BACKGROUND
haemophilus influenzae is a major cause of respiratory tract infections and invasive disease, with encapsulated strains of serotype b  being most virulent  <cit> .
nontypeable isolates  now account for the majority of cases of invasive disease in countries where hib conjugate vaccines have been introduced  <cit> .
nthi vaccines have a huge potential for further reducing the global burden of disease but are not yet available  <cit> .
beta-lactams are first-line drugs for treatment of h. influenzae infections but resistance may develop due to transferable beta-lactamases  or alterations in the transpeptidase domain of penicillin-binding protein  <dig> , encoded by the ftsi gene   <cit> .
traditionally, isolates with the latter resistance mechanism have been denoted beta-lactamase negative ampicillin resistant , whereas isolates with both mechanisms have been denoted beta-lactamase positive amoxicillin-clavulanate resistant .
pbp3-mediated resistance is defined by the presence of particular amino acid substitutions : r517h or n526k near the ktg motif in low-level resistant isolates , and the additional substitution s385t near the ssn motif in high-level resistant isolates   <cit> .
haemophilus influenzae
genotype designations
a
b
category
c
aaccording to ubukata et al.
<cit> , hasegawa et al.
<cit> , garcia-cobos et al.
<cit> , hotomi et al.
<cit>  and this study.
na, not applicable.
bessential amino acid substitutions in pbp <dig>  with the amino acid sequence of h. influenzae rd kw <dig>  as reference.
ssn, ser-ser-asn motif; ktg, lys-thr-gly motif.
crpbp <dig>  isolates with pbp <dig> sequences conferring resistance to beta-lactams ; spbp <dig>  isolates with pbp <dig> sequences conferring wild-type susceptibility to beta-lactams .
doriginally reserved for isolates with the additional substitutions m377i and l389f by ubukata et al.
<cit> , modification proposed by hotomi et al.
<cit> .
eoriginally categorized as group i by ubukata et al.
<cit> , new group assignment proposed by garcia-cobos et al.
<cit> .
an increased prevalence of pbp3-mediated resistance  has been observed worldwide  <cit> .
isolates with high-level resistance  are a major clinical problem in japan and south korea  <cit>  whereas low-level resistant  isolates so far predominate in the rest of the world  <cit> .
group ii isolates with a characteristic substitution pattern, pbp <dig> type a   <cit> , and compatible patterns  are particularly common  <cit> , <cit> .
the mechanisms by which rpbp <dig> isolates emerge are not fully understood.
spontaneous mutations are considered the primary cause of the substitutions r517h, n526k and s385t  <cit>  but horizontal gene transfer  by classical transformation and homologous recombination has been suggested to play an important role in the further development and spread of resistance  <cit> .
clonal spread of rpbp3-nthi is extensively documented  <cit> .
however, knowledge about the molecular epidemiology of rpbp <dig> strains is limited.
previous studies based on pulsed-field gel electrophoresis  and other molecular methods have generated results not easily compared between studies.
multilocus sequence typing  has the advantage of providing objective, unambiguous data, easy to compare and well suited for assessment of phylogenetic relationship in both encapsulated isolates and nthi  <cit> .
the mlst scheme for h. influenzae assigns isolates to sequence types  based on allelic profiles of the seven housekeeping genes adk, atpg, frdb, fuck, mdh, pgi and reca <cit> .
software for phylogenetic analysis and a continuously updated database with sts, serotypes and clinical data  is available on the website http://haemophilus.mlst.net.
mlst has improved our understanding of population structure in h. influenzae <cit> .
a maximum-parsimony analysis of concatenated sequences from all isolates in the database has identified  <dig> phylogenetic groups  with different genetic characteristics, including serotypes and virulence determinants  <cit> .
the objectives of this study were to: 1) estimate the prevalence of rpbp <dig> in eye, ear and respiratory isolates of h. influenzae in norway and map pbp <dig> genotypes and phenotypic beta-lactam susceptibility profiles; 2) examine the molecular epidemiology of rpbp <dig> isolates and seek for evidence of hgt; and 3) explore any associations between phylogeny, resistance genotypes and pathogenicity, as reflected by clinical characteristics .
methods
bacterial isolates
one hundred and seventy-seven h. influenzae isolates with a phenotype suggesting rpbp <dig>  and  <dig> isolates with wild-type susceptibility to beta-lactams  were characterized.
the isolates were selected from a population of  <dig> consecutive eye, ear and respiratory tract isolates, collected as part of standard patient care in january and february  <dig>  included in the norwegian surveillance programme for antimicrobial resistance   <cit> .
selection was based on the susceptibility profiles reported by the primary laboratories .
thirteen isolates were selected but excluded for various reasons.
clinical information  for the  <dig> study isolates and  <dig> isolates in the original population was used for statistical analyses.
pfge band patterns and ftsi sequences for 46 h. influenzae isolates from a comparable population collected in  <dig>  characterized in a previous study  <cit> , were included in the phylogenetic analyses.
species identification and serotyping
isolates were inoculated on chocolate agar and incubated overnight at 35 ± 1°c in ambient air with 5% co <dig>  after control of purity and presumptive identification by smell, colony morphology and dependence of β-nad and haemin, isolates were frozen at −70°c using microbank vials .
species identification was confirmed by outer membrane protein p <dig>  and 16s rrna pcr using primers as described previously  <cit>  and probes designed for this study .
where this test was negative , a 547 bp fragment of the 16s rrna gene was sequenced at gatc biotech  to confirm species identification.
a
asites of modifications in bold.
capsular serotyping was done by bexa pcr and capsule type-specific pcrs for bexa positive isolates as described previously  <cit> , with modifications to the hi- <dig>  hi- <dig> and f <dig> primers.
a new serotype e-specific reverse primer and a bexa probe were designed for this study .
susceptibility testing
mic determination by microbroth dilution  was carried out according to clsi guidelines  <cit> , except that testing of penicillin-beta-lactamase inhibitor combinations was performed with fixed inhibitor concentrations  <cit> .
beta-lactam agents tested were ampicillin, amoxicillin, piperacillin, cefuroxime, cefotaxime  and meropenem .
for beta-lactamase positive isolates, ampicillin, amoxicillin and piperacillin mics were determined in the presence of sulbactam 4 mg/l , clavulanate 2 mg/l and tazobactam 4 mg/l , respectively.
mics were within accepted ranges for h. influenzae atcc  <dig>  and h. influenzae atcc  <dig> , and within the wild type range  for h. influenzae atcc  <dig> .
mics were interpreted according to eucast clinical breakpoints, except for piperacillin and piperacillin-tazobactam where breakpoints are not defined  <cit> .
meningitis breakpoints were used for susceptibility categorization of meropenem to allow quantification of low-level resistance.
data from this study are included in the eucast database for mic distributions of clinical isolates.
resistance genotyping
pcr and sequencing of the transpeptidase domain of the ftsi gene were performed as described previously  <cit> .
dna sequences were analysed using lasergene software  and the sequences  have been deposited in the embl nucleotide sequence database .
an upgma  phylogram of ftsi alleles from this and a previous study  <cit>  was constructed by distance methods using clustalw <dig>  and displayed using treedyn software  with h. parainfluenzae  as outgroup .
clusters of closely related alleles were assigned greek letters  with numbers denominating alleles within each cluster.
according to pbp <dig> substitution patterns , isolates were categorized into resistance genotypes .
group ii rpbp <dig> isolates and isolates lacking essential substitutions  were assigned to pbp <dig> types  according to the previously established system  <cit> , further developed in this study.
resistance genotypes
a
b
n
c
d
e
f
asee table  <dig>
bpbp <dig> types according to skaare et al.
<cit>   and this study .
‘-‘, not designated.
cn, no.
of study isolates.
dsg, no.
of isolates from the susceptible group included in n.
ebla, no.
of beta-lactamase positive isolates  included in n.
famino acid substitutions in pbp <dig>  with the amino acid sequence of h. influenzae rd kw <dig>  as reference .
essential substitutions in bold.
gn526k encoded by the dna triplet aag.
hn526k encoded by the dna triplet aaa.
inovel substitution.
the dna and pbp <dig> sequences of h. influenzae rd kw <dig>  were used as references .
isolates reported as beta-lactamase positive by the primary laboratory and isolates with a phenotype suggesting beta-lactamase production were examined by tem- <dig> and rob- <dig> pcr as described previously  <cit> , with detection of pcr products by probes designed for this study .
molecular strain characterization
mlst was performed by standard procedures with sequencing of internal fragments of the seven housekeeping genes adk, atpg, frdb, fuck, mdh, pgi and reca <cit> .
following registration of sequences at http://haemophilus.mlst.net for assignment of allele numbers and sts, data were analysed using software available on the website, with the construction of an upgma dendrogram based on the pairwise differences in allelic profiles , and division of sts into clonal complexes  using eburstv <dig>  the criterion for assignment to a cc  was sequence identity with another member of the complex at at least six loci  <cit> .
sts were assigned to phylogenetic groups  according to previously performed maximum parsimony analysis of all sts in the mlst database  <cit> .
more recent sts, not encompassed by the parsimony analysis, were indirectly assigned to phylogroups if they belonged to ccs encompassing sts with known phylogroup.
pfge of the  <dig> isolates in the r-group was carried out as described previously  <cit> .
a dendrogram of band patterns, with  <dig> isolates from our previous study included  <cit> , was constructed using gelcompare ii software , dice coefficients of similarity and the upgma algorithm .
clusters of related or possibly related isolates were identified by comparison of band patterns  <cit>  and numbered according to the system established previously  <cit> .
statistics
multivariate regression analysis and fisher’s exact test was performed using predictive analytics software  statistics version  <dig>  .
ethics
the bacterial isolates and patient information used in this study were collected as part of the norwegian surveillance programme for antimicrobial resistance .
the norm programme is warranted in norwegian law  and no further ethical approval was required for the use of isolates and data in this study.
RESULTS
resistance genotypes
in the r-group ,  <dig> isolates  had essential pbp <dig> substitutions and were categorized as rpbp <dig>  the remaining  <dig> isolates in the r-group, and all  <dig> isolates in the s-group, lacked essential substitutions and were categorized as spbp <dig> .
a
c
bla
d
groups of isolates
b
n
n
n
e
anorm  <dig> surveillance population  <cit> , consisting of consecutive routine isolates from patients with eye, ear and respiratory tract infections.
bsee text and figure  <dig> for definition of the study groups .
cpbp3-mediated resistance .
dbeta-lactamase positive.
eproportions of patients hospitalized at the time of sampling.
fassuming that all rpbp <dig> isolates were selected for the resistant group.
gas reported by the primary laboratories.
hthirteen isolates were selected for the resistant group but excluded for various reasons .
most rpbp <dig> isolates were group ii , including seven tem- <dig> positive isolates, but one group iii and two group iii-like high-rpbp <dig> isolates were also identified .
the rpbp <dig> prevalence in the original population was thus 15%  and the prevalence of combined rpbp <dig> and tem- <dig> was  <dig> % .
eighteen pbp <dig> substitution patterns were present in rpbp <dig> isolates, with pbp <dig> types a, b and d accounting for 72%  and pbp <dig> type a alone accounting for 41% .
the n526k substitution was encoded by the dna triplets aaa and aag in 54%  and 46%  of the cases, respectively.
analysis of the  <dig> ftsi alleles in the current study identified  <dig> clusters .
pbp <dig> types a, b and d were confined to distinct clusters , all highly divergent from the reference sequence.
type a was encoded by three closely related alleles  whereas types b  and d  showed no allelic diversity.
several clusters encompassed more than one pbp <dig> type, but only type j appeared in more than one cluster .
the lambda- <dig> and zeta alleles, encoding pbp <dig> types a and b, respectively, were highly prevalent in both sampling periods.
serotypes and phylogeny
except for two serotype f  ear and respiratory tract isolates, all study isolates were nontypeable.
the  <dig> isolates represented  <dig> sts; hereunder  <dig> novel  .
eight sts had > <dig> representatives and accounted for 54%  of the isolates .
by eburst analysis, the sts were grouped into  <dig> clonal complexes  and three singletons.
a
bla
b
c
n
n
n
d
e
f
e
e
apbp3-mediated resistance .
bbeta-lactamase positive .
cproportions for each st were compared with the proportions for other sts  using fisher’s exact test.
characteristics significantly more prevalent in particular sts are indicated .
dproportions of patients hospitalized at the time of sampling.
ep <  <dig> .
fp =  <dig> .
direct assessment of phylogroup was possible for  <dig> sts  and indirect assignment was possible for  <dig> sts .
eight sts  could not be assigned to a phylogroup.
ten out of  <dig> recognized phylogroups  <cit>  were represented, and 69% of the isolates belonged to clade  <dig> , eburst group  <dig>  and clade  <dig> .
the two hif isolates  were in clade  <dig>
the s-group was more diverse than the r-group and differed phylogenetically: fifteen sts were represented among 19 s-group isolates, with only one, st <dig>  being among the eight most frequent sts overall .
two major r-group phylogroups  were absent from the s-group.
eight pfge clusters of > <dig> isolates were identified, with dice coefficients of clustering between 71% and 76% .
pfge clusters corresponded well to ccs, occasionally with a higher or lower resolution level.
additionally, two clusters  suggested genetic relationship  between isolates assigned to phylogroups  and isolates with no phylogroup assignment, probably reflecting distant phylogenetic relationship not detected by the parsimony analysis.
phylogeny and resistance genotypes
the  <dig> rpbp <dig> and  <dig> spbp <dig> isolates were distributed on  <dig> and  <dig> sts, respectively.
six of the  <dig> sts in this study  included both categories.
most rpbp <dig> isolates  belonged to five phylogroups : eburst group  <dig> ; clade  <dig> ; clade  <dig> ; clade  <dig>  or clade  <dig> .
the remaining  <dig> rpbp <dig> isolates lacked phylogroup assignment.
the two group iii-like and the single group iii high-rpbp <dig> isolates were st <dig>  and st <dig> , respectively.
no isolates in clade  <dig> , clade  <dig> , clade  <dig> , clade  <dig>  and clade  <dig>  were rpbp <dig>
the ftsi alleles lambda- <dig>  zeta and omicron, encoding the three most frequent pbp <dig> types a, b and d, respectively, were, with a few notable exceptions, carried by st <dig> , st <dig>  and st <dig>  .
in addition, pbp <dig> type a encoded by the slightly different allele lambda- <dig> was present in st <dig>  a triple locus variant of st <dig> .
these four strains  accounted for 61%  of the rpbp <dig> isolates in the current study.
two strains frequently occurring in this study  had pfge band patterns and ftsi alleles identical to strains in the two most prevalent resistant clones three years earlier    <cit> .
apart from st <dig>  pbp <dig> type a encoded by lambda- <dig> was present in the following unrelated sts: st <dig> , st <dig>  and st <dig> .
similarly, the ftsi allele gamma, encoding pbp <dig> type h, was present in st <dig>  as well as the unrelated st <dig> and st <dig> .
conversely, seven sts hosted more than one pbp <dig> type.
notably, the six st <dig> isolates carried four highly divergent rpbp <dig> types  and the reference sequence .
three st <dig> isolates were tem- <dig> positive but only one isolate had both tem- <dig> and rpbp <dig>  most isolates with both resistance mechanisms  were st <dig>
clinical characteristics
clinical information for the  <dig> study isolates and the  <dig> remaining isolates in the original population is summarized in table  <dig>  for the study isolates, median age and age range of the patients were  <dig>  yrs with a male/female ratio of  <dig> .
the corresponding numbers in the original population were  <dig>  and  <dig> .
multivariate regression analysis of isolates with known hospitalization status  showed that increasing age  and male gender  were significant independent risk factors for hospitalization.
with adjustment for age, gender and beta-lactamase production, there was a borderline significant association between rpbp <dig> and hospitalization .
similarly, multivariate analysis of isolates with known site of isolation  showed a significant association between rpbp <dig> and eye infection  but no association with other localizations.
information about sts was available for study isolates only and thus not included in the regression analysis.
the eight most prevalent sts were highly diverse with respect to resistance genotypes and clinical characteristics .
there was no correlation between rpbp <dig> proportions and hospitalization rates in the various sts.
three sts, two of which consisting entirely of rpbp <dig> isolates  were significantly associated with eye infection .
st <dig> was also significantly associated with the age group 0–3 yrs .
beta-lactam susceptibility
median mics  were generally ≥ <dig> dilution steps higher in group ii rpbp <dig> isolates than in spbp <dig> isolates .
the single group iii high-rpbp <dig> isolate had mics ≥ <dig> steps higher than mic <dig> in group ii isolates.
mic <dig> for cefotaxime differed slightly between isolates with pbp <dig> types a , b  and d .
there were otherwise no significant differences  between mic <dig> in various pbp <dig> types, nor between spbp <dig> isolates in the two study groups.
study groups
a
b
n
50
/mic
<dig>
and susceptibility categorization
c
amp
c
c
c
asee figure  <dig>
bsee table  <dig>
cmics  and susceptibility categorization  according to eucast clinical breakpoints  <cit> .
the following breakpoints were used : ampicillin , 1/1; amoxicillin , 2/2; cefuroxime , 1/2; cefotaxime ,  <dig> / <dig> ; meropenem ,  <dig> / <dig>  clinical breakpoints for piperacillin and piperacillin-tazobactam are not set by eucast.
meningitis breakpoints were used for categorization of meropenem.
dfor beta-lactamase positive isolates, ampicillin, amoxicillin and piperacillin mics were determined in combination with sulbactam , clavulanate  and tazobactam , respectively.
the majority of group ii isolates had mics above the s-breakpoints for ampicillin, amoxicillin and cefuroxime.
significant proportions were resistant to cefotaxime  and non-susceptible to meropenem , with representatives from all four major rpbp <dig> strains.
notably, 12%  of group ii isolates were categorized as susceptible to all agents, whereas 24%  of spbp <dig> isolates were non-susceptible to ≥ <dig> beta-lactam, most commonly intermediately susceptible to cefuroxime .
no association with st or phylogroup was observed.
the prevalences of clinical pbp3-mediated resistance to ampicillin and cefotaxime and non-susceptibility to meropenem in the original population  were 9%,  <dig> % and  <dig> %, respectively.
discussion
resistance epidemiology
we found a 15% prevalence of rpbp <dig> in a nationwide collection of  <dig> eye, ear and respiratory isolates of h. influenzae in norway.
the prevalence of clinical resistance to ampicillin due to rpbp <dig> was 9%, compared to  <dig> % in a similar study three years earlier  <cit> .
despite methodological differences between the two studies, we conclude with a significant increase from  <dig> to  <dig>  national phenotypic surveillance data indicate a further increase to 17% rpbp <dig> in respiratory isolates in  <dig>  <cit>  and a prevalence at 15% rpbp <dig> in invasive isolates in  <dig>   <cit> , consistent with observations in other european countries and in canada  <cit> .
as expected, group ii low-level resistant isolates predominated.
notably, group iii high-rpbp <dig> was identified for the first time in northern europe.
the genotypic distinction between low-level and high-level beta-lactam resistance is clinically relevant: as resistance to cefotaxime is mainly seen in high-rpbp <dig>  <cit> , cefotaxime is suitable for empiric treatment of severe disease only in regions where high-rpbp <dig> is rare.
however, 6% of group ii isolates in the present study were resistant to cefotaxime and 20% were non- susceptible to meropenem in case of meningitis.
these observations underline the importance of confirming susceptibility to beta-lactams in severe infections such as meningitis and septicemia.
when the prevalence of low-rpbp <dig> in japanese respiratory isolates reached 17% in the mid 1990s, group iii isolates increased from zero to 29% in six years  <cit> .
this was followed by a rapid increase in group iii isolates in meningitis  from zero to 70%  <cit> .
a recent report revealed a shift from low-level to high-level resistance in respiratory tract isolates in south korea during the last decade, with an increase in the prevalence of group iii isolates from 1% to 21% in five years  <cit> .
a similar development in other parts of the world would seriously compromise current empiric antibiotic therapy in severe infections.
so far, single group iii isolates have been reported from france  <cit>  and canada  <cit>  whereas group iii-like isolates are slightly more frequent  <cit> .
clusters of group iii and group iii-like high-level resistant isolates were recently observed in norway .
the current epidemiologic situation in europe and canada, with a gradually increase in low-rpbp <dig> and sporadic reports of high-rpbp <dig> isolates, strongly resembles the situation in japan and south korea prior to the shifts in resistance genotypes.
continuous monitoring of susceptibility to cefotaxime and meropenem is necessary to ensure safe empiric treatment.
molecular epidemiology
by comparing the study isolates with isolates from a comparable population collected in  <dig>  <cit> , we were able to study the clonal dynamics of pbp3-mediated resistance.
the increasing prevalence of rpbp <dig> in norway is due to expansion of a few clones.
four sts with characteristic ftsi alleles accounted for 61% of the rpbp <dig> isolates in the present study.
two of these strains were the main contributors to pbp3-mediated resistance in norway three years earlier  <cit> .
interestingly, the replacement of st <dig> by st <dig> as the most prevalent rpbp <dig> strain did not cause a shift in pbp <dig> type nor phylogroup, as both sts carried pbp <dig> type a and belong to eburst group  <dig>
we have previously suggested the existence of one or more widely disseminated rpbp <dig> clones  <cit> .
this is supported by later reports of pbp <dig> type a and compatible substitution patterns  being common in europe  <cit> , canada  <cit> , australia  <cit>  and south korea  <cit> , and by the present study.
pbp <dig> type a is frequently linked to st <dig> and st <dig> in the limited number of previous reports on the molecular epidemiology of rpbp <dig>  studies on invasive h. influenzae in canada in the periods 2000– <dig>  <cit>  and 2008– <dig>  <cit>  revealed an increasing prevalence of rpbp <dig> in nthi, with pbp <dig> type a being common in both sampling periods  <cit> .
st <dig> and st <dig>  respectively, were the most common sts in nthi from two different regions and sampling periods  <cit> .
pbp <dig> type a was by far the most frequent substitution pattern in st <dig> and also appeared in some st <dig> isolates .
furthermore, a study on invasive h. influenzae in sweden  <cit>  identified a cluster of seven nthi isolates of st <dig> and related sts , all carrying pbp <dig> type a and collected in the period 2008– <dig> .
finally, in two recently published spanish studies, st <dig> and/or st <dig> isolates with substitution patterns compatible with pbp <dig> type a were reported in invasive disease   <cit>  and pneumonia   <cit>  in the period 2000– <dig>
the ftsi alleles encoding type a in this and our previous study  <cit>  had high genetic similarity and alleles in separate clusters rarely encoded identical pbp <dig> types.
thus, despite the lack of cross-study comparison of ftsi dna sequences, the examples above indicate that clonal distribution is a more likely explanation for the occurrence of pbp <dig> type a and compatible patterns in separate studies from four continents  <cit> , <cit>  than independent development of this substitution pattern by convergence.
importantly, an invasive high-level resistant rpbp <dig> isolate with the same combination of mlst allelic profile  and pbp <dig> substitution pattern as the two group iii-like isolates in the present study was recently reported from spain  <cit> .
a single-locus variant  with an identical substitution pattern was also reported.
these observations are notable and support the need of global surveillance initiatives.
we here show that combining mlst and pbp <dig> typing provides a tool for cross-study identification of rpbp <dig> strains and clones.
the previously suggested system for subgrouping of group ii isolates  <cit>  does not separate pbp <dig> types  <cit>  and is unsuitable for this purpose.
preferably, mlst should be combined with ftsi dna sequencing.
the ftsi gene is nearly 200 kb from its nearest mlst neighbor  and distortion of the mlst results due to linkage is thus very unlikely.
with recent technological development reducing both costs and analysis time of whole-genome sequencing, and smaller bench-top sequencers becoming readily available, mlst-ftsi typing will probably be possible to perform for surveillance purposes in the near future.
we are aware of a number of previous studies where mlst and ftsi sequencing was performed  <cit> .
to our knowledge, four reports have linked mlst data and pbp <dig> substitution patterns: one presented the allelic profiles of  <dig> group iii respiratory isolates from japan  <cit> ; another presented the substitution pattern of a single group ii st <dig> nthi isolate causing meningitis in italy  <cit> ; and two most recent publications presented the substitution patterns and sts of  <dig> respiratory  <cit>  and  <dig> invasive isolates  <cit>  from spain.
however, the present study is to our knowledge the first to connect sts to ftsi alleles.
pfge is highly discriminative and generally considered suited for assessment of relatedness between epidemiologically connected isolates, particularly in populations with high recombination rates such as nthi  <cit> .
in this study, pfge clusters correlated well to mlst clonal complexes.
band patterns were stable over time and also traced phylogenetic relationship not detected by mlst and parsimony analysis.
combining mlst and pfge for typing of nthi may thus increase both sensitivity and resolution of clone detection.
development of resistance
as discussed above, clonal expansion is important for the spread of rpbp <dig>  however, the pbp <dig> type a-encoding, highly divergent ftsi allele lambda- <dig> was distributed among several unrelated sts.
similar observations are reported previously but only in studies using pfge for strain characterization  <cit> .
exchange of complete alleles by hgt seems the most likely explanation, and has been demonstrated in vitro  <cit> .
the mechanisms for hgt of ftsi sequences in h. influenzae are not completely resolved but involvement of classical transformation and homologous recombination has been suggested  <cit> .
transformational competence varies extensively between h. influenzae strains  <cit> .
this implies that the ability to acquire mutant ftsi alleles encoding rpbp <dig> will vary correspondingly, which may explain the differences in st and phylogroup distribution between rpbp <dig> and spbp <dig> isolates.
it has been suggested that phylogroups are maintained by restriction barriers, preventing recombination between isolates of different heritage  <cit> .
this is challenged by the distribution of lambda- <dig> to several phylogroups.
a simple explanation may be that restriction barriers prevent recombination between some phylogroups and allow recombination between others.
recent studies applying whole-genome sequencing have revealed that transformation in competent strains of h. influenzae is more extensive than previously recognized  <cit>  and that transformational exchange may cause allelic variation involving complete genes between strains of identical sts  <cit> .
however, transfer of complete ftsi alleles is probably less common than exchange of shorter sequences, causing mosaicism  <cit> .
preliminary multiple sequence alignment analysis of ftsi sequences in this study indicated intrageneic recombination .
pbp3-mediated resistance and virulence
the association between rpbp <dig> and virulence is poorly described.
one experimental study reported increased ability of a group iii nthi strain to invade bronchial epithelial cells, and the authors hypothesized that rpbp <dig> may enhance virulence by acting as an adhesion molecule  <cit> .
a more recent retrospective epidemiological study concluded with no difference in pathogenicity between rpbp <dig> and spbp <dig>  but an association between rpbp <dig> and underlying respiratory disease was observed  <cit> .
molecular strain characterization was not performed in any of the two studies.
in the present study, regression analysis  suggested that rpbp <dig> is associated with increased risk of eye infection and hospitalization.
however, st-specific analysis indicated that pathogenicity is correlated with sts rather than with resistance genotypes.
for instance, st <dig>  st <dig> and st <dig> were significantly associated with eye infections but only the two latter sts were associated with pbp3-mediated resistance.
irrespective of resistance genotypes, a number of sts in this study had clinical characteristics recognizable from previous reports: st <dig> was predominantly isolated from the respiratory tract of hospitalized elderly patients, consistent with st <dig> being adapted to infection in chronic obstructive pulmonary disease   <cit> ; and the high proportion of st <dig> in children is consistent with a previously reported association with acute otitis media   <cit> .
finally, the high hospitalization rate of patients with st14-pbp <dig> type a corresponds well with the potential of this strain to cause pneumonia  <cit>  and invasive disease  <cit> .
these observations are in accordance with a recent population study suggesting association between population structure and disease  <cit> .
in conclusion, the association between rpbp <dig> and pathogenicity suggested by the regression analysis most likely reflects that some of the most frequently occurring rpbp <dig> strains in this study also possessed strain-associated virulence properties.
identification of virulence determinants is beyond the scope of this study.
however, our observations underline that studies on the correlation between resistance genotypes and pathogenicity should include molecular strain characterization.
accordingly, the previously reported association between pbp3-mediated resistance and clinical characteristics  <cit>  may be spurious.
CONCLUSIONS
the prevalence of rpbp <dig> in h. influenzae is increasing worldwide, and high-level resistant strains are emerging in new geographic regions.
in this study of eye, ear and respiratory isolates in norway, the rpbp <dig> prevalence was 15%, with four strains accounting for 61% of the resistant isolates.
group ii low-rpbp <dig> isolates predominated, and significant proportions of isolates were non-susceptible to cefotaxime and meropenem.
group iii high-rpbp <dig> was identified for the first time in northern europe.
the results support a role of horizontal gene transfer in the emergence of rpbp <dig> and indicate phylogeny restricted transformation.
comparative analysis with data from previous studies indicates wide dissemination of clonally related rpbp <dig> strains.
notably, two strains highly prevalent in norway  are common in invasive disease in europe and canada.
continuous monitoring of beta-lactam susceptibility is necessary to ensure safe empiric therapy in severe disease and to detect a future shift from low-level to high-level resistance.
the need of a global system for molecular surveillance of rpbp <dig> strains is underlined.
the novel approach of combining mlst and ftsi/pbp <dig> typing is a powerful tool for this purpose.
abbreviations
nthi: nontypeable haemophilus influenzae; hib: haemophilus influenzae serotype b; hif: haemophilus influenzae serotype f; pbp3: penicillin-binding protein 3; blnar: beta-lactamase negative ampicillin resistant; blpacr: beta-lactamase positive amoxicillin-clavulanate resistant; rpbp3: pbp3-mediated resistance present ; spbp3: pbp3-mediated resistance absent ; mlst: multilocus sequence typing; st: sequence type; cc: clonal complex; phylogroup: phylogenetic group; pfge: pulsed-field gel electrophoresis; hgt: horizontal gene transfer; upgma: unweighted pair group method with arithmetic mean; eucast: european committee on antimicrobial susceptibility testing; mic: minimal inhibitory concentration; copd: chronic obstructive pulmonary disease; aom: acute otitis media; norm: norwegian surveillance programme for antimicrobial resistance; r-group: resistant group; s-group: susceptible group.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ds conceived and coordinated the study, performed susceptibility testing, analysed and interpreted data and wrote the first draft; bek, yt, aj, ls and as contributed to study design; ila designed and undertook molecular analyses ; ls analysed the pfge data, dac and ms were responsible for acquisition of mlst data and aj advised on bioinformatics.
all authors participated in interpretation of results, critically revised the draft for intellectual content and approved the final article.

 haemophilus influenzae is a major cause of respiratory tract infections and invasive disease, with encapsulated strains of serotype b being most virulent. nontypeable isolates now account for the majority of cases of invasive disease in countries where hib conjugate vaccines have been introduced. nthi vaccines have a huge potential for further reducing the global burden of disease but are not yet available. beta-lactams are first-line drugs for treatment of h. influenzae infections but resistance may develop due to transferable beta-lactamases or alterations in the transpeptidase domain of penicillin-binding protein, encoded by the ftsi gene. traditionally, isolates with the latter resistance mechanism have been denoted beta-lactamase negative ampicillin resistant, whereas isolates with both mechanisms have been denoted beta-lactamase positive amoxicillin-clavulanate resistant. pbp3-mediated resistance is defined by the presence of particular amino acid substitutions: r517h or n526k near the ktg motif in low-level resistant isolates, and the additional substitution s385t near the ssn motif in high-level resistant isolates. haemophilus influenzae genotype designations a b category c aaccording to ubukata et al., hasegawa et al., garcia-cobos et al., hotomi et al. and this study. na, not applicable. bessential amino acid substitutions in pbp with the amino acid sequence of h. influenzae rd kw as reference. ssn, ser-ser-asn motif; ktg, lys-thr-gly motif. crpbp isolates with pbp sequences conferring resistance to beta-lactams; spbp isolates with pbp sequences conferring wild-type susceptibility to beta-lactams. doriginally reserved for isolates with the additional substitutions m377i and l389f by ubukata et al., modification proposed by hotomi et al.. eoriginally categorized as group i by ubukata et al., new group assignment proposed by garcia-cobos et al.. an increased prevalence of pbp3-mediated resistance has been observed worldwide. isolates with high-level resistance are a major clinical problem in japan and south korea whereas low-level resistant isolates so far predominate in the rest of the world. group ii isolates with a characteristic substitution pattern, pbp type a, and compatible patterns are particularly common,. the mechanisms by which rpbp isolates emerge are not fully understood. spontaneous mutations are considered the primary cause of the substitutions r517h, n526k and s385t but horizontal gene transfer by classical transformation and homologous recombination has been suggested to play an important role in the further development and spread of resistance. clonal spread of rpbp3-nthi is extensively documented. however, knowledge about the molecular epidemiology of rpbp strains is limited. previous studies based on pulsed-field gel electrophoresis and other molecular methods have generated results not easily compared between studies. multilocus sequence typing has the advantage of providing objective, unambiguous data, easy to compare and well suited for assessment of phylogenetic relationship in both encapsulated isolates and nthi. the mlst scheme for h. influenzae assigns isolates to sequence types based on allelic profiles of the seven housekeeping genes adk, atpg, frdb, fuck, mdh, pgi and reca. software for phylogenetic analysis and a continuously updated database with sts, serotypes and clinical data is available on the website http://haemophilus.mlst.net. mlst has improved our understanding of population structure in h. influenzae. a maximum-parsimony analysis of concatenated sequences from all isolates in the database has identified phylogenetic groups with different genetic characteristics, including serotypes and virulence determinants. the objectives of this study were to: 1) estimate the prevalence of rpbp in eye, ear and respiratory isolates of h. influenzae in norway and map pbp genotypes and phenotypic beta-lactam susceptibility profiles; 2) examine the molecular epidemiology of rpbp isolates and seek for evidence of hgt; and 3) explore any associations between phylogeny, resistance genotypes and pathogenicity, as reflected by clinical characteristics. methods bacterial isolates one hundred and seventy-seven h. influenzae isolates with a phenotype suggesting rpbp and isolates with wild-type susceptibility to beta-lactams were characterized. the isolates were selected from a population of consecutive eye, ear and respiratory tract isolates, collected as part of standard patient care in january and february included in the norwegian surveillance programme for antimicrobial resistance. selection was based on the susceptibility profiles reported by the primary laboratories. thirteen isolates were selected but excluded for various reasons. clinical information for the study isolates and isolates in the original population was used for statistical analyses. pfge band patterns and ftsi sequences for 46 h. influenzae isolates from a comparable population collected in characterized in a previous study, were included in the phylogenetic analyses. species identification and serotyping isolates were inoculated on chocolate agar and incubated overnight at 35 ± 1°c in ambient air with 5% co after control of purity and presumptive identification by smell, colony morphology and dependence of β-nad and haemin, isolates were frozen at −70°c using microbank vials. species identification was confirmed by outer membrane protein p and 16s rrna pcr using primers as described previously and probes designed for this study. where this test was negative, a 547 bp fragment of the 16s rrna gene was sequenced at gatc biotech to confirm species identification. a asites of modifications in bold. capsular serotyping was done by bexa pcr and capsule type-specific pcrs for bexa positive isolates as described previously, with modifications to the hi- hi- and f primers. a new serotype e-specific reverse primer and a bexa probe were designed for this study. susceptibility testing mic determination by microbroth dilution was carried out according to clsi guidelines, except that testing of penicillin-beta-lactamase inhibitor combinations was performed with fixed inhibitor concentrations. beta-lactam agents tested were ampicillin, amoxicillin, piperacillin, cefuroxime, cefotaxime and meropenem. for beta-lactamase positive isolates, ampicillin, amoxicillin and piperacillin mics were determined in the presence of sulbactam 4 mg/l, clavulanate 2 mg/l and tazobactam 4 mg/l, respectively. mics were within accepted ranges for h. influenzae atcc and h. influenzae atcc, and within the wild type range for h. influenzae atcc. mics were interpreted according to eucast clinical breakpoints, except for piperacillin and piperacillin-tazobactam where breakpoints are not defined. meningitis breakpoints were used for susceptibility categorization of meropenem to allow quantification of low-level resistance. data from this study are included in the eucast database for mic distributions of clinical isolates. resistance genotyping pcr and sequencing of the transpeptidase domain of the ftsi gene were performed as described previously. dna sequences were analysed using lasergene software and the sequences have been deposited in the embl nucleotide sequence database. an upgma phylogram of ftsi alleles from this and a previous study was constructed by distance methods using clustalw and displayed using treedyn software with h. parainfluenzae as outgroup. clusters of closely related alleles were assigned greek letters with numbers denominating alleles within each cluster. according to pbp substitution patterns, isolates were categorized into resistance genotypes. group ii rpbp isolates and isolates lacking essential substitutions were assigned to pbp types according to the previously established system, further developed in this study. resistance genotypes a b n c d e f asee table  bpbp types according to skaare et al. and this study. ‘-‘, not designated. cn, no. of study isolates. dsg, no. of isolates from the susceptible group included in n. ebla, no. of beta-lactamase positive isolates included in n. famino acid substitutions in pbp with the amino acid sequence of h. influenzae rd kw as reference. essential substitutions in bold. gn526k encoded by the dna triplet aag. hn526k encoded by the dna triplet aaa. inovel substitution. the dna and pbp sequences of h. influenzae rd kw were used as references. isolates reported as beta-lactamase positive by the primary laboratory and isolates with a phenotype suggesting beta-lactamase production were examined by tem- and rob- pcr as described previously, with detection of pcr products by probes designed for this study. molecular strain characterization mlst was performed by standard procedures with sequencing of internal fragments of the seven housekeeping genes adk, atpg, frdb, fuck, mdh, pgi and reca. following registration of sequences at http://haemophilus.mlst.net for assignment of allele numbers and sts, data were analysed using software available on the website, with the construction of an upgma dendrogram based on the pairwise differences in allelic profiles, and division of sts into clonal complexes using eburstv the criterion for assignment to a cc was sequence identity with another member of the complex at at least six loci. sts were assigned to phylogenetic groups according to previously performed maximum parsimony analysis of all sts in the mlst database. more recent sts, not encompassed by the parsimony analysis, were indirectly assigned to phylogroups if they belonged to ccs encompassing sts with known phylogroup. pfge of the isolates in the r-group was carried out as described previously. a dendrogram of band patterns, with isolates from our previous study included, was constructed using gelcompare ii software, dice coefficients of similarity and the upgma algorithm. clusters of related or possibly related isolates were identified by comparison of band patterns and numbered according to the system established previously. statistics multivariate regression analysis and fisher’s exact test was performed using predictive analytics software statistics version. ethics the bacterial isolates and patient information used in this study were collected as part of the norwegian surveillance programme for antimicrobial resistance. the norm programme is warranted in norwegian law and no further ethical approval was required for the use of isolates and data in this study.  resistance genotypes in the r-group, isolates had essential pbp substitutions and were categorized as rpbp the remaining isolates in the r-group, and all isolates in the s-group, lacked essential substitutions and were categorized as spbp. a c bla d groups of isolates b n n n e anorm surveillance population, consisting of consecutive routine isolates from patients with eye, ear and respiratory tract infections. bsee text and figure  for definition of the study groups. cpbp3-mediated resistance. dbeta-lactamase positive. eproportions of patients hospitalized at the time of sampling. fassuming that all rpbp isolates were selected for the resistant group. gas reported by the primary laboratories. hthirteen isolates were selected for the resistant group but excluded for various reasons. most rpbp isolates were group ii, including seven tem- positive isolates, but one group iii and two group iii-like high-rpbp isolates were also identified. the rpbp prevalence in the original population was thus 15% and the prevalence of combined rpbp and tem- was %. eighteen pbp substitution patterns were present in rpbp isolates, with pbp types a, b and d accounting for 72% and pbp type a alone accounting for 41%. the n526k substitution was encoded by the dna triplets aaa and aag in 54% and 46% of the cases, respectively. analysis of the ftsi alleles in the current study identified clusters. pbp types a, b and d were confined to distinct clusters, all highly divergent from the reference sequence. type a was encoded by three closely related alleles whereas types b and d showed no allelic diversity. several clusters encompassed more than one pbp type, but only type j appeared in more than one cluster. the lambda- and zeta alleles, encoding pbp types a and b, respectively, were highly prevalent in both sampling periods. serotypes and phylogeny except for two serotype f ear and respiratory tract isolates, all study isolates were nontypeable. the isolates represented sts; hereunder novel. eight sts had > representatives and accounted for 54% of the isolates. by eburst analysis, the sts were grouped into clonal complexes and three singletons. a bla b c n n n d e f e e apbp3-mediated resistance. bbeta-lactamase positive. cproportions for each st were compared with the proportions for other sts using fisher’s exact test. characteristics significantly more prevalent in particular sts are indicated. dproportions of patients hospitalized at the time of sampling. ep < . fp = . direct assessment of phylogroup was possible for sts and indirect assignment was possible for sts. eight sts could not be assigned to a phylogroup. ten out of recognized phylogroups were represented, and 69% of the isolates belonged to clade, eburst group and clade. the two hif isolates were in clade the s-group was more diverse than the r-group and differed phylogenetically: fifteen sts were represented among 19 s-group isolates, with only one, st being among the eight most frequent sts overall. two major r-group phylogroups were absent from the s-group. eight pfge clusters of > isolates were identified, with dice coefficients of clustering between 71% and 76%. pfge clusters corresponded well to ccs, occasionally with a higher or lower resolution level. additionally, two clusters suggested genetic relationship between isolates assigned to phylogroups and isolates with no phylogroup assignment, probably reflecting distant phylogenetic relationship not detected by the parsimony analysis. phylogeny and resistance genotypes the rpbp and spbp isolates were distributed on and sts, respectively. six of the sts in this study included both categories. most rpbp isolates belonged to five phylogroups: eburst group; clade; clade; clade or clade. the remaining rpbp isolates lacked phylogroup assignment. the two group iii-like and the single group iii high-rpbp isolates were st and st, respectively. no isolates in clade, clade, clade, clade and clade were rpbp the ftsi alleles lambda- zeta and omicron, encoding the three most frequent pbp types a, b and d, respectively, were, with a few notable exceptions, carried by st, st and st. in addition, pbp type a encoded by the slightly different allele lambda- was present in st a triple locus variant of st. these four strains accounted for 61% of the rpbp isolates in the current study. two strains frequently occurring in this study had pfge band patterns and ftsi alleles identical to strains in the two most prevalent resistant clones three years earlier. apart from st pbp type a encoded by lambda- was present in the following unrelated sts: st, st and st. similarly, the ftsi allele gamma, encoding pbp type h, was present in st as well as the unrelated st and st. conversely, seven sts hosted more than one pbp type. notably, the six st isolates carried four highly divergent rpbp types and the reference sequence. three st isolates were tem- positive but only one isolate had both tem- and rpbp most isolates with both resistance mechanisms were st clinical characteristics clinical information for the study isolates and the remaining isolates in the original population is summarized in table  for the study isolates, median age and age range of the patients were yrs with a male/female ratio of. the corresponding numbers in the original population were and. multivariate regression analysis of isolates with known hospitalization status showed that increasing age and male gender were significant independent risk factors for hospitalization. with adjustment for age, gender and beta-lactamase production, there was a borderline significant association between rpbp and hospitalization. similarly, multivariate analysis of isolates with known site of isolation showed a significant association between rpbp and eye infection but no association with other localizations. information about sts was available for study isolates only and thus not included in the regression analysis. the eight most prevalent sts were highly diverse with respect to resistance genotypes and clinical characteristics. there was no correlation between rpbp proportions and hospitalization rates in the various sts. three sts, two of which consisting entirely of rpbp isolates were significantly associated with eye infection. st was also significantly associated with the age group 0–3 yrs. beta-lactam susceptibility median mics were generally ≥ dilution steps higher in group ii rpbp isolates than in spbp isolates. the single group iii high-rpbp isolate had mics ≥ steps higher than mic in group ii isolates. mic for cefotaxime differed slightly between isolates with pbp types a, b and d. there were otherwise no significant differences between mic in various pbp types, nor between spbp isolates in the two study groups. study groups a b n 50 /mic and susceptibility categorization c amp c c c asee figure  bsee table  cmics and susceptibility categorization according to eucast clinical breakpoints. the following breakpoints were used: ampicillin, 1/1; amoxicillin, 2/2; cefuroxime, 1/2; cefotaxime, /; meropenem, / clinical breakpoints for piperacillin and piperacillin-tazobactam are not set by eucast. meningitis breakpoints were used for categorization of meropenem. dfor beta-lactamase positive isolates, ampicillin, amoxicillin and piperacillin mics were determined in combination with sulbactam, clavulanate and tazobactam, respectively. the majority of group ii isolates had mics above the s-breakpoints for ampicillin, amoxicillin and cefuroxime. significant proportions were resistant to cefotaxime and non-susceptible to meropenem, with representatives from all four major rpbp strains. notably, 12% of group ii isolates were categorized as susceptible to all agents, whereas 24% of spbp isolates were non-susceptible to ≥ beta-lactam, most commonly intermediately susceptible to cefuroxime. no association with st or phylogroup was observed. the prevalences of clinical pbp3-mediated resistance to ampicillin and cefotaxime and non-susceptibility to meropenem in the original population were 9%, % and %, respectively. discussion resistance epidemiology we found a 15% prevalence of rpbp in a nationwide collection of eye, ear and respiratory isolates of h. influenzae in norway. the prevalence of clinical resistance to ampicillin due to rpbp was 9%, compared to % in a similar study three years earlier. despite methodological differences between the two studies, we conclude with a significant increase from to national phenotypic surveillance data indicate a further increase to 17% rpbp in respiratory isolates in and a prevalence at 15% rpbp in invasive isolates in, consistent with observations in other european countries and in canada. as expected, group ii low-level resistant isolates predominated. notably, group iii high-rpbp was identified for the first time in northern europe. the genotypic distinction between low-level and high-level beta-lactam resistance is clinically relevant: as resistance to cefotaxime is mainly seen in high-rpbp, cefotaxime is suitable for empiric treatment of severe disease only in regions where high-rpbp is rare. however, 6% of group ii isolates in the present study were resistant to cefotaxime and 20% were non- susceptible to meropenem in case of meningitis. these observations underline the importance of confirming susceptibility to beta-lactams in severe infections such as meningitis and septicemia. when the prevalence of low-rpbp in japanese respiratory isolates reached 17% in the mid 1990s, group iii isolates increased from zero to 29% in six years. this was followed by a rapid increase in group iii isolates in meningitis from zero to 70%. a recent report revealed a shift from low-level to high-level resistance in respiratory tract isolates in south korea during the last decade, with an increase in the prevalence of group iii isolates from 1% to 21% in five years. a similar development in other parts of the world would seriously compromise current empiric antibiotic therapy in severe infections. so far, single group iii isolates have been reported from france and canada whereas group iii-like isolates are slightly more frequent. clusters of group iii and group iii-like high-level resistant isolates were recently observed in norway. the current epidemiologic situation in europe and canada, with a gradually increase in low-rpbp and sporadic reports of high-rpbp isolates, strongly resembles the situation in japan and south korea prior to the shifts in resistance genotypes. continuous monitoring of susceptibility to cefotaxime and meropenem is necessary to ensure safe empiric treatment. molecular epidemiology by comparing the study isolates with isolates from a comparable population collected in, we were able to study the clonal dynamics of pbp3-mediated resistance. the increasing prevalence of rpbp in norway is due to expansion of a few clones. four sts with characteristic ftsi alleles accounted for 61% of the rpbp isolates in the present study. two of these strains were the main contributors to pbp3-mediated resistance in norway three years earlier. interestingly, the replacement of st by st as the most prevalent rpbp strain did not cause a shift in pbp type nor phylogroup, as both sts carried pbp type a and belong to eburst group we have previously suggested the existence of one or more widely disseminated rpbp clones. this is supported by later reports of pbp type a and compatible substitution patterns being common in europe, canada, australia and south korea, and by the present study. pbp type a is frequently linked to st and st in the limited number of previous reports on the molecular epidemiology of rpbp studies on invasive h. influenzae in canada in the periods 2000– and 2008– revealed an increasing prevalence of rpbp in nthi, with pbp type a being common in both sampling periods. st and st respectively, were the most common sts in nthi from two different regions and sampling periods. pbp type a was by far the most frequent substitution pattern in st and also appeared in some st isolates. furthermore, a study on invasive h. influenzae in sweden identified a cluster of seven nthi isolates of st and related sts, all carrying pbp type a and collected in the period 2008–. finally, in two recently published spanish studies, st and/or st isolates with substitution patterns compatible with pbp type a were reported in invasive disease and pneumonia in the period 2000– the ftsi alleles encoding type a in this and our previous study had high genetic similarity and alleles in separate clusters rarely encoded identical pbp types. thus, despite the lack of cross-study comparison of ftsi dna sequences, the examples above indicate that clonal distribution is a more likely explanation for the occurrence of pbp type a and compatible patterns in separate studies from four continents, than independent development of this substitution pattern by convergence. importantly, an invasive high-level resistant rpbp isolate with the same combination of mlst allelic profile and pbp substitution pattern as the two group iii-like isolates in the present study was recently reported from spain. a single-locus variant with an identical substitution pattern was also reported. these observations are notable and support the need of global surveillance initiatives. we here show that combining mlst and pbp typing provides a tool for cross-study identification of rpbp strains and clones. the previously suggested system for subgrouping of group ii isolates does not separate pbp types and is unsuitable for this purpose. preferably, mlst should be combined with ftsi dna sequencing. the ftsi gene is nearly 200 kb from its nearest mlst neighbor and distortion of the mlst results due to linkage is thus very unlikely. with recent technological development reducing both costs and analysis time of whole-genome sequencing, and smaller bench-top sequencers becoming readily available, mlst-ftsi typing will probably be possible to perform for surveillance purposes in the near future. we are aware of a number of previous studies where mlst and ftsi sequencing was performed. to our knowledge, four reports have linked mlst data and pbp substitution patterns: one presented the allelic profiles of group iii respiratory isolates from japan; another presented the substitution pattern of a single group ii st nthi isolate causing meningitis in italy; and two most recent publications presented the substitution patterns and sts of respiratory and invasive isolates from spain. however, the present study is to our knowledge the first to connect sts to ftsi alleles. pfge is highly discriminative and generally considered suited for assessment of relatedness between epidemiologically connected isolates, particularly in populations with high recombination rates such as nthi. in this study, pfge clusters correlated well to mlst clonal complexes. band patterns were stable over time and also traced phylogenetic relationship not detected by mlst and parsimony analysis. combining mlst and pfge for typing of nthi may thus increase both sensitivity and resolution of clone detection. development of resistance as discussed above, clonal expansion is important for the spread of rpbp however, the pbp type a-encoding, highly divergent ftsi allele lambda- was distributed among several unrelated sts. similar observations are reported previously but only in studies using pfge for strain characterization. exchange of complete alleles by hgt seems the most likely explanation, and has been demonstrated in vitro. the mechanisms for hgt of ftsi sequences in h. influenzae are not completely resolved but involvement of classical transformation and homologous recombination has been suggested. transformational competence varies extensively between h. influenzae strains. this implies that the ability to acquire mutant ftsi alleles encoding rpbp will vary correspondingly, which may explain the differences in st and phylogroup distribution between rpbp and spbp isolates. it has been suggested that phylogroups are maintained by restriction barriers, preventing recombination between isolates of different heritage. this is challenged by the distribution of lambda- to several phylogroups. a simple explanation may be that restriction barriers prevent recombination between some phylogroups and allow recombination between others. recent studies applying whole-genome sequencing have revealed that transformation in competent strains of h. influenzae is more extensive than previously recognized and that transformational exchange may cause allelic variation involving complete genes between strains of identical sts. however, transfer of complete ftsi alleles is probably less common than exchange of shorter sequences, causing mosaicism. preliminary multiple sequence alignment analysis of ftsi sequences in this study indicated intrageneic recombination. pbp3-mediated resistance and virulence the association between rpbp and virulence is poorly described. one experimental study reported increased ability of a group iii nthi strain to invade bronchial epithelial cells, and the authors hypothesized that rpbp may enhance virulence by acting as an adhesion molecule. a more recent retrospective epidemiological study concluded with no difference in pathogenicity between rpbp and spbp but an association between rpbp and underlying respiratory disease was observed. molecular strain characterization was not performed in any of the two studies. in the present study, regression analysis suggested that rpbp is associated with increased risk of eye infection and hospitalization. however, st-specific analysis indicated that pathogenicity is correlated with sts rather than with resistance genotypes. for instance, st st and st were significantly associated with eye infections but only the two latter sts were associated with pbp3-mediated resistance. irrespective of resistance genotypes, a number of sts in this study had clinical characteristics recognizable from previous reports: st was predominantly isolated from the respiratory tract of hospitalized elderly patients, consistent with st being adapted to infection in chronic obstructive pulmonary disease; and the high proportion of st in children is consistent with a previously reported association with acute otitis media. finally, the high hospitalization rate of patients with st14-pbp type a corresponds well with the potential of this strain to cause pneumonia and invasive disease. these observations are in accordance with a recent population study suggesting association between population structure and disease. in conclusion, the association between rpbp and pathogenicity suggested by the regression analysis most likely reflects that some of the most frequently occurring rpbp strains in this study also possessed strain-associated virulence properties. identification of virulence determinants is beyond the scope of this study. however, our observations underline that studies on the correlation between resistance genotypes and pathogenicity should include molecular strain characterization. accordingly, the previously reported association between pbp3-mediated resistance and clinical characteristics may be spurious.  the prevalence of rpbp in h. influenzae is increasing worldwide, and high-level resistant strains are emerging in new geographic regions. in this study of eye, ear and respiratory isolates in norway, the rpbp prevalence was 15%, with four strains accounting for 61% of the resistant isolates. group ii low-rpbp isolates predominated, and significant proportions of isolates were non-susceptible to cefotaxime and meropenem. group iii high-rpbp was identified for the first time in northern europe. the results support a role of horizontal gene transfer in the emergence of rpbp and indicate phylogeny restricted transformation. comparative analysis with data from previous studies indicates wide dissemination of clonally related rpbp strains. notably, two strains highly prevalent in norway are common in invasive disease in europe and canada. continuous monitoring of beta-lactam susceptibility is necessary to ensure safe empiric therapy in severe disease and to detect a future shift from low-level to high-level resistance. the need of a global system for molecular surveillance of rpbp strains is underlined. the novel approach of combining mlst and ftsi/pbp typing is a powerful tool for this purpose. abbreviations nthi: nontypeable haemophilus influenzae; hib: haemophilus influenzae serotype b; hif: haemophilus influenzae serotype f; pbp3: penicillin-binding protein 3; blnar: beta-lactamase negative ampicillin resistant; blpacr: beta-lactamase positive amoxicillin-clavulanate resistant; rpbp3: pbp3-mediated resistance present; spbp3: pbp3-mediated resistance absent; mlst: multilocus sequence typing; st: sequence type; cc: clonal complex; phylogroup: phylogenetic group; pfge: pulsed-field gel electrophoresis; hgt: horizontal gene transfer; upgma: unweighted pair group method with arithmetic mean; eucast: european committee on antimicrobial susceptibility testing; mic: minimal inhibitory concentration; copd: chronic obstructive pulmonary disease; aom: acute otitis media; norm: norwegian surveillance programme for antimicrobial resistance; r-group: resistant group; s-group: susceptible group. competing interests the authors declare that they have no competing interests. authors’ contributions ds conceived and coordinated the study, performed susceptibility testing, analysed and interpreted data and wrote the first draft; bek, yt, aj, ls and as contributed to study design; ila designed and undertook molecular analyses; ls analysed the pfge data, dac and ms were responsible for acquisition of mlst data and aj advised on bioinformatics. all authors participated in interpretation of results, critically revised the draft for intellectual content and approved the final article. 
the results indicate that horizontal gene transfer contributes to the emergence of rpbp by phylogeny restricted transformation. group ii low-rpbp predominated, with significant proportions of isolates non-susceptible to cefotaxime and meropenem. we combined multilocus sequence typing and ftsi/pbp typing to study the emergence and spread of rpbp in nontypeable h. influenzae in norway. this study is the first to link ftsi alleles to sts in h. influenzae. the need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rpbp strains is underlined. four mlst sequence types with characteristic, highly diverging ftsi alleles accounted for 61% of the rpbp isolates. the prevalence of rpbp in a population of eye, ear and respiratory isolates from was 15%. infection sites, age groups, hospitalization rates and rpbp frequencies differed between sts and phylogenetic groups. beta-lactam resistance in haemophilus influenzae due to ftsi mutations causing altered penicillin-binding protein is increasing worldwide. knowledge about the molecular epidemiology of rpbp strains is limited. low-level resistant isolates with the n526k substitution predominate in most geographical regions, while high-level resistant isolates with the additional s385t substitution are common in japan and south korea. clonally related virulent rpbp strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. combining mlst and ftsi/pbp typing is a powerful tool for this purpose. group iii high-rpbp was identified for the first time in northern europe.
 beta-lactam resistance in haemophilus influenzae due to ftsi mutations causing altered penicillin-binding protein is increasing worldwide. low-level resistant isolates with the n526k substitution predominate in most geographical regions, while high-level resistant isolates with the additional s385t substitution are common in japan and south korea. knowledge about the molecular epidemiology of rpbp strains is limited. we combined multilocus sequence typing and ftsi/pbp typing to study the emergence and spread of rpbp in nontypeable h. influenzae in norway.  the prevalence of rpbp in a population of eye, ear and respiratory isolates from was 15%. the prevalence of clinical pbp3-mediated resistance to ampicillin was 9%, compared to % three years earlier. group ii low-rpbp predominated, with significant proportions of isolates non-susceptible to cefotaxime and meropenem. group iii high-rpbp was identified for the first time in northern europe. four mlst sequence types with characteristic, highly diverging ftsi alleles accounted for 61% of the rpbp isolates. the most prevalent substitution pattern was present in 41% of rpbp isolates, mainly carried by st and st several unrelated sts possessed identical copies of the ftsi allele encoding pbp type a. infection sites, age groups, hospitalization rates and rpbp frequencies differed between sts and phylogenetic groups.  this study is the first to link ftsi alleles to sts in h. influenzae. the results indicate that horizontal gene transfer contributes to the emergence of rpbp by phylogeny restricted transformation. clonally related virulent rpbp strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. the need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rpbp strains is underlined. combining mlst and ftsi/pbp typing is a powerful tool for this purpose. haemophilus influenzaebeta-lactam resistanceftsipbp3blnarmlstpfgehorizontal gene transferrecombinationsurveillance 