clostridium difficile , a gram - positive and spore - forming anaerobic bacillus , is a leading cause of healthcare - associated diarrhoea in industrialized countries . transmission occurs primarily in healthcare facilities , where exposure to antibiotics and environmental contamination by c.  difficile spores are common . antibiotic treatment , advanced age ( > 65 years ) , comorbidities and hospitalization have been identified as major risk factors for c.  difficile infection ( cdi ) . however , antimicrobial therapy is the single most important risk factor for cdi . it leads to the disruption of the normal intestinal flora , creating favourable conditions for acquisition and proliferation of c.  difficile 
 . clindamycin , amoxicillin , third - generation cephalosporins and more recently fluoroquinolones have been shown to be associated with a particularly high risk of developing cdi . the first whole genome sequence of c.  difficile was obtained in 2006 ; sequencing revealed a large circular chromosome of 4  290  252  bp likely coding for 3776 proteins and an extracircular plasmid 7881  bp in size . these studies have shown that the c.  difficile genome contains a high proportion ( over 10% ) of mobile genetic elements including bacteriophages , repeat elements and genomic islands , as well as transposable and conjugative elements . several typing methods have been used to study the epidemiology , genetic diversity and evolution of c.  difficile . in europe pcr ribotyping has been one of the most widely adopted methods , and over 600 c.  difficile ribotypes have been identified . a particular subtype , c.  difficile ribotype 027 , has been associated with severe disease and hospital outbreaks in north america and europe , , . global emergence of this moxifloxacin - resistant c.  difficile type 027 prompted establishment of national surveillance programs in several european countries , , . as a result of national surveillance , a clonal aggregation of a newly emerging , moxifloxacin - resistant c.  difficile ribotype 231 ( rt231 ) was uncovered in sweden in 2008 . this type was initially geographically limited to the stockholm area , where over 70% of all moxifloxacin - resistant c.  difficile strains were characterized as rt231 in 2008 ( 125/144 ) . despite an increasing awareness of cdi and enhanced infection control policies , rt231 has continued to spread in sweden ; by the end of the year 2015 it had been identified in 13 of 21 counties , suggesting interhospital transmission and possible local outbreaks . although no studies have shown whether moxifloxacin use implies higher risk for cdi than other commonly used antibiotics , the restriction of moxifloxacin use has been associated with a reduction in the number of cdis in northern ireland and england , . similarly , a study from vienna demonstrated that enhanced antibiotic stewardship , including restriction of moxifloxacin use , was associated with a reduction in the number of cdi in population with a high rate of multidrug - resistant c.  difficile 
 . we used whole genome sequencing ( wgs ) and phylogenetic analysis to investigate the spread of this newly emerging moxifloxacin - resistant c.  difficile rt231 in sweden between 2006 and 2015 . we also evaluated the potential association between the extent of moxifloxacin prescription rates and the epidemiology of rt231 . all 189 c.  difficile rt231 isolates identified in sweden between 2006 and 2015 were included in this study ( table  1 ) . the strains collected between 2009 and 2015 were obtained through the national surveillance program for c.  difficile ; before 2009 samples were obtained via a nationwide surveillance study . pcr ribotyping between 2006 and 2012 was performed by a gel - based method   and between 2013 and 2015 by capillary - gel electrophoresis . all moxifloxacin - sensitive strains were included , and moxifloxacin - resistant isolates were selected to cover each isolation region per year to obtain a geographically and temporally representative sample collection . isolates were grown on mueller - hinton fastidious agar , and minimum inhibitory concentration ( mic ) values for moxifloxacin , erythromycin , clindamycin , metronidazole and vancomycin were determined using etests ( biomrieux , marcy letoile , france ) as described previously . the epidemiologic cutoff breakpoints for resistance were as follows : metronidazole mic > 2  mg / l , vancomycin mic > 2  mg / l , erythromycin mic > 2  mg / l , clindamycin mic > 16  mg / l and moxifloxacin mic > 4  mg / l , according to the european committee on antimicrobial susceptibility testing ( eucast ) . isolates were recultured on blood agar plates , inoculated into sterile peptone yeast liquid medium with 0.5  mg / ml cysteine and grown overnight anaerobically at 35c . cells were pelleted , suspended into lysis buffer ( 1  mm tris , 0.1  mm edta , 0.1% triton - x and 20  mg / ml lysozyme ) with proteinase k ( 180 l of buffer and 20 l of proteinase k ( 20 l / ml ) ) and incubated for 18 hours at 56c . genomic dna was extracted using magattract dna mini m48 kit and biorobot m48 according to the manufacturer 's recommendations ( qiagen , sollentuna , sweden ) . wgs was performed using the ion torrent platform , aiming to read lengths of 200  bp and coverage of 30. the sequence data were analysed by clc assembly cell 4.4.2 software . sequence type ( st ) was determined for comparison using mapping and the basic local alignment search tool ( blast ) against the c.  difficile multilocus sequence typing ( mlst ) database ( http://pubmlst.org/cdifficile/ ) . strain mx213 was used as the reference genome for single nucleotide polymorphism ( snp ) analysis . positions with low coverage ( < 10 ) or ambiguous calls ( < 90% ) in one or more samples were filtered out . snps within 500  bp of each other were filtered out as single events from the snps in the phylogenetic analysis to remove nonrandomly distributed mutations ; these are collectively termed recombination events . the analysis was repeated excluding the four nonrelated strains ; the remaining individual snp positions were compiled into an alignment and used for phylogenetic analysis . neighbour - joining phylogenetic trees were constructed on the basis of the snp alignments using mega 6.0 and 100 bootstrap replicates . the genomic relatedness of isolates was assessed by snp analysis : up to three individual snps per genome was expected to be seen between related isolates collected up to 1 year apart , whereas strains which differed more than ten individual snps were classified as unrelated . data on moxifloxacin prescriptions were obtained from apotekens service ab ( 20032013 ) and from the swedish ehealth agency ( 20142015 ) . the data include moxifloxacin prescribed to patients as well as sales to hospitals and other healthcare providers . a total of 51 of 189 c.  difficile rt231 isolates selected for wgs were geographically and temporally representative ; they included isolates from all 13 counties where rt231 strains had been isolated over a 10-year period ( table  1 , table  2 ) . an average minimum coverage of 60.07 ( range 20.52120.13 ) reads per nucleotide and an average genome length of 4.14 ( range 3.924.30 ) mbp were obtained for all samples . no previous wgs data were available for c.  difficile rt231 , and thus the first whole genome sequence of rt231 was obtained by a de novo assembly of strain mox213 ( 3.92  mbp ) . mlst data obtained from whole genome sequence representing approximately 0.09% of the whole genome ( 3501  nt ) were almost consistent with ribotyping data ; all studied isolates belonged to st133 . one isolate ( nt949 ) , determined to be pcr rt231 by a gel - based method , belonged to st133 , but pcr ribotyping analysis by capillary - gel electrophoresis with higher resolution suggested a new , unknown ribotype . a total of 4091 nucleotide differences were identified within the 51 isolates ; 3225 nucleotide differences were classified as recombination events , whereas the remaining 866 nucleotide differences were classed as individual snps . the results are presented in a minimal spanning tree in fig .  1 ( 88.56% of the genome was used as the dynamic core genome ) . a total of 3496 nucleotide differences distinguished four single isolates ( three moxifloxacin - sensitive isolates nt049/jnkping_2009 , nt513/vstragtaland_2010 and nt1619/skne_2012 and one moxifloxacin - resistant isolate nt949/norrbotten_2011 ) from the remaining 47 isolates ( all moxifloxacin - resistant isolates ) . a total of 2681 nucleotide differences out of 3496 were filtered as 447 recombination events . within the remaining 47 isolates , five strains ( mx405/stockholm_2008 , mx381/stockholm_2008 , nt422/gotland_2009 , nt116/stockholm_2009 and nt099/stockholm_2009 ) differed by 554 nucleotides from the closest main cluster of 42 isolates . a total of 544 nucleotide differences out of 554 were filtered as three recombination events . in order to study potential transmission of moxifloxacin - resistant c.  difficile rt231 between different counties during the 10-year study period , a phylogenetic comparison of 47 strains based on the core genome snp analysis was performed ( excluding the unrelated isolates nt049 , nt513 , nt949 and nt1619 ) . a total of 93.2% of the genome was used in the analysis as the dynamic core genome . a total of 566 nucleotide differences were identified , of which 510 were classified as recombination events . seventeen isolates collected between 2007 and 2012 in the counties of stockholm , uppsala and blekinge formed the first cluster , with only 3 snps ' difference between them ( cluster 1 , fig .  2 ) . a clonal strain ( defined as 1 snp difference ) was identified in five different hospitals in the counties of stockholm and uppsala during 2008 and 2009 . the remaining stockholm isolates from 2008 and 2009 ( n  =  4 ; all from the same hospital ) formed their own bootstrap supported cluster together with a single isolate identified in gotland during the same time period ( cluster 2 , fig .  2 ) . the third cluster was composed of the oldest isolate identified in stockholm in 2006 , another stockholm isolate from 2008 and all the other isolates collected in uppsala ( n  =  7 ) , vstmanland ( n =  5 ) , sdermanland ( n  =  8) , rebro ( n  =  2 ) and dalarna ( n  =  1 ) , with a maximum difference of 3 snps between the isolates ( cluster 3 , fig .  2 ) . a second clonal strain was circulating in the counties of uppsala , sdermanland , vstmanland and rebro between 2009 and 2015 . in total , 48 of the 51 c.  difficile rt231 isolates were resistant to moxifloxacin according to eucast epidemiologic cutoff mic breakpoints . all moxifloxacin - resistant c.  difficile rt231 isolates identified between 2009 and 2015 were also resistant to clindamycin and erythromycin . all moxifloxacin - resistant strains harboured an amino acid substitution in the gyra gene ( thr82  >  ile ) ; the unrelated moxifloxacin - resistant nt949 strain had an additional amino acid substitution in the gyrb gene ( asp426  >  asn ) . we screened the isolates for the presence of the ermb gene known to confer macrolide and lincosamide resistance . interestingly all isolates carried most of the tn5398 transposon with a deletion of the ermb locus ( fig . all isolates were also negative for the other predominant erm genes found in anaerobes ( a , c , e , f and q ) . both sensitive and resistant strains carried the cme gene , which encodes an efflux pump similar to mefa , shown to confer erythromycin resistance . furthermore , the 23s rrna sequences were analysed , but no differences were found between sensitive and resistant strains . a mutation in the rpld gene ( g211a , ala71  >  thr ) , encoding the l4 protein of the 50s ribosomal subunit , was found in 25 of 47 resistant isolates , with all isolates belonging to cluster 3 . all five isolates in cluster 2 , on the other hand , carried a mutation in the rplv gene ( g313a , val105  >  ile ) encoding the l22 protein of the 50s ribosomal subunit . to evaluate a possible link between moxifloxacin consumption and the spread of rt231 , we analysed the county - specific consumption rates for moxifloxacin . the mean community prescription rate of moxifloxacin in sweden decreased from 9.2 defined daily doses ( ddd ) per 1000 inhabitants per year in 2003 to 4.46 in 2015 , whereas the mean hospital prescription rates increased from 1.17 ddd per 1000 inhabitants per year in 2003 to 2.59 in 2015 . the differences in the community moxifloxacin prescription between counties became smaller over time ( range in community 0.2423.26 ( 2003 ) and 0.5411.53 ( 2014 ) ddd per 1000 inhabitants per year ) , whereas the opposite was seen in the hospital prescription rates between counties ( range in hospital 0.133.25 ( 2003 ) and 0.189.13 ( 2014 ) ddd per 1000 inhabitants per year ) . in 2014 the highest prescription rates , both in community and hospital settings , were identified in uppsala county ( 11.53 and 9.13 ddd per 1000 inhabitants per year , respectively ) ( fig . hospital use of moxifloxacin was also high in sdermanland county in 2014 ( 9.02 ddd per 1000 inhabitants per year ) . two other counties , jnkping and kronoberg , were noted also to have had high overall prescription rates of moxifloxacin in both community and hospital settings . we investigated the emergence and epidemiology of moxifloxacin - resistant c.  difficile rt231 in sweden between 2006 and 2015 by wgs and compared the geographical spread of this moxifloxacin - resistant type to county - specific moxifloxacin prescription rates . a majority of rt231 isolates identified in sweden since 2006 have been moxifloxacin resistant ( 98% , 186/189 ) . the proportion of moxifloxacin resistance among all c.  difficile isolates has been around 15% in sweden between 2009 and 2015 ( range , 1021% ) , which is less than previously reported : 40% in europe . the amino acid substitution ( thr82  >  ile in gyra gene ) is the most frequently described mutation associated with fluoroquinolone - resistant c.  difficile , including the hypervirulent ribotype 027 ; it was also present in all moxifloxacin - resistant rt231 isolates studied . although association between multiple substitutions and high - level moxifloxacin resistance have been previously demonstrated , all resistant isolates of rt231 included in our study harboured only a single mutation and showed high - level resistance ( mic > 32 interestingly , most of the tn5398 transposon was detected , but with a deletion of the ermb gene locus , known to confer macrolide and lincosamide resistance . macrolide and lincosamide resistance is thus most likely conferred by an as - yet unknown mechanism . although isolates belonging to clusters 2 and 3 carried mutations in the l4 or l22 proteins of the 50s ribosomal subunit proteins associated with macrolide resistance , the closely related isolates in cluster 1 have l4 and l22 proteins identical to the erythromycin - sensitive strains , and we believe that an evolution of separate resistance mechanisms is unlikely . although c.  difficile rt231 was first identified in a stockholm hospital as early as in 1999 , it remained a rare type until the outbreak was identified in stockholm county in 2008 , . on the basis of the previously estimated evolutionary rate of 0.74 snp per year for c.  difficile , up to 3 snps per genome would be expected to be seen between related isolates collected up to 365 days apart . by using these previous estimates of c.  difficile evolution , we concluded that the first two rt231 strains are likely related and originated from a common ancestor . these isolates were isolated almost by a 2-year gap in stockholm ( cd901 , cluster 2 ; mx3 , cluster 1 ) and harbour 6 snp differences in between . in contrast , two genetically distinct c.  difficile genotypes were also shown to be circulating in stockholm in 2008 , as demonstrated by more than 10 snps ' difference between the isolates ( mx70 and mx381 ) . interestingly , a third genetically distinct c.  difficile genotype ( mx165 ) differing from mx70 by five nucleotides was also identified in stockholm during the same year . this shows that at least two divergent sublineages of moxifloxacin - resistant c.  difficile rt231 were already circulating in the stockholm area when the outbreak was found in 2008 , and other strains related to these outbreak strains had likely been circulating in the area years before that . the continued circulation of a clonal strain , demonstrated both in the stockholm and uppsala  rebro healthcare regions , suggests that rt231 strains can persist for a long time in healthcare settings . because the spread of clonal strains ( defined as 1 snp difference ) occurred mainly within a healthcare region , patient movement through hospitals with specialized care is presumably the main transmission route for spreading c.  difficile rt231 . it is therefore important to implement strict cleaning procedures and isolation routines for all patients infected with moxifloxacin - resistant strains to stop further transmission . spread of rt231 was noted in stockholm county in 2008 , but the emergence had likely already occurred few years before that . it is unclear why it emerged particularly in the stockholm area , where the prescription rate of moxifloxacin has not been the highest in sweden . however , it is important to note that the reduced use of moxifloxacin in stockholm county has coincided with a reduction in the spread of rt231 in the area and the increased moxifloxacin use in uppsala county with the increased spread of this moxifloxacin - resistant type . similarly , the counties of jnkping and kronoberg , which had high prescription rates of moxifloxacin between 2005 and 2010 , were affected by outbreaks of moxifloxacin - resistant c.  difficile other than rt231 : rt046 and rt027 respectively , . in the county of jnkping , as prescription rates dropped and infection control was implemented , rt046 has virtually been eliminated from the area . interestingly , in a case  control study in stockholm county , fluoroquinolones were identified as a statistically significant risk factor for acquiring rt231 cdi , but only a small proportion of individuals infected with this strain had actually received moxifloxacin ( zomer et  al . , unpublished data ) . this is keeping with the idea that general antibiotic pressure in the community plays a major role in driving the emergence of resistant strains of c.  difficile , and , as in the case of moxifloxacin - resistant rt231 , further use of any fluoroquinolone will likely lead to cdi problems in infected individuals . a qualitative goal for limiting fluoroquinolone prescribing in outpatient care in sweden was set in 2007 , but none of the counties has reached the target set ( https://www.folkhalsomyndigheten.se/pagefiles/24127/swedres-svarm-2015-15099.pdf ) . a limitation of this study is the lack of information regarding the number of cases and patient hospitalizations ( i.e. a move to another hospital ) . this study may also contain multiple isolates from the same individuals , so the real number of cases can not be determined . in addition , because routine surveillance was established in sweden in 2009 , isolates collected before 2009 were not systematically collected . as a further limitation , age - specific moxifloxacin and general fluoroquinolone consumption rates were unavailable for study . in conclusion , we used snp analysis based on wgs to investigate the emergence and spread of moxifloxacin - resistant c.  difficile rt231 in sweden over a 10-year study period . we demonstrated the continued transmission of rt231 within and between different close - proximity counties . the outbreak emerged first in the stockholm area and spread to surrounding counties in the uppsala  rebro healthcare region . our results support the need for careful monitoring of hospitalized individuals infected with moxifloxacin - resistant c.  difficile types as well as for stricter limits on fluoroquinolone prescriptions in sweden . although rt231 has only been described in sweden so far , it can potentially spread to countries where other moxifloxacin - resistant c.  difficile types have also recently emerged . the factors associated with the emergence of new ribotypes within a certain country remain largely unexplored .