Effect of Plant-Derived Molecules on Acinetobacter baumannii Biofilm on Abiotic Surfaces

42 Pages·2014·0.58 MB·English

by  Ryan P. Pelletier

University of Connecticut
OpenCommons@UConn
Honors Scholar Theses Honors Scholar Program
Spring 5-6-2012
Effect of Plant-Derived Molecules on
Acinetobacter baumannii Biofilm on Abiotic
Surfaces
Ryan P. Pelletier
University of Connecticut - Storrs, [email protected]
Follow this and additional works at:https://opencommons.uconn.edu/srhonors_theses
Part of theAnimal Sciences Commons, and theOther Life Sciences Commons
Recommended Citation
Pelletier, Ryan P., "Effect of Plant-Derived Molecules on Acinetobacter baumannii Biofilm on Abiotic Surfaces" (2012).Honors Scholar
Theses. 257.
https://opencommons.uconn.edu/srhonors_theses/257
Honors Thesis

"Effect of Plant-Derived Molecules on Acinetobacter baumannii
Biofilm on Abiotic Surfaces"

By: Ryan Pelletier
Department of Animal Science
College of Agriculture and Natural Resources
University of Connecticut

Honors Thesis and Academic Advisor:
Dr. Kumar Venkitanarayanan
Professor, Department of Animal Science
College of Agriculture and Natural Resources
University of Connecticut
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Table of Contents

Abstract 3
Introduction 4-5
Literature Review 6-17
Materials and Methods 17-20
Results 20-21
Discussion 21-22
Conclusion 22
References 23-36
Figures 37-41
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ABSTRACT
The emergence of multiple drug resistant (MDR) Acinetobacter baumannii has lead to an
increased interest in finding alternative antimicrobial compounds for controlling the pathogen.
The present study investigated the effect of three plant-derived antimicrobials, namely eugenol
(EUG), carvacrol (CAR), and thymol (THY), on A. baumannii biofilms. Using concentrations
equal to or greater than the minimum bactericidal concentration (MBC) of each compound, the
ability of EUG, CAR, and THY to inactivate the mature biofilms of three strains of MDR A.
baumannii on polystyrene microtiter plates and stainless steel coupons was examined. All three
plant-derived compounds significantly inactivated the bioflims of all three A. baumannii strains
tested (P < 0.05). After 10 min of incubation on polystyrene microtiter plates at 23°C, all three
compounds eliminated all viable biofilm-associated A. baumannii cells, reducing the biofilm-
associated bacterial population by >7.0 log CFU/ml. Eugenol and CAR exerted a similar effect
at 37°C, while THY reduced the viable biofilm-associated bacterial population to 0.45 log
CFU/ml. On stainless steel coupons incubated at 23°C, all three compounds reduced the viable
biofilm-associated bacterial population by 6.5 log CFU/ml. Results indicate that EUG, CAR, and
THY could potentially be utilized to control biofilms of A. baumannii on abiotic surfaces, thus
preventing its persistence in the hospital environment. However, detailed follow up studies are
needed.
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INTRODUCTION
Acinetobacter baumannii is a Gram-negative, nosocomial pathogen (58). It has been
documented as the causative agent in cases of ventilator-associated pneumonia, wound infection,
urinary tract infection, and catheter-associated septicemia (5, 25). A. baumannii has also been
implicated in cases of endocarditis, osteomyelitis, and corneal perforation (66). In recent years,
A. baumannii has emerged as a prevalent nosocomial pathogen in military field hospitals in Iraq
and Afghanistan (57).
The emergence of multiple drug resistant (MDR) strains of A. baumannii constitutes a
significant public health threat. A. baumannii strains resistant to major classes of traditional
antibiotics including β-lactams, aminoglycosides, quinolones, tetracyclines and glycylcyclines,
polymyxins, trimethoprim-sulfamethoxazole, and chloramphenicol have been isolated (58). A
wide variety of mechanisms of antibiotic resistance have been identified in A. baumannii,
including drug-inactivating enzymes, efflux pumps, alteration in outer membrane proteins
(OMPs) and binding sites, and mechanisms of ribosomal protection (28, 58). The ability of A.
baumannii to rapidly acquire foreign genetic elements has contributed to the development of
multiple drug resistance in the bacterium (17, 31). The acquisition of mobile genetic elements
such as plasmids (18), transposons (70), and integrons (2, 18, 69, 71) has been reported.
The ability of A. baumannii to form biofilms also contributes to its pathogenesis. Biofilm
formation contributes to increased persistence in the hospital environment (58). A. baumannii
has been shown to survive for up to 26.5 days on desiccated abiotic surfaces (38). It has been
isolated from hospital suctioning equipment, washbasins, bed rails, bedside tables, ventilators,
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sinks, pillows, mattresses, and resuscitation equipment (65). A comprehensive literature review
conducted by Moultrie et al. (51) found that 73% of studies evaluated identified the environment
as the source of A.baumannii infection. A. baumannii strains which form substantial biofilms
have also been found to have greater resistance to both antibiotics (43, 64, 78) and disinfectants
(70).
Plant-derived essential oils are traditionally used as food additives, both to preserve foods
and enhance flavor (39). Components of several such essential oils include eugenol (EUG) and
trans-cinnamaldehyde found in clove and cinnamon leaf oils, respectively (23). Similarly,
carvacrol (CAR) and thymol (THY) are found in the oil from oregano (13) and thyme (74).
Previous studies have demonstrated the antimicrobial properties of several plant-derived
compounds. Eugenol, CAR, and THY have been shown to exert antimicrobial effects against the
Gram-negative bacteria Salmanella Enteritidis and Campylobacter jejuni (31). These plant-
derived compounds have also been shown to be effective against biofilm-associated bacterial
populations in particular. Carvacrol and THY have demonstrated the ability to inactivate the
biofilms of Staphylococcus aureus and Staphylococcus epidermidis (56), while EUG and CAR
have been shown to inactivate biofilms of Listeria monocytogenes and Escherichia coli O157:
H7 (59). However, no studies to our knowledge have been conducted to investigate the
antibacterial effects of these compounds on MDR A. baumannii. Therefore, the current study
was conducted to investigate the ability of EUG, CAR, and THY to inactivate mature A.
baumannii biofilms for potential use as disinfectants to reduce the persistence of A. baumannii
on abiotic surfaces.
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LITERATURE REVIEW
History
The current genus description and name Acinetobacter were first used in 1954 to
differentiate between motile and non-motile organisms of the genus Achromobacter (10, 58).
The designation was accepted more widely following the work of Baumann et al. in 1968 (4, 58).
A. baumannii was first identified through DNA-DNA hybridizations studies conducted by
Bouvet and Crimont in 1986 (9).
Biology
Acinetobacter spp. are gram-negative, aerobic, catalase-positive, oxidase-negative,
nonmotile, nonfermenting coccobacilli (58) which typically grow at 20-30°C on traditional
laboratory media (27). They are short gram-negative rods that are difficult to destain, leading to
possible misidentification as gram-negative or gram-positive cocci (58). Attempts to
phenotypically differentiate A. baumannii from other Acinetobacter spp. have proven ineffective
(31, 58). Acinetobacter genomic species 13TU, Acinetobacter genomic species 3, Acinetobacter
calcoaceticus, and A. baumannii are often referred to as either the A. calcoaceticus-A. baumannii
(ACB) complex or the A. baumannii-A. calcoaceticus (ABC) complex (57). Molecular methods
of differentiating these species are labor-intensive, expensive, and largely conducted only by a
few reference laboratories (58). The most accepted of these methods are DNA-DNA
hybridization (9), amplified 16S rRNA gene restriction analysis (ARDRA) (77), and high-
resolution fingerprint analysis by amplified fragment length polymorphism (AFLP) (37).
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Role as a Nosocomial Pathogen
A. baumannii poses a significant threat as a nosocomial pathogen. In survey studies from
75 countries, A. baumannii caused approximately 9% of ICU infections (81). It has been
implicated as the causative agent in cases of ventilator-associated pneumonia, wound infection,
urinary tract infection, and catheter-associated septicemia (5, 25). Less frequently, infections
have also resulted in meningitis, endocarditis, osteomyelitis, and corneal perforation (66).
Current data indicate that critically ill patients with ventilator-associated pneumonia and
bloodstream infections caused by A. baumannii experience increased mortality and prolonged
lengths of stay in the hospital (38).
Once present, A. baumannii is capable of persisting in the hospital environment for
extended periods. It has been isolated from hospital suctioning equipment, washbasins, bed rails,
bedside tables, ventilators, sinks, pillows, mattresses, and resuscitation equipment (65).
Persistent environmental contamination in the rooms of patients suffering Acinetobacter spp.
infections has been documented up to 13 days after discharge from the hospital (5). Jawad et al.
(38) showed that 22 strains of A. baumannii isolated from 8 hospital outbreaks in the German
metropolitan area were capable of surviving an average of 26.5 days on a desiccated abiotic
surface, namely glass cover slips. The hospital environment has been demonstrated as a mode of
transmission. A comprehensive literature review conducted by Moultrie et al. (51) found that
73% of studies evaluated identified the environment as the source of A.baumannii infection.
Hospital outbreaks have been traced back to various surfaces (5), including machinery used for
respiratory therapy (14), countertops (51), bedding (82), and improperly autoclaved reusable
needles (40). Martínez-Pellús et al. (49) identified environmental sources as frequent bacterial
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reservoirs of A. baumannii in a Spanish ICU. The study also reported frequent patient
colonization, as 35% had skin colonization, 43% had tracheal colonization, and 31% had rectal
colonization during their stay. Patient colonization can also contribute to further dissemination
of the pathogen through contact with healthcare providers. Morgan et al. (50) found that 20.5%
of healthcare worker interactions with patients in 6 different ICUs resulted in the contamination
of healthcare workers' gowns or gloves. MDR A. baumannii accounted for 32.9% of these
contaminations, the most abundant pathogen detected during the study. Healthcare workers
served as the source of infection in 36% of studies reviewed by Moultrie et al. (51). The
presence of A. baumannii in air samples collected from hospitals has also been reported. A
university hospital in Turkey identified 3 different genotypes of A. baumannii in air samples
from various locations throughout the facility (1). The fact that 1 of these genotypes was also
detected from clinical samples suggests that airborne transmission may contribute to infection.
A. baumannii in Modern Military Conflict
War wound infections by former Achromobacter spp. were first described as early as the
Korean War (47). In recent years, A. baumannii has emerged as a prevalent nosocomial
pathogen in military field hospitals in Iraq and Afghanistan (57). Approximately 14,000
American and British soldiers have been evacuated to medical facilities in their own countries
following wounds in Afghanistan and Iraq since 2001 (57). Consequently, the prevalence of A.
baumannii in domestic military medical facilities has increased as well. The United States
Centers for Disease Control and Prevention identified 102 cases of A. baumannii bloodstream
infections in 5 American military field medical facilities in Iraq and Afghanistan during the
period from 2002 to 2004, compared to 3 during the period from 2000 to 2002 (15, 57). Peterson
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et al. found that Acinetobacter spp. accounted for 36% of wound and blood stream infections in
trauma victims evacuated from Iraq to the naval hospital ship USNS Comfort during a study
period in 2003 (62). In a study of Acinetobacter spp. infection at the National Naval Medical
Center in Bethesda, MD, A. baumannii was isolated in 96% of cases (60).
Acinetobacter spp. are ubiquitous in the environment, commonly isolated from the soil,
water, and the skin and gastrointestinal tracts of healthy individuals (3, 28, 57). However, A.
baumannii has not been isolated from these environmental sources as frequently. Berlau et al.
(6) reported that, although 40% of healthy individuals had skin colonized with Acinetobacter
spp., only 1 of 192 individuals was found to have skin colonized with A. baumannii. Dijkshoorn
et al. (20) isolated A. baumannii from fecal samples in only 1% of healthy individuals examined
in the UK, and only 0.7% of healthy individuals examined in the Netherlands. In the case of
military personal in Iraq and Afghanistan, infection directly from these sources is unlikely to be a
significant factor (57, 60). Of 49 soil samples collected by Scott et al. (67) around 7 field
hospitals in Kuwait and Iraq, only 1 contained ABC complex. Murray et al. (52) cultured
samples from 61 open wounds in American military casualties immediately upon arrival at a
field hospital in Baghdad, none of which were colonized with A. baumannii, and only 3 of which
were colonized with any Acinetobacter spp. Current evidence suggests that non-U.S. personnel
admitted to U.S. military field medical facilities have served as a reservoir for ABC complex
(57). From November 2006 to October 2007, 97.5% of patients in an American Army combat
hospital in Baghdad from which A. baumannii was isolated were Iraqi natives (33). From
September 2007 to August 2008, 91% of patients positive for multi-drug resistant (MDR) Gram-
negative bacteria at the largest U.S. military field hospital in Afghanistan were Afghan (72).