as part of a study on fecal carriage of esbl - producing e. coli or klebsiella pneumonia associated with uti , supragingival plaque and repeat fecal samples were collected from known fecal carriers of these bacteria . the study was conducted at the department of medical microbiology at vestre viken hospital trust in eastern norway and the department of oral biology , university of oslo . the study was approved by the south - eastern norway regional committee for medical and health research ethics following the declaration of helsinki principles ( 1975 , 1983 ) ( reference number : 2009/2037 bs-08901b ) . written informed consent was obtained from all participants and donation of the supragingival plaque sample was not mandatory for participation in the other parts of the study . the participants were adult volunteers from a study on fecal carriage of esbl and risk factors for uti caused by esbl - producing e. coli or k. pneumoniae ( 21 , 22 ) . in this study , inclusion criteria were patients with community - acquired uti caused by these bacteria or their household members . the patients were recruited after their urine had been submitted to a microbiological laboratory for analysis . participants were interviewed ; they submitted fecal samples and data were collected from the norwegian prescription registry and patient medical records . during one time period in 2010 and one in 2011 participants with a fecal sample yielding esbl - producing e. coli or k. pneumoniae the following exclusion criteria were used : 1 ) patients who had lived in norway for < 1 year , 2 ) were unable to answer our questionnaire , 3 ) had a previously diagnosed infection caused by esbl - producing bacteria , and 4 ) patients with health care associated uti ( i.e. hospitalized or residing in a nursing home for > 24 hours during the last 31 days ) . fecal samples were collected by the participants themselves using a sterile cotton swab applied to the toilet paper after defecation . the sample containers were returned by ordinary mail and cultured aerobically on esbl selective agar plates ( chromid esbl , biomerieux , marcy l'etoile , france ) , enriched in a selective broth with 2.5 mg / ml of cefotaxime and then grown on the esbl selective agar plates and on a non - selective lactose agar plate as growth control . species identification was based on characteristic pink to burgundy oxidase - negative colonies for e. coli . species identification of green colonies was obtained using the vitek-2 system ( gn i d card , biomerieux ) . genotypic verification of esbl and phylogroup assignment of fecal esbl - positive isolates was performed using polymerase chain reaction ( pcr ) as described earlier ( 23 ) . sub - typing of fecal esbl - producing e. coli isolates was performed by ten - loci multiple - locus variable - number tandem - repeat analysis ( mlva ) as described earlier ( 24 , 25 ) . supragingival plaque samples were collected at the department of oral biology , university of oslo by a non - calibrated , but clinically experienced dentist with sterile gracey - designed steel curettes ( stalan gmbh , ahrensburg , germany ) . samples were taken from the mesio - buccal site of every tooth , with one vertical stroke in participants with their own teeth intact . patients were not screened for periodontal disease site by site , but periodontal probing was done to make sure what pockets were healthy ( <3 mm ) . anamnestic occurrence of periodontal disease was recorded . from participants with dental prostheses or implants , biofilms were collected from these restorations . the samples were immediately transferred to one 5.5 ml vial of a pre - reduced anaerobically sterilized transport medium ( dental transport medium , anaerobic system , morgan hill , ca ) per patient ( i.e. all samples from each patient were pooled ) . the samples were then homogenized within four hours by vortex and plated onto mcconkey agar plates , chromid esbl ( biomerieux ) and blood agar plates ( nonselective trypticase soy agar supplemented with 5% defibrinated human blood , hemin 5 mg / ml , and menadione 0.05 mg / ml ) and incubated at 37c for 2448 hours in aerobic ( mcconkey and chromid esbl ) and anaerobic ( 90% n2 , 5% h2 , 5% co2 ) conditions ( blood agar plates ) . species identification of suspected enterobacteriaceae was obtained using the vitek-2 system ( gn i d card , biomerieux ) . for pcr , 100 l of the inoculated transport medium dna was extracted using masterpure dna purification kit ( epicentre , madison , wi ) according to the instructions of the manufacturer . pcr was performed in duplicate using the same procedure as for the fecal samples ( 23 ) . to semi - quantitatively assess the sensitivity of the dna extraction and pcr procedure described for oral plaque samples , 10-fold dilutions of ctx - m 1 producing e. coli ( ccug 55971 ) were inoculated in dental transport medium . the detection limit of the dna extraction- and pcr procedures from the dental samples were found to be 330 gene copies per pcr run indicating a lower detection limit of approximately 1,60016,000 esbl gene copies per supragingival plaque sampling procedure . the approximation assumed the same copy number of blactx - m genes in the clinical isolates as in the reference strain e. coli ( ccug 55971 ) . as part of a study on fecal carriage of esbl - producing e. coli or klebsiella pneumonia associated with uti , supragingival plaque and repeat fecal samples were collected from known fecal carriers of these bacteria . the study was conducted at the department of medical microbiology at vestre viken hospital trust in eastern norway and the department of oral biology , university of oslo . the study was approved by the south - eastern norway regional committee for medical and health research ethics following the declaration of helsinki principles ( 1975 , 1983 ) ( reference number : 2009/2037 bs-08901b ) . written informed consent was obtained from all participants and donation of the supragingival plaque sample was not mandatory for participation in the other parts of the study . the participants were adult volunteers from a study on fecal carriage of esbl and risk factors for uti caused by esbl - producing e. coli or k. pneumoniae ( 21 , 22 ) . in this study , inclusion criteria were patients with community - acquired uti caused by these bacteria or their household members . the patients were recruited after their urine had been submitted to a microbiological laboratory for analysis . participants were interviewed ; they submitted fecal samples and data were collected from the norwegian prescription registry and patient medical records . during one time period in 2010 and one in 2011 participants with a fecal sample yielding esbl - producing e. coli or k. pneumoniae the following exclusion criteria were used : 1 ) patients who had lived in norway for < 1 year , 2 ) were unable to answer our questionnaire , 3 ) had a previously diagnosed infection caused by esbl - producing bacteria , and 4 ) patients with health care associated uti ( i.e. hospitalized or residing in a nursing home for > 24 hours during the last 31 days ) . fecal samples were collected by the participants themselves using a sterile cotton swab applied to the toilet paper after defecation . the sample containers were returned by ordinary mail and cultured aerobically on esbl selective agar plates ( chromid esbl , biomerieux , marcy l'etoile , france ) , enriched in a selective broth with 2.5 mg / ml of cefotaxime and then grown on the esbl selective agar plates and on a non - selective lactose agar plate as growth control . species identification was based on characteristic pink to burgundy oxidase - negative colonies for e. coli . species identification of green colonies was obtained using the vitek-2 system ( gn i d card , biomerieux ) . genotypic verification of esbl and phylogroup assignment of fecal esbl - positive isolates was performed using polymerase chain reaction ( pcr ) as described earlier ( 23 ) . sub - typing of fecal esbl - producing e. coli isolates was performed by ten - loci multiple - locus variable - number tandem - repeat analysis ( mlva ) as described earlier ( 24 , 25 ) . supragingival plaque samples were collected at the department of oral biology , university of oslo by a non - calibrated , but clinically experienced dentist with sterile gracey - designed steel curettes ( stalan gmbh , ahrensburg , germany ) . samples were taken from the mesio - buccal site of every tooth , with one vertical stroke in participants with their own teeth intact . patients were not screened for periodontal disease site by site , but periodontal probing was done to make sure what pockets were healthy ( <3 mm ) . anamnestic occurrence of periodontal disease was recorded . from participants with dental prostheses or implants , biofilms were collected from these restorations . the samples were immediately transferred to one 5.5 ml vial of a pre - reduced anaerobically sterilized transport medium ( dental transport medium , anaerobic system , morgan hill , ca ) per patient ( i.e. all samples from each patient were pooled ) . the samples were then homogenized within four hours by vortex and plated onto mcconkey agar plates , chromid esbl ( biomerieux ) and blood agar plates ( nonselective trypticase soy agar supplemented with 5% defibrinated human blood , hemin 5 mg / ml , and menadione 0.05 mg / ml ) and incubated at 37c for 2448 hours in aerobic ( mcconkey and chromid esbl ) and anaerobic ( 90% n2 , 5% h2 , 5% co2 ) conditions ( blood agar plates ) . species identification of suspected enterobacteriaceae was obtained using the vitek-2 system ( gn i d card , biomerieux ) . for pcr , 100 l of the inoculated transport medium dna was extracted using masterpure dna purification kit ( epicentre , madison , wi ) according to the instructions of the manufacturer . pcr was performed in duplicate using the same procedure as for the fecal samples ( 23 ) . to semi - quantitatively assess the sensitivity of the dna extraction and pcr procedure described for oral plaque samples , 10-fold dilutions of ctx - m 1 producing e. coli ( ccug 55971 ) were inoculated in dental transport medium . the detection limit of the dna extraction- and pcr procedures from the dental samples were found to be 330 gene copies per pcr run indicating a lower detection limit of approximately 1,60016,000 esbl gene copies per supragingival plaque sampling procedure . the approximation assumed the same copy number of blactx - m genes in the clinical isolates as in the reference strain e. coli ( ccug 55971 ) . fecal samples were collected by the participants themselves using a sterile cotton swab applied to the toilet paper after defecation . the sample containers were returned by ordinary mail and cultured aerobically on esbl selective agar plates ( chromid esbl , biomerieux , marcy l'etoile , france ) , enriched in a selective broth with 2.5 mg / ml of cefotaxime and then grown on the esbl selective agar plates and on a non - selective lactose agar plate as growth control . species identification was based on characteristic pink to burgundy oxidase - negative colonies for e. coli . species identification of green colonies was obtained using the vitek-2 system ( gn i d card , biomerieux ) . genotypic verification of esbl and phylogroup assignment of fecal esbl - positive isolates was performed using polymerase chain reaction ( pcr ) as described earlier ( 23 ) . sub - typing of fecal esbl - producing e. coli isolates was performed by ten - loci multiple - locus variable - number tandem - repeat analysis ( mlva ) as described earlier ( 24 , 25 ) . supragingival plaque samples were collected at the department of oral biology , university of oslo by a non - calibrated , but clinically experienced dentist with sterile gracey - designed steel curettes ( stalan gmbh , ahrensburg , germany ) . samples were taken from the mesio - buccal site of every tooth , with one vertical stroke in participants with their own teeth intact . patients were not screened for periodontal disease site by site , but periodontal probing was done to make sure what pockets were healthy ( <3 mm ) . anamnestic occurrence of periodontal disease was recorded . from participants with dental prostheses or implants , biofilms were collected from these restorations . the samples were immediately transferred to one 5.5 ml vial of a pre - reduced anaerobically sterilized transport medium ( dental transport medium , anaerobic system , morgan hill , ca ) per patient ( i.e. all samples from each patient were pooled ) . the samples were then homogenized within four hours by vortex and plated onto mcconkey agar plates , chromid esbl ( biomerieux ) and blood agar plates ( nonselective trypticase soy agar supplemented with 5% defibrinated human blood , hemin 5 mg / ml , and menadione 0.05 mg / ml ) and incubated at 37c for 2448 hours in aerobic ( mcconkey and chromid esbl ) and anaerobic ( 90% n2 , 5% h2 , 5% co2 ) conditions ( blood agar plates ) . species identification of suspected enterobacteriaceae was obtained using the vitek-2 system ( gn i d card , biomerieux ) . for pcr , 100 l of the inoculated transport medium dna was extracted using masterpure dna purification kit ( epicentre , madison , wi ) according to the instructions of the manufacturer . pcr was performed in duplicate using the same procedure as for the fecal samples ( 23 ) . to semi - quantitatively assess the sensitivity of the dna extraction and pcr procedure described for oral plaque samples , 10-fold dilutions of ctx - m 1 producing e. coli ( ccug 55971 ) were inoculated in dental transport medium . the detection limit of the dna extraction- and pcr procedures from the dental samples were found to be 330 gene copies per pcr run indicating a lower detection limit of approximately 1,60016,000 esbl gene copies per supragingival plaque sampling procedure . the approximation assumed the same copy number of blactx - m genes in the clinical isolates as in the reference strain e. coli ( ccug 55971 ) . patient characteristics and results from culture- and molecular analyses of fecal and oral samples are presented in table 1 . the mean age was 61 years ( range 2384 ) and 13 ( 76% ) participants were female . the median charlson index score was 1 and data from the prescription registry showed that seven out of 17 participants ( 59% ) had been on one or more courses of antibiotics during the 3 months before the oral sample was obtained . background information about participants and time of fecal sample before and after the oral sampling procedure and results from these samples i d : study participant number ; f / m : female or male gender ; ctx - m : cefotaximase - munich ; mlva : ten - loci multiple - locus variable - number tandem - repeat analysis ; na : not available . all the ctx - m genes identified prior to , and directly after oral sampling were harbored in e. coli . mlva was used for comparison of e. coli isolates in patients with more than one isolate . a low number of diverging loci indicate that the compared strains were identical or closely related . 97 days later ctx - m 1 was detected and 145 days later ctx - m 1 and 9 were detected . fecal carriage of esbl - e was a prerequisite for inclusion and therefore present in all participants . at least one follow - up fecal sample was obtained after dental sampling from 14 of the 17 ( 82% ) participants . the esbl determinants in inclusion- and follow - up samples were harbored exclusively in e. coli and of the 17 inclusion isolates , 12 belonged to ctx - m phylogroup 1 and five to ctx - m phylogroup 9 . the median duration from the inclusion sample to dental sampling was 64 days ( range 8326 days ) . the first follow - up sample from each participant was obtained at median 55 days ( range 3768 days ) after dental sampling . 
 in six ( 35% ) participants , the same esbl phylogroup and mlva profile were identified in both the inclusion and follow - up sample.in two ( 12% ) participants with the same phylogroup , the mlva profile diverged in four and seven loci.in one ( 6% ) participant , the same phylogroup was only detected in the third fecal sample after dental sampling and at that point the mlva profile diverged in four loci.six ( 35% ) participants did not donate a follow - up fecal sample or donated a sample more than 700 days after the dental procedure and all of these were negative.two ( 12% ) participants donated fecal samples promptly after the dental procedure , but these and later samples were ctx - m - negative . 
  in six ( 35% ) participants , the same esbl phylogroup and mlva profile were identified in both the inclusion and follow - up sample . in two ( 12% ) participants with the same phylogroup , the mlva profile diverged in four and seven loci . in one ( 6% ) participant , the same phylogroup was only detected in the third fecal sample after dental sampling and at that point the mlva profile diverged in four loci . six ( 35% ) participants did not donate a follow - up fecal sample or donated a sample more than 700 days after the dental procedure and all of these were negative . two ( 12% ) participants donated fecal samples promptly after the dental procedure , but these and later samples were ctx - m - negative . of the nine fecal esbl culture - positive isolates obtained after the dental procedure , seven isolates belonged to ctx - m phylogroup 1 , one to ctx - m phylogroup 9 and in one participant both ctx - m 1 and 9 were identified . of the 17 participants , 15 had their own teeth , one had a combination of own teeth and implants and one had a dental prosthesis only . no esbl - producing oral bacteria were detected by culturing on the chromid esbl . enterobacteriaceae were isolated from the mcconkey cultures in three patients ( 17.6% ) . from these three participants , four different enterobacteriaceae strains were identified , whereas none of the patients suffered from periodontitis . classical esbls were not identified by pcr in any of the oral plaque samples , but the r. aquatilis strain harbored the class a esbl gene blarahn-1/2 by pcr ( 26 ) . fecal carriage of esbl - e was a prerequisite for inclusion and therefore present in all participants . at least one follow - up fecal sample was obtained after dental sampling from 14 of the 17 ( 82% ) participants . the esbl determinants in inclusion- and follow - up samples were harbored exclusively in e. coli and of the 17 inclusion isolates , 12 belonged to ctx - m phylogroup 1 and five to ctx - m phylogroup 9 . the median duration from the inclusion sample to dental sampling was 64 days ( range 8326 days ) . the first follow - up sample from each participant was obtained at median 55 days ( range 3768 days ) after dental sampling . in six ( 35% ) participants , the same esbl phylogroup and mlva profile were identified in both the inclusion and follow - up sample.in two ( 12% ) participants with the same phylogroup , the mlva profile diverged in four and seven loci.in one ( 6% ) participant , the same phylogroup was only detected in the third fecal sample after dental sampling and at that point the mlva profile diverged in four loci.six ( 35% ) participants did not donate a follow - up fecal sample or donated a sample more than 700 days after the dental procedure and all of these were negative.two ( 12% ) participants donated fecal samples promptly after the dental procedure , but these and later samples were ctx - m - negative . 
  in six ( 35% ) participants , the same esbl phylogroup and mlva profile were identified in both the inclusion and follow - up sample . in two ( 12% ) participants with the same phylogroup , the mlva profile diverged in four and seven loci . in one ( 6% ) participant , the same phylogroup was only detected in the third fecal sample after dental sampling and at that point the mlva profile diverged in four loci . six ( 35% ) participants did not donate a follow - up fecal sample or donated a sample more than 700 days after the dental procedure and all of these were negative . two ( 12% ) participants donated fecal samples promptly after the dental procedure , but these and later samples were ctx - m - negative . of the nine fecal esbl culture - positive isolates obtained after the dental procedure , seven isolates belonged to ctx - m phylogroup 1 , one to ctx - m phylogroup 9 and in one participant both ctx - m 1 and 9 were identified . of the 17 participants , 15 had their own teeth , one had a combination of own teeth and implants and one had a dental prosthesis only . no esbl - producing oral bacteria were detected by culturing on the chromid esbl . enterobacteriaceae were isolated from the mcconkey cultures in three patients ( 17.6% ) . from these three participants , four different enterobacteriaceae strains were identified , whereas none of the patients suffered from periodontitis . classical esbls were not identified by pcr in any of the oral plaque samples , but the r. aquatilis strain harbored the class a esbl gene blarahn-1/2 by pcr ( 26 ) . of the 17 participants , 15 had their own teeth , one had a combination of own teeth and implants and one had a dental prosthesis only . no esbl - producing oral bacteria were detected by culturing on the chromid esbl . enterobacteriaceae were isolated from the mcconkey cultures in three patients ( 17.6% ) . from these three participants , four different enterobacteriaceae strains were identified , whereas none of the patients suffered from periodontitis . classical esbls were not identified by pcr in any of the oral plaque samples , but the r. aquatilis strain harbored the class a esbl gene blarahn-1/2 by pcr ( 26 ) . we have investigated the oral cavity as a potential reservoir for the ctx - m - type esbl resistance determinants in adults with fecal carriage of ctx - m producing e. coli using culture - based and molecular methods . the negative results obtained do not support the oral cavity as a significant reservoir of ctx - m - type esbl . the sensitivity of the molecular methods and chromid esbl agar plates used for detection of esbl - e is high ( 27 ) . furthermore , we sampled supragingival plaque because this is the oral location we considered most likely to harbor enterobacteriaceae . thus , esbl - producing strains or blactx - m should have been detected , if present in the oral samples from our participants . the negative results indicate the absence or presence in low concentrations only , of oral esbls in intestinal carriers and corroborate previous studies that have found a relatively low abundance of enterobacteriaceae in the healthy oral cavity . the present study does not indicate that the oral carriage dynamics of esbl - e is different from non - esbl - e . the negative pcr results also suggest that classical ctx - m genes are not harbored in any of the many different oral bacterial species present in oral samples . our result on oral carriage elucidates the difference between fecal and oral carriage of esbl - e . a recent study by titelman et al . investigated the duration of fecal carriage after esbl - e infections and found a gastrointestinal carriage rate of 66% three months after , and 43% one year after infection ( 28 ) the difference between oral and fecal carriage duration is probably a result of the oral cavity being a microbiological site distinctly different from the gastrointestinal tract . in one study , 509 different bacteria were detected in periodontal samples , but only 29 of these were found among the 560 intestinal- or fecal bacteria isolated , illustrating this point ( 29 ) . furthermore , to our knowledge only narrow spectrum beta - lactamases , ampc beta - lactamases or unspecified tem - genes have been detected in plaque of patients with periodontitis or in plaque samples from the normal population ( 20 , 3032 ) . the r. aquatilis with the esbl gene blarahn-1/2 identified in the present material is thus , to our knowledge , a novelty . however , r. aquatilis is probably a transient bacterium in the mouth originating from drinking water ( 33 ) . supporting this , it was not identified in the patient 's fecal samples , and this finding does not change the overall picture of intestinal esbl carriage as not related to oral carriage . the present study does not support the oral cavity as a source of transmission of esbl . but worryingly , after doxycycline therapy more than a 10-fold increase was seen in subgingival numbers of enterobacter aerogenes and e. coli ( 13 ) . another study showed an increasing frequency of nasal and pharyngeal carriage of enterobacteriaceae during the hospitalized period in patients admitted to general wards and intensive care units ( 10 ) . thus , esbl - e may under certain circumstances be more frequent in the oropharyngeal area than our study demonstrated . possible transmission related to oropharyngeal carriage was demonstrated in another study by branger et al . , which identified an outbreak of esbl - e possibly related to a poorly maintained bronchoscope ( 34 ) . thus , there are suggestions that periods of oral- or upper airway carriage may facilitate the transfer of esbl - e , but this remains to be documented . in hospitals , where oral and oropharyngeal carriage appears to be more frequent , the present results apply to non - hospitalized fecal carriers of esbl - e and should not discourage infectious control measures that take into account a possible oropharyngeal source of esbl - e ( 10 ) . furthermore , the intestinal colonization status of the participants at the exact time of the dental sampling procedure was unknown since fecal samples were not collected at that time . however , esbl was detected in 9 of 11 ( 82% ) participants that donated a fecal sample within 90 days after dental sampling . this is in line with published data on the duration of fecal esbl carriage after infection ( 28 ) . the exact same esbl or e. coli mlva sub - type was not identified in all nine participants . this may be explained by re - colonization and thus it is a possibility that these participants were not fecal carriers at the time of the dental procedure . since norway is a country with a low prevalence of esbl , this seems unlikely . other explanations like initial co - colonization with several esbls , transfer of plasmids between e. coli strains and esbl below the detection threshold seem better explanations for diverging phylogroups and sub - types before and after dental sampling . it 's not possible to know how many participants were actually fecal carriers at the time of oral sampling , yet an estimate based on the 11 participants who did donate a fecal sample within 90 days of the oral sampling is 14 of 17 . given that 20% of fecal esbl - e carriers are also oral carriers , the chance of a negative result in 14 individuals is less than 5% ( 0.80<0.05 ) , indicating that the true oral carriage rate in fecal esbl - e carriers is lower than this . furthermore , none of the 17 previously known fecal carriers of esbl were found to be oral carriers at a median of 64 days after fecal sampling . at this point in time , at least 66% of patients infected with esbl - e would be expected to be fecal carriers according to one earlier study ( 28 ) . thus , we may make a cautious conclusion that the frequency of fecal carriage is significantly higher than oral carriage after a uti caused by esbl - e ( p < 0.001 , fisher 's exact test ) . the results do not rule out transient oral esbl - carriage in fecal carriers or oral carriage in high - risk groups like hospitalized patients . because of the limitations of this pilot study and the importance of the issue investigated , further studies of oral carriage of esbl are warranted .
backgroundthe prevalence of infections caused by cefotaximase - munich ( ctx - m)-type extended - spectrum beta - lactamase - producing enterobacteriaceae ( esbl - e ) has rapidly increased during the past 15 years . 
 enterobacteriaceae are commonly found in the gastrointestinal tract and long - term intestinal carriage is considered important for the spread of esbl and as a source of clinical infections . 
 oral biofilm such as supragingival plaque is known to contain numerous antibiotic resistance determinants and may also represent a poorly investigated site for esbl carriage and further spread.objectiveto investigate possible carriage of esbl - producing bacteria in supragingival plaque of known fecal carriers of these bacteria.designwe screened for the presence of aerobic and anaerobic esbl - producing bacteria and bla 
 ctx - m in supragingival plaque samples from healthy human adults with culture - verified fecal carriage of ctx - m - producing escherichia coli . the presence or absence of enterobacteriaceae and esbl - producing bacteria in plaque samples 
 was evaluated using culture - based methods and consensus ctx - m pcr.resultsoral samples were obtained from 17 participants with known previous carriage of esbl - producing e. coli . 
 no esbl - producing bacteria or esbl genes were detected using culture - based and molecular methods . 
 one colony of rahnella aquatilis harboring the class a esbl gene blarahn-1/2 was identified in an oral sample from one of the participants.conclusionthis pilot study supports the notion that the presence of ctx - m - producing bacteria is uncommon in oral plaque of healthy human adult fecal carriers . due to the limited number of persons tested , a low prevalence of oral esbl - carriage in healthy adults or carriage in selected groups of patients can not be excluded . to our knowledge , this is the first description of an r. aquatilis with the rahn-1/2 gene in the oral cavity .