Novel role of Acinetobacter baumannii RND efflux transporters
in mediating decreased susceptibility to biocides
Govindan Rajamohan1,2, Vijaya Bharathi Srinivasan1and Wondwossen A. Gebreyes1*
1Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA;
2Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh, India
*Corresponding author. Tel: þ1-614-292-9559; Fax: þ1-614-292-4142; E-mail: email@example.com
Received 28 August 2009; returned 28 September 2009; revised 26 October 2009; accepted 4 November 2009
Objectives: Biocides and dyes are commonly employed in hospital and laboratory settings. We investigated the
biocide susceptibilities of a rapidly emerging pathogen, Acinetobacter baumannii, and the underlying molecular
mechanisms, with a primary focus on resistance–nodulation–cell division (RND) efflux systems.
Methods: Biocide susceptibilities, efflux and in vitro inactivation profiles were monitored in the presence/
absence of efflux pump inhibitors. The RND transporters encoded by adeB and adeJ were detected by PCR;
null mutants were constructed in the native host. Expression of adeB and adeJ in clinical isolates was
assayed by semi-quantitative RT–PCR.
Results: Susceptibility testing and phenotypic assays demonstrated the role of active efflux in mediating
decreased susceptibility to biocides. Inactivation of either the adeB or adeJ transporter gene led to increased
susceptibility to biocides. RT–PCR analysis exhibited increased adeB and adeJ expression in clinical isolates.
Conclusions: This is the first study demonstrating the role of efflux pumps in mediating decreased susceptibility
to disinfectants and other chemical substrates in A. baumannii.
Keywords: antimicrobial resistance, multidrug efflux, RND efflux pump
Biocides are an integral component in the practice of clinical
medicine, serving to prevent the dissemination of pathogenic
organisms in the hospital environment.1Reduced susceptibility
to biocides in bacterial species arises from various intrinsic and
acquired (such as qac genes) genetic determinants, but efflux
is increasingly implicated as a major resistance mechanism.2
An association between resistance to antibiotics and cross-
resistance to biocides has been reported for clinically important
One such important human pathogen is Acinetobacter
baumannii, a Gram-negative bacillus that causes numerous
healthcare-associated infections worldwide, with a remarkable
propensity for nosocomial cross-transmission.4Reports have
demonstrated the involvement of intrinsic and acquired resist-
ance determinants as well as efflux pumps (AdeABC and
AdeIJK) in conferring multidrug resistance.5
Despite the fact that these organisms have been linked to
hospital environment contamination,4,5to date only a few
studies have investigated the susceptibility profile of this bacillus
to structurally unrelated compounds, antiseptics and biocides.
The underlying genetic mechanisms responsible for mediating
decreased susceptibility to disinfectants in A. baumannii still
remain unknown. Prompted by the paucity of such information,
a systematic study was initiated using 86 multidrug-resistant
The biocide susceptibilities and underlying molecular mech-
anisms were investigated, with a primary focus on resistance–
nodulation–cell division (RND) efflux systems. To our knowledge,
this is the first study demonstrating the role of active extrusion
and involvement of AdeABC and AdeIJK in mediating decreased
susceptibility to biocides in A. baumannii.
Materials and methods
Bacterial strains, growth media and reagents
Eighty-six A. baumannii strains isolated during 2005–07 were obtained
from two sources, i.e. the Ohio Department of Health (n¼38) and The
Ohio State University Medical Center (n¼48).6All isolates were cultured
in Luria–Bertani (LB) agar and LB broth (Difco, Sparks, MD, USA).
MICs were determined by a broth dilution method following CLSI guide-
lines.7The concentration with no visible growth was considered the MIC.
The isolates were defined to exhibit decreased susceptibility if their MIC
was found to be ?2-fold higher than that for the A. baumannii
# The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: firstname.lastname@example.org
J Antimicrob Chemother 2010; 65: 228–232
doi:10.1093/jac/dkp427 Advance publication 11 December 2009
by guest on October 27, 2015
susceptible strain. The minimum bactericidal concentration (MBC) of bio-
cides was determined by broth dilution in Mueller–Hinton broth (MHB;
Difco), as described previously.8Biocides were diluted with broth for
determination of MICs and this technique is known to have limitations.
However, it is the method of choice for screening large numbers of iso-
lates and was used in this study. The time–kill studies were performed
according to CLSI guidelines.7
In vitro studies to elucidate the occurrence of active
The accumulation of ethidium bromide (EtBr) or acriflavine (AF) and
growth inhibition assays were examined as described previously,
usingthe effluxpump inhibitors
chlorophenylhydrazone (CCCP) (an uncoupler of oxidative phosphoryl-
ation that disrupts the proton gradient of the membrane), phenyl-
arginine-b-naphthylamide (PABN), reserpine and verapamil (Sigma,
St Louis, MO, USA).6The antibacterial activity of EPIs has been investi-
gated and it was found that EPIs have no intrinsic antibacterial activity
against clinical isolates at the concentration (final concentration
25 mg/L) used in different experiments.
PCR amplifications and sequence analyses
Genomic DNA was extracted using a DNeasy Tissue Kit (Qiagen, Valencia,
CA, USA). PCRs were performed using specific primers as described pre-
viously,6,8and confirmed by sequencing (CEQ 8000 capillary electrophor-
esis system; Beckman Coulter Instruments Inc., Palo Alto, CA, USA) and
analysed by BLAST at www.ncbi.nlm.nih.gov. A detailed list of primers
used for this study can be accessed at http://vet.osu.edu/IDMEL.
Construction of adeB and adeJ deletion mutants
The kanamycin resistance gene was retrieved from pUC4K upon digesting
with BamHI and subsequently ligated with a pUC18 vector (New England
Biolabs, MA, USA).
The resulting plasmid, pUC18Kan, was modified by cloning a
PCR-amplified 0.981 or 1.8 kb internal fragment of adeB (50-GTATG
AATTGATGCTGC-30and 50-CACTCGTAGCCAATACC-30) or adeJ (50-GCGAT
respectively, into the SmaI site of pUC18Kan. Obtained recombinant plas-
mids were introduced into clinical isolate AC0037 (kanamycin suscep-
tible) by electrotransformation.9,10Ticarcillin- and kanamycin-resistant
transformants were selected and the appropriate deletions were con-
firmed by PCR and genomic DNA sequencing. The resulting derivatives
with inactivated adeB or adeJ were designated AC0037DadeB::kan and
To detect adeB and adeJ gene expression, RT–PCR was performed. Total
RNA was extracted using an Absolutely RNA Miniprep Kit (Stratagene, La
Jolla, CA, USA). Quantification of adeB and adeJ RNA transcripts was done
using a Titan One Tube RT–PCR System (Roche Inc., USA). The primers
used for adeB were 50-GTATGAATTGATGCTGC-30and 50-GACTTTCAGA
TTCAAGATAT-30, the primers used for adeB were 50-GATATTGCACA
CCT-30, and the primers used for 16s rRNA were 50-TCGATGCAACGCGAA
GAACC-30and 50-CGTAAGGGCCATGATGACTT-30. The bands were subjected
to densitometric analysis using image scanning software Quantity One
4.1.1 (Bio-Rad). The expression levels were standardized relative to the
transcription levels of 16s rRNA (a housekeeping gene) for each isolate.
P values of ,0.05 were considered statistically significant.
Analysis of the MICs of different compounds clearly indicated
that they varied. The clinical isolate A. baumannii AC0040 (refer-
ence for grouping) in our collection was an antibiotic- and
Therefore, for grouping, the MICs of benzalkonium chloride
(BZK) andEtBr(two representative
clinical isolates were compared with the respective MICs for
A. baumannii AC0040 (Table 1). The isolates that exhibited
3-fold and 2-fold higher MICs of BZK and EtBr, respectively,
with respect to that for the susceptible strain were classified as
Group I. The isolates that exhibited 6-fold and .4-fold higher
MICs of BZK and EtBr, respectively, were classified as Group II.
The isolates that exhibited 24-fold and .8-fold higher MICs of
BZK and EtBr, respectively, were classified as Group III (Table 1).
Of the total 86 isolates, 13% (n¼11) of isolates had an MIC of
BZK of ,30 mg/L, 45% (n¼39) of isolates had an MIC of 30 mg/L
and 41% (n¼35) had an MIC of 120 mg/L. About 67% of isolates
had an MIC of AF of .200 mg/L (Table 1). The MICs of other
compounds are shown in Table 1. Recently, we reported the
resistance profiles of these clinical isolates and it was interesting
to note that Group I isolates were either single drug resistant
(chloramphenicol) or susceptible.6Isolates in Groups II and III
were those that exhibited MDR, with MICs of ciprofloxacin
being .32 or .72 mg/L, respectively.
The MBCs were determined for all the isolates. The MBC of a
biocide is the concentration that kills 99.9% of the bacterial
inoculum.8Data obtained in this study indicate that the MBCs
of different biocides for Group III were higher than those for
Groups I and II (Table 1). The susceptibility profiles of representa-
tive clinical isolates from Groups I, II and III to different biocides
were found to be similar, both in terms of MBC testing and time–
kill studies (data not shown).
Functional assay to demonstrate the occurrence
of active efflux
Using the well-known efflux pump substrates EtBr and AF, a
fluorimetric efflux experiment was performed on representative
isolates from Groups II and III (high MICs) to elucidate the occur-
rence of active efflux. In the absence of CCCP, Group II/III strains
accumulated 5- or 10-fold less EtBr, or AF, when compared with
the susceptible strain. However, addition of CCCP increased the
EtBr and AFaccumulation in Group II/III strains, which eventually
reached a plateau in all the strains (data not shown). These find-
ings strongly demonstrate the occurrence of active efflux in
these clinical isolates.
In vitro inactivation profiles
The effect of CCCP on the biocide susceptibility profile of the clini-
cal isolates was tested. Interestingly, addition of CCCP (final con-
centration 25 mg/L) greatly reduced the MICs of various biocides
from 2-fold up to 12-fold (Table 1). Reduction in the MICs of
fluorescent dyes such as EtBr (17- to 26-fold) and AF (5- to
12-fold) was also observed (Table 1). Similar reductions in the
MICs were found upon using PABN (data not shown). The EPIs
Efflux as mechanism for decreased susceptibility to biocides
by guest on October 27, 2015
Table 1. Biocide susceptibility and carriage of efflux genes in clinical isolates of A. baumannii
Biocides and dyesEfflux genes
I (11)2 MICd
III (35)8 MICd
120 (12–16) .64 (10–12)
.1024 (60–120) .240 (20–40)
.1024 (60–120) .240 (20–40)
The different classes of disinfectant formulations used were: Virkon S (an oxidizing agent: peroxygen blend and organic acid; Antec International, UK); Wex-Cide 128 (a phenolic agent;
Wexford Laboratories, USA); Synergize [a glutaraldehyde disinfectant-containing quaternary ammonium compound (QAC); Preserve International, USA]; chlorhexidine gluconate (MP
Biomedicals, LLC); benzalkonium chloride (a QAC; MP Biomedicals, LLC); and Parvosol II RTU (a synergized QAC with inert ingredients; Hess & Clark Inc., USA).
BZK, benzalkonium chloride; CHX, chlorhexidine; VKS, Virkon S; Par, Parvosol; Syn, Synergize; Wex, Wex-Cide; EtBr, ethidium bromide; AF, acriflavine.
an, Number of isolates.
bMIC and MBC values are represented as mg/L.
cMIC and MBC values are represented as %.
dValues in parentheses are MICs in the presence of the efflux pump inhibitor CCCP at 25 mg/L.
Rajamohan et al.
by guest on October 27, 2015http://jac.oxfordjournals.org/ Downloaded from
verapamil and reserpine had no effect on these isolates (data
not shown). The EPIs (final concentration 25 mg/L) had no intrin-
sic antibacterial activity against the clinical isolates in this study.
In order to confirm the role of active efflux, we monitored the
growth inactivation profiles of A. baumannii from Groups II and
III in the presence and absence of EPIs using different concen-
trations of biocides. On the addition of CCCP, the susceptibilities
of clinical isolates to biocides were highly increased (while no
change in growth curve was found when verapamil and reserpine
were used); we observed .5-fold decreases in MICs of chlorhex-
idine and Synergize, and 3-fold decreases in MICs of Virkon S and
BZK (data not shown). These findings strongly imply that active
efflux is involved in mediating high MICs of biocides.
PCR detection of efflux genes
PCR testing revealed that 41% (35/86) of the isolates harboured
the chromosomal qacE disinfectant resistance gene (Table 1).
None of the other qac alleles (qacA, B, C, G, H and J) were
detected in this study. It is important to note that all qacE-
positive isolates belonged to Group III.
Specific PCR assays were carried out to detect the efflux trans-
porter encoded by adeJ, in an attempt to determine the distri-
bution of this efflux system among different isolates with
varying MICs of biocides. The adeJ gene was found in all Group
I, II and III isolates (Table 1).
In our previous study, we found that adeB, adeR and adeS
were present in 55% of the isolates in this collection.6The
majority of the isolates that carried adeB belonged to Group III
(Table 1). It is worthy to state here that the specific point
mutations in adeR and adeS previously reported to cause
AdeABC overexpression were not identified in these isolates.6,9
Role of adeABC and adeIJK in conferring antimicrobial
To determine the contribution of adeJ and adeB efflux systems in
disinfectant (biocide) susceptibility, adeJ or adeB was deleted in a
clinical isolate, A. baumannii AC0037. Inactivation of either adeB
or adeJ resulted in decreased susceptibility to different com-
pounds. The following were the fold reductions in the MICs of
various chemical agents in deletion mutants of adeB and adeJ
genes, respectively: Acridine Orange (1, 4.4); acriflavine (8, 16);
BZK (4,6);chlorhexidine (8,
40,6-diamidine-2-phenylindole (2, 4); deoxycholate (2, 2); EtBr
(16, 8); Methyl Viologen (4, 16); pyronin Y (2, 4); rhodamine
123 (8, 16); SDS (6, 12); Synergize (2, 2); tetraphenylphospho-
nium chloride (12, 36); Virkon S (1, 1.5); and Wex-Cide (2, 2).
A fluorimetric experiment indicated that after 20 min (before
adding CCCP) of incubation with EtBr, the DadeB::kan mutant
showed a 2.2-fold increase in the rate of accumulation com-
pared with its parental strain AC0037 (Figure 1a). Independent
studies performed using the DadeJ::kan mutant demonstrated
a1.9-foldincrease in the
(Figure 1a). However, upon adding CCCP, the levels of accumu-
lation in both parent and mutant strains were found to be
almost similar (Figure 1a). In a separate set of experiments,
the AF accumulation rates were also monitored. After 20 min,
a 3-fold increase in AF accumulation was observed for the
2); CrystalViolet(1, 10);
Time of incubation (min)
0 10 20 3040
0 1020 30 40
Time of incubation (min)
AC0030 AC0036 AC0050 AC0068 AC0037 AC0055
Relative RNA expression levels
Figure 1. (a) Accumulation studies using ethidium bromide for wild-type
A. baumannii and adeB and adeJ null mutants. The relative fluorescence
intensity along the y-axis represents the level of accumulated ethidium
bromide in the wild-type A. baumannii, isogenic mutants and E. coli
KAM32. The graph shows the difference in the fluorescence shown by
the bacterial cell in the presence and absence of the inhibitor CCCP.
The arrow indicates the time of addition of CCCP to a final
concentration of 25 mg/L. Each datapoint represents the mean+SD of
acriflavine. (c) Relative expression of adeB and adeJ genes from
different clinical strains of A. baumannii (AC0030, AC0036, AC0050,
AC0068, AC0037 and AC0055). The expression of the 16s rRNA gene
was used as the internal control. Each bar represents the average
value of two independent experiments and the error bars represent the
(b) Accumulationstudies using
Efflux as mechanism for decreased susceptibility to biocides
by guest on October 27, 2015
DadeJ::kan mutant compared with AC0037; however, a 2.2-fold
increased accumulation rate was seen in the DadeB::kan
mutant (Figure 1b).
Analysis of adeB and adeJ expression
We investigated the expression of membrane transporters
encoded by adeB or adeJ in a range of isolates from all three
groups using RT–PCR. Though the adeB gene was detected by
PCR, we did not find any evidence for its expression in Group I
(AC0030 and AC0036) strains. However, .4- and .8-fold
increases in adeB expression were noticed in isolates from
Groups II (AC0050 and AC0068) and III (AC0037 and AC0055),
respectively (Figure 1c). The expression of adeJ was ?6-fold
higher in Group II and III isolates when compared with that of
Group I isolates (Figure 1c).
Considering the importance of disinfection in the prevention of
nosocomial infection, the aim of this study was to evaluate the
biocide susceptibilities of a set of MDR A. baumannii clinical iso-
lates and to delineate the role of efflux pumps in mediating
decreased biocide susceptibility. Notably, the majority of the iso-
lates exhibited decreased susceptibility to various commonly
used biocidal agents. Consistent with our findings, several
studies have reported the increased resistance of nosocomial
pathogens towards these agents.4,5It is important to state
here that A. baumannii strains with decreased susceptibility to
biocides may live through a non-optimal cleaning regimen or
biocide challenge for a longer period, making them clinically
more problematic to treat.
Phenotypic assays demonstrated that efflux pumps contrib-
ute as an important mechanism for the high-level biocide and
antimicrobial resistance in A. baumannii. Various studies in Gram-
negative bacteria have demonstrated that efflux pumps play an
important role in intrinsic resistance to disinfectants including
quaternary ammonium compounds.3,4,8The disinfectant gene
qacE was found in more than one-third of the clinical isolates.
Resistance to antiseptics and disinfectants associated with inte-
grons carrying efflux-related transporters such as QacE has been
Analysis indicated that the structurally unrelated compounds
were also substrates for AdeIJK and AdeABC, and overexpression
of these efflux pumps correlated well with the increased biocide
MICs for Ohio clinical isolates. However, upon inactivating a
single operon we did not observe a complete loss in resistance
profile. Thus, it is important to state here that other efflux
pumps in the genome of the bacteria may also have a role in
concern that low-level biocide challenge can select for an MDR
population, it is important to determine the susceptibility of clini-
cal A. baumannii to various disinfectants and to promote strict
intervention of control and preventive measures.
to biocides. With the
In this study, we have shown that adeABC and adeIJK transport
systems can extrude disinfectants, structurally unrelated com-
pounds and detergents, providing experimental evidence for
their broad substrate specificity and an additional role in
We are grateful to Drs Tomofusa Tsuchiya, Craig Altier, Preeti Pancholi,
Kurt Stevenson and Mario Marcon for graciously providing plasmids,
E. coli KAM32 and A. baumannii clinical isolates for this study. We
would like to thank members of the Infectious Diseases Molecular
Epidemiology Laboratory team for technical assistance.
This study was funded by The Ohio State University (to W. A. G.).
None to declare.
1 Rutala WA, Weber DJ. Control: the role of disinfectants and sterilization.
J Hosp Infect 1999; 43: S43–55.
2 Piddock LJV. Clinically relevant chromosomally encoded multidrug
resistance efflux pumps in bacteria. Clin Microbiol Rev 2006; 19: 382–402.
3 Poole K. Mechanisms of bacterial biocide and antibiotic resistance.
J Appl Microbiol 2002; 92: 55S–64S.
4 Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals:
multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 2007; 5:
5 Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence
of a successful pathogen. Clin Microbiol Rev 2008; 21: 538–82.
6 Srinivasan VB, Rajamohan G, Preeti P et al. Genetic relatedness and
molecular characterization of resistance determinants in multidrug
resistant Acinetobacter baumannii isolated in central Ohio, USA. Ann
Clin Microbiol Antimicrob 2009; 8: 21.
7 Clinical and Laboratory Standards Institute. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—
Seventh Edition: Approved Standard M7-A7. CLSI, Wayne, PA, USA, 2006.
8 Smith K, Gemmell CG, Hunter IS. The association between biocide
tolerance and thepresenceor absence
hospital-acquired and community-acquired MRSA isolates. J Antimicrob
Chemother 2008; 61: 78–84.
9 Daniels C, Ramos JL. Adaptive drug resistance mediated by root–
nodulation–cell division efflux pumps. Clin Microbiol Infect 2009; 15
Suppl 1: 32–6.
10 Damier-Piolle L, Magnet S, Bre ´mont S et al. AdeIJK, a resistance–
Acinetobacter baumannii. Antimicrob Agents Chemother 2008; 52:
of qac genesamong
effluxing multipleantibiotics in
Rajamohan et al.
by guest on October 27, 2015