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1.  Use of Subtractive Hybridization To Identify a Diagnostic Probe for a Cystic Fibrosis Epidemic Strain of Pseudomonas aeruginosa 
Journal of Clinical Microbiology  2002;40(12):4607-4611.
A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.
PMCID: PMC154653  PMID: 12454160
2.  Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen 
A synthetic genetic system is designed and characterized that allows Escherichia coli to sense and eradicate Pseudomonas aeruginosa, providing a novel antimicrobial strategy that could potentially be applied to fighting infectious pathogens.
We have engineered and demonstrated a novel genetic circuit that enables Escherichia coli to produce and release pyocin upon quorum sensing detection of Pseudomonas aeruginosa, which in turn kills P. aeruginosa.The quorum sensing device, which comprises an LasR transcription factor constitutively expressed by a pTetR promoter and a downstream pLuxR inducible promoter, has a switch point of 1.2 × 10E-7 M 3OC12HSL and is able to sense 3OC12HSL natively produced by P. aeruginosa.The E7 lysis device when coupled downstream of the quorum sensing device enhances pyocin release eight-fold.The engineered E. coli, which carries the sensing, lysing, and killing devices, effectively inhibits the growth of planktonic and biofilm P. aeruginosa by 99 and 90%, respectively.
In this study, we have made progress toward developing a novel antimicrobial strategy, based on an engineered microbial system, using the synthetic biology framework. Our final system was designed to (i) detect AHLs produced by P. aeruginosa; (ii) produce pyocin S5 upon the detection; and (iii) lyse the E. coli cells by E7 lysis protein so that the produced pyocin S5 is released from the cells, leading to the killing of P. aeruginosa.
Figure 1 shows a schematic of our sensing and killing genetic system. The sensing device was designed based on the Type I quorum sensing mechanism of P. aeruginosa. The tetR promoter, which is constitutively on, produces a transcriptional factor, LasR, that binds to AHL 3OC12HSL. The luxR promoter, to which LasR-3OC12HSL activator complex reportedly binds, was adopted as the inducible promoter in our sensing device (Gray et al, 1994). Next, the formation of the LasR-3OC12HSL complex, which binds to the luxR promoter, activates the killing and lysing devices, leading to the production of pyocin S5 and lysis E7 proteins within the E. coli chassis. Upon reaching a threshold concentration, the lysis E7 protein perforates membrane of the E. coli host and releases the accumulated pyocin S5. Pyocin S5, which is a soluble protein, then diffuses toward the target pathogen and damages its cellular integrity, thereby killing it.
To evaluate and characterize the sensing device, the gene encoding the green fluorescent protein (GFP) was fused to the sensing device and the GFP expression was monitored at a range of concentrations of 3OC12HSL. From the measured GFP synthesis rates, we observed a basal expression level of 0.216 RFU per OD per minute without induction, followed by a sharp increase in GFP production rate as the concentration of 3OC12HSL was increased beyond 1.0E-7 M. A transfer function that describes the static relationship between the input (3OC12HSL) and output (GFP production rate) of the sensing device was determined by fitting an empirical mathematical model (Hill equation) to the experimental data where the input 3OC12HSL concentration is <1.0E-6 M. The resulting best fit model demonstrated that the static performance of the sensing device follows a Hill equation below the input concentration of 1.0E-6 M 3OC12HSL. The model showed that the sensing device saturated at a maximum output of 1.96 RFU per OD per minute at input concentration >3.3E-7 M but <1.0E-6 M 3OC12HSL, and the switch point for the sensing device was 1.2E-7 M 3OC12HSL, the input concentration at which output is at half-maximal. Since this switch point concentration is smaller than the concentration of 3OC12HSL present (1.0E-6 to 1.0E-4 M) within proximity to the site of P. aeruginosa infection as earlier reported in the literature (Pearson et al, 1995; Charlton et al, 2000), the sensing device would be sensitive enough to detect the amount of 3OC12HSL natively produced by P. aeruginosa.
In line with the objective of the E7 lysis device in mediating the export of pyocin, we studied the efficiency of the lysis device in the final system by measuring the amount of the released protein. While distinct bands that corresponded to pyocin S5 were observed on the SDS–PAGE of the final system, no bands were seen in lanes without the lysis device. We further validated the results by estimating the protein concentrations in the supernatant with Bradford assay and showed that the amount of pyocin released by our final system was eight times higher than the system without the lysis device.
To verify that our engineered E. coli can inhibit P. aeruginosa in a mixed culture, we monitored the growth of P. aeruginosa co-cultured with the engineered E. coli in the ratio 1:4 by CFU count. The result shows that our engineered E. coli with the final system effectively inhibited the growth of P. aeruginosa by 99% while continuous growths were apparent in P. aeruginosa co-cultured with incomplete E. coli systems missing either the pyocin S5 or E7 lysis devices.
To examine the potential application of our engineered system against a pseudo disease state of Pseudomonas, a static biofilm inhibition assay was performed. Figure 6A shows that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%. This observation is in stark contrast to the pyocin-resistant control strain PAO1 and pyocin-sensitive clinical isolate ln7 subjected to treatment with E. coli having the systems missing either the pyocin S5 or E7 lysis devices. To visualize the extent of biofilm inhibition, biofilm cells with green fluorescence were grown in the presence of engineered E. coli on glass slide substrate and examined with confocal laser scanning microscopy. Figure 6B shows that the morphology of Pseudomonas biofilm treated with the engineered E. coli appeared sparse, while elaborated honey-combed structures were apparent in the control experiments. Collectively, our results suggest that our engineered E. coli carrying the final system, which contains the sensing, killing, and lysing devices, can effectively inhibit the growth of P. aeruginosa in both planktonic and sessile states.
In summary, we engineered a novel biological system, which comprises sensing, killing, and lysing devices, that enables E. coli to sense and eradicate pathogenic P. aeruginosa strains by exploiting the synthetic biology framework. More importantly, our study presents the possibility of engineering potentially beneficial microbiota into therapeutic bioagents to arrest Pseudomonas infection. Given the stalled development of new antibiotics and the increasing emergence of multidrug-resistant pathogens, this study provides the foundational basis for a novel synthetic biology-driven antimicrobial strategy that could be extended to include other pathogens such as Vibrio cholera and Helicobacter pylori.
Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology-driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.
PMCID: PMC3202794  PMID: 21847113
genetic circuits; Pseudomonas aeruginosa; pyocin; quorum sensing; synthetic biology
3.  Comparison of Three Molecular Techniques for Typing Pseudomonas aeruginosa Isolates in Sputum Samples from Patients with Cystic Fibrosis ▿ †  
Journal of Clinical Microbiology  2010;49(1):263-268.
Monitoring the emergence and transmission of Pseudomonas aeruginosa strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of P. aeruginosa outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of P. aeruginosa strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control P. aeruginosa isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by ≤6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing P. aeruginosa. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.
PMCID: PMC3020435  PMID: 21084517
4.  Pseudomonas aeruginosa AES-1 Exhibits Increased Virulence Gene Expression during Chronic Infection of Cystic Fibrosis Lung 
PLoS ONE  2011;6(9):e24526.
Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.
PMCID: PMC3174184  PMID: 21935417
5.  A comparison of two informative SNP-based strategies for typing Pseudomonas aeruginosa isolates from patients with cystic fibrosis 
BMC Infectious Diseases  2014;14:307.
Molecular typing is integral for identifying Pseudomonas aeruginosa strains that may be shared between patients with cystic fibrosis (CF). We conducted a side-by-side comparison of two P. aeruginosa genotyping methods utilising informative-single nucleotide polymorphism (SNP) methods; one targeting 10 P. aeruginosa SNPs and using real-time polymerase chain reaction technology (HRM10SNP) and the other targeting 20 SNPs and based on the Sequenom MassARRAY platform (iPLEX20SNP).
An in-silico analysis of the 20 SNPs used for the iPLEX20SNP method was initially conducted using sequence type (ST) data on the P. aeruginosa PubMLST website. A total of 506 clinical isolates collected from patients attending 11 CF centres throughout Australia were then tested by both the HRM10SNP and iPLEX20SNP assays. Type-ability and discriminatory power of the methods, as well as their ability to identify commonly shared P. aeruginosa strains, were compared.
The in-silico analyses showed that the 1401 STs available on the PubMLST website could be divided into 927 different 20-SNP profiles (D-value = 0.999), and that most STs of national or international importance in CF could be distinguished either individually or as belonging to closely related single- or double-locus variant groups. When applied to the 506 clinical isolates, the iPLEX20SNP provided better discrimination over the HRM10SNP method with 147 different 20-SNP and 92 different 10-SNP profiles observed, respectively. For detecting the three most commonly shared Australian P. aeruginosa strains AUST-01, AUST-02 and AUST-06, the two methods were in agreement for 80/81 (98.8%), 48/49 (97.8%) and 11/12 (91.7%) isolates, respectively.
The iPLEX20SNP is a superior new method for broader SNP-based MLST-style investigations of P. aeruginosa. However, because of convenience and availability, the HRM10SNP method remains better suited for clinical microbiology laboratories that only utilise real-time PCR technology and where the main interest is detection of the most highly-prevalent P. aeruginosa CF strains within Australian clinics.
PMCID: PMC4053291  PMID: 24902856
Pseudomonas aeruginosa; Typing; Cystic fibrosis; MLST; SNP
6.  PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients 
Journal of Clinical Microbiology  2004;42(5):2074-2079.
Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.
PMCID: PMC404678  PMID: 15131172
7.  Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis 
BMC Microbiology  2013;13:143.
The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients.
We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes.
Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did.
Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.
PMCID: PMC3691768  PMID: 24088260
Pseudomonas aeruginosa; Cystic fibrosis; qPCR; Early detection
8.  Immunoglobulin A and immunoglobulin G antibody responses to alginates from Pseudomonas aeruginosa in patients with cystic fibrosis. 
Journal of Clinical Microbiology  1990;28(4):747-755.
Patients with cystic fibrosis have a high prevalence of mucoid, alginate-producing Pseudomonas aeruginosa that causes chronic infection of the mucosal surface of the lungs. We developed enzyme-linked immunosorbent assays (ELISAs) for determination in serum of immunoglobulin A (IgA) and IgG antibodies to alginate purified from P. aeruginosa and an ELISA for detection of IgA antibodies to a polyvalent P. aeruginosa standard antigen. Absorption experiments indicated that the assays were antigen and antibody specific and had analytical variations that ranged from 7 to 19%. Serum samples from 207 patients with cystic fibrosis, 100 healthy children, and 94 healthy adults were examined. The patients responded to P. aeruginosa infection with early IgA and IgG antibody responses that were significantly higher than in controls and noncolonized patients. Analysis of paired serum samples showed that infected patients had an increase in specific IgG and IgA antibodies that was significantly higher than in noncolonized patients. The serological data were analyzed for correlation with clinical condition; poor lung function was significantly associated with increased levels of IgA and IgG antibodies to P. aeruginosa alginate and to the standard antigen and with a relative excess of IgA antibodies to the standard antigen compared with IgA antibodies to P. aeruginosa alginate. The assays showed high predictive values if positive, but a negative test did not exclude infection, and the ELISAs should not be used for diagnostic purposes. Mucoid strains were present initially in the sputa of 28 of 54 infected patients with paired serum samples. These patients had a significant increase in anti-alginate antibodies, but it was not different from the increase seen in patients infected only with nonmucoid strains. Therefore, alginate may also be produced in vivo by nonmucoid P. aeruginosa. The study showed that early formation of IgA and IgG antibodies to P. aeruginosa alginate did not prevent development of chronic infection and that P. aeruginosa-specific IgA antibodies correlate with poor lung function.
PMCID: PMC267788  PMID: 2110181
9.  Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis 
Journal of Bacteriology  2002;184(4):952-961.
Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.
PMCID: PMC134824  PMID: 11807055
10.  Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean▿  
Applied and Environmental Microbiology  2008;74(20):6194-6205.
Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.
PMCID: PMC2570286  PMID: 18757570
11.  Binding of nonmucoid Pseudomonas aeruginosa to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis. 
Journal of Clinical Investigation  1992;89(2):657-665.
Lung infections due to Pseudomonas aeruginosa and Pseudomonas cepacia are common in patients with cystic fibrosis. Initial colonization is due to nonmucoid P. aeruginosa, while later mucoid variants emerge and are associated with chronic infection. P. cepacia colonization tends to be more prevalent in older patients. The present study was conducted to discover whether highly purified mucins (from cystic fibrosis sputum and control intestinal secretions) exhibited specific binding of nonmucoid P. aeruginosa. In vitro solid phase microtiter binding assays (with or without a blocking agent) as well as solution phase assays were conducted. Bacteria bound to both mucins via bacterial pili, but no differences in binding capacity were noted between the mucins. Unlike P. cepacia (described in the accompanying manuscript) there was also no preferential binding of P. aeruginosa to mucins versus bovine serum albumin, casein, gelatin, or a host of structurally unrelated proteins and glycoproteins. Carbohydrate hapten inhibition studies did not suggest the existence of specific mucin carbohydrate receptors for P. aeruginosa. In solid phase assays a low concentration (0.05 M) of tetramethylurea abolished P. aeruginosa bacterial binding to both mucins as well as to BSA, whereas in solution phase assays mucin binding to bacteria was not completely disrupted by tetramethylurea. Specific monoclonal antipilus antibodies did not inhibit binding to a greater extent than did Fab fragments of normal mouse IgG. Binding of strains PAO1 and PAK (and isolated PAK pili) to buccal epithelial cells was not influenced by the presence of mucin in binding assay mixtures. Our findings do not support the widely held notion that specific mucin receptors are responsible for the attachment of P. aeruginosa pili, nor do they support the idea that there is a competitive interference by mucins of bacterial binding to respiratory cells. In patients with cystic fibrosis, it would seem unlikely therefore that initial colonization of the lungs by P. aeruginosa is due to a 'selective tropism' of these bacteria for respiratory mucin.
PMCID: PMC442899  PMID: 1737853
12.  Extensive Genomic Plasticity in Pseudomonas aeruginosa Revealed by Identification and Distribution Studies of Novel Genes among Clinical Isolates  
Infection and Immunity  2006;74(9):5272-5283.
The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, ∼1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.
PMCID: PMC1594838  PMID: 16926421
13.  Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR 
Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR) assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs).
Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification).
Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%.
This reliable technique may offer a rapid (<1.5 h) tool that would help clinicians to initiate an appropriate treatment earlier. Further investigations are needed to assess the clinical benefit of this novel strategy as compared to phenotypic methods.
PMCID: PMC2928764  PMID: 20684778
14.  Pseudomonas aeruginosa: Immune Status in Patients with Cystic Fibrosis 
Infection and Immunity  1972;6(4):628-635.
In order to have a better understanding of the clinical significance of Pseudomonas aeruginosa, circulating and secretory antibodies were measured. Of 100 patients diagnosed as having cystic fibrosis (CF) and an atypical mucoid P. aeruginosa cultured from their sputum, each possessed serum precipitins. These immunoprecipitates, however, were not detected in the sera of 40 CF patients, some of whom were chronically ill with pulmonary colonization by typically rough-smooth strains of P. aeruginosa. The sera of 46 CF patients and 27 CF patient parents not colonized by P. aeruginosa were negative for the precipitins. The sera from 15 of 45 chronically ill patients not having CF, however, but harboring P. aeruginosa, also possessed serum precipitins. The sera from 85 subjects not having CF and not clinically infected with P. aeruginosa were negative for precipitins. Serum hemagglutination titers as high as 1:4096 were measured in older CF patients having advanced pulmonary disease and who were infected with mucoid P. aeruginosa. Salivary titers ranged from 1:8 to 1:64. Increased levels of both circulating and secretory antibodies of the immunoglobulin A and G classes were demonstrated in patients with CF. Once a patient with CF becomes colonized with P. aeruginosa a process of conversion from the rough and smooth forms to the mucoid form is almost inevitable. Although the mucoid form predominates in the sputum, intermediates of the various colony types are often present. Serum precipitins were demonstrable only after the appearance of mucoid strains in the sputum of patients with CF. Although antibiotics tend to reduce the number of mucoid microorganisms, they are rarely, if ever, eradicated from these patients' lungs. Recurrent episodes of servere pulmonary infection and the evidence of increasing antibody formation to mucoid strains indicates the invasiveness of these particular strains.
PMCID: PMC422584  PMID: 4628901
15.  Identification of infectious Pseudomonas aeruginosa strains in an occupational saturation diving environment 
OBJECTIVES: Occupational saturation divers have various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated from skin infections in divers. The purpose of the present work was (a) to report the occurrence of P aeruginosa in skin infections in operational saturation diving in the North Sea from 1987 to 1995; (b) to report the environmental occurrence of P aeruginosa in saturation diving systems, and finally (c) to identify possible relations between infection related to strains of P aeruginosa and environmental isolates of the microbe. RESULTS: During the period 1987-95, P aeruginosa was isolated from 257 skin infections in operational saturation divers. Most of the isolates related to infection by P aeruginosa show a unique growth inhibition pattern towards the normal skin flora, and the serotype pattern of P aeruginosa from skin infections is limited compared with similar infections in non-divers. In a mini-epidemiological study on board one diving vessel during one operational diving period, five significantly different DNA fragment profiles were found among the 12 isolates related to infection by P aeruginosa obtained from the saturation system. In two cases the infectious genotypes were detected in the fresh water for the saturation chambers weeks before the arrival of the infected diver. CONCLUSIONS: The most commonly used epidemiological marker for P aeruginosa world wide, also used in earlier studies, is serotyping, but with pulsed field gel electrophoresis (PFGE) miniepidemiology it was shown to be insufficient for epidemiological purposes in saturation environments. PFGE analyses were shown to be superior both to antibacterial factor and to serotyping in epidemiological analyses of P aeruginosa infections in saturation diving.
PMCID: PMC1757612  PMID: 9816382
16.  Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains 
BMC Microbiology  2008;8:109.
Pseudomonas aeruginosa is one of the most relevant human opportunistic bacterial pathogens. Two strains (PAO1 and PA14) have been mainly used as models for studying virulence of P. aeruginosa. The strain PA14 is more virulent than PAO1 in a wide range of hosts including insects, nematodes and plants. Whereas some of the differences might be attributable to concerted action of determinants encoded in pathogenicity islands present in the genome of PA14, a global analysis of the differential host responses to these P. aeruginosa strains has not been addressed. Little is known about the host response to infection with P. aeruginosa and whether or not the global host transcription is being affected as a defense mechanism or altered in the benefit of the pathogen. Since the social amoeba Dictyostelium discoideum is a suitable host to study virulence of P. aeruginosa and other pathogens, we used available genomic tools in this model system to study the transcriptional host response to P. aeruginosa infection.
We have compared the virulence of the P. aeruginosa PAO1 and PA14 using D. discoideum and studied the transcriptional response of the amoeba upon infection. Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays. For studying the differential response of the host to infection by these model strains, D. discoideum cells were exposed to either P. aeruginosa PAO1 or P. aeruginosa PA14 (mixed with an excess of the non-pathogenic bacterium Klebsiella aerogenes as food supply) and after 4 hours, cellular RNA extracted. A three-way comparison was made using whole-genome D. discoideum microarrays between RNA samples from cells treated with the two different strains and control cells exposed only to K. aerogenes. The transcriptomic analyses have shown the existence of common and specific responses to infection. The expression of 364 genes changed in a similar way upon infection with one or another strain, whereas 169 genes were differentially regulated depending on whether the infecting strain was either P. aeruginosa PAO1 or PA14. Effects on metabolism, signalling, stress response and cell cycle can be inferred from the genes affected.
Our results show that pathogenic Pseudomonas strains invoke both a common transcriptional response from Dictyostelium and a strain specific one, indicating that the infective process of bacterial pathogens can be strain-specific and is more complex than previously thought.
PMCID: PMC2474670  PMID: 18590548
17.  Molecular studies of Pseudomonas exotoxin A gene. 
Infection and Immunity  1986;52(2):538-548.
A 2.7-kilobase DNA fragment carrying the entire exotoxin A (ETA) structural gene was divided into three nonoverlapping probes. Two probes covering the ETA structural gene were used in colony hybridization experiments to determine whether sequences homologous to the ETA gene could be detected in genera other than Pseudomonas or in Pseudomonas species other than Pseudomonas aeruginosa. The majority of strains examined other than the P. aeruginosa strains failed to react in the colony hybridization assays. Some Pseudomonas spp. other than P. aeruginosa and some Bordetella spp. did react in colony hybridization assays with the probes. However, additional studies in which we used Southern hybridization methods indicated that these reactions were apparently nonspecific and that the ETA gene is limited to P. aeruginosa. Studies in which we used all three ETA-related probes in Southern hybridization experiments to analyze the ETA gene and surrounding sequences in P. aeruginosa strains isolated from diverse sources revealed the following: (i) the incidence of the ETA gene in P. aeruginosa is approximately equal to 95%; (ii) there are strains which have been isolated from human infections that do not carry the ETA structural gene; (iii) there is a maximum of one copy of the ETA gene per genome in any given strain; (iv) sequences within and 4 to 5 kilobases downstream of the ETA structural gene appear to be well conserved in different strains of P. aeruginosa; and (v) in contrast, sequences immediately upstream of the ETA structural gene are considerably rearranged from strain to strain. A multicopy plasmid carrying the entire cloned ETA gene was transferred to a tox- P. aeruginosa strain. This strain synthesized and secreted mature, full-length ETA, but the amount produced was small considering the multicopy nature of the plasmid. Synthesis of toxin in this strain was only minimally affected by iron. Our data suggest that the synthesis of ETA is positively regulated. Finally, we found that the presence of the ETA gene is independent of the ability of P. aeruginosa to produce several other recognized virulence factors, supporting the concept of the multifactorial nature of P. aeruginosa virulence.
PMCID: PMC261034  PMID: 3009329
18.  Identification of a Genomic Island Present in the Majority of Pathogenic Isolates of Pseudomonas aeruginosa 
Journal of Bacteriology  2001;183(3):843-853.
Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for “P. aeruginosa genomic island 1”). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.
PMCID: PMC94950  PMID: 11208781
19.  Screening and Rapid Molecular Diagnosis of Tuberculosis in Prisons in Russia and Eastern Europe: A Cost-Effectiveness Analysis 
PLoS Medicine  2012;9(11):e1001348.
Daniel Winetsky and colleagues investigate eight strategies for screening and diagnosis of tuberculosis within prisons of the former Soviet Union.
Prisons of the former Soviet Union (FSU) have high rates of multidrug-resistant tuberculosis (MDR-TB) and are thought to drive general population tuberculosis (TB) epidemics. Effective prison case detection, though employing more expensive technologies, may reduce long-term treatment costs and slow MDR-TB transmission.
Methods and Findings
We developed a dynamic transmission model of TB and drug resistance matched to the epidemiology and costs in FSU prisons. We evaluated eight strategies for TB screening and diagnosis involving, alone or in combination, self-referral, symptom screening, mass miniature radiography (MMR), and sputum PCR with probes for rifampin resistance (Xpert MTB/RIF). Over a 10-y horizon, we projected costs, quality-adjusted life years (QALYs), and TB and MDR-TB prevalence. Using sputum PCR as an annual primary screening tool among the general prison population most effectively reduced overall TB prevalence (from 2.78% to 2.31%) and MDR-TB prevalence (from 0.74% to 0.63%), and cost US$543/QALY for additional QALYs gained compared to MMR screening with sputum PCR reserved for rapid detection of MDR-TB. Adding sputum PCR to the currently used strategy of annual MMR screening was cost-saving over 10 y compared to MMR screening alone, but produced only a modest reduction in MDR-TB prevalence (from 0.74% to 0.69%) and had minimal effect on overall TB prevalence (from 2.78% to 2.74%). Strategies based on symptom screening alone were less effective and more expensive than MMR-based strategies. Study limitations included scarce primary TB time-series data in FSU prisons and uncertainties regarding screening test characteristics.
In prisons of the FSU, annual screening of the general inmate population with sputum PCR most effectively reduces TB and MDR-TB prevalence, doing so cost-effectively. If this approach is not feasible, the current strategy of annual MMR is both more effective and less expensive than strategies using self-referral or symptom screening alone, and the addition of sputum PCR for rapid MDR-TB detection may be cost-saving over time.
Please see later in the article for the Editors' Summary
Editors' Summary
Tuberculosis (TB)—a contagious bacterial disease—is a major public health problem, particularly in low- and middle-income countries. In 2010, about nine million people developed TB, and about 1.5 million people died from the disease. Mycobacterium tuberculosis, the bacterium that causes TB, is spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of TB include fever, a persistent cough, and night sweats. Diagnostic tests include sputum smear microscopy (examination of mucus from the lungs for M. tuberculosis bacilli), mycobacterial culture (growth of M. tuberculosis from sputum), and chest X-rays. TB can also be diagnosed by looking for fragments of the M. tuberculosis genetic blueprint in sputum samples (sputum PCR). Importantly, sputum PCR can detect the genetic changes that make M. tuberculosis resistant to rifampicin, a constituent of the cocktail of antibiotics that is used to cure TB. Rifampicin resistance is an indicator of multidrug-resistant TB (MDR-TB), the emergence of which is thwarting ongoing global efforts to control TB.
Why Was This Study Done?
Prisons present unique challenges for TB control. Overcrowding, poor ventilation, and inadequate medical care increase the spread of TB among prisoners, who often come from disadvantaged populations where the prevalence of TB (the proportion of the population with TB) is already high. Prisons also act as reservoirs for TB, recycling the disease back into the civilian population. The prisons of the former Soviet Union, for example, which have extremely high rates of MDR-TB, are thought to drive TB epidemics in the general population. Because effective identification of active TB among prison inmates has the potential to improve TB control outside prisons, the World Health Organization recommends active TB case finding among prisoners using self-referral, screening with symptom questionnaires, or screening with chest X-rays or mass miniature radiography (MMR). But which of these strategies will reduce the prevalence of TB in prisons most effectively, and which is most cost-effective? Here, the researchers evaluate the relative effectiveness and cost-effectiveness of alternative strategies for screening and diagnosis of TB in prisons by modeling TB and MDR-TB epidemics in prisons of the former Soviet Union.
What Did the Researchers Do and Find?
The researchers used a dynamic transmission model of TB that simulates the movement of individuals in prisons in the former Soviet Union through different stages of TB infection to estimate the costs, quality-adjusted life years (QALYs; a measure of disease burden that includes both the quantity and quality of life) saved, and TB and MDR-TB prevalence for eight TB screening/diagnostic strategies over a ten-year period. Compared to annual MMR alone (the current strategy), annual screening with sputum PCR produced the greatest reduction in the prevalence of TB and of MDR-TB among the prison population. Adding sputum PCR for detection of MDR-TB to annual MMR screening did not affect the overall TB prevalence but slightly reduced the MDR-TB prevalence and saved nearly US$2,000 over ten years per model prison of 1,000 inmates, compared to MMR screening alone. Annual sputum PCR was the most cost-effective strategy, costing US$543/QALY for additional QALYs gained compared to MMR screening plus sputum PCR for MDR-TB detection. Other strategies tested, including symptom screening alone or combined with sputum PCR, were either more expensive and less effective or less cost-effective than these two options.
What Do These Findings Mean?
These findings suggest that, in prisons in the former Soviet Union, annual screening with sputum PCR will most effectively reduce TB and MDR-TB prevalence and will be cost-effective. That is, the cost per QALY saved of this strategy is less than the per-capita gross domestic product of any of the former Soviet Union countries. The paucity of primary data on some facets of TB epidemiology in prisons in the former Soviet Union and the assumptions built into the mathematical model limit the accuracy of these findings. Moreover, because most of the benefits of sputum PCR screening come from treating the MDR-TB cases that are detected using this screening approach, these findings cannot be generalized to prison settings without a functioning MDR-TB treatment program or with a very low MDR-TB prevalence. Despite these and other limitations, these findings provide valuable information about the screening strategies that are most likely to interrupt the TB cycle in prisons, thereby saving resources and averting preventable deaths both inside and outside prisons.
Additional Information
Please access these websites via the online version of this summary at
The World Health Organization provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and on tuberculosis in prisons; a report published in the Bulletin of the World Health Organization in 2006 describes tough measures taken in Russian prisons to slow the spread of TB
The Stop TB Partnership is working towards tuberculosis elimination; patient stories about tuberculosis are available (in English and Spanish)
The US Centers for Disease Control and Prevention has information about tuberculosis, about its diagnosis, and about tuberculosis in prisons (some information in English and Spanish)
A PLOS Medicine Research Article by Iacapo Baussano et al. describes a systematic review of tuberculosis incidence in prisons; a linked editorial entitled The Health Crisis of Tuberculosis in Prisons Extends beyond the Prison Walls is also available
The Tuberculosis Survival Project, which aims to raise awareness of tuberculosis and provide support for people with tuberculosis, provides personal stories about treatment for tuberculosis; the Tuberculosis Vaccine Initiative also provides personal stories about dealing with tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
PMCID: PMC3507963  PMID: 23209384
20.  Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL. 
Journal of Clinical Microbiology  1997;35(6):1295-1299.
A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.
PMCID: PMC229737  PMID: 9163432
21.  Cross-Sectional Analysis of Clinical and Environmental Isolates of Pseudomonas aeruginosa: Biofilm Formation, Virulence, and Genome Diversity  
Infection and Immunity  2004;72(1):133-144.
Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.
PMCID: PMC343948  PMID: 14688090
22.  Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction 
Critical Care  2000;4(4):255-262.
We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.
Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler™ technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples.
Materials and methods:
Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods.
Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods.
Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C.
The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl2, diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp® PCR System 2400. An aliquot of the reaction mixture was put on an agarose gel for electrophoresis and visualisation of the PCR product. An image of the gel was made using a digital camera. Image analysis software was used to calculate the band mass of the experimental bands.
An aliquot of the digoxigenin labelling reaction was denatured and then hybridized with the biotinylated capture probe PrL. Some of the resultant solution was transferred to a well of a streptavidin-coated microtitre plate (MTP) and incubated for 3 h at 45°C. The solution was discarded. Peroxidase conjugated antidigoxigenin was added and the MTP was incubated for 30 min at 37°C. The solution was discarded and ABTS substrate was added. The MTP was incubated for 30 min at 37°C. Absorbance was read at 405 nm.
The RTD-PCR mixture contained PCR grade sterile water, diluted template DNA, primers PAL1 and PAL2, 3' fluorescein (FL)-labelled probe oprL-FL, 5' LC Red 640-labelled and 3' phosphorylated probe oprL-LC, MgCl2, and LC DNA Master Hybridisation Probes, containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and MgCl2. The amplification was performed in a LightCycler™. The fluorescence signal of LC Red 640 was measured during the annealing phase. The measured fluorescence data was processed with analysis software.
Results and discussion:
The three methods showed a good concordance with the culture results. Conventional PCR was at least 100 times less sensitive than bacterial culture and had a low dynamic range (2 logs). With a lower detection limit of 103 CFU/g tissue, ELISA-PCR was ten times more sensitive than conventional PCR. The dynamic range, however, did not increase. ELISA-PCR is very time consuming (8 h). The RTD-PCR produced a linear quantitative detection range of 7 logs with a lower detection limit of 103 CFU/g tissue. More important, however, was that the time from sample collection to result was less than 1 h. Two biopsy specimens scored significantly higher in ELISA-PCR and RTD-PCR than in bacterial culture. This could indicate that DNA from dead bacteria was amplified. One out of ten culture positive biopsy samples was found negative by all PCR-based methods. Topical antimicrobial agents possibly inhibited PCR. These results show that RTD-PCR has potential for the rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Further study is required to assess the reliability of this new technology, and its impact on patient outcome and hospital costs.
PMCID: PMC29046  PMID: 11056755
burn wound; polymerase chain reaction; Pseudomonas aeruginosa; quantitation; sepsis
23.  Cystic Fibrosis-Niche Adaptation of Pseudomonas aeruginosa Reduces Virulence in Multiple Infection Hosts 
PLoS ONE  2012;7(4):e35648.
The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.
PMCID: PMC3338451  PMID: 22558188
24.  Occurrence of a Multidrug-Resistant Pseudomonas aeruginosa Clone in Different Hospitals in Rio de Janeiro, Brazil 
Journal of Clinical Microbiology  2002;40(7):2420-2424.
Multidrug-resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. The existence of metallo-β-lactamase- and extended-spectrum β-lactamase-producing isolates exhibiting resistance to most β-lactam antimicrobial agents greatly complicates the clinical management of patients infected with such isolates. Since 1998, P. aeruginosa isolates resistant to all commercially available antimicrobial agents have been detected at a university-affiliated public hospital in Rio de Janeiro, Brazil. The present study was designed to characterize the antimicrobial resistance profiles and the genetic diversity of the P. aeruginosa strains isolated at this hospital and four private hospitals in Rio de Janeiro. Between April 1999 and March 2000, 200 consecutive isolates were obtained and analyzed for antimicrobial resistance. The genetic diversity of a selected number of them was evaluated by pulsed-field gel electrophoresis and PCR with the ERIC-2 primer. A predominant genotype, designated genotype A, was identified among isolates from four of the five hospitals evaluated. Eighty-four ceftazidime-resistant isolates were evaluated for metallo-β-lactamase production, which was detected in 20 (91%) of 22 genotype A isolates and 11 (18%) of 62 isolates belonging to other genotypes (P < 0.05). Two metallo-β-lactamase-producing genotype A isolates also produced an extended-spectrum β-lactamase. The occurrence of multidrug-resistant P. aeruginosa strains belonging to a unique genotype in different hospitals in Rio de Janeiro underscores the importance of the contribution of a single clone to the increase in the incidence of multidrug-resistant P. aeruginosa nosocomial infections.
PMCID: PMC120547  PMID: 12089256
25.  In vitro inhibition of lymphocyte proliferation by Pseudomonas aeruginosa phenazine pigments. 
Infection and Immunity  1983;41(1):321-330.
Human lymphocyte proliferation is inhibited in vitro in the presence of killed Pseudomonas aeruginosa or cell-free P. aeruginosa culture supernatants. A comparison of culture supernatants obtained under similar conditions from Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Pseudomonas cepacia strains demonstrated that all P. aeruginosa supernatants were strongly inhibitory, whereas supernatants from other bacteria were mildly inhibitory or not inhibitory at all. These P. aeruginosa inhibitors prevent proliferative responses of resting cells upon mitogen activation and decrease [3H]thymidine uptake when added to human lymphocytes undergoing active proliferation in culture. The inhibitory effect is reversible and not due to cytotoxicity. Most of the inhibitory activity present in crude supernatants was detected in ultrafiltrates of molecular weights below 2,000. Purified P. aeruginosa pyocyanine, a low-molecular-weight phenazine pigment present in culture supernatant, was strongly inhibitory for lymphocyte proliferation. Extraction of pyocyanine and phenazine pigments from inhibitory P. aeruginosa supernatants eliminated their inhibitory activity. Inhibitors were recovered from reverse-phase chromatographic cartridges by both chloroform and methanol elution, indicating that pyocyanine and other phenazine pigments present in P. aeruginosa supernatants are responsible for the inhibition of lymphocyte proliferation. In addition to the identification of phenazine pigments as lymphocyte proliferation inhibitors, several criteria ruled out major contributions of P. aeruginosa polysaccharide, exotoxin A, and proteases to this phenomenon. P. aeruginosa strains selected for very low protease production or for very low exotoxin A production produced supernatants as inhibitory for lymphocyte proliferation as supernatants obtained from clinical P. aeruginosa isolates. Purified P. aeruginosa lipopolysaccharide and protease preparations failed to induce reversible lymphocyte proliferation inhibition. Finally, heat inactivation of P. aeruginosa supernatants at 100 degrees C for 60 min inactivates exotoxin A and proteases but produced only a moderate decrease of the inhibitory activity for lymphocyte proliferation.
PMCID: PMC264781  PMID: 6408002

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