bacterial isolation and identification : a collection of 60 s. pseudintermedius isolates from canine pyoderma were included in this study . bacteria were isolated from clinical specimens submitted to the bacteriology laboratory at the department of veterinary medicine ( university of perugia ) . upon arrival at the laboratory , specimens were cultured on 5% defibrinated sheep blood agar and mannitol salt agar and incubated aerobically at 37c for 24 hr . suspect colonies were identified using standard techniques : colony morphology , gram stain , coagulase and catalase test and api staph system ( biomerieux , marcy - ltoile , france ) . s. pseudintermedius isolates were identified using a polymerase chain reaction ( pcr ) restriction fragment length polymorphism ( rflp ) assay based on the mboi digestion pattern of a pcr amplified internal fragment of the pta gene as previously described . phenotypic characterization of biofilm - producing ability on congo red agar ( cra ) : to examine bacterial slime production , which is a step for biofilm formation , all the strains were cultured on congo red agar . cra plates were prepared by adding 0.8 g of congo red and 36 g of saccharose ( sigma , st . louis , mo , u.s.a . ) to 1 l of brain heart infusion agar ( oxoid , strada rivoltana , italy ) . after inoculation , the plates were incubated for 24 hr at 37c and subsequently overnight at room temperature . on cra , black colonies were considered slime - producing strains , almost black colonies were considered weakly slime producing , and red colonies were classified as strains unable to produce biofilm , slightly modifying the procedure of arciola et al . . phenotypic characterization of biofilm formation by tube adherence test : the qualitative assay for biofilm formation was performed according to previous authors   with minor modifications . glass tubes filled with 2.6 ml of tryptic soy broth ( tsb ) ( oxoid ) containing 1% glucose were inoculated with a loopful of a pure culture of a strain from tryptic soy agar ( tsa ) plates containing 1% glucose . after overnight incubation at 37c in air , the contents of each tube was carefully removed with a pipette , and 2 ml of a 0.25% safranin solution was added . after 1 min , the tubes were emptied with a pipette and placed upside down without a wash step . the test was considered positive when there was a layer of stained material on the inner surface of 
 the tube . phenotypic characterization of biofilm formation by microtiter plate test ( mtp ) : the ability of the isolates to form biofilms was determined by the ability to adhere to 96-well polystyrene microtiter plates ( nunc , rochester , ny , u.s.a . ) using the method described by previous authors   with minor modifications . briefly , isolates were subcultured onto blood agar , and pure 24 hr growth was used for testing . each isolate was suspended in 5.0 ml of tryptic soy broth ( tsb ) supplemented with 1% glucose to achieve a turbidity equivalent to a 0.5 mcfarland standard ( ~10cfu / ml ) . a 200 l bacterial suspension was transferred in triplicate into microtiter plate wells , with the negative control containing growth medium only . the plates were incubated at 37c for 24 hr , washed with water to remove non - adhered cells and then dried at room temperature before the addition 
 of hucker crystal violet solution ( sigma ) . the plates were subsequently incubated at room temperature for 30 min before excess dye was removed by rinsing with tap water . after the plates were air - dried , the dye bound to the adherent cells was resolubilized with 160 ml of 33% ( v / v ) glacial acetic acid per well . the result was calculated by subtracting the median od570 of triplicate determinations for the negative control ( test broth only ) from the median od570 of triplicate determination for the samples . adherent , strains with od values between 0.065 and 0.130 were classified as weakly adherent , strains with od values between and 0.130 and 0.260 were classified as moderately adherent , and strains with an od > 0.260 were classified as strongly adherent . dna extraction and pcr for detection of icaa and icad genes : for pcr analyses , dna was extracted from bacterial colonies using a qiaamp dna mini kit according to the manufacturer s instructions ( qiagen , hilden , germany ) . primers used in this study and pcr conditionsgeneprimer namesequence ( 53)amplicon lengthpcr conditionsicaaicaa - fwactgtttcggggacaagcat134 bp94c  3 min , 35  ( 94c  15 sec , 60c  20 sec , 72c  20 sec ) 4c icaa - revattgaggctgtagggcgttgicadicad- fwcgttaatgccttctttcttattgcg166 bp94c  3 min , 35  ( 94c  15 sec , 56c  20 sec , 72c  20 sec ) 4c icad - revattagcgcacattcggtgtt ) genes were designed from the staphylococcus pseudintermedius ed99 genome ( genbank accession : cp002478 ) using the primer - blast suite of the ncbi   in order to limit the amplification of unintended targets . briefly , the 25 l reaction volume consisted of 10  pcr buffer , 1.5 mm mgcl2 , 200 m of each deoxyribonucleotide triphosphate , 10 m of each primer ( table 1 ) , 1 u of taq polymerase and 100 ng of template dna . the dna of each sample was tested in duplicate . the presence and size of the amplified products pcr products were purified using a wizard sv gel and pcr clean - up system ( promega , madison , wi , u.s.a . ) in accordance with the manufacturer s recommended protocol and subjected to direct sequencing ( primm srl , milan , italy ) . the final sequences were subjected to a blast analysis against prokaryotic databases ( ncbi ) to confirm specific amplification . statistical analysis : data derived from laboratory examinations were stored in microsoft excel . agreement was calculated using cohen s kappa values ( kappa ) for dichotomous data and squared weighted kappa values for categorical data . the strength of the agreement was interpreted according to landis and koch , who classified agreement with a kappa value of 00.20 as poor , 0.210.40 as fair , 0.410.60 as moderate , 0.610.80 as good and 0.811 as very good . the analysis was performed using the statsdirect software ( version 2.7 , statsdirec ltd . , phenotypic and genotypic characterization of canine s. pseudintermedius isolatesisolatepcr icaa / icadcramtptube test1+/+redabsentabsent2+/+blackmoderateweak3+/+blackmoderateweak4+/+blackmoderateweak5+/+blackmoderatemoderate6+/+blackmoderatemoderate7+/+blackmoderateweak8+/+almost blackweakweak9+/+blackstrongweak10+/+blackmoderateweak11+/+blackstrongmoderate12+/+almost blackweakweak13+/+redabsentabsent14+/+blackmoderateweak15+/+blackweakweak16+/+almost blackweakweak17+/+blackmoderatemoderate18+/+almost blackweakweak19+/+blackmoderatemoderate20/redweakmoderate21+/+almost blackweakweek22+/+blackmoderateweak23+/+blackweakweak24+/+blackmoderateweak25+/+almost blackweekweak26+/+blackweakweak27+/+blackmoderateweak28/redmoderateweak29+/+almost blackweakweak30+/+almost blackweakweak31+/+almost blackweakweak32+/+blackmoderateweak33/redmoderateweak34+/+almost blackmoderateweak35+/+blackmoderateweak36/redweakweak37+/+blackweakweak38+/+blackweakweak39+/+blackmoderateweak40/redabsentabsent41+/+blackweakweak42+/+redabsentabsent43+/+blackweakweak44+/+almost blackweakweak45+/+almost blackweakweak46+/+blackmoderateweak47+/+almost blackweakweak48+/+almost blackweakweak49+/+blackmoderatemoderate50+/+blackstrongmoderate51+/+blackstrongmoderate52+/+blackmoderatemoderate53+/+blackmoderatemoderate54+/+blackstrongweak55+/+blackweakweak56+/+blackstrongweak57+/+blackstrongweak58+/+blackstrongweak59+/+blackstrongweak60+/+blackstrongweak . the tube adherence test results showed that 45 of the 60 isolates ( 75% ) were weak biofilm producers , 11 of the 60 isolates ( 18.3% ) were moderate biofilm producers , and 4 of the 60 isolates ( 6.6% ) were biofilm non - producers ( fig . non - biofilm - producing isolate ( a ) , weak biofilm - producing isolate ( b ) and moderate biofilm - producing isolate ( c ) . ) , for a total of 56 ( 93.3% ) positive isolates and 4 ( 6.6% ) negative isolates . non - biofilm - producing isolate ( a ) , weak biofilm - producing isolate ( b ) and moderate biofilm - producing isolate ( c ) . by mtp , 25 out of 60 ( 41.6% ) were classified as weekly adherent , 21 ( 35% ) were classified as moderately adherent , 10 ( 16.6% ) were classified as strongly adherent , and 4 ( 6.6% ) were classified as non - adherent , for a total of 56 ( 93.3% ) positive isolates and 4 ( 6.6% ) negative isolates . investigation of biofilm production by cra showed that 38 of the 60 ( 63.3% ) s. pseudintermedius isolates were found to produce typical black colonies , 14 ( 23.3% ) of the 60 strains produced almost black colonies , and the remaining 8 ( 13.3% ) strains produced red colonies even after 48 hr of incubation ( fig . ( b ) almost black colonies of biofilm - producing s.pseudintermedius isolates , ( c ) red colonies of non - biofilm - producing s. pseudintermedius isolates . ) , for a total of 52 ( 86.6% ) positive isolates and 8 ( 13.3% ) negative isolates . ( b ) almost black colonies of biofilm - producing s.pseudintermedius isolates , ( c ) red colonies of non - biofilm - producing s. pseudintermedius isolates . the presence of the icaa ( 134 bp ) and icad ( 166 bp ) genes was demonstrated by amplification of the corresponding fragments ( fig . 3fig . lane 1 , 134 bp ( ica - a ) band from s. pseudintermedius ; lane m , 100 bp molecular weight marker ; lane 2 , 166 bp ( ica - d ) band from s. pseudintermedius . ) . icaa and icad genes were detected concomitantly in 55 ( 91.6% ) of 60 isolates . lane 1 , 134 bp ( ica - a ) band from s. pseudintermedius ; lane m , 100 bp molecular weight marker ; lane 2 , 166 bp ( ica - d ) band from s. pseudintermedius . statistical analysis : the agreement between the tube adherence test and mtp ( table 3a 
 table 3.a . comparison of mtp test and tube adherence test ) and between the cra and tube adherence test ( table 3b ) using categorical data was fair ( kappa=0.3 ) , while it was moderate ( kappa=0.5 ) between the cra and mpt ( table 3c ) . however , weak slime - producing strains by cra ( almost black ) ( n=14 ) were regularly classified in the same manner by tube adherence ( n=14 ) and mpt ( n=13 ) . the agreement obtained when grouping the results as dichotomous between cra vs tube adherence and cra vs mpt was good ( kappa=0.6 ) and very good ( kappa=1 ) between tube adherence test vs mpt . the percentage of matching results ( considering 2 classes : producers and non - producers ( np ) ) between the cra and tube adherence test and between cra and mpt was 93.3% ( table 3b and 3c ) . the percentage of matching results ( considering 2 classes : producers and np ) between mpt and 
 the tube adherence test was 100% ( table 3a ) . all positive samples were classified in the same way by the three phenotypic tests , while four out of the eight negative results by the cra were classified as positive with the other two tests . the results of the icaa and icad gene pcrs showed good agreement ( kappa=0.7 ) with the cra results . the percentage of matching results ( considering 2 classes : producers and np ) between these two tests was 95% ( table 3d ) . the percentage of matching results ( considering 2 classes : producers and np ) between pcr and mtp and between pcr and tube adherence was 88.3% ( table 3e and 3f ) . biofilm production by s. pseudintermedius may play an important role in the pathophysiology of disease and potentially colonization and could be a contributing factor in the rapid worldwide emergence of mrsp [ 25 , 26 ] . the phenotypic data reported in our study demonstrate that the majority of s. pseudintermedius isolates ( 96% ) were able to produce biofilms . moreover , pcr revealed the concomitant presence of icaa and icad genes in 91% of the 60 isolates studied . all isolates had either both icaa and icad genes or had neither gene ; no strain showed the presence of one gene . these data confirm the fact that both genes are part of one operon , so the entire operon was either present or absent . these results agree with those of other studies of s. epidermidis and s. aureus 
 that demonstrated that biofilm production occurs only when both icaa and icad genes are expressed [ 1 , 8 , 17 ] . our results differ from those reported in a study   in which the biofilm forming ability of staphylococcus pseudintermedius from dogs was characterized . in that study , icaa was detected in 109/140 ( 77.9% ) s. pseudintermedius isolates , and icad was detected in 106/140 ( 75.7% ) isolates , demonstrating that some isolates had a single icaa or icad gene . however , the authors found that 96% ( 136/140 ) of s. pseudintermedius isolates were classified as strong or moderate biofilm producers by mpt and concluded that there was no association between the presence of ica genes and biofilm formation . whether the difference in results obtained can be explained by the different primers and pcr conditions used 88.3% of all the strains screened had matching results by the tube adherence test , mtp and pcr analysis . good concordance ( 95% ) was found between the pcr - based analysis and the cra . this association was further confirmed by the good agreement ( kappa=0.7 ) between the cra and icaa and icad gene pcr results . this finding is in agreement with what was previously described by other authors [ 3 , 34 ] . comparing the phenotypic methods and considering the two classes ( biofilm producer and biofilm non - producer ) , the percent of matching results between the cra method and the tube adherence test and between the cra method and mtp was 93.3% . several authors consider the tube adherence test and mtp reliable and sensitive quantitative techniques for biofilm screening [ 12 , 22 , 24 , 29 ] . interestingly , three of 55 icaa / icad positive strains were biofilm non - producers according to all three phenotypic methods . similarly , a previous study   observed that only 24 of 35 s. aureus mastitis bovine isolates positive for icaa / icad produced biofilms in vitro . other authors   observed that s. aureus strains , despite having the ica locus , may fail to form biofilms in vitro , as biofilm formation on inert surfaces is highly sensitive to growth conditions . another study   reported that cra red variants of slime - positive s. aureus and s. epidermidis were found to lack the icaa and icad genes as well as the whole ica 
 locus . the authors suggested that the altered phenotype might be associated with deletion of the entire ica locus . however , using the cra method , we observed that the red colonies of the three isolates that failed to turn black even after 48 hr of incubation carried the icaa and icad genes . one of the common mechanisms that gives rise to slime - negative variants in s. epidermidis is insertional inactivation of the ica locus by the insertion sequence is256 , which has been detected in s. pseudintermedius as well . it has also been reported that failure of staphylococcal strains that possess the ica locus to form a biofilm in vitro could be due to point mutations in the locus and/or other yet unidentified factors that negatively regulate pia production or influence biofilm formation . nevertheless , our results showed that four isolates determined to be biofilm producers by tube adherence test and microtiter plate test did not possess the icaa and icad genes . these four strains are very interesting , since they imply the possibility that these isolates could form a biofilm in vitro not containing pia . recent studies have shown that additional components , such as accumulation - associated protein ( aap ) , independent of dna and rna or in cooperation with the ica operon may be important in biofilm production by staphylococci [ 8 , 16 ] . bap ( biofilm - associated protein ) and ica  independent mechanisms have been shown to be involved in the biofilm formation of s.aureus [ 11 , 15 ] . interestingly , bap homologue protein 
 ( bhp ) is found in human s. epidermidis strains and induces an alternative mechanism of biofilm formation that does not depend on pia . the four ica - negative isolates could be false positives by the tube adherence test and microtiter plate test . even though the microtiter plate method and tube adherence assay are considered reliable and sensitive quantitative methods for biofilm screening , there are certain limitations to both methods . reading the results of the tube adherence method may be difficult , especially if the observer is not familiar with this technique . moreover , observers frequently disagree with regard to interpretation of the results of the tube adherence test , particularly when interpreting weak reactions . consequently , high variability has been observed , and classification as biofilm positive and negative has been difficult by this method [ 9 , 14 , 18 , 21 , 31 ] . the microtiter plate method 
 has day - to - day and to some extent well - to - well technical variations with occasional outliers . it has been demonstrated that phenotypic expression of biofilm formation is highly susceptible to in vitro conditions , and hence , variability can be detected by different methods . therefore , a combination of various methods ( phenotypic and genotypic ) is useful for identifying biofilm - producing s. pseudintermedius . this is especially important considering the role of biofilms in evasion of the immunological defenses and resistance to antibiotic therapy and the difficulty of pathogen eradication . however , s. pseudintemedius biofilm production needs to be further investigated , since several authors have obtained conflicting results .
biofilm - forming ability is increasingly being recognized as an important virulence factor in several staphylococcus species . 
 this study evaluated the biofilm - forming ability of sixty canine derived clinical isolates of s. pseudintermedius , using three phenotypic methods , microtiter plate test ( mtp ) , congo red agar method ( cra ) and tube adherence test , and the presence and impact of biofilm - associated genes ( icaa and icad ) . 
 the results showed that icaa and icad genes were detected concomitantly in 55 ( 91.7% ) of 60 isolates . 
 a majority ( 88.3% ) of the strains screened had matching results by the tube adherence test , mtp and pcr analysis . 
 better agreement ( 95% ) was found between the pcr - based analysis and the cra . 
 results of the icaa and icad gene pcrs showed good agreement with cra results , with a kappa of 0.7 . comparing the phenotypic methods , 
 the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good . considering two classes ( biofilm producer and biofilm non - producer ) , 
 the percentage of matching results between the cra method and the tube adherence test and between the cra method and the mtp was 93.3% . 
 a concordance of 100% was revealed between the mtp and the tube adherence test . 
 the results indicate a high prevalence of the ica genes within s. pseudintermedius isolates , and their presence is associated with in vitro formation of a biofilm . a combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in s. pseudintermedius .