FENOTIPURILE DE REZISTENÞÃ LA ANTIBIOTICE ALE … · fenotipurile de rezistenÞÃ la antibiotice ale tulpinilor bacteriene izolate din ulcere gambiere, pe durata a 5 ani, Într-un

FENOTIPURILE DE REZISTENÞÃ LA ANTIBIOTICE ALE TULPINILOR BACTERIENE IZOLATE DIN ULCERE

GAMBIERE, PE DURATA A 5 ANI, ÎNTR-UNDEPARTAMENT DE DERMATOLOGIE

ANTIBIOTICS RESISTANCE PHENOTYPES OF THE BACTERIAL STRAINS ISOLATED

FROM LEG ULCERS DURING 5 YEARS IN A DERMATOLOGY DEPARTMENT

MARA MÃDÃLINA MIHAI*, MIRCEA IOAN POPA*, LILIANA GABRIELA POPA*,**, ALEXANDRA

VICTORIA ION**, ANDREEA CÃLUGÃREANU**, IULIA SOLOMON**, CÃLIN GIURCÃNEANU*,**

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Rezumat

Rezistenþa la antibiotice reprezintã o realã problemã lanivel internaþional. Date recente epidemiologiceevidenþiazã o prevalenþã mai înaltã a infecþiilor cumicroorganisme multi-rezistente în mediu spitalicesc înRomânia comparativ cu alte state europene. Vindecareaîntârziatã a ulcerelor gambiere determinã spitalizãrifrecvente, iar curele repetate cu antibiotice cresc risculdezvoltãrii rezistenþei. Cu scopul de a identifica profilulfenotipic de sensibilitate la antibiotice al microorganismelorizolate de la nivelul ulcerelor gambiere, s-a efectuat unstudiu retrospectiv în Departamentul de Dermatologie alSpitalului Universitar de Urgenþã „Elias”, în perioadaianuarie 2011 - decembrie 2015. 340 de pacienþi au fostdiagnosticaþi cu ulcere gambiere (venoase, arteriale,diabetice sau cu etiologie mixtã). 227 de examenebacteriologice efectuate din prelevate au avut un rezultatpozitiv, cu identificarea a 257 de tulpini bacteriene. Profilulde sensibilitate la antibiotice a fost testat conform ghiduluiInstitutului de Standardizare pentru Laboratoare ºi Clinici(CLSI) ºi cu ajutorul sistemelor automate (Vitek2C/

Summary

Antibiotics resistance is a real problem at theinternational level. Recent epidemiological data show ahigher incidence of infections with multiresistant hospitalmicroorganisms in Romania than in the other Europeancountries. The delayed healing of the leg ulcers causesfrequent hospitalizations and the repeated antibiotictreatments increase the risk of developing resistance. Inorder to identify the antibiotic sensitivity phenotypicalprofile of the microorganisms that were isolated from legulcers, a study was performed retrospectively at theDermatology Department of the Emergency UniversityHospital „Elias“ from January 2011 to December 2015.340 patients were diagnosed with leg ulcers (venous,arterial, diabetics or with mixed etiology). 227 bacteri-ological examinations had a positive result with theidentification of 257 bacterial strains. The antibioticsensitivity profile was tested according to the guide of theClinical and Laboratory Standards Institute (CLSI) and byusing automated systems (Vitek2C/Phoenix BD). A D-testwas performed as well. Out of a total of 22 bacterial species,

* Universitatea de Medicinã ºi Farmacie „Carol Davila“, Bucureºti, România.„Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.

** Departamentul de Dermatologie „Elias“, Spitalul Universitar de Urgenþã Bucureºti, România.Dermatology Department, „Elias” University Emergency Hospital, Bucharest, Romania

STUDII CLINICE ªI EXPERIMENTALECLINICAL AND EXPERIMENTAL STUDIES

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DermatoVenerol. (Buc.), 62: 7-23

Introducere

Ulcerele membrelor inferioare sunt patologiifrecvente, cu un impact semnificativ asupracalitãþii vieþii pacienþilor, atât prin evoluþia delungã duratã, cât ºi prin dezvoltarea unorcomplicaþii severe. Tratamentul presupunecosturi crescute, iar dezvoltarea rezistenþei laantibiotice ºi antiseptice a microorganismelor cecolonizeazã leziunile comportã un înalt riscepidemiologic.

În contextul spitalizãrilor repetate ºi alterapiei îndelungate cu antibiotice la pacienþii cuulcere cronice se ridicã o altã problemã impor-tantã, aceea a emergenþei tulpinilor rezistente lamultiple clase de antibiotice.

Grupate sub acronimul ESKAPE, Enterococcusfaecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumanii, Pseudomonas aeruginosa,Enterobacter spp., alãturi de alte specii bacterieneprecum Escherichia coli, Proteus spp.Morganellamorganii, Serratia marcescens, sunt recunoscutepentru capacitatea de a dezvolta rezistenþãmultiplã la antibiotice ºi de a determina infecþiipersistente, care nu rãspund la schemeleterapeutice standard. Aceste microorganismereprezintã izolate frecvente de la nivelul plãgilorcutanate cronice, atât la nivel internaþional [1], cât

Introduction

Leg ulcers are high-incidence pathologieswith a significant impact on the patient’s lifequality, both through the long-term evolution,and through the development of severecomplications. The treatment involves high costs,and the development of antibiotic and antisepticresistance of the microorganisms colonizing thelesions represents a high epidemiological risk.

Through repeated hospitalizations and pro-longed antibiotic therapies, another importantissue arises for the chronic ulcers patients: theemergence of multiple class antibiotic resistantstrains.

Also known as ESKAPE, Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumanii, Pseudomonas aeruginosa,Enterobacter spp., together with other bacterialspecies such as Escherichia coli, Proteus spp.Morganella morganii, Serratia marcescens, , have arecognized capability to develop multipleresistance towards antibiotics and to causepersistant infections that do not respond tostandard therapeutic schemes. These micro-organisms are frequent isolates from the chroniccutaneous plagues, internationally [1] and inRomania as well [1,2,3]. Antibiotic multiresistant

Phoenix BD). S-a efectuat ºi testul D. Dintr-un total de 22de specii bacteriene, cele mai frecvent izolate au fostStaphylococcus aureus (121 tulpini, 47%) ºi Pseudomonasaeruginosa (51 tulpini, 19.8%). Asocierea acestor speciiîntr-un prelevat a fost mai frecventã comparativ cu alteasocieri microbiene. Aproximativ o jumãtate dintretulpinile S. aureus a prezentat un fenotip de multi-rezistenþã la antibiotice (56 tulpini, 46.3%), iar 35.5% aufost meticilino- rezistente (MRSA). Rezistenþa lamacrolide, lincosamide ºi streptograminã B a fost mai înaltãla tulpinile MRSA (69.7%), comparativ cu cea a tulpinilorde S. aureus meticilino-sensibile. Pe durata studiului s-aobservat o rezistenþã în creºtere la meticilinã. Mai mult, afost detectatã o prevalenþã înaltã a rezistenþei la antibioticea tulpinilor izolate. Rezultatele acestui studiu evidenþiazãimportanþa monitorizãrii sensibilitãþii la antibiotice, carepermite administrarea unui tratament adecvat, dar ºicontrolul epidemiologic al infecþiilor nozocomiale.

Cuvinte cheie: Rezistenþã la antibiotice, MRSA, ulcergambier.

the most frequently isolated were Staphylococcus aureus(121 strains, 47%) and Pseudomonas aeruginosa (51strains, 19.8%). The association of these species in a samplewas more frequent than other microbial associations.Almost half of the S. aureus strains showed an antibioticmultiresistant phenotype (56 strains, 46.3%), and 35.5%were methicillin- resistant (MRSA). There was a higherresistance towards macrolides, lincosamides andstreptogramin B for the MRSA strains (69.7%) than for themethicillin-sensitive S. aureus strains. During the studyan increasing resistance towards methicillin was observed.Moreover, a high incidence of antibiotic resistance of theisolated strains was detected. The results of this studyhighlight the importance of monitoring the antibioticsensitivity, which allows the administration of an adequatetreatment and the epidemiological control of the nosocomialinfections.

Key-words: antibiotic resistance, MRSA, leg ulcer.

Intrat în redacþie: 25.11.2016Acceptat: 15.12.2016

Received: 25.11.2016Accepted: 15.12.2016

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ºi în România [1,2,3]. Tulpinile bacterienemultirezistente la antibiotice pot contribui ladezvoltarea infecþiilor cronice cutanate, afectândcapacitatea de vindecare a ulcerelor gambiere.Mai mult, pacienþii cu potenþial de apãrare imunãredus, pot dezvolta complicaþii sistemice severe.Modalitãþile de abordare terapeuticã suntlimitate, având un impact important asupraºanselor de vindecare ºi/ sau supravieþuire.

Cele mai recente date cu privire la incidenþainfecþiilor sistemice cu tulpini multirezistente înRomânia este îngrijorãtoare: mai mult de 50%dintre tulpinile de P. aeruginosa raportate au fostrezistente la ceftazidim, piperacilinã/ tazo-bactam, carbapeneme ºi/sau fluorochinolone ºimai mult de 50% dintre tulpinile de S. aureus aufost rezistente la Oxacilinã/ Meticilinã (MRSA)[4]. Agenþii antimicrobieni enumeraþi suntutilizaþi frecvent în tratamentul pacienþilordiagnosticaþi cu ulcere gambiere suprainfectate.

Material ºi metodã

Pe baza foilor de observaþie ºi a informaþiilorexistente în baza electronicã de date a SpitaluluiUniversitar de Urgenþã ”Elias”, au fostidentificaþi pacienþii diagnosticaþi cu ulcere alemembrelor inferioare, care s-au prezentat înClinica de Dermatologie ºi Alergologie, înperioada ianuarie 2011-decembrie 2015. A fostluatã în considerare fiecare reinternare apacienþilor, dacã aceasta a avut loc pe duratadesfãºurãrii studiului. Pacienþii au fost clasificaþiîn funcþie de diagnosticul stabilit la internare în 4categorii: ulcer venos, ulcer arterial, ulcer mixt,ulcer gambier de altã etiologie. Au fost analizatedate epidemiologice despre patologia pacienþilorincluºi în studiu precum: prevalenþa fiecãruidiagnostic de boalã care a necesitat internare,incidenþa internãrilor în funcþie de anotimp,frecvenþa internãrilor în spital. Au fost analizatedate demografice precum sexul, vârsta laprezentare, mediul de provenienþã.

S-au analizat rezultatele examenelor micro-biologice ale produselor patologice prelevate dela nivelul plãgilor cutanate, în perioada ianuarie2011-decembrie 2015, din arhiva computerizatã aSpitalului Universitar de Urgenþã ”Elias”. Au fostidentificate rezultatele pozitive ale examenelorbacteriologice ºi tulpinile izolate din secreþiileprelevate de la pacienþii incluºi în studiu.

bacterial strains can contribute to thedevelopment of the chronic cutaneous infections,affecting the healing ability of the leg ulcers.Moreover, the patients with low immune defensecan develop severe systemic complications. Thetherapeutic approaches are limited, significantlyimpacting the healing and/or surviving chances.

The most recent data regarding the incidenceof systemic infections with mutiresistant strainsin Romania is concerning: more than 50% of thereported P. aeruginosa strains were resistanttowards ceftazidime, piperacillin/tazobactam,carbapenems and/or fluoroquinolones and morethan 50% of the S. aureus strains were resistanttowards oxacillin/ methicillin (MRSA) [4]. Thementioned antimicrobial agents are frequentlyused for the treatment of the patients diagnosedwith superinfected leg ulcers.

Material and methods

Based on the observation sheets and on theinformation available in the electronic data bankof the „Elias“ University Emergency Hospital,there were identified patients who were diag-nosed with leg ulcers and investigated at theClinic for Dermatology and Allergology fromJanuary 2011 to December 2015. Each rehos-pitalization was taken into account, if it tookplace within the study’s time frame. Based on thediagnostic set at the hospitalization, the patientswere classified in 4 categories: venous ulcer,arterial ulcer, mixed ulcer, leg ulcer of differentetiology. The epidemiological data about thepathology of the patients included in the studywas analysed, i.e. the incidence of each diseasediagnostic that required hospitalization, theseason when the hospitalization took place, thefrequency of hospitalizations. Demographic datasuch as gender, age and background wasanalysed.

The results of the microbiological exam-inations of the samples from cutaneous plaguesthat were present in the digital archive of „Elias“University Emergency Hospital were analysed.The positive results of the bacteriologicalexaminations and the isolated strains from thesecretions collected from the patients included inthe study were identified. The bacterial strainswere isolated and identified at the Laboratory forMicrobiology at „Elias“ University Emergency

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Tulpinile bacteriene au fost izolate ºi identificateîn Laboratorul de Microbiologie al SpitaluluiUniversitar de Urgenþã „Elias”, conform sche-melor standard de diagnostic microbiologic,identificarea biochimicã utilizând sistemulautomat Vitek 2 ºi truse API. În vederea stu-diului fenotipic al profilurilor de rezistenþã laantibiotice s-au interpretat rezultatele obþinuteprin metoda antibiogramei difuzimetrice stan-dardizate Kirby-Bauer, pe agar Mueller-Hinton,conform ghidului CLSI (Clinical and LaboratoryStandards Institute)[5] ºi cu sisteme automate(Vitek2C / Phoenix BD). Au fost de asemeneaefectuate D-teste pentru evaluarea rezistenþeiinductibile la clindamicinã pentru S. aureus,conform recomandãrilor producãtorului.

Pentru a stabili gradul de rezistenþã laantibiotice al fiecãrei tulpini au fost detectatemicroorganismele MDR (Multi Drug Resistant;multirezistente la antibiotice), XDR (ExtensivelyDrug Resistant; extensiv rezistente la antibiotice)ºi PDR (Pan Drug Resistant; pan-rezistente laantibiotice) conform definiþiilor ghidului ECDC(European Centre for Disease Prevention andControl) [6]. Termenul de MDR este definit carezistenþa concomitentã a unei tulpini la cel puþinun agent antimicrobian din trei sau mai multeclase de antibiotice, stabilite pentru fiecare specieîn parte, excluzând rezistenþa intrinsecã a fiecãreispecii [6]. Rezistenþa extinsã (XDR) reprezintãrezistenþa unei tulpini microbiene la cel puþin unagent antimicrobian din mai multe clase deantibiotice, stabilite pentru fiecare specie în parte,cu conservarea sensibilitãþii la una sau maximdouã clase de antibiotice [6]. Panrezistenþa (PDR)reprezintã rezistenþa unei tulpini bacteriene latoþi agenþii antimicrobieni din toate clasele deantibiotice recomandate pentru a fi testate pentruspecia respectivã [6].

Rezultate

1. Date epidemiologice

S-au identificat 340 pacienþi diagnosticaþi cuulcer al membrelor inferioare, internaþi în Clinicade Dermatologie ºi Alergologie, a SpitaluluiUniversitar de Urgenþã ”Elias”, în perioadaianuarie 2011-decembrie 2015. Din aceºtia 206 aufost diagnosticaþi cu ulcer venos gambier.

Hospital, according to the standard micro-biological diagnostic schemes, and for thebiochemical identification, the automated systemVitek 2 and API test kits were used. In order toperform the phenotypical study of the antibioticresistance profiles, the results obtained throughthe standardized diffusion antibiogram Kirby-Bauer method on Mueller-Hinton agar plates andwith the automatized systems (Vitek2C/ PhoenixBD) were interpreted according to the CLSI guide(Clinical and Laboratory Standards Institute)[5].D-tests were performed as well, in order toevaluate the inducible clindamycin resistance inS. aureus, according to the recommendations ofthe manufacturer.

To identify the degree of the antibioticresistance of each strain, the MDR (Multi DrugResistant), XDR (Extensively Drug Resistant) andPDR (Pan Drug Resistant) micro-organisms weredetected according to the definitions of the ECDCguide (European Centre for Disease Preventionand Control) [6]. MDR is defined as thesimultaneous resistance of a strain towards atleast an antimicrobial agent from three or moredifferent antibiotic classes that are specific foreach particular species, excepting the intrinsicresistance of each species [6]. Extensive DrugResistance (XDR) represents the resistance of amicrobial strain towards at least an antimicrobialagent from more antibiotic classes that arespecific for each particular species, while thesensitivity towards one or maximum twoantibiotic classes is preserved [6]. Panresistance(PDR) represents the resistance of a bacterialstrain towards all the antimicrobial agents fromall the antibiotic classes recommended for testingfor that particular species [6].

Results

1. Epidemiological data

340 patients diagnosed with leg ulcers andhospitalized from January 2011 to December 2015in the Clinic for Dermatology and Allergology ofthe „Elias“ University Emergency Hospital wereidentified. 206 of these were diagnosed with legvenous ulcer.

The patients distribution according to genderwas very balanced with a 169 to 171 female tomale ratio. It was noticed that most of the

Distribuþia pacienþilor în funcþie de sex a fostfoarte echilibratã cu un raport sex feminin/sexmasculin de 169/171. S-a constatat cã majoritateapacienþilor provin din mediul urban (71%) de 2.4ori mai mult comparativ cu mediul rural (29%).Vârsta medie la internare a fost de 64.6 ani(medianã 64 ani) la pacienþii de sex feminin, 68.4ani (medianã 71 ani) la pacienþii de sex masculin.Vârsta maximã a fost de 95 de ani, iar cea minimãde 31 de ani.

Pe durata celor 5 ani, pacienþii incluºi înstudiu au fost internaþi într-un numãr total de 646de ori, incluzând 415 prezentãri pentrudiagnosticul ºi/sau tratamentul ulcerelor deetiologie venoasã, 145 prezentãri pentrucomplicaþii ale insuficienþei venoase cronice(celulitã gambierã, eczemã de stazã, trombozãvenoasã), 2 pentru complicaþii neoplazice aleulcerelor venoase ºi 84 de prezentãri pentruulcere ale membrelor inferioare de altã etiologie(ulcere mixte, ulcere arteriale, ulcere diabetice).

Aproximativ douã treimi (67%) din pacienþiau fost internaþi o singurã datã. O proporþiesemnificativã (10%) a prezentat un numãr deinternãri mai mare de 4 în Clinica deDermatologie ºi Alergologie, numãrul maximfiind de 18 internãri la un pacient cu ulcer venosce a suferit recurenþe infecþioase frecvente.

Pacienþii cu ulcere venoase cronice au avut oproporþie mai mare a recurenþelor, comparativ cupacienþii care sufereau de ulcere gambiere deetiologie diferitã: 10.4% din pacienþi au revenit laspital de 2 ori, faþã de 7.8% în a doua categorie;alþi 15.6% au revenit de cel puþin 3 ori în perioadaianuarie 2011-decembrie 2016, raportat la 11.7%.

În ceea ce priveºte perioada anului în care s-ainternat pacienþii incluºi în studiu, a existat opredominanþã în sezonul cald, 30% din pacienþiau fost internaþi vara, 24.34% primãvara ºi 24.92%toamna, comparativ cu 20.28% iarna.

2. Identificarea rezultatelor examenelormicrobiologice de interes

În perioada ianuarie 2012-decembrie 2015, aufost efectuate 227 de examene microbiologice aleprelevatelor de la nivelul plãgilor cutanate,izolându-se 257 tulpini bacteriene, aparþinândunui numãr total de 22 specii bacteriene. 33 derezultate bacteriologice au fost negative. Cea maifrecventã specie bacterianã a fost S. aureus,

patients come from urban areas (71%),representing 2.4 times more than the ones comingfrom rural areas (29%). The average hospitaladmission age was 64.6 years (64 years median)for female patients and 68.4 years (71 yearsmedian) for male patients. The maximal age was95, and the minimal one 31.

During the 5 years, the patients included inthe study were admitted in the hospital 646times, including 415 visits for the diagnosticand/or the treatment of the ulcers of venousetiology, 145 visits for complications of thechronic venous insufficiency (calf cellulite, stasisdermatitis, venous thrombosis), 2 for neoplasticcomplications of the venous ulcers and 84 for legulcers of different etiology (mixed ulcers, arterialulcers, diabetis ulcers).

Approximately two thirds (67%) of thepatients were only once hospitalized. Asignificant percentage (10%) showed more than 4hospital admissions in the Clinic forDermatology and Allergology, with a maximumof 18 hospital admissions for a patient withvenous ulcer suffering frequent reinfections.

The chronic venous ulcer patients had ahigher recurrence than the patients with calvulcers of different etiology: 10.4% of the patientscame again twice to the hospital, whereas only7.8% from the second group; other 15.6% cameagain at least 3 times in the period January 2011-December 2016, in comparison to 11.7%,respectively.

With respect to the season when the patientsincluded in the study were admitted in thehospital, there was a higher incidence in thewarm than in the cold season: Summer (30%),Spring (24.34%), Autumn (24.92%) and only20.28% in Winter.

2. Identifying the relevant microbiologicalexaminations results

From January 2012 to December 2015, 227microbiological examinations were performed onsamples collected from cutaneous plagues, fromwhich 257 bacterial strains belonging to 22bacterial species were isolated. 33 bacteriologicalresults were negative. The most frequentbacterial species was S. aureus, representingapproximately half of the isolates from the legplagues (121 strains, 47%). The second frequent

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reprezentând aproximativ o jumãtate dintreizolatele de la nivelul plãgilor membrelorinferioare (121 tulpini, 47%). A fost urmatã de P.aeruginosa (51 tulpini, 19.8%), cu o proporþie deaproximativ 1/5 din totalul tulpinilor bacteriene.Enterobacteriile au fost reprezentate de E. coli (22tulpini), Klebsiella spp. din care 13 tulpini de K.pneumoniae ºi 1 tulpinã K. oxytoca, 9 tulpini deProteus spp.,urmate în proporþii mai mici deEnterobacter spp.(3) Morganella morganii (3),Citrobacter freundii (2), Serratia marcescens (2) ºiProvidencia spp. (1). Speciile de Enterococcus aufost depistate la 12 pacienþi cu ulcere cutanate, iarspeciile de Streptococcus au fost reprezentatepredominant de Streptococcus agalactiae, urmatede streptococii β-hemolitici de grup G (3 tulpini)ºi de grup A (1 tulpinã). Alte specii bacterieneidentificate mai puþin frecvent au fostCorynebacterium spp. (2), Acinetobacter baumanii(2) ºi Delfia acidovorans (1) (Fig. 1).

Din punctul de vedere al numãrului de speciibacteriene identificate în cadrul unui examenmicrobiologic, s-a observat faptul cã în

was P. aeruginosa (51 strains, 19.8%), a fifth of thetotal bacterial strains. The identifiedEnterobacteriaceae were E. coli (22 strains),Klebsiella spp. of which 13 strains of K. pneumoniaeand one strain of K. oxytoca and 9 strains ofProteus spp. The following were present with alower incidence: Enterobacter spp (3 strains)Morganella morganii (3), Citrobacter freundii (2),Serratia marcescens (2) and Providencia spp (1). TheEnterococcus species were identified at 12 patientswith cutaneous ulcer, while the Streptococcusspecies were mostly represented by Streptococcusagalactiae, followed by Group G β-hemolyticstreptococcus (3 strains) and Group A β-hemolytic streptococcus (1strain). Other bacterialspecies that were identified with a lowerincidence were Corynebacterium spp. (2 strains),Acinetobacter baumanii (2) and Delfia acidovorans(1) (Fig. 1).

With respect to the number of the identifiedbacterial species by a microbiological exam-ination, in most of the cases a single strain wasidentified (135, 59.5%), followed by the

Bacterial species isolated from chronic skin wounds

Delfia acidovoransAcinetobacter baumannii

Corynebacterium spp.Streptococcus pyogenes_betahemolitic grup A

Streptococcus_betahemolitic grup CStreptococcus agalactiae

Enterococcus spp.Providencia spp.

Serratia marcescensCitrobacter freundii

Morganella morganiiEnterobacter spp.

Proteus spp.Klebsiella spp.

Escherichia coliPseudomonas aeruginosa

Staphylococcus aureus

1221

12233

914

2251

121

0 20 40 60 80 100 120 140

3812

Fig. 1. Specii bacteriene identificate în prelevatele din ulcere cronice gambiereFig. 1. Bacterial species isolated from chronic lower limb wounds

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majoritatea cazurilor a fost identificatã o singurãtulpinã (135, 59.5%), urmatã de asocierea a douãmicroorganisme (50, 22%) ºi, mai rar, a trei speciibacteriene (8, 3.5%). Într-un singur caz de ulcervenos au fost identificate 4 specii bacterienediferite. Cea mai frecvent întâlnitã asocieremicrobianã a fost P. aeruginosa ºi S. aureus înrezultate bimicrobiene (12 din totalul de 50),trimicrobiene (4 din totalul de 8), cât ºi însingurul rezultat cu 4 tulpini bacteriene (S.aureus, P. aeruginosa, C. freundii, streptococ beta-hemolitic de grup G). Aceste asocieri pot avea unimpact important atât asupra rezistenþei, cât ºi atoleranþei la tratamentul antibiotic.

3. Studiul fenotipic al profilurilor derezistenþã la antibiotice al tulpinilorbacteriene identificate

3.1. Staphylococcus aureusÎn perioada ianuarie 2011- decembrie 2015, au

fost identificate 121 tulpini de S. aureus înprelevate ce au provenit de la nivelul ulcerelorcutanate ale membrelor inferioare. Din total,aproximativ douã treimi au fost sensibile lameticilinã (78 tulpini, 64.4%). Mai mult, 7 tulpiniau fost sensibile la penicilinã, antibiotic care nueste recomandat în mod standard în infecþiilestafilococice, considerându-se ineficient datoritãfrecvenþei crescute a rezistenþei (Fig. 2).

Din totalul izolatelor S. aureus, aproapejumãtate au prezentat un fenotip demultirezistenþã la antibiotice MDR, conform

association of two microorganisms (50, 22%) andin rather rare cases, the association of threebacterial species (8, 3.5%). In a single case ofvenous ulcer 4 different bacterial species wereidentified. The most common microbialassociation was that of P. aeruginosa and S. aureusin bimicrobial (12 out of the total of 50) andtrimicrobial results (4 out of the total of 8), with asingle result with 4 bacterial strains (S. aureus, P.aeruginosa, C. freundii, Group G beta-homolyticstreptococcus). These associations can have animportant impact on the resistance and on thetolerance of the antibiotic treatment as well.

3. The phenotypical study of the antibioticresistance profiles of the identifiedbacterial strains

3.1. Staphylococcus aureusIn the time-frame January 2011- December

2015, 121 strains of S. aureus were identified insamples collected from cutaneous leg ulcer.Approximately two thirds of the total weremethicillin sensitive (78 strains, 64.4%).Moreover, 7 strains were penicillin sensitive,antibiotic that is not commonly recommended instaphylococcal infections, since it is consideredineffective because of its frequent resistance (Fig. 2).

Almost half of the S. aureus isolates showed amultiresistance phenotype towards MDRantibiotics, according to the definition above (56strains, 46.3%). 43 of these strains (35.53%) were

Fig. 2. Sensibilitatea la antibiotice a tulpinilor de S. aureus izolate din ulcere cronice gambiereFig. 2. Antibiotic susceptibility for S. aureus strains isolated from chronic lower limb wounds

Antibiotic susceptibility S. aureus R

0 20 40 60 80 100 120 0 10 20 30 40

S Antibiotic susceptibility MRSA

LinezolidTeicoplanin

VancomycinCo-trimoxazole

RifampicinDoxycyclineTetracycline

CiprofloxacinumErythromycinClindamycinGentamicinOxacillinumPenicillinum

LinezolidTeicoplanin

VancomycinCo-trimoxazole

RifampicinDoxycyclineTetracycline

CiprofloxacinumErythromycinClindamycinGentamicinOxacillinumPenicillinum

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definiþiei de mai sus (56 tulpini, 46.3%). Dinacestea, 43 de tulpini au fost rezistente lameticilinã (MRSA) (35.53% din total). Toatetulpinile de stafilococ meticilino-sensibil, darMDR (13 tulpini) au fost izolate de la pacienþidiagnosticaþi cu ulcere venoase. Nu au existattulpini XDR sau PDR (Figura nr. 4).

Aproximativ o jumãtate din tulpinile de S.aureus (61 tulpini, 50.4%) a prezentat fenotipul derezistenþã inductibilã la macrolide-lincosamide-streptogramine (MLSbi), evidenþiat prin testul D.Aceastã rezistenþã încruciºatã a fost exprimatã la69.7% din tulpinile de MRSA, mai intenscomparativ cu tulpinile meticilino-sensibile,unde s-a observat o frecvenþã de 39.7%.

MRSA au fost rezistente la alte 4 clase deantibiotice în urmãtoarele proporþii: amino-glicozide (9/43), fluorochinolone (8/43), tetra-cicline (12/43), ansamicine (rifampicinã) (6/43).Toate tulpinile MRSA au fost sensibile lainhibitorii cãii folaþilor (Cotrimoxazol/ Tri-metoprim-Sulfametoxazol), glicopeptidele Van-comicinã ºi Teicoplanin, precum ºi la oxazo-lidinone (Linezolid) (Figura nr. 5).

Analizând evoluþia în timp a marker-ilor derezistenþã la antibiotice a tulpinilor de S. aureus, s-a observat o frecvenþã de aproximativ 5 ori maimare, a izolatelor MRSA în perioada mai 2014-decembrie 2015 (un an ºi jumãtate), faþã deperioada ianuarie 2011-aprilie 2014 (trei ani ºijumãtate), cu un raport de 25 la 6 tulpini. Acestrezultat evidenþiazã o rezistenþã în creºtere atulpinilor S. aureus, la pacienþii spitalizaþi înClinica de Dermatologie, a Spitalului Universitarde Urgenþã ”Elias”, pe durata ultimilor doi ani(Fig. 3).

3.2. Pseudomonas aeruginosa

În perioada ianuarie 2011- decembrie 2015, 51tulpini de P. aeruginosa au fost identificate înprelevate de la nivelul ulcerelor cutanate alemembrelor inferioare, dintre care 42 au provenitde la nivelul ulcerelor venoase.

Evaluarea profilurilor de rezistenþã pentrutulpinile de P. aeruginosa a arãtat cã majoritatea aumanifestat un nivel de rezistenþã scãzut la claselede antibiotice testate. 18 tulpini bacteriene auprezentat rezistenþã la cel puþin un agentantimicrobian testat, pe când 33 de tulpini au fostsensibile la toate antibioticele testate. 4 tulpini P.

methicillin- resistant (MRSA) . All the methicillinsensitive staphylococcal strains were isolatedfrom patients diagnosed with venous ulcers. NoXDR or PDR strains were present (Fig. 4).

Almost half of the S. aureus strains (61 strains,50.4%) showed a macrolide-lincosamide-streptogramine (MLSbi) inductible resistancephenotype, highlighted through the D-test. Thiscross-resistance was expressed at 69.7% of theMRSA strains in a more intensive way incomparison to the methicillin-sensitive strains,where an occurrence rate of 39.7% was observed.

MRSA were resistant towards other 4antibiotic classes as following: aminoglycosides(9/43), fluroquinolones (8/43), tetracyclines(12/43), ansamycins (rifampicin) (6/43). All theMRSA strains were sensitive towards theinhibitors of the folate pathway (cotrimoxazol/trimethoprim-sulfamethoxazole), the glycopep-tides vancomycin and teicoplanin, and towardsoxazolidones (linezolid) (Fig. 5).

By analysing the time evolution of theantibiotic resistance markers of the S. aureusstrains, a 5 times higher occurrence of the MRSAisolates was observed from May 2014 toDecember 2015 (1.5 years) than from January2011 to April 2014 (3.5 years), with a ratio of 25 to6 strains. This result highlights an increasingresistance of the S. aureus strains at the patientshospitalized in the Clinic for Dermatology of the„Elias“ University Emergency Hospital duringthe last two years (Fig. 3).

3.2. Pseudomonas aeruginosa

From January 2011 to December 2015, 51 P.aeruginosa strains were identified in samplescollected from the cutaneous leg ulcers, of which42 were venous ulcers.

The evaluation of the resistance profiles for P.aeruginosa strains demonstrated that most ofthem showed low resistance towards the testedantibiotic classes. 18 bacterial strains showedresistance towards at least one testedantimicrobial agent, whereas 33 strains weresensitive towards all the tested antibiotics. 4 P.aeruginosa strains isolated from venous ulcersshowed an extended antibiotic resistancespectrum (XDR). These were resistant towardsantibacterial agents from 6 antibiotic classes thatare definitory for this profile: aminoglycosides,

aeruginosa izolate de la nivelul ulcerelor venoase,au prezentat spectru extins de rezistenþã laantibiotice (XDR). Acestea au fost rezistente laagenþi antibacterieni din 6 clase de antibioticedefinitorii pentru acest profil: aminoglicozide,carbapeneme anti- Pseudomonas, cefalosporineanti- Pseudomonas, peniciline anti- Pseudomonas ºiinhibitori de β-lactamaze, monobactami, fluoro-chinolone anti- Pseudomonas [6].Singurulantibiotic testat faþã de care microorganismele ºi-au conservat sensibilitatea este Colistin (Fig. 4,Fig. 5).

anti- Pseudomonas carbapenems, anti- Pseudomonascephalosporins, anti- Pseudomonas penicillins andβ-lactamases, monobactams, anti- Pseudomonasfluoroquinolones [6]. The only antibiotic that wastested against the microorganisms that preservedtheir sensitivity is colistin (Fig. 4, Fig. 5).

It is very probable that the two P. aeruginosaXDR strains that were isolated during thehospital admission of a female patient diagnosedwith venous ulcer are identical, given the fact thatthe patient’s evolution under the antibiotictreatment was severe and lead to death by sepsis.

15


Fig 3. Evoluþia în timp a marker-ilor de rezistenþã la antibiotice la tulpini de S. aureus.Fig 3. Evolution of S. aureus antibiotic susceptibility profiles.

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12

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No. of antibiotics withdocumentedresistance_S. aureus

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Staphylococcusaureus

Evolution of S. aureus antibiotic susceptibility profiles

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0-15

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2-15

Fig. 4. Sensibilitatea la antibiotice a tulpinilor de P. aeruginosa ºi Enterobacteriaceae izolate din ulcere cronice gambiereFig. 4. Antibiotic susceptibility for P. aeruginosa and Enterobacteriaceae strains isolated from chronic lower limb wounds

Antibiotic susceptibility P. aeruginosa Antibiotic susceptibility EnterobacteriaceaeR

0 10 20 30 40 50 0 10 20 30 40 50 60

S

Colistin

Aztreonam

Meropenem

Imipenem

Pefloxacin

Levofloxacin

Ciprofloxacin

Amikacin

Tobramycin

Gentamicin

Ticarcillin

Piperacillin

Ticarcillin/Clavulanate

Piperacillin/Tazobactam

Cefepime

Ceftazidime

Co-trimoxazole

Levofloxacin

Ciprofloxacin

Amikacin

Gentamicin

Aztreonam

Meropenem

Imipenem

Ertapenem

Piperacillin/Tazobactam

4th gen. cephalosporin

3rd gen. cephalosporin

2nd gen. cephalosporin

Ampicillin

Amoxicillin/Clavulanic acid

16


Este foarte probabil ca cele douã tulpini de P.aeruginosa XDR izolate pe durata internãrii la opacientã diagnosticatã cu ulcer venos sã fieidentice, important cu atât mai mult cu câtevoluþia pacientei sub tratament antibiotic a fostseverã, fiind soldatã cu deces prin sepsis. S-aobservat faptul cã tulpinile izolate de la pacienþicu ulcere venoase au prezentat un grad mai înaltde rezistenþã la antibiotice comparativ cu plãgilecutanate de altã etiologie. Din ultima categorie,doar douã tulpini au prezentat rezistenþã laCeftazidim, respectiv Ceftazidim ºi Levofloxacinºi, interesant, au provenit din ulcere de etiologiemixtã venoasã ºi arterialã. Toate tulpinile testateau fost sensibile la Colistin.

Analizând evoluþia în timp a profilurilor derezistenþã pentru tulpinile de P. aeruginosa, lapacienþi internaþi în aceeaºi Clinicã deDermatologie, s-a observat cã nu a existat ocreºtere constantã a spectrului de rezistenþã laantibiotice, ci, periodic, cazuri izolate de tulpinicu rezistenþã înaltã (XDR) au determinat infecþiila pacienþii cu ulcere ale membrelor inferioare.

3.1.3.3. Enterobacteriaceae

În perioada ianuarie 2011- decembrie 2015, aufost identificate în prelevate de la nivelul

It was observed that the strains which wereisolated from patients with venous ulcers showeda higher resistance towards antibiotics incomparison to the cutaneous plagues of differentetiology. From the last category, only two strainsshowed resistance towards cephtazidime,cephtazidime plus levofloxacin, respectively, andmore interesting, they were both collected fromulcers of mixed venous and arterial etiology. Allthe tested strains were sensitive towards colistin.

By analyzing the time evolution of theresistance profiles for the P. aeruginosa strainsfrom the patients hospitalized in the same Clinicfor Dermatology, it was noticed that there was noconstant expansion of the antibiotic resistancespectrum. However, periodically isolated cases ofhigh resistance (XRD) strains caused infections tothe patients with leg ulcers.

3.1.3.3. Enterobacteriaceae

From January 2011 to December 2015, 56strains collected from cutaneous leg ulcers andbelonging to the Enterobacteriaceae family wereidentified, of which 40 were collected fromvenous ulcers. E. coli was most frequentlyisolated (22 strains, 39%), followed by Klebsiellaspp. (14 strains, of which 13 strains were K.

Fig. 5. Profilurile de rezistenþã la antibiotic ale tulpinilor isolate din ulcere cronice cutanate Fig. 5. Antibiotic resistance profiles of isolates from chronic skin ulcers

Antibiotic resistance profilesof isolates from chronic skin ulcers

Antibiotic resistantstrains,non-MDR, non-XDR

XDR

65

3

56 20

0 20 40 60 80 100 120 140 160

48 36

MDR

S. aureus P. aeruginosa Enterobacteriaceae

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ulcerelor cutanate ale membrelor inferioare, 56tulpini care au aparþinut familiei Entero-bacteriaceae dintre care 40 au provenit de lanivelul ulcerelor venoase. Din total, E.coli a fostizolatã cel mai frecvent (22 tulpini, 39%), urmatãde Klebsiella spp. (14 tulpini, din care 13 tulpini aufost K. pneumoniae, 25%), Proteus spp. (9 tulpini,16%), Enterobacter spp. ºi Morganella morganii înproporþii egale (3.5%) ºi altele.

Majoritatea enterobacteriilor identificate aufost rezistente la Ampicilinã (45 tulpini, 80.4%),urmatã de rezistenþa la combinaþia peniciline ºiinhibitori de β-lactamaze, în particular,Amoxicilinã-Clavulanat (41.1%). De asemeneamicroorganismele au prezentat rezistenþã laaminoglicozide (Gentamicinã-12 tulpini), fluoro-chinolone (Ciprofloxacin-12 tulpini, Levofloxacin6 tulpini), cefalosporine fãrã un spectru extins, degeneraþia 2 (11 tulpini), cefalosporine cu spectruextins, de generaþii 3 ºi 4 (11, respectiv 6 tulpini),monobactami (Aztreonam- 6 tulpini) ºi inhibitoriai cãii folaþilor (Trimetoprim-Sulfametoxazol, 9tulpini). Toate tulpinile studiate au fost sensibilela carbapeneme (Meropenem, Imipenem,Ertapenem) (Fig. 4).

Microorganisme producãtoare de ESBL, cuun numãr total de 9 tulpini, au aparþinut, înordinea descrescãtoare a frecvenþei, speciilorbacteriene E.coli (5 tulpini), Proteus mirabilis (2tulpini), K. pneumoniae (1 tulpinã) ºi Morganellamorganii (1 tulpinã). Microorganisme MDR, într-un numãr total de 20 au aparþinut, în ordineadescrescãtoare a frecvenþei, urmãtoarelor speciibacteriene E.coli (11 tulpini), K. pneumoniae (4tulpini), Proteus mirabilis (3 tulpini), Morganellamorganii (1 tulpinã) ºi Enterobacter spp. (1tulpinã).

La pacienþii diagnosticaþi cu ulcere venoase s-a observat un numãr de 3 ori mai mare alenterobacteriilor MDR, comparativ cu acelea careau avut drept origine ulcere ale membrelorinferioare de altã etiologie (15/5) (Fig. 5).Microorganisme MDR, într-un numãr total de 20au aparþinut, în ordinea descrescãtoare afrecvenþei, urmãtoarelor specii bacteriene E.coli(11 tulpini), K. pneumoniae (4 tulpini), Proteusmirabilis (3 tulpini), Morganella morganii (1tulpinã) ºi Enterobacter spp. (1 tulpinã).

Din punctul de vedere al evoluþiei în timp aprofilurilor de rezistenþã pentru tulpinile de

pneumoniae, 25%), Proteus spp. (9 strains, 16%),Enterobacter spp. and Morganella morganii in equalpercentages (3.5%) and others.

Most of the identified enterobacteria wereampicillin-resistant (45 strains, 80.4%), followedby resistance towards the penicilline and β-lactamases inhibitors mixture, in particular,amoxycillin-clavulanat (41.1%). The micro-organisms also showed resistance towardsaminoglycosides (gentamicin- 12 strains), fluoro-quinones (ciprofloxacin- 12 strains, levofloxacin-6 strains), cephalosporines without an extendedspectrum of second generation (11 strains),extendedspectrum cephalosporines of third andfourth generation (11 and 6 strains, respectively),monobactams (aztreonam- 6 strains) and folatepathway inhibitors (trimetroprim-sulfame-toxazol, 9 strains). All the studied strains weresensitive towards carbapenems (meropenem,imipenem, ertapenem) (Figure 4).

ESBL producing microorganisms with a totalof 9 strains belonged to the following bacterialspecies: E.coli (5 strains), Proteus mirabilis (2strains), K. pneumoniae (1 strain) and Morganellamorganii (1 strain). The MDR microorganisms, intotally 20 strains, belonged to the followingbacterial species: E.coli (11 strains), K. pneumoniae(4 strains), Proteus mirabilis (3 strains), Morganellamorganii (1 strain) and Enterobacter spp. (1 strain).

At patients diagnosed with venous ulcers, 3times more MDR enterobacteria were observedthan at the ones with leg ulcers of differentetiology (15/5) (Fig. 5). The MDR micro-organisms, in totally 20 strains, belonged to thefollowing bacterial species: E.coli (11 strains), K.pneumoniae (4 strains), Proteus mirabilis (3 strains),Morganella morganii (1 strain) and Enterobacterspp. (1 strain).

From the time evolution perspective of theEnterobacteriaceae strains resistance profiles at thepatients hospitalized in the same Clinic forDermatology, it was noticed a relatively uniformtime distribution of the MDR strains, except forthe period July 2014- July 2015, when suchmicroorgamisms were not isolated from legulcers. In this study, no information regarding thechanges in the treatment schemes of thesepatients or in the antiseptic and asepsis protocolfollowed in that period was analysed, although

18


Enterobacteriaceae, la pacienþi internaþi în aceeaºiClinicã de Dermatologie, s-a observat distribuþiarelativ uniformã în timp a tulpinilor MDR, cuexcepþia perioadei iulie 2014- iulie 2015, când nuau existat astfel de microorganisme izolate de lanivelul ulcerelor membrelor inferioare. În aceststudiu nu au fost analizate informaþii cu privire lamodificarea schemelor de tratament la aceºtipacienþi sau a protocolului de asepsie ºiantisepsie utilizat din acea perioadã, factori carear putea influenþa evoluþia microbiologicã apacienþilor.

Discuþii

Patologia venoasã a reprezentat cauzamajoritarã a ulcerelor membrelor inferioare.

Pacienþii au avut adeseori recurenþe, ceea ceevidenþiazã atât impactul individual al ulcerelorcronice, care prin internãrile repetate în spital ºiîntreruperea activitãþii profesionale afecteazãsemnificativ calitatea vieþii, cât ºi impactuleconomic important asupra sistemului sanitar pecare îl determinã recidivele sau complicaþiileulcerelor membrelor inferioare.

În ceea ce priveºte perioada anului în care s-au internat pacienþii incluºi în studiu, a existat opredominanþã în sezonul cald. Aceastã fluctuaþiedependentã de anotimp este confirmatã deobservaþii din literatura de specialitate în ceea cepriveºte frecvenþa semnificativ mai mare adezvoltãrii ulcerelor venoase în perioada aprilie-octombrie [7]. Mai mult a fost sugeratã ºi oevoluþie diferitã în funcþie de anotimpul în care adebutat boala, mai severã la pacienþii care audezvoltat ulcere venoase vara sau iarna ºi ovindecare mai rapidã la cei care s-au îmbolnãvitprimãvara sau toamna [7]. Aceste variaþiisezoniere pot fi explicate atât de agravareainsuficienþei venoase în sezonul cald, cât ºi demodificarea rãspunsului imun în funcþie deanotimp [7]. Alþi factori implicaþi suntreprezentaþi de expunerea crescutã în sezonulcald la traumatisme (lovituri, înþepãturã deinsectã etc.) ºi la agenþi infecþioºi (îmbrãcãminteacare nu protejeazã membrele inferioare).

Distribuþia pacienþilor în funcþie de sex a fostfoarte echilibratã cu un raport aproximativ egalsex feminin/sex masculin. Aproximativ o treimedin pacienþi au avut vârsta mai micã de 62 de ani,fiind în continuare activi din punct de vedere

these factors could have influenced themicrobiological evolution of the patients.

Discussion

The venous pathology represented the majorcause of leg ulcers.

The patients showed often recurrences,which highlights both the individual impact ofthe chronic ulcers, that through rehospitalizationand interruption of the professional activitiessignificantly affect the life quality, and theimportant economic impact on the healthcaresystem determined by recurrences or by thecomplications of the leg ulcers.

Regarding the season when the hospi-talizations of the patients included in this studytook place, there was a higher occurrence in thewarm season. This season dependent fluctuationis confirmed by observations included in thescientific literature, that highlight a significantlyhigher frequency of the venous ulcersdevelopment from April to October [7].Moreover, a different evolution depending on theseason in which the disease started was alsosuggested: more severe for the patients whosevenous ulcers started in Summer and in Winterand a faster healing of the patients whose diseasestarted in Spring and in Autumn [7]. Theseseason variations can be explained both throughthe aggravation of the venous insufficiencyduring the warm season, and through the changein the immune system depending on the season[7]. Other factors are represented by the increasedexposure towards traumatism (hits, insect bitesetc.) and towards infectious agents (the clothingthat does not protect the legs) during the warmseason.

The patients distribution according to thegender was balanced with an approximativelyequal female to male ratio. Approximatively athird of the patients were younger than 62 yearsold, all professionally active, fact that highlightsthe important impact of venous ulcers on lifequality.

The scientific literature comprises numerousstudies on cutaneous samples, noticing variableincidences of the different isolated bacterialspecies. However, these are very similar to thepresent study.

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profesional, ceea ce evidenþiazã impactulimportant pe care ulcerele venoase îl au asupracalitãþii vieþii.

În literatura de specialitate existã numeroasestudii care au observat prelevatele de la nivelululcerelor cutanate, observându-se incidenþevariabile ale diferitor specii bacteriene izolate,dar asemãnãtoare cu rezultatele prezentuluistudiu.

Într-un studiu retrospectiv din Italia,coordonat de Bessa LJ ºi colab. (2015), s-au izolatdin 312 prelevate de la nivelul plãgilor de diverseetiologii 28 de specii bacteriene, dintre care celemai frecvente au fost S. aureus (37%), urmatã de P.aeruginosa (17%) ºi de enterobacteriile Proteusmirabilis (10%) ºi E. coli (6%). În aproximativ unsfert din rezultate a fost depistatã infecþiapolimicrobianã, cea mai frecventã asociere fiindS. aureus ºi P. aeruginosa[8].

Într-un studiu din Polonia, în care s-auanalizat rezultatele a 173 examene microbiologiceale secreþiilor ulcerelor membrelor inferioare,efectuate pe o duratã de 5 ani (1998-2002), S.aureus a fost de asemenea principalulcontaminant al leziunilor cutanate, în mai multde jumãtate din cazuri (56.57%), urmat de P.aeruginosa (37.14%), de Enterococcus faecalis(22.29%) ºi enterobacteriile Proteus mirabilis(13.71%) ºi E. coli (12.57%) [9]. A fost raportatã ºiprezenþa fungilor Candida spp. în 6.86% dincazuri [9].

În România, într-un studiu prospectiv, dinperioada iulie 2013- aprilie 2014, Georgescu ºicolab. (2014) au identificat speciile bacteriene dinprelevate de la nivelul ulcerelor cronice cutanate,observând o predominanþã a tulpinilor de S.aureus (14 tulpini), Enterococcus faecalis (8 tulpini)ºi a speciilor de stafilococi coagulazo-negativi (11tulpini) [10]. P. aeruginosa a fost identificatã în 4%din prelevate [10]. Lotul studiat a fost unul redus,de 40 de pacienþi, ceea ce poate explica ordineadiferitã în funcþie de frecvenþã a speciilorbacteriene.

Într-o altã clinicã de Dermatologie dinBucureºti, s-a efectuat un studiu retrospectiv carea inclus un numãr de 259 de rezultate aleexamenelor bacteriologice ale prelevatelor de lanivelul ulcerelor cutanate, în perioada ianuarie2009 - decembrie 2011 [3]. S-a evidenþiaturmãtoarea distribuþie a speciilor bacteriene

In a retrospective study performed in Italyunder the coordination of Bessa LJ et al. (2015),from 312 samples collected from plagues ofdifferent etiologies, 28 bacterial species wereisolated, from which the most frequent were thefollowing: S. aureus (37%), P. aeruginosa (17%) andenterobacteria Proteus mirabilis (10%) and E. coli(6%). In almost a quarter of the results thepolimicrobial infection was identified, with S.aureus and P. aeruginosa as the most commonassociation [8].

In a study from Poland, where the results of173 microbiological examinations of secretionsfrom leg ulcers, that were performed during 5years (1998-2002), were analysed, S. aureus wasalso the main contaminant of the cutaneouslesions in more than half of the cases (56.57%),followed by P. aeruginosa (37.14%), Enterococcusfaecalis (22.29%) and the enterobacteria Proteusmirabilis (13.71%) and E. coli (12.57%) [9]. Thepresence of Candida spp. in 6.86% of the cases wasas well reported [9].

In a retrospective study from July 2013 toApril 2014, Georgescu et al. (2014) identified thebacterial species in the samples collected fromchronic cutaneous ulcers, noticing a higheroccurency of S. aureus (14 strains), Enterococcusfaecalis (8 strains) and of coagulase-negativestaphylococci (11 strains) [10]. P. aeruginosa wasidentified in 4% of the samples [10]. The studiedlot was small with only 40 patients, fact thatcould explain the different order according tofrequency of the bacterial species.

A retrospective study including 259 results ofthe bacteriological examinations of samplescollected from cutaneous ulcers was performedin another clinic for Dermatology in Bucharest,Romania, from January 2009 to December 2011[3]. The following distribution of the identifiedbacterial species was observed: S. aureus (55patients, 26.3%), followed by the enterobacteriaEnterobacter spp.(17.2%), Proteus spp. (15.8%), E.coli (14.8%) and P. aeruginosa (15.3%) [3]. Therewere only 2 cases with Enterococcus spp. and onlyone case with Candida albicans infection at apatient suffering from diabetes [3]. There is norecent data that allows the observation of themicrobiological evolution of the patients withcutaneous ulcers from the period January 2012-December 2015.

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identificate: S. aureus (55 pacienþi, 26.3%), urmatde enterobacteriile Enterobacter spp.(17.2%),Proteus spp. (15.8%), E. coli (14.8%) ºi de P.aeruginosa (15.3%) [3]. Au existat doar 2 cazuri cuEnterococcus spp. ºi un singur caz de infecþieCandida albicans la un pacient diabetic [3]. Nuexistã date mai recente prin care sã fiemonitorizatã evoluþia microbiologicã apacienþilor cu ulcere cutanate în perioadaianuarie 2012- decembrie 2015.

Din punctul de vedere al depistãrii infecþiilorpolimicrobiene la nivelul ulcerelor venoase, s-aobservat faptul cã acestea au fost mai frecvente înlotul de interes, comparativ cu ulcerelemembrelor inferioare de altã etiologie, cea maifrecvent întâlnitã fiind asocierea speciilor P.aeruginosa ºi S. aureus.

În literatura de specialitate aceasta esteraportatã drept cea mai frecventã infecþiepolimicrobianã a ulcerelor cronice [11]. DeLeon Sºi colab. (2014) au observat o creºtere a graduluide toleranþã la antibiotice al coinfecþiei P.aeruginosa ºi S. aureus, atât in vitro prin cultivareaconcomitentã planctonicã, cât ºi in vivo printestarea într-un model de plagã conceput deautori[11]. Mai mult, aceastã infecþie poli-microbianã poate sã întârzie vindecarea plãgilorcutanate, prin reducerea producþiei factorului decreºtere keratinocitar 1 (keratinocyte growthfactor 1- KGF1) [12,13]. Existã date în literaturãcare sugereazã o creºtere a virulenþei S. aureus înprezenþa P. aeruginosa, in vitro, prin exprimareagenelor pvl ºi hla, fapt care ar putea confirmacorelaþia observatã in vivo dintre patogenitateabacterianã ºi cronicitatea ulcerelor infectate[12,13].

S-a observat un fenotip de rezistenþã crescutla antibiotice al tulpinilor bacteriene izolate de lanivelul ulcerelor venoase. Aproximativ jumãtatedin tulpinile de S. aureus,identificate la pacienþiicu ulcere venoase, au prezentat un fenotip demultirezistenþã la antibiotice MDR, ºi o treimerezistenþã la meticilinã (MRSA). Deºi evaluareaprofilurilor de rezistenþã pentru tulpinile de P.aeruginosa a arãtat cã majoritatea au manifestatun nivel de rezistenþã scãzut la clasele deantibiotice testate, 4 tulpini de P. aeruginosa,izolate de la nivelul ulcerelor venoase, auprezentat spectru extins de rezistenþã laantibiotice (XDR). Mai mult, o treime din

With respect to discovering the polimicrobialinfections in venous ulcers, it was noticed thatthese were more frequent in the lot of interestthan in the leg ulcers of different etiology, havingP. aeruginosa and S. aureus as the most commonassociation.

The scientific literature also reports this as themost common polimicrobial infection of thechronic ulcers [11]. DeLeon S et al. (2014) noticedan antibiotic tolerance increase of the co-infectionP. aeruginosa and S. aureus, both in vitro, bysimultaneous plankton culture, and in vivo bytests in a plague model designed by the authors[11]. Moreover, this polimicrobial infection candelay the healing of the cutaneous plagues bydecreasing the production of the keratinocytegrowth factor 1- KGF1 [12,13]. There is literaturedata that suggests an increase of the S. aureusvirulence in the presence of P. aeruginosa, in vitro,by expressing the pvl and hla genes, which couldconfirm the correlation observed in vivo betweenthe bacterial pathogenicity and the chronicity ofthe infected ulcers [12,13].

An increased antibiotic resistance phenotypewas observed at the strains isolated from thevenous ulcers. Almost half of the S. aureus strainsthat were identified at patients with venousulcers, showed an antibiotic multiresistancephenotype (MDR) and a third resistance towardsmethicillin (MRSA). Although the evaluation ofthe resistance profiles for P. aeruginosa strainsshowed that most of them had a low resistancetowards the tested antibiotic classes, 4 P.aeruginosa strains, that were isolated from venousulcers, showed an extended antibiotic resistancespectrum (XDR). Moreover, a third of theEnterobacteriaceae strains identified athospitalized patients were MDR. All these resultsraise the alarm about the danger of spreading ofthese bacteria inside the hospitals, with severeimplications both on the health of theimmunosupressed patients who could developacute infections, and on the epidemiologicallevel, through the nosocomial spread of theresistance.

From the point of view of the evolution of themicroorganisms resistance, we noticed anincreasing general resistance of the S. aureusstrains at patients diagnosed with leg ulcer

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tulpinile de Enterobacteriaceae identificate lapacienþi spitalizaþi au fost MDR. Toate acesterezultate ridicã un semnal de alarmã asuprapericolului de rãspândire în mediu spitalicesc alacestor bacterii, cu implicaþii grave atât asuprastãrii de sãnãtate a pacienþilor imunosupresaþi,care ar putea dezvolta infecþii severe, cât ºi dinpunct de vedere epidemiologic, prin rãspândireanosocomialã a rezistenþei.

Din punctul de vedere al evoluþiei rezistenþeimicroorganismelor, am observat o rezistenþãgeneralã în creºtere a tulpinilor S. aureus, lapacienþii diagnosticaþi cu ulcere ale membrelorinferioare, spitalizaþi în Clinica de Dermatologie,a Spitalului Universitar de Urgenþã ”Elias”, pedurata ultimelor 20 luni de studiu.

Nu a existat o creºtere constantã a spectruluide rezistenþã la antibiotice al P. aeruginosa, ci,periodic, cazuri izolate de tulpini cu rezistenþãînaltã (XDR) au determinat infecþii la pacienþii cuulcere ale membrelor inferioare. Aceasta ar puteasugera absenþa factorului nozocomial îndezvoltarea rezistenþei.

Prin analiza comparativã a prelevatelor de lanivelul plãgilor cutanate, în mediu spitalicescversus ambulatoriu, un grup de cercetãtori dinSerbia a observat recent faptul cã nu au existatdiferenþe semnificative în ceea ce priveºtenumãrul tulpinilor de P. aeruginosa izolate ºi niciîn profilul acestora de rezistenþã la antibiotice[14], ceea ce corespunde observaþiilor noastre.

Analiza din punctul de vedere al rezistenþeila antibiotice a izolatelor de P. aeruginosa, pe oduratã de 10 ani, într-un spital din Portugalia arelevat o scãdere în timp a frecvenþei tulpinilorMDR ºi o creºtere a celor XDR, proporþiile dinfiecare tip fiind de 39.9%, respectiv 2.9% [15].

Conform celui mai recent raport ECDC, înRomânia, ponderea tulpinilor rezistente laantibiotice, din totalul tulpinilor de P. aeruginosaizolate din sânge ºi lichid cefalorahidian,depãºeºte valoarea de 50%, pentru principaleleclase de substanþe antimicrobiene [4].

În studiul nostru nu au fost depistate P.aeruginosa MDR, iar microorganismele XDR aufost identificate în ultimii 3 ani, perioada ianuarie2013- septembrie 2015, reflectând, posibil, oasociere cu incidenþa generalã în creºtere atulpinilor bacteriene extensiv rezistente.

hospitalized at the Clinic for Dermatology of the„Elias“ University Emergency Hospital over thelast 20 months of the study.

There was no constant enlargement of theantibiotic resistance spectrum of P. aeruginosa, butperiodically, isolated cases of high resistancestrains (XDR) caused infections at the patientswith leg ulcers. This can suggest the absence ofthe nosocomial factor on the development of theresistance.

Through the comparative analysis of thesamples from the cutaneous plagues in hospitalsversus ambulatory care, a group of researchers inSerbia observed recently that there are nosignificant differences with respect to the numberof P. aeruginosa strains isolated, nor in theirantibiotic resistance profile [14], whichcorresponds to our observations.

The analysis with respect to antibioticresistance of the isolated strains of P. aeruginosaduring 10 years in a hospital in Portugal revealeda decrease in time of the MDR incidences(representing 39.9%) and an increase of the XDR(representing 2.9%) [15].

According to the most recent ECDC report,the percentage of the antibiotic resistant strainsfrom all the P. aeruginosa strains isolated fromblood and cerebrospinal fluid, is above 50% forthe main classes of antimicrobial substances [4].

In the present study P. aeruginosa MDR werenot identified, whereas the XRD microorganismswere identified in the last 3 years, from January2013 to September 2015, revealing a possibleassociation with the increasing general incidenceof the extensively resistant bacterial straints.

Joseph NM et al. (2013) observed thelowering incidence of the P. aeruginosa resistantstrains, including MDR, isolated from thecutaneous plagues of different etiologies at thehospitalized patients from June 2007 to June 2012from 37.9% to 23.7%. There are no more recentdata to observe the microbiological evolution in2012-2015 [16].

A decreasing resistance of theEnterobacteriaceae strains was noticed at thepatients diagnosed with leg ulcers who werehospitalized in the Clinic for Dermatology of the„Elias“ University Emergency Hospital duringthe last 18 months of the study.

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Joseph NM ºi colab. (2013) au observat oscãdere a frecvenþei tulpinilor rezistente P.aeruginosa, inclusiv MDR, izolate de la nivelulplãgilor cutanate de diferite etiologii, la pacienþiinternaþi în spital, în perioada iunie 2007- iunie2012, de la 37.9% la 23.7%. Nu existã date mairecente pentru a observa evoluþia microbiologicãîn perioada 2012-2015 [16].

S-a observat o rezistenþã în scãdere atulpinilor de Enterobacteriaceae, la pacienþiidiagnosticaþi cu ulcere ale membrelor inferioare,spitalizaþi în Clinica de Dermatologie, aSpitalului Universitar de Urgenþã ”Elias”, pedurata ultimelor 18 luni de studiu.

Concluzii

Lipsa datelor din þara noastrã, evidenþiazãnecesitatea studierii aprofundate a compoziþieimicrobiotei ulcerelor cutanate, pe un lot maimare de pacienþi români.

Patogeneza infecþiilor de la nivelul ulcerelorcronice este multifactorialã prin implicarea încolonizarea ºi invazia microbianã a unui numãrmare de factori de virulenþã parietali sauextracelulari. Elucidarea mecanismelor pato-genice, la nivel molecular este crucialã în luptaîmpotriva agenþilor bacterieni care determinãinfecþii cronice ºi împiedicã procesul fiziologic devindecare al ulcerelor cutanate. Þinta finalã esteaceea de a descoperi noi agenþi terapeutici care sãdetermine evoluþia favorabilã a patologieipacienþilor.

Rezultatele acestui studiu evidenþiazãimportanþa monitorizãrii sensibilitãþii laantibiotice, care permite administrarea unuitratament adecvat, dar ºi controlul epidemiological infecþiilor nozocomiale.

Menþiuni

Aceastã lucrare este parþial sprijinitã decãtre Universitatea de Medicinã ºi Farmacie„Carol Davila”, Bucureºti, România, prin bursade cercetare „Proiecte Tineri Cercetãtori”. Autoriiar dori sã mulþumeascã Dr. Marilena Filipov ºiDr. Mariana Buzea pentru efectuarea testelormicrobiologice.

Conclusions

The lack of data from our country highlightsthe need of an in-depth study of the microbiotacomposition of the cutaneous ulcers on morepatients.

The pathogenicity of the infections in chroniculcers is multifactorial by implying the microbialcolonization and invasion of a high number ofparietal or extracellular virulence factors.Clarifying the pathogenic mechanisms at themolecular level is critical in the battle against thebacterial agents that cause chronic infections andhinder the physiological healing process of thecutaneous ulcers. The final target is the discoveryof new therapeutic agents that can favorablyinfluence the evolution of the patientphatogenicity.

The results of this study highlight theimportance of monitoring the antibioticsensitivity, which allows the administration of anadequate treatment and the epidemiologicalcontrol of the nosocomial infections.

Acknowledgements

This work is partially supported by „CarolDavila“ University of Medicine and Pharmacy,Bucharest, Romania through the research grant„Young Researchers Projects“. The authorswould like to thank Dr. Marilena Filipov and Dr.Mariana Buzea for performing the micro-biological tests.

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Bibliografie/Bibliography1. Rhoads D D, Cox S B, Rees E Jet al. Clinical identification of bacteria in human chronic wound infections:

culturing vs. 16S ribosomal DNA sequencing. BMC Infect Dis 2012; p 321.2. Mihai M M, Holban A M, Giurcaneanu Cet al. Identification and phenotypic characterization of the most frequent

bacterial etiologies in chronic skin ulcers. Rom J Morphol Embryol 2014; p 1401-1408.3. Salavastru C M, Nedelcu L E,Tiplica G S. Management of leg ulcers in patients with chronic venous insufficiency:

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16. Joseph N M, Devi S, Shashikala Pet al. Changing Trend in the Antibiotic Resistance Pattern of PseudomonasAeruginosa Isolated from Wound Swabs of Out-Patients and in-Patients of a Tertiary Care Hospital. J Clin DiagnRes 2013; p 2170-2172.

Conflict de interese Conflict of interestNEDECLARATE NONE DECLARED

Adresa de corespondenþã: Mara Mãdãlina Mihai, Str. Dionisie Lupu nr. 37, Bucureºti, Sector 2, CP 020021, Romania; Tel.: 0040213180719; Fax: 0040213180730 E-mail: [email protected]

Correspondance address: Mara Mãdãlina Mihai, Dionysius Lupu Street. 37, Bucharest, District 2CP 020021, Romania; Tel.: 0040213180719; Fax: 0040213180730 E-mail: [email protected]

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