Cystic Fibrosis Isolates Research Articles

A novel signal transduction pathway that modulates rhl quorum sensing and bacterial virulence in Pseudomonas aeruginosa.

The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmSL181P, BfmSL181P/E376Q, and BfmSR393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients.

To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance.

Persistent cystic fibrosis isolate Pseudomonas aeruginosa strain RP73 exhibits an under-acylated LPS structure responsible of its low inflammatory activity

Pseudomonas aeruginosa, the major pathogen involved in lethal infections in cystic fibrosis (CF) population, is able to cause permanent chronic infections that can persist over the years. This ability to chronic colonize CF airways is related to a series of adaptive bacterial changes involving the immunostimulant lipopolysaccharide (LPS) molecule. The structure of LPSs isolated from several P. aeruginosa strains showed conserved features that can undergo chemical changes during the establishment of the chronic infection. In the present paper, we report the elucidation of the structure and the biological activity of the R-LPS (lipooligosaccharide, LOS) isolated from the persistent CF isolate P. aeruginosa strain RP73, in order to give further insights in the adaptation mechanism of the pathogen in the CF environment. The complete structural analysis of P. aeruginosa RP73 LOS was achieved by chemical analyses, NMR spectroscopy and MALDI MS spectrometry, while the assessment of the biological activity was attained testing the in vivo pro-inflammatory capacity of the isolated LOS molecule. While a typical CF LPS is able to trigger a high immune response and production of pro-inflammatory molecules, this P. aeruginosa RP73 LOS showed to possess a low pro-inflammatory capacity. This was possible due to a singular chemical structure possessing an under-acylated lipid A very similar to the LPS of P. aeruginosa found in chronic lung diseases such as bronchiectstasis.

Pseudomonas aeruginosa Genome Evolution in Patients and under the Hospital Environment.

Pseudomonas aeruginosa is a Gram-negative environmental species and an opportunistic microorganism, establishing itself in vulnerable patients, such as those with cystic fibrosis (CF) or those hospitalized in intensive care units (ICU). It has become a major cause of nosocomial infections worldwide and a serious threat to Public Health because of overuse and misuse of antibiotics that have selected highly resistant strains against which very few therapeutic options exist. Herein is illustrated the intraclonal evolution of the genome of sequential isolates collected in a single CF patient from the early phase of pulmonary colonization to the fatal outcome. We also examined at the whole genome scale a pair of genotypically-related strains made of a drug susceptible, environmental isolate recovered from an ICU sink and of its multidrug resistant counterpart found to infect an ICU patient. Multiple genetic changes accumulated in the CF isolates over the disease time course including SNPs, deletion events and reduction of whole genome size. The strain isolated from the ICU patient displayed an increase in the genome size of 4.8% with major genetic rearrangements as compared to the initial environmental strain. The annotated genomes are given in free access in an interactive web application WallGene designed to facilitate large-scale comparative analysis and thus allowing investigators to explore homologies and syntenies between P. aeruginosa strains, here PAO1 and the five clinical strains described.

Conditions Associated with the Cystic Fibrosis Defect Promote ChronicPseudomonas aeruginosaInfection

Progress has been made in understanding how the cystic fibrosis (CF) basic defect produces lung infection susceptibility. However, it remains unclear why CF exclusively leads to chronic infections that are noninvasive and highly resistant to eradication. Although biofilm formation has been suggested as a mechanism, recent work raises questions about the role of biofilms in CF. To learn how airway conditions attributed to CF transmembrane regulator dysfunction could lead to chronic infection, and to determine if biofilm-inhibiting genetic adaptations that are common in CF isolates affect the capacity of Pseudomonas aeruginosa to develop chronic infection phenotypes. We studied P. aeruginosa isolates grown in agar and mucus gels containing sputum from patients with CF and measured their susceptibility to killing by antibiotics and host defenses. We also measured the invasive virulence of P. aeruginosa grown in sputum gels using airway epithelial cells and a murine infection model. We found that conditions likely to result from increased mucus density, hyperinflammation, and defective bacterial killing could all cause P. aeruginosa to grow in bacterial aggregates. Aggregated growth markedly increased the resistance of bacteria to killing by host defenses and antibiotics, and reduced their invasiveness. In addition, we found that biofilm-inhibiting mutations do not impede aggregate formation in gel growth environments. Our findings suggest that conditions associated with several CF pathogenesis hypotheses could cause the noninvasive and resistant infection phenotype, independently of the bacterial functions needed for biofilm formation.

NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase–DNA and neutrophil elastase–DNA complexes

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO–DNA complexes and established a new HNE–DNA ELISA. We show that these methods reliably quantify MPO–DNA and HNE–DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired “early” and “late” bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.

High-level Relatedness amongMycobacterium abscessussubsp.massilienseStrains from Widely Separated Outbreaks

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.

Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

ABSTRACTPseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections.

Comparative Analyses of a Cystic Fibrosis Isolate of Bordetella bronchiseptica Reveal Differences in Important Pathogenic Phenotypes

Bordetella bronchiseptica is a Gram-negative bacterium that infects and causes disease in a wide variety of animals. B. bronchiseptica also infects humans, thereby demonstrating zoonotic transmission. An extensive characterization of human B. bronchiseptica isolates is needed to better understand the distinct genetic and phenotypic traits associated with these zoonotic transmission events. Using whole-genome transcriptome and CGH analysis, we report that a B. bronchiseptica cystic fibrosis isolate, T44625, contains a distinct genomic content of virulence-associated genes and differentially expresses these genes compared to the sequenced model laboratory strain RB50, a rabbit isolate. The differential gene expression pattern correlated with unique phenotypes exhibited by T44625, which included lower motility, increased aggregation, hyperbiofilm formation, and an increased in vitro capacity to adhere to respiratory epithelial cells. Using a mouse intranasal infection model, we found that although defective in establishing high bacterial burdens early during the infection process, T44625 persisted efficiently in the mouse nose. By documenting the unique genomic and phenotypic attributes of T44625, this report provides a blueprint for understanding the successful zoonotic potential of B. bronchiseptica and other zoonotic bacteria.

Panton-Valentine leukocidin and biofilm production of Staphylococcus aureus isolated from respiratory tract

Staphylococcus aureus is one of the first bacteria colonizing in cystic fibrosis (CF) respiratory tract and different virulence factors are responsible for disease progression. It is not clear if CF S. aureus strains are more virulent than strains isolated from non-CF patients. Biofilm production was detected by a modified tissue culture plate method, presence of genes encoding for Panton-Valentine leukocidin (PVL) was investigated by a signal amplified sandwich hybridization assay and antimicrobial susceptibility patterns were detected by disk diffusion method. Staphylococcus aureus clinical isolates (n = 88) recovered from respiratory tract specimens in which 31 of them were from cystic fibrosis (CF) patients were analysed. Biofilm production was detected in 96.8% of CF isolates in which 32.3% exhibited strong positive phenotype and in 47.4% of non-CF isolates in which strong positive phenotype was not observed (p <0.05). All CF isolates were methicillin susceptible, whereas 53.4% of non-CF isolates (n = 31) were methicillin resistant. No resistance was observed for vancomycin, chloramphenicol and trimethoprim/sulfamethoxazole in any of the isolates. PVL genes were detected only in two isolates (2.3%), one from each group, CF and non-CF, which both were methicillin susceptible. Biofilm rather than PVL production appears to be an important virulence factor in CF patients.

Developing an international Pseudomonas aeruginosa reference panel

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

Draft Genome Sequence of the Antibiotic-Producing Cystic Fibrosis Isolate Pantoea agglomerans Tx10.

Pantoea agglomerans is an enteric bacterium that is capable of causing both plant and human disease. Here, we report the genome sequence of a cystic fibrosis isolate, P. agglomerans Tx10, which produces an antibiotic that is effective against Staphylococcus aureus.

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa

BackgroundAlginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ22) to initiate transcription of the alginate biosynthetic operon.ResultsIn the current study, we mapped the mucE transcriptional start site, and determined that PmucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in PmucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE.ConclusionsOur data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Draft Genome Sequences of Burkholderia cenocepacia ET12 Lineage Strains K56-2 and BC7

The Burkholderia cepacia complex (BCC) is a group of closely related bacteria that are responsible for respiratory infections in immunocompromised humans, most notably those with cystic fibrosis (CF). We report the genome sequences for Burkholderia cenocepacia ET12 lineage CF isolates K56-2 and BC7.

Secretome of Transmissible Pseudomonas aeruginosa AES-1R Grown in a Cystic Fibrosis Lung-Like Environment

Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.

Amoxicillin–clavulanic acid resistance in fecal Enterobacteriaceae from patients with cystic fibrosis and healthy siblings

The present study set out to detect and identify amoxicillin-clavulanic acid (AMC)-resistant Enterobacteriaceae in fecal samples of two patients with cystic fibrosis (CF) and their respective siblings. Fecal Enterobacteriaceae were enumerated onto EMB agar containing amoxicillin (AMX). A total of 173 CF isolates and 41 sibling isolates were grouped into seven Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) clusters and identified through 16S rRNA and rpoB sequence analysis. The fecal microbiota of patients with CF revealed a higher prevalence of AMX resistant Enterobacteriaceae compared to that of their healthy siblings. Whereas all selected isolates of healthy siblings were assigned to Escherichia coli, isolates of patients with CF belonged to Klebsiella oxytoca (58.4%), E. coli (28.3%), Klebsiella variicola (7.5%) or Citrobacter sp. (5.8%). All tested CF isolates showed a high resistance rate to AMX, and a lower level of resistance to the combination with clavulanic acid. In contrast, all tested sibling isolates were susceptible for both AMX and AMC. The higher abundance of AMX resistance in the investigated patients with CF suggests that frequent AMC administration may be one of the major contributing factors in the proliferation of Enterobacteriaceae and the development of resistant strains in the gastrointestinal tract.

Antibiotic resistance in Prevotella species isolated from patients with cystic fibrosis

To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non-CF patients and analyse the impact of antibiotic prescribing in the preceding year on resistance amongst CF isolates. The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls. All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P < 0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were β-lactamase positive. This study profiles antibiotic susceptibility in Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.

Diversity of -lactam resistance mechanisms in cystic fibrosis isolates of Pseudomonas aeruginosa: a French multicentre study

To investigate the resistance mechanisms of β-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France. Two-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to β-lactams were investigated. Their β-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic β-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations. MLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to β-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to β-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary β-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene. β-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of β-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.

High-throughput single-nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96% agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24%) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

Comparison of in Vitro Activity of Doripenem versus Old Carbapenems against Pseudomonas Aeruginosa Clinical Isolates from both CF and Burn Patients.

The antimicrobial activity of doripenem in comparison of imipenem, meropenem and ertapenem among Pseudomonas aeruginosa isolated from burn and Cystic Fibrosis (CF) patients were determined. Metallo-β-lactamase (MBL) genes in imipenem non susceptible P. aeruginosa isolates were detected using PCR method. The in vitro susceptibilities of doripenem, imipenem, meropenem and ertapenem were determined by Etests. MIC50 and MIC90 for corresponding antibiotics were determined individually in burn and CF isolates. Among isolates which were resistant to imipenem, 16 isolates were positive for the bla IMP gene. All isolates had no bla VIM gene. All MBL producing isolates were excluded. MIC50/MIC90 of doripenem in CF and burn isolates were 0.75/>32 and >32/>32 mg/L respectively. The corresponding values for imipenem in CF and burn isolates were 2/>32 and >32/>32 mg/L, respectively. The susceptibility rate of doripenem is higher than that of imipenem and meropenem among P.aeruginosa isolated from CF patients, whereas, there is no difference between the efficiency of doripenem and old carbapenems in non MBL producing P.aeruginosa isolates in burn patients.