Verification and application of a modified carbapenem inactivation method (mCIM) on Pseudomonas aeruginosa: a potential screening methodology on carbapenemases phenotype in Bacillus cereus.
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SUBMITTER: Liu J
PROVIDER: S-EPMC9275876 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We report here the emergence of VIM-2 and IMP-15 carbapenemases in a series of clinical isolates of carbapenem-resistant Pseudomonas aeruginosa in Lebanon. We also describe the disruption of the oprD gene by either mutations or insertion sequence (IS) elements ISPa1328 and ISPre2 isoform. Our study reemphasizes a rapid dissemination of the VIM-2 carbapenemase-encoding gene in clinical isolates of P. aeruginosa in the Mediterranean basin.
| S-EPMC4136035 | biostudies-literature
Project description:The worldwide emergence and spread of antimicrobial resistance in Gram-negative bacteria are severely limiting therapeutic options and thus constitute a major public health threat. The timely accurate detection of carbapenemase producers and the determination of carbapenemase class according to the Ambler classification can guide antimicrobial therapy and facilitate infection control measures. A modified version of the carbapenemase inactivation method (CIM), mCIM, was described and approved by the CLSI in 2017. We evaluated the performance of a faster new mCIM-based assay, mCIMplus, which can detect carbapenemase activity within 8 h and characterize the carbapenemase according to the Ambler classification in 20 h. A panel of 137 isolates producing carbapenemases (GES, IMP, KPC, NDM, OXA-48, OXA-48-like, and VIM enzymes) and 22 non-carbapenemase-producing isolates was used to evaluate the performance of mCIMplus. We evaluated the detection of carbapenemase activity at 8 and 20 h. Carbapenemase class was determined, with specific inhibitors, at 20 h. The sensitivities of mCIMplus were 99.3% at 8 h and 98.5% at 20 h. Its specificity was 100% regardless of culture time. Based on a decision algorithm, this test successfully identified the carbapenemase class for 98.4% of the tested isolates (127/129). Characterization was correct for 100, 95, and 100% of Ambler class A, B, and D isolates, respectively. This test can, therefore, be used to detect carbapenemase activity within 8 h and to determine carbapenemase class within 20 h. It constitutes a very affordable (<€1 per isolate) and reliable technique requiring only basic laboratory equipment.
| S-EPMC7587109 | biostudies-literature
Project description:Carbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.
| S-EPMC8557820 | biostudies-literature
Project description:BackgroundCarbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the distribution and clinical significance of carbapenemases are unclear. The aim of this study was to define characteristics and outcomes of CRPA infections and the global frequency and clinical impact of carbapenemases harboured by CRPA.MethodsWe conducted an observational, prospective cohort study of CRPA isolated from bloodstream, respiratory, urine, or wound cultures of patients at 44 hospitals (10 countries) between Dec 1, 2018, and Nov 30, 2019. Clinical data were abstracted from health records and CRPA isolates were whole-genome sequenced. The primary outcome was 30-day mortality from the day the index culture was collected. We compared outcomes of patients with CRPA infections by infection type and across geographic regions and performed an inverse probability weighted analysis to assess the association between carbapenemase production and 30-day mortality.FindingsWe enrolled 972 patients (USA n=527, China n=171, south and central America n=127, Middle East n=91, Australia and Singapore n=56), of whom 581 (60%) had CRPA infections. 30-day mortality differed by infection type (bloodstream 21 [30%] of 69, respiratory 69 [19%] of 358, wound nine [14%] of 66, urine six [7%] of 88; p=0·0012) and geographical region (Middle East 15 [29%] of 52, south and central America 20 [27%] of 73, USA 60 [19%] of 308, Australia and Singapore three [11%] of 28, China seven [6%] of 120; p=0·0002). Prevalence of carbapenemase genes among CRPA isolates also varied by region (south and central America 88 [69%] of 127, Australia and Singapore 32 [57%] of 56, China 54 [32%] of 171, Middle East 27 [30%] of 91, USA ten [2%] of 527; p<0·0001). KPC-2 (n=103 [49%]) and VIM-2 (n=75 [36%]) were the most common carbapenemases in 211 carbapenemase-producing isolates. After excluding USA patients, because few US isolates had carbapenemases, patients with carbapenemase-producing CRPA infections had higher 30-day mortality than those with non-carbapenemase-producing CRPA infections in both unadjusted (26 [22%] of 120 vs 19 [12%] of 153; difference 9%, 95% CI 3-16) and adjusted (difference 7%, 95% CI 1-14) analyses.InterpretationThe emergence of different carbapenemases among CRPA isolates in different geographical regions and the increased mortality associated with carbapenemase-producing CRPA infections highlight the therapeutic challenges posed by these organisms.FundingNational Institutes of Health.
| S-EPMC10016089 | biostudies-literature
Project description:The purpose of this study was to develop the modified carbapenem inactivation method (mCIM) for the detection of carbapenemase-producing Pseudomonas aeruginosa (CP-PA) and carbapenemase-producing Acinetobacter baumannii (CP-AB) and perform a multicenter evaluation of the mCIM and Carba NP tests for these nonfermenters. Thirty P. aeruginosa and 30 A. baumannii isolates previously characterized by whole-genome sequencing from the CDC-FDA Antibiotic Resistance Isolate Bank were evaluated, including CP isolates (Ambler class A, B, and D), non-carbapenemase-producing (non-CP) carbapenem-resistant isolates, and carbapenem-susceptible isolates. Initial comparison of a 1-?l versus 10-?l loop inoculum for the mCIM was performed by two testing sites and showed that 10 ?l was required for reliable detection of carbapenemase production among P. aeruginosa and A. baumannii Ten testing sites then evaluated the mCIM using a 10-?l loop inoculum. Overall, the mean sensitivity and specificity of the mCIM for detection of CP-PA across all 10 sites were 98.0% (95% confidence interval [CI], 94.3 to 99.6; range, 86.7 to 100) and 95% (95% CI, 89.8 to 97.7; range, 93.3 to 100), whereas the mean sensitivity and specificity among CP-AB were 79.8% (95% CI, 74.0 to 84.9; range, 36.3 to 95.7) and 52.9% (95% CI, 40.6 to 64.9; range, 28.6 to 100), respectively. At three sites that evaluated the performance of the Carba NP test using the same set of isolates, the mean sensitivity and specificity of the Carba NP test were 97.8% (95% CI, 88.2 to 99.9; range, 93.3 to 100) and 97.8% (95% CI, 88.2 to 99.9; range, 93.3 to 100) for P. aeruginosa and 18.8% (95% CI, 10.4 to 30.1; range, 8.7 to 26.1) and 100% (95% CI, 83.9 to 100; range, 100) for A. baumannii Overall, we found both the mCIM and the Carba NP test to be accurate for detection of carbapenemase production among P. aeruginosa isolates and less reliable for use with A. baumannii isolates.
| S-EPMC5744225 | biostudies-literature
Project description:Concerns arising from accidental and occasional releases of novel industrial nanomaterials to the environment and waterbodies are rapidly increasing as the production and utilization levels of nanomaterials increase every day. In particular, two-dimensional nanosheets are one of the most significant emerging classes of nanomaterials used or considered for use in numerous applications and devices. This study deals with the interactions between 2D molybdenum disulfide (MoS2) nanosheets and beneficial soil bacteria. It was found that the log-reduction in the survival of Gram-positive Bacillus cereus was 2.8 (99.83%) and 4.9 (99.9988%) upon exposure to 16.0 mg/mL bulk MoS2 (macroscale) and 2D MoS2 nanosheets (nanoscale), respectively. For the case of Gram-negative Pseudomonas aeruginosa, the log-reduction values in bacterial survival were 1.9 (98.60%) and 5.4 (99.9996%) for the same concentration of bulk MoS2 and MoS2 nanosheets, respectively. Based on these findings, it is important to consider the potential toxicity of MoS2 nanosheets on beneficial soil bacteria responsible for nitrate reduction and nitrogen fixation, soil formation, decomposition of dead and decayed natural materials, and transformation of toxic compounds into nontoxic compounds to adequately assess the environmental impact of 2D nanosheets and nanomaterials.
| S-EPMC8229097 | biostudies-literature
Project description:In this study, we report the insertion sequence ISPpu21 in the oprD porin gene of carbapenem-resistant Pseudomonas aeruginosa isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for P. aeruginosa isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of oprD and mex efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS-PAGE. The genetic relationship between the P. aeruginosa isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of P. aeruginosa harboured the ISPpu21 insertion element in the oprD gene. The extended-spectrum β-lactamase-encoding gene in ISPpu21-carrying isolates was blaTEM. PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The rmtA, aadA, aadB and armA genes were positive in all ISPpu21 harbouring isolates. The relative expression levels of the mexX, mexB, mexT and mexD genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the oprD in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain P. aeruginosa PAO1. Evaluation of the outer membrane protein by SDS-PAGE suggested that oprD was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing ISPpu21 were shown to be clonally related. The present study describes a novel molecular mechanism, ISPpu21 insertion of the oprD gene, associated with carbapenem resistance in clinical P. aeruginosa isolates.
| S-EPMC5722278 | biostudies-literature
Project description:The rise of antimicrobial resistant bacteria has fast-tracked the exploration for novel antimicrobial compounds. Reports on antimicrobial producing soil anaerobes such as Clostridium spp. are very limited. In the present study, the antimicrobial activity of soil Clostridium enriched conditioned/spent media (CMs) against Bacillus mycoides, Bacillus cereus and Pseudomonas aeruginosa was assessed by turbidimetric growth inhibition assay. Our results highlighted the antimicrobial potential of soil Clostridium enriched conditioned media against pathogenic and spoilage bacteria. Farm 4 soil conditioned medium (F4SCM) demonstrated a greater growth inhibition activity against all three tested microorganisms in comparison to other soil conditioned media. Non-targeted metabolite profiling of all soil conditioned media revealed distinctive polar and intermediate-polar metabolites in F4SCM, consistent with its strong antimicrobial property. Moreover, 539 significantly abundant metabolites including some unique features were detected in F4SCM suggesting its substantial and specialized chemical diversity. This study putatively identified seven significantly high metabolites in F4SCM; 3-hydroxyphenylacetic acid, γ-aminobutyric acid, creatine, tryptamine, and 2-hydroxyisocaproic acid. Tryptamine and 2-hydroxyisocaproic acid were previously reported to have antimicrobial properties. The present study shows that soil Clostridium spp. are a promising group of bacteria producing metabolites with antimicrobial activity and provides future prospects for clostridial antimicrobial discovery within their metabolic diversity.
| S-EPMC7746556 | biostudies-literature
Project description:Background: Quorum quenching, the interference of a Quorum sensing (QS) system that contributes to the pathogenesis through triggering the production of various virulence determinants, is among the newly suggested antivirulence strategies. Purpose: This study aimed at screening of N-Acyl homoserine lactonase activity from local bacterial isolate, and investigating its effect on Pseudomonas aeruginosa (P. aeruginosa) virulence and biofilm formation. Materials and methods: Soil bacteria were screened for aiiA gene coding for lactonase enzyme by Polymerase Chain reaction and sequencing of aiiA gene homologs. Lactonase activity and spectrum were assessed in the cell-free lysate by well diffusion assay using Agrobacterium tumafaciens KYC55. A bacterial isolate showing the highest N-acyl-homoserine lactones degradation percentage was identified by gene amplification and sequencing of the 16S rRNA gene and its aiiA gene homolog. High performance liquid chromatography was used to confirm N-acyl-homoserine lactone degradation. The effect of cell-free lysate on the biofilm formation ability and cytotoxicity of P. aeruginosa PAO1 and P. aeruginosa clinical isolates from different clinical sources were assessed by static microtiter plate and viability assay, respectively Results: Lactonase gene and activity were identified in three Bacillus spp. isolates. They showed broad catalytic activities against tested N-acyl-homoserine lactones. However, The lactonase activity in the cell- free lysate of isolate 30b showed the highest significant degradation percentage on all tested signals; N-butanoyl-L-homoserine lactone (71%), N-hexanoyl-l-homoserine lactone (100%), N-decanoyl-homoserine lactone (100%), N-(3-oxohexanoyl)-L-homoserine lactone (37.5%), N-(oxodecanoyl)-L-homoserine lactone (100%), and N-(3-oxododecanoyl)-L-homoserine lactone (100%). Alignment of the amino acid sequences of AiiA protein of isolate 30b showed 96% identity with Bacillus cereus (B. cereus) homologous lactonases in the GenBank database, and the isolate was designated as B. cereus isolate 30b. Cell-free lysate of B. cereus isolate 30b reduced biofilm formation significantly in 93% of P. aeruginosa isolates. The highest mean percentage of reduction in the biofilm was 86%. Moreover, the viability percentage of human lung carcinoma A549 cells infected by P. aeruginosa and treated with cell-free lysate of B. cereus isolate 30b increased up to 15%. Conclusion: The results of this study highlight the potential of lactonases as a promising strategy to combat Pseudomonas aeruginosa virulence.
| S-EPMC6559722 | biostudies-literature
Project description:The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN (?(54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a ?rpoN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a ?rpoN ?pqsA mutant, in which the carbapenem-tolerant phenotype of the ?rpoN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the ?rpoN mutant is mediated through pqsE Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the ?rpoN ?pqsA mutant, and overexpression of pqsE failed to alter the susceptibility of the ?rpoN ?pqsA mutant to biapenem. The mutations in the ?rpoN ?rhlR mutant and the ?rpoN ?pqsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the ?rpoN mutant and the ?rpoN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.
| S-EPMC5038263 | biostudies-other