1

National multicenter survey of multidrug-resistant Pseudomonas aeruginosa

and Acinetobacter baumannii in Belgian hospitals in 2013

Y. Glupczynski, C. Berhin, C. Bauraing, T.-D Huang, P. Bogaerts

Laboratoire de bactériologie, CHU Dinant-Godinne UCL I Namur, 5530 Yvoir

A. Deplano, C. Nonhoff, R. de Mendonça, S. Roisin, O. Denis

Laboratoire de bactériologie, Hôpital Universitaire Erasme – ULB, 1070 Bruxelles

B. Jans, B. Catry

Healthcare Associated Infections (NSIH), Public Health & Surveillance,

Scientific Institute of Public Health, 1050 Brussels

Objectives:

We conducted a large national multicenter survey conducted in Belgian acute care

hospitals during the first semester of 2013, with the following objectives:

(1) To characterize the most prevalent mechanisms of acquired transferable

resistance to carbapenems and to other beta-lactams Pseudomonas

aeruginosa and Acinetobacter baumannii isolates by phenotypic and

genotypic methods and to determine their prevalence among MDR clinical

isolates belonging to these species.

(2) To determine the MIC values and activity of a panel of antimicrobial agents by

the reference microdilution method against MDR P. aeruginosa and A.

baumannii.

(3) To study the epidemiological relatedness of these MDR isolates by several

molecular typing methods including rep-PCR, PFGE and by Multilocus

sequence typing (MLST). For Pseudomonas aeruginosa, O serotyping and

characterization of the variable regions of class 1 integrons by PCR-mapping

and sequencing was also carried out.

Materials and Methods:

2

Study design

All acute care hospital-affiliated laboratories who participate to the NSIH network

were invited to participate to the survey by mailing in November 2012. Participants

were asked to send a maximum of up to 5 isolates of multi-drug resistant (MDR) P.

aeruginosa and 5 MDR A. baumannii isolates (total maximum of 10 isolates per

centre).

Isolates were collected prospectively between 01/2013 and 05/2013. Participants

were requested to fill in a short questionnaire with demographic data (age, gender),

source of isolation (i.e. anatomic isolation site), hospitalization (ward/unit of

hospitalization at the time of sampling) or ambulatory status of the patient. Clinical

specimens obtained routinely for diagnostic or from screening specimens (e.g. rectal

swabs, upper respiratory tract) were accepted but duplicate isolates collected from

the same patients were excluded.

The isolates were stored at -20°C or at -80°C locally and were referred to the national

reference center in batch at the end of the collection period (1) for confirmation of

species identification, (2) for phenotypic and genotypic characterization of

transferable class A, B, and D penicillinases (including small-spectrum penicillinases,

ESBLs and/or carbapenemases), (3) and for further characterization of MDR isolates

(O serotyping for P. aeruginosa, PFGE and MLST. Molecular epidemiology of class 1

integrons (variable sequences between the 5’ CS and 3’ CS sequences of the

integrons) was investigated by PCR mapping and by sequencing of the amplicons in

all MBL- and OXA- producing P. aeruginosa isolates

Case definitions

In P. aeruginosa, multidrug resistance (MDR) was defined as non-susceptibility (i.e:

resistance or intermediate resistance by CLSI or EUCAST interpretative criteria) to >

one representative antibiotic from at least four out of five of the following classes of

antimicrobial agents: (broad-spectrum penicillins [ticarcillin +/- clavulanate,

piperacillin +/- tazobactam]; anti-pseudomonal cephalosporins [ceftazidime,

cefepime]; carbapenems [imipenem, meropenem]; fluoroquinolones [levofloxacin,

ciprofloxacin]; aminoglycosides [gentamicin, tobramycin, amikacin]. For

Acinetobacter baumannii, MDR was defined for isolates intermediate or resistant to

meropenem. This simplified definition was used since resistance to carbapenems in

3

A. baumannii is almost systematically found in association with resistance to most

other classes of antimicrobial agents including broad-spectrum penicillins and

cephalosporins, aminoglycosides and fluoroquinolones in contrast to the situation

observed in P. aeruginosa) in which OprD deficient mutants account for the large

majority of carbapenem-resistant strains often without any associated resistance

mechanisms.

Identification and susceptibility testing

Identification of all isolates was verified by MALDI-TOF MS on a microflex LT (Bruker

Daltonik GmbH, Leipzig, Germany) for verification of the species identification. The

acquired bacterial spectra were analysed in the MALDI Biotyper 3.0 software with the

database version 3.1.2. In vitro antimicrobial susceptibility testing was checked for all

isolates by disc diffusion on Mueller-Hinton agar using CLSI guidelines and

interpretative criteria (CLSI M100-S23 document; January 2013).

MICs of 12 antibiotics were determined against all confirmed MDR P. aeruginosa and

A. baumannii isolates whatever the underlying resistance mechanisms (i.e. presence

or absence of carbapenemase and/or other transferable acquired β-lactamases)

ESBL-producing isolates) by microdilution using Sensititre® plates (GNX2F panels,

Trek Diagnostic Systems, UK). For Acinetobacter spp. isolates, the MIC value of

tigecycline was also determined and in the absence of any CLSI interpretative

guidelines, susceptibility categorization was determined according to the breakpoint

tables for interpretation of MICs of EUCAST (Version 4.0, valid from 2014-01-01).

Phenotypic screening and confirmatory tests for the presence of

carbapenemase

Detection of putative metallo-β-lactamases (MBLs) was carried out by inhibitor based

combination disk test on Mueller-Hinton II agar plates (BD) with an inoculum of 0.5

McFarland (+/- 1.5 x 108 CFU/ml) using the total MBL confirm kit (ref. N° 98006;

ROSCO Diagnostica Neosensitabs, A/S Taastrup, Denmark) including imipenem (10

µg) with and without EDTA (750 µg) imipenem (10 µg) with and without DPA (1000

µg) and meropenem (10 µg) with and without DPA (1000 µg) tablets.1

Interpretative criteria for a positive test result (presence of an MBL) were applied

according to the manufacturer’s recommandations (i.e.: a difference of diameter > 5

mm of imipenem and of meropenem in the presence of DPA versus one or both

4

carbapenem indicators and a difference of diameter > 10 mm of imipenem in the

presence of EDTA [ROSCO Diagnostica User guide; Detection of carbapenemases;

Document 1.5.0.; last date of access 17/05/2014]). The phenotypic detection of

carbapenemase-producing P. aeruginosa was also assessed by means of the Carba

NP test, a rapid imipenem hydrolysis-based colorimetric assay, according to the

method reported by Dortet et al.2

Molecular techniques

All MDR isolates were systematically screened (whatever the results of the

phenotypic tests) by means of several in house validated multiplex endpoint PCR

protocols targeting some of the epidemiologically most relevant carbapenemases

(PCR CARBA targeting blaVIM, blaKPC, blaNDM, blaOXA-48) and PCR OXACARBA

targeting blaOXA-23, blaOXA-24, blaOXA-58 and blaOXA-143 genes) and the minor extended

spectrum β-lactamases (PCR MINOR ESBL targeting blaBEL, blaGES, blaPER and

blaVEB) according to the method reported by Bogaerts et al.3,4 Further, β-lactamase

genes coding for OXA-1, OXA-2, OXA-10 group, OXA-9, OXA-18, OXA-20, OXA-198

and CARB-1/4-6, CARB-2 (PSE-1) were sought by multiplex PCR in MDR P.

aeruginosa only. The genetic context of the targeted genes was assessed by PCR

mapping and sequencing of the variable region of the integrons by using primers

designed on the basis of the 5’ and 3’ conserved segments of class 1 integron in

combination with specific primers for blaVIM, blaIMP, blaBEL and blaOXA genes according

to methods previously reported.5 Selected PCR products were sequenced on an ABI

3100 Genetic Analyser (Applied Biosystems). The presence of OprD

mutations/deficiencies, of active efflux and of AmpC cephalosporinase

overexpression was inferred from phenotypical resistance patterns by disc diffusion

AST also including an in house disk combination test of imipenem (10 µg) alone vs

imipenem 10 µg disk plus cloxacillin (4000 µg/disk) (restoration of imipenem activity

by cloxacillin in the presence of overexpression of AmpC combined with OprD

deficiency) as reported by Fournier et al.6

PFGE and serotyping

PFGE of SpeI-digested genomic DNA was used to examine the clonal relatedness of

the isolates. Computer-assisted analysis of PFGE profiles was performed using

BioNumerics software (Applied Maths, Kortrijk, Belgium) and clonal relatedness was

5

determined following the classification criteria previously described by Deplano et al.7

PFGE type included profiles showing up to six DNA fragment differences

corresponding to a > 80% level of Dice similarity.

O serotyping was determined by slide agglutination test using polyvalent antisera and

16 monovalent antisera numbered O1-O16 according to the manufacturer’s

instructions (Bio-Rad, Marnes-La-Coquette, France).

Multilocus sequence typing

Multilocus sequence typing (MLST) was performed according to the protocol

published by Curran et al.8 Nucleotide sequences were determined for internal

fragments of the acsA, aroE, guaA, mutL, nuoD, ppsA and trpE genes on both

strands and searched against the MLST database (http//:pubmlst.org/paeruginosa/)

for assignment of allelic numbers and sequence type (ST). The eBURST software

was used for phylogenetic analysis as described by Feil EJ et al.9 Clonal complexes

(CCs) were defined as a group of isolates with either identical STs or STs that varied

at one or two loci (single- or double-locus variants).

Epidemiology of resistance

The frequency and distribution of the types of resistance mechanisms, serotyping

and clonal diversity among the MDR P. aeruginosa clinical isolates collected were

assessed in order to monitor the extension of the spread of previously known

epidemic strains (so-called high-risk clones) as well as the possible emergence of

new high-risk MDR clones and/or of novel genetic resistance determinants across

Belgian hospitals.

Results

On the whole, 69 participants (regional distribution: 33 from Flanders, 26 from

Wallonia and 10 from the Brussels area) sent a total of 229 putative MDR P.

aeruginosa and 47 putative MDR A. baumannii isolates collected between Feb. 1th

and June 30th 2013. Out of these 276 isolates, 265 were viable at culture, did match

with the expected identification (P. aeruginosa [n=221]; A. baumannii [n=44]) and

were thus further characterized.

A. Epidemiology of MDR P. aeruginosa in Belgian hospitals

Among

confirm

and/or

(resista

were h

131 MD

About

roughly

specim

isolates

percen

ICU or

urine a

only ag

isolates

g 221 P. ae

med as non

meropene

ance to >3

hospitalized

DR P. aeru

half of the

y 25% of t

mens. Spec

s (of note

t of the pa

r in medici

mong patie

gent which

s (MIC90= 2

eruginosa i

n-susceptib

em) while

3 classes o

d at the tim

uginosa iso

e isolates o

the MDR P

cimens fro

, 5% MDR

tients colo

ine wards

ents from i

h still displa

2 µg/ml) (T

isolates co

ble to carb

e 131 (59

of antimicr

me of sam

olates are s

originated

P. aerugin

m deep-se

R isolates

onized/infec

(mostly re

internal me

ayed cons

Table 2).

ollected du

apenems (

%) were

robial agen

mpling and

shown in T

from the l

nosa isolate

eated sites

recovered

cted by MD

espiratory

edicine wa

tant in vitr

ring the st

(intermedia

indeed co

nts). The w

the anato

Table 1.

ower resp

es were a

s accounte

d from pos

DR P. aeru

tract spec

ards). By m

ro activity a

udy period

ate or resis

onfirmed a

ward/units

omic isolate

iratory trac

associated

ed for less

sitive bloo

uginosa we

cimens in

microdilutio

against MD

d, 179 (81%

stant to im

as MDR

in which p

es sites fo

ct specime

with urina

s than 20%

od cultures

ere hospita

ICU patien

n, colistin w

DR P. aeru

6

%) were

mipenem

isolates

patients

or these

ens and

ary tract

% of the

s). Sixty

alized in

nts and

was the

uginosa

Fifty-th

aerugin

lactama

excepti

strains

positive

and of

Fourty-

various

carben

(cf. Tab

ESBLs

carbap

one sin

oxacilli

modera

false-ne

combin

respect

*Second

ree of the

nosa) wer

ases (MB

ionnaly of

were foun

e by IMI +/

98%, resp

-two of th

s types

icillinases)

ble 3). Mo

(without

enem hyro

ngle centre

nase with

ate level o

egative by

nation inh

tively.

dary (acquire

179 (30%)

re found

BLs), mos

f IMP type

nd to expre

/- DPA or

ectively).

he 179 (2

of ot

) either alo

st of the is

the asso

olysis by th

e and of sim

broadene

of hydrolys

y carbap N

ibitor disk

d) β-lactama

) carbapen

to harbou

stly of V

e (IMP-13

ess a carb

by the IMI

24%) carb

her narr

one or in a

solates ca

ociation w

he carba N

milar clona

d spectrum

sis of carb

NP test. T

ks with D

ases with bro

nem-resista

ur resistan

VIM-types

[n=1]). F

bapenemas

I +/- EDTA

bapenem-r

row-spectr

ssociation

rrying narr

with an M

NP test. Ho

al origin) ca

m that has

bapenems1

The specifi

DPA or E

oad-spectrum

ant isolate

nce genes

(VIM-2 [

Fifty-one o

se by the

A synergy

resistant M

rum pen

with MBL

row-spectr

MBL) yield

owever, thr

arried a ge

s been pre10 and co

city of the

DTA were

m are shown

s (all of the

s coding

n=51], VI

f these 53

carba NP

disk test (

MDR isola

nicillinases

s or some

rum beta-la

ded a ne

ree isolates

ene coding

eviously sh

ould thus b

e carba NP

e of 100

in the table

em being M

for metal

IM-4 [n=1

3 MBL-pro

test and 5

sensitivity

ates co-pr

(oxacil

times with

actamases

egative res

s (originati

g for OXA-

hown to e

be conside

P test and

% and o

in bold chara

7

MDR P.

lo-beta-

1]) and

oducing

52 were

of 96%

roduced

linases,

ESBLs

s and/or

sult for

ng from

198, an

exhibit a

ered as

d of the

of 95%,

acters.

By PFG

clone ty

that O1

60% of

were O

in 7% o

Fig. 1.

The 53

differen

PFGE

predom

Interes

almost

of type

the inte

countrie

France

produc

GE typing,

ypes and

12 and O1

f the MDR

O6, O10 an

of the isola

Serotypin

3 MBL-pro

nt cities (in

into 12 m

minant bein

tingly, PFG

exclusivel

s X and Q

ernational

es throug

e, Spain,

cing P. aeru

N=125

65 of the

19 isolates

11 were th

P. aerugin

nd O15. N

ates (Fig. 1

ng of MDR

ducing P.

n 9 out of

ajor clone

ng isolated

GE type X

ly found in

Q were foun

clonal clus

ghout Euro

UK). Thes

uginosa in

MDR P. a

s were sing

he two mo

nosa isolat

on aggluti

).

R P. aerugi

aeruginos

10 provin

s of which

d in 16 an

was largel

n the Bruss

nd to be al

ster ST111

ope (Gree

se two clo

Belgium in

aeruginosa

gleton (spo

st frequen

es. Other l

nable or p

inosa stra

sa isolates

ces of Be

h two type

nd in 5 dif

ly spread t

sels area.

most exclu

1 which is

ece, Hung

ones were

n 2005 and

a were del

oradic isol

nt serotype

less freque

polyagglutin

ains collec

were coll

lgium) (Fig

s, X and Q

fferent hos

throughout

VIM-2 pro

usively ser

s currently

gary, Sca

e already

d in 2010.1

inated in 2

ates). O s

es altogeth

ently encou

nable sero

cted in Be

ected in 3

g. 2) and

Q were fo

spitals res

t Belgium w

oducing MD

rotype O12

highly wid

andinavian

predomin1,12

29 differen

erotyping s

her accoun

untered se

otypes wer

lgium in 2

34 hospital

they cluste

ound to be

spectively

while clone

DR P. aeru

2 and belo

despread i

countries

nant amon

8

t PFGE

showed

nting for

erotypes

e found

2013

ls in 24

ered by

largely

(Fig 3).

e Q was

uginosa

nged to

n many

s, Italy,

ng VIM-

Fig. 2. Belgia

Altoget

of sero

did bel

aerugin

marked

244) d

produc

These

interna

similari

Remar

with ST

includin

IMP, N

Belgium

for whic

Distributin hospital

ther, three

type O12 w

long to ST

nosa in Be

dly decreas

id emerge

cing P. aer

were mo

tional clon

ty patterns

kably the S

T 111 in t

ng Belgium

DM, PER,

m5. The pro

ch MLST ty

on of MBLls in 2013

other VIM

were close

T 244. Wh

elgium in

sed and tw

e in 2012.

ruginosa (A

ostly of se

nal cluster,

s below cu

ST 235 clo

the presen

m to be co

BEL, SHV

oportion of

yping coul

L- and ES

M-2 produc

ely related

hile clone

200512 an

wo new re

Likewise,

AO, AS, A

erotype O

, ST 235 w

t-off of 80%

onal lineage

nt survey h

oupled with

V, GES, O

f the differ

d be deter

BL-produ

cing clones

to each ot

W was th

nd it was

lated clone

several o

AW, AT, ZG

O11 and b

which enc

% (cf. Fig.

e which ap

has been

h the spre

OXA) in ma

rent ST typ

rmined is s

cing P. ae

s of P. aer

ther (PFGE

he third m

also found

es (AU an

ther new

G) were a

belong to

compassed

3).

ppeared as

frequently

ead of sev

any countri

pes of 59 M

hown in th

eruginosa

ruginosa a

E types W

ajor clone

d in 2010

d AP, also

clones of

lso identifi

another

d multiple

s the most

found in

veral beta-

ies13 includ

MDR P. ae

he Fig. 4.

isolates i

lso predom

, AU and A

type of M

,11 its prev

o belongin

VIM-2 (or

ed in this

very wide

PFGE typ

frequent t

several co

lactamase

ding Franc

eruginosa

9

n

minantly

AP) and

MDR P.

valence

g to ST

VIM-4)

survey.

espread

pes with

ogether

ountries

es (VIM,

ce14 and

isolates

Fig. 3.

PFGE typping of 65

MDR P. aeeruginosaa isolates collected in 2013

10

Fig. 4 M

Moreov

among

(PFGE

located

blaIMP-1

Luxem

reporte

closely

Africa c

of large

The ST

in Belg

Argenti

epidem

5. blaVI

In59 c

the mo

previou

aminog

MLST typi

ver, two clo

MBL-prod

types AF

d to each

3 serotyp

bourg. VIM

ed in Norw

y related V

countries (

er outbreak

T 621, IMP

gium betw

inia, Austr

mic clone to

M and blaIM

arrying an

ost commo

usly obse

glycoside

3

14%

ing of MD

ones that w

ducing MD

F and AX)

other in B

e O4, ST

M-2 produc

way in 2006

VIM-2 stra

Ghana, Ivo

ks.16

P-13 produ

ween 2006

ria and Ro

o widely sp

MP gene ca

aacA29a

on structur

rved in

modifying

37%

%

5

R P. aerug

were not fo

DR P. aer

was dete

Brussels w

T621 was

cing P. aer

6 after med

ins have

ory coast,

cing P. ae

6 and 200

omania, a

pread.17 Th

assettes we

gene cass

re being f

Belgium

g enzyme

5%2%

ginosa str

ound in the

ruginosa: A

ected for t

where it ca

s found in

ruginosa be

dical repat

also been

Kenya) eit

eruginosa c

08 and it

also illustra

he genetic

ere carried

sette down

found in a

in 2010.

e (aacA2

rains colle

e 2010 surv

A blaVIM-2

he first tim

aused a n

n one ho

elonging to

triation of a

n isolated

her as spo

clone was

has been

ating the s

context of

d by five dis

stream the

ll Q and X

The aac

9a) whic

ected in Be

vey were e

serotype O

me in two

nosocomia

ospital in

o the ST23

a patient f

from. seve

oradic case

already pr

n also rep

successful

f the blaMB

stinct class

e blaVIM-2 g

X PFGE t

cA29a ge

h confers

42%

elgium in

evidenced

O6, ST233

hospitals

l outbreak

the prov

33 clone ha

from Ghan

eral sub-S

es or in the

reviously r

ported from

l potential

BL is shown

s 1 integro

gene casse

types (ST1

ene encod

s resistan

ST 11

ST 23

ST 24

ST 23

ST 62

11

2013

in 2012

3 strain

closely

k and a

ince of

as been

a15 and

Saharan

e setting

reported

m Italy,

of this

n in Fig.

ons. The

ette was

111) as

des an

nce to

11

35

44

33

21

12

tobramycin, amikacin and isepamicin but not gentamicin. The frequent co-resistance

of VIM-producing P. aeruginosa isolates to all aminoglycosides including gentamicin

results from the fact that other aminoglycoside resistance genes can also be present

but most probably located on other genetic elements in such isolates (data not

shown).

Overall the blaVIM

genes were found to be located on the chromosome in most

isolates and transfer of these to P. aeruginosa PAO1 or to E. coli J53 by

transformation and/or by conjugation were not successful. The fact that VIM and IMP

coding genes were carried mostly on distinct integrons with different gene cassettes

structures altogether suggests and supports the horizontal transfer of the blaVIM gene

in different clonal lineages including in successful spreading international clones of

MDR P. aeruginosa (e.g. ST111 and ST235).

Fig. 5. Genetic context surrounding the blaVIM and blaIMP genes in MBL producing P. aeruginosa isolates collected in Belgium in 2013

The susceptibility of the VIM- or IMP-producing Pseudomonas aeruginosa isolates is

shown in the Figure 6. As expected, isolates carrying these resistance mechanisms

exhibited a high resistance level to all carbapenems and to all other beta-lactam

IntI1 blaVIM-2aacA29a qacE1sul-1

IntI1 blaVIM-2aacA7 qacE1sul-1

IntI1 aadA13blaVIM-4 qacE1sul-1

A

B

C

D

E

blaVIM-2IntI1 aadA5 qacE1sul-1

500 bp

IntI1 aacA4blaIMP-13 qacE1sul-1

PCR and sequencing of the variable region of the class 1 integron associated with MBL genes showed 5 different structure types. Open reading frames are represented by arrows indicating the orientations. MBL genes are shown in grey. The 59-be of each gene cassette is represented by an open circle and the attI site by a grey oval.

agents

interme

resistan

constan

fluoroq

Fig. 6.

except az

ediately su

nt to genta

ntly obser

uinolones

Susceptibantibiotics

ztreonam t

usceptible

amicin wh

rved. Furt

(MIC of >

bility of 53s (microd

to which a

level (MIC

ile resistan

ther all s

32 mg/L).

3 VIM- or Iilution MIC

almost 90%

C of 8-16 m

nce to tob

strains we

Colistin re

MP- produC /CLSI in

% of the is

mg/L). Onl

bramycin a

ere also h

mained the

ucing P. anterpretativ

solates we

y 25% of

and to ami

high-level

e only activ

aeruginosave criteria

ere suscep

the isolate

kacin was

resistant

ve agent.

a to 12 a).

13

ptible or

es were

almost

to the

14

B. Epidemiology of MDR Acinetobacter baumannii in Belgian hospitals

Of 44 A. baumannii isolates collected during the study period, 31 were confirmed as

non-susceptible (intermediate or resistant) to carbapenems (imipenem and/or

meropenem) by microdilution MIC method using CLSI interpretive criteria (M100-S23,

CLSI 2013). These isolates all of which were multi-drug resistant (resistant to >3

classes of antimicrobial agents) were collected in 21 (30%) of the 69 participating

centers. These isolates were mostly collected from clinical specimens of patients

hospitalized in ICU (n=14) or in medical wards (n=13) and originated from specimens

of the lower respiratory tract (n=13), from wounds (n=7) and less commonly from

urines (n=5). For the majority of the reported cases, no data concerning the

colonization vs infection status of the patients were available. In 4 of 21 hospitals

(19%), MDR A. baumannii isolates were detected through screening samples (rectal

swab) of patients who were predominantly hospitalized in ICUs and other at risk units

(oncohematology wards).

By microdilution, colistin and tigecycline were the only two agents which retained in

vitro activity against MDR carbapenem-resistant A. baumannii (MIC90=2 µg/ml)

(Table 1).

Table 1: Microdilution MIC of MDR A. baumannii [n=31] (CLSI M100-S23 guidelines)

Percentage of susceptibility per CLSI interpretive criteria are shown in Table 2.

Ninety percent of the isolates were susceptible to colistin (3 isolates with high level

resistance to colistin; MIC=32 µg/ml). For tigecycline, 90% of the isolates were

Antimicrobial agents MIC range (µg/ml)

MIC50 (µg/ml)

MIC90 (µg/ml)

Imipenem 4 - >256 32 >256 Meropenem 4 - > 256 32 >256 Piperacillin-tazob. 16 ->256 >256 >256 Ceftazidime 4 - >256 128 >256 Cefepime 2 - >256 64 >256 Aztreonam 1 - >256 32 >256 Amikacin 4 ->128 >128 >128 Tobramycin 1 - >32 >32 >32 Gentamicin 1 - >32 >32 >32 Ciprofloxacin 0.25 - >64 >64 >64 Tigecycline 0.12 – 8 1 2 Colistin 0.5 – 32 1 2

15

susceptible at MIC level of 2 µg/ml (no CLSI breakpoints available; However, if

considering the EUCAST breakpoints applied for Enterobacteriaceae (Susceptible

and resistance category limits at <1 µg/ml and > 2 µg/ml, respectively); only 52% of

the MDR A. baumannii isolates would have been considered susceptible to

tigecycline and 10% as resistant.

Table 2: Susceptibility categorization of MDR A. baumannii [n=31] by CLSI interpretive criteria (M100-S23 guidelines) Concerning the resistance mechanisms, the majority of the MDR Acinetobacter

baumannii isolates carried an OXA-23 carbapenemase coding gene either alone

(n=23) or associated to NDM-1 (n=2); Other OXA-class D carbapenemases found in

this survey were OXA-58 (n=3) and OXA-24/-40 group (n=1). Finally, two strains

harboured an MBL alone: VIM-4 and NDM-1 each. The genetic environment

(transposons, integrons) and their support (chromosome vs plasmid) have as yet not

been determined and this work remains to be remain to be done.

Molecular typing by PFGE delineated the 31 carbapenemase-producing A.

baumannii in 15 different types one of which was found to be spread in 5 different

hospitals in the province of Liège. Incidently, it has been shown that this particular

clone type (called #23) had been imported in Belgium in summer 2011 following the

transfer of one patient from Sicily and that it was at the source of one major outbreak

in the admission hospital with subsequent loco-regional spread following repeated

inter-institution transfer of patients. A second, less common OXA-23 producing A.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Per

cen

tag

e

Imi

Merop

PtzCaz

FepAzt

Amik

Tobra

Genta

Cipro

TigeColi

Resistant Intermediate Susceptible

16

baumannii clone type (#40) was also found to be present in clinical specimens from

patients hospitalized in three different hospitals (in three different provinces) while

most other clone types were different from each other and were found to be present

in single hospitals (either sporadically or as local clusters).

In comparison to previous studies carried out in Belgium, major shifts have occurred

in the distribution of the resistance mechanisms in Acinetobacter. The first cases of

carbapenemase-producing A. baumannii reported in Belgium date back from 2005.

These were at that time essentially associated with OXA-58 and they caused

nosocomial outbreaks in several hospitals. From 2008 onwards, the proportion of

OXA-58 markedly decreased and this mechanism was gradually replaced by OXA-23

which nowadays accounts as the most frequent mechanism of carbapenem

resistance in A. baumannii in Belgium and elsewhere worldwide. The level of

resistance to carbapenem of OXA-23 and OXA-24/40 is usually higher than the one

found in OXA-58 producing isolates. It can be speculated that the increased usage of

carbapenems over the last decade may have contributed to this shift in resistance

mechanisms over time although this is not proven.

When comparing the molecular epidemiology of A. baumannii over time it is also very

obvious that there has been a replacement of the earlier epidemic clones by other

more recently emerging isolates, suggesting the local and regional spread of

successive waves of different clusters in several Belgian hospitals. Currently, it

seems that many cases of MDR A. baumannii and of MDR P. aeruginosa

infection/colonization are not associated anymore with importation following

international travel and hospitalization in countries abroad but rather may have been

acquired locally. However, our perception and understanding of the current

epidemiology of MDR Acinetobacter and P. aeruginosa is still very fragmentary.

17

Conclusions and perspectives

This multicenter survey carried out in 2013 confirmed the importance of MDR P.

aeruginosa and to a lesser extent of MDR A. baumannii in Belgium.

Assuming a proportion of MDR P.aeruginosa of 8.3% in 2012 in Belgian hospitals11

and 30% of these MDR isolates being confirmed as carbapenemase producers (as

found in the present survey), the estimated prevalence of MBL producing isolates

would be approximately 2 to 3% of all clinical P. aeruginosa. Multi-drug resistant P.

aeruginosa isolates were largely distributed and were found in 50% of the

participating centers throughout the countries and included both large tertiary referral

university hospitals as well as smaller general hospitals. In comparison to a similar

survey performed in 2009-2010, the distribution of the different types of acquired

resistance mechanisms remained fairly constant with VIM-2 being largely

predominant. However, we found a diversification in the clones with the emergence

of clone lineages of MDR P. aeruginosa in 2013 that had not been detected in

Belgium before (or only very occasionally). Local and interregional spread of similar

clonal lineages was also well documented in different part of the countries, most

probably reflecting the transfer of patients across different institutions. The fact that

identical gene cassettes and integrons were found in isolates of different genetic

background (genotypes) strongly suggests that carbapenem- and MDR resistance

might increase in P. aeruginosa both by clonal expansion and by horizontal transfer

of resistance genes.

The smaller number of MDR A. baumannii isolates collected in this survey over the

same time period may indicate that the burden caused by this organism may be less

important in Belgium than P. aeruginosa. Indeed, carbapenemase-producing A.

baumannii (mostly OXA-23) were found in about 25% of the participating hospitals.

Although nosocomial outbreaks of variable scale have been reported in several

hospitals over the last six years there is very little available information in Belgium

about the epidemiology and evolution trends of MDR A. baumannii and it is probable

that the collected data most probably lack exhaustiveness. Additional studies

(determination of the MLST patterns and more in depth characterization of the

resistance determinants and genetic supports are still ongoing and results should be

available very soon.

18

In parallel to this microbiological survey which will be repeated every 2 to 3 years, a

new collaborative active surveillance programme aiming to follow the epidemiology of

resistance in P. aeruginosa and in Acinetobacter spp. will be launched and start early

in 2015 in collaboration with the Scientific Institute of Public Health aims. This yearly

survey would run in parallel with the CPE surveillance, and we wish to stimulate

laboratories to actively take part to these two programmes in order to gain a better

insight of the epidemiology of these organisms (risk factors and of the associated

resistance mechanisms).

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