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Abstract
P.aeruginosa
is a significant pathogen associated with nosocomial and
community-acquired chronic infection. Presumptive
P.aeruginosa isolates were obtained from two Irish
hospitals. The aim of this study was to phenotypically and
genotypically characterise the isolates, and assess their
innate virulence in response to antibiotic treatment. The
clinical strains were characterised biochemically by API NE
and Biolog GN systems, and subsequently confirmed as
P.aeruginosa by 16s rRNA sequence analysis. Their
genetic relationship was established by Phylogenetic
analysis of the 16s rRNA, which confirmed individuality
amongst the strains but more significantly, genetic
similarity to known clinical isolates of P.aeruginosa
and Burkholderia cepacia, the cystic fibrosis
pathogen. Antibiotic resistance profiles were completed on
all the isolates to obtain MIC values for numerous
antibiotics. Extrapolation of the MIC profiles identified
one multi-resistant strain, characterised by high-level
resistance to gentamicin, the topically administered
treatment for invasive P.aeruginosa infection. This
strain was chosen for further study. Biochemical
tests identified all the clinical isolates as
P.aeruginosa, which with the exception of subtle
metabolic differences were indistinguishable from each
other. Phenotypic differences were confirmed by 16s rRNA
sequencing which identified genetic relationship with other
clinical pathogens. MIC profiling identified one
multi-resistant clinical isolate, P.aeruginosa PA13,
resistant to all classes of antibiotic, specifically
aminoglycosides. This study demonstrated that the
occurrence of chronic P.aeruginosa infection is not
restricted to one genotype, as confirmed by phylogenetic and
molecular epidemiology. It also illustrates the potential
for cross-resistance between clinical isolates in nosocomial
environments, confirming the need for more rigorous
infection control protocols.
Introduction
Opportunistic pathogens presenting broad-spectrum antibiotic
resistance have emerged extensively in hospital
environments, causing serious infections in
immunocompromised hosts. Organisms resistant to antibiotic
treatment; such as MRSA (Methicillin-resistant
Staphylococcus aureus), VRE (Vancomycin-resistant
Enterococcus) (Kim, Chamkamnoetkanok et al. 2005;
Stevenson, Searle et al. 2005) and aminoglycoside
resistant Pseudomonas aeruginosa are now endemic,
causing serious nosocomial infections (Van Eldere, 2003;
Poole, 2005), especially in neutropenic pateints.
Pseudomonas aeruginosa, has become one of the most
prevalent bacterial pathogens identified in cystic fibrosis
patients enumerating approximately 80% of all adult cases of
CF (Spilker, Coenye et al. 2004). The organism can
cause a wide range of infections including bacterial
meningitis, endocarditis (Venkatesan, Spalding et al.
2005), otitis media (Lee, Remtulla et al. 2005) ,
chronic pulmonary colonisation and pneumonia (Tramper-Stranders,
van der Ent et al. 2005), urinary tract infections (Harjai,
Mittal et al. 2005) and osteomyelitis (Andonian,
Rabah et al. 2002). Primary treatment of most P.
aeruginosa infections involves the use of
aminoglycosides, either alone or in combination with
b-lactams.
P.aeruginosa has been reported to have an innate
resistance to several antibiotics due to the presence of
lipopolysaccharides in the outer membrane, but persistent
administration of antimicrobial agents, has resulted in the
emergence of multi-resistant strains of P.aeruginosa
(Van Eldere, 2003). This acquired resistance is
characteristic of high-level resistance to almost all
aminoglycosides but more importantly to the clinically used
tobramycin, netilimicin and specifically gentamicin (Wright,
1999).
The evolution of multi-resistant P.aeruginosa and its
mechanisms of antibiotic resistance have been examined.
Primary mechanisms include reduced cell permeability, efflux
pumps, changes in the target enzymes and inactivation of the
antibiotics (Lambert, 2002; Matsuo, Eda et al. 2004).
Aminoglycoside-resistance in Pseudomonas sp. is
primarily due to the latter mechanism, by the genetic
expression of enzymes responsible for the modification of
the aminoglycosides. There are three specific classes of
aminoglycoside-modifying enzymes (AMEs), that have been
identified, the N’acetyltransferases (AAC), O-phosphotransferases
(APH) and O-adenyltrnsferases (ANT) (Shaw, Rahter
et al. 1993; Poole, 2005; Wright, 1999). The genes
encoding these enzymes have also been identified fused in a
“gene-cassette” type structures called integrons, typically
fused with
b-lactamase
genes thus conferring a multiple resistance on the organism
(Laurent, 2001). These novel gene-cassettes are thought to
migrate between strains carried on small circular plasmids
that incorporate into the cell’s genome. As a result, strain
diversity and the evolution of new Pseudomonas
species equipped with varying degrees of antibiotic
resistance exist, emerging as serious infections in
nosocomial environments. To investigate this, we obtained
several clinical isolates from two hospitals in Ireland,
collected from various sites of infections. To identify and
characterise each isolate, a series of tests were completed
including, morphological and biochemical analyses such as;
API20NE (bioMerieux, Marcy-l’Ecoile, France), and the Biolog
GN (Biolog Inc., Hayward, Calif). Taxonomic classification
was subsequently verified by 16s rRNA sequencing to confirm
identification. Virulence was assessed by antibiotic
resistance profiling of each isolate, to obtain MIC values
for a numerous antimicrobial agents, primarily
aminoglycosides.
Materials and Methods
Strains and Isolates
Clinical isolates were obtained from,
two Irish hospitals
and assigned the codes;
PA 1, 3, 5, 7-13, 16 and 17 for identification.
Pseudomonas
aeruginosa
PA01 was used as a reference strain in all tests completed.
All isolates were maintained on cetrimide agar (Merck)
supplemented with naldixic acid (15
mg/ml),
nutrient agar slants at 4°C
and in 80% glycerol stocks at -80°C.
Phenotypic and biochemical characterisation
Morphological analysis, gram & spore staining, catalase and
oxidase activities, oxidation-fermentation tests and
motility, starch hydrolysis, arginine, tween 80 and malonate
utilisation tests were completed according to standard
methods according to Harrigon & McCance (Harrigan & McCance,
1976). Cultures were also streaked onto Difco Pseudomonas
agar F to detect fluorescein production and Difco
Pseudomonas agar P to detect pyocyanin production.
Plates were incubated at 37ºC for 1-2 days.
Automated Identification by API20NE
The API identification system API20NE (bioMerieux, Marcy-l’Ecoile,
France), for non-enteric Gram-negative rods was used for
identification of the Pseudomonas aeruginosa
isolates. The identification systems were used according to
the manufactures’ instructions. Inocula from overnight
nutrient broth cultures (10 ml) were harvested in a Labofuge
6000 bench-top centrifuge (5000 rpm for 10 minutes) and
washed once with sterile 0.01 M sodium phosphate buffer.
Pellets were resuspended in 0.85% (w/v) NaCl (10 ml) and
used to inoculate a portion of the tests. For assimilation
tests, 200
ml
of this suspension was used to inoculate auxiliary medium
supplied by the manufacturer and this was then used to
inoculate the remaining tests.
Automated Identification and characterisation by Biolog GN
Identification was also carried out by a Biolog microlog
system (BIOLOG Inc., Hayward, Calif., USA) according to the
manufacturers’ instructions. GN microplates for Gram
negative organisms were used. Isolated colonies were
transferred to Biolog inoculating fluid using sterile swabs
to give the correct cell density required by the system.
Cell densities were compared against GN-NENT (Gram negative
Non-enteric) turbidity standards supplied by the company. To
each of the 96 test wells in the microtitre plate, 150
ml
of the resulting inoculum was added. The plates were
incubated for 16-24 hours at 37ºC before results were
interpreted using Biolog automated microlog software.
16s rRNA Sequencing
Total genomic DNA was isolated from all isolates by the
method of Chen and Kuo, 1993 {Chen, 1993 #27}. Initially,
amplicons of 956bp were amplified for all the classified
isolates, using P.aeruginosa specific primers;
PA-SS-F (5’-GGG GGA TCT TCG GAC CTC A-3’) and PA-SS-R (5’TCC
TTA GAG TGC CCA CCC G-3’) (MWG Biotech) (Spilker, Coenye
et al. 2004) and Red Taq PCR Mix (Sigma, UK). PCR
conditions included an initial denaturation stage of 95oC
for 2 mins, followed by 25 cycles of 1 min at 95oC,
1 min at 58oC, and 1min at 72oC. A
final extension of 1min at 72oC completed the
protocol. PCR products were purified and sequenced by Qiagen,
Germany. Resultant sequences were visualised by Chromas
version 2.3 (Technelysium Pty. Ltd, Australia). BLASTN
search analysis was used to compare the obtained sequence
data with sequences available in the NCBI database (www.ncbi.nlm.nih.gov/BLAST)
{Altschul, Gish et al. 1990). Following analysis of
the amplicons, total 16s rRNA subunits were amplified using
the standard 63f (5′-CAG GCC TAA CAC ATG CAA GTC-3′) and
reverse primer 1387r (5′-GGG CGG WGT GTA CAA GGC-3′) (MWG
Biotech) (Marchesi, Sato et al. 1998). Resultant PCR
products were purified, sequenced and verified by BLASTN
analysis. Phylogenetic analysis of the sequences was
performed following alignment of the total 16s rRNA
sequences using ClustalX software (Thompson, Gibson et
al. 1997) and visualised using Treeview software
package.
MIC and antibiotic resistance profiles
Susceptibility of the isolates was assessed by two methods,
the multi-disc diffusion method and the microtitre broth
dilution method. The disc diffusion tests were completed
using antibiotic sensitivity disks (Mast Diagnostics
Germany; Oxoid, UK). 0.1 ml of overnight cultures were,
spread plated on nutrient agar and horse blood agar. The
antibiotic susceptibility discs were placed on the agar
surface using a sterile tweezers. The plates were then
incubated at 37°C
for 24 hours. The clear zones (zones of inhibition) around
the discs were noted and measured.
The microtitre broth dilution method was used to assess
aminoglycoside resistance in the isolates. The
aminoglycsosides; amikacin, apramycin, butirosin A,
gentamicin hygromycin, kanamycin, lividomycin A, neomycin,
netilmicin, paromomycin, spectinomycin and tobramycin
(Sigma, UK), were prepared in individual 96 well plates at
final concentrations of 0-120mg/ml.
Strains were grown on nutrient broth overnight. Using a
multipippetter, 100
ml
of nutrient broth was dispensed into all wells of a
microtitre plate. An aliquot of 100
ml
of each 2x antibiotic solution was pipetted into the wells
in column 1 (far left of plate the antibiotics were mixed
into the wells in column 1 by pipetting up and down 6-8
times without splashing. A serial dilution was performed
between columns 1-11. Column 12 was used as a sterility
control and blank for the plate scanner.The plates were
incubated at 37°C. The bacterial cultures were streaked on
plates to check their purity. After 24-48 hours growth on
the plates was notes and recorded.
Results
Morphological and Phenotypic characterisation
Morphological and phenotypic characteristics were examined
for each isolate all of which were consistent with the
description of typical Pseudomonads according to
Bergey’s Manual for systematic Bacteriology (Bergey,
Holt et al. 1984), which describes the genus as being
Gram-negative, non-spore forming, motile, catalase positive
and oxidase positive, due to the enzyme indophenol oxidase,
straight or slightly curved rods. The morphological
characteristics of all isolates were identical and
consistent with P. aeruginosa. Only one isolate, PA12
displayed mucoid characteristics, all other isolates were
non-mucoid. Generally, common to all constituent species of
the genus Pseudomonas are certain physiological
properties such as chemoorganotrophic nutrition, aerobic
metabolism, absence of fermentation, absence of
photosynthesis, inability to fix nitrogen, and capacity for
growth at the expense of a large variety of organic
substrates. To examine the metabolic abilities and substrate
utilisation of all the isolates, a series of non-automated
conventional tests were completed. The metabolic profiles of
the isolates, were identical and consistent with P.
aeruginosa metabolism. All strains oxidised glucose,
indicated by results on OF (Hugh & Liefson) medium, a
classic positive trait of pathogenic P. aeruginosa (Gilardi,
1968).
Other classic positive results shared by all the
P.aeruginosa isolates, including the reference strain
PAO1 included oxidase positive reactions due to the presence
of cytochrome c-oxidase in the electron transport chain,
thus producing indopenol oxidase. This is one of the
distinguishing characteristics in the identification of
Pseudomonas sp. apart from other Enterobacteriae
(Hampton & Wasilauskas, 1979). Catalase, malonate
utilisation and arginine hydrolysis, encoded by the
“arginine dihydrolase” system consisting of arginine
deiminase, catabolic ornithine caramoyl-transferase, and
carbanate kinase (Haas, Evans et al. 1979). Selective
plating on centrimide (cetyltrimethylammonium
bromide)
agar (Brown & Lowbury, 1965), designed specifically to
identify P.aeruginosa and prevent the growth of other
organisms, with the addition of naldixic acid (Goto &
Enomoto, 1970) again confirmed all isolates as
P.aeruginosa, as did fluorescence on Pseudomonas
agar F. With the exception of colour, the colony
characteristics of twelve of the strains were identical. The
strains gave positive results for arginine hydrolysis,
growth on centrimide agar and fluorescence on Pseudomonas
agar F. All strains possessed lipolytic activity (Tween
80 hydrolysis). None of the strains were, capable of
hydrolysing starch. These tests were selected as
conventional phenotypic tests used to identify
Pseudomonas organisms (Costas, 1992) and selectively
identified all isolates as strains of Pseudomonas
aeruginosa. The strains differed however in their
respective pigment production, characterised for
P.aeruginosa by the production of fluorescent pigments,
particularly pyocyanin.
Automated phenotypic analysis
In addition to the classic biochemical tests carried out,
identification was carried out using the commercially
available API 20NE and Biolog identification systems.
Identification using a Biolog GN system identified all the
strains as being Pseudomonas aeruginosa strains,
while identification using API 20NE tests gave 91.4%-99.99%
i.d., and good to excellent identification.
The results of the AP1 20NE tests showed that there is great
similarity among all the strains in their ability to utilise
the various carbon sources.
The results of the AP1 20NE tests showed great similarity
among all the strains in their ability to utilise the
various carbon sources. All thirteen strains had the same
reaction thirteen of the twenty tests on the API. There
were however some exceptions. PA3 was unable to produce
indole, it could acidify glucose and it was positive for
b-galactosidase.
PA8, PA9, PA10 and PA16 were all negative for urease. PA10
and PA16 could not assimilate N-acetyl-glucosamine, PA10 and
PA17 could not, assimilate adipate and PA10 could not
assimilate citrate. However all isolates scored >98%
identification as strains of P.aeruginosa.
The Biolog results, also showed similarities between the
metabolic abilities of the isolates in their ability to
either utilise or inability to utilise sixty-two of the
ninety-six carbon sources on the Biolog plates. However,
there were quite a number of exceptions.
PA11 and PA12 were able to use
a-cyclodextrin.
PA3, PA5, PA7, PA8, PA16, PA17 were unable to assimilate
dextrin. PA5, PA16 and PA17 were unable to use N-acetyl-D-glucosamine
or D-fructose. PA5, PA9, PA11, PA12, PA16 and PA17 were
unable to utilise L-arabinose. PA5, PA9, PA12, PA13, PA16,
PA17 were not able to use D-aribitol. PA12 had the ability
to utilise cellobiose, melibiose and D-glucosaminic acid.
PA1, PA7, PA11 and PA12 could use
b-methyl
D-glucoside. PA3, PA5, PA9 and PA17 were unable to utilize
D-psicose. PA7 was able to use L-rhamnose and both PA7 and
PA8 were able to use sucrose. PA1, PA9 and PA11 had the
ability to utilise trehalose. PA1 was unable to use
a-ketovaleric
acid, D, L lactic acid, propionic acid, D-saccharic acid or
succinic acid. PA9 was able to utilize L-phenylamine and
unable to utilize D, L carnitine. Neither PA1 nor PA9 were
able to use quinic acid. Both PA1 and PA3 had the ability to
use uridine. PA 17 was unable to use L-ornithine or L-threonine.
PA1, PA7, PA8, PA16 and PAO1 were able to use sebacic acid.
PA5, PA9, PA13 and PA17 were not able to utilise L-leucine.
Both PA7 and PA10 were able to use D-serine. PA3, PA5, PA8,
PA10, PA16 and PA17 were unable to use i-erythritol. PA1,
PA7, PA8, PA10 and PAO1 were able to utilize D-mannose. Both
PA10 and PA12 were able to use L-alanyl-glycine. PAO1 was
able to use glycyl-L-glutamic acid.
16s rRNA sequencing
16s analysis was performed on all the PA isolates, initially
using species-specific oligonucleotide primers designed by
Spilker et al, 2004. The primer set, based on
P.aeruginosa conserved regions of the 16s rRNA and
designed for the differentiation of P.aeruginosa from
other Pseudomonas species, specifically in sputum
samples taken from cystic fibrosis patients. PCR
amplification products of
»956bp
were obtained for all the PA isolates and the reference
organism PA01 (Figure 1). These amplicons were sequenced,
the resultant sequences analysed using BLASTN analysis (Altschul,
Gish et al. 1990) on the NCBI website (www.ncbi.nlm.nih.gov).
All of the amplicons were identified with 99% homology to
16s rRNA from various P.aeruginosa isolates.
For PCR based assay procedures, the amplification of
species-specific conserved regions, of the 16s rRNA is
sufficient, however for phylogenetic comparison of the
isolates, the amplification of the entire 16 rRNA was
required. To facilitate this, a set of universal
oligonucleotide primers, designed by Marchesi et al,
1998 were used. The resultant 1.3kb products (data not
shown) were definitively identified as P.aeruginosa
isolates by nucleotide sequence analysis. The DNA analytical
software tools; ClustalW (www.ebi.ac.uk)
(Thompson, Higgins et al. 1994) and ClustalX
(Thompson, Gibson et al. 1997), were used to compare
the 16s rRNA sequences. From the results, a Phylogenetic
tree (Figure 2) was generated to analyse the relationship
between the P.aeruginosa isolates. This tree confirms
that although the strains displayed significant metabolic
similarities each isolate differs genotypically.
For a more comprehensive analysis, the PA isolates were then
genetically compared with other strains within the DNA
database including P.aeruginosa and B.cepacia,
a significant pathogen in CF patients, the relationship
illustrated in Figure 3. The phylogenetic tree in Figure 3
represents the comparison, which verified the close
relationship between all the PA isolates and various
hospital-acquired pathogens, especially B.cepacia.
Figure 2.
Phylogenetic tree, illustrating the genetic relationship
between the nosocomial isolates of P.aeruginosa
PA1-PA17 and the reference strain PA01.
0.1=nucleotide differences. 1000=bootstrap operations
Antibiotic resistance profiles and MICs.
Antibiotic resistance profiles were obtained for each
isolate, using the method described previously to generate
MIC values for numerous antibiotics, the results of which
are shown in Table 1.
To investigate the extent of antibiotic resistance in the PA
isolates, numerous classes of antibiotics were initially
examined (Table 1).
Analysis of the antibiotic resistance profiles shows
consistencies amongst all the isolates, with the exceptions
of PA7, which was inhibited by tetracycline, PA12 which
appeared to be resistant to spectinomycin but most
importantly, PA13, which showed multiple drug resistance
especially to gentamicin, the most commonly administered
aminoglycoside in the treatment of P. aeruginosa
infections (Akiyoshi, Intetsu et al.
2005).Aminoglycoside resistance profiles were subsequently
completed (Table 2).
Susceptibility to
amikacin, apramycin, butirosin A and lividomycin A and
hygromycin B was common
among all the isolates.
except for PA3, which was resistant to the latter.
Resistance varied from 32mg/ml
(for PA8, PA9, PA11, PA12, PA13, PA16 and PAO1) to 62.5mg/ml
(for PA1, PA5, PA7, PA10 and PA17).
Resistance to paromomycin, was common among all of the
strains, (32mg/ml
(PA8) to 500mg/ml
(PA13 and PA17), except PA3 and PA9. All strains except PA9
and PA11 were found to be resistant to spectinomycin. PA1
was highly resistant to the antibiotic with an MIC of 1000mg/ml.
PA7, PA8 and PA10 could grow up to a concentration of 125mg/ml.
PA5 had an MIC of 62.5mg/ml,
whereas PA3, PA12, PA13, PA16, PA17 and PAO1 grew up to a
concentration of 32mg/ml.
All the strains were susceptible to streptomycin except for
PA13, which had an MIC of 62.5mg/ml.
PA12 had an MIC of 15.6mg/ml,
which means it is susceptible to streptomycin. This result
is in contrast to the result obtained using the antibiotic
susceptibility disc, which showed PA12 to be resistant to
streptomycin.
In comparison to all the other isolates, the ARP of PA13
showed significant differences in the levels of resistance
to the various aminoglycosides. PA13 did respond to amikacin
(13.9mg/ml),
apramycin (15.6mg/ml),
butirosin A (12.5mg/ml)
and lividomycin A (15.6mg/ml).
At 32mg/ml,
PA13 displayed medium resistance to hygromycin B, neomycin,
spectinomycin and tobyramycin. No other PA isolate was
responsive to tobramycin. High-level resistance was
identified in the presence of streptomycin (62.5mg/ml)
and paromomycin (500mg/ml).
PA13 was extremely resistance to netilimicin up to a
concentration of 1250mg/ml.
Crucially, PA13 showed high-level resistance to gentamicin
up to a concentration of 120mg/ml.
Resistance to gentamicin, being the most commonly
administered aminoglycoside in topical treatment, poses
significant difficulties in patients with immunocompromised
conditions. The other strains were susceptible to gentamicin
and could not grow in the antibiotic above a concentration
of 3.9mg/ml.
Discussion
Identification systematics and analytical techniques have
evolved due to the advancement of molecular diagnostics.
These techniques particularly, combined with conventional
analyses have enabled rapid and definitive identification of
unknown isolates from nosocomial and/or community-acquired
origin. These techniques were employed to identify the 12
clinical isolates submitted for analysis by two Irish
hospitals.
Although preliminary identification completed prior
to our analysis identified the isolates as P.aeruginosa,
problems with misidentification occurs frequently due to
strain similarity with Burkholderia cepacia, formerly
Pseudomonas cepacia, re-classified in 1996 due to
DNA-DNA hybridisation and 16sRNA analyses (Govan, Hughes
et al. 1996; Moore, Millar et al. 2002). This is
significant as B. cepacia is a frequent pathogen
associated with hospital acquired infections in
immunocompromised patients especially CF patients (McMenamin,
Zaccone et al. 2000).
As such, a combination of comprehensive biochemical and
molecular techniques provided definitive identification of
all isolates but also identified diversity amongst the
group. All the isolates presented similar characteristics
consistent with Bergey’s manual description of
P.aeruginosa (Bergey, Holt et al. 1984).
However, the distinguishing biochemical characteristic that
differed between the strains, therefore indicating diversity
among the group, was the production of diffusible pigments.
Pigment production is a contributory phenotypic
characteristic in the classification of P.aeruginosa.
P. aeruginosa, has an innate ability to produce
specific fluorescent phenazine pigments, genetically encoded
by two operons for the production of metabolites such as
pyocyanin or PCN (blue-green) (Kanner, Gerber et al.
1978; Mavrodi, Bonsall et al. 2001), pyoverdin or
fluorescein (greenish-yellow),
pyomelanin (red-brown) and pyorubin (red).
Production of phenazine pigments by the isolates was
confirmed by selective streaking on the two specialist
Pseudomonas agars, Pseudomonas agar P and F. Both
selective media stimulate the production of either pyocyanin
or pyoverdine but inhibit the production of the alternate
pigment.
Results from the selective plating on Pseudomonas
agar P, identified nine of the twelve isolates with the
ability to produce pyocyanin, Pyocyanin, a redox-active
secondary metabolite is synthesised from chromate due to the
action of the phzABCDEFG operon and is regulated by
quorum sensing (Fuqua, Parsek et al. 2001; Mavrodi,
Bonsall et al. 2001|). The pigment has been
determined to display antibiotic, antifungal and cytotoxic
properties therefore contributes to the pathogenesis of
P.aeruginosa, as a human pathogen (Baron & Rowe, 1981;
Kerr, Taylor et al. 1999; Reszka, O’Malley et al.
2004). Significantly, the compound has been identified in
sputum samples from patients with chronic pulmonary
infections, especially CF patients (Lau, Hassett et al.
2004; Lee, Haagensen et al. 2005), thus is
considered to be an infection-associated virulence factor.
Similarly, pyoverdin (fluorescein), produced by all thirteen
strains, is another virulence factor produced by
Pseudomonas sp, especially P.aeruginosa (Meyer,
Neely et al 1996). Pyoverdin, encoded by the pvd
genes (Lamont & Martin 2003 ) acts as a siderophore,
involved in a complex iron acquisition system tightly
binding and transporting soluble iron (Fe III) from the
environment under iron-deficient conditions, which has been
determined to be an essential component in biofilm formation
(Stintzi, Evans et al. 1998; Banin, Vasil et al.
2005).
A brown pigment, thought to be pyomelanin, produced by PA9
is not commonly produced, but where it is, it is usually
associated with isolates from urinary tract infections and
patients in burn units (De Vos, Lim et al. 1997;
Masahisa, Hiroyuki et al. 2005).
This pigment, in common with other melanins, is produced
from aromatic amino acids such as tyrosine or phenylalanine
(Sanchez-Amat, Ruzafa et al. 1998).
The function of pyomelanin is unknown but it is thought to
confer benefits including protection against oxidative
stress (Nosanchuk & Casadevall 2003). A red pigment was
produced in PA3, PA11 and PA12. This pigment is known as
either pyorubin or aeruginosin A or B. Pyorubin is also
believed to be involved in protection of the organism
against oxidative stress.
Although these conventional tests preliminarily identified
the isolates as P.aeruginosa misidentification,
especially due to its similarity to the cystic fibrosis
pathogen Burkholderia cepacia (Campana, Taccetti
et al. 2005), more comprehensive automated biochemical
tests, including the API20NE and the Biolog GN systems were
used to identify subtle metabolic differences amongst the
group. Both commercially available the systems operate on
the basis of, the organisms metabolism of different
substrates. The API20NE (Analytical Profile Index) test (Janin,
1976), similar to the Enterotube is designed specifically
for the identification of Enterobacteriaceae and
incorporates 8 conventional tests and 12 assimilation tests.
Interpretation of the metabolic results generates a
numerical code or “profile”, unique to the isolate, leading
to definitive identification of the organism. This kit
differs from the Biolog, which is designed to classify
gram-negative organisms on the basis of their carbon
oxidation of approximately 95 carbon sources, which
generates a metabolic profile for each organism identified
by the accompanying data analysis software (Bochner,
1989). However,useful for identification, both systems
have innate problems, especially, leading to
misidentification of strains. To circumvent
misidentification issues, several PCR-based assays have been
developed for rapid identification of bacteria from clinical
samples {Gee, Sacchi et al. 2003; Wellinghausen,
Kothe et al. 2005).
16s rRNA sequencing is therefore now considered to be the
hierarchy in phenotypic identification (Woese, 1987). It was
for this reason that the 16s rRNA sequencing was performed
on all the “P.aeruginosa” isolates for definitive
identification, as shown. 16s rRNA genes are conserved among
all organisms however possess various unique species regions
that allow bacterial identification (Gobel, Geiser et al.
1987). Advances in molecular analysis and DNA manipulations
have facilitated the development of rapid identification
systems in clinical and environmental analyses.
Species-specific oligonucleotide probes have enabled the
development of efficient diagnostic methodologies,
especially where pathogenic organisms are difficult to type
and culture. Cystic fibrosis is one such area where
bacterial identification systems were previously difficult,
especially when employing API and/or Biolog systems.
However, the advancement in 16s rRNA analysis has resulted
in the development of rapid diagnostic techniques for the
identification of P.aeruginosa (O'Callaghan, Tanner
et al. 1994: LiPuma, Dulaney et al. 1999;
Spilker, Coenye et al. 2004).
The relationship between the isolates is clearly indicated
in Figure 2, having performed a phylogenetic comparison of
the 16s sequences. However, the difference between the
isolates is also apparent, conclusive of having been
isolated from a variety of sources.
A more comprehensive phylogenetic comparison of the 16s
sequences, compared with a variety of Pseudomonas and
Burkholderia isolates (Figure 3), shows the
relationship of our twelve strains with clinically isolated
strains. All twelve clearly, cluster together similar to but
not clustered with the Burkholderia strains, thus
confirmed definitively the identification as Pseudomonas
aeruginosa strains.
As P.aeruginosa is recognised as a significant
opportunistic pathogen in clinical situations, presenting as
infections in patients with cystic fibrosis (Cantón, Cobos
et al. 2005; Chambers, 2005), neutropenia (Ying-Wei,
Souichi et al. 2003) and cancer (Andremont, Marang
et al. 2003; Norio, Hend et al 2005).
Consequently, patients now present with exacerbated
conditions, due to the innate and evolved drug-resistance
expressed by the causative organisms.
Treatment for Pseudomonas infections frequently
involves the administeration of aminoglycosides as primary
treatment of Pseudomonas infections in clinical
situations therefore susceptibility to aminoglysosides is
cruical in therapeutics. Aminoglycoside-resistance in the
isolates was assessed using the microtitre broth dilution
method outlined above. Minimum Inhibitory concentrations
(MIC’s) were then be established for each isolate and to
generate aminoglycoside-resistance profiles (ARPs) for each
isolate and hence the extent of aminoglycoside resistance in
the isolate PA13. Recommended standards classify medium
resistance as between 8-32
mg/ml,
with significant resistance considered to be from 32mg/ml
and above.
PA13’s AME characterised resistance to gentamicin,
netilmicin and tobramycin, the antibiotics regularly used to
treat Pseudomonas aeruginosa infections, was found to
be resistant to eight of the twelve aminoglycoside
antibiotics tested. It’s profile was quiet different to all
of the other isolates, therefore the strain was chosen for
further studies including genetic analysis to probe for
genes encoding aminoglycoside-modifying enzymes. Also,
during culturing of the strain in high concentrations of
gentamicin, a unique phenotypic change was observed. PA13
cells were seen to spontaneously and transiently aggregate
and subsequently disperse. It was postulated that a dual
resistance was possibly being expressed by the strain, thus
contributing to it’s broad and high-level aminoglycoside
resistance.
Conclusion
The problems posed by hospital borne infections and
opportunistic pathogens now present as one of the most
difficult challenges in the medical world. This problem
compounded by the development of broad-spectrum antibiotic
resistance amongst these organisms has escalated
antibiotic-resistance research in clinical settings.
We analysed 12 nosocomial isolates of P.aeruginiosa
from two primary hospitals in Ireland. The strains were
“typed” initially by conventional biochemical analysis
followed by comprehensive phenotypic identification using
commercially available automated identification systems.
Definitive taxonomic classification was completed by 16srRNA
analysis and all the isolates were identified as novel
strains of P.aeruginosa. Due to the nosocomial origin
of the isolates, all were assessed for their individual
responsiveness to antibiotics and more importantly
aminoglycosides. Each strain presented some low-level
resistance to various drugs. However, one extremely
drug-resistance Pseudomonas aeruginosa isolate, PA13
was identified. PA13’s AME resistance profile, characterised
by high-level resistance to crucial clinically administered
drugs, suggested it expressed a unique resistance mechanism
not common to all the isolates. The strain is now the focus
of further studies to investigate and identify the
regulation of the expressed mechanisms of aminoglycoside-resistance.
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