Pseudomonas

Introduction. Indwelling medical devices such as endotracheal tubes (ETTs), urinary catheters, vascular access devices, tracheostomies and feeding tubes are often associated with hospital-acquired infections. Bacterial biofilm formed on the ETTs in intubated patients is a significant risk factor associated with ventilator-associated pneumonia. Pseudomonas aeruginosa is one of the four frequently encountered bacteria responsible for causing pneumonia, and the biofilm formation on ETTs. However, understanding of biofilm formation on ETT and interventions to prevent biofilm remains lagging. The ability to sense and adapt to external cues contributes to their success. Thus, the biofilm formation is likely to be influenced by the two-component systems (TCSs) that are composed of a membrane-associated sensor kinase and an intracellular response regulator.

Aim. This study aims to establish an in vitro method to analyse the P. aeruginosa biofilm formation on ETTs, and identify the TCSs that contribute to this process.

Methodology. In total, 112 P. aeruginosa PA14 TCS mutants were tested for their ability to form biofilm on ETTs, their effect on quorum sensing (QS) and motility.

Results. Out of 112 TCS mutants studied, 56 had altered biofilm biomass on ETTs. Although the biofilm formation on ETTs is QS-dependent, none of the 56 loci controlled quorum signal. Of these, 18 novel TCSs specific to ETT biofilm were identified, namely, AauS, AgtS, ColR, CopS, CprR, NasT, KdpD, ParS, PmrB, PprA, PvrS, RcsC, PA14_11120, PA14_32580, PA14_45880, PA14_49420, PA14_52240, PA14_70790. The set of 56 included the GacS network, TCS proteins involved in fimbriae synthesis, TCS proteins involved in antimicrobial peptide resistance, and surface-sensing. Additionally, several of the TCS-encoding genes involved in biofilm formation on ETTs were found to be linked to flagellum-dependent swimming motility.

Conclusions. Our study established an in vitro method for studying P. aeruginosa biofilm formation on the ETT surfaces. We also identified novel ETT-specific TCSs that could serve as targets to prevent biofilm formation on indwelling devices frequently used in clinical settings.

Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors such as lectin A, pyocyanin, elastase and rhamnolipids. These compounds are controlled transcriptionally by three quorum-sensing circuits, two based on the synthesis and detection of N-acyl-homoserine-lactone termed the Las and Rhl system and a third system named the Pseudomonas quinolone signal (PQS) system, which is responsible for generating 2-alkyl-4(1 h)-quinolones (AQs). The transcriptional regulator called PqsR binds to the promoter of pqsABCDE in the presence of PQS or HHQ creating a positive feedback-loop. PqsE, encoded in the operon for AQ synthesis, is a crucial protein for pyocyanin production, activating the Rhl system by a still not fully understood mechanism. In turn, the regulation of the PQS system is modulated by Las and Rhl systems, which act positively and negatively, respectively. This review focuses on the PQS system, from its discovery to its role in Pseudomonas pathogenesis, such as iron depletion and pyocyanin synthesis that involves the PqsE protein – an intriguing player of this system.

Introduction. One of the most important resistant mechanisms in Gram-negative bacteria is extended spectrum β-lactamases (ESBLs). Harbour-related genes on plasmids, increase the risk of resistance transmission among commonly reported hospital infections.

Aim. This study was designed to explore the dissemination of Pseudomonas aeruginosa producing ESBLs on their plasmids recovered from the different wards of Amir-Al-Momenin burn center, Affiliated with Shiraz University of Medical Sciences.

Methodology. Among 256 isolates, 88 (34.38 %) P. aeruginosa strains were isolated from burn hospitalized patients. Samples were processed for antibiotic resistance using the Kirby–Bauer method while MIC was performed for colistin. MIC was used by the microdilution broth method as recommended by Clinical and Laboratory Standards Institute guidelines. Related studied genes were evaluated on extracted plasmids by the PCR method.

Results. According to the phenotypic and molecular steps, a total of 58 (65.91 %) and 74 (84.10 %) strains detected positive ESBLs, respectively. Based on antibiogram tests, a total of 63 (71.59 %) isolates were detected as multidrug resistant. All ESBL P. aeruginosa isolates showed identical antimicrobial susceptibility profiles. The genotypic prevalence of ESBLs for bla SHV, bla TEM, bla GES, bla OXA-10 and bla PSE genes was 47.73, 78.41, 5.58, 3.41, 4.55 %, respectively.

Conclusion. All P. aeruginosa strains producing ESBLs had plasmids containing related genes. The data indicated a high prevalence of ESBL among P. aeruginosa isolates in the southwest of the Iran burn center and their enzyme types were diverse.

The antibiotic ciprofloxacin is used extensively to treat a wide range of infections caused by the opportunistic pathogen Pseudomonas aeruginosa. Due to its extensive use, the proportion of ciprofloxacin-resistant P. aeruginosa isolates is rapidly increasing. Ciprofloxacin resistance can arise through the acquisition of mutations in genes encoding the target proteins of ciprofloxacin and regulators of efflux pumps, which leads to overexpression of these pumps. However, understanding of the basis of ciprofloxacin resistance is not yet complete. Recent advances using high-throughput screens and experimental evolution combined with whole-genome sequencing and protein analysis are enhancing our understanding of the genetic and biochemical mechanisms involved in ciprofloxacin resistance. Better insights into the mechanisms of ciprofloxacin resistance may facilitate the development of new or improved therapeutic regimes effective against P. aeruginosa. In this review we discuss the current understanding of the mechanisms of ciprofloxacin resistance and summarize the genetic basis of ciprofloxacin resistance in P. aeruginosa, in the context of current and future use of this antibiotic.

A prevailing opinion is that the strains of Pseudomonas aeruginosa that infects both plants and humans are two separate species. This study strongly disputes that notion until the modern molecular technology proves otherwise. This paper examines a spectrum of strains occurring in nature, their habitats, dissemination, their relationship to clinical strains, and the environmental conditions that favor their colonization of plants. The isolates were obtained from clinical specimens, plants, soil, and water. The identity of these strains was confirmed using pyocin typing and biochemical assays. The data reveal that agricultural soils, potted ornamental plants, hoses, fountains, and faucets frequently harbored P. aeruginosa. However, it was not commonly found in semi-arid areas, suggesting that moisture and high humidity is necessary for colonization and survival. Though found in soil, P. aeruginosa was seldom isolated on edible plant parts. The pathogenicity of various strains on plants was tested by inoculating vegetables, lettuce slices (Lactuca sativa L. "Great Lakes"), celery stalks (Apium graveolens L. var. Dulce], potato tuber slices (Solanum tuberosum L. "Whiterose"), tomato (Lycopersicon esculentum L. Mill), cucumber (Cucumis sativus L.), rutabaga (Brassica campestris L.), and carrot (Daucus carota L. var sativa). There was considerable variation in the strains' ability to cause rot, but no difference was observed between clinical isolates and others from agricultural fields, water, and soil. Two of the clinical isolates from burn patients, P. aeruginosa PA13 and PA14, exhibited the greatest virulence in causing rot in all the plants that were tested, especially on cucumber, lettuce, potato, and tomato. The study discusses how closely the epidemiology of P. aeruginosa relates to many plant pathogens, and the ability of human isolates to colonize plants and food material under favorable conditions. The biochemical and phenotypic similarity among strains from the clinical and agricultural material is strongly indicative that they are the same species and that plants and soil are natural reservoirs for P. aeruginosa.

Purpose. The presence of alginate-overproducing (Alg+) strains of Pseudomonas aeruginosa in cystic fibrosis patients is indicative of chronic infection. The Alg+ phenotype is generally due to a mutation in the mucA gene, encoding an innermembrane protein that sequesters AlgT/U, the alginate-specific sigma factor. AlgT/U release from the anti-sigma factor MucA is orchestrated via a complex cascade called regulated intramembrane proteolysis. The goal of this study is to identify new players involved in the regulation of alginate production.

Methodology. Previously, a mutant with a second-site suppressor of alginate production (sap), sap27, was isolated from the constitutively Alg+ PDO300 that harbours the mucA22 allele. A cosmid from a P. aeruginosa minimum tiling path library was identified via en masse complementation of sap27. The cosmid was transposon mutagenized to map the contributing gene involved in the alginate production. The identified gene was sequenced in sap27 along with algT/U, mucA, algO and mucP. The role of the novel gene was explored using precise in-frame algO and algW deletion mutants of PAO1 and PDO300.

Results/Key findings. The gene responsible for restoring the mucoid phenotype was mapped to lptD encoding an outer-membrane protein. However, the sequencing of sap27 revealed a mutation in algO, but not in lptD. In addition, we demonstrate that lipopolysaccharide transport protein D (LptD)-dependent alginate production requires AlgW in PAO1 and AlgO in PDO300.

Conclusion. LptD plays a specific role in alginate production. Our findings suggest that there are two pathways for the production of alginate in P. aeruginosa, one involving AlgW in the wild-type, and one involving AlgO in the mucA22 mutant.

Purpose. This study examined the risk factors for, and molecular mechanisms underlying, the increase in carbapenem minimum inhibitory concentrations (MICs) in clinical isolates of Pseudomonas aeruginosa.

Methodology. Consecutive clinical isolates of P. aeruginosa were collected. The MicroScan WalkAway system detected more than fourfold increases in the MICs of carbapenems in P. aeruginosa isolates serially recovered from some patients during their clinical course. The clinical risk factors associated with this increase were examined by multiple logistic regression analysis. Western blot analysis and nucleotide sequencing of the oprD gene of 19 clonally related and paired P. aeruginosa isolates from the same patients were undertaken to examine the mechanisms underlying the increase in MICs.

Results. The results showed that prior use of carbapenems (OR, 2.799; 95 % CI, 1.088–7.200; P=0.033) and the use of ventilators or tracheostomies (OR, 2.648; 95 % CI, 1.051–6.671; P=0.039) were risk factors for increased carbapenem MICs. Analysis of the underlying mechanisms revealed that loss of functional OprD protein due to mutation of the oprD gene tended to occur in P. aeruginosa isolates with imipenem MICs of more than 8 µg ml−1; a reduction in OprD expression was observed in P. aeruginosa isolates with imipenem MICs of 4 or 8 µg ml−1. This difference in the resistance mechanism was not correlated with the MICs of meropenem.

Conclusion. This difference in the resistance mechanism of P. aeruginosa indicates a critical breakpoint at an imipenem MIC of 8 µg ml−1, in accordance with EUCAST criteria. Reducing carbapenem use will prevent P. aeruginosa clinical isolates from developing resistance to carbapenems.

Purpose. Pseudomonas aeruginosa is one of the agents that are commonly implicated in nosocomial infections. However, it is also present as a commensal in various body sites of healthy persons, making the diagnosis of infection by culture difficult. A number of virulence factors expressed by the organism have been implicated in its pathogenicity. We undertook this study to identify the host and organism factors associated with infection.

Methodology. Pathogenic, colonizing and environmental isolates were tested for apr, lasB, the T3SS effector exoenzymes (exoS, exoT, exoU and exoY) and toxA genes, biofilm production and antimicrobial susceptibility. The isolates were further typed by RAPD.

Results. Eighty-seven isolates from 61 patients, including 11 environmental isolates, were obtained. None of the virulence factors were found to be significantly associated with infection, and nor was the antimicrobial susceptibility. The presence of the exoU gene and infection by MDR strains correlated significantly with the duration of hospital stay. Positivity for exoS and exoU genes was found to be strongly correlated with multi-drug resistance. exoU positivity correlated strongly with fluoroquinolone resistance. Sinks in the ward and intensive care unit were found to be a niche for XDR P. aeruginosa. Eighty-five isolates were typeable using the ERIC2 primer, showing 71 distinct RAPD patterns with >15 % difference in UPGMA-generated dice coefficients.

Conclusions. exoU positivity is associated with severe disease, as evidenced by the longer duration of hospital stay of these patients. However, the presence of virulence factors or multi-drug resistance in the cultured strain should not prompt the administration of anti-pseudomonal chemotherapy.

Purpose. It is known that the arnB (or pmrH) gene encoding uridine 5′-(beta-1-threo-pentapyranosyl-4-ulose diphosphate) aminotransferase plays a critical role in colistin resistance in Pseudomonas aeruginosa through the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A. In this study, we attempted to obtain a colistin-resistant mutant from an arnB-deleted mutant through exposure to colistin.

Methodology. We constructed an arnB deletion mutant (P5ΔarnB :: nptIII) from a colistin-susceptible strain (P5) by allelic replacement mutagenesis, and colistin-resistant mutants were selected in vitro using P5 and P5ΔarnB :: nptIII. The growth rate, lipid A structure, biofilm-forming activity and cell viability in diverse stressful conditions (osmotic, oxidative, acidic and heat stress) were investigated. Expression of phoP, pmrA, parR, and cprR was evaluated by qRT-PCR.

Results. An arnB deletion mutant was shown to develop colistin resistance through the addition of l-Ara4N to lipid A, despite a low survival rate (over 1000-fold lower than that of the wild-type strain) in the media with colistin. Two colistin-resistant mutants showed higher survival rates than colistin-susceptible strains against 5 % NaCl. In the presence of acidic and heat stress, P5ΔarnB :: nptIII-CstR exhibited higher survival rates during conditions of 1 % HCl and 42 °C than the other strains. Both phoP and pmrA genes were overexpressed significantly in both colistin-resistant mutants, but parR and cprR genes were not.

Conclusion. We revealed that colistin resistance could be developed despite arnB deletion in P. aeruginosa through the addition of l-Ara4N to lipid A, which was accompanied by diverse physiological changes.

Purpose. The molecular epidemiology of Pseudomonas aeruginosa bloodstream infection (BSI) isolates has received limited attention. This study aims to characterize the molecular relationship of P. aeruginosa BSI isolates in the non-outbreak setting at a single tertiary healthcare facility.

Methodology.  P. aeruginosa BSI isolates from patients who were admitted to the Royal Brisbane and Women’s Hospital over a 13 month period from November 2009 were identified retrospectively from the Pathology Queensland Clinical and Scientific Information System. The isolates were typed by the iPLEX MassARRAY matrix assisted lazer desorption/isonisation time of flight (MALDI-TOF) MS genotyping. The DiversiLab automated rapid strain typing platform (bioMérieux) was used to assess the genotypic relationships between study isolates that showed indistinguishable iPLEX20SNP profiles. Clinical data was also collected retrospectively from patient notes.

Results. Fifty-three P. aeruginosa BSI episodes were available for study. Thirty-five different clones or clonal complexes were identified by the iPLEX MassARRAY MALDI-TOF MS genotyping. Seventeen BSI isolates with indistinguishable iPLEX20SNP profiles underwent further DiversiLab genotyping and were found to belong to a further 13 different genotypes. There was no relationship between clonality and acquisition type, source of infection or length of stay in the setting of hospital-acquired infection.

Conclusion. The non-clonal population structure suggests that there is ongoing environmental exposure of inpatients to P. aeruginosa. In clinical areas dealing with at-risk patients, routine attention to mechanism of environmental colonization is important and should be addressed even in the non-outbreak setting.