scholarly journals Presence of Chromosomal crpP-like Genes Is Not Always Associated with Ciprofloxacin Resistance in Pseudomonas aeruginosa Clinical Isolates Recovered in ICU Patients from Portugal and Spain

2021 ◽  
Vol 9 (2) ◽  
pp. 388
Author(s):  
Marta Hernández-García ◽  
María García-Castillo ◽  
Sergio García-Fernández ◽  
Diego López-Mendoza ◽  
Jazmín Díaz-Regañón ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Function ◽  
Resistance Mechanism ◽  
Amino Acid Position ◽  
Genomic Islands ◽  
Missense Mutations ◽  
The Novel ◽  
Surveillance Studies ◽  
Ciprofloxacin Resistance ◽  
Chromosomal Mutations

CrpP enzymes have been recently described as a novel ciprofloxacin-resistance mechanism. We investigated by whole genome sequencing the presence of crpP-genes and other mechanisms involved in quinolone resistance in MDR/XDR-Pseudomonas aeruginosa isolates (n = 55) with both ceftolozane-tazobactam susceptible or resistant profiles recovered from intensive care unit patients during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Ciprofloxacin resistance was associated with mutations in the gyrA and parC genes. Additionally, plasmid-mediated genes (qnrS2 and aac(6′)-Ib-cr) were eventually detected. Ten chromosomal crpP-like genes contained in related pathogenicity genomic islands and 6 different CrpP (CrpP1-CrpP6) proteins were found in 65% (36/55) of the isolates. Dissemination of CrpP variants was observed among non-related clones of both countries, including the CC175 (Spain) high-risk clone and CC348 (Portugal) clone. Interestingly, 5 of 6 variants (CrpP1-CrpP5) carried missense mutations in an amino acid position (Gly7) previously defined as essential conferring ciprofloxacin resistance, and decreased ciprofloxacin susceptibility was only associated with the novel CrpP6 protein. In our collection, ciprofloxacin resistance was mainly due to chromosomal mutations in the gyrA and parC genes. However, crpP genes carrying mutations essential for protein function (G7, I26) and associated with a restored ciprofloxacin susceptibility were predominant. Despite the presence of crpP genes is not always associated with ciprofloxacin resistance, the risk of emergence of novel CrpP variants with a higher ability to affect quinolones is increasing. Furthermore, the spread of crpP genes in highly mobilizable genomic islands among related and non-related P. aeruginosa clones alert the dispersion of MDR pathogens in hospital settings.

GPC-1, a novel class A carbapenemase detected in a clinical Pseudomonas aeruginosa isolate

2020 ◽  
Vol 75 (4) ◽  
pp. 911-916 ◽  
Author(s):  
Jennifer Schauer ◽  
Sören G Gatermann ◽  
Daniel Hoffmann ◽  
Lars Hupfeld ◽  
Niels Pfennigwerth
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heterologous Expression ◽  
Biochemical Characterization ◽  
Resistance Mechanism ◽  
Carbapenem Resistance ◽  
Amino Acid Identity ◽  
The Novel ◽  
Bacterial Strains ◽  
Class A ◽  
Carbapenem Resistant

Abstract Objectives To investigate the carbapenem resistance mechanism of a carbapenem-resistant clinical Pseudomonas aeruginosa isolate. Methods A carbapenem-resistant P. aeruginosa isolate was recovered from a tracheal swab from a patient of a general ward in central Germany. Various phenotypic tests confirmed production of a carbapenemase that could not be identified further by PCR. A novel bla gene was identified by WGS and its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain. Kinetic parameters of the novel β-lactamase were determined by spectrophotometric measurements using purified enzyme. Results WGS confirmed the presence of a novel class A carbapenemase. The novel bla gene was named GPC-1 (GPC standing for German Pseudomonas Carbapenemase) and exhibited 77% amino acid identity to BKC-1. WGS also showed that blaGPC-1 was located on the chromosome surrounded by multiple ISs as part of a 26 kb genetic island. Heterologous expression of GPC-1 in E. coli TOP10 led to increased MICs of penicillins, oxyimino-cephalosporins, aztreonam and imipenem, but not of meropenem or ertapenem. Spectrophotometric measurements supported the MIC studies, but detected a slight hydrolysis of ertapenem and meropenem when using high concentrations of purified enzyme. Conclusions The biochemical characterization of GPC-1 emphasizes the ongoing emergence of novel carbapenemases. Strains expressing a weak carbapenemase like GPC-1 might go unrecognized by routine diagnostics due to low MICs for the bacterial strains producing such enzymes.

scholarly journals ICEs are the main reservoirs of the ciprofloxacin-modifying crpP gene in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
João Botelho ◽  
Filipa Grosso ◽  
Luísa Peixe
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Direct Repeats ◽  
Bacterial Genomes ◽  
Genome Database ◽  
Integrative And Conjugative Elements ◽  
Data Files ◽  
Supplementary Material ◽  
Ciprofloxacin Resistance ◽  
Chromosomal Mutations ◽  
Sequence Types

AbstractThe ciprofloxacin-modifying crpP gene was recently identified in a plasmid isolated from a clinical Pseudomonas aeruginosa clinical isolate. Homologues of this gene were also identified in Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. We set out to explore the mobile genetic elements involved in the acquisition and spread of this gene in publicly available and complete genomes of Pseudomonas. The crpP gene was identified only in P. aeruginosa, in more than half of the complete chromosomes (61.9%, n=133/215) belonging to 52 sequence types, of which the high-risk clone ST111 was the most frequent. We identified 136 crpP-harboring ICEs, with 93.4% belonging to the mating-pair formation G (MPFG) family. The ICEs were integrated at the end of a tRNALys gene and were all flanked by highly conserved 45-bp direct repeats. The core ICEome contains 26 genes (2.2% of all genes), which are present in 99% or more of the crpP-harboring ICEs. The most frequently encoded traits on these ICEs include replication, transcription, intracellular trafficking and cell motility. Our work reveals that ICEs are the main vectors promoting the dissemination of the ciprofloxacin-modifying crpP gene in P. aeruginosa.Author NotesAll supporting data has been provided within the article or through supplementary data files. Supplementary material is available with the online version of this article.Impact StatementA high proportion of Pseudomonas aeruginosa clinical isolates are resistant to ciprofloxacin. Resistance to this antibiotic is often mediated by chromosomal mutations, but recently horizontally transferred genes have been identified. We assessed the repartition of the ciprofloxacin-modifying crpP gene among Pseudomonas genomes and we characterized the mobile elements associated with its acquisition. We found that this gene is prevalent in P. aeruginosa and frequently associated with integrative and conjugative elements (ICEs). Importantly, we also identified highly conserved direct repeats that can be used to accurately delimit crpP-carrying ICEs in P. aeruginosa genomes.Data SummaryAll the bacterial genomes scanned in this study have been deposited previously in the National Center for Biotechnology Information genome database and are listed on the supplementary tables. The newick files used to create the trees in Figures 1 and 4 are deposited on figshare at https://figshare.com/projects/ICEs_are_the_main_reservoirs_of_the_ciprofloxacin-modifying_crpP_gene_in_Pseudomonas_aeruginosa/79308.

scholarly journals Emergence of resistance to novel cephalosporin-β-lactamase inhibitor combinations through the modification of the Pseudomonas aeruginosa MexCD-OprJ efflux pump

2021 ◽  
Author(s):  
María A. Gomis-Font ◽  
Cristina Pitart ◽  
Ester del Barrio-Tofiño ◽  
Yuliya Zboromyrska ◽  
Sara Cortes-Lara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Negative Regulator ◽  
Whole Genome ◽  
Ventilator Associated Pneumonia ◽  
Missense Mutations ◽  
The Novel ◽  
Wild Type ◽  
Emergence Of Resistance ◽  
Lactamase Inhibitor

A ceftolozane/tazobactam and ceftazime/avibactam resistant Pseudomonas aeruginosa isolate was recovered after treatment (including azithromycin, meropenem and ceftolozane/tazobactam) from a patient that had developed ventilator associated pneumonia after Covid-19 infection. Whole genome sequencing revealed that the strain, belonging to ST274, had acquired a nonsense mutation leading to a truncated carbapenem porin OprD (W277X), a 7-bp deletion (nt213Δ7) in NfxB (negative regulator of the efflux pump MexCD-OprJ), and two missense mutations (Q178R, S133G) located within the first Large Periplasmic Loop of MexD. Through the construction of mexD mutants and complementation assays with wild-type nfxB , it was evidenced that resistance to the novel cephalosporin-β-lactamase inhibitor combinations was caused by the modification of MexD substrate specificity.

scholarly journals Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Syed A. K. Shifat Ahmed ◽  
Michelle Rudden ◽  
Sabrina M. Elias ◽  
Thomas J. Smyth ◽  
Roger Marchant ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Genomic Islands ◽  
Clinical Strain ◽  
Whole Genome ◽  
Synonymous Mutations ◽  
Wide Range

AbstractPseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

scholarly journals Ciprofloxacin Resistance in Clinical Isolates of Pseudomonas aeruginosa from Italian Patients

Drugs ◽  
1995 ◽  
Vol 49 (Supplement 2) ◽  
pp. 175-176 ◽  
Author(s):  
G. Corti ◽  
F. Paradisi ◽  
E. Giganti ◽  
G. Buffini ◽  
E. Tortoli ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolates ◽  
Ciprofloxacin Resistance

scholarly journals Genotype-Phenotype correlation in Dravet Syndrome with SCN1A mutation increase efficiency of molecular diagnosis

2012 ◽  
Vol 18 (2) ◽  
pp. 60-62
Author(s):  
MC Gonsales ◽  
P Preto ◽  
MA Montenegro ◽  
MM Guerreiro ◽  
I Lopes-Cendes
Keyword(s):  
Protein Function ◽  
Copy Number ◽  
Mutation Screening ◽  
Copy Number Variants ◽  
Febrile Seizures ◽  
Copy Number Variations ◽  
Dravet Syndrome ◽  
Missense Mutations ◽  
The Impact ◽  
Doose Syndrome

OBJECTIVES: The purpose of this study was to advance the knowledge on the clinical use of SCN1A testing for severe epilepsies within the spectrum of generalized epilepsy with febrile seizures plus by performing genetic screening in patients with Dravet and Doose syndromes and establishing genotype-phenotype correlations. METHODS: Mutation screening in SCN1A was performed in 15 patients with Dravet syndrome and 13 with Doose syndrome. Eight prediction algorithms were used to analyze the impact of the mutations in putative protein function. Furthermore, all SCN1A mutations previously published were compiled and analyzed. In addition, Multiplex Ligation-Dependent Probe Amplification (MLPA) technique was used to detect possible copy number variations within SCN1A. RESULTS: Twelve mutations were identified in patients with Dravet syndrome, while patients with Doose syndrome showed no mutations. Our results show that the most common type of mutation found is missense, and that they are mostly located in the pore region and the N- and C-terminal of the protein. No copy number variants in SCN1A were identified in our cohort. CONCLUSIONS: SCN1A testing is clinically useful for patients with Dravet syndrome, but not for those with Doose syndrome, since both syndromes do not seem to share the same genetic basis. Our results indicate that indeed missense mutations can cause severe phenotypes depending on its location and the type of amino-acid substitution. Moreover, our strategy for predicting deleterious effect of mutations using multiple computation algorithms was efficient for most of the mutations identified.

scholarly journals Development of Novel Protective Polyvalent Irradiated Pseudomonas aeruginosa Vaccine for Immuno-Compromised Patients

2021 ◽  
Vol 16 ◽  
pp. 1-10
Author(s):  
Manal M.E. Ahmed ◽  
Jakeen Eljakee ◽  
Tarek Mahran
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protective Efficacy ◽  
Challenge Test ◽  
Opportunistic Pathogen ◽  
Effective Vaccine ◽  
The Novel ◽  
Therapeutic Approaches ◽  
Promising Strategy ◽  
Antigenic Expression ◽  
Compromised Patients

Pseudomonas aeruginosa is an opportunistic pathogen affecting immuno-compromised patients; however, no effective vaccine is currently available in the market. Here, we developed novel polyvalent irradiated P. aeruginosa vaccine using cobalt 60 that inhibited pathogen viability but retained antigenic expression functionally. Mice were vaccinated by the developed vaccine by intranasal, intramuscular and subcutaneous route of administration followed by challenge test. The protective efficacy of the novel vaccine reached up to 95%. This significant protection was mainly associated with measurable antiserum opsonic killing activity. In conclusion, the novel vaccine provides a promising strategy of both prophylactic and therapeutic approaches for immuno-compromised patients against MDR P. aeruginosa.

scholarly journals Novel compound heterozygous EYS variants may be associated with arRP in a large Chinese pedigree

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
Chunli Wei ◽  
Ting Xiao ◽  
Jingliang Cheng ◽  
Jiewen Fu ◽  
Qi Zhou ◽  
...  
Keyword(s):  
Novel Mutation ◽  
Gene Mutations ◽  
Chinese Family ◽  
Compound Heterozygous ◽  
Missense Mutations ◽  
The Novel ◽  
Pathogenic Variants ◽  
Pathogenic Genes ◽  
Compound Heterozygous Variants ◽  
Normal Controls

Abstract As a genetically heterogeneous ocular dystrophy, gene mutations with autosomal recessive retinitis pigmentosa (arRP) in patients have not been well described. We aimed to detect the disease-causing genes and variants in a Chinese arRP family. In the present study, a large Chinese pedigree consisting of 31 members including a proband and another two patients was recruited; clinical examinations were conducted; next-generation sequencing using a gene panel was used for identifying pathogenic genes, and Sanger sequencing was performed for verification of mutations. Novel compound heterozygous variants c.G2504A (p.C835Y) and c.G6557A (p.G2186E) for the EYS gene were identified, which co-segregated with the clinical RP phenotypes. Sequencing of 100 ethnically matched normal controls didn’t found these mutations in EYS. Therefore, our study identified pathogenic variants in EYS that may cause arRP in this Chinese family. This is the first study to reveal the novel mutation in the EYS gene (c.G2504A, p.C835Y), extending its mutation spectrum. Thus, the EYS c.G2504A (p.C835Y) and c.G6557A (p.G2186E) variants may be the disease-causing missense mutations for RP in this large arRP family. These findings should be helpful for molecular diagnosis, genetic counseling and clinical management of arRP disease.

scholarly journals Attenuated enzootic (pestoides) isolates of Yersinia pestis express active aspartase

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 198-209 ◽  
Author(s):  
Scott W. Bearden ◽  
Christopher Sexton ◽  
Joshua Pare ◽  
Janet M. Fowler ◽  
Cindy G. Arvidson ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Yersinia Pseudotuberculosis ◽  
Amino Acid Position ◽  
Enzymic Activity ◽  
Broad Host Range ◽  
Missense Mutations ◽  
Tissue Invasion ◽  
Muroid Rodents ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Reduced Activity

It is established that Yersinia pestis, the causative agent of bubonic plague, recently evolved from enteropathogenic Yersinia pseudotuberculosis by undergoing chromosomal degeneration while acquiring two unique plasmids that facilitate tissue invasion (pPCP) and dissemination by fleabite (pMT). Thereafter, plague bacilli spread from central Asia to sylvatic foci throughout the world. These epidemic isolates exhibit a broad host range including man as opposed to enzootic (pestoides) variants that remain in ancient reservoirs where infection is limited to muroid rodents. Cells of Y. pseudotuberculosis are known to express glucose-6-phosphate dehydrogenase (Zwf) and aspartase (AspA); these activities are not detectable in epidemic Y. pestis due to missense mutations (substitution of proline for serine at amino position 155 of Zwf and leucine for valine at position 363 of AspA). In this study, functional Zwf was found in pestoides strains E, F and G but not seven other enzootic isolates; enzymic activity was associated with retention of serine at amino acid position 155. Essentially, full AspA activity occurred in pestoides isolates where valine (pestoides A, B, C and D) or serine (pestoides E, F, G and I) occupied position 363. Reduced activity occurred in strains Angola and A16, which contained phenylalanine at this position. The k cat but not K m of purified AspA from strain Angola was significantly reduced. In this context, aspA of the recently described attenuated enzootic microtus biovar encodes active valine at position 363, further indicating that functional AspA is a biomarker for avirulence of Y. pestis in man.

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