BACKGROUND
molecular typing is an important tool in epidemiologic studies of infectious diseases, for identifying identical or closely related strains, sources of infection, and for detecting cross-transmissions in the nosocomial environment.
epidemiological outbreaks of bacterial infections are usually caused by initial exposure to a single etiologic agent.
therefore, the bacteria identified in the outbreak are often genetically identical or clonally related as a consequence of microevolutions events  of an ancestor strain  <cit> .
molecular typing represents a tool to elucidate the genetic diversity underlying important phenotypic features such as host specificity, pathogenicity, antibiotic resistance and virulence  <cit> .
through molecular typing it is also possible to monitor the spread and the genetic diversity of nosocomial pathogens such as pseudomonas aeruginosa.
this bacterium represents a threat for its antibiotic resistance and its capability to cause a wide range of infections in human, including urinary tract, blood, and skin infections, but predominantly respiratory tract infections.
this opportunistic pathogen plays a particularly detrimental role in cystic fibrosis  patients, causing chronic respiratory infections leading to high infection rate and morbidity  <cit> .
the genome complexity of p. aeruginosa is assumed to be the major reason for the adaptation skills of this bacterium to various environmental niches and its ability to cause a wide range of infections.
its large genome  includes core genes, necessary for survival, and a wide set of accessory genes conferring functional peculiarities to individual strains  <cit> .
such genomic variability derives from the extended capability of this species to acquire or discard genomic segments via horizontal gene transfer and recombination  <cit> .
several comprehensive molecular typing techniques for discriminating among p. aeruginosa strains have been developed, based either on dna banding patterns  and pulsed-field gel electrophoresis ), on dna sequencing  and genome sequencing) or on dna hybridization   <cit> .
pfge typing is considered the “gold standard” dna banding pattern-based method, being the most discriminative for hospital epidemiologists, who need to monitor the effectiveness of infection control measures  <cit> .
the pfge method, generating genome-wide dna fingerprints with rare-cutter restriction enzymes, is also a cost-effective method.
nevertheless, it is extremely labor-intensive and lacks comparability between laboratories  <cit> .
nowadays, a viable pfge pulsotype database for p. aeruginosa is not available, as a consequence of the unsuccessful efforts to standardize protocols worldwide.
after pfge, mlst has become one of the most popular genotyping techniques  <cit> .
the mlst is a sequencing-based method, which identifies snps as well as genomic rearrangements in six or seven conserved genes.
its significant advantage over pfge typing is to be high-throughput and highly reproducible, allowing reliable data comparison to public global databases.
however, to date it is still an expensive method and it bears the in silico complexity associated to sequencing output.
overall, both dna-banding pattern-based and sequencing-based methods present drawbacks, showing either low reproducibility  or high realization costs .
dna hybridization-based methods have recently become a promising alternative for high-throughput investigation of genetic markers defining bacterial genetic diversity and relatedness  <cit> .
dna macro- and micro-arrays methods represent in fact the optimal compromise between the cost-effectiveness of dna banding pattern-based methods and the reproducibility of sequencing-based methods.
for p. aeruginosa typing, a species-specific oligonucleotide-microarray has been developed by wiehlmann and collaborators  <cit> .
this microarray is based on the arraytube  platform  and allows the genotyping of p. aeruginosa strains using  <dig> informative single nucleotide polymorphisms  at conserved loci, the flica/flicb multiallelic locus and the presence or absence of the exos/exou marker gene.
<cit> .
these reference alleles are based on the p. aeruginosa pao <dig> chromosome and are described to be informative with a frequency of > =15% for the rarer allele in the p. aeruginosa population  <cit> .
in contrast to pfge-based fingerprinting, the discrimination between isolates based on pao1- and non pao1-like alleles represent a limit for performing phylogenetic analyses since these alleles are based on few core genome loci subjected to diversifying selection and mutation rate is not fast enough to investigate evolutionary relationships.
similarly to mlst, which is based on housekeeping genes with high sequence conservation, the pao1-based at profiles are sufficiently stable over time to make the at approach ideal for defining relatedness of isolates for epidemiologic purposes.
in order to define the relatedness between genotypes, the eburst algorithm can be applied  <cit> , which divides bacterial populations into cluster of clones and potentially identifies the ancestral strain.
this clustering algorithm is commonly applied to mlst data  <cit> , but it is suitable to any typing method based on defined genetic elements  <cit> .
unlike mlst, which scans only genetic informative traits of the core genome, the at multimarker microarray also analyzes the composition of the accessory genome through a set of  <dig> genetic markers, so defining the intra-clonal diversity and epidemiological gene pattern  <cit> .
moreover, the at typing, as the mlst, produces a robust and informative genotyping identifying isolates to the strain level and allowing easy and reliable data comparison between laboratories worldwide  <cit> .
the arraytube has been already employed for molecular typing of p. aeruginosa populations isolated from various environments  <cit>  and it has been shown to be adequate even when other typing techniques produced inconsistent results  <cit> .
this work reports the molecular typing of an italian p. aeruginosa clinical collection , performed with the at microarray, and the investigation on the virulence genes/gene islands correlating to the strain source .
data from a set of strains were compared with the pfge and mlst methods, focusing on the adequateness for epidemiological studies.
the prevalence of specific virulence genes from the accessory genome in the identified cluster of clones was defined.
at data of our local population on independent isolates  were clustered according to their genetic similarity and analyzed together with publicly available p. aeruginosa worldwide at datasets.
RESULTS
genotyping by arraytube and comparison to reference typing techniques
<dig> p. aeruginosa isolates were collected from three italian hospitals, located in rovereto, trento, and verona.
all strains were typed with the arraytube , a dna-based multimarker microarray.
the at array design and array experiments are available in the arrayexpress database  under accession numbers e_mtab_ <dig> and a-mexp- <dig>  respectively.
excluding all isolates with identical at-profile collected from individual patients, although from different body sites,  <dig> independent-strains could be selected.
besides at-typing, pfge and mlst were performed on up to  <dig> strains of our collection, for comparison purposes.
the at-genotypes and virulence markers profiles, pfge-clone types and mlst genotypes are provided as supplementary material .
concerning the at-dataset, the at-genotype was derived from the  <dig> snps markers plus the flica/b multiallelic locus and the exos/exou markers, as described by wielhmann and collaborators  <cit> .
isolates with identical at-genotype  and also identical pattern of at virulence markers were defined as at-clones, since they are genetically indistinguishable according to the at approach.
isolates with identical at-genotype but different pattern of virulence markers were referred to as isolates belonging to the same at-clonal complex.
finally, isolates with different at-genotypes but related, according to eburst analysis, were defined as isolates belonging to the same at-cluster of clones  <cit> .
the at-genotyping analysis revealed that the  <dig> collected strains belonged to  <dig> different at-genotypes.
the relative low genomic variation observed in strain-specific regions within the core genome was concordant with the high genetic conservation previously found by genomic sequencing for p. aeruginosa strains  <cit> .
each clonal complex, i.e.
group of isolates with identical at-genotype, comprised  <dig>  +/−  <dig>  isolates.
a set of strains of our collection was analyzed also with two genotyping techniques commonly used in microbiology, which are renowned as high resolution reference methods, i.e.
pulsed-field gel electrophoresis  and multi-locus sequence typing   <cit> .
comparison with these techniques was performed to gain insights into differences/similarities between approaches and to verify results of previous research groups underlining the feasibility of the at approach for epidemic strains  <cit> .
the pfge/spei typing was performed on  <dig> independent strains of our collection, and resolved  <dig> different fingerprints, defined as different pfge clones or pulsotypes , against the  <dig> at-genotypes identified by microarray typing within the same set of isolates.
only  <dig> % pfge/spei clones appeared to be clonal complexes, according to the phylogenetic analysis, whereas at-typing identified  <dig> multi-isolates at-genotypes out of  <dig> .
the determined simpson’s diversity index  simpson’s index of diversitywas higher for pfge than at ; di =  <dig>  for at  and the global congruence between the typing methods was low ).
the displayed greater discriminatory power of the pfge technique compared to at-typing was concordant with published data  <cit>  and it is a consequence of the different definition of a clone on which these two techniques are based.
pfge/spei typing resolves isolates by their spei macrorestriction pattern, thus focusing on presence or absence of recognition sites for the selected genome-wide rare-cutter restriction endonuclease .
differently, the arraytube genotyping is based on the knowledge of p. aeruginosa genome organization, and it recognizes presence or absence of  <dig> a priori well-known snps or gene markers  and  <dig> marker genes)  <cit> .
being the at-markers less numerous than spei restriction sites and based solely on the pao1-genome, they do not allow to perform phylogenetic analyses.
however, they are well suitable for epidemiological studies, since they are not affected by the genome instability exhibited by some epidemic strains, which bias the discrimination power when routine methods are used  <cit> .
for example, the isolates with genotype 4b9a, mostly found in cf patients, were dispersed in  <dig> different pfge clones  .
another example is represented by genotype 6c <dig>  comprising isolates from the same hospital  and even department .
according to the pfge typing, they belonged to  <dig> different clones, hh and ii although closely related .
this example shows how the microarray typing, despite being less discriminative than pfge provides a genotype definition which is particularly suitable for epidemiological studies.
this finding is striking looking at isolates from the same patient.
for example,  <dig> isolates from patient p <dig>  presenting genotype 1bae and identical virulence profile, were defined as the same epidemiological clone according to at approach, but showed  <dig> different pfge fingerprints.
besides the evaluation of the discriminatory power of at versus pfge typing, we tested whether there was concordance between clusters of clones defined by those techniques.
out of  <dig> at clusters of clones identified, only the  <dig> small clusters had the at least 50% of the clones defined as part of the same cluster by both at and pfge .
the isolates from the main at cluster instead  were spread over  <dig> different pfge pulsotypes.
mlst was also applied to a set of independent isolates .
among them,  <dig> showed a known sequence type  profile and were grouped in  <dig> mlst sts with  <dig> to  <dig> strains .
the remaining  <dig> isolates were defined as new by the mlst public database  .
however, these  <dig> new mlst-profiles included  <dig> profiles deriving from a combination of known alleles not described in the public database,  <dig> profiles due to the presence of at least one novel allele, and the remaining  <dig> profiles with medium-quality sequence within one or multiple alleles, for which the allele type could not be univocally determined.
excluding the two isolates with >  <dig> alleles with medium-quality sequences, overall  <dig> mlst sts could be identified among the remaining  <dig> isolates, the majority of which were single-isolate st groups .
the at-approach identified within the same set of isolates a smaller number of at-genotypes, precisely  <dig>  more than half of which  with multiple isolates .
this data suggested a higher discriminatory power of mlst in comparison to at-typing, which could be explained by the much higher information content of sequence data on the  <dig> mlst-marker genes versus presence/absence of polymorphisms in single nucleotides within the  <dig> arraytube snps-markers.
the simpson’s index of diversity , calculated on all  <dig> isolates, was indeed higher for mslt than at microarray typing ; di =  <dig>  for at ), indicating a higher discriminatory ability of mlstversus at.
however, the difference in discrimination ability was lower than for pfge versus at.
also, the global congruence between mlst and at ) was higher than for pfge versus at.
focusing on the  <dig> at-groups with the most mlst-typed isolates, i.e.
f <dig>  4b9a and ec2a, we observed that within each of these groups, more than 62% of the isolates  had an identical mlst-profile, whereas the other isolates differed for  <dig> to  <dig> mlst-alleles from the dominant clone of the group.
by computing the genetic distances between the mlst dna sequences of the three at-types, we observed that highest genetic distance was equivalent to  <dig>   within the f469-group,  <dig>   in the 4b9a-group and  <dig>   within the ec2a-group .
looking at the three larger at-groups, the exclusion of all isolates with medium-quality allele sequences increased the number of isolates with identical mlst st within each group.
in detail, all  <dig> isolates from the f <dig> group had an identical mlst-profile, i.e.
st <dig>  all  <dig> isolates from ec2a had mlst st <dig> and  <dig> out of  <dig> isolates within the 4b9a-group corresponded to a unique sequence type, i.e.
st <dig>
we observed that excluding all isolates with one or more medium-quality allele sequences, the disagreement between the two techniques further decreased, as shown by the similarly high simpson’s index of diversity and the higher global congruence between methods calculated on the  <dig> isolates with good quality allele sequences ; di =  <dig>  for at ; adjust rand coefficient =  <dig>  ).
overall, the at-approach was comparably informative to mlst for genotype definition and additionally provided information on the accessory genome.
thus, we employed the at multimarker microarray to define genotype and virulence profile for all strains of our collection, identify potential correlations between strain source and at-genotype or virulence gene pattern, and relate our data to the global at population.
correlation between at-genotype and strain source
the strains were collected from three hospitals and were isolated from patients affected by one of these two different infection-types: chronic infections  and acute infections  or other hospital departments ).
to investigate whether strain at-genotype correlated with strain source, we grouped the 124-independent isolates of our collection according to their at-type, infection type or hospital location.
overall,  <dig> out of  <dig> at-genotypes were exclusively found in either cf or non-cf  and, among the multi-isolate clones,  <dig> out of  <dig> at-types showed to be prevalent  in either chronic or acute infections , supporting previous evidence of an association of clones to a particular source  <cit> .
the existence of infection-type specific clones is still under debate  <cit>  and the reduced size of some of our clonal complexes did not allow us to draw statistically significant conclusions on the overall behaviour but rather to gather information on individual genotypes.
among the  <dig> multi-isolates at-genotypes of our collection 4b9a, ec2a, 3c2a were more frequently  associated to chronic infections, whereas f <dig>  2c1a, 6c <dig> to acute infections .
despite the unbalanced distribution of isolates from chronic and acute infections in our settings depending from the hospital location , we assumed that a similar distribution of clones would be observed in the three hospitals, given the short geographical distance between their locations.
thus, we attributed the observed prevalence of specific at-genotypes exclusively to the infection type.
also, some of the individually identified correlations confirmed findings of other research groups  <cit> .
at-genotypes were clustered according to their genetic similarity, using the eburst algorithm on the  <dig> at-markers designed for genotyping   <cit> .
by excluding the exou and exos markers,  <dig> clones collapsed into others, precisely clones f <dig> into f <dig> and ec <dig>  ec <dig> into ec2a .
focusing on chronic associated isolates, the 4b9a at-genotype belonged to the largest at clonal complex and correlated to chronic infections, being  <dig> % of its isolates collected from cf patients , in contradiction with other collections in which this at-genotype was described within keratitis, environmental and copd samples  <cit> .
as for the 4b9a at-genotype, ec2a, known as cha strain  <cit> , was also mostly associated to the cf patient cohort .
the identified correlation is supported by previous studies and the mechanism of action of strains with this at-genotype on human blood cells has been already elucidated  <cit> .
3c2a was exclusively cf-associated, but it has been previously described as a frequent at-type both in cf and non-cf patients  <cit> .
among the multi-isolate at-genotypes, only one novel one out of  <dig> novel genotypes was identified also in cf patients, although in 50% of the cases only.
an investigation on co-infections events, taking in account the 124-independent isolates collection, revealed that almost 40% of our cf patients were colonized by more than one at-genotype, among which the most frequent were again 4b9a and ec2a but also the 2c <dig> at-types .
interestingly, isolates typed as 4b9a and ec2a, when present, were always co-colonizers .
according to the eburst analysis shown in figure  <dig>  these two at-genotypes showed low snp-profile similarity and were classified as unrelated by the eburst analysis of our collection being part of different cluster of clones.
looking at the accessory-genome markers, the isolates with 4b9a and ec2a at-type presented an identical pattern of virulence genes/gene islands .
among the  <dig> patients infected by more than one at-genotype, only an individual patient  was co-infected by two strains from the same cluster of clones, with ec2a and 2c <dig> at-genotype.
despite the similar snp-pattern characterizing these two strains, they differ in the set of virulence markers for the presence of the fla-glycosylation island .
another patient  may be infected by two highly similar strains, being typed as ec <dig> and 2c <dig>  excluding the exos/exou at core-genome marker, the ec <dig> isolate was in fact genotypically identical to the ec2a one, thus becoming part of the cluster of clone  <dig>  together with the co-infecting 2c <dig> strain.
as for chronic infections, acute infections were also found to be dominated by specific at-genotypes.
in particular, f <dig>  the absolute most frequent at-type within our collection, was exclusively associated to acute infection .
f <dig> isolates were primarily found  in patients from the intensive care unit , carrying severe acute infections, and secondly  in patients from the hematology unit , affected as well by acute infections .
the correlation between f <dig> and acute infections is well supported by other at studies, identifying this at-genotype within environmental samples and keratitis patients  <cit>  .
the at-genotypes identified in our study were search in other published at-databases  <cit> .
genotypes were grouped according to the at-databases sharing them.
the 2c1a at-genotype, better known as midlands  <dig>  <cit> , was also exclusively identified in acute infection and predominantly  in patients affected by an acute infection of the respiratory apparatus .
our finding is in contrast with previous data, describing the midlands  <dig> as the second most common clone in cf centres in great britain  <cit> .
the 6c <dig> at-type was exclusively isolated from blood infections in verona, and it has been previously mainly reported as environmental  <cit> .
besides known at-genotypes,  <dig> novel ones, b46a and 4d <dig>  were identified.
multiple isolates belonged to these at-types which were exclusively found in patients affected by acute infections, precisely within the intensive care unit  of the hospital located in verona.
few co-infection events  could be observed in patients with acute infections, in comparison to those observed in patients affected by chronic infections  .
moreover, the co-infecting strains differed in their at-type in each patient and, according to the eburst analysis of our collection, only one patient  was co-colonized by two strains with at-genotypes belonging to the same cluster of clones .
b46a showed a different set of virulence genes and gene islands than f <dig>  precisely for the absence of exou and the presence of the papi1-island.
correlation between genes or gene islands of the accessory genome and strain source
the arraytube multimarker microarray allowed not only discriminating among p. aeruginosa genotypes with proper resolution for epidemiological investigations, but also defining a molecular profile of key accessory genes and gene islands and their correlation to infection type or department.
the prevalence of each accessory genome marker was determined among at-genotypes belonging to the  <dig> cluster of clones identified by eburst analysis in our collection of independent isolates  .
the main cluster of clones within our strain collection  was characterized by genes and gene islands shared by all at-genotypes of the cluster , but also by at-type specific genomic regions such as the exou gene, the les-specific mutations or the fla-glycosilation island.
a statistical analysis  <cit>  revealed that the presence of the exou gene positively correlated  with the icu department, which hosted patients with severe acute infections.
this finding was concordant with the known function of the protein encoded by the exou gene, a potent cytotoxin causing damages in lung tissue, thus not compatible with chronic infections  <cit> .
on the contrary, the exos gene, described as mutually exclusive with the exou gene  <cit> , was associated in this study to cf strains .
besides the exou gene, a positive correlation was also identified between the genes belonging to the pklc102-like island, in particular genes encoding for pkl- <dig>  pkl- <dig>  pklc adhesion, pklc fatty acid synthase , the pklc conserved hypothetical protein  and the infection-type .
these  <dig> genes were prevalent in cf strains, not only in our strain collection but also in the global population   <cit>  and copd isolates   <cit> .
the function of most of the genes belonging to this island has not been deciphered yet, but it is known that the papi-1/pklc102-like members encode virulence factors, such as cytotoxins, pili, fimbriae and regulators of biofilm synthesis and antibiotic resistance  <cit> .
given the known functions of this island, the identified positive correlation to chronic infections was unexpected, as it has been demonstrated that p. aeruginosa reduces its acute virulence during the adaptation to the cf lung environment  <cit> .
nevertheless, rakhimova and collaborators  <cit>  showed that the pkl- <dig> gene was associated to a prolonged colonization time in a minority of p. aeruginosa strains in copd patients  <cit> , whose lung colonization pattern by pseudomonas strains is comparable to the one observed in cf patients.
analysis of the at-genotypes identified within the publicly available population studies
an intrinsic feature of the at technology is to be standardized and therefore to guarantee reliable data comparison between genotyping studies performed worldwide in different laboratories  <cit> .
in order to gain further information on the 124-independent strains of our collection, we compared them with a global database, obtained by retrieving information from  <dig> publicly available at-datasets, comprising a total of  <dig> isolates  <cit> .
these datasets comprised  <dig> strains of diverse habitat and geographic origin  <cit> ,  <dig> strains collected from patients affected by chronic obstructive pulmonary disease  <cit> ,  <dig> strains isolated from keratitis  <cit> , and  <dig> environmental isolates from rivers  <cit> .
our 124-independent strain collection included  <dig> genotypes previously described  <cit>  and  <dig> which have never been previously reported .
among the  <dig> already described at-genotypes, it is interesting to notice that  <dig> of them  were shared by all collections  <cit>  and were all among the  <dig> most abundant in the global p. aeruginosa population  <cit> .
an eburst analysis using  <dig> markers  was performed to illustrate the similarities between snp profiles of our and other collections, typed by the at method.
as shown in additional file  <dig>  the eburst analysis revealed the presence of  <dig> main clusters of clones and  <dig> small ones .
most at clones also previously described  belonged to the  <dig> large clusters,  <dig> of which were among the  <dig> most abundant clones in the global p. aeruginosa population, namely d <dig>  f <dig>  1bae, 2c1a, 0c2e, 239a,  <dig>  c40a, e <dig>  ec <dig>  f <dig> and 3c2a  <cit> .
all novel at clones except one  were part of the  <dig> large clusters or gave rise to a small cluster including a previously described strain  .
overall,  <dig> % clones of the local population  were equally divided into the two large clusters of clones and almost 30%  were primary founders, i.e.
e <dig>  e <dig>  d <dig>  f <dig>  c40a, ec2a, 0c2e,  <dig>  2c1a, 239a, and 1bae .
among the  <dig> primary founders identified within our collection,  <dig> were known to be abundant clones in the global p. aeruginosa population  <cit> , confirming their dominant role in the global p. aeruginosa population.
CONCLUSIONS
the arraytube multimarker-microarray represented a reliable and reproducible tool for p. aeruginosa molecular typing.
genotypic data was readily comparable to public databases and allowed to draw conclusions on the correlation between isolates and infection type or department.
a comparison with reference genotyping techniques showed how the at provides a genotypic profile which is not biased by genome variations within unknown or not informative regions, and defines additionally epidemiological features to identifying the causative strain and transmission pattern in epidemiological outbreaks.

 molecular typing is an important tool in epidemiologic studies of infectious diseases, for identifying identical or closely related strains, sources of infection, and for detecting cross-transmissions in the nosocomial environment. epidemiological outbreaks of bacterial infections are usually caused by initial exposure to a single etiologic agent. therefore, the bacteria identified in the outbreak are often genetically identical or clonally related as a consequence of microevolutions events of an ancestor strain. molecular typing represents a tool to elucidate the genetic diversity underlying important phenotypic features such as host specificity, pathogenicity, antibiotic resistance and virulence. through molecular typing it is also possible to monitor the spread and the genetic diversity of nosocomial pathogens such as pseudomonas aeruginosa. this bacterium represents a threat for its antibiotic resistance and its capability to cause a wide range of infections in human, including urinary tract, blood, and skin infections, but predominantly respiratory tract infections. this opportunistic pathogen plays a particularly detrimental role in cystic fibrosis patients, causing chronic respiratory infections leading to high infection rate and morbidity. the genome complexity of p. aeruginosa is assumed to be the major reason for the adaptation skills of this bacterium to various environmental niches and its ability to cause a wide range of infections. its large genome includes core genes, necessary for survival, and a wide set of accessory genes conferring functional peculiarities to individual strains. such genomic variability derives from the extended capability of this species to acquire or discard genomic segments via horizontal gene transfer and recombination. several comprehensive molecular typing techniques for discriminating among p. aeruginosa strains have been developed, based either on dna banding patterns and pulsed-field gel electrophoresis ), on dna sequencing and genome sequencing) or on dna hybridization. pfge typing is considered the “gold standard” dna banding pattern-based method, being the most discriminative for hospital epidemiologists, who need to monitor the effectiveness of infection control measures. the pfge method, generating genome-wide dna fingerprints with rare-cutter restriction enzymes, is also a cost-effective method. nevertheless, it is extremely labor-intensive and lacks comparability between laboratories. nowadays, a viable pfge pulsotype database for p. aeruginosa is not available, as a consequence of the unsuccessful efforts to standardize protocols worldwide. after pfge, mlst has become one of the most popular genotyping techniques. the mlst is a sequencing-based method, which identifies snps as well as genomic rearrangements in six or seven conserved genes. its significant advantage over pfge typing is to be high-throughput and highly reproducible, allowing reliable data comparison to public global databases. however, to date it is still an expensive method and it bears the in silico complexity associated to sequencing output. overall, both dna-banding pattern-based and sequencing-based methods present drawbacks, showing either low reproducibility or high realization costs. dna hybridization-based methods have recently become a promising alternative for high-throughput investigation of genetic markers defining bacterial genetic diversity and relatedness. dna macro- and micro-arrays methods represent in fact the optimal compromise between the cost-effectiveness of dna banding pattern-based methods and the reproducibility of sequencing-based methods. for p. aeruginosa typing, a species-specific oligonucleotide-microarray has been developed by wiehlmann and collaborators. this microarray is based on the arraytube platform and allows the genotyping of p. aeruginosa strains using informative single nucleotide polymorphisms at conserved loci, the flica/flicb multiallelic locus and the presence or absence of the exos/exou marker gene.. these reference alleles are based on the p. aeruginosa pao chromosome and are described to be informative with a frequency of > =15% for the rarer allele in the p. aeruginosa population. in contrast to pfge-based fingerprinting, the discrimination between isolates based on pao1- and non pao1-like alleles represent a limit for performing phylogenetic analyses since these alleles are based on few core genome loci subjected to diversifying selection and mutation rate is not fast enough to investigate evolutionary relationships. similarly to mlst, which is based on housekeeping genes with high sequence conservation, the pao1-based at profiles are sufficiently stable over time to make the at approach ideal for defining relatedness of isolates for epidemiologic purposes. in order to define the relatedness between genotypes, the eburst algorithm can be applied, which divides bacterial populations into cluster of clones and potentially identifies the ancestral strain. this clustering algorithm is commonly applied to mlst data, but it is suitable to any typing method based on defined genetic elements. unlike mlst, which scans only genetic informative traits of the core genome, the at multimarker microarray also analyzes the composition of the accessory genome through a set of genetic markers, so defining the intra-clonal diversity and epidemiological gene pattern. moreover, the at typing, as the mlst, produces a robust and informative genotyping identifying isolates to the strain level and allowing easy and reliable data comparison between laboratories worldwide. the arraytube has been already employed for molecular typing of p. aeruginosa populations isolated from various environments and it has been shown to be adequate even when other typing techniques produced inconsistent results. this work reports the molecular typing of an italian p. aeruginosa clinical collection, performed with the at microarray, and the investigation on the virulence genes/gene islands correlating to the strain source. data from a set of strains were compared with the pfge and mlst methods, focusing on the adequateness for epidemiological studies. the prevalence of specific virulence genes from the accessory genome in the identified cluster of clones was defined. at data of our local population on independent isolates were clustered according to their genetic similarity and analyzed together with publicly available p. aeruginosa worldwide at datasets.  genotyping by arraytube and comparison to reference typing techniques p. aeruginosa isolates were collected from three italian hospitals, located in rovereto, trento, and verona. all strains were typed with the arraytube, a dna-based multimarker microarray. the at array design and array experiments are available in the arrayexpress database under accession numbers e_mtab_ and a-mexp- respectively. excluding all isolates with identical at-profile collected from individual patients, although from different body sites, independent-strains could be selected. besides at-typing, pfge and mlst were performed on up to strains of our collection, for comparison purposes. the at-genotypes and virulence markers profiles, pfge-clone types and mlst genotypes are provided as supplementary material. concerning the at-dataset, the at-genotype was derived from the snps markers plus the flica/b multiallelic locus and the exos/exou markers, as described by wielhmann and collaborators. isolates with identical at-genotype and also identical pattern of at virulence markers were defined as at-clones, since they are genetically indistinguishable according to the at approach. isolates with identical at-genotype but different pattern of virulence markers were referred to as isolates belonging to the same at-clonal complex. finally, isolates with different at-genotypes but related, according to eburst analysis, were defined as isolates belonging to the same at-cluster of clones. the at-genotyping analysis revealed that the collected strains belonged to different at-genotypes. the relative low genomic variation observed in strain-specific regions within the core genome was concordant with the high genetic conservation previously found by genomic sequencing for p. aeruginosa strains. each clonal complex, i.e. group of isolates with identical at-genotype, comprised +/− isolates. a set of strains of our collection was analyzed also with two genotyping techniques commonly used in microbiology, which are renowned as high resolution reference methods, i.e. pulsed-field gel electrophoresis and multi-locus sequence typing. comparison with these techniques was performed to gain insights into differences/similarities between approaches and to verify results of previous research groups underlining the feasibility of the at approach for epidemic strains. the pfge/spei typing was performed on independent strains of our collection, and resolved different fingerprints, defined as different pfge clones or pulsotypes, against the at-genotypes identified by microarray typing within the same set of isolates. only % pfge/spei clones appeared to be clonal complexes, according to the phylogenetic analysis, whereas at-typing identified multi-isolates at-genotypes out of. the determined simpson’s diversity index simpson’s index of diversitywas higher for pfge than at; di = for at and the global congruence between the typing methods was low ). the displayed greater discriminatory power of the pfge technique compared to at-typing was concordant with published data and it is a consequence of the different definition of a clone on which these two techniques are based. pfge/spei typing resolves isolates by their spei macrorestriction pattern, thus focusing on presence or absence of recognition sites for the selected genome-wide rare-cutter restriction endonuclease. differently, the arraytube genotyping is based on the knowledge of p. aeruginosa genome organization, and it recognizes presence or absence of a priori well-known snps or gene markers and marker genes). being the at-markers less numerous than spei restriction sites and based solely on the pao1-genome, they do not allow to perform phylogenetic analyses. however, they are well suitable for epidemiological studies, since they are not affected by the genome instability exhibited by some epidemic strains, which bias the discrimination power when routine methods are used. for example, the isolates with genotype 4b9a, mostly found in cf patients, were dispersed in different pfge clones. another example is represented by genotype 6c comprising isolates from the same hospital and even department. according to the pfge typing, they belonged to different clones, hh and ii although closely related. this example shows how the microarray typing, despite being less discriminative than pfge provides a genotype definition which is particularly suitable for epidemiological studies. this finding is striking looking at isolates from the same patient. for example, isolates from patient p presenting genotype 1bae and identical virulence profile, were defined as the same epidemiological clone according to at approach, but showed different pfge fingerprints. besides the evaluation of the discriminatory power of at versus pfge typing, we tested whether there was concordance between clusters of clones defined by those techniques. out of at clusters of clones identified, only the small clusters had the at least 50% of the clones defined as part of the same cluster by both at and pfge. the isolates from the main at cluster instead were spread over different pfge pulsotypes. mlst was also applied to a set of independent isolates. among them, showed a known sequence type profile and were grouped in mlst sts with to strains. the remaining isolates were defined as new by the mlst public database. however, these new mlst-profiles included profiles deriving from a combination of known alleles not described in the public database, profiles due to the presence of at least one novel allele, and the remaining profiles with medium-quality sequence within one or multiple alleles, for which the allele type could not be univocally determined. excluding the two isolates with > alleles with medium-quality sequences, overall mlst sts could be identified among the remaining isolates, the majority of which were single-isolate st groups. the at-approach identified within the same set of isolates a smaller number of at-genotypes, precisely more than half of which with multiple isolates. this data suggested a higher discriminatory power of mlst in comparison to at-typing, which could be explained by the much higher information content of sequence data on the mlst-marker genes versus presence/absence of polymorphisms in single nucleotides within the arraytube snps-markers. the simpson’s index of diversity, calculated on all isolates, was indeed higher for mslt than at microarray typing; di = for at ), indicating a higher discriminatory ability of mlstversus at. however, the difference in discrimination ability was lower than for pfge versus at. also, the global congruence between mlst and at ) was higher than for pfge versus at. focusing on the at-groups with the most mlst-typed isolates, i.e. f 4b9a and ec2a, we observed that within each of these groups, more than 62% of the isolates had an identical mlst-profile, whereas the other isolates differed for to mlst-alleles from the dominant clone of the group. by computing the genetic distances between the mlst dna sequences of the three at-types, we observed that highest genetic distance was equivalent to within the f469-group, in the 4b9a-group and within the ec2a-group. looking at the three larger at-groups, the exclusion of all isolates with medium-quality allele sequences increased the number of isolates with identical mlst st within each group. in detail, all isolates from the f group had an identical mlst-profile, i.e. st all isolates from ec2a had mlst st and out of isolates within the 4b9a-group corresponded to a unique sequence type, i.e. st we observed that excluding all isolates with one or more medium-quality allele sequences, the disagreement between the two techniques further decreased, as shown by the similarly high simpson’s index of diversity and the higher global congruence between methods calculated on the isolates with good quality allele sequences; di = for at; adjust rand coefficient = ). overall, the at-approach was comparably informative to mlst for genotype definition and additionally provided information on the accessory genome. thus, we employed the at multimarker microarray to define genotype and virulence profile for all strains of our collection, identify potential correlations between strain source and at-genotype or virulence gene pattern, and relate our data to the global at population. correlation between at-genotype and strain source the strains were collected from three hospitals and were isolated from patients affected by one of these two different infection-types: chronic infections and acute infections or other hospital departments ). to investigate whether strain at-genotype correlated with strain source, we grouped the 124-independent isolates of our collection according to their at-type, infection type or hospital location. overall, out of at-genotypes were exclusively found in either cf or non-cf and, among the multi-isolate clones, out of at-types showed to be prevalent in either chronic or acute infections, supporting previous evidence of an association of clones to a particular source. the existence of infection-type specific clones is still under debate and the reduced size of some of our clonal complexes did not allow us to draw statistically significant conclusions on the overall behaviour but rather to gather information on individual genotypes. among the multi-isolates at-genotypes of our collection 4b9a, ec2a, 3c2a were more frequently associated to chronic infections, whereas f 2c1a, 6c to acute infections. despite the unbalanced distribution of isolates from chronic and acute infections in our settings depending from the hospital location, we assumed that a similar distribution of clones would be observed in the three hospitals, given the short geographical distance between their locations. thus, we attributed the observed prevalence of specific at-genotypes exclusively to the infection type. also, some of the individually identified correlations confirmed findings of other research groups. at-genotypes were clustered according to their genetic similarity, using the eburst algorithm on the at-markers designed for genotyping. by excluding the exou and exos markers, clones collapsed into others, precisely clones f into f and ec ec into ec2a. focusing on chronic associated isolates, the 4b9a at-genotype belonged to the largest at clonal complex and correlated to chronic infections, being % of its isolates collected from cf patients, in contradiction with other collections in which this at-genotype was described within keratitis, environmental and copd samples. as for the 4b9a at-genotype, ec2a, known as cha strain, was also mostly associated to the cf patient cohort. the identified correlation is supported by previous studies and the mechanism of action of strains with this at-genotype on human blood cells has been already elucidated. 3c2a was exclusively cf-associated, but it has been previously described as a frequent at-type both in cf and non-cf patients. among the multi-isolate at-genotypes, only one novel one out of novel genotypes was identified also in cf patients, although in 50% of the cases only. an investigation on co-infections events, taking in account the 124-independent isolates collection, revealed that almost 40% of our cf patients were colonized by more than one at-genotype, among which the most frequent were again 4b9a and ec2a but also the 2c at-types. interestingly, isolates typed as 4b9a and ec2a, when present, were always co-colonizers. according to the eburst analysis shown in figure  these two at-genotypes showed low snp-profile similarity and were classified as unrelated by the eburst analysis of our collection being part of different cluster of clones. looking at the accessory-genome markers, the isolates with 4b9a and ec2a at-type presented an identical pattern of virulence genes/gene islands. among the patients infected by more than one at-genotype, only an individual patient was co-infected by two strains from the same cluster of clones, with ec2a and 2c at-genotype. despite the similar snp-pattern characterizing these two strains, they differ in the set of virulence markers for the presence of the fla-glycosylation island. another patient may be infected by two highly similar strains, being typed as ec and 2c excluding the exos/exou at core-genome marker, the ec isolate was in fact genotypically identical to the ec2a one, thus becoming part of the cluster of clone together with the co-infecting 2c strain. as for chronic infections, acute infections were also found to be dominated by specific at-genotypes. in particular, f the absolute most frequent at-type within our collection, was exclusively associated to acute infection. f isolates were primarily found in patients from the intensive care unit, carrying severe acute infections, and secondly in patients from the hematology unit, affected as well by acute infections. the correlation between f and acute infections is well supported by other at studies, identifying this at-genotype within environmental samples and keratitis patients. the at-genotypes identified in our study were search in other published at-databases. genotypes were grouped according to the at-databases sharing them. the 2c1a at-genotype, better known as midlands, was also exclusively identified in acute infection and predominantly in patients affected by an acute infection of the respiratory apparatus. our finding is in contrast with previous data, describing the midlands as the second most common clone in cf centres in great britain. the 6c at-type was exclusively isolated from blood infections in verona, and it has been previously mainly reported as environmental. besides known at-genotypes, novel ones, b46a and 4d were identified. multiple isolates belonged to these at-types which were exclusively found in patients affected by acute infections, precisely within the intensive care unit of the hospital located in verona. few co-infection events could be observed in patients with acute infections, in comparison to those observed in patients affected by chronic infections. moreover, the co-infecting strains differed in their at-type in each patient and, according to the eburst analysis of our collection, only one patient was co-colonized by two strains with at-genotypes belonging to the same cluster of clones. b46a showed a different set of virulence genes and gene islands than f precisely for the absence of exou and the presence of the papi1-island. correlation between genes or gene islands of the accessory genome and strain source the arraytube multimarker microarray allowed not only discriminating among p. aeruginosa genotypes with proper resolution for epidemiological investigations, but also defining a molecular profile of key accessory genes and gene islands and their correlation to infection type or department. the prevalence of each accessory genome marker was determined among at-genotypes belonging to the cluster of clones identified by eburst analysis in our collection of independent isolates. the main cluster of clones within our strain collection was characterized by genes and gene islands shared by all at-genotypes of the cluster, but also by at-type specific genomic regions such as the exou gene, the les-specific mutations or the fla-glycosilation island. a statistical analysis revealed that the presence of the exou gene positively correlated with the icu department, which hosted patients with severe acute infections. this finding was concordant with the known function of the protein encoded by the exou gene, a potent cytotoxin causing damages in lung tissue, thus not compatible with chronic infections. on the contrary, the exos gene, described as mutually exclusive with the exou gene, was associated in this study to cf strains. besides the exou gene, a positive correlation was also identified between the genes belonging to the pklc102-like island, in particular genes encoding for pkl- pkl- pklc adhesion, pklc fatty acid synthase, the pklc conserved hypothetical protein and the infection-type. these genes were prevalent in cf strains, not only in our strain collection but also in the global population and copd isolates. the function of most of the genes belonging to this island has not been deciphered yet, but it is known that the papi-1/pklc102-like members encode virulence factors, such as cytotoxins, pili, fimbriae and regulators of biofilm synthesis and antibiotic resistance. given the known functions of this island, the identified positive correlation to chronic infections was unexpected, as it has been demonstrated that p. aeruginosa reduces its acute virulence during the adaptation to the cf lung environment. nevertheless, rakhimova and collaborators showed that the pkl- gene was associated to a prolonged colonization time in a minority of p. aeruginosa strains in copd patients, whose lung colonization pattern by pseudomonas strains is comparable to the one observed in cf patients. analysis of the at-genotypes identified within the publicly available population studies an intrinsic feature of the at technology is to be standardized and therefore to guarantee reliable data comparison between genotyping studies performed worldwide in different laboratories. in order to gain further information on the 124-independent strains of our collection, we compared them with a global database, obtained by retrieving information from publicly available at-datasets, comprising a total of isolates. these datasets comprised strains of diverse habitat and geographic origin, strains collected from patients affected by chronic obstructive pulmonary disease, strains isolated from keratitis, and environmental isolates from rivers. our 124-independent strain collection included genotypes previously described and which have never been previously reported. among the already described at-genotypes, it is interesting to notice that of them were shared by all collections and were all among the most abundant in the global p. aeruginosa population. an eburst analysis using markers was performed to illustrate the similarities between snp profiles of our and other collections, typed by the at method. as shown in additional file the eburst analysis revealed the presence of main clusters of clones and small ones. most at clones also previously described belonged to the large clusters, of which were among the most abundant clones in the global p. aeruginosa population, namely d f 1bae, 2c1a, 0c2e, 239a, c40a, e ec f and 3c2a. all novel at clones except one were part of the large clusters or gave rise to a small cluster including a previously described strain. overall, % clones of the local population were equally divided into the two large clusters of clones and almost 30% were primary founders, i.e. e e d f c40a, ec2a, 0c2e, 2c1a, 239a, and 1bae. among the primary founders identified within our collection, were known to be abundant clones in the global p. aeruginosa population, confirming their dominant role in the global p. aeruginosa population.  the arraytube multimarker-microarray represented a reliable and reproducible tool for p. aeruginosa molecular typing. genotypic data was readily comparable to public databases and allowed to draw conclusions on the correlation between isolates and infection type or department. a comparison with reference genotyping techniques showed how the at provides a genotypic profile which is not biased by genome variations within unknown or not informative regions, and defines additionally epidemiological features to identifying the causative strain and transmission pattern in epidemiological outbreaks. 
pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis patients, who are easily affected by chronic lung infections. at typing of this italian collection could be easily integrated with the global p. aeruginosa at-typed population, uncovering that most at-genotypes identified belonged to two large clonal clusters, and included among the most abundant clones of the global population. the arraytube multimarker array represented a robust and portable alternative to reference techniques for performing p. aeruginosa molecular typing, and allowed us to draw conclusions especially suitable for epidemiologists on an italian clinical collection from chronic and acute infections. for epidemiological purposes, discriminating p.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. the presence of the exou gene correlated with non-cf patients within the intensive care unit whereas the pklc102-like island appeared to be prevalent in the cf centre. an investigation on co-infections events revealed that almost 40% of cf patients were colonized by more than one genotype, whereas less than 4% were observed in non cf patients. a collection of p. aeruginosa clinical strains isolated within italian hospitals from patients with chronic infections, i.e. cystic fibrosis patients, and with acute infections were genotyped. pulsed-field gel electrophoresis and multilocus sequence typing were employed as reference genotyping techniques to estimate the arraytube resolution power. almost 30% of the genotypes belonged to a main cluster of clones.
 pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis patients, who are easily affected by chronic lung infections. for epidemiological purposes, discriminating p.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. a collection of p. aeruginosa clinical strains isolated within italian hospitals from patients with chronic infections, i.e. cystic fibrosis patients, and with acute infections were genotyped. molecular typing was performed with the arraytube multimarker microarray, a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory p.aeruginosa genome. pulsed-field gel electrophoresis and multilocus sequence typing were employed as reference genotyping techniques to estimate the arraytube resolution power.  at-genotypes were identified within our collection, among which were novel and had been previously described in publicly available at-databases. almost 30% of the genotypes belonged to a main cluster of clones. 4b9a, ec2a, 3c2a were mostly associated to cf-patients whereas f 2c1a, 6c to non cf. an investigation on co-infections events revealed that almost 40% of cf patients were colonized by more than one genotype, whereas less than 4% were observed in non cf patients. the presence of the exou gene correlated with non-cf patients within the intensive care unit whereas the pklc102-like island appeared to be prevalent in the cf centre. the congruence between the arraytube typing and pfge or mlst was and, respectively. at typing of this italian collection could be easily integrated with the global p. aeruginosa at-typed population, uncovering that most at-genotypes identified belonged to two large clonal clusters, and included among the most abundant clones of the global population.  the arraytube multimarker array represented a robust and portable alternative to reference techniques for performing p. aeruginosa molecular typing, and allowed us to draw conclusions especially suitable for epidemiologists on an italian clinical collection from chronic and acute infections. 