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Achromobacter xylosoxidans Genomic Characterization and Correlation of Randomly Amplified Polymorphic DNA Profiles with Relevant Clinical Features of Cystic Fibrosis Patients

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Achromobacter xylosoxidans Genomic Characterization and Correlation of Randomly Amplified Polymorphic DNA Profiles with Relevant Clinical Features of Cystic Fibrosis Patients
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  J OURNAL OF  C LINICAL   M ICROBIOLOGY , Apr. 2010, p. 1035–1039 Vol. 48, No. 40095-1137/10/$12.00 doi:10.1128/JCM.02060-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.  Achromobacter xylosoxidans  Genomic Characterization andCorrelation of Randomly Amplified Polymorphic DNA Profiles of Cystic Fibrosis Patients   Annarita Magni, 1 †* Maria Trancassini, 1 † Paola Varesi, 3 Valerio Iebba, 2  Anna Curci, 1 Claudia Pecoraro, 3 Giuseppe Cimino, 3 Serena Schippa, 1 ‡ and Serena Quattrucci 3 ‡  Department of Public Health Sciences, Sapienza University, Rome, Italy 1  ; Department of Pediatrics, Sapienza University, Rome, Italy 2  ; and Regional Cystic Fibrosis Center, Pediatrics Department, Sapienza University, Rome, Italy 3 Received 20 October 2009/Returned for modification 21 December 2009/Accepted 15 January 2010  Achromobacter xylosoxidans  is an emerging pathogen increasingly being isolated from respiratory samples of cystic fibrosis (CF) patients. Its role and clinical significance in lung pathogenesis have not yet been clarified.The aim of the present study was to genetically characterize  A. xylosoxidans  strains isolated from CF patientsby use of randomly amplified polymorphic DNA (RAPD) profiles and to look for a possible correlation betweenRAPD profiles and the patients’ clinical features, such as their spirometry values, the presence of concomitantchronic bacterial flora at the time of isolation, and the persistent or intermittent presence of   A. xylosoxidans strains. A set of 106 strains of   A. xylosoxidans  were typed by RAPD analysis, and their profiles were analyzedby agglomerative hierarchical classification (AHC) and associated with the patient characteristics mentionedabove by factorial discriminant analysis (FDA). The overall results obtained in this study showed that (i) thereis a marked genetic relationship between strains isolated from the same patients at different times, (ii)characteristic RAPD profiles are associated with different predicted classes for forced expiratory volume in 1 s(FEV1%), (iii) some characteristic RAPD profiles are associated with different concomitant chronic flora(CCF) profiles, and (iv) there is a significant division of RAPD profiles into “persistent strains” and “inter-mittent strains” of   A. xylosoxidans . These findings seem to imply that the lung habitats found in CF patientsare capable of shaping and selecting the colonizing bacterial flora, as seems to be the case for the  A. xylosoxidans  strains studied. Cystic fibrosis (CF) is the most common lethal genetic dis-ease, causing a chronic infection of the respiratory tract, whichin turn leads to progressive respiratory deficiency (6, 15).  Pseudomonas aeruginosa  is the most frequently found Gram-negative pathogen in the sputa of patients with CF, while Staphylococcus aureus  is the most frequently found Gram-pos-itive one. Recently, new pathogens have also emerged, such as  Burkholderia cepacia  complex,  Stenotrophomonas maltophilia ,and  Achromobacter xylosoxidans  (3, 9, 18, 19, 20, 24). Althoughthe clinical significance of   A. xylosoxidans  is not yet clear, it isincreasingly being isolated from the sputum cultures of CFpatients. Tan et al. (22) found that 2.3% of CF patients had atleast 3 positive cultures for  A. xylosoxidans  during a 6-monthperiod. The U.S. Cystic Fibrosis Foundation’s National PatientRegistry reported an increase of 4.5%, from 1995 to 2002 (1,2), in the frequency of isolation of this microorganism from CFpatients. Recently,  A. xylosoxidans  has been considered a noso-comial pathogen, particularly in immunocompromised pa-tients, causing a variety of infections, including bacteremia,meningitis, pneumonia, and peritonitis (8, 23, 25).  Achromo- bacter   spp. are aerobic, nonfermentative, Gram-negative bacilli(5, 7, 21) that are frequently misidentified by routine labora-tory tests, thus seriously compromising control measures re-lated to epidemiology studies. These microorganisms are oftenhighly resistant to various antibiotics, including   -lactams,quinolones, aminoglycosides, and carbapenems, all commonlyused for the management of lung infection in CF patients.Considering the importance of bacterial lung infections inCF patients, our goals were (i) to assess the genetic relation-ships among isolated  A. xylosoxidans  strains by randomly am-plified polymorphic DNA (RAPD) analysis and (ii) to usemultivariate analysis techniques to look for possible correla-tions between  A. xylosoxidans  RAPD profiles and patients’clinical features (predicted classes for forced expiratory vol-ume in 1 s [FEV1%], presence of concomitant chronic flora[CCF] during the isolation step, and persistent or intermittentpresence of   A. xylosoxidans ). The study was conceived in orderto give a picture of adaptive changes of   A. xylosoxidans  duringlung infection in patients with CF and to improve our knowl-edge about this emerging pathogenic species, highlighting itspotential role in CF disease. MATERIALS AND METHODSEthics.  All patients were involved in the study after providing written consent.The study protocol was approved by the Committee on Ethical Practice of thePoliclinico Umberto I, Rome, Italy. Patients.  From January 2005 to January 2007, our laboratory cultured respi-ratory samples from 450 patients attending the Cystic Fibrosis Centre of thePediatric Department of Policlinico Umberto I of Rome.  A. xylosoxidans  wasisolated from the sputum cultures of 40 of these 450 patients. For the presentstudy, we selected the 16 patients among these 40 for whom  A. xylosoxidans * Corresponding author. Mailing address: Department of PublicHealth Sciences, Sapienza University, Piazzale Aldo Moro 5, 00185Rome, Italy. Phone: 390649914610. Fax: 390649914641. E-mail:annarita.magni@uniroma1.it.† A. Magni and M. Trancassini contributed equally to this research.‡ S. Quattrucci and S. Schippa contributed equally to this research.  Published ahead of print on 27 January 2010.1035  strains were isolated more than once. We divided the 16 selected patients intothe following three FEV1% classes (following the European Respiratory Soci-ety’s criteria): class 3, mild obstruction or normal (  70%); class 2, moderateobstruction (  40% and   70%); and class 1, severe obstruction (  40%). A further subdivision was based on the persistent or intermittent presence of   A. xylosoxidans . Table 1 summarizes the patients’ demographic and clinical features. Microbiological methods.  All samples were cultured by using appropriatemedia, including  Burkholderia cepacia  selective agar (BCSA) (bioMe´rieux,Marcy l’Etoile, France) for  B. cepacia  complex isolates. All Gram-negative iso-lates were identified with an API 20NE system (bioMe´rieux, Marcy l’Etoile,France) and with an automated Vitek2 system (bioMe´rieux, Marcy l’Etoile,France). The biochemical results of the API 20NE system (bioMe´rieux, Marcyl’Etoile, France) were read after 48 and 72 h of incubation at 30°C. Oxidaseactivity was checked with dimethyl-paraphenylenediamine disks (bioMe´rieux,Marcy l’Etoile, France). The results of the API 20NE tests and oxidase reaction were further interpreted with the Apilab Plus software package (bioMe´rieux,Marcy l’Etoile, France). All  Achromobacter   sp. strains were cryopreserved at  80°C before use. DNA extraction.  Each strain was grown overnight at 37°C in brain heartinfusion broth (Becton Dickinson, NJ), and DNA extraction was performed byuse of a Wizard genomic DNA purification kit (Promega Corporation, Madison,WI). DNA was resuspended in RNA-free deionized water and quantified byspectrophotometry. Species-specific PCR assay.  Species-specific PCR was performed as describedelsewhere (11). Negative-control PCRs were employed for every experiment.PCR products were separated by electrophoresis in a 2% agarose gel (InvitrogenCorporation, CA), stained with ethidium bromide (EtBr; Invitrogen Corpora-tion), and captured with a DigiDoc-It (UVP, Cambridge, United Kingdom)photographic system. RAPD typing.  The RAPD amplification mixture and cycling conditions were asdescribed elsewhere (13). The primer used was primer 270 (5  -TGCGCGCGGG-3  ). RAPD products were separated by electrophoresis in a 1.5% agarose gel(Invitrogen Corporation, CA). Molecular size markers (Invitrogen Corporation)and a negative control were included in all gels. The gels were stained with EtBr(Invitrogen Corporation) and captured with a DigiDoc-It (UVP) photographicsystem. Data analysis. (i) AHC.  Agglomerative hierarchical classification (AHC), anunsupervised method, was performed on RAPD profiles by means of a binarymatrix generated by the presence/absence of RAPD bands, using Doc-It LSsoftware (UVP), and the subsequent dendrogram was generated with XLStat 7.5(Addinsoft), using a Euclidean distance dissimilarity matrix and the agglomera-tion method of Ward. (ii) PCA.  Using XLStat 7.5 software, linearly dependent data (absence/pres-ence of RAPD bands) were transformed into independent variables (factorialaxes; F1, F2. . .F  n ) through principal component analysis (PCA), an unsuper- vised method. The coordinates of the observations on the factorial axes weretaken into account as the new variables for the subsequent factorial discriminantanalysis (FDA). (iii) FDA.  FDA, a supervised method closely linked to multivariate analysis of  variance, was employed by means of XLStat 7.5 software. Explanatory variables were automatically verified to be linearly independent by calculating the multiplecorrelation of each variable with all the others. Wilks’s lambda test was used tocompare the patients’ clinical features with  A. xylosoxidans  RAPD profiles, anda  P   value of   0.05 was considered statistically significant. RESULTS  A. xylosoxidans  isolates.  During the period of study (January2005 to January 2007), 40 patients among the 450 patientsattending the Regional Centre of Cystic Fibrosis of the Poli-clinico Umberto I of Rome showed the presence of   A. xylosoxi- dans  in sputum samples. In this study, we followed the out-comes of 16 patients showing repeated isolations of   A. xylosoxidans , from July 2005 to January 2007. The isolationrange was 2 to 15 bacterial strains per patient, with a total of 106 strains of   A. xylosoxidans . Among the isolated strains, iden-tified by API 20NE, 56 (52.8%) showed a very good identifi-cation, 38 (35.8%) a good identification, and 12 (11.4%) a lowlevel of discrimination. The  A. xylosoxidans  strains were oftenassociated with CCF. The most frequently isolated concomi-tant strains were  P. aeruginosa  mucoid and rough strains and  S. aureus . The CCF profiles found during the culturing step arereported in Table 2. As Table 2 shows, we obtained six CCFprofiles: ppp, ppa, paa, apa, aap, and aaa. Species-specific PCR assay.  A species-specific PCR assay was performed to validate the chemical identification of   A. xylosoxidans  isolates. For this purpose, oligonucleotides AX-F1and AX-B1 were used. A 163-bp PCR product was detected forall 106  A. xylosoxidans  isolates (data not shown). The resultsobtained by species-specific PCR assay were in agreement with biochemical identification performed with bioMe´rieux systems. Data analysis.  In this study, 106 strains of   A. xylosoxidans  were typed by RAPD analysis. The profiles obtained weresubsequently analyzed by AHC and FDA. These approaches TABLE 1. CF patient characteristics Patient  a Strains Age (yr) FEV1% class Intraindividual similarity(%) (mean  SD) 1M 123, 124, 145 33 1 34.34  3.372M 2, 126, 143 19 3 53.74  13.353F 28, 55, 127 5 3  b 65.69  12.604F 99, 122 40 2 83.33  1.405M 7, 37, 114 24 1 80.45  6.116F 6, 35, 52, 63, 73, 75, 79, 86, 92, 108, 120, 135 33 1 31.99  16.337M 12, 34, 56, 78, 98, 139 34 2 41.50  46.198F 5, 26, 47, 77 32 1/2 53.58  8.319M 25, 46, 71, 83, 90 25 2 34.99  21.1610M 13, 18, 36, 53, 65, 87 30 3 36.43  24.4411F 30, 49, 50, 57, 60, 91, 134 14 3 54.29  22.8412F 11, 22, 27, 45, 64, 89, 133 22 3 56.57  13.8813F 23, 40g, 40p, 59, 80, 100, 112, 118 27 1 58.97  14.8614M 4, 14, 19, 29, 38, 48, 96, 104, 115, 138 38 1 42.22  16.4815M 3, 32, 43, 51, 74, 76, 97, 102, 110, 111, 136, 137 21 1 41.59  20.0216F 1, 8, 9, 10, 15, 21, 31, 41, 81, 82, 93, 94, 95, 119, 121 27 1 32.69  22.65  a M, male patient; F, female patient.  b The FEV1% class was arbitrarily assigned. 1036 MAGNI ET AL. J. C LIN . M ICROBIOL  .   were employed to look for genetic relationships between iso-lated strains and for putative associations between RAPD pro-files and different CF patient clinical features.The RAPD profiles were first analyzed by XLStat software,using hierarchical cluster analysis. Four major well-definedclusters (Fig. 1) were obtained, as shown in the resulting den-drogram. The first cluster (A) grouped 41/106 bacterial strains, while the second one (B) grouped 8/106 bacterial strains, allfrom the same patients, the third (C) grouped 30/106 bacterialstrains, and the fourth (D) grouped 27/106 bacterial strains.Intraindividual similarity was calculated by means of the Diceindex, with values ranging from 31.99% to 83.33% (Table 1), with a mean value of 40.86%    22.81% (95% confidenceinterval [CI]  2.21).FDA of RAPD profiles showed that characteristic genomicprofiles were associated with predicted FEV1% classes (Fig.2). Predicted FEV1% classes 1 and 3, as well as classes 2 and3, were significantly separated (  P     0.0001), indicating thatthere were significantly different RAPD profiles associated with these spirometry classes. Of particular interest are strains47 and 137, isolated from two patients, of FEV1% class 2 andFEV1% class 1, respectively. FDA reclassified these strains asFEV1% class 1 (  a posteriori  probability, 73%) and FEV1%class 2 (  a posteriori  probability, 54%), respectively.FDA of RAPD profiles also showed a correlation betweenthe RAPD profiles and some of the CCF profiles (Table 2).RAPD profiles significantly differed between almost all theCCF profiles, as shown in Fig. 3.  A. xylosoxidans  RAPD profiles were also analyzed by FDA inorder to study a possible relationship between the genotypes of the strains and their persistence or intermittence in CF pa- TABLE 2. Concomitant chronic bacterial flora profiles Profile  a Presence of bacterium  Pseudomonas aeruginosa mucoid strain  Pseudomonas aeruginosa rough strain Staphylococcus aureus ppp     ppa     paa     apa     aap     aaa      a p, presence; a, absence. FIG. 1. AHC. Genetic relationships between  A. xylosoxidans  strains are shown, as estimated by clustering analysis of genomic RAPD profiles.The Euclidean distance dissimilarity method and the agglomeration method of Ward were employed. The threshold defining a cluster was set at80% similarity. A comprehensive text string with strain number and patient ID was added at the bottom of each RAPD profile.V OL  . 48, 2010 GENOMIC CHARACTERIZATION OF  ACHROMOBACTER  SPP. 1037  tients. The classification of   A. xylosoxidans  RADP profilesshowed a significant division (  P   0.0004) into two groups, withthe first one grouping RAPD profiles of persistent strains andthe second one grouping those of intermittent strains (Fig. 4).Strain 137, isolated from a persistently colonized patient, wasreclassified as intermittent. DISCUSSION This is the first study focused on the genetic characterizationof   A. xylosoxidans  strains isolated from CF patients and itspossible correlation with their clinical features. The overallresults obtained in this study, although the patient number waslow, showed a genetic relationship among  A. xylosoxidans strains isolated from the same patient and a strong associationconnecting the RAPD profiles of   A. xylosoxidans  and CF pa-tients’ clinical characteristics. Our findings could be related tothe particular lung habitat present in CF patients, which iscapable of shaping and selecting the colonizing bacterial flora(14), generating significantly different RAPD profiles of indig-enous bacterial flora. The reclassification of some strains, suchas strains 137 and 47, supports our results.Strain 137 was reclassified as belonging to FEV1% predictedclass 2 and as an intermittent strain, in contrast to the otherstrains isolated from the same patient. This could be explainedby the fact that strain 137 was acquired by the patient at a latertime than the other ones, and thus its pathoadaptive evolutioncould be different from that of the other (older) colonizingstrains.Strain 47, isolated from patient 8F, was reclassified as aFEV1% class 1 strain. This patient had a borderline predictedFEV1% value of 35% to 45% during the study period, so it wasnot surprising that this RAPD profile was reclassified.Finally, strains 28, 55, and 127, isolated from patient 3F, who was too young to be subjected to spirometry assay, were cor-rectly classified as FEV1% class 3 strains (mild obstruction ornormal status), in agreement with the patient’s clinical condi-tions, as she was not hospitalized or subjected to intravenoustherapy. This also seems to support the discriminatory powerof RAPD profiles and FDA.The clinical characteristics selected for the study are factorsinfluencing and clinically characterizing the pulmonary habitat.The capability of different habitats to produce different forcesand selective pressures, promoting colonization of some bac-terial species or strains in place of others, has been reported inmany recent studies (10, 12, 17). Strains with related RAPDprofiles share common genetic traits, and it is possible thatthese common traits are phenotypic characters important fortheir survival in CF patients’ lung tissue. In the future, it wouldbe of great interest to understand, through DNA sequencing,the phenotypic traits that allow these bacteria to survive and tobe at an advantage in these habitats. The survival of speciesdepends on a balance between fidelity of DNA replication andrepair, and the generation of variation allows adaptation tonovel environmental challenges. The obtained partition be-tween RAPD profiles of isolated persistent strains and RAPD FIG. 2.  A. xylosoxidans  RAPD profiles grouped by spirometricclass. The percentages of variation described by the factorial axes (F1and F2) are given in parentheses. The center of gravity for each groupis reported with a filled symbol. The Mahalanobis distances (  D 2 ) be-tween the three centers of gravity were as follows: for FEV1% class 1 versus FEV1% class 2, 11.2; for FEV1% class 1 versus FEV1% class 3,25.6; and for FEV1% class 3 versus FEV1% class 2,  27.8. Compar-isons of the aforementioned distances were statistically significant(Fisher tests;  P     0.0001) for FEV1% class 1 versus FEV1% class 3and FEV1% class 2 versus FEV1% class 3. The predictability of themodel is 96.2%, and Wilks’s lambda value  0.075.FIG. 3.  A. xylosoxidans  RAPD profiles grouped by the presence of chronic concomitant bacterial flora. Profiles were colored as follows:ppp  blue, aaa  red, apa   yellow, paa  green, aap  orange, andppa    light cyan. The Mahalanobis distances (  D 2 ) between the six centers of gravity were as follows: for ppp versus ppa, 124.9; for ppp versus paa, 152.5; for ppp versus apa, 130.2; for ppp versus aap, 160.4;for ppp versus aaa, 116.1; for ppa versus paa, 32.2; for ppa versus apa,19.5; for ppa versus aap, 25.5; for ppa versus aaa, 26.6; for paa versusapa, 34.4; for paa versus aap, 13.7; for paa versus aaa, 24.6; for apa versus aap, 28.5; for apa versus aaa, 30.1; and for aap versus aaa, 25.9.Comparisons of the aforementioned distances were statistically signif-icant (Fisher tests;  P   0.0001) for all the groups, except for profile aap versus ppa, aap versus paa, apa versus ppa, and aaa versus paa (  P    0.78). The predictability of the model is 93.4%, and Wilks’s lambda value  0.003.1038 MAGNI ET AL. J. C LIN . M ICROBIOL  .  profiles of isolated intermittent strains could be explained bydifferent pathoadaptive evolutionary rates (4).Individuals with CF are particularly susceptible to lung in-fection by a limited spectrum of microbial pathogens, among which  A. xylosoxidans  is an important emergent one (16).Chronic airway bacterial infection in patients with CF leads toprogressive damage of lung tissue and, ultimately, to the deathof patients. During long-standing persistence of particular bac-terial strains, such as  A. xylosoxidans , the environmental pres-sures are likely to select bacterial mutants more suited to theinflamed lung tissue. Our results could be explained by adap-tive changes in the physiology of   A. xylosoxidans  during chroniclung infection. These changes could result in a genome adap-tive shift to support the growth under nutritional and mi-croaerobic conditions created by the suppurative secretions inthe lungs of patients with CF.We can suppose that the biotypes found associated withdifferent degrees of lung function could be the result of alter-native genome shaping due to various lung habitats. Moreover,the different biotypes could also play an important role in lunginflammation/damage. It was recently reported (9) that  A. xylosoxidans  can cause a level of inflammation similar to thatinduced by  P. aeruginosa  in chronically infected CF patients,supporting the observation that this bacterial species is anemerging pathogen in CF.These results provide important clues about the persistencestrategies used by  A. xylosoxidans  during progressive CF lungdisease. Further biomolecular studies will be performed togain more comprehensive knowledge about the involvement of   A. xylosoxidans  in CF disease. 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Comparison of the distances was statistically sig-nificant (Fisher tests;  P   0.004) between the two groups of patients.The predictability of the model is 97.2%, and Wilks’s lambda value  0.306. Data were manually scattered along the  y  axis to avoid overlap-ping points.V OL  . 48, 2010 GENOMIC CHARACTERIZATION OF  ACHROMOBACTER  SPP. 1039
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