and acinetobacter baumannii in belgian hospitals in 2013 · rosco diagnostica neosensitabs, a/s...
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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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