American Journal of Infection Control xxx (2014) 1-5

Contents lists available at ScienceDirect

American Journal of Infection Control American Journal of

Infection Control

journal homepage: www.ajicjournal.org

Major article

Influence of whole-body washing of critically ill patients

with chlorhexidine on Acinetobacter baumannii isolates
Soraya Mendoza-Olazarán MSc a, Adrian Camacho-Ortiz MD b,
Michel Fernando Martínez-Reséndez MD b, Jorge Martín Llaca-Díaz MSP c,
Edelmiro Pérez-Rodríguez MD d, Elvira Garza-González PhD a, c, *
a
Servicio de Gastroenterología, Hospital Universitario Dr. José Eleuterio González, Universidad Autónoma de Nuevo León, Monterrey,
Nuevo Leon, Mexico
b
Servicio de Infectología, Hospital Universitario Dr. José Eleuterio González, Universidad Autonoma de Nuevo León, Monterrey, Nuevo Leon, Mexico
c
Departamento de Patología Clínica, Hospital Universitario Dr. José Eleuterio González, Universidad Autónoma de Nuevo León, Monterrey,
Nuevo Leon, Mexico
d
Servicio de Trasplantes, Hospital Universitario Dr. José Eleuterio González, Universidad Autónoma de Nuevo León, Monterrey, Nuevo Leon, Mexico

Key Words: Background: Acinetobacter baumannii is 1 of the most important nosocomial pathogens and the caus-
Pulsed-field gel electrophoresis ative agent of numerous types of infections, especially in intensive care units (ICUs). Our aim was to
Oxacillinase evaluate the effect of 2% chlorhexidine gluconate (CHG) whole-body washing of ICU patients on
VIM (Verona integron-encoded metallo-ß-
A baumannii in a tertiary care hospital.
lactamase)
Methods: During the 6-month intervention period, 327 patients were subjected to whole-body bath with
IMP (active on imipenem)
Disinfectant 2% CHG-impregnated wipes. blaIMP (active on imipenem), blaVIM (Verona integron-encoded metallo-ß-
lactamase), and blaoxacillinase (OXA) of A baumannii were typed. Isolates were genotyped by pulsed-field
gel electrophoresis. Minimum inhibitory concentrations (MIC) to CHG were determined by the agar
dilution method and drug susceptibility determined using the broth microdilution method. Biofilm
formation was determined by crystal violet staining.
Results: We analyzed 80 isolates during the baseline period and 69 isolates during the intervention
period. There was a decrease in the MIC50 and MIC90 values for CHG for isolates (8 mg/L and 16 mg/L,
respectively). All isolates typed positive for OXA51-like and 86% typed positive for OXA24-like pulsed-field
gel electrophoresis identified 2 main clone types. During the intervention period the frequency of clone A
decreased and that of clone B increased. Both clones were OXA24-like positive.
Conclusions: The A baumannii isolates recovered from patients who received body washing with 2% CHG
presented with a significant decrease in CHG MIC values associated with a change in clonality correlating
with increased biofilm production.
Copyright Ó 2014 by the Association for Professionals in Infection Control and Epidemiology, Inc.
Published by Elsevier Inc. All rights reserved.

Acinetobacter baumannii is 1 of the most important nosocomial
pathogens and the causative agent of numerous types of infections,
especially in intensive care units (ICUs). This gram-negative bac-
teria species is exceptionally resistant to most antibiotics,1 with
carbapenem resistance being >50% in some countries2 and
pandrug-resistant isolates have been described.3 In addition, this
bacterium is resistant to disinfectants; has long survival times on
dry surfaces;4 and possesses the potential of producing biofilms,5
making it a serious concern in hospital environments.
* Address correspondence to Elvira Garza-González, PhD, Av Madero s/n Colonia
Mitras Centro, Edificio Barragán, Segundo Piso, Monterrey, Nuevo Leon, Mexico.
E-mail address: elvira_garza_gzz@yahoo.com (E. Garza-González).
Conflicts of interest: None to report.
Due to the emergence of multidrug resistance the use of anti-
septics for skin disinfection has been reevaluated. Several studies
have reported that chlorhexidine gluconate (CHG) body washing
effectively prevented carriage and reduced bloodstream infections
caused by methicillin-resistant Staphylococcus aureus (MRSA)6-8
and vancomycin-resistant enterococci (VRE)9 in ICU patients.
It has been further reported that daily whole-body disinfection
with 4% CHG significantly reduces A baumannii skin colonization
and that this regimen may be considered in combination with other
traditional infection control measures.10 A review of the literature
suggested that CHG body washing of patients in an ICU effectively
prevented carriage and bloodstream infections with MRSA and VRE
in different ICUs. The reviewed studies used multiple interventions
simultaneously and a wide variation in patient characteristics,

0196-6553/$36.00 - Copyright Ó 2014 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajic.2014.04.009
2 S. Mendoza-Olazarán et al. / American Journal of Infection Control xxx (2014) 1-5
which made it difficult to accurately establish the efficacy of CHG
body washing in controlling infections.11
Our study evaluated the effect of 2% CHG whole-body washing
of ICU patients on A baumannii in a tertiary care hospital in Mon-
terrey, Mexico.
METHODS
Study design
The study was conducted at the Hospital Universitario Dr. José
Eleuterio González, a 460-bed tertiary care hospital in Monterrey,
Mexico. A baumannii is endemic in this hospital and 69% of isolates
are meropenem-resistant.12,13 Our hospital is equipped with 4 ICUs
(neonatal, pediatric, medical, and surgical ICUs, respectively). This
study was performed in the adult medical and surgical ICUs with a
combined 20-bed area.
The hospital ICU has an infection control program that is based
on proper handwashing practices that are supervised by the hos-
pital’s epidemiology unit based on the recommendations of the
World Health Organization. All patients with potential or proven
colonization-infection by multidrug resistant A baumannii are
placed on contact precautions. Cohorting of patients was imple-
mented when necessary.
During the 1-year study period we included a baseline period
(between January 1, 2012, and June 30, 2012) during which 80
isolates were collected and a 6-month intervention period (July 1,
2012-December 31, 2012) during which 69 isolates were collected.
In total, 149 clinical isolates were obtained from both ICUs. One
isolate per patient was included. To minimize bias, we included all
specimens from patients with suspected infection according to the
criteria proposed by the Centers for Disease Control and Preven-
tion/National Health Surveillance Network.14 This project was
approved by the institutional ethics committee.
Study population and whole-body washing
For samples collected during the intervention period, exclusion
criteria for participation were history of allergy to CHG, burns to
>20% of the total skin surface, pregnancy, or age < 18 years. During
the 6-month intervention period, 346 patients were enrolled and
19 were excluded (11 were younger than age 18 years, 4 were
pregnant, and 4 had burns) leaving 327 participants. Daily bathing
of patients with soap and water was replaced with no-rinse skin
cleansing with 2% CHG-impregnated wipes (Clorhexi-wipes, G70
Antisepsis, Leon, Gto, Mexico). Whole-body bathing with 2% CHG-
impregnated wipes excluded the face. Hair and scalp were washed
daily with a no-rinse 2% CHG shampoo. This intervention began on
the first day of admission to the hospital and continued daily until
discharge. Cultures were performed by standard procedures.
Identification of isolates
A baumannii complex identification was performed using Sen-
sititre panels (TEK Diagnostic Systems Inc, Cleveland, Ohio) as
described by the manufacturer. Species-level identification was
performed by polymerase chain reaction (PCR) as previously
described.15
CHG minimal inhibitory concentration (MIC) determination
MICs to CHG were determined using the agar dilution method
according to the protocol recommended by the Clinical and Labo-
ratory Standards Institute in documents M07-A9 and M100-
S20.16,17 Each bacterial culture was adjusted to a turbidity
equivalent to that of a 0.5 McFarland standard and then diluted 1:10
in sterile Mueller-Hinton broth (Becton Dickinson & Co, Franklin
Lakes, NJ). A 5 mL aliquot of each diluted bacterial suspension was
spotted onto the agar surface and the plates were incubated at 36
C
for 20 hours. The isolates that presented MIC values higher than the
MIC90 value were classified as disinfectant-reduced susceptibility
isolates as proposed by Kawamura-Sato et al.18
Susceptibility testing
Drug susceptibility of all isolates collected was determined us-
ing the broth microdilution method as previously described17 using
Sensititre panels as described by the manufacturer (Trek Diagnostic
Systems, Cleveland, OH). For tigecycline, the US Food and Drug
Administration-approved breakpoint (ie, susceptibility at  2 mg/mL)
provided in the package insert were used.19 Multidrug resistance
was defined as resistance to at least 3 different antibiotic classes.
Determination of biofilm production
Semiquantitative determination of biofilm formation was per-
formed using crystal violet staining as previously described.20,21 All
isolates were tested in quadruplicate in 2 different experiments
conducted on different days. These assays were conducted on
polystyrene 96-well flat bottom, untreated plates with a low
evaporation lid. Biofilm-forming capacity of all isolates was tested
by culturing respective isolates in trypticase soy broth supple-
mented with 1% glucose. Suspensions were prepared in trypticase
soy broth supplemented with 1% glucose from overnight cultures
and adjusted to optical density (OD) 600 of 0.1 (w 107 CFU/mL).
Aliquots of bacterial suspension (100 mL) were then inoculated
into individual wells and incubated at 37
C for 48 hours.
Following incubation, the culture medium was discarded from
the biofilm microtiter plates, and the biofilms were washed
twice with 200 mL 1 phosphate buffered saline (pH 7.4) and air
dried. The dried biofilms were stained with 100 mL of 0.1% crystal
violet for 30 minutes at room temperature. Excess crystal violet
was removed and the wells washed 5 times with 200 mL phos-
phate buffered saline at pH 7.4. The stained biofilm was
resuspended to homogeneity in 200 mL 95% ethanol. The optical
density of respective wells was measured in a microplate absor-
bance reader at 595 nm. To simplify the data we used the ordinal
classification for the level of biofilm production proposed by
Christensen et al20 because at present there are no methods for
defining an established biofilm for A baumannii. Isolates with optical
densities  0.25, between 0.15 and 0.24, and  0.14 were considered
strong, weak, or nonbiofilm producers, respectively. Staphylococcus
saprophyticus ATCC 15305 (positive biofilm) and Staphylococcus
hominis ATCC 27844 (negative biofilm) were used as control
organisms.
Genetic typing
Template DNA was prepared by thermal lysis at 95
C for 10 mi-
nutes. PCR designed to amplify blaIMP (active on imipenem)22 and
blaVIM (Verona integron-encoded metallo-ß-lactamase)23 sequences
were performed using primer sequences previously described.
Multiplex PCR was performed to detect blaoxacillinase(OXA)-like carba-
penemase (including blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like).24
Pulsed field gel electrophoresis (PFGE)
All isolates were genotyped by PFGE.25 Genomic DNA was iso-
lated in an agarose-embedded form and subjected to enzymatic
digestion with 10 U SmaI. Electrophoresis was performed using a
 S. Mendoza-Olazarán et al. / American Journal of Infection Control xxx (2014) 1-5
CHEF III Mapper system (Bio-Rad Laboratories, Inc, Richmond, Calif)
with pulses ranging from 0.5-15 seconds and voltage of 6 V/cm at
14
C for 20 hours.26 Band patterns were generated by visual anal-
ysis using Labworks 4.5 software (UVP Company, Upland, CA) with
1% tolerance and interpreted using the Tenover criteria.27 Similarity
coefficients were generated from a similarity matrix calculated
using the Jaccard coefficient (SPSS version 20.0; IBM-SPSS Inc,
Armonk, NY).
Statistical analyses
Data were analyzed using the statistical program SPSS for
Windows version 20.0 (IBM-SPSS Inc). The c2 test was performed to
determine the association of the clinical condition (infection/
colonization groups) vs biofilm production and antibiotic suscep-
tibility. The correlation between CHG MIC values and antimicrobial
agents and the production of biofilm were determined using the
Spearman rank correlation test. One-way analysis of variance with
Holm-Sidak post hoc evaluation was performed for comparison
between MIC of CHG, PFGE types, and biofilm production (optical
density595 values). P  .05 was considered statistically significant.
RESULTS
Isolates
Of the 157 isolates examined, 149 were identified at the species
level as A baumannii and only these isolates were further analyzed.
Specimens were isolated from 110 respiratory samples (tracheal
aspirate, n ¼ 84; bronchial lavage, n ¼ 23; pleural fluid, n ¼ 1; and
sputum, n ¼ 2), 24 blood samples, 6 urine samples, 3 central
catheters, 4 wounds, and 2 abdominal abscesses. Isolates were
collected between January and June (n ¼ 80) (baseline isolates)
with sample distribution by month as follows: July (n ¼ 12), August
(n ¼ 12), September (n ¼ 6), October (n ¼ 20), November (n ¼ 11),
and December (n ¼ 8). The mean age of patients was 44 years
(range, 17-85 years) with 28 women and 58 men.
From all patients studied, 73 of 149 (49%) were colonized and 76
of 149 (51%) were infected with A baumannii. We found no asso-
ciation between the clinical condition (infected/noninfected) and
biofilm production and antibiotic susceptibility.
MICs of CHG and antibiotics
During the first 5 months of the baseline period (January-May)
the MICs to CHG (both MIC50 and MIC90) were 64 mg/mL and
increased to 128 mg/mL in June.
During July and August (the intervention period), the MIC50 to
CHG was 128 mg/mL and the MIC90 was 256 and 128 mg/mL,
respectively. From October to December, the MIC50 and MIC90
decreased to 8 and 16 mg/mL, respectively.
During all months of the study the MIC50 and MIC90 values for
cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem,
meropenem, levofloxacin, norfloxacin, ticarcillin, tobramycin,
trimethoprim/sulfametoxazol, and tigecycline were > 32 mg/mL, >
16 mg/mL, > 2 mg/mL, > 8 mg/mL, > 8 mg/mL, > 8 mg/mL, > 4 mg/mL,
> 8 mg/mL, > 64 mg/mL, > 8 mg/mL, > 2 mg/mL, and < 2 mg/mL,
respectively. The MIC50 and MIC90 for amikacin were > 32 in all
months except August (during which MIC50 was 32 mg/mL). For
cefepime the MIC50 and MIC90 were 16 mg/mL and > 16 mg/mL,
respectively, in all months except August (during which MIC50 was
 8 and MIC90 was 16 mg/mL). Samples collected during September
were not included because only 6 isolates were collected during
that period.
3
Ninety-four percent of all isolates identified were multidrug-
resistant. Most isolates showed a high drug resistance to all anti-
biotics tested except tigecycline. Resistance rates for amikacin,
cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imi-
penem, levofloxacin, meropenem, norfloxacin, ticarcillin, tobra-
mycin, and trimethoprim/sulfametoxazol were 75%, 40%, 94%, 94%,
94%, 90%, 86%, 93%, 86%, 94%, 87%, 91%, and 93%, respectively. No
correlation was found between the resistance phenotype and the
MIC for CHG.
Phenotypic biofilm assay
Among the isolates examined 97% (144 of 149) were biofilm
producers with 94% (140 of 149) of isolates having a OD595  0.25
and defined as strong biofilm producers based on the classification
system established by Christensen et al.20 Three percent of isolates
(4 of 149) were weak biofilm producers and 5 isolates did not
produce biofilm. Significant differences in biofilm production be-
tween isolates collected at baseline and during the intervention
period were not observed. No correlation was observed between
biofilm production levels and the antibiotic resistance phenotype of
any isolate examined.
A baumannii genotyping
All isolates typed positive for blaoxa-51 and none were positive
for blaoxa58-like, blaoxa23-like, blaVIM, or blaIMP. Eighty-six percent
(128 of 149) typed positive for blaoxa24-like. All carbapenem-
resistant isolates were blaoxa24-like positive. Only 21 isolates were
carbapenem susceptible and none were positive for the carbape-
nem resistance genes tested in this study.
Analysis of clonal relatedness
PFGE identified several clusters designated as follows
(n ¼ baseline/intervention): pulsotype A (n ¼ 41/18), B (n ¼ 3/30),
A1 (n ¼ 1/0), A2 (n ¼ 2/2), A3 (n ¼ 2/1), A4 (n ¼ 0/1), A5 (n ¼ 0/1),
A6 (n ¼ 0/1), C (n ¼ 10/0), D (n ¼ 5/1), and D1 (n ¼ 1/0). These were
the clones that had a higher number of isolates, thus possibly are
related to the cross-transmission between medical personnel, fo-
mites, and patients. The remaining A baumannii isolates identified
during the baseline and intervention periods (n ¼ 29) had a clonal
relatedness lower than 75%. Clone A isolates presented with a fre-
quency of 63% and 35% in isolates collected during the baseline and
intervention periods, respectively. Clone B isolates presented with a
frequency of 9% and 44% during the baseline and intervention pe-
riods, respectively (Fig 1). The mean biofilm production ODs in both
study periods for clone A, B, C, and D isolates were OD595 ¼ 0.511,
0.758, 0.561, and 0.688, respectively. All clone A, B, C, and D isolates
were blaoxa24-like positive.
DISCUSSION
Due to the high prevalence rates of disinfectant-resistant bac-
teria in ICUs, several studies have evaluated the effectiveness of
disinfectants in preventing infections in this clinical setting. It has
been previously reported that body washing with CHG significantly
reduced MRSA and VRE skin colonization rates. However, data
regarding the efficacy of this strategy as a means of preventing
A baumannii infections in the ICU are not available to date.11 The
few studies examining the effect of body washing in the context of
A baumannii have assessed skin colonization rates and the clonal
relatedness of the isolates.10,28 To improve infection control it is
necessary to know more about microorganism susceptibility to
antiseptics, particularly in ICUs where A baumannii is endemic. In
4 S. Mendoza-Olazarán et al. / American Journal of Infection Control xxx (2014) 1-5
Fig 1. Distribution of pulsed-field gel electrophoresis (PFGE) types. Each bar represents a bimonthly period (January-February, March-April, May-June, July-August, September-
October, and November-December). Percentages represent the each PFGE type frequency every 2 months.
our study we evaluated the influence of CHG whole-body washing
of critically ill patients on the clonal distribution, CHG resistance,
antibiotic resistance, and biofilm production of A baumannii isolates
in an ICU.
Although resistance and reduced susceptibility have been
previously reported4,18 we did not detect drug-resistant isolates
after the 6-month baseline period. In addition, we observed an
interesting decrease in the CHG MICs during the intervention
period. This suggested that extending antiseptic use time in the
ICU could increase its usefulness in controlling A baumannii in-
fections. Despite the increased use of CHG, the CHG MICs were
encouraging; however, it is important to constantly monitor the
susceptibility to disinfectants of A baumannii isolates due to possible
development of resistance and therefore loss of effectiveness.
The high rate of antibiotic resistance observed in our study was
not a new observation in relation to A baumannii isolates. However,
the steady increase in the prevalence of carbapenem resistance in
our hospital was alarming.12,13 Several mechanisms are associated
with resistance to carbapenems in A baumannii, but the most
common are the production of serine OXA (Ambler class D OXA
type) and the metallo-b-lactamases (MBLs) (Ambler class B).
Among these carbapenem-hydrolysing OXAs have been distin-
guished, the intrinsic OXA-51-like enzymes, OXA-23, -24, and -58. The
MBLs have been identified less frequently in A baumannii OXA-type
carbapenemase; however, their hydrolytic activities toward car-
bapenems are significantly more potent (100- to 1,000-fold). Of the
5 MBL groups described to date only 3 have been identified in
A baumannii, including IMP, VIM, and SIM (Seoul imipenemase)
types, the latter much less frequently. The results of our study
showed that 86% of A baumannii isolates (128 of 149) were carba-
penem resistant and all of these isolates were OXA51-like and OXA24-
29
like positive. A previous multicenter study that included clinical
isolates from Mexico also reported high carbapenem resistance
rates (67%) and 16 positive-OXA24-like isolates. However, screening
for OXA in that study was not performed for all carbapenem-
resistant isolates identified.
The ability of A baumannii to form biofilms contributes to the
bacteria’s ability to colonize skin and survive in hospital envi-
ronments.1,5 We found that most isolates were also strong
biofilm-producers (94%), suggesting that this virulence mecha-
nism may be a factor complicating the eradication of endemic
strains in our ICU. Although biofilm production by A baumannii
has been previously reported30 there has been significant vari-
ability between studies. Strong biofilm production and high drug-
resistance profiles of respective isolates make them a significant
health risk to patients in hospitals, particularly in the ICU.
One of the most relevant results of our study was the observation
that CHG bathing affected clonal displacement; that is, clone A
predominated baseline cultures but was displaced by clone B, which
predominated during the intervention period. The main difference
between clones was biofilm production. Clone B showed higher
biofilm production (OD595 ¼ 0.758) than clone A (OD595 ¼ 0.511).
Both clones were positive for OXA51-like and OXA24-like and were
resistant to the antibiotics tested. Contrary to what was expected it
seemed that bathing patients with CHG facilitated the establish-
ment of a “more virulent” A baumannii clone.
To explain the observed decreasing MIC values following CHG
administration during the intervention period and the replacement
of baseline clones with intervention period clones, we hypothe-
sized that microorganisms infecting/colonizing our patients during
the intervention period were not colonizing the skin of patients
(where only CHG-resistant A baumannii strains would be expected),
but were transmitted to patients via fomites that facilitated bac-
terial survival due to strong biofilm production. It seemed that the
presence of A baumannii on fomites located in close proximity to
patients were CHG-susceptible strains. At this time we do not have
evidence to confirm this hypothesis, but work is currently under-
way to further substantiate this possibility. We are also analyzing
clinical data to further define the rate of infection/colonization in
this study, not only in relation to A baumannii but also with respect
to other important bacterial species such as S aureus, Klebsiella
pneumoniae, Pseudomonas aeruginosa, and all bacteria in the
ESKAPE group (i.e. Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa and Enterobacter species).
Overall, A baumannii isolates recovered from patients who
received body washing with 2% CHG presented with a significant
decrease in CHG MICs associated with a change in clonality asso-
ciated with increased biofilm production.
Acknowledgments
The authors thank Dr. Sergio Lozano, from the Hospital Uni-
versitario, for his help in reviewing the manuscript.
 S. Mendoza-Olazarán et al. / American Journal of Infection Control xxx (2014) 1-5
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