Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.     

Detection of Pseudomonas aeruginosa carried a new array of gene cassettes within class 1 integron isolated from a teaching hospital in Nanjing, China

We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6′)-II-aadA13-cmlA8-oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about 15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile indicated those clinical strains were clonally related. These authors contributed equally to this work.     

Chemotherapy-mediated p53-dependent DNA damage response in clear cell renal cell carcinoma: role of the mTORC1/2 and hypoxia-inducible factor pathways

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel–Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.     

Genomic rearrangements in BRCA1 and BRCA2: A literature review

Women with mutations in the breast cancer genes BRCA1 or BRCA2 have an increased lifetime risk of developing breast, ovarian and other BRCA-associated cancers. However, the number of detected germline mutations in families with hereditary breast and ovarian cancer (HBOC) syndrome is lower than expected based upon genetic linkage data. Undetected deleterious mutations in the BRCA genes in some high-risk families are due to the presence of intragenic rearrangements such as deletions, duplications or insertions that span whole exons. This article reviews the molecular aspects of BRCA1 and BRCA2 rearrangements and their frequency among different populations. An overview of the techniques used to screen for large rearrangements in BRCA1 and BRCA2 is also presented. The detection of rearrangements in BRCA genes, especially BRCA1, offers a promising outlook for mutation screening in clinical practice, particularly in HBOC families that test negative for a germline mutation assessed by traditional methods.     

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ²²). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.     

Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease

Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection. A series of full-length and truncation mutants were purified for structural and functional studies to evaluate the role of Ca(2+) in AP folding and activation. We find that Ca(2+) binding induces RTX folding, which serves to chaperone the folding of the protease domain. Subsequent association of the RTX domain with an N-terminal α-helix stabilizes AP. These results provide a basis for the Ca(2+)-mediated regulation of AP and suggest mechanisms by which Ca(2+) regulates the RTX family of virulence factors.     

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1^R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1''s substrate α-KG. The mutant IDH1^R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC''s treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.     

siRNA-mediated gene silencing of MexB from the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa

To gain insights into the effect of MexB gene under the short interfering RNA (siRNA), we synthesized 21 bp siRNA duplexes against the MexB gene. RT-PCR was performed to determine whether the siRNA inhibited the expression of MexB mRNA. Changes in antibiotic susceptibility in response to siRNA were measured by the E-test method. The efficacy of siRNAs was determined in a murine model of chronic P. aeruginosa lung infection. MexB-siRNAs inhibited both mRNA expression and the activity of P. aeruginosain vitro. In vivo, siRNA was effective in reducing the bacterial load in the model of chronic lung infection and the P. aeruginosa-induced pathological changes. MexB-siRNA treatment enhanced the production of inflammatory cytokines in the early infection stage (P ＜ 0.05). Our results suggest that targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. [BMB Reports 2014; 47(4): 203-208]     

An efflux pump is involved in secretion of newly synthesized siderophore by Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes the fluorescent siderophore, pyoverdine (PVD), to enable iron acquisition. Epifluorescence microscopy and cellular fractionation were used to investigate the role of an efflux pump, PvdRT-OpmQ, in PVD secretion. Bacteria lacking this efflux pump accumulated PVD, or a fluorescent precursor, in the periplasm, due to their inability to efficiently secrete into the media newly synthesized PVD. PvdRT-OpmQ is only the second system identified for secretion of newly synthesized siderophores by Gram negative bacteria.     

α1-antitrypsin promotes SPLUNC1-mediated lung defense against Pseudomonas aeruginosa infection in mice

Background

Pseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection.

Method

SPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2 hrs after the PA infection, and mice were sacrificed 24 hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot.

Results

In WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production.

Conclusion

A1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation.     

Pesticidal and pest repellency activities of a plant derived triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against Tribolium castaneum

Background

Tribolium castaneum (Herbst) is a major pest of stored grain-based products, and cause severe damage to cereal grains throughout the world. The present investigation was aimed to determine the pesticidal and pest repellent activities of 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against T. castaneum. The compound 2α,3β,21β,23,28-penta hydroxyl 12-oleanene is a triterpenoid which was isolated from the roots of Laportea crenulata Gaud. Surface film technique was used for pesticidal screening, whereas, pest repellency property of the triterpenoid was determined by filter paper disc method.

Results

At 24 hours of exposure duration, significant mortality records (80% and 86%) were observed at doses 0.88 and 1.77 mg/cm². No significant change in mortality records was observed when duration of exposure was increased up to 48 hours. The triterpenoid showed significant repellency activity at doses 0.47 and 0.94 mg/cm².

Conclusion

These data suggest that the triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene possess both pesticidal and pest repellency activities against T. castaneum and can be used in controlling the pest of grain-based products.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-68) contains supplementary material, which is available to authorized users.     

Analysis of the Sam50 translocase of Excavate organisms supports evolution of divergent organelles from a common endosymbiotic event

As free-living organisms the ancestors of mitochondria and plastids encoded complete genomes, proteomes and metabolomes. As these symbionts became organelles all these aspects were reduced – genomes have degenerated with the host nucleus now encoding the most of the remaining endosymbiont proteome, while the metabolic processes of the symbiont have been streamlined to the functions of the emerging organelle. By contrast, the topology of the endosymbiont membrane has been preserved, necessitating the development of complex pathways for membrane insertion and translocation. In this study, we examine the characteristics of the endosymbiont-derived β-barrel insertase Sam50¹ in the excavate super-group. A candidate is further characterized in Trichomonas vaginalis, an unusual eukaryote possessing degenerate hydrogen-producing mitochondria called hydrogenosomes. This information supports a mitochondriate eukaryotic common ancestor with a similarly evolved β-barrel insertase, which has continued to be conserved in degenerate mitochondria.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.     

Prevalence of multidrug resistant and extended spectrum beta-lactamase producing Pseudomonas aeruginosa in a tertiary care hospital

Resistance to broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamase enzymes (ESBL), is an increasing problem worldwide. The present study was undertaken to determine the incidence of ESBL-production among the clinical isolates of Pseudomonas aeruginosa and their susceptibility to selected antimicrobials. A total of one eighty-seven clinical specimens were tested for the presence of ESBL production using the double-disc synergy test. Of these, 25.13% (n = 47) isolates of P. aeruginosa were observed as ESBL positive. The maximum number of ESBL-producing strains were found in sputum (41.67%; n = 24) followed by pus (28.36%; n = 19), cerebrospinal fluid and other body fluids (21.74%; n = 5), urine (20.45%; n = 9) and blood (13.79%; n = 4). ESBL producing isolates exhibited co-resistance to an array of antibiotics tested. Imipenem and meropenem can be suggested as the drugs of choice in our study.     

Autophagy is a key tolerance mechanism during Staphylococcus aureus infection

Defense strategies against infectious threats can be divided into resistance and tolerance mechanisms. Resistance mechanisms involve reduction of pathogen burden and include many established examples, one of them being the destruction of intracellular pathogens through autophagy (xenophagy). Tolerance mechanisms protect the host from damage caused by the pathogen or the immune response independent of pathogen load. The role of autophagy in maintaining homeostasis in response to environmental stress suggests that this pathway is involved in tolerance to a variety of infectious agents. However, demonstrating that autophagy promotes tolerance independent of its role in resistance has been a challenge, especially during infection by clinically relevant pathogens. We have found that autophagy protects against Staphylococcus aureus infection by maintaining tolerance toward a pore forming toxin secreted by the bacteria, α-toxin.