Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients.P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10%) were positive by both culture and qPCR.In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86%) remained P. aeruginosa positive both by culture and qPCR.Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa infection for only a limited number of patients.Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending  Pseudomonas aeruginosa and Staphylococcus aureus are the most important. Chronic lung infection with P. aeruginosa is a major cause of morbidity and mortality among the CF patients [P. aeruginosa is successful in postponing chronic infection. Hence, it is important to detect new infection with P. aeruginosa as early as possible so that eradication treatment can be started as soon as possible [P. aeruginosa in respiratory samples is done by conventional methods such as culture and biochemical characteristics. Misidentification can occur due to the variable phenotypic characteristics of this species [P. aeruginosa from CF patients [Cystic fibrosis (CF) is one of the most common genetic disorders, caused by mutations in the CFTR gene, coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein . Mutatiopatients . It is npossible -7. Curre species . Moreovepatients .P. aeruginosa in the respiratory samples from CF patients, not chronically infected by P. aeruginosa.In this national study, we followed CF patients during periods between 1 to 15 months and we compared the sensitivity of conventional culture techniques with qPCR for the detection of P. aeruginosa free and not chronically infected according to the criteria used by the different Belgian CF centers, i.e., the European Consensus criteria [et al. [P. aeruginosa positive culture were treated according to the standard antibacterial treatment protocols of each center, patients with only a PCR positive result were not treated.From January 2008 until May 2009, sputum, nasopharyngeal or throat swab samples were routinely collected from 397 CF patients attending all but one of Belgian CF-centres, i.e. Ghent University Hospital , Universitair Ziekenhuis Brussel , St Luc University Hospital , Queen Fabiola Children's University Hospital and Erasme University Hospital , Antwerp University Hospital , CF Center Liege . Patients were seen every three months and sputum or nasopharyngeal aspirate/throat swab samples were cultured at every visit. Nasopharyngeal aspirates/throat swab samples were collected in case the patients could not expectorate. All 397 included patients, , were considered as criteria  or those [et al. . For theAfter arrival at the Laboratory Bacteriology Research (LBR), sputum and nasopharyngeal samples were liquefied with Sputasol  . Throat swabs  were vortexed in the liquid transport medium present in the Eswab tube. For microbiological culture, samples were immediately processed after arrival. For qPCR, at least 200 μl of each sample was stored at -80°C prior to DNA-extraction.Fifty μl of the samples were inoculated onto MacConkey Agar plates  and 100 μl into 4 ml Cetrimide Broth  and incubated for at least 24 h at 37°C at ambient atmosphere before examination. Cetrimide Broth was subcultured by inoculating 50 μl onto a Sheep Blood Agar plate (Becton Dickinson), which was also incubated for at least 24 h at 37°C before examination.After a maximum of 5 days incubation, lactose negative colonies on MacConkey Agar were picked, subcultured onto a 5% Sheep Blood Agar plate (Becton Dickinson) and identified using tDNA-PCR .Before DNA-extraction, respiratory samples were pre-incubated with proteinase K, i.e. incubation of 200 μl of each sample during 1 h at 55°C in 200 μl proteinase K buffer  with vortexing every 15 min. DNA was extracted using the protocol Generic 2.0.1 on the bioMérieux easyMAG Nuclisens extractor . Final elution volume was 110 μl. This DNA-extraction protocol had been shown previously to be the most sensitive of five different methods .oprL gene (NP_249664), was performed using primers PAO1 A (5' CAGGTCGGAGCTGTCGTACTC 3') and PAO1 S (5' ACCCGAACGCAGGCTATG 3') and hydrolysis probe oprL TM (5' FAM-AGAAGGTGGTGATCGCACGCAGA-BBQ 3'), manufactured by TIB Molbiol , as described previously [Quantitative PCR (qPCR), targeting the eviously . The reaP. aeruginosa in the respiratory sample is determined as the cycle number whereby the fluorescence signal intensity crosses the detection threshold. This value is expressed as the quantification cycle (Cq). The number of cycles is inversely correlated to the concentration of P. aeruginosa in the sample, e.g. a high cycle number indicates a low the initial concentration of P. aeruginosa in the sputum.Using qPCR, the concentration of et al. [5 Jelly Fish oligonucleotides (105 bp) (IAC-oligo), 0.4 μM forward primer (IAC fw) and 0.4 μM reversed primer (IAC rev) primers were added to the reaction mix, and a separate qPCR experiment, using the SybrGreen kit, was carried out with primers hybridizing to the target DNA. When compared to a set of control samples, i.e. culture and qPCR P. aeruginosa positive samples to which the same amount of IAC had been added, the PCR was considered as inhibited by (the DNA extract of) the sample, when an increase of 3 Cqs could be observed.To exclude PCR inhibition as an explanation for the PCR negative, culture positive samples, the PCR mix, containing the DNA extract of the sample, was spiked with an internal amplification control (IAC), as described by Khot et al. . BrieflyoprL gene of the P. aeruginosa isolates for culture positive, PCR negative samples, oprL PCR was carried out on DNA extracted from the P. aeruginosa isolates, cultured from the same samples.To exclude that PCR negativity was due to primer mismatch with the The study was approved by the ethics committee from Ghent University Hospital (project nr. 2007/503). Written informed consent was obtained from the patients > 18 years, or from the parents for the children.q values were examined using the Mann-Whitney U test and p values of < 0.05 were considered as significant.Differences in CP. aeruginosa negative by culture and by P. aeruginosa specific qPCR and 89 samples (10%) from 64 CF patients were both P. aeruginosa culture and qPCR positive  from 307 patients remained  samples 0% from 6 samples 0% from 6Twenty-six samples (3%), obtained from 26 CF patients, were culture negative but qPCR positive . The follow-up samples of these three patients were culture and qPCR negative. The average qPCR Cq value (31.7) for these 26 samples was significantly higher, compared with the Cq value of culture and qPCR positive samples (26.4)  was/were culture and qPCR negative, whereas for two patients the follow-up sample(s) was/were culture and qPCR positive.Ten samples, obtained from 9 patients, were When taking culture as the gold standard, the PCR had a sensitivity of 90%, a specificity of 85%, a positive predictive value of 77% and a negative predictive value of 99%.For the samples with a dissimilar culture and qPCR result, there was no relation with the presence of other bacterial species isolated from the respiratory samples (data not presented) and there was no linkage with the sample type (data not presented).Pseudomonas aeruginosa in respiratory samples of CF patients has become of utmost importance, taking into account that it is now possible to postpone chronic infection with the use of early aggressive antibiotic treatment [P. aeruginosa. However, other detection methods that might be more sensitive than microbiological culture still need evaluation and validation [Early detection of reatment -7. In molidation .P. aeruginosa antibodies has been proposed as an alternative to culture for the early establishment of new infection episodes. Several groups reported that anti-P. aeruginosa antibodies can be detected prior to P. aeruginosa detection by culture and prior to the onset of chronic infection [Serological testing for nfection -18. Howenfection  found moP. aeruginosa in respiratory samples from CF patients, not yet chronically infected with this organism.In this prospective study, we evaluated whether qPCR can improve early detection of P. aeruginosa have been developed [P. aeruginosa from respiratory samples of CF patients [oprL gene [P. aeruginosa in sputum from CF patients. In our hands, using a dilution series of P. aeruginosa in sputum, the three culture methods were equally sensitive to each other but also to the combination of the most sensitive DNA extraction method and the most sensitive amplification assay, i.e. probe based qPCR.During the last decade, several PCR formats and other molecular methods for the detection of eveloped ,20-30. Spatients ,19, whilpatients  or a lowpatients . In thisprL gene ,21, prevprL gene , we compP. aeruginosa detection by culture and by qPCR is compared in a long term study [P. aeruginosa on average 4.5 months prior to culture. In our opinion, this conclusion should be interpreted with caution, because also in their study only 5 of the 10 culture negative, PCR positive patients became P. aeruginosa culture positive during the follow-up period. It can also be argued whether the cultured strain was identical as the one causing PCR positivity 4-17 months prior to culture positivity, given the long follow-up period and the fact that the average conversion rate to culture positivity among CF patients can be considered as relatively high. Finally, the authors also found 5 culture positive, PCR negative samples, for which PCR might have become positive later on, however no follow-up data were reported. In our study, we found that out of the 26 qPCR positive, culture negative samples, only 5 follow-up samples became also P. aeruginosa culture positive, of which one became double positive only in the third follow-up episode after initial PCR positivity. The significantly higher Cq values of these 26 samples, compared to the Cq values of double positive samples, suggest a low P. aeruginosa inoculum in the respiratory sample and may explain why the presence of P. aeruginosa was missed by culture. Thus, PCR positivity may have had a predictive value for impending infection in only 5 of the 26 patients, whereas in 21 patients a positive PCR signal became negative again and did not predict a positive culture at the next follow-up sample. For three of the 26 qPCR positive, culture negative patients, the previous sample was P. aeruginosa culture and qPCR positive but the follow-up samples were culture and qPCR negative. This may indicate that qPCR still detected DNA of already killed bacteria. Another 10 samples (1%) were P. aeruginosa qPCR negative but culture positive. False negativity of the qPCR was not the reason for the negative qPCR result, because qPCR inhibition and primer mismatch could be excluded. Interestingly, for 5 of these 10 patients, there was discordance between both culture techniques, suggestive for borderline detection by culture and thus a low inoculum of the pathogen. Such discordance between culture results was observed in only 11 out of 89 qPCR positive samples.Surprisingly, there is at present only one published study in which rm study . These aP. aeruginosa isolate might differentially influence the ease with which P. aeruginosa can be detected by culture versus qPCR. Further research is warranted on a larger set of samples with discordant qPCR - bacterial culture results to determine the influence of some of these factors.For many samples with discordant qPCR and culture results, a low bacterial inoculum may be the explanation. Based on our results in this study and a previous study , both apP. aeruginosa in respiratory samples of CF patients, not chronically infected with P. aeruginosa. Looking at it from a different angle, qPCR was both sensitive and specific compared with a gold standard of culture.The present study indicates that the currently used routine culture techniques perform equally well as DNA amplification techniques for detection of These data, gathered on clinical samples, confirm the results of our previous laboratory study in which culture methods were equally sensitive to the combination of the most sensitive DNA extraction method and the most sensitive amplification assay, i.e. probe based qPCR .P. aeruginosa may have a predictive value for impending P. aeruginosa infection in only a limited number of cases.Therefore, we may conclude that for this study, based on a large amount of patients and samples, qPCR for MV, FDB, SVD, PS and PD conceived the study and designed the experiments. MV, FDB, PD, PS, SVD wrote the manuscript. PD, LVS, GLdSS performed the experiments. Authors from other universities provided patient samples and helped with the manuscript discussion. All authors have read and approved the final manuscript.Table S1: Overview of the culture positive and qPCR positive samples. Table S2: Overview of the culture negative and qPCR positive samples. Table S3: Overview of the culture positive and qPCR negative samples. Overview of all samples with at least a P. aeruginosa positive qPCR or a P. aeruginosa positive culture result.Click here for file


Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa  has been found to be widespread in CF patients in eastern Australia.Chronic lung infection with the bacterium P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources.Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples.We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the  P. aeruginosa in cystic fibrosis (CF) clinics in Australia, the UK, Europe and Canada has been reported [P. aeruginosa was first detected following the death of five unrelated children attending the CF Clinic at the Royal Children's Hospital, Melbourne [P. aeruginosa had an identical or closely related strain to that of the five young children [The emergence of "epidemic" strains of reported -8. In Auelbourne ,10. Ensuchildren . We alsochildren . This stchildren . To avoiTwo hospital environmental studies in 1995 and 1999 failed to identify a common source for AES-I, suggesting person-to-person transmission or cross-infection . To haltAES-I strains isolated from CF patient sputa are currently identified via pulsed field gel electrophoresis (PFGE). PFGE is an expensive, time consuming technique requiring highly skilled personnel and thus cannot be used as a means of routine surveillance for the presence of the AES-I strain in patients attending CF clinics. There is an urgent need for the development of a simple, rapid diagnostic method that will enable routine surveillance for AES-I in Australian CF clinics so that appropriate segregation measures can be instituted in an expeditious manner.P. aeruginosa strains [The development of diagnostic PCR-based assays have proven to be effective tools for the rapid detection of pathogens in infectious diseases, including other known clonal  strains ,15-20. OPseudomonas aeruginosa strains used in this study are listed in Table Eschericia coli strain DH5α was used for transformation of the suppression subtractive hybridization (SSH) library.® Elute cards. Miniprep DNA of SSH clones was prepared using the QIAprep Spin Miniprep Kit (Qiagen Sciences).Chromosomal DNA for SSH was isolated using the Masterpure DNA Purification Kit (Epicentre Technologies). Chromosomal DNA used for PCR was isolated using Masterpure DNA Purification Kit, DNeasy Tissue Purification Kit (Qiagen Sciences) or Whatman FTARsaI fragments according to manufacturer's instructions but with a hybridization temperature of 73°C. The library of SSH PCR amplicons was cloned in pGEM-T Easy (Promega). Individual clones were miniprepped and sequenced. Nucleotide sequences were determined by use of a PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer's instructions and separated by capillary electrophoresis on an Applied Biosystems 3730S Genetic Analyser at the Monash University Micromon sequencing facility. Sequence data was analysed using MacVector (Accelrys). To determine the presence or absence of the SSH clone sequence in the PAO1 genome, sequences were used in BLASTN searches at the Pseudomonas genome project web-site [http://www.ncbi.nlm.nih.gov.The AES-I isolate (strain 973) was used as the tester strain for SSH. This strain was chosen from a collection obtained from the Royal Children's Hospital, Melbourne and had been previously identified as the clonal strain AES-I . The driweb-site . AES-I sweb-site  of the NoprL gene have been described previously [Oligonucleotides (Sigma Proligo) and annealing temperatures used in the PCR assays are listed in Additional File eviously . AmplifiSpeI and DraI was performed as previously described [P. aeruginosa strain 973 [Molecular typing by PFGE following digestion with escribed . An isolrain 973 .® Elute cards and allowed to air dry. DNA from a 2 mm diameter disc from each card was eluted into 50 μL of water as per the manufacturer's instructions and added to PCR tubes containing reaction mixtures for the PCR amplification of either oprL or the SSH-identified AES-1 genomic locus HW2. P. aeruginosa strains were also isolated from the sputum samples and typed by PFGE to validate the PCR diagnosis.Samples of sputum were swabbed onto Whatman FTA® MGB probes (Applied Biosystems) were designed from regions of the sequences of the HW2 locus and the oprL gene using the Primer Express® Software v1.0 program (Applied Biosystems). Probe RTHW2-P  of template DNA. To monitor PCR inhibition the TaqMan® Exogenous Internal Positive Control (IPC) (Applied Biosystems) was multiplexed in the HW2 assay. The oprL assay was performed as a singleplex reaction. Amplification and detection were performed with the Eppendorf Realplex Mastercycler using the following program: 1 cycle of 95°C for 15 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Negative controls were included in each assay. PCR products were also visualized by agarose gel electrophoresis to verify product amplification of the expected size. Cycle thresholds (Ct) from experiments to determine method sensitivity were reported as the average and standard deviation of three biological repeats. Correlation coefficients between Ct and DNA template concentration for each TaqMan assay were calculated by regression analysis and amplification efficiency was calculated using the formula: efficiency = -1 + 10^(-1/slope). Primers and probes for quantitative real-time PCR for AES-I are available as a kit through Cardinal Bioresearch http://www.cardinalbioresearch.com.au.Primers and TaqManP. aeruginosa. We sequenced 20 clones from the SSH clone library and identified 14 that contained AES-I sequences not present in PAO1  of DNA from AES-I isolate 973 against PAO1, the sequenced reference strain of  cluster  and thus cluster . Other hWe designed PCR primer pairs to amplify each of the 9 different AES-I genetic sequences that were not found in PAO1 and that did not localise to the O6 antigen gene cluster. We first performed PCRs with each primer set using the AES-I SSH driver strain (isolate 973) and PAO1 genomic DNA to confirm that the PCR reaction worked and was specific to AES-I. One of the primers sets (HW1/11/20) amplified a non-specific product in PAO1 and was not used in further assays.oprL gene that is present in all P. aeruginosa strains [We next performed an initial PCR profiling with the remaining 8 AES-I PCR primer sets on a collection of 9 AES-I strains (including driver strain), 9 non-AES-I CF isolates, 7 non-CF clinical isolates, 6 environmental isolates and the common laboratory strains PAO1, PA103, PAK and ATCC 27853  or non-AES-I (20) [P. aeruginosa strains tested whereas HW3 was found to be present in all but 4 AES-I strains and none of the other P. aeruginosa strains  , 9 repre0 , 9 repS-I (20)  as well P. aeruginosa by standard techniques [P. aeruginosa isolates of which 8 were identified to be AES-I. PCR with the oprL, HW2 and HW3 primer sets was performed on DNA purified from the same set of P. aeruginosa isolates , has been shown to be an efficient method to store, transport and isolate DNA from sputum samples suspected to harbor Mycobacterium tuberculosis [® Elute technology has further simplified the elution of DNA for PCR and is readily adaptable for routine pathology testing. We used PCR assays for the oprL and HW2 loci to determine if we could detect the presence of P. aeruginosa and AES-I directly from sputum samples stored on Whatman FTA® Elute cards. Sputum samples from Monash Medical Centre CF clinic patients that were positive or negative for the AES-I strain as determined by PFGE, were swabbed onto Whatman FTA® Elute cards, DNA eluted and used in the oprL PCR assay to determine the presence of P. aeruginosa in the sputum sample and in a 2nd PCR assay with the HW2 AES-I primers to determine if the P. aeruginosa present in the sputum was AES-I .To improve the diagnostic utility of the HW2 PCR we then developed real-time PCR TaqMan assays for rL Table . Based oP. aeruginosa PAO1, P. putida and P. fluorescens. The HW2 TaqMan assay was negative for these samples while oprL was positive for both P. aeruginosa and P. putida. DNA was then extracted from Whatman FTA® Elute cards spiked with AES-I positive and unique strains of P. aeruginosa. As predicted, all extracts were oprL positive and only extracts from P. aeruginosa AES-I strains were HW2 and oprL positive. The same pattern of results was obtained with DNA extracted from the AES-I positive and negative sputum samples stored on Whatman FTA® Elute cards used in the conventional PCR assay  indicating that this locus is present in a region of some variability between AES-I isolates. The HW2 locus was found to be present in all 35 strains determined by PFGE to be AES-I and absent in all 153 non AES-I P. aeruginosa strains tested. These results indicate that PCR for the HW2 locus is 100% specific and 100% sensitive for detection of the AES-I epidemic P. aeruginosa strain.AES-I is a highly transmissible virulent strain of troduced ,10. We htroduced . There iThe collection of strains used in this study included isolates from CF individuals that were identified in our initial 1999 clinic surveillance to harbour the AES-I strain  as well oprL PCR assay in our study was to act as a positive control for DNA quality to avoid false negative results in our PCR screens. Since we commenced our clinical surveillance study in 2005, it has been shown that the ecfX locus is more specific than oprL to identify P. aeruginosa from various environmental and clinical extracts [ecfX locus could be substituted for the oprL assay to provide greater specificity for the presence of P. aeruginosa in sputum samples if desired.The main purpose of the extracts . AccordioprL and HW2 loci can be used for the direct detection of P. aeruginosa and AES-I in CF sputum swabbed onto Whatman FTA® Elute cards. We are now further assessing the potential for these assays for use as routine surveillance tools for P. aeruginosa and AES-I in CF sputum. The use of Whatman FTA® Elute cards for sputum storage, transport and DNA template purification for PCR is simple and relatively inexpensive and could be easily adapted for detection of other bacterial pathogens from a broad range of respiratory and other infections.Compared with PFGE which can take 6 to 8 days to obtain a diagnosis from time of receipt of patient sputum, PCR-based assays have many benefits as potential diagnostic tools as they are simple, relatively inexpensive, do not require highly skilled personnel, reduce handling and detection errors and can significantly reduce the time required to identify patients harbouring the AES-I strain. We have shown that PCR amplification of the Monash University has a patent application pending for the AES-I sequences identified in this study. CBW will receive a percentage of royalties arising from commercialisation of the AES-I diagnostic kit according to Monash University policy. The other authors of this manuscript declare no personal, professional or financial relationships that cause a conflict of interest that could bias this work.HLW carried out the subtractive hybridization study, designed the PCR screen and performed the initial screening of lab and clinical isolates. LT participated in the design of the study, carried out screening of the clinical isolates, analysed the data and helped to draft the manuscript. SJT screened additional clinical isolates and carried out the real-time PCR conversion of the assay and screened clinical isolates. AM co-ordinated collection of samples from patients and helped with data analysis and interpretation. TS participated in design and co-ordination of the real-time PCR assay conversion and helped draft the manuscript. DSA participated in the concept and design of the study and data interpretation. CBW conceived, designed and co-ordinated the study and drafted the manuscript. All authors have read and approved the final manuscript.PCR Primer sequences used in this study.Click here for fileHomologies of AES-I genomic sequences identified by suppression subtractive hybridization (SSH).Click here for file


In acute lung injury, repair of the damaged alveolar-capillary barrier is an essential part of recovery. Endostatin is a 20 to 28 kDa proteolytic fragment of the basement membrane collagen XVIII, which has been shown to inhibit angiogenesis via action on endothelial cells. We hypothesised that endostatin may have a role in inhibiting lung repair in patients with lung injury. The aims of the study were to determine if endostatin is elevated in the plasma/bronchoalveolar lavage fluid of patients with acute lung injury and ascertain whether the levels reflect the severity of injury and alveolar inflammation, and to assess if endostatin changes occur early after the injurious lung stimuli of one lung ventilation and lipopolysaccharide (LPS) challenge.Endostatin was measured by ELISA and western blotting.Endostatin is elevated within the plasma and bronchoalveolar lavage fluid of patients with acute lung injury. Lavage endostatin reflected the degree of alveolar neutrophilia and the extent of the loss of protein selectivity of the alveolar-capillary barrier. Plasma levels of endostatin correlated with the severity of physiological derangement. Western blotting confirmed elevated type XVIII collagen precursor levels in the plasma and lavage and multiple endostatin-like fragments in the lavage of patients. One lung ventilation and LPS challenge rapidly induce increases in lung endostatin levels.Endostatin may adversely affect both alveolar barrier endothelial and epithelial cells, so its presence within both the circulation and the lung may have a pathophysiological role in acute lung injury that warrants further evaluation. Acute lung injury (ALI) is characterised by neutrophilic inflammation of the alveolar-capillary barrier. ALI has multiple aetiologies, but appears to follow a uniform pattern of injury at a cellular level. Extensive damage to the alveolar-capillary barrier leads to the influx of a protein-rich oedema fluid and accompanying inflammatory cells into the alveoli. A complex cascade of both inflammatory and anti-inflammatory cytokines is triggered and inflammatory cells, including neutrophils and monocytes, are recruited to the alveoli. Studying these processes early in the course of the disease can be challenging because most insults causing lung injury are not predictably timed.Two human models of lung injury allow assessment of the early phases of ALI. One lung ventilation (OLV) during oesophagectomy is associated with a significant post-operative risk of ALI with proposed causative mechanisms including the ischaemic/reperfusion insult experienced by the collapsed lung, oxidative stress injury and barotrauma to the ventilated lung . SystemiThe neutrophilic inflammation of the alveolar capillary barrier in ALI and the models of OLV or LPS challenge result in the release of proteases including collagenases . The balin vitro [There are at least 27 different species of collagen. Types I and III predominate within both healthy and fibrotic lung . Perivasin vitro ,18.Resolution of ALI is dependent on the successful repair of a confluent barrier of alveolar epithelial and endothelial cells . This prAll patients enrolled in the study gave written informed consent themselves at the time of enrollment, or it was given by the consultant in charge of the intensive care unit (ICU) (not part of the research team) as their legal guardian if they were unable to give consent because of sedation and ventilation. In addition, in all cases, patients' next of kin gave informed signed assent to inclusion. In the UK. patients' relatives cannot consent for incapacitated relatives. In addition, those patients who recovered mental competency during their inpatient stay retrospectively provided signed consent for their inclusion in the study. This study was fully approved by the local National Health Service trusts and ethical review committee.Patients were studied within 48 hours of admission to the ICU of Birmingham Heartlands and University Hospital, Birmingham, UK, between 2006 and 2008. 38 patients  16 years, 12 with direct and 21 with remote lung injury) were identified as having ALI according to the American-European consensus statement [2): fraction of inspired oxygen (FiO2) ratio was recorded on the day of bronchoscopy. A summary of patient physiological severity is shown in Table Patient demographic characteristics were recorded at baseline. The acute physiology and chronic health evaluation II (APACHE II) and simplified acute physiology score II (SAPS II) were recorded as global markers of disease severity. The Murray lung injury score, partial pressure of arterial oxgen  1 94% predicted, mean 25 pack-year smoking history) undergoing elective oesophagectomy for oesophageal carcinoma underwent BAL of the collapsed lung at the end of the operation, which as such potentially suffers from ischaemia-reperfusion injury. Ventilation was performed with pressure control for eight patients and two patients with volume control. Mean peak airway pressures were 25.8 cm, standard error of the mean (SEM) 0.88 cm, and a mean tidal volume of 430 ml was achieved. Mean duration of OLV was 189 minutes, SEM 22.29 minutes. The preoperative PaOWe include data from a human LPS challenge study assessing the anti-inflammatory effects of simvastatin versus placebo in healthy volunteers. Data are presented from 10 patients treated with placebo  who underwent bronchoscopy six hours post-inhalation of LPS. Full details of the patient characteristics and lavage fluid abnormalities are available in another paper .Bronchoscopy was performed by instilling three 50 ml aliquots into the right middle lobe . Bronchoalveolar lavage fluid (BALF) was processed as described previously . Into liEscherichia coli serotype O26:B6; Sigma Chemicals, Poole, Dorset, UK) was dissolved in endotoxin-free sterile 0.9% saline and inhaled via an automatic inhalation–synchronised dosimeter nebuliser , which delivers particles of a mass median aerodynamic diameter of 10 mm, as described previously. The dosimeter produces a calibrated aerosol of 8 μl at each slow inhalation starting from functional residual capacity to total lung capacity. Each subject performed five successive inhalations of the LPS solution (1.25 mg/ml) through the mouthpiece with a nose clip in place. The total dose of inhaled LPS was 50 μg. BAL was performed six hours after LPS inhalation according to standard guidelines by a single researcher (DM).LPS . The epithelial lining fluid (ELF) levels of endostatin were estimated using the following formula: ELF endostatin = BAL urea (mmol/L) × BAL endostatin/plasma urea concentration (mmol/L), as described previously . BALF prEndostatin and IL-8 were measured by using an ELISA kit  according to manufacturer's instructions. CV for intra-assay variability in BALF of endostatin is 3.6% and inter-assay variability was 8%. Spiking ALI BALF with recombinant endostatin standard (2.5 ng/ml) has a 96% recovery rate.Immunoprecipitation and western blotting of plasma and BALF was performed using methods described previously ,28. The A 25 μl sample of BALF was loaded onto polyacrylamide gel and the proteins were separated on a 7 to 12% SDS-PAGE under reducing conditions, electrotransferred to a nitrocellulose membrane  and probed with rabbit polyclonal antibodies against endostatin (HES.6)  and humaCollagen XVIII was immunoprecipitated from human plasma using a monoclonal anti-ALL type XVIII antibody, DB144-N2 bound to anti-mouse IgG-coated magnetic beads , as described elsewhere . The bouP < 0.05.The Ryan-Joiner normality test was used to test the distribution of the data. Non-parametric Mann-Whitney U tests were used for data that was not normally distributed (ELISA endostatin ALI plasma data and BALF data) and are quoted as medians (interquartile ranges (IQR)). Normally distributed data  are quoted as mean (SEM) and compared using analysis of variance (ANOVA). Statistical significance assumed at P = 0.008) and patients at risk from ALI . On day four, plasma levels in ALI patients remained elevated  compared with normal controls  and SAPS II  but not with lung injury score  (data not shown). Plasma endostatin levels at the onset of ALI or at day four did not predict whether patients subsequently survived or died.There were significant linear associations between plasma endostatin at the onset of ALI and the global severity of illness markers APACHE II  and day 4  than healthy individuals (0.08 ng/ml). ALI day 0 endostatin levels (2.6 ng/ml) were higher than those at risk of ALI . There was no difference between BALF levels at day 0 or day 4 between patients who died or survived, or those with direct or remote lung injury. BALF endostatin did not correlate with APACHE II, SAPS II or lung injury score at day 0 or day 4.Endostatin levels in ALI BALF fell from day 0 to day 4 . In addition, day 0 BALF endostatin levels correlated with the degree of BAL neutrophilia  but not with total cell count or BALF IL-8. At day 4 endostatin also correlated strongly with protein permeability index  and total neutrophil counts  (data not shown).There was also a significant relationship between day 0 BALF endostatin levels and protein permeability index . When ELF levels were lower than plasma levels there was a significant correlation  between plasma and ELF endostatin at the onset of ALI (data not shown). However, in some patients, calculated ELF levels were actually higher than plasma suggesting intra-alveolar generation of endostatin. Interestingly, at day 4 ELF endostatin correlated with lung injury score  but not in ALI patients at day 0.Plasma levels of endostatin were significantly higher than calculated ELF levels in the lung  diff = 18 to 169; P = 0.013 vs normal; PPI in LPS: median = 0.0025, IQR = 0.0013 to 0.003, P = 0.011 versus normal; normal PPI = 0.0014, IQR = 0.0008 to 0.0024). Percentage of neutrophils in BAL rose from 1.1% (IQR = 0.0 to 2.65) in normal BALF to median 11% (IQR = 2 to 60.5) in OLV (P = 0.011), and 41% (IQR = 24 to 57) in LPS challenge as described previously [OLV and LPS challenge induced a BAL neutrophilia and an increase in protein permeability index (PPI) compared with normal controls  in LPS-challenged patients. OLV increased BAL endostatin  but not in OLV patients. In contrast, plasma levels of endostatin were not altered by LPS challenge or OLV (data not shown).Inhalation challenge with LPS increased BALF endostatin from a median of 0.08 ng/ml in normal individuals to 0.385 ng/ml   = 198, SE 38) compared with normal plasma  in normal controls  (data not shown). The main bands of 55 kDa, also detectable with anti-LONG antibody, and 72 kDa were interpreted to represent proteolytic N-terminal fragments from the middle form and the short form of collagen XVIII, respectively  in ALI compared with 41.21 (SE 4.8)  end of type XVIII collagen revealed increased levels of C-terminal endostatin-containing fragments of collagen XVIII are present within the BALF of ALI patients with a fragmentation pattern that was different to normal controls.P = 0.02) . Whole lane densitometric analysis showed that significantly elevated amounts of type XVII carboxy terminal endostatin-like fragments were present in ALI samples  compared with normal BALF , which have been implicated in the pathogenesis of ALI ,34,35. WWhat are the implications of our findings for alveolar capillary repair in ALI? Previous studies using the animal corneal micropocket assay have demonstrated that BALF has a strong angiogenic potential that is related to elevated CXC chemokine levels . In contin vitro wound repair responses of both distal small airway epithelial cells, and primary human type II epithelial cells [In addition to effects on endothelial cells, we have recently reported that endostatin inhibits the proliferation and al cells . Thus elThis study has several limitations. Firstly, despite several attempts, we were unable to detect plasma endostatin fragments by immunoprecipitation and western blotting with the HES.6 antibody, which appears unsuitable for plasma endostatin estimation.Secondly, there were a number of drop outs in our sequential assessments, for clinical reasons . We do not believe the results have been biased by this because there were no baseline differences in endostatin between those re-studied at day 4 and those not. Thirdly, it would be interesting to observe the effect of neutralising endostatin bioactivity in BALF on endothelial cell viability. Unfortunately, no effective specific inhibitor for the bioactivity of endostatin is currently available as endostatin has a wide variety of effects on endothelial cells influencing nearly 12% of the genome via a variety of mechanisms . ClearlyTo our knowledge, this is the first report of the presence of endostatin in plasma and BALF of humans with ALI. It is clear that ALI patients have persistently elevated alveolar endostatin levels during the early course of the disease. Our models of the early onset of lung injury suggest these changes occur very early in the injurious process. As endostatin may adversely affect both alveolar-barrier endothelial and epithelial cells, its presence within both the circulation and the lung may have a pathophysiological role in ALI that warrants further evaluation.• Endostatin is elevated within the plasma and BALF of patients with ALI.• Endostatin levels in ALI BALF reflected the degree of neutrophilia and the extent of the loss of protein selectivity of the alveolar-capillary barrier.• Plasma levels of endostatin at the onset of ALI were associated with the severity of physiological derangement.• Western blotting confirmed elevated type XVIII collagen precursor levels and multiple amino- and carboxy-terminal fragments in plasma and BALF.• Increases in endostatin occur early after OLV and LPS challenge in human volunteers but this is compartmentalised to the lung.2: fraction of inspired oxygen; ICU: intensive care unit; Ig: immunoglobulin; IL: interleukin; IQR: interquartile range; LPS: lipopolysaccharide; MAPK: mitogen activated protein kinase; MMP: matrix metalloproteinases; OD: optical density; OLV: one lung ventilation; PaO2: partial pressureof arterial oxygen; PBS: phosphate-buffered saline; SAPS II: simplified acute physiology score II; SD: standard deviation; SEM: standard error of the mean; TNF: tumour necrosis factor; VEGF: vascular endothelial growth factor.ANOVA: analysis of variance; ALI: acute lung injury; APACHE II: acute physiology and chronic health evaluation II; BAL: bronchoalveolar lavage; BALF: bronchoalveolar lavage fluid; ELF: epithelial lining fluid; ELISA: enzyme-linked immunosorbent assay; FEV: forced expiratory volume; FiOThe authors declare that they have no competing interests.GDP, NN, SS, DM, AR, WT, MM and DRT recruited patients and performed bronchoscopy. GDP, NN and AR performed ELISA measurements. RH performed the western blot analyses. All authors contributed to writing the paper. GDP NN and DRT performed the statistical analyses. NN and GDP contributed equally to the paper.


Pseudomonas aeruginosa, a non-fermentative, gram-negative rod, is responsible for a wide variety of clinical syndromes in NICU patients, including sepsis, pneumonia, meningitis, diarrhea, conjunctivitis and skin infections. An increased number of infections and colonisations by P. aeruginosa has been observed in the neonatal intensive care unit (NICU) of our university hospital between 2005 and 2007.Hand disinfection compliance before and after an educational programme on hand hygiene was evaluated. Identification of microrganisms was performed using conventional methods. Antibiotic susceptibility was evaluated by MIC microdilution. Genotyping was performed by PFGE analysis.Pseudomonas aeruginosa in the NICU of the Federico II University hospital  and the infection control measures adopted to stop the spreading of P. aeruginosa in the ward were described. From July 2005 to June 2007, P. aeruginosa was isolated from 135 neonates and caused severe infections in 11 of them. Macrorestriction analysis of clinical isolates from 90 neonates identified 20 distinct genotypes, one major PFGE type (A) being isolated from 48 patients and responsible for 4 infections in 4 of them, four other distinct recurrent genotypes being isolated in 6 to 4 patients. Seven environmental strains were isolated from the hand of a nurse and from three sinks on two occasions, two of these showing PFGE profiles A and G identical to two clinical isolates responsible for infection. The successful control of the outbreak was achieved through implementation of active surveillance of healthcare-associated infections in the ward together with environmental microbiological sampling and an intense educational programme on hand disinfection among the staff members.The molecular epidemiology of P. aeruginosa infections in the NICU were caused by the cross-transmission of an epidemic clone in 4 neonates, and by the selection of sporadic clones in 7 others. An infection control programme that included active surveillance and strict adherence to hand disinfection policies was effective in controlling NICU-acquired infections and colonisations caused by P. aeruginosa. Klebsiella pneumoniae and Serratia marcescens in the NICU of the Federico II University Hospital of Naples between 2002 and 2004 [Gram-negative bacteria have become relevant causes of healthcare-associated infections in the neonatal intensive care unit (NICU) environment. ,2. Outbrand 2004 .Pseudomonas aeruginosa, a non-fermentative, gram-negative rod, is responsible for a wide variety of clinical syndromes in NICU patients, including sepsis, pneumonia, meningitis, diarrhea, conjunctivitis and skin infections [P. aeruginosa in NICU settings have been much less reported up-to-date and have been associated with both environmental reservoirs and healthcare workers' carriage [fections . Neverthcarriage -10.P. aeruginosa has been observed in the NICU of our university hospital between 2005 and 2007. The aims of this study were: i) to analyze the molecular epidemiology and antimicrobial susceptibility patterns of P. aeruginosa isolates; ii) to describe the infection control measures undertaken to limit P. aeruginosa spread in the ward.An increased number of infections and colonisations by P. aeruginosa isolated from surveillance swabs and from clinical samples  of babies in the NICU were included in the study. Environmental samples were obtained from the following sites: air, room surfaces, sinks, hand disinfectants, baby incubators, monitors, and staff hands. All three sinks present in the ward were sampled.The tertiary-level NICU of the University 'Federico II' hospital of Naples, Italy serves approximately 350 admissions per year including both inborn and outborn patients and consists of three rooms with a maximum capacity of eight neonates per room. Sinks, chlorhexidine/alcohol hand disinfectants and gloves are available in each room. Sinks, 4% chlorhexidine hand disinfectants, and gloves are available in each room, together with hand disinfection instructions for staff members and visiting parents. Healthcare-associated infections were defined using standard Centers for Disease Control and Prevention definitions adapted to neonatal pathology . InfectiAn educational programme on hand disinfection was repeatedly performed as part of the ward's plan for healthcare-associated infections control. It involved both medical and nursing staffs, it was carried out each time for one week, and it consisted of a 30 minutes review of the main topics on hand disinfection according to the US Centers for Disease Control and Prevention (CDC) recommendations . Hand diHand disinfection compliance was defined as hand-washing with the appropriate quantity of 4% chlorhexidine disinfectant and water for the recommended time, before and after each patient contact and it was evaluated by carrying out an observational study. Moreover, surveillance cultures from 30 randomly selected healthcare workers' hands were obtained by means of contact plates before and after each educational programme.A trained, disguised observer monitored staff's (doctors and nurses) compliance to hand disinfection for the two weeks preceding and the two weeks following each series of educational meetings. Four patients were randomly selected each day (Monday through Friday) and all healthcare workers (HCWs) who cared for the target neonates were observed for 1 hour period during morning shifts and their compliance to hand disinfection procedures before and after each patient contact was recorded on a dedicated form.Data were analysed using SPSS 11.0 . Pearson's Chi-squared test was used to compare hand hygiene compliance before the beginning of the educational programme and after the first and the last series of educational meetings. Pearson's correlation coefficient between hand disinfection compliance in the same periods and colonisation rates was also calculated. All results were considered to be statistically significant at p < 0.05.Isolates were identified using the API NE manual identification system  or the Phoenix automatic system . The susceptibililty of isolates to 12 selected antimicrobials was determined by the standard antimicrobial susceptibility methods . The antXbaI enzyme and pulsed-field gel electrophoresis (PFGE) was performed on all P. aeruginosa infection isolates and on available surveillance culture isolates as previously described [DNA macrorestriction with escribed .P. aeruginosa has circulated in the ward in an endemic fashion, being responsible for a mean colonisation rate of 10.6% and for two ocular infections (data not shown). During the study period (July 2005 to June 2007), 616 neonates were prospectively surveilled for healthcare-associated infections  and 568 of them had their weekly surveillance swabs taken at least once. One hundred-thirty-five neonates  became colonised by P. aeruginosa during their stay in the ward , after Candida spp. (21.8%) and Escherichia coli (16.8%). Moreover, no pathogen was identified in 18.8% of infants diagnosed as having an infection. P. aeruginosa was responsible for 11 severe infections in 11 neonates, with 3 and 4 of them having their birth-weights below 1000 and 750 grams, respectively , all 11 infected patients had positive cultures for P. aeruginosa at nasal/pharynx or rectum surveillance swabs.In the two years preceding the outbreak period P. aeruginosa isolates) identified twenty PFGE types, named A through T, which showed up to six fragments variation in macrorestriction pattern , gentamicin (MIC > 8) and ciprofloxacin (MIC > 2); in addition, PFGE type G appeared to be resistant to piperacillin/tazobactam (MIC > 64/4). Moreover, such resistant profiles were responsible for two sepsis in two neonates with extremely low birth-weight, both having a fatal outcome Table .P. aeruginosa during the fourth quarter of 2005, a combination of targeted infection control measures were undertaken .Although no further isolations of multi-drug resistant d Figure . ReportiP. aeruginosa at the following sites: three sinks on both occasions and a nurse's hand on the second sampling. Genotyping of such strains demonstrated that the isolate recovered from one of the sinks on the first environmental sampling displayed an A profile and the one from the nurse's hand on the second environmental sampling displayed a G profile. The other environmental isolates showed different profiles, not corresponding to any of the profiles isolated from patients' surveillance swabs or clinical specimens.Environmental microbiological sampling identified P. aeruginosa were diagnosed and colonisation rate returned to pre-epidemic values.No further targeted control measures were undertaken after June 2007 as no other infections by P. aeruginosa of PFGE profile G was isolated only once from surveillance cultures of a total of 90 randomly selected healthcare workers' hands before and after each educational programme, as described above. In addition, other 16 surveillance cultures proved to be inappropriate . Hand disinfection compliance of HCWs before the first educational programme proved to be 23.4% and 11.7% before and after each patient contact, respectively . Conversely, no significant correlation was found between the latter and improvement in hand disinfection compliance after patient contact . Finally, hand disinfection compliance of HCWs before and after patient contact proved to be significantly different at all times of observation  and 7 of them were of extremely low birth weight (ELBW), i.e. below 1000 grams. ELBW neonates have actually been shown to have a significantly increased risk of acquiring P. aeruginosa when compared to higher birth-weight infants [P. aeruginosa was of 57%. Although no attributable mortality rate was calculated, three of the four neonates died soon after (0–24 hours) the infection was diagnosed, thus our data indirectly confirm other Authors' findings regarding the very high mortality rates related to P. aeruginosa infections [P. aeruginosa antibiotypes, both displaying resistance to imipenem, meropenem, gentamicin and ciprofloxacin, have probably affected the final outcome in two of the four fatal cases. We did not analyse mechanisms of resistance as no further such phenotypes were identified. Nevertheless, to our knowledge, this is one of the first accounts on two carbapenem-resistant P. aeruginosa genetically unrelated strains which caused two sepsis in a NICU.The overall incidence rate of tely 10% ,15, whiltely 10% . Higher tely 10% ,17, but  infants . MoreoveP. aeruginosa frequently causes multi-clone outbreaks, with the concurrent isolation of genetically distinct strains among patients and healthcare workers (HCWs) and in the environment. During a 15 months-long epidemic in a NICU, Moolenaar et al. [P. aeruginosa genotypes A, B, and C, isolated in 75%, 15%, and 10% of case-patients, respectively. Such genotypes were also found on three nurses' hands, while two positive environmental isolates showed two distinct genotypes, unrelated to any human isolate. Moreover, Foca et al. [P. aeruginosa PFGE profiles over a 33 months-period in the NICU, showing the presence of a major clone, which was also isolated from the hands of a nurse, of two other PFGE types, and of eight unique clones. No environmental specimen proved to be positive for P. aeruginosa. Our study, covering a 24 months time span, identified a predominant PFGE type which was responsible for 36% of infections by P. aeruginosa and at least 35% of colonisations by the same pathogen. Such PFGE profile was also found in one sink, but not on any nurse's hand and circulated in the ward together with less recurrent and with sporadic strains, which caused the remaining infections and colonisations. Other five environmental samples proved to be positive for distinct P. aeruginosa PFGE types, unrelated to the ones colonising or infecting the patients. Transmission of P. aeruginosa from environmental sources to patients and HCWs has been thoroughly described [P. aeruginosa reservoirs, both environmental and human, and, owing to the long time period between the appearance in neonates (July 2005) and the environmental isolation (2nd quarter 2006), we are not able to understand whether the only sink sample found to be positive for P. aeruginosa PFGE profile A has been a result, rather than the origin, of the pathogen's circulation in the ward.r et al.  identifia et al.  describeescribed ,20. Our P. aeruginosa in the NICU setting [P. aeruginosa, displaying the G genotype. The culture was drawn when the epidemic was at its peak value, with nearly 50% of neonates being colonised. The detection of HCWs who are colonised at any body site by P. aeruginosa epidemic clones may sometimes identify the pathogen's reservoir, thus enabling a successful and timely outbreak containment [P. aeruginosa epidemic clone has not been recognized as an independent risk factor for acquiring P. aeruginosa colonization or infection [P. aeruginosa epidemic clone on multivariate analysis [P. aeruginosa may be underestimated during outbreaks investigations and that reinforcement of hand disinfection and of correct gloves use should always be promptly initiated when an increased number of P. aeruginosa isolations is detected. In agreement with previous data [P. aeruginosa circulation of in the ward, the hand disinfection educational programme was the most effective one. The programme started during the fourth quarter of 2006, when the outbreak was at its peak value, and by the end of the second quarter of 2007 the outbreak was over. Owing to the programme's success, the meaning of different hand disinfection compliance rates before and after patient contact and how this may have affected P. aeruginosa circulation in the ward were not further investigated. A possible explanation for the partial ineffectiveness of the traditional infection control interventions may be the molecular heterogeneity of P. aeruginosa outbreaks. Thus, the timely identification of increased isolation of this pathogen, achieved by means of active surveillance, appears to be crucial to limit the spreading of P. aeruginosa in NICU settings.Healthcare workers' (HCWs) hands have been frequently implicated in the spreading of  setting ,10,17. Anfection . In turnous data , our stuP. aeruginosa outbreaks.This study suggests that an infection control programme based on active surveillance and strict adherence to hand disinfection and gloves use policies and supported by environmental sampling and molecular analysis is effective in controlling NICU multi-clone The authors declare that they have no competing interests.P. aeruginosa strains and carried out the antimicrobial susceptibility experiments, ADP performed the PFGE experiments, VC, MT and RZ conceived the study and participated in its design and coordination, VC and RZ drafted the manuscript. All authors read and approved the final manuscript.VC, AS, AC and MDR carried out the active surveillance of healthcare-associated infections and infection control interventions in the NICU, AL, ADP and TB isolated the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/70/prepub


Pseudomonas aeruginosa and Acinetobacter baumanii are important nosocomial pathogens with wide intrinsic resistance. However, due to the dissemination of the acquired resistance mechanisms, such as extended-spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production, multidrug resistant strains have been isolated more often.P. aeruginosa, PER-1 producing A. baumannii, SHV-5-producing Klebsiella pneumoniae and VIM-2-producing P. aeruginosa isolates were subcultured from the patient's samples in Hungary. Comparing the pulsed-field gel electrophoresis (PFGE) patterns of the P. aeruginosa strains from the patient to the P. aeruginosa strains occurring in this hospital, we can state that the PER-1-producing P. aeruginosa and VIM-2-producing P. aeruginosa had external origin.We report a case of a Hungarian tourist, who was initially hospitalized in Egypt and later transferred to Hungary. On the day of admission PER-1-producing P. aeruginosa,and PER-1-producing A. baumanii strains in Hungary. This case highlights the importance of spreading of the beta-lactamase-mediated resistance mechanisms between countries and continents, showing the importance of careful screening and the isolation of patients arriving from a different country.This is the first report of PER-1-producing  Pseudomonas aeruginosa and Acinetobacter baumanii are very important nosocomial pathogens mainly in intensive care units, being responsible for various types of infections with more and more limited therapeutic options [ options .P. aeruginosa have significant intrinsic resistance to antibiotics [P. aeruginosa which are resistant to third-generation cephalosporins produce a chromosomally mediated molecular class C beta-lactamase, the AmpC enzyme [P. aeruginosa and in Acinetobacter spp. as well. Acquired resistance to beta-lactams can lead to therapeutic failure, especially when it is associated with resistance to other classes of drugs, such as aminoglycosides and fluoroquinolones. Among these enzymes acquired, PER (Pseudomonas extended resistance), a class A extended-spectrum beta-lactamase (ESBL), occurring less frequently has clinical importance by conferring resistance to oxyimino beta-lactams [P. aeruginosa among which the VIM-type enzymes appear to be the most prevalent [The ibiotics . TherefoC enzyme . However-lactams . Poor ou-lactams . The alsrevalent .P. aeruginosa, A. baumannii isolates and VIM-producing P. aeruginosa in Hungary from a patient, who was hospitalized in Egypt and transferred to Hungary. This work illustrates the dissemination of bacteria carrying PER-type ESBL and VIM-type MBL enzymes.Here we report the first detection of PER-producing P. aeruginosa strains – an ESBL-producing P. aeruginosa (PA1), an imipenem-resistant P. aeruginosa (PA2), an MBL-producing P. aeruignosa (PA3), were observed and furthermore ESBL-producing Klebsiella pneumoniae (ESBL-KP), methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis (EF) were isolated. Next day ESBL-producing A. baumanii (ESBL-AB) and PA2 were cultured from the patient's canul. During his hospitalization from his nose PA1, ESBL-AB, ESBL-KP, MRSA, from his throat PA1, ESBL-AB, ESBL-KP, from his trachea PA3, ESBL-AB, ESBL.KP,-MRSA and from his wound PA1, PA3, ESBL-AB, ESBL-KP, MRSA and EF were isolated. Blood cultures were taken nine occasions and PA1, ESBL-KP, MRSA and EF were subcultured. The patient received adequate supportive treatment and empirically cefepime, subsequently meropenem and vancomycin were administered intravenously at high dosage. The aminoglycosides were synergy resistant. On the 8th day of the hospitalization in Hungary the patient died.In April, 2006 a 53-year-old Hungarian tourist was involved in a severe terror-attack in Egypt. He was initially hospitalized because of his burn, mechanical injuries and sepsis syndrome in Egypt. Five days later he was transferred in comatose, hypoxic and hypothermic condition to the Burn Unit of State Health Center, Budapest, Hungary. On the day of admission, bacterial cultures taken from burn wound. Based on the different colony morphology and antibiotic susceptibility patterns three different  in K. pneumoniae and in A. baumanii strains but no synergy was observed in P. aeruginosa strains. The minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by E-test  and by using the interpretative criteria of the Clinical and Laboratory Standards Institute [SpeI-digested  genomic DNA of P. aeruginosa was performed and analyzed as described previously [blaTEM [blaSHV [blaPER [blaOXAs [blaIMP, blaVIM and blaclassI genes [The isolates were identified by VITEK 2 . On a routine antibiogram synergy was observed between amoxycillin/clavulanic acid and cefepime or ceftazidime disks nstitute . Pulsed-eviously . Isoeleceviously . In the  [blaTEM , blaSHV  [blaSHV , blaPER  [blaPER , blaOXAs[blaOXAs , blaIMP,sI genes  were useP. aeruginosa strains isolated from the patient on the day of admission  was sensitive only to meropenem and both PA1 and PA2 were resistant to all the other tested antibiotics. The third P. aeruginosa (PA3) strain was sensitive to aztreonam and netilmicin and resistant to the all cephalosporins and carbepenems. However, the imipenem MIC value decreased in the presence of EDTA from > 256 to 12 μg/ml suggesting the presence of an MBL enzyme. The A. baumanii isolate was resistant to all cephalosporins and aztreonam, but the ceftazidime and cefotaxime MIC values decreased in the presence of clavulanic acid suggesting the presence of an ESBL enzyme. The K. pneumoniae strain had also ESBL phenotype based on the MIC values and it was sensitive to carbapenems, amikacin and ciprofloxacin as well.Three different antibiotic susceptibility patterns were observed in on Table . All of A. baumanii and K. pneumoniae. OXA genes were recognized in PA1 and PA3 strains. PER-1 genes were found in PA1 and A. baumanii isolates and SHV-5 was found in K. pneumoniae. Only the PA3 isolate harbored the VIM-2 gene. The OXA-10 and VIM-2 genes were located on the same class I integrons, as the sequencing result of class I integron showed.The PCR and sequencing results are summarized in Table According to IEF test results, all the isolates contained β-lactamases. Isoelectric focusing confirmed the expression of PER-1 (pI 5.3), TEM-1 (pI 5.4), OXA-1 (pI 7.4), OXA-10 (pI 6.1), SHV-5 pI 8.2), VIM-2 (pI 5.3) and the chromosomal AmpC cephalosporinase (pI 8 or > 8.5) enzymes in the different strains  suggesting that they belonged to different clones , the OprD-loss P. aeruginosa (PA2) and VIM-2-producing P. aeruginosa (PA3) strains – had external origin and could be transferred from Egypt to Hungary. The PER-1 producing P. aeruginosa and A. baumanii strains disappeared from the hospital, no more infections have been detected with these strains since then.Furthermore the result of the PFGE analysis of the PA1, PA2 and PA3 confirms, that the three P. aeruginosa strains. The CLSI recommendation for screening the ESBL-production in different bacteria species is uncomplete, exist just for Klebsiella spp. and Escherichia coli strains [The emergence and subsequently spread of PER-producing and MBL-producing strains are alarming. Supposing, that these resistance mechanisms might also exist in other countries the dissemination of PER-enzyme could be more prevalent over the world, particularly due to the unsolved problem of routine screening for ESBL-production in  strains .P. aeruginosa and A. baumannii isolate, VIM-2 producing P. aeruginosa strains in Hungary. Furthermore it illustrates the possibility of the inter-country and the inter-continent spread of the beta-lactamase-mediated resistance mechanisms. Our study features the intercontinental spread of antimicrobial resistance, showing the importance of careful screening and the isolation patients arriving from a different country.In conclusion, this work confirms the emergence of PER-1-producing The authors declare that they have no competing interests.LR and ZJ provided clinical care, literature search and edited the manuscript, JS and KK identified the bacteria, performed the antibiotic susceptibility tests, DS carried out the molecular studies, characterized the bacteria and drafted the manuscript, NK drafted the manuscript. All authors read and approved the final manuscript.Written informed consent was obtained from the patient's relative for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA , LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.Survival of  Pseudomonas aeruginosa is the leading cause of morbidity and mortality in CF patients, producing chronic pulmonary infections that can persist over decades P. aeruginosa, generally acquired from environmental reservoirs P. aeruginosa lineage diversifies into phenotypes specifically adapted to the hostile environment of the CF lung, thus allowing its long-term persistence P. aeruginosa to the CF lung The opportunistic pathogen P. aeruginosa CF adaptive phenotypes is mainly based on the occurrence (and further selection) of loss-of-function mutations in specific genes. Among these, the most commonly mutated is mucA, which encodes for a negative regulator of the biosynthesis of the exopolysaccharide alginate and whose inactivation is the basis of the conversion to the mucoid phenotype P. aeruginosa CF infections, is lasR, which is the main quorum sensing regulator controlling the expression of several virulence traits mexZ, a negative regulator of the MexXY-OprM multidrug-efflux pump, whose inactivation increases the resistance to aminoglycoside and other drugs The acquisition of these P. aeruginosa isolates from CF patients is the hypermutator phenotype P. aeruginosa infections. In fact, the link between the acquisition of antimicrobial resistance and hypermutators has been well-established by in vitro and in vivo approaches P. aeruginosa MRS-deficient strain in vitro, we previously established an association between hypermutability and both mucoid conversion lasR inactivation P. aeruginosa infections in the CF airways. Similarly, Waine et al. P. aeruginosa isolates from adult CF patients with chronic lung infections. Nevertheless, the role of hypermutability in P. aeruginosa phenotypic diversification in the natural scenario of a human infection has only very recently been investigated by Mena et al. lasR, the latter of which showed a higher number of mutations). Hence, these results show that the involvement of hypermutability in the acquisition of the mutations which drive the adaptive process of P. aeruginosa into the CF airways is complex. Also, exactly how P. aeruginosa acquires those mutations which enable it to persist into the CF lung environment is still an open question. These issues need to be resolved in order to shed light on the mutational mechanisms involved in P. aeruginosa adaptation.Another highly prevalent phenotype observed in mucA (mucoid phenotype), lasR (quorum sensing deficient phenotype), mexZ (multi-drug resistant phenotype) and the MRS genes (hypermutator phenotype) in a collection of P. aeruginosa isolates obtained from Argentinean CF patients. mexZ turned out to be the most frequently mutated gene followed by mucA and lasR, but mutations in all these three genes were highly prevalent in our collection. Also, the occurrence of hypermutators among Argentinean CF patients was very high, with hypermutability being a consequence of mutations affecting the MRS genes . However, the mutational spectra of each gene were clearly different, suggesting that more than one mechanism could be involved in the mutational process of each gene. Our results show a lack of association between hypermutability and the frequency of adaptive mutations occurring in mucA, lasR and mexZ, and, although hypermutability was associated with an increased resistance to most of the antibiotics tested on P. aeruginosa isolates from the Argentinean CF population, this association was not observed for mucoid variants, quorum sensing deficiency or the mexZ related multi-drug resistant phenotype.In the present work, we carried out an exhaustive survey and characterization of mutations in P. aeruginosa CF isolates in Latin America which is focused on mucoidy, lasR-deficiency, hypermutability and antibiotic resistance, critical issues regarding CF chronic infections, and provides new insights into the mutational mechanisms required for bacterial persistence.This is the first detailed characterization of P. aeruginosa isolates were collected from the sputum of twenty-six CF patients during the course of chronic pulmonary infection . Moreover, the strains from different patients were epidemiologically unrelated  as well as with those from the MPAOMS (2.8×10−6±1.6×10−6) and MPAOML (1.3×10−6±0.6×10−6) hypermutator mutant strains. In this sense, clinical isolates were considered hypermutators when their mutation frequencies were ≥2×10−7, with this being an arbitrary value which has been previously established to be reached/surpassed mostly by MRS-defficient strains P. aeruginosa isolates (42%), with these hypermutable P. aeruginosa strains being obtained from 12 patients (46%). It is important to mention that the prevalence of P. aeruginosa hypermutator strains was very high in the adult population as well as in the child/teenaged population.Thus, the results were compared with those obtained for the PAO1 reference strain  were responsible for the hypermutator phenotype in both the mutS and mutL genes, including deletions and insertions . This amino acid is one of the last 10 C-terminal residues that abolishe MutL homodimerization upon deletion, a phenomenon that has a key function in communicating mismatch recognition by MutS to downstream repair processes. To our knowledge, these three missense mutations have not been previously reported.It has been described that the stable hypermutators found among sertions . FurthermutL, respectively. In addition, a third hypermutator isolate (3c) was obtained from the same patient in 2008, which possessed an unaltered mutL sequence but which failed to amplify a region of the mutS gene. This result indicates that during four years of chronic infection in patient 3, three different hypermutator lineages emerged, suggesting a strong selection for hypermutability in the CF lung.Finally, it is interesting to highlight the results obtained for patients 1 and 3. From patient 1, we isolated a previous hypermutator strain in 2004 (1a) and a subsequent hypermutator strain in 2007 (1b) . Both thmucA, lasR and mexZ genes were analyzed in our collection of isolates to score for mutations that could be involved in the phenotypic switches to mucoidy, deficiency in the quorum sensing and antimicrobial (aminoglycoside) resistance respectively, three phenotypes considered to be characteristics of P.aeruginosa chronic CF infections. Thus, only nonsynonymous mutations which are expected to cause partial or complete loss of function in these genes were considered in the analysis . This was followed by mucA, which showed mutations in 24 of the 38 isolates (63%), and finally lasR, which was mutated in 15 isolates (39%). Taken together, these results and the data from the MRS gene mutational inactivation (42%) confirm that the occurrence of mutations in all the analyzed genes was highly prevalent among Argentinean CF patients.As shown in mucA, lasR, mexZ, and mutS/mutL, which were particular to each gene  with the rest of the mutations consisting of different kinds of transitions (25%) and transversions (8.3%) , with a 2∶1 ratio for transitions (57%) over transversions (29%) , of which there was a high prevalence of large deletions (>4 bp) (50%) respect to small deletions (1–4 bp) (15%), followed by transitions (20%) and transversions (15%)  . Notablys (8.3%) , indicatns (29%) , which mns (29%) . In conteletions . mexZ, ons (15%) . Howeverp codons . As menttivation , includitivation .mucA, lasR, mexZ and MRS, indicating that each gene has a particular way of being mutated. Taken together, these observations suggest that different mechanisms of mutagenesis are responsible for the loss-of-function mutations in the analyzed genes.By comparing the mutational spectra obtained in the Argentinean isolates with those reported in other studies P. aeruginosa chronic lung infection, it has been proposed that the proliferation of the hypermutators in the population infecting an individual patient may be sustained by their association (hitchhike) with mutations adaptive to growth in the lung in vitro that the MRS loss-of-function was a key determinant in targeting mucA for mucoid conversion and lasR for quorum sensing deficiency Theoretical studies suggest that, under stressful conditions, selective pressure favors hypermutator strains over nonmutator strains mucA, lasR and mexZ genes in the Argentinean CF isolate collection, we analyzed the proportion of mutations that occurred in each gene in the hypermutator and nonmutator isolates. As shown in mexZ and mucA genes was not significantly different to that observed in the nonmutator isolates (P = 0.17 and P = 1.00 respectively). In the case of lasR, we saw that among the hypermutators, the number of mutations per isolate was higher (0.56) than nonmutators (0.27), although this difference was not statistically significant (P = 0.09), indicating that, as in the case of mexZ and mucA, it was not possible to establish an association between the occurrence of mutations in lasR and the hypermutator phenotype. Furthermore, as shown in In order to investigate if an association could be established between hypermutability and the mutations observed in the mucA, lasR and mexZ genes. It was observed that although the hypermutator isolates showed a tendency towards substitutions (53%), which were either transversions or transitions, with nonmutator isolates displaying a higher number of deletions (62%) , there was not a statistically significant difference between hypermutators and nonmutators with respect to the kind of mutations occurring in the analyzed genes. Furthermore, whatever the type of mutations, their distribution among any particular analyzed gene in hypermutators and nonmutators isolates did not show any statistically significant differences  to any of the specific adaptive traits studied.We now investigated if hypermutability could be associated with any particular kind of mutations in ferences . TherefoP. aeruginosa strains isolated from CF patients not only possess a substantially higher antibiotic resistance than nonmutator isolates P. aeruginosa CF isolates, we tested the 38 isolates by measuring the inhibition zone diameters and scoring for RMS within the inhibition zones of five different antimicrobial agents . As shown in P. aeruginosa with detectable levels of ciprofloxacin and ceftazidime resistance was significantly higher than the proportion observed in non-mutator strains (P = 0.02 and P = 0.02 respectively), indicating that in the Argentinean CF population, hypermutability correlated with ciprofloxacin and ceftazidime resistance. In fact, 62.5% of the hypermutators were ciprofloxacin resistant compared to a proportion of 22.7% found among nonmutators. Although a smaller proportion of hypermutators (25%) were ceftazidime resistant, all nonmutator strains were susceptible to this antibiotic . Nevertheless, it is important to point out that only one isolate (isolate 19a) that was a hypermutator showed resistance to all the antibiotics tested  (By quantifying the emergence of the resistant mutant subpopulation (RMS) P<0.001) . These oFinally, it is important to remark that in the Argentinean population, the disk diffusion tests showed similar results for the child/teenage and the adult groups of CF patients.P. aeruginosa isolated from sputa of CF patients mexZ inactivation is involved in the development of stable aminoglycoside resistance among CF strains of P. aeruginosalasR, which was not sufficient to alter the MIC, but resulted in a greater production of spontaneous resistant colonies lasR loss-of-function mutations confered resistance to ciprofloxacin and tobramycin when oxygen was limited and there was increased nitrate utilization mucA, lasR or mexZ could be associated with an altered antibiotic resistance in our collection. As shown in mucA, lasR or mexZ. Moreover, scoring for RMS within the inhibition zones of the five antimicrobial agents tested showed that the presence of resistant mutants was not a phenomenon related to the inactivation of any of these three regulators, not even when the lasR mutants were tested for β-lactam antibiotics (not shown).Previous studies have reported an enhanced resistance to antibiotics for mucoid strains of P. aeruginosa establishes chronically in the CF airways by a diversification process that allows fine tuning-adaptation and long-term survival within the host. Since this process is mainly mutagenic P. aeruginosa acquires these mutations may help to identify the functions required for bacterial persistence.P. aeruginosa to the CF lung, due to their being the most mutated among CF patients mucA, lasR and mexZ genes as well those implicated in the inactivation of MRS, mutS and mutL, were analyzed in a collection of P. aeruginosa isolates obtained from Argentinean CF patients, with this constituting the first study of this nature in Latin America. According to our results, mutations in the first three genes were highly prevalent in the Argentinean population, with mexZ being the most frequently mutated followed by mucA and lasR , our analysis was focused on strong MRS deficient hypermutators, not considering weak mutators  which can also be found among the CF P. aeruginosa isolates In this study, we carried out a survey and a molecular characterization of the mutations that occurred in the genes which had been previously described as important determinants for the adaptation of and lasR . In addiand lasR , in agreary role . It is wP. aeruginosa in the airways of CF patients is a consequence of their second-order selection with other adaptive mutations, since a higher mutation rate would increase their probability to be acquired lasR quorum sensing-deficient phenotype mexZmucA/lasR/mexZ, may confer a level of antibiotic resistance detectable by disk diffusion, which is the methodology used in this study.It is proposed that the high prevalence of stable hypermutator strains of in vitro that MRS-deficiency accelerates the acquisition of mucAlasRin vivo. In line with this, Waine et alP. aeruginosa isolates obtained from CF patients. However, in the present study, by scoring for mutations in hypermutator and nonmutator CF isolates of our collection, no association could be established between hypermutability and mutagenesis, not only for mucA but also for the lasR and mexZ mutations. A possible explanation for this discrepancy is that in Waine et al., the phenomenon was not characterized at the genetic level, and that the reversion phenomenon of the mucoid to a nonmucoid phenotype, which is highly frequent in isolates recovered from CF patients mucA, indicating that they had previously been mucoid before reverting to the phenotype. Furthermore, contrary to our isolates which were clonally different, almost half of the isolates in Waine et al. corresponded to epidemic clones, a feature that increases dissimilarities among collections.In a previous recent work, we determined mucA, lasR and mexZ genes of hypermutator and nonmutator isolates. However and most interestingly, these mutational spectra vary in a gene dependant manner, with mucA being dominated by small deletions (1 bp), lasR by base substitutions and mexZ by large deletions (>4 bp). This observation is intriguing, since it suggests the involvement of different mechanisms of mutagenesis, possibly operating simultaneously, during the process of CF chronic infection. mucA and lasR, but not mexZ mutational spectra coincide well not only with the spectrum generated by a MRS deficiency P. aeruginosa to this environment. Consistent with this hypothesis, we have recently shown that Pol IV activity is an essential ingredient to establish mucA as the main target for mutagenesis in mucoid conversion, with this factor having a prominent role in the generation of -1 deletion in a monomeric simple sequence repeats of five Gs (G5-SSR426) one of the most prevalent mutations among CF mucoid isolates 5SSR426 by site directed mutagenesis not only significantly reduces the mutation frequency in mucA, but also makes mucA to be no longer the major pathway for mucoid conversion mucA and lasR mutations was also found lasR-deficient isolates occur prior to the appearance of hypermutator strains, which had previously been shown to accumulate at later stages of the infection mucA, lasR and mexZ arise by different mechanisms in each gene, with these being independent of MRS-deficiency. However, further research is required to achieve a better understanding of the complex and variable mutagenic strategies used by P. aeruginosa to continuously adapt to the environment of the CF lung.It has been recently shown that hypermutator strains accelerate the mutagenic genetic adaptation thus enhancing the accumulation of new mutations P. aeruginosa isolates, representing different colony morphotypes , were collected from sputum samples of 26 Argentinean CF patients with chronic P. aeruginosa lung infection (with at least four years of continuous isolation of P. aeruginosa). These patients were either children/teenagers or adults, who had been treated at two local Hospitals: Hospital de Niños Santísima Trinidad (Córdoba city)  or Hospital Alemán (Buenos Aires city) . The P. aeruginosa reference strain PAO1 and hypermutable strains MPAOMS and MPAOML were used as controls  and Hospital Alemán (Buenos Aires), Argentina, as byproducts of the routine established for bacterial typing and antimicrobial susceptibility testing. In this sense, sputa sampling was not performed with the aim to fulfill the study described in this work, and isolates recovered from these sputa were just a derivative of the habitual CF patient therapeutic controls. Thus, we obtained a collection of 38 P.aeruginosa isolates which was characterized in this work. It is important to clarify that bacterial isolates were processed anonymously, there was no contact with the patients or access to medical records, and that the therapeutic treatments of the patients were not modified in any case as a consequence of the results obtained in this study. The research protocols followed in this study were approved and reviewed by the Institutional Review Board of the Biological Chemistry Research Center of Córdoba , which waived the need for a written consent from the patients involved.The SpeI enzyme as described elsewhere et al.All isolates were genotyped by PFGE using the P. aeruginosa CF isolates, we performed the quantification of rifampin-resistant mutant frequencies which is considered to be proportional to the estimation of mutation frequencies. Mutation frequencies were determined as previously described P. aeruginosa isolate were cultured in LB medium at 37°C for 24 h with shaking performed at 225 r.p.m. Appropriate dilutions of the cultures were plated on LB agar plates to determine the total number of viable cells, or on LB agar supplemented with 100 µg ml−1 rifampin to count the number of rifampin-resistant cells, following incubation overnight at 37°C. Then, the mutation frequency was determined as the ratio of the number of rifampin–resistant cells and the number of viable cells. Strains were considered hypermutators when their mutation frequencies were ≥2×10−7, which corresponds to a mutation frequency approximately 20 fold higher than that obtained from the reference P. aeruginosa PAO1 strain (2.4±1.3×10−8) and which has been shown to score for strong MRS-deficient mutators Mutation rates determined via fluctuation analysis can provide good estimations of mutagenesis mucA, lasR, mexZ, mutS and mutL genes are shown in mucA, mexZ, mutS and mutL) or at 50°C (lasR) 2 min at 72°C, and a final extension of 10 min at 72°C. The PCR products were cleaned with a Gel Purification kit (QIAGEN) and both strands were directly sequenced by using their respective PCR primers . To score for mutations within the genes, the sequencing results were compared with the corresponding gene sequences of the PAO1 strain (www.pseudomonas.com) by using the BLAST program of the NCBI database (www.ncbi.nlm.nih.gov/blast/).Primers for PCR amplification and DNA sequencing of mucA, lasR and mexZ included only nonsynonymous mutations, which are expected to cause partial or complete loss of function in the genes by generation of premature stop codons, shifts of reading frames or deletions/insertions in the coding sequence. To predict the effect of nonsynonymous (missense) mutations on protein function, the SIFT software was P<0.05.The analyses of the linkage between hypermutability and mutations observed in P. aeruginosa wild-type mutS and mutL genes  agar plates by using conventional disks (Rosco). Approximately 0.5 McFarland standard suspensions were used for the inocula standardization of each isolate, and the diameters of the inhibition zones were measured after 24 h and 48 h of incubation at 37°C. The relative number  of resistant mutant subpopulations growing within the inhibition zones was determined for each antibiotic after 48 h of incubation at 37°C, as described previously P. aeruginosa PAO1 strain and the hypermutable MPAOMS and MPAOML strains (The inhibition-zone diameters of the thirty eight  strains  were useAntibiotic multiresistance was defined as resistance to at least three antibiotics.P value of >0.05 as significant.Statistical analyses were performed using Fisher's exact test, considering a