Pseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum.Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus.Adult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media , we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months.By sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The oprL gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.This study indicates that molecular detection of PA in sputum employing the  Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. However, with modern antibiotic management with improved antimicrobial agents, such as the aminoglycosides and carbapenems, CF patients have an improved survival, resulting in more adults in employment.Cystic fibrosis [CF] is the most commonly inherited fatal disease in persons originating from a white and European background, currently affecting approximately 30,000 adults and children in the US . The defPseudomonas aeruginosa in CF patients.[P. aeruginosa,[P. aeruginosa in CF patients [P. aeruginosa directly from the sputum of CF patients in the UK and Ireland is uncommon. Most clinical microbiology laboratories in the UK and Ireland, which support a CF centre, have developed standard operating procedures (SOPs) for the isolation of this organism from patients' sputum. Hence, it was the aim of this study to compare the conventional and molecular detection of P. aeruginosa from the sputum of adult patients attending the adult centre in Northern Ireland, as well as to estimate the lag time of conventional culture to detection, compared with molecular (PCR) detection.Previous studies have described alternative laboratory markers to conventional bacteriological culture for the detection of patients.,5 These ruginosa, as well Pseudomonas aeruginosa. Processed sputa (10 μ l) were inoculated and incubated, onto several selective media for the isolation of Pseudomonas aeruginosa, including:- Columbia Blood Agar (Oxoid CM0331) supplemented with 5% (v/v) defribinated horse blood, MacConkey Agar (Oxoid CM0007) and Pseudomonas Isolation Agar (PIA) (Oxoid CM0559 + SR0102). All media were incubated aerobically at 37°C for 48 h, unless otherwise stated. The PIA plates were incubated at room temperature for a further three days following initial 48 hrs incubation. In addition, all different phenotypes from the sputum of each patient were identified phenotypically employing a combination of conventional identification methods (e.g. oxidase), as well as the API Identification schemes  .Duplicate sputa (1 ml minimum) specimens were collected from 57 adult patients with a well characterized history of CF in sterile (100 ml) plastic disposable containers. Sputum was collected immediately after a standardized session of physiotherapy and was stored at ambient temperature and was processed within 4 h from collection. Fresh sputum (1 ml min) was mixed with an equal mass (1:1) of Sputasol  and was incubated in a water bath at 37°C for 15 min, before further qualitative processing for the detection of et al[Pseudomonas aeruginosa  ATCC 27853, by employment of the Roche High Purity PCR Template Preparation Kit , in accordance with the manufacturer's instructions. Extracted DNA was stored at -80°C prior to PCR amplification. For each batch of extractions, a negative extraction control containing all reagents minus sputum, was performed, as well as an extraction positive control with P. aeruginosa.All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet  in a room physically separated from that used to set up nucleic acid amplification reaction mixes and also from the "post-PCR" room in accordance with the Good Molecular Diagnostic Procedures (GMDP) guidelines of Millar et al, in orde2, 200 μM (each) dATP, dCTP, dGTP and dTTP; 1.25 U of Taq DNA polymerase , 0.1 μM (each) of the each set of primers (exoA &oprL) (Table P. aeruginosa ATCC 27853 DNA) and multiple negative (water) amplification controls were included in every set of PCR reactions. In addition, a broad-range/universal PCR was employed with each sputum to demonstrate successful extraction of bacterial DNA from the specimen, as well as lack of PCR inhibition, by employing the highly conserved 16S rDNA primers, PSL/PSR  were set up as follows:-10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl00 μl werexoA or oprL loci, the specimen was considered positive for P. aeruginosa.Following amplification, aliquots (10 μl) were removed from each reaction mixture and examined by electrophoresis  in gels composed of 2% (w/v) agarose  in TAE buffer , stained with ethidium bromide (5 μg/100 ml). Gels were visualised under UV illumination using a gel image analysis system  and all images archived as digital graphic files (*.bmp). Where a band was visualized at the correct expected size for either P. aeruginosa in patients' sputum by conventional and molecular techniques is shown , there was complete agreement between molecular and conventional detection techniques. The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative, However, there were five patients who were culture positive but PCR -ve by both gene targets. Given that the universal 16S rDNA PCR was positive for these sputum specimens and that the appropriate molecular controls were working optimally, this may accounted for by potential phenotypic misidentification of P. aeruginosa, which has been recently described.[P. aeruginosa colonization with a false-negative culture result due to sample overgrowth by other bacteria or to the presence of non-cultivable organisms or auxotrophic mutations in the organism. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that five of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months, whereas the remaining five patients remained negative for P. aeruginosa.A comparison of the occurrence of wn Table . By cultP. aeruginosa directly from the sputum of patients with CF. Two gene loci were targeted, as specific markers of P. aeruginosa, namely the exotoxin A (ETA) gene and the outer membrane lipoprotein (oprL) gene. ETA is produced by the majority of P. aeruginosa strains and can inhibit eucaryotic protein biosynthesis at the level of polypeptide chain elongation factor 2, similarly to diphtheria toxin.[In this study, two PCR assays were performed individually for the molecular detection of ia toxin. OprL is ia toxin.Pseudomonas aeruginosa is the most important bacterial pathogen in patients with CF [Pseudomonas colonisation of the major airways leading to delibating exacerbations of pulmonary infection, is the major cause of morbidity and mortality in patients with CF, hence it is important to be able to reliably detect P. aeruginosa from patients' sputum. Emerson et al. [P. aeruginosa was a major predictor of morbidity and mortality, whereby the 8-year risk of death parameter was 2.6 times higher in patients who had positive sputum cultures for this organism, as well as having a significantly lower percent predicted forced expiratory volume (FEV1). These workers suggested that early interventions may help decrease associated morbidity and mortality of young patients with CF. with CF , as demon et al.  recentlyet al. [P. aeruginosa infection in CF. These workers suggested that chronic lower airway infection with P. aeruginosa is associated with significant morbidity and mortality among CF patients [P. aeruginosa does not appear to cause an immediate and rapid decline in lung function, as early isolates are generally non-mucoid, antibiotic-sensitive and present at low densities, suggesting a possible "window of opportunity" for early intervention, as they describe [More recently, Rosenfeld et al.  describepatients . Howeverdescribe . This stdescribe Pseudomonas colonization as early as possible, so that (i) aggressive antibiotic regimes may be considered,(ii). the patient is managed optimally, in an attempt to avoid early biofilm formation and chronic colonization with Pseudomonas and (iii) appropriate infection control precautions are considered. More recently, West et al. showed by a combination of serum IgG, IgA and IgM anti P. aeruginosa antibodies, in conjunction with these authors' Wisconsis Cystic Fibrosis Radiograph score, that P. aeruginosa infections occurred approximately 6–12 months before the organism was recovered from respiratory secretions.[P. aeruginosa acquisition.It is therefore important that primary diagnostic bacteriology laboratories have the ability to detect transient and early cretions. In additPseudomonas aeruginosa, either from culture plates or patients' sputum, small numbers of Pseudomonas colonies (n = 1–2) may therefore be missed when present in the early stages of colonization preceding infection of the patient's airways, particularly where such single colonies are mixed alongside other phenotypically similar genera on the primary culture plate.[Pseudomonas colonization. Although such assays are not generally available in most clinical diagnostic laboratories, these laboratories generally do have access to such technology at regional specialist microbiology centres and it may therefore be prudent to establish routine analysis of children's sputum, particularly from patients with no or an intermittent history by culture of Pseudomonas colonization, at annual review. Furthermore, it is very important to follow up any such patients, which demonstrate a PCR +ve/culture -ve finding from their sputum, in order to establish whether there is transient infection in patients, which never result in established colonization leading to chronic infection. Additionally, it is important to monitor such PCR +ve/culture -ve patients in terms of optimal antibiotic management and infection control.Given that not all laboratories employ molecular detection methods for re plate. PragmatiP. aeruginosa were able to detect P. aeruginosa at an early stage, on average 4.5 months sooner than culture detection. However, it remains unclear what this "window of opportunity" potentially offers in terms of a decrease in P. aeruginosa-associated morbidity and mortality.Overall, our study demonstrated that molecular assays for the detection of JEM, PGM and JSE jointly conceived the study and designed the experiments. JX executed and analyzed all practical aspects of the study, as well as analyzing the data. JEM executed certain molecular components of experimentation, analyzed the data and prepared the manuscript. BCM helped guide the molecular aspects of experimentation and was involved in drafting of the manuscript. PGM provided expert microbiological analysis and interpretation of data. JSE provided clinical expertise, interacted with the CF patients and was involved in interpretation of data, as well as critically reviewing the final manuscript. All authors read and approved the final manuscript.


Many studies associated nosocomial infections with increased hospital costs due to extra days in hospital, staff time, extra investigations and drug treatment. The cost of antibiotic treatment for these infections represents a significant part of hospital expenditure. This prospective observational study was designed to determine the daily antibiotic cost of nosocomial infections per infected adult patient in Akdeniz University Hospital.All adult patients admitted to the ICUs between January 1, 2000, and June 30, 2003 who had only one nosocomial infection during their stay were included in the study. Infection sites and pathogens, antimicrobial treatment of patient and it's cost were recorded. Daily antibiotic costs were calculated per infected patient.Pseudomonas aeruginosa infections was the most expensive infection treated. Piperacillin-tazobactam and amikacin were the most prescribed antibiotics, and meropenem was the most expensive drug for treatment of the nosocomial infections in the ICU.Among the 8460 study patients, 817 (16.6%) developed 1407 episodes of nosocomial infection. Two hundred thirty three (2.7%) presented with only one nosocomial infection. Mean daily antibiotic cost was $89.64. Daily antibiotic cost was $99.02 for pneumonia, $94.32 for bloodstream infection, $94.31 for surgical site infection, $52.37 for urinary tract infection, and $162.35 for the other infections per patient. The treatment of Daily antibiotic cost of nosocomial infections is an important part of extra costs that should be reduced providing rational antibiotic usage in hospitals. Nosocomial infections are frequent complications of hospitalization and also an important public health problem in developing countries, as well as in developed ones. The socioeconomic impact, ie, prolongation of hospitalization, mortality, and cost of these infections adversely effects patients and nations' economic well-being . The cosThe aim of our study was to determine daily antibiotic cost of nosocomial infection per infected patient in a university hospital.Akdeniz University Hospital is a 600-bed tertiary referral centre in Antalya, Turkey, treating 27 000 patients per year. The study was conducted in six adult medical and surgical intensive care units (ICUs) with a total of 51 ICU beds. Neonatal ICU was not included in the study. Since 1993, the institutional policies of hospital infection control have been implemented by infection control team.In our hospital, routine prospective, active surveillance of nosocomial infections in all ICUs is performed by one infection control nurse, supervised by an infection control physician. Nosocomial infections are defined using the Centers for Disease Control and Prevention criteria ,6. We doBetween January 1, 2000 and June 30, 2003, all inpatients hospitalized in one of the adult ICUs were included in this study. Data on antimicrobial treatment were recorded for patients aged 15 or above presenting with only one nosocomial infection. For all patients included in the study, the following were recorded: age, sex, infection site, microbiologic data, antimicrobial therapy and antibiotic cost.In our ICUs, all antimicrobial prescriptions are recommended by an infectious disease consultant. Antimicrobial agents prescribed only for therapeutic indications were recorded. The daily antibiotic cost was calculated in US dollars based on June 2003 prices of antimicrobial agents provided by the hospital pharmacy. The daily antibiotic cost per infected patient was calculated by the multiplication of box price and number of daily doses that was used for that infection. Two costs were calculated for each antimicrobial; the minimal cost (min) was based on the lowest recommended parenteral daily dose and the maximal cost (max) was based on the highest recommended parenteral daily dose.Between January 1, 2000, and June 30, 2003, a total of 8460 patients were admitted to the adult ICUs. Overall, 817 patients developed 1407 episodes of nosocomial infections, accounting for an infection rate of 16,6%. Among them, 233 patients  had only one nosocomial infection. Mean daily antibiotic cost was found $89,64 per infected patient .Among the sites of nosocomial infections, urinary tract infections had the lowest daily antibiotic cost per infected patient Table . The meaPseudomonas aeruginosa was the most prevalent bacteria followed by Klebsiella spp. and Acinetobacter spp.. P. aeruginosa infections had the highest overall daily antibiotic cost per infected patient than other pathogens. Among 21 Staphylococcus aureus strains 15 (72%) were resistant to methicillin. The overall daily antibiotic cost of methicillin resistant S. aureus (MRSA) infections was two to three times higher than infections with susceptible strains. However, median daily cost per infected patient for MRSA infections were lower than susceptible strain infections.Out of 233 patients, 206 patients had microbiologically documented infections, 177 patients were infected by a single pathogen while 29 patients were diagnosed as having polymicrobial infections  declare that they have no competing interests.DI collated and analyzed the data, participated in the study design and was principal writer of manuscript. ANY conceived the study. GO carried out the laboratory studies. RS, FG, OT and LM participated in the patient management. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Pseudomonas aeruginosa frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules  3-oxo-C12-HSL and C4-HSL, regulates the production by P. aeruginosa of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear.P. aeruginosa isolates growing inside the biofilm covering the intubation devices and those resident in the lungs of colonized patients differ in their QS-dependent phenotypes. We collected the intubation devices of eight patients colonized by P. aeruginosa. We detected 3-oxo-C12-HSL on eight, and C4-HSL on six of these devices. In three of these patients we also obtained P. aeruginosa isolates from tracheal aspirates at the time of extubation (n = 18), as well as isolates from the intubation devices (n = 25). We genotyped these isolates, quantified their AIs production, and determined three QS-dependent phenotypes . The production of 3-oxo-C12-HSL was consistently increased for isolates from the intubation devices, whereas the production of C4-HSL was significantly higher for isolates from tracheal aspirates. Isolates from tracheal aspirates produced significantly higher amounts of elastase but less biofilm, and had a marginally reduced adhesion capacity than isolates from the intubation devices. Levels of 3-oxo-C12-HSL and elastase production correlated statistically for tracheal intubation isolates, whereas levels of 3-oxo-C12-HSL production and adhesion ability, as well as biofilm production, correlated weakly amongst intubation device isolates.We wondered whether QS is active during colonization of intubated patients, and whether P. aeruginosa. The microenvironment, in which P. aeruginosa grows, may select for bacteria with different capacities to produce autoinducers and certain QS-dependent phenotypes. QS-inhibition might therefore affect differently isolates growing inside the biofilm covering intubation devices and those resident in the lungs.Our findings demonstrate that autoinducers are produced during the colonization of intubated patients by  Pseudomonas aeruginosa is an opportunistic pathogen implicated in a wide variety of infections, particularly in burn victims, cancer patients and cystic fibrosis (CF) patients. In addition P. aeruginosa is a major cause of severe pneumonia in mechanically ventilated patients . factors ,6 but ha factors -10.P. aeruginosa has been clearly established in vivo in animal models [P. aeruginosa infections.The importance of a functional QS-circuit for the virulence of l models . As a coP. aeruginosa isolates from CF patients produce AIs in vitro [P. aeruginosa produces the same signals ex vivo [in situ in CF lung tissue [12-HSL and C4-HSL have also been detected in biofilms, both in vivo and in situ [However the precise role during human infections remains speculative. Most studies so far have focused on the capacity of clinical isolates to produce the two autoinducers and on the detection of the two AIs directly in clinical samples. in vitro , and spu ex vivo . AIs havg tissue , in the g tissue ,16, and g tissue . Recentlg tissue . Both 3- in situ ,16,19; m in situ .P.aeruginosa. Because of the multitude of phenotypes depending on QS we then wondered whether P. aeruginosa isolates growing in different microenvironments might vary in their capacity to produce AIs and differ in their expression of QS-dependent phenotypes. We therefore collected P. aeruginosa isolates from the surface of intubation devices and from tracheal aspirates obtained from the same patients on the day of extubation. For each patient, we grouped strains according to their genotype, and characterized them in terms of production of autoinducers, and capacity to adhere to PVC, and produce elastase and biofilm. Our results suggest that the relative production of autoinducers, and the QS-dependent phenotypes of P. aeruginosa vary according to their site of isolation.Because much remains to be understood about the role of QS during human infections, and the potential of QS-inhibition in their prevention and/or treatment, we examined the behavior of QS during the colonization of intubated patients. To determine whether QS is active during the colonization of intubated patients, we first extracted and quantified AIs directly from the intubation devices of patients colonized with P. aeruginosa isolates were collected both from the intubation device and from a tracheal aspirate on the day of extubation:Patients' clinical characteristics are presented in Table P. aeruginosa was cultured for the first time from his tracheal aspirates 7 days after his intubation. Therapeutic retreat was decided after 15 days of mechanical ventilation and he died without having received any antibiotics. The presence of P. aeruginosa in his tracheal aspirates was considered as colonization. The tracheal aspirate used in the present study was collected shortly before his death and extubation.Patient 8 was a 27-year-old male who suffered a severe polytrauma including cerebral contusion and hemorrhage. P. aeruginosa was first cultured in bronchoalveolar lavage fluid four days after her intubation. P. aeruginosa pneumonia was diagnosed two days later and she was treated with an imipenem – tobramycin combination therapy. The clinical response was satisfying and she was extubated 3 days later still on antimicrobial therapy. A tracheal aspirate was obtained on the day of extubation.Patient 12 was a 21-year-old female who suffered a severe polytrauma including a cranio-cerebral trauma with diffuse axonal lesions and multiple rib fractures. P. aeruginosa was first cultured from a tracheal aspirate three days after her intubation. P. aeruginosa pneumonia was treated with cefepime during 10 days starting 6 days after intubation. She remained colonized by P. aeruginosa and was extubated after 17 days of mechanical ventilation.Patient 13 suffered severe congestive heart failure with a cardiac arrest. Materials and Methods. 3-oxo-C12-HSL was detected on all cuffs, the amounts varying between 0.4 and more than 10 nmoles per cuff (Table 4-HSL was detected on six of the eight devices tested and its amount varied between 17 and 300 nmoles per cuff (Table in situ isolation of autoinducers from biofilms of the majority of the intubation devices suggests that QS might play a role during colonization by P. aeruginosa of such devices. The fact that we detected C4-HSL only from 6 devices could result from the lower sensitivity of our C4-HSL bioassay (2 nmoles/cuff) compared to the one used to quantify 3-oxo-C12-HSL (0.01 nmoles/cuff).The cuff of each endotracheal intubation device was removed and autoinducers were extracted from the biofilm as described in P. aeruginosa isolates directly from the cuff of the intubation device (ID) as described in Material and Methods. The complete collection consisted of 18 TA isolates and 25 ID isolates partitioned as shown in Table For the three patients in whom we obtained tracheal aspirates (TA) at the time of extubation we also collected in vitro, though levels varied according to the site of isolation , compared to the TA group  without reaching statistical significance, possibly due to the small sample size. In contrast the TA isolates of all patients produced higher amounts of C4-HSL than the ID isolates  than for the ID isolates group . Interestingly all isolates (including genotypes C and P), whatever their site of collection, produced higher levels of C4-HSL than of 3-oxo-C12-HSL. The mean of the C4-HSL/3-oxo-C12-HSL ratios was 38.5 for the TA group compared to 5.1 for the ID group  as for determination of AIs production. For each patient the isolates obtained from the TA displayed higher elastolytic activities than those obtained from the ID . For each individual patient P. aeruginosa isolates recovered from the surface of the ID adhered more efficiently than their counterparts collected from the TA . This positive correlation became statistically significant when genotypes C and P were included . Furthermore, C4-HSL production was not correlated with elastase activity, whatever the isolate group studied . These results suggest that the level of 3-oxo-C12-HSL is positively correlated to elastase production for isolates from tracheal aspirates of colonized patients, but not for P. aeruginosa isolates from biofilms covering the intubation devices.-systems ,9. In spnscripts . Other iisolates . We wond12-HSL production and adhesion capacity for isolates from the ID group   and TA isolates .We then examined correlations between autoinducer production and adhesion capacity. There was a weak, statistically not significant, correlation between 3-oxo-C14) Fig. . No such12-HSL and biofilm production for isolates of the ID group , and TA isolates , despite the inclusion of genotypes C and P . Together these observations suggest that the production of 3-oxo-C12-HSL but not of C4-HSL, might be associated with in vitro adherence and biofilm formation capacities among isolates recovered from the biofilm on intubation devices.Similarly we observed a weak, statistically not significant, correlation between 3-oxo-CP. aeruginosa is initiated by the colonization of the intubation device by bacteria either originating from the digestive flora of the patient (endogenous acquisition) or transmitted via handling of the device by health care workers (exogenous acquisition) [P. aeruginosa then reach the respiratory tract either by leakage around the cuff of the intubation device, by shearing of secretions containing embedded bacteria from the device during the inspiratory air flow, or by contamination and direct inoculation of the respiratory tract by suction tubes introduced through the intubation device by health care workers [Respiratory tract colonization of intubated patients by isition) . This coisition) . P. aeru workers -25.in vitro studies and animal models have suggested that QS plays a major role in the virulence of P. aeruginosa [in situ and in patient data concerning the potential role of QS during colonization and infection by P. aeruginosa in humans remain scarce. QS-activity has been linked to the detection of P. aeruginosa AIs in the lungs of cystic fibrosis patients [Both ruginosa . Howeverpatients -16, as wpatients . So far patients ,22.P. aeruginosa autoinducers, 3-oxo-C12-HSL and C4-HSL, can be detected in situ in biofilms covering intubation devices retrieved from patients colonized by P. aeruginosa. Moreover all P. aeruginosa isolates collected either from these biofilms or from tracheal aspirates produced these AIs in vitro. Whereas 3-oxo-C12-HSL has been previously shown to play a role in the differentiation of P. aeruginosa biofilms [4-HSL seems to be important during the maturation stage of biofilm development [4-HSL than 3-oxo-C12-HSL in vitro. Previous observations have suggested that C4-HSL is produced in higher quantities than 3-oxo-C12-HSL in P. aeruginosa biofilms in the lungs of CF patients [4-HSL as compared to 3-oxo-C12-HSL was observed when isolates were grown in liquid cultures, suggesting that this ratio is not useful as a biomarker for biofilm growth for these isolates. None of the isolates collected from our patients had a mucoid phenotype. This discrepancy could therefore be explained by inherent differences in biofilm development patterns between mucoid and non-mucoid strains. So far there is no evidence that non-mucoid P. aeruginosa colonizing the lung of intubated patients grows in biofilms. Such a hypothesis deserves further investigations.In the present study we show that the two biofilms ,26, C4-Helopment , for theelopment , and forelopment . In our patients ,14. Singpatients . In contAutoinducers have been previously recovered from biofilms formed on urinary tract catheters  and our in vitro production of both AIs varied with the site of collection. Isolates from tracheal aspirates produced higher levels of C4-HSL but lower levels of 3-oxo-C12-HSL than their genotypically identical counter-parts isolated from intubation devices, even after several in vitro passages. This observation suggests that different microenvironments select P. aeruginosa isolates with various AI production capacities. Moreover the production of elastase, and the capacity to adhere to an inert surface and to produce a biofilm also varied with the site of isolation. Tracheal aspirate isolates produced higher levels of elastase, but were less able to adhere and produce biofilm than their counter-parts recovered from intubation devices. LasB elastase is one of the major virulence factors controlled by the P. aeruginosa QS-circuit [In vitro its production is regulated by the QS-circuit and depends mainly on the production of 3-oxo-C12-HSL [P. aeruginosa to invade surrounding tissues by its broad range enzymatic activity and to facilitate blood stream invasion by degradation of elastin fibers in the lamina propria of blood vessels [Intriguingly, the -C12-HSL ,10. LasB12-HSL production correlated positively with the capacity to produce elastase. In contrast for isolates originating from the intubation device, 3-oxo-C12-HSL levels did not correlate with elastase production, but correlated weakly with the capacity to adhere and to form biofilms on inert surfaces. These observations suggest that the previously described control of LasR on elastase production might not apply to all isolates depending on the site they were collected from. It seems that the microenvironment on the intubation devices selects for phenotypes that produce high levels of 3-oxo-C12-HSL but without concomitant increased elastase production. In contrast the lower lung environment seems to select for isolates that produce less 3-oxo-C12-HSL but with a positive control of this AI on elastase production. Two of our patients (12 and 13) received antimicrobial therapies during their mechanical ventilation. We can therefore not exclude that different concentrations of these drugs at the two sites might have selected different phenotypes. However this seems unlikely as isolates of patient 8 presented the same differences in phenotype in the absence of any exposure to antimicrobial therapies.The observed differences in phenotypes between isolates obtained from tracheal aspirates, compared to those from the intubation devices, make sense. Isolates growing in the biofilm on intubation devices might not invest energy in producing elastase, but might be primed to adhere and form biofilms, whereas isolates growing in the lungs might find an advantage in producing elastase. For tracheal aspirate isolates, levels of 3-oxo-C4-HSL production and elastase activity, adhesion or biofilm formation. Strikingly, these phenotypes remained stable for each isolate independently of its origin even after several in vitro passages. This suggests that mutations in specific regulators, that affect the production of AIs and the expression of QS-dependent phenotypes might be selected in particular microenvironments. Indeed lasR and rhlR mutants of P. aeruginosa strains isolated from intubated patients have been previously described [lasR and rhR in 6 TA and 6 ID isolates . A total of 25 ID isolates were collected from patients 8, 12 and 13, and stored at -70°C  either on the day before, or on the day of extubation. The tracheal aspirates (TA) were plated on selective agar plates (Cetrimide 0.03%) in order to identify isolates . SimilarFor further assays both ID and TA isolates were taken from the frozen stocks and grown in liquid cultures.Cultures for extraction of cell-to-cell signaling molecules and production of elastase were grown in Luria-Bertani (LB) broth. M63 medium  was used12-HSL and C4-HSL determined using the reporter strains E. coli MGλ1,4 (pPCS1) [P.aeruginosa PAO-JP2 (pECP61.5) [Previous studies have suggested that secretions colonized by bacteria accumulate above the cuff of intubation devices and form biofilms -36. Leak (pPCS1)  and P.aeECP61.5)  respectiBacterial strains were genotyped by Random Amplified Polymorphic DNA (RAPD) analysis as previously described using the Ready-to-Go Kit (Amersham)  using prTotal extracellular protease  and rhamin vitro was determined in a static model as previously described [660 of 0.5 and incubated for a further 6 hours at 37°C with shaking until cells had entered early stationary phase. Sterile calibrated coupons of PVC tracheal tubing were immersed in the suspension and incubated statically at 37°C for 1 h, then the PVC coupons were transferred to 6-well cell culture plates and incubated statically at 37°C for 72 h in AP medium. The extent of biofilm formation was measured using crystal violet staining [Adhesion capacity was measured for both ID and TA isolates as the ability of bacteria to adhere to the wells of 96-well microtiter plates. The assay was performed as previously described . Resultsescribed . Brieflystaining . ResultsWe used the Student's t test to compare mean production of AIs and virulence factors by isolates from tracheal aspirates (TA) and the cuff of the intubation device (ID). Correlations between AIs production and QS-dependent phenotypes were calculated using a robust estimator of the Pearson's correlation coefficient. To account for the correlation among multiple observations in the same patient, standard errors and p values were computed using a first-order Taylor-series linearization method. All analyses were conducted using Stata 6.0 statistical software . p < 0.05 was considered statistically significant.SFB carried out the experiments and drafted the manuscript. CE conducted the statistical analysis. TK participated in the design of the study and participated in the analysis of the results. JAR organized the sample collection. CVD conceived the study, supervised the experiments and their analysis, and wrote the final manuscript. All authors read and approved the final manuscript.


Pseudomonas aeruginosa isolates from clinical specimens to those of environmental isolates encountered in the same units of a medical center. Antibiotic susceptibility testing, RAPD analysis and detection of enzymatic activities of extracellular virulence factors, were done on these isolates.We correlated genotypes, virulence factors and antimicrobial susceptibility patterns of nosocomially identified Data showed that most of the clinical and environmental isolates were susceptible to tested antimicrobial agents. RAPD analysis determined the presence of 31 genotypes, with genotype 1 detected in 42% of the clinical isolates and 43% of the environmental isolates. Enzymatic activity testing showed that genotype 1 produced all virulence factors tested for.P. aeruginosa genotype, circulating in a number of units of the medical center and emphasize the need to reinforce infection control measures.In conclusion, our data demonstrated the predominant prevalence of a potentially virulent  Pseudomonas aeruginosa continues to be a major nosocomial pathogen particularly in patients who suffer from immunosuppression [P. aeruginosa is a ubiquitous pathogen prevalent in the hospital environments, and can cause severe nosocomial infections [P. aeruginosa infection in hospitals have been identified; such as tap water, medical equipment, hospital personnel and other patients [P. aeruginosa accounts for 10% of all hospital acquired infections, a site specific prevalence which may vary from one unit to another and from study to study [P. aeruginosa appears to be the major cause of ventilator-associated pneumonia with a high rate of attributable mortality [P. aeruginosa nosocomial infections in our medical center was 18%. Such a high rate prompted us to study the P. aeruginosa genotypes circulating in the various units to reveal the clonal relationship between clinical and environmental isolates and to allow elucidating the source and mode of transmission of this important bacterium at this medical center.Despite the advances in hospital care and the introduction of a wide variety of antimicrobial agents, pression . P. aerufections . The latfections . Variouspatients ,4. P. aeto study . Among dortality . Moreoveortality ,8. DurinP. aeruginosa infection in our patients with 55% due to mechanical ventilation and the remaining 45% due to polysite catheters, Foley catheters and different types of surgery.Our data have shown that several mechanical devices were associated with RAPD analysis have shown 31 genotypes present among the clinical and the environmental isolates figure . Table 1P. aeruginosa spread and infection especially after identification of a potentially virulent clone (genotype 1) of this organism that had been propagated in various units over a period of 9 months. This could indicate that the patients were continuously infected with a strain originating from an exogenous source. The importance of cross colonization of P. aeruginosa in nosocomial infections was previously reported, by Bergmans et al who studied 100 patients admitted to an ICU ward; cross colonization accounted for 50% of all cases of acquired P. aeruginosa colonization, the other 50% of patients were probably colonized from endogenous sources [The strain distribution based on RAPD analysis showed that clinical and environmental isolates distributed in one or more than one unit, included genotypes 1–8 and 9, with predominance of genotype 1 in all investigated units. This may indicate that cross contamination among patients lead to the spread of this genotype among the various units, possibly through transient hand carriage by health care personnel due to contact with contaminated surfaces or by patient contact with contaminated surfaces or medical equipment . Our fin sources .P. aeruginosa from endogenous sources occurs mainly in the respiratory and gastrointestinal tracts [The remaining genotypes were isolated exclusively from the clinical specimens of patients and were not detected among environmental isolates. Most of these genotypes did not harbor all the virulence factors tested for. Not being encountered in environmental sources may indicate that these strains may have been endogenously acquired. High rate of colonization with l tracts . Hospital tracts ,15,16.Prevalence of strains with resistance to all antimicrobial agents will constitute a major risk for hospitalized patients. In our study, though, the most prevalent strain of genotype 1 was susceptible to all antimicrobial agents and does not constitute a problem in treatment. However, its potential of producing all virulence factors and being spread by various means in the hospital, unlike other genotypes that harbor all virulence factors, may render genotype 1 a high risk pathogen specially in the immunosuppressed and debilitated patients. The fact that genotype 1 remained susceptible to all antibiotics lead to the conclusion that patients could be continuously infected with a strain originating from an exogenous source.In addition to mechanical spread by personnel, this genotype may carry a number of adhesins that enhance its colonization in the hospital environment and render it more accessible to patients. Studies are underway in our laboratory to detect by PCR, genes encoding a number of adhesins and determine their transcription levels in genotype 1, in comparison to other encountered genotypes. This will shed light on the possible role of adhesins on the prevalence of this genotype in the hospital environment. The extracellular enzymes or toxins produced, on the other hand, will contribute to the breaking down physical barriers and help the organism to penetrate, impair host defenses, and render its new milieu more conducive to its physical, nutritional and reproductive requirements .P. aeruginosa genotype 1 in clinical and environmental specimens, circulating in the various hospital units. From a practical point of view, the results of our study emphasize the need to reinforce implementation of infection control measures, and to limit the transmission of P. aeruginosa among patients and from environmental sources to patients. Screening for P. aeruginosa carriage in all patients with nosocomial colonization or infection should also be done in research settings using both rectal and respiratory tract specimens to determine the source of colonization/infection and hence get informed on whether it was acquired endogenously or exogenously.In summary, our data have shown the predominant prevalence of a potentially virulent P. aeruginosa genotype 1 in clinical and environmental specimens, circulating in the various medical center units.In conclusion, our data have shown the predominant prevalence of a potentially virulent P. aeruginosa (90) isolates were recovered from different patients specimens (one per patient) submitted for bacteriological investigations at the Clinical Microbiology Laboratory at the American University of Beirut Medical Center (AUBMC), between September 2003 and May 2004. Patients acquiring a nosocomial infection due to P. aeruginosa as determined by clinical and laboratory testing and indicated in their medical records, were only considered in this study. Fever, and recovery of P. aeruginosa from the site of infection during stay at the medical center, constituted the most important criteria that defined patient's infection with this organism. Patients' data collected from the medical records, included age (2 to 91 years), sex , admission date, admission diagnosis, invasive procedures used on the patients, symptoms, and the date of the first positive culture for P. aeruginosa. The drugs used in the treatment of these patients, mainly included: amikacin, azactam, gentamicin, tobramycin, and tazocin. Patients were distributed within ten different units in the medical center, mainly the Respiratory Care Unit (RCU), Intensive Care Unit (ICU), Coronary Care Unit (CCU), the Surgery Unit, as well as other units. Twenty three isolates were also collected from different environmental sources, such as, respirators, respirators' filters, water irrigation, tap water, basins, trays, bed side tables, side rails, and sink sides. Statistical analysis determined the sample size required to estimate the true proportion (percentage of P. aeruginosa infections during a given period of time) to within 0.10, with 95% confidence was calculated. Calculations estimated the minimum number of samples required to be 54 [Consecutive ruginosa 0 isolateP. aeruginosa on culture based on colonial morphology, Gram stain microscopy, and oxidase test was further confirmed by the API NE Kits and growth at 42°C. Susceptibility of all isolates to a panel of antimicrobial agents  was determined according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) [P. aeruginosa ATCC 25321 strain was used as positive control in all tests.The presumptive identification of  (NCCLS) . P. aeruP. aeruginosa ATCC strain and from all isolates of P. aeruginosa by the GFX™ Genomic Blood DNA Purification Kit  according to the manufacturers' specifications. Random amplified polymorphic DNA (RAPD) analysis of the clinical and environmental isolates using two in-house oligonucleotide primers, Pa1 (5'AGGGGTCTTG 3') and Pa2 (5' CTTCTTCAGCTCGACGCGACG 3') was done. RAPD was carried out according to Matar et al [2), 1 μl of primer 1 (0.5 μM), 1 μl of primer 2 (0.3 μg/ μl), 2.5 U of Taq DNA polymerase and 61.5 μl of nanopure sterile water. The amplification program, included the following steps: denaturation at 94°C for 3s, annealing at 53°C for 1 min and extension at 72°C for 1 min, for 44 cycles. The cycles were followed by a final extension step at 72°C for 10 min. Amplicons were subjected to electrophoresis on 2% agarose gels at 107 volts for 2 hours. Patterns that had the same number of bands and similar fragments size were considered identical.DNA was extracted from ar et al  using thP. aeruginosa were evaluated by spot inoculation containing 106 CFU/ml of the organisms in various media [The enzymatic activities of the isolates of us media . Media uGM supervised the study and wrote the manuscript. MC did the bench work and helped in writing the manuscript. GA provided bacterial isolates. ZS provided help on wards. GJ provided clinical support. UH provided clinical support. All authors read and approved the final manuscript.


Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. In this study, we focused on the virulence of multi-drug resistant clinical strains P. aeruginosa against the intestinal epithelial barrier, since P. aeruginosa can cause lethal sepsis from within the intestinal tract of critically ill and immuno-compromised patients via mechanisms involving disruption of epithelial barrier function.Multi-drug resistant P. aeruginosa clinical strains for their ability to disrupt the integrity of human cultured intestinal epithelial cells (Caco-2) and correlated these finding to related virulence phenotypes such as adhesiveness, motility, biofilm formation, and cytotoxicity.We screened consecutively isolated multi-drug resistant P. aeruginosa clinical strains were attenuated in their ability to disrupt the barrier function of cultured intestinal epithelial cells. Three distinct genotypes were found that displayed an extreme epithelial barrier-disrupting phenotype. These strains were characterized and found to harbor the exoU gene and to display high swimming motility and adhesiveness.Results demonstrated that the majority of the multi-drug resistant P. aeruginosa against the intestinal epithelium has the potential to identify strains most likely to place patients at risk for lethal gut-derived sepsis. Surveillance of colonizing strains of P. aeruginosa in critically ill patients beyond antibiotic sensitivity is warranted.These data suggest that detailed phenotypic analysis of the behavior of multi-drug resistant  Pseudomonas aeruginosa, is a major cause of infectious-related mortality among the critically ill patients, and carriers the highest case fatality rate of all gram-negative infections [P. aeruginosa infection among critically ill patients, a significant number of these infections arise as a result of direct contamination of the airways by the gastrointestinal flora or by hematogenous dissemination from the intestine to the lung parenchyma [P. aeruginosa within the intestinal tract alone can be a major source of systemic sepsis and death among immuno-compromised patients [P. aeruginosa in the critically ill patients have identified the intestinal tract to be the single most important reservoir for this pathogen in cases of severe life-threatening sepsis [P. aeruginosa lies in its ability to adhere to and disrupt the intestinal epithelial barrier [The human opportunistic pathogen, fections . Althougrenchyma ,3. Yet epatients ,5. Exteng sepsis ,7. Work  barrier .P. aeruginosa with up to 30% of these strains being antibiotic resistant [P. aeruginosa alone has been associated with a 3-fold increase in mortality in critically ill patients [P. aeruginosa as a cause of mortality in critically ill patients was recently demonstrated by a randomized prospective study in which selective antibiotic decontamination of the digestive tract (SDD) in critically ill patients with oral non-absorbable antibiotics decreased mortality associated with a decrease in fecal P. aeruginosa [Within as little as 3 days in an intensive care unit, the feces of more than 50% of patients will culture positive for esistant . In suchpatients . In factruginosa .P. aeruginosa clinical isolates behave against the human intestinal epithelium is unknown. Therefore the purpose of this study was to determine the ability of MDR P. aeruginosa to disrupt epithelial integrity of Caco-2 monolayers and to correlate these findings to other relevant virulence features of P. aeruginosa including adhesiveness, motility, ability to form biofilm, and the presence of specific type III secretion related genes exoU and exoS.How multi-drug resistant (MDR) P. aeruginosa were consecutively obtained from the clinical microbiology laboratory from those selectively screened for gentamicin (Gm) resistance. We initially screened consecutive P. aeruginosa isolates that were resistant to Gm since Gm resistance has been shown to be the most common feature of MDR P. aeruginosa [P. aeruginosa on subsequent culture. Therefore 31 clinical strains were available for phenotype and genotype analysis. Most isolates identified as P. aeruginosa were oxidase positive, hydrolyzed acetamide and arginine, oxidized glucose, and grew on cetrimide agar. Remaining isolates were identified by the Vitek 2 system . Additionally, isolates were verified by amplification of 16S DNA using primers forward 5'-GGACGGGTGAGTAATGCCTA-3' and reverse 5'-CGTAAGGGCCATGATGACTT-3', and genome DNAs of clinical isolates as templates. Susceptibility testing was performed by testing on the Vitek 2 or by disk diffusion. Susceptibility results were interpreted using Clinical Laboratory Standards Institute (CLSI) guidelines. Single colonies were picked up from Columbia SB agarized plates , grown in Pseudomonas broth containing Gm, 50 μg.ml-1 and kept at -80°C as frozen stocks containing 8% glycerol. The isolates were routinely subcultured from frozen stocks on Pseudomonas isolation agar (PIA) containing Gm, 50 μg.ml-1. P. aeruginosa strains PAOI, ATCC 27853, PA103, and the environmental isolates PA190 and PA180 [Under IRB protocol #11646B, University of Chicago, 35 strains of ruginosa . Among tnd PA180  were useP. aeruginosa isolates was determined using the random amplified polymorphic DNA (RAPD) PCR fingerprinting, described previously [-1) and visualized using UV radiation. Fingerprints were considered distinct if they differed by at least three bands.The clonality of eviously -16. Prim2 transwells (Costar) in HEPES buffered (15 mM) DMEM media containing 10% FBS for 20 days, and electrophysiological measurements were done using agar bridges and Ag-AgCl-calomel electrodes and a voltage clamp  as previously described [P. aeruginosa strains against Caco-2 monolayers, overnight culture was added to the apical well (volume = 200 μl) to achieve a final bacterial concentration of ~107 CFU/ml. Media from the apical wells was then quantitatively cultured on PIA plates to determine the final bacterial count. Caco-2 monolayers were co-incubated with bacteria for up to 8 hours at 37°C, 5% CO2, and TER was measured each hour. All experiments were performed in triplicate.The Caco-2bbe (brush border-expressing) cell line was used in bacterial-cell culture experiments. Caco-2 cells were grown in 0.3 cmescribed . Fixed cSwimming assay was performed as previously described by Rashid and Kornberg . BrieflyTwitching motility was determined by the method of Rashid and Kornberg . Fresh pP. aeruginosa strains were grown in 96-well plates in M63 supplemented with 0.5% casamino acids and 0.2% glucose. Plates were incubated at 37°C under mild shaking at 50 rpm  for 8 hrs. The wells were then rinsed thoroughly with water and the attached material was stained with 0.1% crystal violet, washed with water, and solubilized in ethanol. Solubilized fractions were collected and absorbance measured at 550 nm with a Plate Reader. All experiments were performed in triplicate.Biofilm formation was assayed as described with modifications . BrieflyP. aeruginosa were added to the apical side of Caco-2 cells to a final concentration of 107 CFU/ml and co-incubated for 1 hour at 37°C, 5% CO2. Following the one hour incubation, the media was removed and ten-fold dilutions were plated on PIA plates to quantify non-adherent bacteria. Wells were then washed with a continuous flow of 35 ml of PBS. A final single washing with 200 μl was diluted and plated on PIA to quantify the final amount of remaining non-adherent bacteria. Caco-2 cells were then trypsinized with 200 μl Trypsin-EDTA (Gibco), incubated for 20 min at 37°C, 5% CO2, and lysed with 400 μl of a lysis mixture  [Caco-2 cells were grown to confluence in 24-well plates using HEPES-buffered DMEM media containing 10% fetal bovine serum. Overnight cultures of P. aeruginosa clinical isolate #1 was diluted as 1:100 in fresh M63 media supplemented with 0.5% casamino acids and 0.2% glucose and grown for 2 hours. After that, culture was spitted for control (no Gm) and Gm-variant that was added by Gm to a desirable concentration. 300 μl aliquots (in triplicates) were loaded in 96-well plate, and absorbance at OD550 nm was measured dynamically during growth at 37°C, 200 rpm. All experiments were performed in triplicate.Overnight culture of exoU and exoS genes were performed using intact P. aeruginosa grown on PIA as a source of template chromosomal DNA as described [exoU: exoU2998, 5'-GCTAAGGCTTGGCGGAATA-3' and exoU3182, 5'-AGATCACACCCAGCGGTAAC-3'; for exoS: exoS 1106, 5'-ATGTCAGCGGGATATCGAAC-3', and exoS 1335, 5'-CAGGCGTACATCCTGTTCCT-3'.PCR assays for detection of the escribed . AmplifiP. aeruginosa to the final concentration of 107 CFU/ml. Cells were incubated at 37°C, 5% CO2, for 8 hours, and released lactate dehydrogenase was determined by CytoTox 96 assay (Promega). All experiments were performed in triplicate.Caco-2 cells were grown to confluence in 96-well plates, and inoculated apically by Statistical analysis of the data was performed using Student t-test. Regression analysis was performed using Sigmaplot software.P. aeruginosa strains were consecutively collected based on their resistance to gentamicin (Gm), however most clinical isolates displayed multiple antibiotic resistances to various antibiotics clinical used against P. aeruginosa. Most strains were obtained from sputum and tracheal aspirates while few were from tissues and urine. Significant variation was noted in colony morphology among the various strains. Environmental strains PA190 and PA180 were also tested for antibiotic resistance. Results indicated that PA190 was sensitive to all of the antibiotics routinely used for P. aeruginosa infection, whereas PA180 was resistant to Gm.Morphological and demographic data are displayed in Table P. aeruginosa clinical isolates were typed by RAPD analysis with primers 208 . There was no correlation however between TER changes and twitching motility (r = 0.44)  Fig. , or biof44) Fig. . High sw-1) of growth , whereas strain #1 grown in the presence of Gm showed a dose-dependent stimulation  and displayed cytotoxicity against Caco-2 monolayers. Clinical isolates harboring the exoS gene were not cytotoxic to Caco-2 cells.The cytotoxic effect of the various clinical isolates following 8 hours of bacterial exposure is shown in Figure P. aeruginosa on the intestinal epithelial barrier since intestinal P. aeruginosa has been shown to be a major cause of morbidity and mortality among immuno-compromised patients [Numerous reports have documented that the rise in multi-drug resistant nosocomial pathogens continues to threaten hospitalized patients despite various countermeasures including isolation techniques and antibiotic de-escalation therapy ,23. In tpatients ,24,25.P. aeruginosa to subvert and erode an already compromised epithelial defense system exists [Caco-2 cells are an ideal cell model for these studies since they express several markers that are characteristic of normal intestinal epithelial cells including the presence of a brush border and the ability to maintain a highly resistant barrier to bacterial pathogens ,26. As pm exists ,32.P. aeruginosa [P. aeruginosa against the intestinal epithelium is unknown, its high prevalence in the intestinal tract of critically ill and immuno-compromised patients begs a better understanding of the degree to which certain strains can disrupt the intestinal epithelial barrier. For example the apical side of the intestinal epithelium is highly resistant to various toxic and cytolytic exoproducts of P. aeruginosa including exotoxin A and elastase [P. aeruginosa infection and pathogenesis cannot be directly extrapolated to the intestinal model. Interestingly, data from the present study establish that among the MDR P. aeruginosa isolates tested in the Caco-2 model, most display a minor to minimal ability to disrupt the intestinal epithelium in both motile and non-motile strains.Whether MDR ruginosa -36 straielastase ,11,37, wWe identified 8 MDR clinical isolates with 3 distinct RAPD fingerprints that display a disruptive phenotype against the intestinal epithelial barrier. The presence of such strains within the intestinal tract of critically ill patients has the potential to induce a state of gut-derived sepsis with a high mortality rate as their presence in this site is often difficult to detect and eradicate.exoU gene. ExoU, an effector protein of the type III secretion machinery, has been previously shown to play a major role in mediating a cytotoxic phenotype of P. aeruginosa [P. aeruginosa strains harboring the exoU-genotype may be associated with poor outcome in patients colonized by such strains. Although the presence of ExoS has been previously reported to play a role in the virulence of P. aeruginosa in a lung model [Common features of these highly disruptive strains include high swimming motility, increased adhesiveness to intestinal epithelium, and the presence of the ruginosa ,39 againruginosa . That Exng model , we founng model , motilitP. aeruginosa that pose a significant threat to the patient. The ability of multi-drug resistant strains to persist for prolonged periods in such patients may allow for the development of extremely virulent phenotypes [As we and others have suggested, bacteria are fully capable of changing their virulence phenotype in direct response to host illness ,44. The enotypes .exoU gene could provide a significant prognostic input to identify multi-drug resistant P. aeruginosa strains most likely to place patients at risk for lethal gut-derived sepsis. Further characterization of strains 1, 13 and those of G20 RAPD genotype will be necessary to better understand the precise mechanism by which these strains disrupt the intestinal epithelium to a degree not previously reported for any intestinal pathogen.In conclusion, heterogeneity among critically ill humans, variability in immune response, and antibiotic use could explain the extremely polar phenotypes identified in the series of multi-drug resistant isolates collected in the present study: from phenotypes that are essentially inert with respect to the intestinal epithelium to highly motile, adhesive, and destructive phenotypes. Phenotypic assays such as motility and adhesiveness, and genotyping for the Pseudomonas isolation agar, PIA.Multi-drug resistance, MDR; transepithelial resistance, TER; random amplified polymorphic DNA PCR fingerprinting, RAPD; phosphate buffered saline, PBS; lactate dehydrogenase, LDH; The author(s) declare that they have no competing interests.OZ performed experimental design, most experimental work, and drafting/revising the manuscript. JEK had developed and carried out the adhesiveness assay. YW was responsible for cultivation of Caco-2 cells and growing them on transwells. CB isolated and identified clinical isolates. OS participated in adherence and RAPD analyses. LW participated in adherence analyses. JRT was involved in the experimental design and discussion of experiments and manuscript revision. JCA performed experimental design, experimental data discussion, drafting/revising the manuscript, and is the PI of the NIH funding mechanism of the study. All authors read and approved the final manuscript.


P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC β-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance.Aztreonam exhibited the highest in vitro activity against the P. aeruginosa clinical isolates.Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary β-lactamases play also an important role in the multi-drug resistance phenotype among  Pseudomonas aeruginosa is an aerobic gram-negative pathogen and a common etiologic agent of nosocomial infections, especially pneumonia, in seriously ill patients [patients ,2. This patients .Bacterial efflux systems capable of ejecting antimicrobials are mostly encoded by chromosomal genes and generally fall into five classes, the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) family, the small multi-drug resistance (SMR) family, the multi-drug and toxic compound extrusion (MATE) family and the resistance-nodulation-division (RND) family . The RNDP. aeruginosa genome revealed the presence of several RND efflux systems. Of those, MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM are able to pump out multiple antipseudomonal compounds [P. aeruginosa. However, when overexpressed, these efflux systems confer reduced susceptibility to different classes of antimicrobial agents [P. aeruginosa, their overexpression may also contribute to the acquired multi-drug resistance phenotype in mutant isolates [Sequencing of ompounds ,4,6. Stuompounds . The Mexompounds . MexAB-Ol agents ,8. Althoisolates .P. aeruginosa clinical isolates [P. aeruginosa clinical isolates.Overexpression of efflux systems generally confers modest levels of antimicrobial resistance ,10. Howeisolates . The assisolates . For thiP. aeruginosa isolates were collected from bloodstream infections between June and December 2005. The majority of isolates was collected from patients hospitalized in intensive care units (64.4%), followed by the emergency room ward (28.8%) and pediatric oncology unit (6.8%). Aztreonam showed the highest susceptibility rate against the isolates studied (64.4%), followed by cefepime (49.2%), meropenem (47.2%), imipenem (47.2%), ceftazidime (44.1%), amikacin (40.7%), ciprofloxacin (35.6%) and gentamicin  were susceptible to all tested antimicrobial.Fifty-nine non-repetitive idime 44.%, amikacP. aeruginosa clinical isolates studied. Five P. aeruginosa isolates could not be typed by PFGE using SpeI. Although 38 isolates were clustered in six PFGE patterns, 16 isolates showed distinct PFGE patterns.A total of 23 distinct PFGE patterns were detected among the 59 P. aeruginosa, representing 25.4% of the whole collection and 48.4% of the imipenem-resistant isolates. These isolates had their carbapenemase activity inhibited by EDTA, and the presence of the MBL-encoding genes blaSPM-1 and blaIMP-like was confirmed by multiplex PCR, in 14 and 1 isolates, respectively. Among the SPM-producing P. aeruginosa studied, 13 showed the same PFGE pattern, whereas one isolate could not be typed using Spe I. ESBL-encoding genes were present in five isolates: blaGES-1 (n = 3), blaGES-5 (n = 1) and blaCTX-M-2 (n = 1). GES-type producers belonged to the same genotype, whereas CTX-M-2-producer showed a unique PFGE profile.Carbapenem hydrolysis was detected in 15 P. aeruginosa isolates that were non-susceptible to antimicrobials and demonstrated overexpression of efflux genes and ampC, coupled with oprD down-regulation is shown in Table The percentage of n = 30) and 27.1% (n = 16) of P. aeruginosa clinical isolates demonstrated increased mexY (from 2.2- to 41.0-fold) and mexB (from 2.1- to 10.0-fold) transcription mRNA levels, respectively, compared to those of PAO1. In addition, 11 P. aeruginosa isolates (18.6%) showed overexpression of both mexB and mexY efflux genes. Overexpression of MexCD-OprJ and MexEF-OprN were not observed among the clinical isolates of P. aeruginosa evaluated in this study. Overall, 69.5% and 11.9% of P. aeruginosa clinical isolates studied showed decreased oprD expression (from 0.1- to 0.7-fold compared to PAO1), and overexpression of ampC (from 14- to 402-fold compared to PAO1), respectively. None of the investigated resistance determinants was identified in 11.8% of clinical isolates . Among the cefepime non-susceptible isolates that did not show mexY overexpression, 33.3% produced SPM-1, 33.3% overexpressed ampC, 16.7% produced the ESBL CTX-M-2, and 16.7% produced GES-5, an ESBL with carbapenemase activity.Among the isolates overexpressing the mexB (from 2.1- to 5.5-fold higher than PAO1). Of those, 90.0% showed decreased oprD expression, 40.0% were MBL producers, 20.0% overexpressed ampC and 10.0% were GES-5 producers (data not shown). As expected, all meropenem-susceptible isolates that overexpressed mexB, presented normal expression of both ampC and oprD when compared to that of PAO1. Higher percentage of mexB overexpression was observed among isolates that were also not susceptible to cefepime, amikacin, gentamicin and ciprofloxacin. Of note, 85.7% and 28.6% of SPM-producing P. aeruginosa showed increased transcriptional levels of mexY and mexB, respectively.Meropenem non-susceptibility was observed among 62.5% of isolates overexpressing P. aeruginosa usually confers modest increase in the MICs of antimicrobial agents that are ejected by these systems.It is worth to mention that MexAB-OprM and/or MexXY-OprM overexpression was observed among isolates that were susceptible to most antimicrobials tested. This finding was expected since efflux pump overexpression in P. aeruginosa is the fifth most frequent pathogen of bloodstream infections and the first one causing pneumonia in Latin America according to the SENTRY Antimicrobial Surveillance Program [P. aeruginosa has been observed worldwide. Some of antimicrobial agents have become less effective against these organisms reducing the available therapeutic options for treatment of these infections. Program . In the P. aeruginosa isolates studied were resistant to carbapenems. Our findings are in accordance of previous studies that showed high rates of antimicrobial resistance, including carbapenems, among P. aeruginosa clinical isolates collected from Brazilian institutions [P. aeruginosa isolates studied indicates that spread of clones and emergency of distinct genotypes have occurred in our hospital. The high rate of carbapenem resistance can be partially explained by the spread of an endemic SPM-producing clone. It also justifies the susceptibility rate to aztreonam since MBL producers are not able to hydrolyze this antimicrobial agent. This finding corroborates with those previously reported that described a single SPM producer clone spread out in the Brazilian territory [In this study 52.5% of the itutions . The generritory .P. aeruginosa infections since they are capable of pumping out many classes of antimicrobial agents used for treatment of these infections [The overexpression of efflux systems may impact on clinical outcome of fections . Howeverfections .ampC and oprD among 59 P. aeruginosa clinical isolates. This collection represents the total number of patients with bloodstream infection due to P. aeruginosa in a six-month period in Hospital São Paulo, Brazil. We also aimed to evaluate the frequency of isolates presenting different mechanisms of β-lactam resistance and their association.In the present study, we have evaluated the transcriptional levels of four efflux-encoding genes as well as P. aeruginosa isolates. Since MexAB-OprM and MexXY-OprM are constitutively expressed in wild type P. aeruginosa isolates, the antimicrobial policy in use in each individual institution may interfere with the selection of the most overexpressed efflux system. Aminoglycosides are important substrates of MexXY-OprM and might have exerted a role in selecting P. aeruginosa that overexpressed this system [P. aeruginosa infections. These facts could in part justify why MexXY-OprM was the most frequent overexpressed efflux system, since mexXY expression may be induced by these antimicrobial class [The overexpression of the MexAB-OprM and MexXY-OprM efflux systems were more frequent among antimicrobial resistant s system . The exps system . In our al class . InteresWe did not notice a strict correlation between antimicrobial resistance and efflux genes overexpression. However, efflux overexpressing isolates often presented higher antimicrobial MICs than did PAO1 and those isolates in which no antimicrobial resistance determinant was found.Our findings clearly demonstrate that β-lactamase production increase antimicrobial MICs more efficiently than do efflux overexpression or porin down-regulation alone. However, these chromosomal resistance mechanisms were frequently present among acquired β-lactamase producers. These findings suggest that efflux overexpression and porin down-regulation may favor the bacterial survival under selective pressure, increasing its chance to acquire further resistance determinants.P. aeruginosa, but represents an adjuvant mechanism for antimicrobial resistance. The association of distinct mechanisms such as the porin down-regulation and AmpC overproduction play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates studied. In addition, our findings indicate that spread of clones and emergency of distinct genotypes have occurred in our institution and implementation of control measures is extremely necessary to modify this scenario.In the present study we have observed that efflux pump overexpression do not appear to be the main mechanism of drug resistance among the studied clinical isolates of P. aeruginosa were evaluated, regardless of their antimicrobial susceptibility profile. These isolates were consecutively collected between June and December 2005 from blood culture of patients hospitalized at Hospital São Paulo, a tertiary teaching hospital located in São Paulo, Brazil. Only a single bacterial isolate per patient was evaluated. MICs for ceftazidime, cefepime, aztreonam, imipenem, meropenem, gentamicin, amikacin and ciprofloxacin were determined by agar dilution and interpreted according to Clinical Laboratory Standards Institute [P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 strains were used as quality control strains.With the approval of the local Ethics in Research committee , a total of 59 clinical isolates of nstitute . P. aeruGenomic DNA of isolates was prepared in agarose blocks and digested with the restriction enzyme SpeI . Electrophoresis was performed on CHEF-DR III , with the following conditions: 0.5 × TBE, 1% agarose, 13°C, 200 V, for 24 h with switch time ramped from 5 to 90 s. The band patterns were interpreted as previously recommended .P. aeruginosa 48-1997A [blaTEM, blaSHV, blaCTX-M, blaGES, blaVEB and blaPER was investigated by PCR, as previously reported [Investigation of carbapenemase activity in crude extracts was performed by UV spectrophotometric assays. Briefly, a full 10 μl loop of the test organism was inoculated into 500 μl of phosphate buffer 100 mM (pH 7.0) and disrupted by sonication. The cells were removed by centrifugation and the supernatants were used for further experiments. Protein quantification in the crude extracts was performed using the Bradford stain. Hydrolytic activity of crude extracts was determined against 100 μM imipenem and 100 μM meropenem in 100 mM phosphate buffer (pH 7.0). Measurements were carried out at a 297 nm wavelength. Positive control included SPM-1-producing 48-1997A . Carbape48-1997A . The prereported ,25.mexB, mexD, mexF, mexY, ampC and oprD were determined with Mastercycler Realplex2 . In brief, total RNA was extracted using the RNase Mini Kit, following the manufacturer recommendations . Five micrograms of total RNA was submitted to cDNA synthesis using High Capacity cDNA Archive Kit . Quantitative RT-PCR was performed with Platinum SYBR Green Supermix , using specific primers for mexB, mexD, mexF, mexY, ampC and oprD as previously described [http://bibiserv.techfak.uni-bielefeld.de/genefisher/old.html , by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) that conceded a grant to DEX and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) that provides a researcher grant to ACG. (307714/2006-3).


Pseudomonas aeruginosa.Cystic fibrosis (CF) is an inherited multi-system disorder characterised by chronic airway infection with pathogens such as P. aeruginosa by patients with CF is usually from the environment, but recent studies have demonstrated patient to patient transmission of certain epidemic strains, possibly via an airborne route. This study was designed to examine the survival of P. aeruginosa within artificially generated aerosols.Acquisition of P. aeruginosa were able to survive within the aerosols, but strains expressing a mucoid phenotype had a survival advantage.Survival was effected by the solution used for aerosol generation. Within the aerosols it was adversely affected by an increase in air temperature. Both epidemic and non-epidemic strains of P. aeruginosa from those with chronic P. aeruginosa infection who are more likely to be infected with mucoid strains may help reduce the risk of cross-infection. Environmental factors also appear to influence bacterial survival. Warming and drying the air within clinical areas and avoidance of humidification devices may also be beneficial in reducing the risk of cross-infection.This would suggest that segregating individuals free of  Pseudomonas aeruginosa.Cystic fibrosis (CF) is an inherited multi-system disorder characterised by chronic airway infection, pancreatic insufficiency, elevated sweat chloride concentration, impaired fertility and hepatobiliary disease. The condition is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Lack of CFTR function results in reduced fluid secretion and excessive fluid absorption, with a net effect of producing a thickening of the mucous component of the airway surface liquid. This causes plugging of the sub-mucosal glands and impairment of mucociliary clearance leading to infection, inflammation, and tissue damage resulting in bronchiectasis and a predisposition to infection with pathogens such as P. aeruginosa is the most common and clinically important pathogen in patients with CF. The organism is a Gram-negative, non-fermentative, aerobic bacillus belonging to the family Pseudomonadaceae. Although classified as an aerobic organism P. aeruginosa is a facultative anaerobe which may allow it to survive within the relatively hypoxic lungs of patients with CF. It is ubiquitous within the environment and is particularly isolated from moist areas such as soil and water. The bacterium is frequently found in environmental reservoirs, such as the drains of hospital ward wash basins [P. aeruginosa can be detected when opening taps [P. aeruginosa from tap water follows contamination of the taps rather than the mains water supply [h basins  and aeroing taps ,3. The ir supply .P. aeruginosa infection in patients with CF can occur at any age. During early stages of P. aeruginosa infection in patients with CF the bacteria are not usually mucoid and can be cleared with aggressive antibiotic treatment [P. aeruginosa infection and increased mortality [P. aeruginosa infection by patients with CF is usually from the environment, but recent studies have demonstrated patient to patient transmission of certain strains of P. aeruginosa [P. aeruginosa remains unclear, there is evidence that airborne transmission may be important [P. aeruginosa within artificially generated aerosols; and 2) identify differences in airborne survival of epidemic and non-epidemic strains under controlled environmental conditions.Acquisition of reatment . Once threatment . A numbeortality -9. Acquiruginosa -13. Moreruginosa ,15. Whilmportant ,17 and tP. aeruginosa (NCIMB 10848) was obtained from the National Collection of Industrial and Marine Bacteria. Isolates of both epidemic and non-epidemic strains of P. aeruginosa were obtained from stored strains at the Department of Microbiology, Leeds General Infirmary and the Centre for Infectious Diseases, University of Edinburgh  glycerol solution. The concentrated bacterial suspensions were stored at -20°C until required.Bacteria of each strain examined were harvested from 200 ml of overnight, stationary liquid broth culture, washed, and stored in 5 × 1 mL Eppendorf tubes with 40%  through an Andersen 6-stage impactor [During each sampling event 56.6 L of air was drawn (i.e. at a rate of 28.3 L minimpactor  (Anderseimpactor . The con-1) foetal bovine serum (FBS) containing 105 CFU mL-1 of P. aeruginosa (NCIMB 10848) inoculated from the same frozen sample.In order to assess the impact of variations in the nebuliser suspension fluid, aerosols were generated into the laminar flow apparatus using variously 100 mL of distilled water, 1/8 × Ringers, 1/4 × Ringers, 1/2 × Ringers, 1 × Ringers and 2 × Ringers solutions, and 10% (v v5 CFU mL-1 of P. aeruginosa (NCIMB 10848).In order to assess the impact of variations in temperature on microbial survival, the temperature of the air within the laminar flow model was maintained at either 22 ± 2°C or 27 ± 2°C, and 45% Relative Humidity (RH) by adjustment of the air conditioning controls of the class II aerobiological chamber. Aerosols were generated into the laminar flow model using 100 mL of 1/4 × Ringers containing 105 CFU mL-1 of P. aeruginosa (NCIMB 10848).In order to assess the impact of variations in air humidity on microbial survival, the relative humidity of the air within the laminar flow model was maintained at either 45% RH or 67% RH, and 22 ± 2°C by adjustment of the air conditioning controls of the class II aerobiological chamber. Aerosols were generated into the laminar flow model using 100 mL of 1/4 × Ringers containing 10P. aeruginosa in the laminar flow model aerosols were generated into the laminar flow model using 100 mL of 1/4 × Ringers containing 105 CFU mL-1 of each strain of P. aeruginosa.In order to examine the survival of strains of All data was expressed as a mean and standard error of mean (SEM) The t-Test and one-way ANOVA was used for analysis of different groups . A p-value of < 0.05 was taken to be significant.P. aeruginosa (NCIMB 10848) when generated using distilled water, 1/8 × Ringers, 1/4 × Ringers, 1/2 × Ringers, 1 × Ringers and 2 × Ringers solutions, and 10% FBS predominantly produce aerosols containing particles of less than 2.0 μm in diameter  in the aerosols generated using 1/4 × Ringers and 1/2 × Ringers (p = 0.0705), or 1 × Ringers (p = 0.5198), or 2 × Ringers (p = 0.055). Generation of aerosols using hypotonic solutions resulted in a significant reduction in the concentration of viable P. aeruginosa (NCIMB 10848) isolated from the aerosols compared to the use of isotonic Ringer's solution . The concentration of viable P. aeruginosa (NCIMB 10848) was significantly increased when the aerosols generated using 10% FBS compared to 1/4 × Ringers (p = 0.0040) , compared with conditions of high temperature  and high humidity (22°C. 67%RH). Under all three environmental conditions the aerosols generated predominately contained particles less than 2.0 μm diameter . The concentration of viable P. aeruginosa was greater under conditions of high humidity, but this did not reach statistical significance (p = 0.1340)  (See Figure The mucoid strains of P. aeruginosa can only survive in droplets and not in droplet nuclei, the implication being that respiratory droplet nuclei are not implicated in the transmission of this pathogen [P. aeruginosa can survive within droplet nuclei in artificially generated aerosols, with mucoid strains in particular surviving well in an aerosolised state. In so doing, we have demonstrated that aerial dissemination may be a plausible route of transmission for P. aeruginosa infection.The laminar flow apparatus used in this study enabled comparison of recovered concentrations of viable bacteria, during steady state nebulisation, under controlled environmental conditions. In addition to concentration data, the study produced data regarding the size distribution of the aerosol particles containing viable bacteria in the apparatus. Liquid particles suspended in an aerosol are categorized as being either droplets or droplet nuclei. Droplets are generally considered to be greater than 10 μm in diameter , whereaspathogen . The resP. aeruginosa from bioaerosols using an Andersen 6-stage impactor, it is important to remember that the concentration cultured using the sampler may not be a true representation of the actual concentration of bacteria within the air. It has been estimated that the quantity of bacteria cultured from an air sample represents only approximately 10% of the actual bacterial burden, with the remaining 90% in a viable but non-culturable state [The Andersen 6-stage sampler is a standard piece of equipment and is probably the most widely used aerobiological sampler . While ole state . Gram-neOur experiments demonstrated that the solution used for nebulisation is critical for the survival of the bacteria within aerosols. The use of hypotonic solutions produced a significant reduction in the ability of the bacteria to survive within the aerosol. Inoculation of bacteria into a hypotonic solution will result in an osmotic shock. However, in our experiments we found that this was not sufficient to kill the bacteria, as the control counts from the nebuliser remained stable pre- and post-nebulisation; it may however be that the osmotic shock is enough to leave the bacteria in a weakened state so that subsequent survival in an aerosol is significantly reduced. The reduction in survival may also have been due to the increase in the boiling temperature that occurs with the addition of a solute to a solution. An increase in the boiling temperature will result in a reduced rate of evaporation and hence protect the bacteria from the lethal effects of desiccation. The solution which allowed for the greatest survival of bacteria was 10% FBS. This solution will not only protect the bacteria from osmotic shock and desiccation, but also contains nutrients that will allow the bacteria to continue metabolising whilst suspended in the aerosol. In many respects this represents the case for bioaerosols produced by patients with CF, who will tend to liberate microorganisms in aerosols containing salts and proteinaceous material.P. aeruginosa is adversely effected by a small increase in temperature. However, increased humidity did not statistically improve the survival of P. aeruginosa within the aerosol. Both of these effects may have potential clinical implications as it would suggest patients with CF should avoid cool and damp air conditions in an effort to minimise infection with P. aeruginosa. Consideration should be made to the environmental air conditions when CF units are been designed. Potentially the use of warm and dry air may reduce the risk of cross-infection by P. aeruginosa between patients with CF. It would also seem reasonable to advise patients with CF to avoid the use of devices such as humidifiers as they may promote the survival of P. aeruginosa within aerosols.The environmental conditions of the aerosol are important for survival of bacteria within droplet nuclei. Bacteria are more rapidly desiccated in conditions of high temperature or low relative humidity. The data presented here confirms that the survival of P. aeruginosa with an aerosol. Isolates of the Liverpool, Leeds Paediatric and Unique CF strains expressing both a mucoid and non-mucoid phenotype were studied in the laminar flow apparatus. For all the strains the presence of a mucoid phenotype resulted in significantly increased concentrations in the aerosols. The mucoid strains over-produce the exopolysaccaride alginate which has been implicated in the persistence and pathogenesis of chronic P. aeruginosa infection in patients with CF [P. aeruginosa in aerosols by providing a protective barrier which reduces the lethal effects of desiccation upon the bacteria.The presence of a mucoid phenotype seems to be particularly important for the survival of  with CF . The algP. aeruginosa between patients with CF. There have been two studies that have demonstrated that patients with CF can produce aerosols containing viable P. aeruginosa [Burkholderia cepacia complex can be disseminated from patients with CF during physiotherapy [P. aeruginosa could be cultured from the air 3 hours after a patient had left the area. To survive this length of time suspended within an aerosol the bacteria must be in a droplet nuclei, because larger droplets would have fallen to the floor due the effect of gravity.The data presented from this study provides support for the hypothesis that airborne dissemination may be important for the cross-infection of ruginosa ,17 as weotherapy . A furthotherapy . The samP. aeruginosa used in the present study were lower than those commonly found in sputum samples from patients with CF which can contain approximately 107 CFU g-1 [P. aeruginosa required to cause pulmonary infection in patients with CF is not knownAlthough this study is limited in that the aerosol residency time for the laminar flow apparatus was relatively short at only just over 1 minute, this was however long enough for the bacteria to travel 4 m – conclusively demonstrating that the bacteria were transported in droplet nuclei and not in droplet form. The concentrations of  CFU g-1 . AlthougP. aeruginosa expressing a non-mucoid phenotype. However the expression of the mucoid phenotype does seem important for survival within droplet nuclei. This suggests that particular emphasis should be made on segregating individuals free from P. aeruginosa from those with chronic infection who are more likely to be infected with mucoid strains of P. aeruginosa.There appears to be little difference in airborne survival between the epidemic and non-epidemic strains of P. aeruginosa between patients could include warming and drying the air within clinical areas and avoidance of humidification devices.Finally, our study suggests that environment appears to influence bacterial survival and optimal conditions for reducing airborne transmission of CF: Cystic fibrosis; CFTR: Cystic fibrosis transmembrane conductance regulator; FBS: Foetal bovine serum; NCIMB: National Collection of Industrial and Marine Bacteria; RH: Relative humidity.IJC undertook the studies, performed the statistical analysis and drafted the manuscript. LAF participated in the design of the study and assisted in undertaking the studies, CBB, MD, and DGP participated in the design and coordination of the study. All authors read and approved the manuscript.


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).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%.Thirty-three strains of 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. Pseudomonas aeruginosa is a major human opportunistic pathogen responsible for numerous nosocomial infections, especially within intensive care units [P. aeruginosa is the seventh (i.e. 4.3%) most frequently isolated pathogen from the bloodstream, with a crude mortality exceeding 25% [P. aeruginosa represents 6.8% of bacteremia caused by gram-negative rods [P. aeruginosa bacteremia is clinically indistinguishable from other gram-negative bacterial bloodstream infections, it has been demonstrated that an inappropriate initial antimicrobial therapy was associated with severe adverse outcomes [re units . In US hh i.e. 4.% most frP. aeruginosa, especially in respiratory samples from cystic fibrosis patients. Different molecular targets have been employed such as 16 S rRNA, algD, oprI, oprL, toxA, gyrB, and ecfX genes [oprI genes) as well as false-negative results  have been reported, the ecfX gene is a suitable target for species-specific identification of P. aeruginosa isolates [Because standard phenotypic methods are time consuming and most have inherent limitations, molecular techniques have shown to be a rapid and reliable approach for the identification of bacterial pathogens . TherefofX genes -13. Sincisolates ,13.P. aeruginosa from a positive blood culture (BC) takes at least 24 h using conventional techniques, molecular identification directly from positive BCs could be an interesting alternative leading to a rapid and accurate species-level identification with subsequent adequate empirical treatment. However, PCR detection of P. aeruginosa from positive BCs has been poorly investigated [oprI and oprL as target genes, described previously as non-100% specific [While identification of stigated ,15, and specific ,11.ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive BCsIn this study, we have developed a novel quantitative PCR (qPCR) assay, using P. aeruginosa. Positive bottles were inoculated aerobically and anaerobically at 37°C for 24-48 h onto trypticase-soy agar, Drigalski agar, 5% horse blood agar and chocolate agar plates, and the concentration of bacteria was determined by quantitative culture . Strains were identified by colony morphology, oxidase reaction, and results of the API 20 E system (bioMérieux).From August 2008 to June 2009, a total of 100 positive BCs from 100 inpatients were included. For each patient, a pair of aerobic (BacT/ALERT FA) and anaerobic (BacT/ALERT FN) bottles was taken, then incubated in the BacT/ALERT automated continuous monitoring system . The distribution of positive BCs by bottle type was as follows: 87 aerobic (87%) and 13 (13%) anaerobic. All these BCs showed gram-negative rods at the direct examination, and most of them presented a Gram-staining and/or a motility compatible with those of the species From a 0.5-ml aliquot of blood, template DNA was prepared by using a simple and rapid boiling procedure, taking less than 20 min ,17. BrieecfX gene . Oligonucleotide primers and fluorescent-labeled hybridization probes were designed for amplification and sequence-specific detection of a 152-bp fragment within the P. aeruginosa ATCC 27853 into the commercial plasmid vector, pCR2.1-TOPO plasmid vector from the TOPO TA Cloning Kit  following the manufacturer's recommendations to produce plasmid pRT-ecfX. This plasmid was transformed into competent TOP10 Escherichia coli cells (Invitrogen), and transformants were selected on media containing 30 μg/mL kanamycin. Plasmid DNA was extracted and purified from one transformant using the HiSpeed Midi Kit  according to the manufacturer's instructions, resuspended in elution buffer (Qiagen) and sequenced to determine the presence of the ecfX insert and its orientation. Plasmid DNA concentration was determined by using the NanoDrop ND-1000 spectrophotometer , and the plasmid DNA reference material was then serially diluted to obtain 10 to 1010 plasmid genome equivalents for standard curve analysis. Quantitative analysis was carried out with the LightCycler software version 3.5 (Roche). The ratio of signals measured at 705 nm/signals measured at 530 nm was used to calculate the crossing point values.A plasmid standard curve was constructed by ligating the 152-bp PCR product from P. aeruginosa (including 2 recovered in anaerobic bottles), 53 strains of Enterobacteriaceae (including 22 Escherchia coli), 9 strains of Stenotrophomonas maltophilia, 2 other gram-negative species, and 3 BCs were polymicrobial of which one mixture of Klebsiella pneumoniae and P. aeruginosa  as compared with that of K. pneumoniae (108 CFU/ml). Limit of detection was estimated at 102 CFU/ml  tool with high sensitivity and specificity for the identification of The authors declare that they have no competing interests.Each author acknowledges that he has contributed in a substantial way to the work described in the manuscript and its preparation. All authors have read and approved the final manuscript.