study population and sample preparation : one hundred and 24 human subjects 
 were included in the  dog - contact risk factor group  . in detail , the dog - contact risk factor 
 group was composed of 41 dog owners and 43 veterinarians who regularly contact dogs . in this 
 group , 12 veterinarians who were themselves dog owners .
each of the dog owners including 
 veterinarians was matched with their own 39 dogs by labeling for the statistical analyses . 

forty human subjects with no history of pet ownership were included for the negative 
 dog - contact factor ( supplemental fig . 1 
 ) .
the feces and 
 saliva samples were taken by using sterilized cotton swabs and subsequently submerged in 500 
 l of autoclaved phosphate buffered saline .
dna was extracted from 20 to 
 30 l of each sample by using dneasy tissue kit ( qiagen , santa clarita , ca , 
 u.s.a . ) .
the dna samples were eluted in 200 l volume and stored in a 20c 
 freezer until pcr was conducted .
genus - specific pcr : each dna sample was amplified on 
 helicobacter 16s rrna gene using c70 and b37 outer primers30 , and 
 subsequently , the pcr products were conducted nested pcr using c97 and c98 inner primer pair 
 ( supplemental table 1 ) . the pcr mixture , total volume of 
 20 l contained a final primer concentration of 0.5 m , 1 
 l dna samples ( 0.3 l for nested pcr ) and 18 
 l diethyl pyrocarbonate treated water , was added to maxime pcr premix 
 kit ( intron biotechnology inc . ,
the pcr samples were heated at 95c for 5 min 
 followed by 30 cycles of denaturation at 94c for 45 sec , annealing at 53c for 45 sec and 
 extension at 72c for 3 min and finally extended at 72c for 15 min using outer primers by 
 using programmed temperature control system ( pc808 , astec , fukuoka , japan ) . for nested pcr
, 
 the pcr products were heated at 94c for 2.5 min followed by 35 cycles of denaturation at 
 94c , annealing at 50.5c , extension at 72c for 1 min each and a final extension at 72c 
 for 15 min .
the pcr products were electrophoresed by 1.5% agarose gels containing ethidium 
 bromide in 0.5x tbe buffer and visualized on ultraviolet light illuminator .
species- specific pcr : pcr amplifications of h. felis and 
 h. bizzozeronii were performed using primer ( supplemental table 1 ) which amplify the urease b gene of them 
 .
the total volume of pcr mixture 20 l 
 including a final each primer concentration of 0.5 m , 1 
 l dna samples ( 0.3 l for nested pcr ) and 18 
 l diethyl pyrocarbonate treated water was added to maxime pcr premix kit 
 ( intron biotechnology inc . ) .
dna extracts from pure cultures of h. 
 pylori(hpktcc h. pylori strain 114 ) , h. felis(atcc 49179 ) and 
 h. bizzozeronii(atcc 70030 ) served as positive controls . for pcr amplification of h. pylori
, the samples were heated at 95c for 5 
 min and followed by 35 cycles at 94c for 45 sec , at 59c for 45 sec and at 72c for 45 sec 
 and a final extension at 72c for 10 min using outer primes .
second round of pcr was 
 performed at 95c for 5 min , 30 cycles followed at 94c for 45 sec , at 54c for 45 sec and 
 at 72c for 30 sec and final extension at 72c for 10 min . for h. 
 felis - specific pcr ,
samples were heated at 94c for 2.5 min once , followed by 40 
 cycles of denaturation at 94c , annealing at 45c , extension at 72c for 1 min each with a 
 final extension at 72c for 15 min using outer primes .
second round of pcr was performed at 
 94c for 2.5 min , 30 cycles followed at 94c for 45 sec , at 50c for 45 sec and at 72c for 
 45 sec and final extension at 72c for 15 min .
the h. bizzozeronii - specific 
 pcr was carried out following conditions , heated at 94c for 2.5 min once and 33 cycles of 
 at 94c for 1 min , at 57c for 1 min and at 72c for 1 min .
second round of pcr was performed at 94c for 2.5 min , 
 30 cycles followed at 94c for 45 sec , at 55.5c for 45 sec and at 72c for 45 sec and final 
 extension at 72c for 15 min .
the pcr products were electrophoresed on ethidium - bromide 
 stained 1.5% w / v agarose gels in 0.5x tbe buffer and visualized on ultraviolet light 
 illuminator .
nucleotide sequence analysis : in order to confirm the identity of 
 h. pylori , h. felis and h. bizzozeronii 
 specific pcr assay products with their target genes , after the pcr products of the specific 
 size were extracted by commercial gel extraction kit ( megaquick - spin , intron , seoul , korea ) , 
 direct sequencing of the pcr products with specific primer was conducted by abi prism 3730 
 xl dna analyzer ( pe applied biosystems , foster city , ca , u.s.a . ) .
the result of sequencing 
 was compared to those present in databases using blast software .
statistical analysis : pearson s chi - square test and fisher s exact test 
 were used to find the dependence between 2 categories .
to check for the independence of 2 
 categories , including positive / negative pcr detection , the results in human subjects / animal 
 subjects were tested by chi - square analysis using contingency tables . after counting the 
 positive results for helicobacter spp . , h. pylori , h. 
 felis and h. bizzozeronii as the variables , the possibility of 
 risk of transmission between animal and human subjects was assessed using pearson s 
 chi - square test with yates continuity correction or 2-tailed fisher s exact test for count 
 data to rule out a variety of distribution assumptions . for this reason , pearson s 

chi - square test is vague when cell counts are less than 5 . from the contingency tables , odd 
 ratios for each helicobacter spp
statistical analyses were conducted using the conventional 
 statistical software r ( version 2.15.2 ) , and the significance was set at 
 p<0.05 .
risk factor analyses by genus / species - specific pcr : genus / species - specific 
 single pcr was performed using inner primer for 16s rrna gene in 
 helicobacter spp .
h. pylori , h. felis and h. 
 bizzozeronii were detected by nested pcr assay ( supplemental fig .
2 ) . direct sequencing was performed on 2 of each 
 helicobacter species - specific pcr product in both human and animal , and these pcr products 
 were randomly selected . when the result of sequencing was compared to those present in 
 databases using blast software , above 99%
was represented homology to their specific 
 helicobacter species . for the risk factor analyses , 124 eligible human subjects and 39 dogs participated in this 
 study . among the human subjects , 41 dog owners and 43 veterinarians including 12 
 veterinarians who were themselves dog owners
infection diagnosed 
 by genus - specific pcr showed a statistically significant result ( p<0.01 , 
 table 1table 1.transmitted infection possibility of helicobacter spp .
, 
 helicobacter pylori , helicobacter felis and 
 helicobacter bizzozeronii in dog - contact risk factorhelicobacter spp .
helicobacter pylori helicobacter felis helicobacter bizzozeronii 
 species subtotaltotalnegativepositivenegativepositivenegativepositivenegativepositivedog - contactnegative1921346391241640160positive186631536618513384336a ) pearson s  test with yates continuity correction : 
 =7.59 , p < 0.01 , fisher s exact test for count data : 
 odds ratio=3.28 , p < 0.01 .
b ) pearson s  test with 
 yates continuity correction : =23.23 , p = not significant , 
 fisher s exact test for count data : odds ratio=9.50 , p = not 
 significant .
c ) pearson s  test with yates continuity correction : 
 =6.09 , p < 0.05 , fisher s exact test for count data : 
 odds ratio=10.50 , p < 0.01 .
d ) pearson s  test with 
 yates continuity correction : =0 , p = not significant , 
 fisher s exact test for count data : odds ratio=0.97 , p = not 
 significant . ) , but in the relativity analyses between groups of humans with frequent 
 contact with dogs and h. pylori , h. felis and h. 
 bizzozeronii infections diagnosed using species - specific pcr , only h. 
 felis showed a statistically significant result ( table 1 ) .
although h. pylori infection showed a 
 statistically significant relativity , no statistically significant association was found 
 between the veterinarian subjects and helicobacter .
h. 
 felis and h. bizzozeronii infections ( table 2table 2.transmitted infection possibility of helicobacter spp .
, 
 helicobacter pylori , helicobacter felis and 
 helicobacter bizzozeronii in veterinarian risk factorhelicobacter spp.helicobacter pylorihelicobacter felishelicobacter bizzozeronii 
 d)species subtotaltotalnegativepositivenegativepositivenegativepositivenegativepositiveveterinariannegative103120213110281341164positive8351132358232043172a ) pearson s  test with yates continuity correction : 
 =0.14 , p = not significant , fisher s exact test for count 
 data : odds ratio=1.40 , p = not significant.b ) pearson s  test with yates continuity correction : 
 =3.59 , p<0.05 , fisher s exact test for count data : 
 odds ratio=2.73 , p<0.05 .
c ) pearson s  test with 
 yates continuity correction : =0.14 , p = not significant , 
 fisher s exact test for count data : odds ratio=0.71 , p = not 
 significant .
d ) pearson s  test with yates continuity correction : 
 =1.35 , p = not significant , fisher s exact test for 
 count data : odds ratio=1.85 , p = not significant . ) . on performing risk factor analyses of hhlo-2 infection by transmission , 
 using matching species , between hhlo-2-positive dog owners and hhlo-2-positive dogs
, 
 h. felis infection showed an extremely significant relativity 
 ( p<0.0001 , table 
 3table 3.risk factor analyses of helicobacter felis and 
 helicobacter bizzozeronii by matching species between dog - owner and 
 doghelicobacter felis 
 a)dogshelicobacter 
 bizzozeroniidogsnegativepositivenegativepositivedog - ownersnegative230dog - ownersnegative181positive79positive713a ) pearson s  test with yates continuity correction : 
 =13.79 , p<0.0001 , fisher s exact test for count data : 
 odds ratio = inf*. * infinite value describes the faulty cell data by excel program 
 ( 0/0 ) .
b ) pearson s  test with yates continuity correction : 
 =12.62 , p<0.001 , fisher s exact test for count 
 data : odds ratio=30.04 , p < 0.001 . ) , and h. bizzozeronii may also be a possible significant risk 
 factor ( p<0.01 , table 
 3 ) .
a ) pearson s  test with yates continuity correction : 
 =7.59 , p < 0.01 , fisher s exact test for count data : 
 odds ratio=3.28 , p < 0.01 .
b ) pearson s  test with 
 yates continuity correction : =23.23 , p = not significant , 
 fisher s exact test for count data : odds ratio=9.50 , p = not 
 significant .
c ) pearson s  test with yates continuity correction : 
 =6.09 , p < 0.05 , fisher s exact test for count data : 
 odds ratio=10.50 , p < 0.01 .
d ) pearson s  test with 
 yates continuity correction : =0 , p = not significant , 
 fisher s exact test for count data : odds ratio=0.97 , p = not 
 significant .
a ) pearson s  test with yates continuity correction : 
 =0.14 , p = not significant , fisher s exact test for count 
 data : odds ratio=1.40 , p = not significant .
b ) pearson s  test with yates continuity correction : 
 =3.59 , p<0.05 , fisher s exact test for count data : 
 odds ratio=2.73 , p<0.05 .
c ) pearson s  test with 
 yates continuity correction : =0.14 , p = not significant , 
 fisher s exact test for count data : odds ratio=0.71 , p = not 
 significant .
d ) pearson s  test with yates continuity correction : 
 =1.35 , p = not significant , fisher s exact test for 
 count data : odds ratio=1.85 , p = not significant .
a ) pearson s  test with yates continuity correction : 
 =13.79 , p<0.0001 , fisher s exact test for count data : 
 odds ratio = inf*. * infinite value describes the faulty cell data by excel program 
 ( 0/0 ) .
b ) pearson s  test with yates continuity correction : 
 =12.62 , p<0.001 , fisher s exact test for count 
 data : odds ratio=30.04 , p < 0.001 .
the present study investigated the possible role of frequent contact with dogs or dog 
 ownership in the transmission of helicobacter spp .
infection in a 
 representative population sample comprising of dogs , dog owners , non - dog owners and 
 veterinarians in korea .
we specifically tested the hypothesis that hhlo-2 may be a very 
 important agent causing cross infection , because it has attracted attention as a zoonotic 
 agent in recent studies [ 11 , 17 ] .
dog owners and veterinarians were included as the subjects , because 
 of their frequent contact with dogs .
overall , we found strong evidence for an increased risk 
 of hhlo-2 infections , such as h. felis and h. bizzozeronii 
 infections , associated with the presence of dogs in the household ( dog owners group ) , and 
 co - infection rates were relatively high in dog - contact group ( 42.85% ) than no dog - contact 
 group ( 7.50% ) .
in addition , h. felis and h. bizzozeronii 
 co - infection rate was most frequent in both human and animal subject ( 42.85% in human 
 subject and 55.12% in animal subject ) . in the previous study about h. 
 pylori , co - infection rate of h. pylori l - form and vegetable - form 
 in human subject
results indicated that in the group that was in frequent contact with dogs inside or outside 
 the house ( group in frequent contact with dogs ) , genus helicobacter spp . 

( p<0.01 ) and h. felis(p<0.05 ) 
 infection could be considered as a zoonotic infection . however , frequent contact with dogs 
 did not have relativity as a risk factor in h. pylori and h. 
 bizzozeronii infections , and this result supported the result of the previous 
 studies .
interestingly , the veterinary group in 
 korea showed a high prevalence of h. pylori infection with statistical 
 significance as shown in table 2 .
acquisition of hhlo-2 , including h. felis or h. 
 bizzozeronii , may occur through animal contact , contamination of the household 
 environment and also provably through direct or indirect contact inside the house . among 
 these possible transmission routes , possibility of transmission via animal contact through 
 the oral - oral or the fecal - oral route may be highest in dog owners as compared with non - dog 
 owners .
a statistically 
 strong positive relation between h. felis infection and contact with dogs 
 was identified in this study .
therefore , the potential for zoonosis might be considered in 
 h. felis infection , both from dogs to humans and from humans to dogs , 
 indicating that dogs might be a source for this infection ; and this result is in contrast 
 with the result of previous studies that have considered a low possibility of zoonosis in 
 h. pylori infection in germany . 

further research should integrate the approaches to the environmental or cultural factors , 
 including diet and other life - style variables with clear roles of individual factors .
several reports described the isolation of h. pylori from cats , and a 
 possible non - primate reservoir for h. pylori was also identified [ 13 , 17 , 18 ] .
h. pylori has been discovered in 
 the stomach of dogs , and h. pylori was found in 3 dogs with no clinical 
 signs according to our pcr results ( data not shown ) .
this result may suggest that dogs could 
 be the natural host for h. pylori , or it may be transmitted from other 
 sources especially humans .
furthermore , on comparing the type of identified hhlo-2 in owners 
 and their dogs , the dog owners group showed the presence of organisms identical to those in 
 their dogs . it might be speculated that h. felis and h. 
 bizzozeronii could be transmitted both from dogs to humans and from humans to 
 dogs in a strong statistically significant manner .
although the other sources of infection , 
 such as contaminated water or food , were considered , the possibility of these sources of 
 infection is comparatively low in h. pylori infection .
these data support 
 that dog ownership was linked to a high socioeconomic status in the risk of transmitted 
 hhlo-2 infection .
treatment of helicobacter infection consists of combination therapy with 
 antimicrobial and anti - secretory drugs .
it is already well - known that 
 helicobacter infection in dogs and cats can be eradicated with treatment , 
 and it is also known which drugs are the best for use in the veterinary medical field .
a strong incidence of hhlo-2 infection has 
 been reported in dog owners , suggesting that transmission from animals to humans or from 
 humans to animals is strongly possible . in this report
, we have statistically significant 
 supporting results in dog owners and veterinarians of hhlo-2 infection , which should be 
 included as the subjects , because of their frequent contact with dogs .
overall , we found 
 strong evidence for an increased risk of hhlo-2 infections , such as h. 
 felis and h. bizzozeronii infections from this research , 
 although infection - positive subjects showed no clinical signs ( subclinical infection ) 
 mostly . only few human subjects reported extremely sporadic vomit symptoms in the 
 pre - investigation for sampling procedures . in conclusion
, we might suggest that hhlo-2 ( helicobacter heilmannii - like 
 organisms type 2 ) infection might be zoonotic , because continuous contact with dogs was 
 proved to be correlated with human h. felis and h. 
 bizzozeronii infections in this study .
however , this report never intended to 
 criticize pet ownership , but to carry out more intensive prevention , treatment and 
 socio - epidemologic research of helicobacter felis and h. 
 bizzozeronii infections , which should be considered in both the medical and 
 veterinary fields .
 abstracthelicobacter spp . may have multiple routes of transmission . 
 it 
 is unclear , however , whether the agent is zoonotic and therefore transmitted from an 
 animal reservoir , including dogs . 
 the aim of this population - based study was to assess the 
 relationship between pet ownership or frequent exposure to dogs and 
 helicobacter spp . 
 infection , especially focusing on hhlo-2 
 ( helicobacter heilmannii - like organisms type 2 ) in saliva and feces 
 samples in korea , using non - invasive genus / species - specific pcr . 
 one hundred twenty - four 
 eligible human subjects and 39 dogs participated in this study . 
 relativity of contact with 
 dogs and helicobacter spp . 
 infection diagnosed by genus - specific pcr 
 showed a statistically significant result ( p<0.01 ) , but in the 
 relativity analyses between contact with dogs and h. pylori , h. felis and 
 h. bizzozeronii infections diagnosed using species - specific pcr , only 
 helicobacter felis showed a statistically significant result . although 
 h. pylori infection showed a statistically significant relativity , no 
 statistically significant association was found between veterinarian subjects and 
 helicobacter . 
 spp . , 
 h. felis and h. 
 bizzozeronii infections . on performing risk factor analyses of hhlo-2 infection 
 by transmission , using matching species , between hhlo-2-positive dog owners and 
 hhlo-2-positive dogs , helicobacter felis infection showed an extremely 
 significant relativity ( p<0.0001 ) , and helicobacter 
 bizzozeronii may also be a possible significant risk factor 
 ( p<0.01 ) . 
 these results suggest that hhlo-2 infection might be a 
 zoonotic infection , because continuous contact with dogs was proved to be correlated with 
 human h. felis and h. bizzozeronii infections in this 
 study . 
MATERIALS AND METHODS
RESULTS
DISCUSSION
Supplementary 1
Supplementary 2
Supplementary 3