they prevent the establishment of potentially harmful pathogens and assist in improving the immune system . on the other hand
, the microbiota may promote the development of allergic diseases and is a major reservoir for endogenous infections . for bacteria in the nasal habitat ,
the latter has mainly been demonstrated for the role of nasal staphylococcus aureus carriage in the development of nosocomial infections such as bacteraemia , sternal or orthopaedic infections , , , .
recently , noncultural methods have greatly increased knowledge on the communities of microorganisms colonizing different body sites .
hence , analyses of 16s rrna sequences in samples from the nares of healthy individuals demonstrated that > 80% of rrna sequences detected belonged to either actinobacteria ( e.g. cornyebacteria ) or firmicutes ( e.g. staphylococci ) .
in addition , other bacterial taxa including proteobacteria ( e.g. enterobacteriaceae or pseudomonales ) , bacteriodetes or fusobacteria were also found , , .
interestingly , various studies indicated that the composition of the microbiota of different human habitats is influenced by several demographic factors such as gender , age and ethnicity , but also by health - related factors like hospitalization , intake of antibiotics , antipneumococcal vaccination or presence of viral infections as well as by lifestyle - associated factors like frequency of hand washing or smoking , , , , . recognizing the value of culture - independent methods , these techniques have two major limitations .
firstly , they can not assess bacterial antibiotic susceptibilities , and secondly , the overload of information on predominating phyla tends to dissolve information on the presence of important human pathogens as a result of the lack of abundance of these species .
however , it is important to overcome these limitations , as recent studies have indicated changes in the occurrence of antimicrobial resistant pathogens in the healthy general population .
this was demonstrated for extended - spectrum -lactamase ( esbl)- or carbapenemase - producing enterobacteriaceae and methicillin - resistant s.  aureus ( mrsa ) , which were increasingly found as colonizers of the nares and the intestinal tract in association with travel activities , contact with or ingestion of contaminated food items or contact with livestock husbandries , , , , .
in addition , the emergence of antimicrobial resistant gram - negative bacteria among humans has mainly been documented by studies assessing these pathogens in stool samples or rectal swabs .
data on their occurrence and persistence in the nares , which might be important for the probability of transmission to the environment and to other persons , are lacking .
therefore , the objective of this prospective cohort study was to assess the occurrence of nasal colonization of important facultative pathogenic bacteria including s.  aureus , enterobacteriaceae and nonfermentative bacteria in samples from a large number of nonhospitalized volunteers .
in addition , information on associated antimicrobial resistance and lifestyle conditions was obtained and correlated with the nasal bacterial colonization pattern .
nonhospitalized adult participants were recruited in ambulatory departments of public health offices ( offering , e.g. , travel vaccination or health checks ) or were employees of these offices ; dental practices ; a family physician 's practice ; and among students at colleges or universities . at the beginning of the study , participants were informed about the study 's objectives and provided written informed consent .
ethical approval was obtained by the ethical commission of the westphalian wilhelms - university mnster ( 2006 - 268-f - s ) .
samples were taken at three different time points ( june  august 2011 , january  march 2012 , august  october 2012 ) , each separated by at least 4 months . at every time point , participants were asked to provide a nasal swab and to answer a standardized questionnaire .
answers of the participants were directly assessed in a protected data - collection application on a portable usb flash drive .
from all participants , premoistened nasal swabs ( floqswabs ; copan , murrieta , ca , usa ) were used to sample the anterior nares .
after nonselective enrichment in mueller - hinton broth ( 24 hours , 36c ) , swabs were streaked onto columbia blood agar and macconkey agar as well as esbl and s.  aureus screening agars ( all oxoid , wesel , germany ) and incubated for 24 hours at 36c .
all colonies suspicious for s.  aureus , enterobacteriaceae or clinically important nonfermenters ( i.e. acinetobacter baumannii , pseudomonas spp . ,
all isolates were tested by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) .
antimicrobial susceptibility testing was performed using vitek2 automated systems ( and for some nonfermenter species using agar disc diffusion ) with clinical breakpoints and application of expert rules as recommended by the european committee on antimicrobial susceptibility testing ( eucast ) .
all s.  aureus isolates were analysed for the presence of the nuc and meca genes . for s. 
aureus isolates that were cefoxitin resistant , mecc was tested in addition as previously described .
the questionnaire assessed the following data for each participant : year of birth , sex , zip code for place of residence , country of birth , employment in healthcare institutions , employment with contact to livestock animals , number of other persons living in the same household , living with companion animals in the same household , being a smoker , having regular ( i.e. at least weekly ) contact with medical personnel , having regular ( i.e. at least weekly ) contact with persons with employment in livestock production , and travelling abroad ( if yes , which countries ; within the past 12 months ) . in addition , participants were asked whether they had diabetes mellitus , allergies , chronic skin diseases ( e.g. psoriasis , atopic dermatitis , acne ) , chronic diseases of the liver or the airways ( e.g. asthma , chronic obstructive pulmonary disease ) , chronic renal insufficiency , chronic inflammatory diseases of the paranasal sinuses or the bowel , chronic osteomyelitis , solid tumors or haemato - oncologic diseases .
participants provided information on whether they were immunocompromised ( if yes , why ? ) , whether they had implanted devices ( e.g. heart valves , prostheses ) . moreover , information on hospitalization ( within the past 12 months ) was obtained .
participants were asked whether they received antibiotics or antimycotic agents ( if yes , oral / intravenous or topical ) , oral contraceptives or medication against clinical depression or neuroleptic agents . data on density of livestock and human population in different districts as well as geographical data were assessed from the german regional database ( https://www.regionalstatistik.de/ ; codes 116 - 33 - 4 , 173 - 21 - 4 , 449 - 01 - 4 ) .
statistical analysis was performed by spss 22 ( ibm , armonk , ny , usa ) .
chi - square or fisher 's exact tests were used to calculate differences between categorical variable ; t test was used for parametric variables .
p  < 0.05 was considered significant . for assessing independent risk factors associated with nasal colonization of different groups of bacteria , we applied multivariable logistic regression by stepwise backward selection of variables and probabilities of 0.05 for entry into and 0.01 for removal from the model ( spss 22 ) .
all variables associated with p values of 0.2 were entered into the initial regression model .
overall , 1878 persons participated in the study . among these , 1168 ( 62.2% ) participated at all three time points , 283 ( 15.1% ) at two time points and 427 ( 22.7% ) only once ( table  1 ) .
the majority of participants were female ( 58.4% ) , and the median ( mean ) age was 45 years ( 45.1 years ) ( range , 797 years ) .
1 shows that all but six of the participants lived in a total of 38 districts in the german federal states of north rhine  westphalia and lower saxony . of all 1878
participants , 1203 ( 64.0% ) were colonized with at least one of the bacterial pathogens assessed at least at one time point .
the overall prevalence of nasal s.  aureus carriage was 41.0% ( n  =  768 ) ; 33.4% of the participants were nasally colonized with enterobacteriaceae ( n  =  628 ) and 3.7% with nonfermenters ( n  =  69 ) ( fig .  2 ) . at the species level , klebsiella oxytoca ( 6.4% ) , escherichia coli ( 6.1% ) , proteus mirabilis ( 6.0% ) , citrobacter koseri ( 3.6% ) , pantoea agglomerans ( 3.1% ) , enterobacter aerogenes ( 2.6% ) , klebsiella pneumoniae ( 2.4% ) , citrobacter freundii ( 2.2% ) , enterobacter cloacae ( 1.4% ) , acinetobacter baumannii ( 1.1% ) , raoultella ornithinolytica ( 1.1% ) and serratia marcescens ( 1.0% ) were detected in more than 1% of the 1878 participants . among those participants carrying enterobacteria ,
n  =  515 were colonized with one enterobacterial species , 96 with two , 16 with three , and one with four different species , respectively . of the 69 participants colonized by nonfermenters , all carried one nonfermentative species only .
statistically significant differences in the co - colonization patterns were mainly found for s.  aureus carriers , who were less frequently colonized with various enterobacteria and nonfermenters ( fig . 
table  2 shows the persistence of nasal colonization as indicated by detection of the bacteria at the three different time points of investigation . focusing on those participants who provided three nasal swabs at time points separated by four to six months each ,
the persistence of colonization , which was firstly detected at the initial time point of the study , was significantly ( p  < 0.001 ) different for carriers of s.  aureus (
n  =  525 ; 63.8% colonized for more than four months and 39.2% for more than eight months ) , enterobacteriaceae ( n  =  440 ; 54.7% and 32.1% ) and nonfermenters ( n  =  55 ; 5.5% and 0% ) . at the three time points , we obtained a total of 1378 s.  aureus isolates ( all nuc positive ) , 1169 enterobacteriaceae isolates ( from 28 species ) and 73 nonfermenter isolates ( from nine species ) .
enterobacteriaceae mostly belonged to proteus mirabilis ( n  =  211 isolates ; 18.0% of all 1168 enterobacteriaceae isolates ) , e.  coli ( n  =  180 ; 15.4% ) , k.  oxytoca ( n  =  175 ; 15.0% ) , c.  koseri ( n  =  138 ; 11.8% ) , e.  aerogenes ( n  =  86 ; 7.4% ) , c.  freundii ( n  =  64 ; 5.5% ) , p.  agglomerans ( n  =  63 ; 5.4% ) , k.  pneumoniae ( n  =  61 ; 5.2% ) , e. cloacae , r. ornithinolytica , hafnia alvei , and s.  marcescens ( each n  =  27 ; 2.3% ) . among all nonfermenter isolates , the following species were detected : a.  baumannii ( n  =  24 isolates ; 32.9% of all nonfermenter isolates ) , stenotrophomonas maltophilia ( n  =  14 ; 19.2% ) , pseudomonas putida ( n  =  12 ; 16.4% ) , pseudomonas aeruginosa ( n  =  8 ; 11.0% ) , pseudomonas fluorescens ( n  =  6 ; 8.2% ) , pseudomonas stutzeri ( n  =  5 ; 6.8% ) , pseudomonas spp .
( n  =  2 ; 2.7% ) , pseudomonas monteilii ( n  =  1 ; 1.4% ) and achromobacter xylosoxidans ( n  =  1 ; 1.4% ) . the antibiotic susceptibility test results of every first isolate detected per participant are shown in table  3 ; data are shown for those enterobacterial and nonfermenter species , which were detected in more than ten participants , respectively . resistance against linezolid , vancomycin and tigecyclin
thirteen participants ( 0.7% ) were colonized with mrsa , as defined by cefoxitin - resistant isolates harbouring meca .
this represents a proportion of 1.7% mrsa on all 768 s.  aureus first isolates .
in addition , s.  aureus isolates from two participants ( 0.1% ) were cefoxitin resistant , but neither meca nor mecc was detectable , and hyperproduction of -lactamases was excluded . for these isolates ,
the vitek2 esbl screening test was negative for all e.  coli and klebsiella spp .
overall , participants ' first isolates of enterobacteriaceae ( all species ) were resistant to ampicillin , cefuroxime , cefotaxime , ciprofloxacin , trimethoprim  sulfamethoxazole and gentamicin in 67.0 , 11.9 , 1.6 , 0.4 , 2.5 and 2.4% , respectively .
supplementary tables  s1 , s2 and s3 show the association between the risk factors assessed and nasal colonization ( at any time point ) with s.  aureus , enterobacteriaceae and nonfermenters based on univariate analysis and logistic regression .
we found that household sizes of three ( odds ratio ( or ) 1.420 , 95% confidence interval ( ci ) 1.1091.818 ) or more than four persons ( or 1.349 , 95% ci 1.0831.682 ) , living in regions with > 100 livestock animals per km ( or 1.251 , 95% ci 1.0011.562 ) , atopic dermatitis ( or 1.706 , 95% ci 1.0252.841 ) and asthma ( or 1.501 , 95% ci 1.0152.219 ) were independently associated with s.  aureus carriage , while female gender ( or 0.752 , 95% ci 0.6210.911 ) , smoking ( or 0.726 , 95% ci 0.5880.896 ) , colonization with enterobacteriaceae ( or 0.644 , 95% ci 0.5250.789 ) and unreported household size ( or 0.259 , 95% ci 0.0750.886 ) were significantly less frequent among s.  aureus carriers . performing
a separate analysis for participants colonized with meca - positive mrsa showed that only occupational contact with livestock animals ( 6/13 vs. 55/1865 ; p  < 0.001 ) and taking antidepressant / neuroleptic drugs ( 4/13 vs. 125/1865 ; p 0.009 ) were significantly associated with mrsa in univariate analysis . a regression model initially containing the risk factors associated with p values of < 0.2 revealed that occupational livestock contact ( or 31.036 ; 95% ci 8.480113.585 ) , chronic renal insufficiency ( or 40.611 ; 95% ci 3.735441.627 ) , taking antidepressant / neuroleptic medication ( or 4.974 ;
95% ci 1.14021.701 ) and chronic obstructive pulmonary disease ( or 28.111 ; 95% ci 1.808437.103 ) were independent risk factors for mrsa .
nasal colonization with enterobacteriaceae ( supplementary table  s2 ) was associated with age of 7180 years ( or 1.505 , 95% ci 1.0021.78 ) , travel to the united states ( or 2.022 , 95% ci 1.1173.660 ) and topical antimycotic therapy ( or 2.933 , 95% ci 1.5855.428 ) .
the factors s.  aureus carriage ( or 0.671 , 95% ci 0.5470.822 ) , smoking ( or 0.596 , 95% ci 0.4750.748 ) and hemato - oncologic diseases ( or 0.099 , 95% ci 0.0130.760 ) , living in regions with low population densities of < 500 inhabitants per km ( or 0.739 , 95% ci 0.5940.919 ) and with rather low livestock densities of 5099 animals per km ( or 0.779 , 95% ci 0.6280.966 ) were significantly less frequent among participants colonized with enterobacteriaceae .
s3 ) was independently associated with living in areas with a livestock density of < 50 animals per km ( or 2.114 , 95% ci 1.2763.504 ) , travel to the united states ( or 3.756 , 95% ci 1.4189.946 ) and oral antibiotic therapy ( or 1.729 , 95% ci 1.0182.937 ) . for both enterobacteriaceae and nonfermenters , the univariate analysis of lifestyle - related risk factors
was repeated for colonization , with each of the different bacterial species included in these two groups of bacteria as the independent variable ( supplementary table  s4 ) .
in this prospective study , we assessed nasal colonization with important human pathogens among a cohort of > 1800 individuals from the general population .
because of the design of the study , and because the study was carried out in only two federal states , the participants involved are not fully representative for the german general population . however , our approach of recruiting volunteers resulted in the formation of a sufficiently heterogeneous cohort . comparing the demographic data assessed with reference data for the german population ( federal statistical office of germany ( destatis ) ; https://www.destatis.de/ ; data based on 20112012 census ; date of data retrieval , 11 september 2014 )
, we can assume that basic parameters such as gender ( female ; census / cohort : 52%/58% ) , mean age ( census / cohort : 44 years/45 years ) or migration background ( census : 20% ; german nationality 91.7%/cohort : 12.6% birth country other than germany ) are comparable . also , working in the healthcare sector ( 10.9% of all participants ) , which could significantly influence colonization , was representative for the german general population , where  according to destatis data  about 12% of the population were employed in the healthcare sector in 2011 .
surprisingly , we found that 33.4 and 3.7% of all participants were colonized with enterobacteriaceae and nonfermenters , respectively .
data on studies investigating nasal carriage with gram - negative rods are rare because most reports focus on colonization of the intestinal tract . in switzerland ,
gram - negative rods ( klebsiella , e. coli , e. cloacae , citrobacter diversus ) were isolated from nasal samples obtained from 117 ( 38% ) of 534 healthy men . in a cohort of german and eritrean children ,
, more than half of the number of the individuals harboured isolates belonging to the enterobacteriaceae family . for k.  pneumoniae
, some studies even showed that colonization rates in the nares or in the respiratory tract were higher than in stool samples .
hence , our data support that a major part of the general population is nasally colonized with enterobacteriaceae .
antimicrobial susceptibility testing revealed that among nasal e.  coli , k. pneumoniae and k.  oxytoca isolates there was hardly any resistance against second- ( 9398% susceptible ) or third - generation cephalosporins ( 99100% susceptible ) .
this finding is interesting , as clinical isolates of these species are resistant in higher proportions according to german national surveillance data , where cefuroxime / cefotaxime were susceptible in 88.3/92.4% of e. coli , 81.3/90.8% of k.  pneumoniae and 87.5/95.3% of k.  oxytoca isolates ( https://ars.rki.de/ ; reference data for ambulatory care , 2013 ) .
moreover , large studies recently indicated the emergence of resistance against third - generation cephalosporins ( associated with esbl production ) in e.  coli and klebsiella isolates obtained from persons in the general population .
in germany , testing of stool samples from healthy individuals and outpatients revealed esbl colonization rates of 4.16.3% , .
several investigations demonstrated that rectal colonization with esbl - producing enterobacteriaceae was associated with travel . in the netherlands ,
the prevalence of esbl genes detected in stool samples of travellers increased from 0 before to 33.6% after travel ( mainly to southeast asia ) . in a scandinavian study ,
risk factors for esbl carriage were travel to the indian subcontinent ( or 24.8 ) , asia ( or 8.63 ) and northern africa ( or 4.94 ) .
coli identified in samples obtained < 72 hours after admission to a german hospital , asian mother tongue ( or 13.4 ) was a significant risk factor . as we detected no esbl production in any of the e.  coli , k. pneumoniae and k.  oxytoca isolates tested in this study , we assume a divergent antimicrobial resistance pattern between enterobacteriaceae from the nasal and the intestinal habitats with respect to esbl - producing organisms .
the reasons for this are unclear , and it is a limitation of this study that it was not possible to analyse faecal and nasal samples in parallel , which would have enabled direct comparability of these two reservoirs .
hypothetically , the density and variability of enterobacterial species in the intestinal tract could facilitate exchange of resistance determinants .
alternatively , transmission of esbl - producing bacteria could predominantly follow orofaecal transmission pathways , leading to colonization of the intestinal tract rather than droplet or direct smear infection or cross - transmission via the hands enabling nasal colonization .
nevertheless , our findings may have an impact on defining active surveillance strategies for prevention of infections with multiresistant enterobacteriaceae .
there are only few truly population - based studies on the prevalence of colonization with s. aureus . in the united kingdom ,
a review of longitudinal studies found that about 20% ( range 1230% ) of individuals persistently carry s. aureus , while about 30% are intermittently colonized ( range 1670% ) , and about 50% ( range 1669% ) are noncarriers .
interestingly , we found significantly lower rates of persistent nasal carriage for enterobacteriaceae and for nonfermenters , although 54.5% of participants colonized with enterobacteriaceae were carriers for more than 4 and 32.1% for more than 8 months .
however , for drawing more exact conclusions on long - term persistence , genotyping of all isolates would be necessary in order to differentiate between repetitive short - term contamination ( with several bacterial clonal lineages ) and long - term colonization ( with one clonal lineage ) .
thirteen participants ( 0.7% ) were colonized with meca - positive mrsa . unlike the united states , where the prevalence of mrsa carriage in the population was estimated to be 1.5% in 2004 , mrsa colonization in the german general population has rarely been assessed systematically . various studies
interestingly , we detected two participants who carried cefoxitin - resistant , nuc - positive s. aureus , which did not harbour the resistance determinants meca or mecc 
 .
analysis of risk factors confirmed other studies indicating that s.  aureus colonization is more frequent among male subjects , , , and some investigators hypothesized that taking hormonal contraceptives could have a preventive effect . although the latter risk factor was not significantly associated with s.  aureus in our cohort , factors such as larger household size , atopic dermatitis and asthma were more frequent among s.  aureus carriers compared to other participants , which is in line with previous investigations , , , .
we found that smokers were less frequently carriers of s. aureus , putatively due to the bactericidal activity of the smoke , which confirms other studies , .
in addition , we showed that nasal colonization with enterobacteriaceae also prevented ( or 0.64 ) s.  aureus carriage , which was previously described for the competitive effects of corynebacteria and coagulase - negative staphylococci .
the geographic area of this study ( as defined by the participants ' places of residences ) comprises both rural and urban parts of the german federal states of north rhine  westphalia and lower saxony ( fig .  1 ) .
the northwestern areas are represented by a low density of inhabitants and a high density of livestock ( especially pigs ) .
persons with occupational livestock contact had a 31 times higher risk for mrsa carriage compared to the rest of the cohort .
it has been shown that livestock - associated mrsa plays a major role among persons in this part of germany , where up to 29% of human mrsa carriers in hospitals were colonized with mrsa molecular types belonging to the clonal lineage cc398 as defined by multilocus sequence typing , , , which is predominant in european livestock populations . while the risk of acquiring mrsa cc398 via direct livestock contact has been demonstrated in various studies ( reviewed in ) , recent reports from the netherlands indicated that mrsa cc398 is increasingly acquired independently from livestock exposure by persons in the general population living in pig - dense regions . in this context ,
the results of this study clearly show that this phenomenon does not affect major parts of the population , as only 0.7% of the participants were colonized with mrsa .
taking antidepressant / neuroleptic drugs was another risk factor for mrsa carriage in this study .
this raises the question of whether or to what extent pharmaceutical agents contribute to the selection pressure on antibiotic - resistant bacteria .
we are not aware of any studies assessing risk factors for nasal colonization with enterobacteriaceae or nonfermenters in the general population . in this study ,
in addition , factors such as topical antimycotic therapy , age of more than 70 years and oral antibiotic therapy , as well as certain characteristics of the place of residence had an impact of colonization with enterobacteria and nonfermenters .
in contrast , our results that s.  aureus carriage , smoking and hemato - oncologic diseases were significantly less frequent among participants colonized with enterobacteriaceae could be due to previous antibiotic consumption , the bactericidal effects of smoking and bacterial competition in the nasal habitat .
overall , this prospective cohort study provided insight into nasal colonization patterns with s.  aureus , enterobacteriaceae and nonfermenters in the german general population as well as in associated antimicrobial resistance profiles .
however , mrsa carriage was detected in only 0.7% of the participants . although colonization rates with enterobacteriaceae were unexpectedly high ( overall 33% , with k.  oxytoca , e. coli , citrobacter spp . and pantoea spp .
being predominant ) , antimicrobial resistance of enterobacteriaceae was infrequent , and colonization with esbl - producing enterobacteriaceae was not detected .
 the nares represent an important bacterial reservoir for endogenous infections . this study aimed to assess the prevalence of nasal colonization by different important pathogens , the associated antimicrobial susceptibility and risk factors . 
 we performed a prospective cohort study among 1878 nonhospitalized volunteers recruited from the general population in germany . 
 participants provided nasal swabs at three time points ( each separated by 46 months ) . 
 staphylococcus aureus , enterobacteriaceae and important nonfermenters were cultured and subjected to susceptibility testing . 
 factors potentially influencing bacterial colonization patterns were assessed . 
 the overall prevalence of s.  aureus , enterobacteriaceae and nonfermenters was 41.0 , 33.4 and 3.7% , respectively . 
 thirteen participants ( 0.7% ) were colonized with methicillin - resistant s.  aureus . 
 enterobacteriaceae were mostly ( > 99% ) susceptible against ciprofloxacin and carbapenems ( 100% ) . 
 extended - spectrum -lactamase  producing isolates were not detected among klebsiella oxytoca , klebsiella pneumoniae and escherichia coli . 
 several lifestyle- and health - related factors ( e.g. household size , travel , livestock density of the residential area or occupational livestock contact , atopic dermatitis , antidepressant or anti - infective drugs ) were associated with colonization by different microorganisms . 
 this study unexpectedly demonstrated high nasal colonization rates with enterobacteriaceae in the german general population , but rates of antibiotic resistance were low . 
 methicillin - resistant s.  aureus carriage was rare but highly associated with occupational livestock contact . 
Introduction
Methods
Results
Discussion