oprD Genes Detected in Pseudomonas aeruginosa Isolates from a Teaching Hospital but Lost in a Carbapenem-Resistant Strain

Aims:The aims of the study were to evaluate the multidrug resistance profile and mechanisms of carbapenem resistance in Pseudomonas aeruginosaclinical isolates using phenotypic and genotypic methods.Study Design: A descriptive laboratory based study.Place and Duration of Study: Microbiology Laboratory, Ondo State University of Science and Technology, Okitipupa, and Biotechnology Laboratory, Ladoke Akintola University of Technology, Osogbo, Nigeria, between June 2017 and November 2018.Methodology:Ten P. aeruginosa isolates were recovered from patients at Lagos University Teaching Hospital, and susceptibilities to imipenem (10μg), meropenem (10μg) and a panel of antibiotics were performed by the disk diffusion method. Genotypic methods including Polymerase Chain Reactions (PCR) and agarose gel electrophoresis were carried out according to established protocols. oprD and blaIMPgene primers were used for the PCR amplification. Results: Fifty percent (50%) of the isolates showed multiple drug resistance. Four isolates (40%) were carbapenem resistant (CR). oprDgene was detectedin 90% (9/10) of the isolates. 75% (3/4) of CR strains were among the strains showing oprDgene. 25% (1/4) CR strain (PA1421) was oprDnegative. Loss or mutation of oprDgene seems to be the mechanism of carbapenem resistance in strain PA1421. Conclusion: Loss or mutation of oprDgene was identified in this study as a mechanism of carbapenem resistance. oprDgene encodes the outer membrane protein (OprD) porin in P. aeruginosawhose deficiency confers resistance to carbapenems, especially imipenem. Surveillance of the antimicrobial susceptibility patterns ofP. aeruginosais of critical importance in understanding new and emerging resistance trends, reviewing antibiotic policies and informing therapeutic options.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Analysis of oprD gene in imipenem-intermediate clinical isolates of Pseudomonas aeruginosa / 中国感染与化疗杂志

Objective To analyze the mechanism of imipenem resistance in Pseudomonas aeruginosa clinical isolates. Methods? Antibiotic?resistance?was?analyzed?using?VITEK32?system.?Metallo?β-lactamase?activity?was?determined?by?double-disc?synergy?test.?Amp?C?β-lactamase?activity?was?determined?by?Kirby-Bauer?disc?method.?OprD?protein?was?analyzed?by?sodium?dodecyl sulphate-polyacrylamide gel electrophoresis. PCR was performed to amplify gene oprD. The amplified products were subject?to?sequencing?analysis.?The?phylogenetic?relationship?was?determined?using?random?amplified?polymorphic?DNA?(RAPD)?method. Results Membrane protein OprD was analyzed in 7 clinical isolates of imipenem-intermediate P. aeruginosa. Two strains were devoid of OprD proteins and the corresponding oprD genes were found disrupted by the insertion element ISRP10 in the coding regions. Five strains had OprD proteins with different sizes. Sequence analysis showed that the peptides ranged from 427 to 443 amino acids. Multiple amino acid substitutions and / or deletions were found within the Loop 1 through Loop 8 of the OprD secondary structures. Conclusions ISRP10 inactivation and amino acid substitutions in oprD gene confer imipenem resistance in the clinical isolates of P. aeruginosa.

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.

Chromosomal Mutations in oprD, gyrA, and parC in Carbapenem Resistant Pseudomonas aeruginosa / 대한임상미생물학회지

BACKGROUND: Outbreaks of carbapenem resistant P. aeruginosa give rise to significant therapeutic challenges for treating nosocomial infections. In this study, we analyzed carbapenem resistance mechanisms in carbapenem resistant and clonally different P. aeruginosa strains. We analyzed chromosomal alterations in the genes of OprD and efflux system regulatory proteins (MexR, NalC, NalD, MexT, and MexZ). We also investigated chromosomal alterations in the quinolone resistance-determining region (QRDR) for quinolone resistance mechanisms. METHODS: Twenty-one clonally different P. aeruginosa strains were isolated by repetitive extragenic palindromic sequence-based PCR (rep-PCR). PCR and DNA sequencing were conducted for the detection of beta-lactamase genes and chromosomal alterations of efflux pump regulatory genes, oprD, and QRDR in gyrA, gyrB, parC, and parE. RESULTS: Only one (P28) of the 21 strains harbored bla VIM-2. Two isolates had mutations in nalD or mexZ that were associated with efflux pump overexpression. Chromosomal alterations causing loss of OprD were found in 4 out of 21 carbapenem resistant P. aeruginosa strains. Nine of 10 imipenem and ciprofloxacin resistant strains had alterations in gyrA and/or parC. CONCLUSION: Carbapenem resistance in P. aeruginosa was mediated by several mechanisms, including loss of the OprD, overexpression of efflux systems, and production of carbapenemase. Resistance to quinolone is frequently caused by point mutations in gyrA and/or parC.

Analysis of correlation between resistance of carbapenem agents in Pseudomonas aeruginosa and mutation and altered expression of oprD2 / 中华微生物学和免疫学杂志

Objective To investigate the impact on the resistance of carbapenem with the expression of OprD2 or OprD2 mutation in Pseudomonas aeruginosa. Methods One hundred and one clinical strains of Pseudomonas aeruginosa with MIC for imipenem ≥8 μg/ml were studied. MIC were determined by the broth microdilution method, and the antibiotics tested were imipenem(IPM ), biapenem( BPM), meropenem(MEM) and panipenem(PEM). The expression of the oprD2 gene in Pseudomonas aeruginosa were analyzed by real-time reverse transcriptase PCR(RT-PCR). For the Pseudomonas aeruginosa with normal expression of OprD2 and resistance to imipenem, full-length oprD2 gene was amplified by PCR and the products were sequenced. Results According to the result of the expression of oprD2 gene, 101 strains of Pseudomonas aeruginosa were divided into two groups: group1 with diminished expression of OprD2, and group2 with normal expression of OprD2. Comparing isolates with MIC of 4 kinds of carbepenem agents ≥ 16 μg/ml in two groups. Data showed the amount of OprD2 expression were different between two groups(P ＜0.01 or P ＜ 0.05). In group1, there are 28 isolates with MIC ≥ 16 μg/ml of all the 4 kinds of carbapenems, among which 25 isolates have obviously diminished expression of OprD2 ( ＜ 0.4). Negative correlations tendency appeared between the level of OprD2 transcription and MICs of 4 kinds of carbepenem agents in Pseudomonas aeruginosa. In group2, 16 strains with OprD2 mutation divided into 4 types according to the pattern of alteration. Compared with PAO1, these strains have increased MIC with different degree to IPM,BPM, MEM and PEM. Conclusion The deletion or diminished expression of OprD2 resulted in resistance to imipenem in Pseudomonas aeruginosa. The level of OprD2 transcription and antimicrobial activities for carbapenem agents proved to be highly correlated in Pseudomonas aeruginosa. The mutation of OprD2 in Pseudomonas aeruginosa probably decreased the sensitivity of carbapenem agents against Pseudomonas aeruginosa.

Study of resistance to carbapenems of Pseudomonas aeruginosa in 5 Beijing hospitals / 中华检验医学杂志

Objective To investigate the molecular mechanism of resistance to carbapenems in Pseudomonas aeruginasa from 5 teaching hospitals in Beijing. Methods A total of 213 non-duplicate imipenem-resistant Pseudomonas aernginosa (IRPA) isolates were collected from 5 hospitals in Beijing from June 2004 to December 2005. The minimal inhibitory concentrations (MICs) of meropenem, imipenem and others antibiotics were determined by agar dilution method. Disk diffusion test was used for screening metalloenzymes. The bla IMP,bla VIM and OprD2 genes were amplified by PCR and only sequenced. Results Out of 213 isolates, OprD2 loss was detected in 84 isolates and IMP-1 enzyme was detected in 6 isolates simultaneously. Thirteen IRPAs only produced IMP-1 and 2 isolates only produced VIM-2. Conclusion OprD2 loss and metallo-β-lactamuse production are the parts of the mechanisms of resistance to carbapenems in Pseudomonas aeruginosa in Beijing.

Forty Types of Resistant-related Genes in a Pan-resistant Pseudomonas aeruginosa / 中华医院感染学杂志

OBJECTIVE To study 40 kinds of resistant-related genes in a pan-resistant Pseudomonas aeruginosa.METHODS To detect the susceptibility of antimicrobial agents by MIC,40 resistant-related genes including 29 ?-lactamases genes,porin oprD2 genes,6 aminoglycoside-modifying enzymes(AMEs)genes,chlorhexidine/sulfadiazine resistant gene(qacE△1-sul1)and intergron(intⅠ1,2,3),etc,form 1 strain of P.aeruginosa were measured by PCR,and verified by DNA sequencing.RESULTS In the strain,there were positive of 6 kinds of resistant-related genes(blaTEM,blaOXA10,aac(6′)-Ⅱ,aac(3)-Ⅱ,qacE△1-sul1 and intⅠ1),but without oprD2 genes.Twenty-seven kinds of ?-lactamases genes,4 kinds of AMEs(aac(6′)-Ⅰb,aac(3)-Ⅰ,ant(3″)-Ⅰ and ant(2″)-Ⅰ),and 2 kinds of intⅠ(intⅠ2 and intⅠ3) were negative.CONCLUSIONS The multi-resistant mechanisms of pan-resistant P.aeruginosa are mainly related to 7 kinds of resistant-related genes(blaTEM,blaOXA10,oprD2,aac(6′)-Ⅱ,aac(3)-Ⅱ,qacE△1-sul1 and intⅠ1).

Study on loss of OprD_2 in imipenem-resistant Pseudomonas aeruginosa / 临床检验杂志

Objective To investigate the loss of outer membrane protein D2 (OprD2) gene in imipenem-resistant Pseudomonas aeruginosa.Methods The minimal inhibitory concentrations (MIC) of 10 antimicrobial agents against 80 strains of imipenem-resistant clinical isolates were determined by agar dilution method.PCR was used to detect OprD2 gene.Results PCR results showed that 44 of the 80 isolates of imipenem-resistant P.aeruginosa were positive for OprD2 gene.There was significant difference (P

ERIC-PCR Fingerprinting Related with oprD_2 Gene of Pseudomonas aeruginosa Isolates Resistant to Imipenem / 中华医院感染学杂志

0.05).Thirty genotypes were got from 54 strains by ERIC-PCR.CONCLUSIONS There isn′t significant difference on the absence rate of oprD2 gene in Tianjin and it gives us a hint that other factors can also result in the prevalence of IRPA.The existence of prevalence of IRPA clone in some departments advises us to effectively control the IRPA in hospital.

Imipenem-resistant Mechanism of Pseudomonas Aeruginosa / 中国药房

OBJECTIVE:To investigate the mechanism of imipenem-resistance in clinical isolates of Pseudomonas aeruginos (PA). METHODS: 55 strains of PA had been isolated from sputum specimen in clinic and their drug sensitivity to imipenem was tested by K-B slip diffusion method. Their amount of outer membrane protein OprD2 was analyzed by SDS-PAGE with outer membrane protein profiles. Metallo-?-lactamases (MBLs) was detected by double-disk synergy test (DDST). RESULTS: 36 strains of imipenem-resistant and 19 strains of imipenem-sensitive PA isolated were studied. The amount of OprD2 showed a various degree of reduction in all resistant isolates, and the relative amount of OprD2 in resistant isolates were significantly lower than in sensitive isolates (P