Abstract
The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P<0.001 and P<0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P<0.01) when compared to other clinical isolates (P<0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.
INTRODUCTION
To be described as a virulence factor, a molecule should adhere to the molecular Koch's postulates, which state that any virulence factor must be (i) encoded by a gene that is associated with bacteria that cause disease, but is usually absent in or inactive in strains that fail to cause disease, (ii) required for virulence, as evidenced by a loss of virulence when disrupted or a gain in virulence when expressed in an avirulent strain and (iii) usually expressed during infection (Falkow, 1988). Pseudomonas aeruginosa is capable of elaborating an impressive array of virulence factors, which are divided into specific groups dependent upon their mode of action or method of delivery to the host cell. P. aeruginosa virulence factors are described as belonging to adhesins and other secreted toxins. Exotoxins are either passively secreted from the cell or actively secreted via the type I secretion system (T1SS), the type II secretion system (T2SS) or the type III secretion system (T3SS).
The expression of virulence genes in a given infection is of primary importance in the capacity of an individual P. aeruginosa isolate to establish and maintain infection. However, a number of previous studies have investigated potential associations between the presence or absence of known virulence factor genes and differing types of infection. These studies have differed in gene targets, the number of isolates assayed and the geographical and temporal source of isolates used. Debate still exists about the role that individual virulence genes play in determining the type of infection caused by strains of P. aeruginosa.
Virulence genes were selected in this study based upon their varying modes of action and mechanisms of secretion from the P. aeruginosa cell. Alkaline protease (encoded by apr) is a T1SS secreted metallo-protease with a wide range of substrates, including collagen, C1q and C3 of the complement pathway, serum protease inhibitors, fibrin, fibrinogen, laminen and elastin (Pitt, 1998; Engel, 2003). Pulmonary elastase (encoded by lasB) is a powerful T2SS secreted proteolytic enzyme. This enzyme has a wide range of substrates, including elements of connective tissue such as elastin, collagen, fibronectin and laminen, as well as immune and host defence molecules such as fibrin, gastric mucin, transferrin, α-1 proteinase inhibitors, IgG, γ-interferon and components of the complement pathway (Pitt, 1998; Engel, 2003). The phenazine operons (phzI and phzII) and genes (phzH, phzM and phzS) encode precursor proteins involved in the formation of three phenazine compounds passively secreted by P. aeruginosa: pyocyanin, 1-hydroxyphenazine and phenazine-1-carboxamide (Mavrodi et al., 2001; Finnan et al., 2004). Phenazines increase intracellular oxidative stress through intracellular redox cycling of reducing agents and oxygen, producing superoxide and hydrogen peroxide (Mavrodi et al., 2001). The compounds inhibit mitochondrial activity, cell proliferation, cytokine secretion and superoxide production in neutrophils and macrophages (Engel, 2003). High concentrations of pyocyanin may be recovered from the respiratory tract of cystic fibrosis (CF) patients, and is believed to be involved in lung damage through interference with ion transport mechanisms and ciliary beating as well as interference with mucus secretion (Mavrodi et al., 2001).
The four effector enzymes of the T3SS, ExoS, ExoT, ExoU and ExoY, remain the focus of much research. The first identified P. aeruginosa T3SS effector enzymes, ExoT and ExoS, share 75 % homology at the amino acid level (Engel, 2003). It has been found that ExoS is the major cytotoxin involved in colonization, invasion and dissemination during infection, while ExoT protects cultured cells from T3SS-dependent lysis in vitro (Lee et al., 2005). Both enzymes show ADP-ribosyltransferase activity, but ExoT shows only 0.2 % of the activity of ExoS (Engel, 2003). ExoS also targets small Ras-like proteins, inhibiting internalization and DNA synthesis and inducing apoptosis (Engel, 2003; Vance et al., 2005). ExoT targets host kinases involved in focal adhesion and phagocytosis (Vance et al., 2005), and has been associated with dissemination of disease from the lung to the liver in mice (Shaver & Hauser, 2004). The expression of ExoT alone will induce death in Galleria mellonella at near wild-type rates (Miyata et al., 2003) and induce apoptosis in HeLa cells (Shafikhani et al., 2008). ExoY shows little pathology in mouse pneumonia (Lee et al., 2005) but has significant cytotoxicity in MDCK cells (Lin et al., 2006). In contrast, the fourth and most recently described exoenzyme of the P. aeruginosa T3SS, ExoU, shows marked cytotoxic capabilities with remarkably rapid and fulminant cytotoxic effects (Engel, 2003; Finnan et al., 2004; Vance et al., 2005). ExoU causes significant cytotoxicity in MDCK cell culture (Lin et al., 2006) and has been shown to be over 100 times more cytotoxic than ExoS (Lee et al., 2005). Deletion of exoU severely limits the toxicity of P. aeruginosa strains in the lung, and the enzyme has been implicated as an agent associated with septic shock and increased disease severity and mortality in pneumonia (Engel, 2003; Schulert et al., 2003; Vance et al., 2005; Wong-Beringer et al., 2008). An association between exoU and invasive disease has been proposed based upon increased rates of the exoS− exoU+ genotype in isolates causing bloodstream infection (Wareham & Curtis, 2007) but it is not associated with colonization and invasion in BALB/c mice (Lin et al., 2006).
For a greater understanding of why specific strains colonize and cause infection in specific sites and to clarify the results of previous studies, investigation of the prevalence of specific virulence factor genes in alternative types of infections was undertaken. Examination of multiple virulence genes in a large number of P. aeruginosa strains taken from disparate sources and collected from a limited geographical space over a limited period of time was performed.
METHODS
Source and identification of isolates.
One hundred and eighty-four isolates of P. aeruginosa from diverse clinical and environmental sources were employed in this study. CF isolates were sourced from Tasmanian adult CF patients statewide. These isolates included all known isolates of Australian Epidemic Strain 3 (AES 3) and the Tasmanian CF Cluster strain, as well as one clinical isolate of AES 1 from a Tasmanian CF patient (Bradbury et al., 2008). Non-CF in-patient and community clinical isolates and hospital environment isolates (herein referred to as ‘nosocomial’ isolates) were isolated at the major tertiary referral hospital in Tasmania. Community environmental isolates (from swimming pools, streams, sewerage outlets, landfill sites and spas) were sourced from the Tasmanian Public Health Laboratory and represent samples from sites around southern Tasmania (Bradbury et al., 2009). The methods used for isolation of these bacteria have been previously published (Bradbury et al., 2008, 2009). The laboratory control isolates PAO1 and PA103 and clinical isolates of AES 1 and AES 2 were employed as controls in this study. All isolates were confirmed as P. aeruginosa by a combination of a positive oxidase test, capacity for growth at 42 °C, resistance to the compound C390 (Rosco) (Anthony et al., 2002) and by use of a P. aeruginosa-specific PCR (Spilker et al., 2004).
Virulence factor PCRs.
A subset of secreted P. aeruginosa virulence factor genes was chosen based upon their variable presence in the genomes of P. aeruginosa isolates previously studied. These were apr (an alkaline protease gene), lasB (an elastase gene), phzI, phzII, phzH, phzM, phzS (phenazine precursor genes), exoS, exoT, exoU and exoY (the T3SS effector enzyme genes).
All PCR primers used in this study were designed by Finnan et al. (2004), with the exception of the exoU forward primer. The binding site of each primer in the PAO1 genome (NCBI database; ) was determined, and it was ensured that these primers did not bind to other genomes in the database with any significant stringency.
For the exoU PCR, the forward primer previously described by Finnan et al. (2004) was used as a reverse primer, whilst a novel exoU forward primer was designed (5′-ATGCATATCCAATCGTTGG-3′). The binding site of the exoU PCR primers was confirmed using the PA14 genome (NCBI database).
PCR was performed on extracted bacterial DNA. Each specific assay incorporated an appropriate positive control (PAO1 DNA for all but the exoU PCR as this strain is exoU-negative). The positive control used for the exoU PCRs included PA103 DNA. Every set of PCRs performed included ddH2O added to the PCR template as a negative control. All negative PCR results were repeated. A 1 kb molecular mass ladder (Geneworks) was included in one well of each lane of gels in which products were visualized to confirm the size of the PCR products.
To confirm that the newly designed exoU PCR was truly amplifying the exoU gene, a number of exoU PCR products were cut out of an agarose gel, purified using a QIAquick kit (Qiagen) and referred to the Micromon DNA sequencing facility of Monash University (Melbourne, Australia), where the DNA sequences of these products were determined. The resultant product sequences were entered into a blast search on the NCBI database to confirm their identity as the exoU gene of P. aeruginosa.
DNA–DNA hybridization.
A digoxigenin-labelled probe for each virulence factor gene where PCR yielded at least one negative result was prepared using a PAO1 PCR product (with the exception of the exoU PCR, for which a PA103 PCR product was employed) with the Roche Diagnostics Easy Hyb kit, according to the manufacturer's instructions. DNA–DNA hybridization blots were performed on DNA extracts of all isolates yielding a negative result by PCR to confirm absence of homologues of the gene of interest.
Ciprofloxacin resistance testing.
The sensitivity of all isolates to the fluoroquinolone ciprofloxacin was tested using ciprofloxacin 5 μg discs (Oxoid) by the CLSI (2006) method on Mueller–Hinton agar (Oxoid).
Statistical analysis.
Results of individual virulence factor presence in isolates in this study were compared by site of isolation and source of isolate using χ2 analysis in Microsoft Office Excel 2003 (11.8220.8202) SP3.
RESULTS AND DISCUSSION
exoU PCR design and validation
The priming sites of virulence factor gene PCR primers employed in this study were compared with the genomes of P. aeruginosa strains held in the NCBI database. Whilst the majority of these PCR primers bound to points in the P. aeruginosa genome consistent with the gene that they were intended to amplify, discrepancies were identified for the described exoU PCR primers. Firstly, inversion of the published forward and reverse primers was identified. More so, the published reverse primer (actually a forward primer) was found to bind to a site outside of the gene which is less likely to be conserved between bacterial isolates.
In order to ensure accurate amplification of only exoU in this study, a new forward primer was designed and used in combination with the reverse primer developed by Finnan et al. (2004) (normally a forward primer) resulting in a novel exoU-specific PCR primer pair (2415 bp product). As expected, this primer pair resulted in a 2.4 kb product from P. aeruginosa strain PA103 and failed to amplify a product from PAO1.
The exoU PCR products of the control strain PA103 and two hospital environment strains (isolates 123 and 124) were sequenced, resulting in partial sequences of between 145 and 519 bp in length. The returned sequences all provided between 91 and 98 % identity as the P. aeruginosa UCBPP-PA14 exoU in NCBI blast searches (data not shown), confirming that the newly designed PCR does indeed amplify the exoU gene.
Virulence factor genes are highly conserved
The vast majority of P. aeruginosa strains tested in this study were shown to possess all virulence factor genes examined, with the exception of exoU (Table 1⇓). Thus, the presence of these virulence genes was found to be highly conserved across the genome of P. aeruginosa, regardless of the secretion system to which their products belonged. While a small number of individual isolates did show deletions of some of the specific virulence factor genes tested, no pattern was observed with regard to the source or site of infection that these isolates were recovered from. All negative PCR results were confirmed by dot blot hybridization, and no discrepancies between PCR results and DNA hybridization results were observed, indicating that these bacteria were unlikely to contain mutants or homologues of the genes that could not be detected using gene-specific PCR primers.
Results of virulence gene PCRs performed upon clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa
Other ward, non-intensive therapy ward; muc, mucoid on first isolation; non, non-mucoid on first isolation; COPD, chronic obstructive pulmonary disease; S, sensitive; R, resistant; I, intermediate.
All isolates investigated in this study possessed both the apr and lasB genes. These findings were in concordance with those from a comprehensive study carried out on 145 isolates of P. aeruginosa finding these genes to be universally present (Lomholt et al., 2001), but varied significantly from those in another study involving a much smaller sample of eleven CF and five environmental P. aeruginosa isolates (Finnan et al., 2004). This latter study also found two environmental isolates, both from hospital sinks in Belgium, with significant absence of the phenazine group genes (Finnan et al., 2004). No such isolates were identified among our environmental and nosocomial strains, indicating that absence of these genes may be a rare event.
The greatest heterogeneity in gene prevalence was observed in the T3SS effector exoenzyme genes. Regarding the chromosomal T3SS effector genes (exoS, exoT, exoY), the findings of this study were in concordance with other studies, in that a high prevalence of exoT and exoY was identified (Feltman et al., 2001; Lomholt et al., 2001).
Exclusivity of exoU and exoS
Ten isolates in this study were found to harbour both exoS and exoU, including four isolates from the same patient and with identical genotypes. Despite this, a significant (P<0.001) trend towards mutual exclusivity between these two genes was observed, as previously reported (Feltman et al., 2001; Kulasekara et al., 2006; Lomholt et al., 2001; Wong-Beringer et al., 2008). This phenomenon is surprising, as no easily identifiable linkages between these genes are observable in the P. aeruginosa genome and these genes are found at points distant from each other on the P. aeruginosa genome (Feltman et al., 2001; Kulasekara et al., 2006). At present, insufficient is known about the functional role of the exoenzyme products of these genes, their interaction in both the virulence pathways and the global cellular mechanisms of P. aeruginosa to identify a specific reason for this phenomenon. However, it is conceivable that possession of both genes in some way reduces organism fitness, encouraging the loss of one or the other from an individual organism's genome. It may also be possible that simultaneous production of both exoenzyme products results in an increased or upregulated host immune response. Therefore, isolates of this genotype would be less capable of establishing infection in a new host due to their higher immunogenicity and resultant increased clearance from the host. The fact that a high relative percentage of the exoS+ exoU+ isolates in this study was seen in nosocomial isolates but not in clinical isolates in the same hospital provides a tentative basis for this argument, as the genotype of these isolates would not be influenced by any host immune response.
Prevalence of exoU
One exception to the conservation of P. aeruginosa virulence factor genes was found in the T3SS, specifically in retention of exoU and its relationship to the exoS gene. Only 23 of 184 isolates tested (12.5 %) were of genotype exoS− exoU+, whilst 170 isolates (92 %) were exoS+ exoU−. Just 10 (5 %) of the isolates tested were found to have the exoU+ exoS+ genotype (Table 1⇑). Four of the latter isolates were recovered from different sites of infection in the same patient (Table 1⇑) and have previously been shown to be genotypically indistinguishable by both RAPD PCR using primer 272 and by SpeI PFGE (Bradbury et al., 2008). When the relative prevalence of these T3SS exoenzyme genes was compared, it was noted that lack of exoS was significantly (P<0.001) more common in isolates harbouring exoU when compared to the rate of exoS negativity overall. Co-selection of exoU and fluoroquinolone resistance mechanisms in some isolates of P. aeruginosa has previously been suggested (Wong-Beringer et al., 2008). In this study, no significant association between ciprofloxacin resistance and exoU carriage was observed (P=0.166).
When the relative prevalence of the exoU+ strains recovered in this study was investigated by site and source of isolation, a number of significant patterns were observed. Firstly, isolates from the environment were found to be significantly more likely to carry exoU than other isolates (P=0.003). Further analysis of this finding showed that this was due to a relative increase in exoU prevalence in intensive care unit and neurosurgical unit nosocomial isolates specifically (P<0.001 and P=0.032, respectively).
The exoU gene appears to have been recently acquired via horizontal transmission on a plasmid, and then integrated into the P. aeruginosa genome (Kulasekara et al., 2006). Given this, it is unsurprising that this particular gene shows a lower prevalence in the P. aeruginosa genome when compared to other virulence genes. It has been shown to have a significantly lower overall prevalence in P. aeruginosa isolates compared to other T3SS effector genes by a number of studies (Feltman et al., 2001; Lomholt et al., 2001; Wong-Beringer et al., 2008), a finding supported by this investigation. An exception to this was the findings of one study, which found exoU prevalence to be second only to exoS in the T3SS genotype of the isolates tested, as well as a low prevalence of exoT (Finnan et al., 2004).
CF isolates were found to be significantly (P=0.001) less likely to carry exoU when compared to other isolates in this study. Following exclusion of environmental and nosocomial isolates from the analysis, this trend was maintained (P=0.005). This finding is supported by one previous T3SS gene study (Feltman et al., 2001). A different study involving 137 P. aeruginosa isolates, 81 of which were recovered from the CF lung, found a significant decrease in the prevalence of exoS in CF isolates (Lanotte et al., 2004). Unfortunately, no data are available from that study regarding the relative prevalence of exoU in these isolates, but given the demonstrated propensity for the exoS+ exoU− phenotype in P. aeruginosa (Wareham & Curtis, 2007), it is not unreasonable to expect that a conversely decreased prevalence of exoU in the CF strains studied may have occurred. Whilst outpatient isolates were also found to have a significant propensity towards exoU absence, with further analysis excluding the large number of CF isolates in this group, the significance of this result was negated.
Expression of exoenzyme U is known to greatly increase the virulence of P. aeruginosa in vivo generally (Lin et al., 2006; Vance et al., 2005, ), and specifically in lung infections (Schulert et al., 2003). In CF patients, chronic lung infection occurs, and such a condition is not conducive to the presence of bacterial strains elaborating an enzyme known to cause great damage in the lung. In cases where a CF patient is infected with a strain producing significant amounts of ExoU, the infection would either rapidly progress to death, or be cleared by the patient. As the latter scenario is almost unknown in CF patients, greatly decreased patient mortality provides an explanation for the lower number of exoU-carrying strains in the CF population.
Another potential explanation for this phenomenon does however exist. It has been shown that CF isolates of P. aeruginosa often do not express their T3SS effector exoenzymes after prolonged lung colonization (Dacheux et al., 2000; Jain et al., 2004; Roy-Burman et al., 2001). The most probable explanation for the latter phenomenon is that reduction of virulence factor expression results in evasion of the host immune system. This hypothesis explains many of the clinical and scientific observations regarding CF lung infection, such as the alteration in bacterial phenotype and the prolonged duration of infection with relatively little morbidity in comparison to P. aeruginosa lung infection in the acute setting and the incapacity of patients to clear their infections. If this is the mechanism by which P. aeruginosa avoids detection and clearance by the host immune system in CF infections, elaboration of a factor with the degree of virulence attributed to ExoU would have an extremely deleterious effect on the long-term survival prospects of the organism in the host lung. It may also reasonably be assumed that an enzyme causing such extreme damage to host cells would be a powerful ligand for the human immune system. Thus, strains of P. aeruginosa elaborating ExoU would be less capable both of establishing infection due to their early detection by the host immune system, and of persistence in the CF lung. This combined with the probable increased mortality in CF patients should they be infected with such a strain provides a very likely explanation for the relative lack of exoU-positive CF strains of P. aeruginosa.
Conversely to the CF respiratory isolates, nosocomial isolates in this study were found to have an increased prevalence of exoU. Careful statistical analysis of the data showed that this was due to an increased prevalence of the exoU+ genotype in the samples recovered from the ICU and NSU. This increased prevalence may be attributed to the small geographical area and increased selective pressures that will apply in the environment of these highly specialized wards. Relatively little is known about the role which the product of exoU plays in the interactions of P. aeruginosa and environments outside of the mammalian host, and it is possible that possession of exoU provides a selective advantage to P. aeruginosa in this particular environment. Both the ICU and NSU environments are subject to more stringent disinfection protocols than other environments within the hospital, which may facilitate and promote the acquisition of exoU by horizontal transfer between nosocomial strains of P. aeruginosa. It may be speculated that acquisition of other resistance mechanisms which may assist survival in the ICU environment might also positively modify the exoU acquisition rate.
No other significant patterns in virulence factor genotype were noted in the P. aeruginosa strains during this study. The associations found by some other studies between disease type and T3SS virulence gene prevalence were not supported by this particular investigation. This does not decrease the validity of the findings of these studies, as specific geographical areas will have different genotypes of P. aeruginosa present and, as discussed, factors such as antibiotic use and the class of antibiotics used in treatment of specific diseases may significantly alter the genotype of the infecting strains. The relative heterogeneity of the virulence genes studied in this work is concordant with the conserved nature of the P. aeruginosa genome in both disease and the environment. That exoU was the source of all significant variability within the virulence genome correlates with its mobile nature and capacity to be acquired under the selective pressure of antibiotic use.
Conclusions
This study represents the largest and most comprehensive survey of virulence genotype thus far performed upon strains of P. aeruginosa. A number of confusing and inconsistent findings from previous studies have been clarified. The large sample size allowed elucidation of statistically significant results in regard to the prevalence of P. aeruginosa virulence genes in strains from markedly varied sites and sources of isolation.
Acknowledgments
The authors would like to acknowledge the assistance of Mr Roger Latham (Cystic Fibrosis Research Group, Menzies Research Institute, University of Tasmania, Hobart, Australia). They would also like to acknowledge Associate Professor Timothy Inglis (Microbiology Department, PathWest Laboratory Medicine, Nedlands, Western Australia) and Dr Janet Williamson (Department of Molecular Medicine, Royal Hobart Hospital, Hobart, Australia) in the preparation of this work. Dr David Armstrong (Department of Paediatrics, Monash Medical Centre, Victoria, Australia), Associate Professor Scott Bell (Department of Respiratory Medicine, Royal Children's Hospital, Herston, Australia) and Associate Professor Iain Lamont (Biochemistry Department, University of Otago, Dunedin, New Zealand) provided isolates of AES 1, AES 2 and PA103. Thanks go to the staff of the Tasmanian Public Health Laboratory and the Microbiology Department of the Royal Hobart Hospital for their assistance in the provision of many of the isolates used in this study. This study was made possible with a grant from the Royal Hobart Hospital Research Foundation.