REZUMATUL TEZEI DE DOCTORAT

Fenotipuri de rezisten ntlnite

la specii de Enterococcus izolate
din diferite produse patologice

Doctorand LIA SORINA PEPELEA

Conductor de doctorat Prof.dr. LIA MONICA JUNIE

CLUJ-NAPOCA 2015
2 Pepelea Lia Sorina

CUPRINS

INTRODUCERE 13
STADIUL ACTUAL AL CUNOATERII
1. Generaliti despre genul Enterococcus 17
1.1. Scurt istoric 17
1.2. Habitat i implicaii n patologia infecioas 18
1.3. Caractere morfotinctoriale 19
1.4. Caractere de cultur 20
1.5. Caractere de patogenitate 25
1.6. Identificarea unor markeri epidemiologici 26
2. Rezistena enterococilor la antibiotice 28
2.1. Rezistena la beta-lactamine 29
2.1.1. Rezistena intrinsec 29
2.1.2. Tolerana 30
2.1.3. Beta-lactamazele 30
2.2. Rezistena la aminozide 31
2.2.1. Rezistena intrinsec 31
2.2.2. Rezistena dobndit 32
2.2.3. Evidenierea rezistenei de nivel nalt la animozide 33
2.2.3.1. Incorporarea antibioticului n agar 33
2.2.3.2. Microcomprimate cu concentraie crescut de antibiotic 33
2.2.3.3. Metoda diluiilor n bulion 34
2.3. Rezistena la macrolide, lincosamide, streptogramine i ketolide (MLSK) 34
2.4. Rezistena la glicopeptide 35
2.4.1. Metode de screening pentru detectarea enterococilor rezisteni
la vancomicin
37
2.5. Rezistena la oxazolidinone 38
2.6. Sinergismul antibioticelor 38
2.7. Alternative terapeutice pentru enterococii multiplu rezisteni la
antibiotice
39

CONTRIBUIA PERSONAL
1. Ipoteza de lucru/obiective 43
2. Studiul 1. Implicarea tulpinilor de Enterococcus spp. n
45
etiologia infeciilor la pacieni spitalizai i din ambulator
2.1. Introducere 45
2.2. Ipoteza de lucru/obiective 45
2.3. Material i metod 45
2.4. Rezultate 54
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 3

2.5. Discuii 66
2.6. Concluzii 71
3. Studiul 2. Testarea sensibilitaii la antibiotice i evidenierea
73
fenotipurilor de rezisten ale tulpinilor de Enterococcus spp.
3.1. Introducere 73
3.2. Ipoteza de lucru/obiective 73
3.3. Material i metod 74
3.4. Rezultate 80
3.5. Discuii 109
3.6. Concluzii 114
4. Concluzii generale 115
5. Originalitatea i contribuiile inovative ale tezei 117

REFERINE 119

Cuvinte cheie: enterococi, rezisten, vancomicin, infecii

4 Pepelea Lia Sorina

STADIUL ACTUAL AL CUNOATERII

1. GENERALITI DESPRE GENUL ENTEROCOCCUS

Enterococii au fost clasificai iniial drept coci Gram pozitivi aparinnd genului
Streptococcus1.
Numii ulterior streptococi de provenien fecal sau enterococi, au fost
considerai pentru mult timp o subdiviziune a genului Streptococcus (streptococci de
grup D), difereniindu-se de acetia prin rezistena crescut la ageni chimici i fizici 2.
La nceputul anilor 1930, enterococii au fost clasificai ca streptococi de grup D
i au fost difereniai de grupul D de streptococi non-enterococi prin teste biochimice
auxiliare2.
n anii urmtori, Sherman a recomandat ca termenul enterococi s fie utilizat
n mod specific pentru streptococii care se dezvolt att la 10C ct i la 45C, la un
pH de 9.6, n prezena NaCl 6.5%, supravieuiesc la 60C timp de 30 de minute i
hidrolizeaz esculina3.
Diferenierea evident a S.faecalis i S.faecium de ceilali membrii ai genului a
fcut posibil includerea acestor specii ntr-un gen bacterian nou, ale crui
caracteristici au fost foarte bine studiate prin tehnici de biologie molecular2.
Metodele ce stabilesc criteriile de includere a bacteriilor n genul Enterococcus
cuprind recombinarea ADN-ADN, secvenierea genic a subunitii 16S ARNr, analiza
proteic i testele fenotipice obinuite4, 5.
Bacteriile din genul Enterococcus au fost catalogate ca bacterii cu un grad sczut
de patogenitate, dar in anii 1990 au devenit importani ageni etiologici ai infeciilor
nosocomiale6.
La pacienii spitalizai, enterococii pot fi evideniati la nivelul leziunilor
esuturilor moi, ulcerelor i tractului gastro-intestinal7.
Sindroame infecioase care recunosc enterococi n etiologie pot fi reprezentate
de infecii ale tractului urinar la pacienii cu uropatii obstructive, infecii mixte ale
plgilor mai ales la pacienii aflai sub tratament antimicrobian 8, 9.
Pot sa apar bacteriemii la vrstnici i pacienii imunocompromii 10.
Studii efectuate n Statele Unite au demonstrat c majoritatea pacienilor
infectai cu tulpini deosebit de rezistente la antibiotic provin din unitile de terapie
intensiv11.
Factorii de risc ce pot fi adugai celor deja menionai regsim: durata crescut
a spitalizrii, colonizarea gastro-intestinal cu VRE, expunerea la echipamente
medicale contaminate, transplantul medular, afeciunile hematologice, tratamentul cu
cefalosporine de generatia a 3-a i antibiotice cu aciune asupra bacteriilor anaerobe16.
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 5

Enterococii cauzeaz rar infecii ale tractului respirator, osteomielit sau

celulit, n schimb au devenit a treia cauz a infeciilor nosocomiale n Statele Unite12.
Infeciile enterococice pot fi cauzate de cel puin 19 specii incluznd E. avium, E.
casseliflavus, E. durans, E.faecalis, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E.
mundtii, E. pseudoavium, E. raffinosus, E. solitarius13.
O serie de alte specii au fost adugate ulterior acestei liste: E. cecorum,
E.columbae, E. saccharolyticus, E. dispar, E. sulfurous, E. seriolicida i E. flavescens14.
Majoritatea infeciilor clinice sunt cauzate de E. faecalis (80-90%) sau
E. faecium (5-15%)16.
Speciile E. durans, E. avium i E. hirae sunt mai rar intlnite, iar celelalte specii
amintite sunt izolate doar ocazional17.

2. REZISTENA ENTEROCOCILOR LA ANTIBIOTICE

Implicai din ce n ce mai frecvent n infeciile nosocomiale, enterococii

prezint rezisten natural sau dobndit la antibiotice, ceea ce le permite
supravieuirea n faa unor multiple scheme de tratament cu antibiotice15.
Enterococii au dobndit rezisten la un numr de ageni antimicrobieni,
inclusiv tetracicline, eritromicin, cloramfenicol, niveluri ridicate de trimetoprim,
clindamicin, niveluri ridicate de aminoglicozide i peniciline (prin penicilinaz i
mecanisme non-penicilinaz) i mai recent la vancomicin16.
Toate aceste rezistene adaug noi semne de ntrebare terapiei cu antibiotice n
cazul infeciilor enterococice, deoarece apare o scdere a numrului de opiuni
terapeutice17.

CONTRIBUIA PERSONAL

Obiective
Enterococii, dei fac parte dintre comensalii florei intestinale pot deveni
patogeni nosocomiali.
n ultimele 3 decenii s-a constatat o cretere constant a incidenei acestor
infecii, fenomen cauzat probabil de creterea populaiei care prezint factorii de risc
(inflaii cronice, intervenii chirurgicale, neoplazii intestinale)dar i de apariia
tulpinilor de enterococi cu rezisten multipl la antibiotice, n special la Vancomicin.
n aceast lucrare am urmrit evaluarea implicrii tulpinilor de Enterococcus
spp. n etiologia infeciilor i sensibilitatea la antibiotice a acestor microorganisme.
n acest scop, au fost luate n studiu att tulpinile izolate din infecii de pe
seciile spitalului, ct i tulpini izolate de la pacieni din ambulator.
n al doilea rnd, am urmrit evidenierea fenotipurilor de rezisten ale
6 Pepelea Lia Sorina

tulpinilor de enterococi circulante izolate de pe seciile spitalului ct i din ambulator,

cu rol n ameliorarea antibioterapiei empirice a infeciilor produse de aceste tulpini.

Material i metod
Studiul a fost realizat prospectiv, urmrindu-se tulpinile de Enterococcus spp.
implicate n etiologia infeciilor att la pacienii spitalizai ct i la pacienii din
ambulator.
n acest scop, au fost luate n studiu infeciile enterococice diagnosticate la
pacienii internai pe seciile institutului, precum i infeciile diagnosticate n
ambulator n perioada ianuarie 2010- decembrie 2012.
n cadrul spitalului, s-au utilizat date ale Compartimentului de Microbiologie
al Laboratorului Clinic. Pentru colectarea datelor din spital am folosit fia tip de
supraveghere, prezentat n anexa Ordinului Ministerului Sntii 10/2005.
Pentru fiecare an studiat (2010, 2011 i 2012) s-au cules date att despre
tulpinile de enterococi care s-au dovedit a fi agent etiologic unic ct i despre tulpinile
de enterococi izolate n asociere cu alte microorganisme.
n spital, toate tulpinile de enterococi izolate au fost identificate cu sistemul
automat Vitek 2 Compact (bioMrieux).
Speciile de Enterococcus identificate de VITEK 2 GP: E.avium, E. casseliflavus,
E.cecorum, E.columbae, E.durans, E.faecalis, E.faecium, E.gallinarum, E.hirae,
E. raffinosus, E. saccharolyticus.
n ambulator, bacteriile din genul Enterococcus au fost identificate rapid pe baza
prezenei antigenului de grup D, cu truse PASTOREXR STREP (BioRad) i al galeriilor API 20
STREP (bioMrieux).

Metodologia statistic
Datele obinute n urma cercetrii au fost introduse i stocate n baze de date tip
Microsoft Acces 2010 (format *.mdb) i au fost exportate spre prelucrare n fiiere de
tip worksheet Excel 2010 (*.xls).
Prelucrarea statistic a fost efectuat cu ajutorul pachetelor software Microsoft
Excel 2010, EPI-INFO versiunea 6.04 i IBM SPSS versiunea 10, rezultatele prelucrrii
fiind exportate n toate cele trei cazuri n fiiere de tip worksheet Excel 2010.
Variabilele de tip dihotomic, caracterizate prin procentaj, au fost comparate prin
aplicarea tabelelor de contigen, cu calcularea testului 2 i utilizarea, la nevoie, a
coreciei Fisher18.
Toate testele statistice au fost calculate cu 2 extremiti iar valoarea semnificaiei
statistice p s-a luat n considerare la valori 0,01.
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 7

Studiul 1. Implicarea tulpinilor de Enterococcus spp. n

etiologia infeciilor la pacieni spitalizai i din ambulator

1. Cele mai izolate specii de enterococi au fost E. faecalis (74,88%) i

E. faecium (20,09%), att n spital ct i n ambulator.
2. Alte specii de enterococi izolate numai n spital au fost E. gallinarium,
E. durans , E. casseliflavus i E. avium.
3. La pacienii spitalizai enterococii au fost mai frecvent implicai n etiologia
infeciilor postoperatorii (30,72%), urmate de infeciile tractului urinar (29,19%).
4. n spital, cele mai multe infecii enterococice au provenit din seciile de ATI i
chirurgie.
5. Cele mai frecvente infecii enterococice diagnosticate n ambulator au fost
infeciile tractului urinar (75,6%).
6. n timp ce infeciile din ambulator au fost n totalitate monomicrobiene, la
pacienii spitalizai enterococii au fost relativ frecvent asociai bacililor Gram-negativi.

Studiul 2. Testarea sensibilitii la antibiotice a tulpinilor de

Enterococcus spp. i evidenierea fenotipurilor de
rezisten circulante

Tulpinile slbatice sunt extreme de semnificativ statistic mai prezente n

ambulator (47,56%) dect n mediul de spital (11,67%), p<0,001.
Alterarea sensibilitii la beta-lactamine a fost identificat la 43,06% din
tulpinile de Enterococcus spp. provenite din spital i la 29,26% din enterococii izolai
n ambulator.
Studiul rezistenei la aminoglicozide a evideniat 46,71% tulpini de
Enterococcus spp. rezistente n mediul spitalicesc i respectiv 24,39% n ambulator.
Cele 7 tulpini VRE identificate n secia de ATI, pe parcursul studiului,
reprezint 3,19% din totalul enterococilor izolai i respectiv 5,1% din tulpinile
provenite din spital.
Numai 8,02% dintre enterococii testai au fost rezisteni la linezolid, n
timp ce niciuna din tulpinile cuprinse n studiu nu a fost rezistent la tigeciclin, aceste
dou antibiotice fiind recomandate n tratamentul infeciilor produse de tulpini de
enterococi multirezistente.
Numrul mare de tulpini de Enterococcus spp. rezistente la beta-lactamine
i aminoglicozide, izolate n prezentul studiu, este ngrijortor datorit faptului c
aceste antibiotice sunt considerate de prim intenie n tratarea infeciilor
enterococice.
8 Pepelea Lia Sorina

Concluzii generale
Tulpinile de Enterococcus spp. izolate din spital au reprezentat 15,6% din
totalul germenilor implicai n etiologia unor infecii, n timp ce n ambulator 7,05%
din infeciile diagnosticate au fost determinate de enterococi.
Enterococii dei nu sunt printre cei mai frecvent izolai ageni patogeni,
prin rezistena lor natural la cefalosporine, fac parte din flora implicat n etiologia
unor infecii grave la pacieni spitalizai.
Stabilirea fenotipurilor de rezisten circulante n spital dar i n
ambulator este important n elaborarea terapiei antiinfecioase n regim de urgen.
Diferenele semnificative statistic privind rezistena la chimioterapicele
antiinfecioase n favoarea spitalului comparativ cu ambulatorul, sunt legate de
administrarea mai frecvent a antibioticelor la pacienii spitalizai, dar i de prezena
altor factori de risc privind transmiterea tulpinilor multirezistente.
Enterococii dein numeroase mecanisme de rezisten capabile s le
asigure supravieuirea n prezena chimioterapicelor antiinfecioase, ceea ce impune
identificarea unor posibile noi inte de aciune pentru antibiotice.
Controlul emergenei i rspndirii antibiorezistenei reprezint o
prioritate n sntatea public, la nivel mondial.

Elemente de originalitate i contribuii personale

S-a pus n eviden importana utilizrii metodelor moderne de identificare i
testare ale enterococilor prin sistemul automat VITEK 2 COMPACT, ceea ce a permis
ncadrarea tulpinilor n fenotipuri de rezisten, evideniind avantajele introducerii
acestei metode moderne de testare a sensibilitii la antibiotice, uor de utilizat,
performant i cu rezultate rapide.
Pentru izolarea i identificarea bacteriilor au fost utilizate medii moderne,
cromogene, ca i alternativ la metodele clasice de izolare ale bacteriilor, relevndu-se
avantajele utilizrii acestora i necesitatea introducerii lor i n alte laboratoare
pentru identificarea rapid a unor microorganisme.
Aplicarea i compararea metodelor moderne de identificare a bacteriilor cu
metodele clasice, are o deosebit aplicabilitate practic, eficientiznd astfel munca din
laborator.
Precizarea etiologiei infeciilor la pacieni spitalizai i din ambulator,
incidena speciilor i a asocierilor bacteriene, a demonstrat importana cunoaterii
enterococilor, lucrarea de fa fiind un ghid orientativ i pentru alte spitale din Cluj-
Napoca dar i din alte regiuni.
Elucidarea aspectelor epidemiologice privind infeciile enterococice,
urmrindu-se frecvena i repartiia acestor infecii pe diferite servicii medicale i
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 9

ambulator, reprezint o premier pentru spitalele universitare i laboratoarele private

din Cluj-Napoca dar i la nivel naional.
Precizarea frecvenei i a etiologiei diferitelor tipuri de infecii
enterococice (infecii urinare, infecii postoperatorii, infecii respiratorii,
bacteriemii/septicemii, infecii ginecologice, peritonite, colecistite, infecii de CVC)
este de un real folos pentru prevenirea i controlul acestor infecii, n special al celor
produse de tulpini de VRE.
Evidenierea tulpinilor VRE precum i a rezistenei multiple la antibiotice
a acestor microorganisme, izolate de la pacienii spitalizai, este o contribuie
personal important, cu deosebite repercusiuni practice, fiind un ghid orientativ
pentru medicii de laborator i clinicieni.
Precizarea fenotipurilor de rezisten pe clase de antibiotic (beta-
lactamine, aminoglicozide, glicopeptide, MLSB), precum i a fenotipurilor circulante
ale tulpinilor de Enterococccus spp. este de un real folos putnd orienta clinicianul spre
o terapie antimicrobian ct mai eficient.
10 Pepelea Lia Sorina

SUMMARY OF THE PH.D.THESIS

Resistance phenotypes found in

Enterococcus species isolated
from different pathological
products

Doctorand LIA SORINA PEPELEA

Conductor de doctorat Prof.dr. LIA MONICA JUNIE

CLUJ-NAPOCA 2015
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 11

CONTENTS

INTODUCTION 13
THE CURRENT STATE OF KNOWLEDGE
1.Generalities about the genus Enterococcus 17
1.1. Short history 17
1.2. Habitat and implications in infectious pathology 18
1.3. Morfotinctorial characters 19
1.4. Culture characters 20
1.5. Pathogenicity characters 25
1.6. Identifing epidemiological markers
26
2. Resistance of Enterococci to antimicrobial agents 28
2.1. Resistance to beta-lactams 29
2.1.1. Intrinsec resistance 29
2.1.2. Tolerance 30
2.1.3. Beta-lactamase 30
2.2. Resistance to aminoglycoside 31
2.2.1. Intrinsec resistance 31
2.2.2. Acquired resistance 32
2.2.3. High level aminoglycoside resistance 33
2.2.3.1. Incorporating antibiotic in agar 33
2.2.3.2. Microtablets with high concentration of antibiotic 33
2.2.3.3. Broth dilution method 34
2.3. Resistance to macrolides, lincosamides, streptogramines and
Ketolides (MLSK)
34
2.4. Resistance to glycopeptides 35
2.4.1. Screening methods for Vancomycin- resistant enterococci 37
2.5. Resistance to oxazolidinone 38
2.6. Antibiotics synergism 38
2.7. Alternative treatments for multiple antibiotic-resistant 39
enterococci
12 Pepelea Lia Sorina

PERSONAL CONTRIBUTION
1. Assumption of work/Objectives 43
2. Study 1. The Involvement of Enterococcus spp. Strains in the Aetiology of
Infections in Hospitalized Patients and outpacients 45

2.1. Background 45
2.2. Working hypothesis/Objectives
45
2.3. Material and Methods 45
2.4. Results 54
2.5. Discussions 66
2.6. Conclusions 71
3. Studiul 2. Testing the sensitivity of Enterococcus spp. strains to
antibiotics and highlighting the circulating resistance phenotypes. 73

3.1. Background 73
3.2. Working hypothesis/Objectives 73
3.3. Material and Methods 74
3.4. Results 80
3.5. Discussions 109
3.6. Conclusions 114
4. General conclusions 115
5. The originality and innovative contributions of the thesis 117

REFERENCES 119

Keywords: enterococci, resistance, Vancomycin, infections, aetiology, hospital, patients

Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 13

THE CURRENT STATE OF KNOWLEDGE

1. Generalities about the genus Enterococcus

Enterococci were initially classified as gram-positive cocci of the genus
Streptococcus.1
Were mainly related to the streptococci of fecal origin.
They were considered for a long time to be a major division of the Streptococcus
(group D streptococci), differentiated by their higher resistance to chemical and
physicalagents.2
In the early 1930s, the enterococci were splitting as group D streptococci and
they were differentiated from non-enteococcal group D streptococci by auxiliary
biochemical tests.2
Over the next decade, Sherman recommended that the term enterococcus had
been used for streptococci that grow at 10 to 45 C, in 6,5% NaCl and at pH=9.6, and
wich survived 60 from 30 minuets and had the ability to split esculin.3
Definitive evidence of S.faecalis and S.faecium from the other members of the
genus made possible the inclusion into a separate genus, whose characteristics have
been very studied by molecular biology tehniques.2
Current criteria for inclusion in the genus Enterococcus include a combination
of DNA-DNA reassiciation values, 16rRNA gene sequencing, conventional phenotypic
test, whole-cell protein analysis.4,5
Microorganism that are now included in the genus Enterococcus have been
categired as a low pathogenicity bacteria, but in the 1990s the prevalence of these
organsim as nosocomial pathogens is clearly increasing. 6
In the case of hospitalized patients, enterococci can be distinguished at the level
of soft tissue wounds, ulcers, and the gastrointestinal tract.7
Infectious syndromes that recognize enterococci in their aetiology can be
represented by urinary tract infections in the case of patients with obstructive
uropathies and by mixed wound infections, especially in the case of patients
undergoing antimicrobial treatment8,9.
Bacteraemias may occur in the elderly and in immunocompromised patients. 10
Studies conducted in the United States demonstrated that the most pacients
infected with Vancomycin-resistant enterococci strains coming from ICU.11
Among the risk factors that may be added to the ones already mentioned are:
prolonged hospitalization, gastrointestinal colonization with VRE, exposure to
contaminated medical equipment, medullary transplant, haematological conditions,
treatment with third-generation cephalosporins and antibiotics acting on anaerobic
bacteria.11,12
14 Pepelea Lia Sorina

Enterococci rarely cause respiratory tract infections, osteomyelitis or

cellulite; however, they have become the third cause of nosocomial infections in the
United States.13
Enterococcal infections can be caused by at least 19 species including: E.
avium, E. casseliflavus, E. durans, E.faecalis, E. faecium, E. gallinarum, E. hirae, E.
malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. solitarius 14.
A number of other species were subsequently added to this list: E. cecorum,
E.columbae, E. saccharolyticus, E. dispar, E. sulfurous, E. seriolicida i E. flavescens.15
Most clinical infections are caused by E. faecalis (80-90%) or E. faecium (5-
15%) .16

E.durans, E. avium i E. hirae species are less common, and other species
mentioned are only isolated ocazional.17

2. Resistance of Enterococci to antimicrobial agents

Being involved ever more frequently in nosocomial infections, enterococci
present natural or acquired resistance to antibiotics, which allows them to survive in
the face of multiple antibiotic treatment schemes.18
Enterococci have acquired resistance to a number of antimicrobial agents,
including tetracycline, erythromycin, chloramphenicol, high levels of trimethoprim,
clindamycin, high levels of aminoglycosides and penicillins (via penicillinase and non-
penicillinase mechanisms), and, more recently, to vancomycin.
All of these resistance instances only raise new questions in connection with
antibiotic therapy in the case of enterococcal infections, because there occurs a
decrease in the number of therapeutic options 19.

PERSONAL CONTRIBUTION

1. Assumption of work/Objectives

Enterococci, although they are part of the intestinal flora commensals, may
become nosocomial pathogens.
In the past three decades, a constant growth in the number of infections
caused by species of the genus Enterococcus has been reported, a phenomenon
probably caused by a growth in the population that shows risk factors (chronic
inflammations, surgery, intestinal neoplasia), but also by the emergence of enterococci
strains with multiple resistance to antibiotics, especially Vancomycin 20.
The present paper is concerned with the assessment of the involvement of
Enterococcus spp. strains in the aetiology of infections, and these microorganisms
sensitivity to antibiotics.
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 15

To this end, both the strains isolated from hospital ward infections as well as
the strains isolated from outpatients were taken into study.
Secondly, the present paper attempts to highlight the circulating resistance
phenotypes of the enterococci strains isolated from the hospital wards as well as from
outpatients, which phenotypes play a role in the improvement of the empirical
antibiotherapy of the infections caused by these strains.

Material and Methods

The present study was conducted in a prospective manner, in that the
Enterococcus spp. strains involved in the aetiology of infections in hospitalized
patients were observed, by ward, during the period January 2010 December 2012.
The hospitalized patients were from:
The Intensive Care ward (54 beds)
2 General Surgery wards (130 beds)
The Orthopaedics ward (60 beds)
The Gastroenterology ward (50 beds)
The Neonatal ward (40 beds)
The Obstetrics and Gynaecology wards (85 beds)
In the hospital, data from the Clinical Laboratorys Microbiology
Compartment was used. In order to collect data from the hospital, we used the
standard surveillance data sheet, presented in the annex of Health Ministry Order no.
10/2005.
For each year studied (2010, 2011, and 2012), data was collected both about
the enterococci strains that proved to be a unique etiological agent and the
enterococci strains isolated in association with other microorganisms.
The samples were normally collected from patients by specialized hospital staff,
on account of the fact that the observance of sample-collecting and transportation
rules for pathological products intended for bacteriological analysis influences the
different stages and the accuracy of the diagnosis.
Urine, blood, and cephalorachidian fluid samples were collected, as well as
samples from purulent collections, bronchial aspirate, sputum, and central venous
catheters.
The collected samples were sown on Columbia gelose supplemented with sheep
blood (5%), and then incubated at 37 C for 24 hrs.
In order to isolate the enterococci, we also used the Bile Esculin Azide Agar (Sanimed)
medium, CPS (bioMrieux), Trypticase Soy Agar with 5% sheep blood (bioMrieux),
and the Uriselect (BioRad) chromogenic medium21.
The identification of bacteria belonging to the genus Enterococcus was done
based on culture and biochemical characters.
The observance of the culture characters involved the presence of the following
types of colony:
16 Pepelea Lia Sorina

on gelose-blood, type S, small, white-grey, non-haemolytic colonies - E.

faecalis, beta-haemolytic colonies E durans, or alpha-haemolytic colonies;
on the Bile Esculin Azide Agar medium, the enterococci develop white-grey
colonies with a black halo;
by wiping with a wad the 24 hour culture obtained on the Trypticase Soy
Agar medium and 5% sheep blood, one can notice the mediums yellow colouring- E.
casseliflavus, E. mundtii, E. sulfureus;
On the chromogenic CPS medium, the enterococci colonies are green and
small.
In the hospital, the isolated enterococci cultures were identified via the Vitek 2
Compact automatic system (bioMrieux).
The Vitek 2 cards for the identification of Gram-positive cocci are standardized
products that contain miniature biochemical tests.
The Enterococcus species identified by VITEK 2 GP: E.faecalis, E.faecium,
E.avium, E.casseliflavus, E.cecorum, E.columbae, E.durans, E.gallinarum, E.hirae,
E. raffinosus, E. Saccharolyticus.

Statistical methodology
The data obtained as a result of research was entered into and stored in
Microsoft Access 2010-type (*.mdb format) databases and was exported for processing
to Excel 2010 (*.xls) files.
Statistic processing was carried out with the aid of Microsoft Excel 2010, EPI-
INFO version 6.04 and IBM SPSS version 10 packages, and the results of this processing
were exported in all three cases to Excel 2010 worksheet files.
The dichotomous variables, characterized by percentage, were compared by
applying contingency tables and calculating the 2 test, and using the Fisher correction
where necessary 22.
All these statistical tests were calculated with two extremities, and the
statistical significance value p was taken into account in the case of values 0.01.

Study 1. The Involvement of Enterococcus spp. Strains in the

Aetiology of Infections in Hospitalized Patients and
outpacients

The species most frequently isolated in hospitalized and outpatients was

E. faecalis (74.88%), followed by E. faecium (20.09%).
8.04 % were other Enterococcus species encountered only in the hospital
environment: E. durans (2.19%), E. casseliflavus (2.19%), E. gallinarium (2.19%), and
E. avium (1.45%).
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 17

In hospitalized patients Enterococci were more frequently involved in the

etiology of postoperative infections(30.72%) followed by urinary tract infections
( 29.19 %).
In the hospital, most of enterococcal infections came from the ICU and surgery.
The most common enterococcal infections that were diagnosed in outpatient
were urinary tract infections (75.6 %).
While in outpacients infections were fully monomicrobiene, hospitalized
patients Enterococci were relatively commonly associated Gram -negative bacilli.

Studiul 2. Testing the sensitivity of Enterococcus spp. strains

to antibiotics and highlighting the circulating resistance
phenotypes.

Wild strains are more frequently present in outpatients in an extremely

statistically-significant manner (47.56%) than they are in a hospital setting (11.67%),
p<0.001.
The alteration of the sensitivity to beta-lactams was identified in 43.06%
of the Enterococcus spp. strains coming from the hospital and in 29.26% of the
enterococci isolated in outpatients.
The study of resistance to aminoglycosides showed 46.71% resistant
Enterococcus spp. strains in a hospital setting and 24.39% in the case of outpatients.
The 7 VRE strains identified in the Anaesthesia Intensive Care ward, in
the course of the study, represent 3.19% of the total enterococci isolated and 5.1% of
the strains coming from the hospital.
Only 8.02% of the enterococci tested were resistant to linezolid, while
none of the strains comprised in the study was resistant to tigecycline, these two
antibiotics being recommended in the treatment of infections produced by
multiresistant enterococci strains.
The large number of Enterococcus spp. strains resistant to beta-lactams
and aminoglycosides isolated in the present study is alarming due to the fact that
these antibiotics are considered to be the first option in the treatment of enterococcal
infections.

General conclusions

The Enterococcus spp. strains isolated in hospital represented 15.6% of

the total germs involved in the aetiology of certain infections, while in the case of
outpatients, 7.05% of the infections diagnosed were caused by enterococci.
18 Pepelea Lia Sorina

Although they are not among the most frequently isolated pathogenic
agents, enterococci, through their natural resistance to cephalosporins, are part of the
flora involved in the aetiology of certain serious infections in hospitalized patients.
The establishment of the circulating resistance phenotypes in hospital
but also in outpatients is important in the urgent development of the anti-infective
therapy.
The statistically-significant differences regarding the resistance to
antibiotics in favour of the hospital, as opposed to outpatients, are connected to the
more frequent administration of antibiotics in the case of hospitalized patients, but
also to the presence of other risk factors regarding the transmission of multiresistant
strains.
Enterococci hold numerous resistance mechanisms capable of ensuring
their survival in the presence of antibiotics, which calls for the identification of certain
possible new action targets for antibiotics.
The control of the emergence and spread of antibioresistance is a public
health priority, worldwide.

Original elements and personal contributions

The study highlights the importance of using modern methods to identify

and test enterococci through the automatic VITEK 2 Compact system, which allowed
the strains to be classified as resistance phenotypes and showed the advantages of
introducing this modern, easy-to-use, accurate method of testing sensitivity to
antibiotics, which yields quick results.
For the isolation and identification of bacteria, modern, chromogenic
media were used as an alternative to the classical methods of isolating bacteria, and
the study points out the advantages of using them and the necessity of introducing
them in other laboratories as well, for a quick identification of certain
microorganisms.
The application and comparison of modern methods of identifying
bacteria with classical methods has a special practical applicability, in that it makes
laboratory work more efficient.
The specification of the aetiology in hospitalized patients and
outpatients, and of the incidence of species and bacterial associations, has proven the
importance of knowledge on enterococci, the present paper also being also an
indicative guide for other hospitals in Cluj-Napoca and other regions.
The elucidation of the epidemiological aspects regarding enterococcal
infections, which involved tracking the frequency and distribution of these infections
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 19

by different medical and outpatient services, represents an all-time first for university
hospitals and private laboratories in Cluj-Napoca and also nationwide.
Specifying the frequency and aetiology of different types of enterococcal
infections (urinary infections, post-operative infections, respiratory infections,
bacteraemias/septicaemias, gynaecological infections, peritonitis, cholecystitis, CVC
infections) is genuinely useful in the prevention and control of these infections,
especially those caused by VRE strains.
The highlighting of the VRE strains as well as of the multiple resistance
to antibiotics of these microorganisms isolated from hospitalized patients is an
important personal contribution that has outstanding practical repercussions, as it is
an indicative guide for laboratory doctors and clinicians.
Specifying the resistance phenotypes by antibiotic class (beta-lactams,
aminoglycosides, glycopeptides, MLSB), as well as the circulating phenotypes of the
Enterococcus spp. strains is really helpful, because it can point the way for clinicians to
a highly efficient antimicrobial therapy.
20 Pepelea Lia Sorina

Referine
1. Lucia Martins Teixeira, Carvalho M. G. S., Merquior V. L., Steigerwalt A. G. and al., 1997.
Recent approaches on the taxonomy of the enterococci and some related microorganisms.
Adv. Exp. Med. Biol. 418: 397-400
2. Schleifer, K.H., R. Kilper-Balz. 1984, Transfer of Streptococcus faecalis and Streptococcus
faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb.nov. and
Enterococcus faecium comb.nov. Int. J. Syst.Bacteriol. 34:31-34
3. Sherman JM. The streptococci. Bacteriol Rev 1937;1: 3-97
4. Devrise, L. A., Collins M. D., Wirth R., 1992, The genus Enterococcus, p.1465-1481. In A.
Balows, H. G. Truper, Dworkin M., Harder W., Schleifer K. H. (ed), The Prokaryotes. A
Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications,
2nd ed. Springer-Verlag, New York, N.Y
5. Facklam R. R, Carvalho G. S., Teixeira L. M.,2002. History, taxonomy, biochemical
caracteristics and antibiotic susceptibility testing of enterococci, p.1-54. In Gilmore M. S,
Clewell, Courvalin P., Dunny G. M., Murray B. E, Rice L. B (ed), The Enterococci: Pathogenesis,
Molecular Biology and Antibiotic Resistance., ASM Press, Washington, D.C
6. Merquior V. L. C, Peralta J. M., Facklam R. R., Teixeira. 1994. Analysis of electrophoretic
wholecell protein profiles as a tool for characterization of Enterococcus species. Curr.
Microbiol. 28: 149-153
7. Patel R., Piper K. E., Rouse M. S., Steckelberg J. M., Uhl J. R., Kohner M. K., and al., 1998.
Determination of 16 rARN sequences of enterococci and application to species identification
of nonmotile Enterococcus gallinarum isolates. J. Clin. Microbiol. 36:3399-3407
8. Noble, C. J. 1978. Carriage of group D streptococci in the human bowel. J. Clin. Pathol.
31:1182-1186.
9. Kurrie, E., S. Bhaduri, D. Krieger, W. Gaus, H. Heimpel, H.Pflieger, R. Arnold, and E. Vanek.
1981. Risk factors for infections of the oropharynx and the respiratory tract in patients with
acute leukemia. J. Infect Dis. 144:128-136.
10. Smyth, C. J., M. A. Docent, M. K. Halpenny, and S. J. Ballagh. 1987. Carriage rates of
enterococci in the dental plaque of haemodialysis patients in Dublin. Br. J. Oral Maxillofacial
Surg. 25:21-33.
11. Edwards DD. Enterococci attract attention of concerned microbiologists. ASM News 2000;
66: 540-5
12. Warren, R. E. 1988. Difficult streptococci. J. Hosp. Infect.II(Suppl. A):352-357
13. Harding, G. K. M., and A. R. Ronald. 1974. A controlled study of antimicrobial prophylaxis of
recurrent urinary infection inwomen. N. Engl. J. Med. 291:597-601.
14. Warren, J. W., J. H. Tenney, and J. M. Hoopes. 1982. Aprospective microbiologic study of
bacteriuria in patients with chronic indwelling urethral catheters. J. Infect. Dis. 146:719.
15. Maki, D. G., and W. A. Agger. 1988. Enterococcal bacteremia:clinical features, the risk of
endocarditis, and management.Medicine (Baltimore) 67:248-269.
Fenotipuri de rezisten ntlnite la specii de Enterococcus izolate din diferite produse patologice 21

16. Hoffmann, S. A., and R. C. Moellering. 1987. The enterococcus: "putting the bug in our ears."
Ann. Intern. Med. 106:757-761.
17. Schaberg DR, Culver DH, Gaynes RP. Major trends in the microbial etiology of nosocomial
infection. Am J Med 1991; 91:72S-75S
18. Evans, A. C., and A. L. Chinn. 1947. The enterococci: with special reference to their
association with human disease. J.Bacteriol. 54:495-512.
19. Facklam, R. R., and M. D. Collins. 1989. Identification of Enterococcus species isolated from
human infections by a conventional test scheme. J. Clin. Microbiol. 27:731-734.
20. Collins, M. D., D. Jones, and J. A. E. Farrow, R. Kilpper-Balz,and K. H. Schleifer. 1984.
Enterococcus aivium nom. rev.,comb. nov.; E. casseliflavus nom. rev., comb. nov.; E. durans
nom. rev., comb. nov.; E. gallinarurn comb. nov.; and E.malodoratus sp. nov. Int. J. Syst.
Bacteriol. 34:220-223.
21. Barbara E. Murray, Beta-Lactamase-Producing Enterococci. In Antimicrobial Agents And
Chemotherapy, 2002, 36: 2355-2359
22. Sharma, R., L. Sharma, and B. Kapoor. 2005. Antibacterial resistance: current problems and
possible solutions. Indian J. Med. Sci. 59120-129.
23. Fritsche TR, Sader HS, Jones RN. Epidemiology of Antimicrobial Resistance: Species
Prevalence, Susceptibility Profiles, and Resistance Trends. In: Lorian V. Antibiotics in
laboratory medicine. Lippincott Williams &Wilkins. 2005; Chapter 17: 815-850
24. Knapp R, Miller III MC. Test of Statistical Significance: chi-square Procedure in Clinical
Epidemiology and Biostatistics, Williams &Wilkins, Baltimore, Maryland 1992; 209-21
25. Buiuc D, Negu M. Tratat de Microbiologie Clinic. Editura Medical, Bucureti, 1999