstrates that the o el tor strains bearing the sxt/r391-like ice were in circulation in kenya in the 1994- period and have continued to persist in recent outbreaks. this may suggest that the strains isolated from the two outbreaks in 1994- in kwale, a coastal town of kenya, are some of the oldest strains in the region known to harbour this integrating conjugative element in this part of the continent. analysis for mobile genetic elements and vibrio cholerae pathogenicity island all the o strains were positive for all the v. cholerae pathogenic genes except for the nag-specific heat-stable toxin. these strains were also positive for the inti integrase belonging to integron class asuper-integron believed to be important in shuffling the vibrio cholerae genome. it is worth noting that the st gene normally occurs as a cassette within int region in some v. cholerae strains but not in others. besides the st gene, another pathogenicity determinant, mrha, is frequently detected in si region of o and non-o1strains. it is not clear whether the presence of the st gene improves biological fitness of the host but appears to be dispensable without compromising the ability to cause disease in pathogenic strains. besides the sxt elements, other mobile genetic elements implicated in the spread of antibiotic resistance phenotype in v. cholerae from africa include conjugative plasmids belonging to class c, integron class, and integron class. although the isolates we studied carried the sxt element, they lacked the class and integrons and did not harbour any conjugative plasmids. all the strains were negative for the transposase gene belonging to tn but were positive for the trpm gene associated with tn the tn has frequently been detected in gram negative strains containing integron class. on the other hand, tn and its relatives are major agents in the dissemination of mercury resistance and antibiotic resistance genes in gram negative bacteria but not all tn21-like transposons are associated with antibiotic resistance and there are variations in the diversity of antibiotic resistance genes detected in tn21-like transposons that harbour antibiotic resistance markers. pcr analysis of transconjugants did not detect the tn implying that this transposon was not co-transferred with the sxt/r391-like element during conjugation. we were however not able to determine if this element confers mercury resistance to the strains we studied or if it is physically linked to any antibiotic resistance markers. it is also not clear if this transposon has all the other genes responsible for transposition such as tnpa, tnpr, res, and inverted repeats or if it exists as a defective transposon in these strains. however, the presence of the trpm gene suggests that although the strains carrying the sxt/r391-like elements lack multiple resistant integrons, this transposon is genetically ready to accept such elements because integrons are normally located adjacent to this gene. it has been suggested that tn21-like transposons which confer multiple antibiotic resistance descended from an ancestral mercury resistance transposon like tn by successive insertions of antibiotic resistances and/or insertion sequences. it is therefore important to further characterize tn in pathogenic v. cholerae strains. all the strains were positive for the ctxetΦ but negative for all the other ctxΦ phage repressor gene alleles and this contradicts with the study on o el tor strains isolates from mozambique and india which have been reported to harbour the ctxclassΦ repressor. such el tor strains carrying the ctxclassΦ repressor are now designated as the matlab variants of v. cholerae. our finding on the diversity of the ctxetΦ repressor and the absence of the other rstr genes in all the strains further indicate the need for detailed studies on the genetic diversity of v. cholerae strains from different parts of the continent to gain insight into the evolutionary trends of v. cholerae species causing epidemics in africa. pulsed field gel electrophoresis analysis of macro-restricted dna using not demonstrated that all the sxt-carrying strains produced indistinguishable digest patterns regardless of the year and region of isolation. similarly, restriction digest analysis using sfi showed that all strains were clonal. the fact that all our strains showed identical pattern in antibiotic susceptibility patterns, pathogenicity genes, and the diversity of mobile genetic elements strongly suggest that this population of o strains that have caused outbreaks since to as recent as are clonally related. the absence of the st gene and the absence of the classical biotype-specific tcpa and hyla genes in these strains further indicates that genetic exchanges between this population and other v. cholerae serotypes that might be in circulation in kenya have been highly restricted. in a previous study by jiang et al. it was noted that a number of o strains from kenya failed to cluster with those isolated from other parts of the world when using amplified fragment length polymorphism genotyping technique. similarly, the study by pugliese et al. showed that strains that carried the sxt-element alone or in combination with an incc plasmid belonged to a unique rapd cluster iv. in the same study, strains without this ice were shown to belong to other cluster types shared by isolates from ethiopia and somali. it is also interesting to note that none of the isolates from 1998- study shared a rapd cluster with strains isolated in india and bahrain isolated in and such observations have led to a theory that some toxigenic v. cholerae strains circulating in different countries may not have originated from a single clone in asia as is popularly believed, but may have been derived locally from genetic exchange between the asian o strains and the o or non-o strains from local environments.  we observed that antibiotic susceptibility and genomic content of the strains bearing the sxt/r391-like ice that have been in circulation in kenya between and has not changed significantly and there are indications that these strains have undergone minimum genotypic changes during this entire period. in the absence of older isolates for molecular characterization, it is not possible to determine whether other clones of v. cholerae bearing this ice have existed in kenya and is also difficult to determine when the sxt/r391-bearing clone emerged as the major cause of outbreaks in kenya. in order to understand the epidemiological trends of cholera outbreaks in the region, there is need for further studies to determine evolutionary trends among strains isolated from the african region and compare them with those from other parts of the world. authors' contributions jnk designed and coordinated the study, carried out molecular characterization studies and drafted the manuscript. smk participated in the design and supervision of the study and revision of the manuscript. bmg revised the manuscript and supervised the study. ncw participated in manuscript revision. sms provided strains from earlier outbreaks and revised the manuscript. pb participated in the study design, supervision of molecular characterization studies in belgium and revision of manuscript. all authors read and approved the final manuscript. authors' information jnk is a research scientist at the kenya medical research institute and doctoral fellow at katholieke universiteit leuven, belgium. he holds an msc and msc where he is currently pursuing a phd in bioscience engineering at the department of biosystems. his work is supported by a scholarship from the vlaamse interuniversitaire raad, belgium. smk, ncw and sms are scientists at kemri, kenya. smk is a wellcome trust research fellow and an opinion leader in the field of antibiotic resistance in the east african region while ncw is the former director centre for microbiology research, kemri. bmg is professor of immunology at the k.u.leuven and the university of ghent, belgium while pb is a senior research scientist at the veterinary and agrochemical research centre and an expert in the field of antibiotic resistance in belgium. he is also a professor at the faculty of veterinary medicine at ugent. 
pulse field gel electrophoresis showed that regardless of the year of isolation, all the strains bearing the sxt element were clonally related. this study demonstrates that the v. cholerae o strains carrying an sxt/r391-like element implicated in recent cholera outbreaks in kenya has not changed significantly between and and are clonally related. all the strains were negative for integron class and and for transposase gene of transposon tn but were positive for integron class and the trpm gene of transposon tn no plasmids were isolated from any of the strains. a total of vibrio cholerae o el tor serotype inaba isolated between and from various outbreaks in kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements, conjugative plasmids and for their genotypic relatedness. although a number of recent studies describe the epidemiology of cholera in kenya, there is paucity of information concerning the diversity and occurrence of mobile genetic elements in vibrio cholerae strains implicated in these outbreaks. none of the strains were positive for virulence genes associated with the v. cholerae classical biotype. over the last decade, cholera outbreaks in parts of kenya have become common.
 over the last decade, cholera outbreaks in parts of kenya have become common. although a number of recent studies describe the epidemiology of cholera in kenya, there is paucity of information concerning the diversity and occurrence of mobile genetic elements in vibrio cholerae strains implicated in these outbreaks. a total of vibrio cholerae o el tor serotype inaba isolated between and from various outbreaks in kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements, conjugative plasmids and for their genotypic relatedness.  all the strains were haemolytic on 5% sheep blood and positive for the vibrio cholerae el tor-specific haemolysin toxin gene by pcr. they all contained strb, sulii, flor and the dfra genes encoding resistance to streptomycin, sulfamethoxazole, chloramphenicol and trimethoprim respectively. these genes, together with an ice belonging to the sxt/r family were transferable to the rifampicin-resistant e. coli c en bloc. all the strains were negative for integron class and and for transposase gene of transposon tn but were positive for integron class and the trpm gene of transposon tn no plasmids were isolated from any of the strains. all the strains were also positive for all v. cholera el tor pathogenic genes except the nag- specific heat-stable toxin gene. none of the strains were positive for virulence genes associated with the v. cholerae classical biotype. all the strains were positive for el tor-specific ctxphi bacteriophage rstrr repressor gene but negative for the classical, calcutta, and the environmental repressor types. pulse field gel electrophoresis showed that regardless of the year of isolation, all the strains bearing the sxt element were clonally related.  this study demonstrates that the v. cholerae o strains carrying an sxt/r391-like element implicated in recent cholera outbreaks in kenya has not changed significantly between and and are clonally related. 