oprD Gene Research Articles

Меропенем-индуцированное снижение чувствительности к колистину у Pseudomonas aeruginosa ATCC 27853

Antibiotic-resistant strains of Pseudomonas aeruginosa are a global threat to public health. The knowledge of mechanisms underlying antibiotic resistance is essential to counter P. aeruginosa infections. This study describes the phenomenon of meropenem-induced cross-resistance to colistin in the ATCC 27853 strain of P. aeruginosa. The study was conducted in the specimens of P. aeruginosa grown from the reference ATCC 27853 strain in the medium containing meropenem gradients. Susceptibility of the isolates to carbapenems and colistin was assessed using the agar dilution method; susceptibly to colistin was assessed using the broth microdilution method. A total of 93 P. Aeruginosa isolates were analyzed; of them two demonstrated reduced susceptibility to carbapenems (meropenem, imipenem) and colistin. Whole-genome sequencing of the isolates was performed on a MGISEQ-2000 platform. Missense mutations in the oprD and mexD genes and a nonsense mutation in the phoQ gene were detected. We conclude that exposure of P. aeruginosa to meropenem can lead to cross-resistance to colistin, a last resort drug for P. aeruginosa infections.

Genetic polymorphism of opioid receptors and use of opioid substances by high school students of Cotonou and Parakou (Benin)

In Benin, schools become sometimes the hub for dealing and consuming psychoactive substances; opioids in particular. The objectives of this study are to identify the risk factors related to the use of opioids and investigate the genetic polymorphism of mu and delta opioid receptors of teenagers and young adults who consume opioids in schools. To accomplish this, 453 students participated in this study; R diversity 3.6.1 software in the RStudio environment was used to identify students who experience opioids through ASSIST V3.0 scoring. SNPs A118G on the OPRM gene (µ) and T921C on the OPRD gene (d), were searched by PCR on DNA extracts from peripheral blood of individuals. We identified 54 regular opioid users and 399 non users. This experience begins for most with the consumption of alcohol and tobacco and is facilitated by the proximity of marshlands, kiosks, and pubs near high schools and colleges. The aggressive advertisement combined with relative socio-cultural tolerance just worsen this behavioral deviance. We found no difference in the SNP frequencies of the OPRM (µ) and OPRD (d) genes between students opioids consumers and non-consumers.

Computer Program for Detection and Analyzing the Porin-Mediated Antibiotic Resistance of Bacteria.

The aim of this work was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to demonstrate the functionality of this program by detecting porin-mediated resistance to carbapenems in clinical isolates of Pseudomonas aeruginosa.Materials and MethodsThe proposed algorithm is based on searching for a correspondence between the reference and the studied genes. When the sought nucleotide sequence is found in the analyzed genome, it is compared with the reference one and analyzed. The genomic analysis is then verified by comparing between the amino acid sequences encoded by the reference and studied genes. The genes of the susceptible P. aeruginosa ATCC 27853 strain were used as the reference nucleotide sequences encoding for porins (OprD, OpdD, and OpdP) involved in the transport of carbapenems into the bacterial cell. The complete genomes of clinical P. aeruginosa isolates from the PATRIC database 3.6.9 and our own collection were used to test the functionality of the proposed program. The analyzed isolates were phenotypically characterized according to the CLSI standard. The search for carbapenemase genes in the studied genomes of P. aeruginosa was carried out using the ResFinder 4.1.ResultsThe developed program for detecting the genetic determinants of non-plasmid antibiotic resistance made it possible to identify mutations of various types and significance in the porin genes of P. aeruginosa clinical isolates. These mutations led to modifications of the peptide structure of porin proteins. Single amino acid substitutions prevailed in the OpdD and OpdP porins of carbapenem-susceptible and carbapenem-resistant isolates. In the carbapenem-resistant strains, the gene encoding for OprD porin was found heavily modified, including insertions and/or deletions, which led to premature termination of porin synthesis. In several isolates resistant to meropenem, no mutations were detected in the gene encoding for OprD, which might be associated with alternative mechanisms of resistance to carbapenems.ConclusionThe proposed software product can become an effective tool for deciphering the molecular genetic mechanisms of bacterial chromosomal resistance to antibiotics. Testing the program revealed differences between the occurrences of mutations significant for carbapenem resistance in the oprD, opdD, and opdP genes.

Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits.

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

Control of extensively drug-resistant Pseudomonas aeruginosa co-harboring metallo-β-lactamase enzymes with oprD gene downregulation

Purposeto study control and treatment of infection with extensive drug-resistant carbapenem-resistant Pseudomonas aeruginosa (XDR-CRPA). MethodsEleven Pseudomonas aeruginosa (XDR-CRPA) strains used in this study were isolated from a clinical sample, identified, and antibiotics susceptibility recorded in a previous study. Real-time PCR (RT-PCR) was performed to determine the expression level of the OprD gene. Besides, a checkerboard technique was performed to assess the effect of polymyxin-B (POX), colistin (COL), rifampicin (RIF), imipenem (IPM), and meropenem (MEM) during 2 and 3- dimensional antibiotic combinations. Further, the time-kill study was determined for the most potent combination against four representative strains, log10 changes of viable cell counts were expressed as their mean value (±SD) values. ResultsMolecular analysis by Real-time PCR revealed that the diminished expression level of OprD mRNA was overwhelming to various degrees. The checkerboard method demonstrated that the relevant synergism was achieved in 90.9% of strains for both carbapenem antibiotics during the triple combinations. While an additive effect was noted for all the dual regimen assays. Regarding time-kill experiments, a remarkable bactericidal effect with [99.9% killing rate] was observed toward only one strain whilst a bacteriostatic attitude was proven with ≥95% bacterial eradication against the three remaining strains. ConclusionsThese findings underscore the promising implications of these combinations for treatment against XDR-Pseudomonas aeruginosa even they are resistant to carbapenems due to multiple mechanisms of action.

Carbapenem and colistin-resistant bacteria in North Lebanon: Coexistence of mcr-1 and NDM-4 genes in Escherichia coli.

The increasing incidence of infections caused by multidrug-resistant bacteria is considered a global health problem. This study aimed to investigate this resistance in Gram-negative bacteria isolated from patients hospitalized in North-Lebanon. All isolates were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility testing was achieved using disk diffusion, E-test and Broth microdilution methods. Phenotypic detection of carbapenemase was carried out using the CarbaNP test. RT-PCR, standard-PCR and sequencing were performed to detect resistance genes and oprD gene. Conjugal transfer was carried out between our isolates and Escherichia coli J53 to detect the genetic localization of resistance genes. MLST was conducted to determine the genotype of each isolate. Twenty-three carbapenem-resistant Enterobacterales of which eight colistin-resistant Escherichia coli, and Twenty carbapenem-resistant Pseudomonas aeruginosa were isolated. All isolates showed an imipenem MIC greater than 32 mg/mL with MICs for colistin greater than 2 mg/L for E. coli isolates. All the Enterobacterales isolates had at least one carbapenemase-encoding gene, with E. coli isolates coharboring blaNDM-4 and mcr-1 genes. Moreover, 16/20 Pseudomonas aeruginosa harbored the blaVIM-2 gene and 18/20 had mutations in the oprD gene. MLST revealed that the isolates belonged to several clones. We report here the first description in the world of clinical E. coli isolates coharboring blaNDM-4 and mcr-1 genes, and K. pneumoniae isolates producing NDM-6 and OXA-48 carbapenemases. Also, we describe the emergence of NDM-1-producing E. cloacae in Lebanon. Screening for these isolates is necessary to limit the spread of resistant microorganisms in hospitals.

Upstream region of OprD mutations in imipenem-resistant and imipenem-sensitive Pseudomonas isolates

The current study was aimed at investigating the prevalence of the mutations upstream of the oprD coding region and its promoters among imipenem-resistant and sensitive Pseudomonas aeruginosa isolated from educational hospitals in Yazd City, Iran. All isolates were identified by the conventional biochemical tests. Then, the antibiotic resistance of these isolates was determined using the disk diffusion method according to the CLSI guidelines. Also, the E.test was performed to determine the minimum inhibitory concentrations (MIC) of imipenem. The mutations of this gene were recognized by the amplification of this region and subsequently sequenced. Sequencing of the genomic region upstream of oprD these regions were done in the 29 clinical strains. Statistical analysis was done by the statistical software SPSS-18. Seventy (77.7%) of isolates had MIC ≥ 16 and were resistant to imipenem. Mutations of the upstream of the oprD gene and its promoters were seen in 25 (86.2%) isolates and 4 isolates had no mutation. One isolate had a base substitution A→Cat nt 25 in the coding region and this isolate had a point mutation leading to an amino acid change at positions 9 (I→L). Our study results indicated that none of the strains had mutation in Shine-Dalgarno and the point mutations were the most common mutations upstream of the oprD coding region among P. aeruginosa isolates. Mutations were observed in imipenem-resistant isolates and it seems this mechanism is effective in resistance of isolates to imipenem and this confirmed that the indiscriminate use of antibiotic should be controlled.

High Carbapenem Resistance Caused by VIM and NDM Enzymes and OprD Alteration in Nonfermenter Bacteria Isolated from a Libyan Hospital.

Acinetobacter baumannii and Pseudomonas aeruginosa are among the most prevalent pathogens causing a wide range of serious infections in hospitalized patients and contaminating intensive care units and inanimate surfaces. The purpose of this study was to investigate the mechanism of carbapenem resistance in clinical and hospital environmental isolates of A. baumannii and P. aeruginosa recovered from a Libyan hospital. From a total of 82 Gram-negative bacteria, 8 isolates of A. baumannii and 3 isolates of P. aeruginosa exhibited resistance to imipenem with minimum inhibitory concentrations ranging from 16 to >32 μg/mL. Five isolates of A. baumannii harbored blaOXA-23 gene, from which three isolates were collected from patients and two from hospital environment. Only one isolate harbored blaNDM-1 gene, which was responsible for carbapenem resistance in A. baumannii. The OprD gene seems to be disturbed by an insertion sequence (IS) in two isolates and affected by polymorphism in one isolate. Pulsed-field gel electrophoresis results showed high genetic diversity among carbapenemase producing A. baumannii. This study highlights the dissemination of blaOXA-23 and blaNDM-1 genes in a Libyan setting. Therefore, infection prevention and control practices, antimicrobial stewardship initiatives, and antimicrobial resistance surveillance systems should be implemented to prevent the wide spread of antimicrobial resistance.

Characterization of antibiotic resistance profiles in Pseudomonas aeruginosa isolates from burn patients

ObjectiveTo investigate the prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) producing extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria. MethodsBetween April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR). ResultsAmong the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for six groups of bla genes encoding ESBL-type enzymes, namely blaCTX-M2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Out of the 28 ESBL-producing strains, 23 (82.14%) were blaCTX-M2 positive; 18 (38.29%) were blaPER positive, and 16 (34.04%) were blaTEM positive, while 5 (17.9%) were co-harboring blaCTX-M2, blaTEM, and blaPER genes. The blaSHV, blaVEB, and blaGES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the blaNDM-1, blaIMP, blaVIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the oprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 11 distinct PA clones among the blaCTX-M2 positive strains. ConclusionThis is the first study to report the presence of CTX-M2-producing PA in the North Africa region and the first to detect blaCTX-M2-positive and blaPER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains to this part of the world. Identification of bacterial genotypic alterations that confer antibiotic resistance is critical in determining the most effective antimicrobial strategies to be employed. Therefore, our findings could potentially facilitate clinical decision making regarding the antibiotics of choice for the treatment of burn patients that suffer from PA infections in Algeria.

Location of OprD porin in Pseudomonas aeruginosa clinical isolates

Multidrug-resistant Pseudomonas aeruginosa is one of the main opportunistic pathogens causing severe infection. One of the mechanisms involved in the resistance to imipenem in clinical isolates is the loss of the OprD porin. Changes like substitutions, deletions, insertions, or mutations in the oprD gene can modify the conformation of OprD porin or inhibit its presence and generate resistance to carbapenems. The aim of this work was to obtain anti-OprD polyclonal antibodies and to determine by both immunofluorescence microscopy (IFI) and Western blot assays, the presence of the OprD porin in resistant-carbapenem P.aeruginosa strains with different changes in the oprD gene. Changes in the gene oprD were identified in clinical isolates of P.aeruginosa. When proteins were translated, several polymorphisms were found; however, these did not affect the presence of OprD porin (PCM25, PCM36, and PCM78). Also it was detected an insertion sequence ISPa1328 (PCM52) and a premature stop codon (PCM91), which inhibited the presence of the OprD porin. This study shows how changes in the oprD gene of P.aeruginosa clinical isolates affect the presence of the OprD porin detected by Western blot and indirect immunofluorescence assays using specific polyclonal anti-OprD antibodies generated in this work.

Molecular epidemiology and carbapenem resistance of Pseudomonas aeruginosa isolated from patients with burns.

The aim of this study was to investigate the molecular epidemiology and carbapenem resistance mechanisms of Pseudomonas aeruginosa isolated from patients with burns in Azerbaijan, Iran. Pseudomonas aeruginosa was isolated from 38 patients with burns. Disk diffusion and agar dilution methods were used to determine antibiotic susceptibility patterns. The overproduction of AmpC β-lactamase and efflux pumps were detected by phenotypic methods. The presence of carbapenemase-encoding genes was detected by multiplex polymerase chain reaction (PCR). Expression of the OprD gene and MexAB efflux pumps were also evaluated with real-time PCR. Random amplified polymorphic DNA typing (RAPD-PCR) was used for genotyping of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Minimum inhibitory concentration (MIC) assays demonstrated high levels of resistance to all classes of antibiotics except colistin and polymyxin B. The initial screening by carbapenem disks indicated 24 isolates (63.15%) as CRPA. Different mechanisms of carbapenem resistance were observed, including carbapenemase production (8.4%), overexpression of AmpC (25%) and decreased expression of OprD (75%). The overexpression of MexAB efflux pumps was detected in 19 (79.1%) isolates by phenotypic assay or real-time PCR. The resistance to carbapenem was multifactorial in most cases (58.3%). The RAPD genotyping revealed different patterns with nine clusters. According to our results, the prevalence of CRPA is at an alarming level. Our results did not demonstrate an epidemic clone. The most common mechanism of carbapenem resistance was decreased expression of OprD. Therefore, we suggest a reconsideration in the management of CRPA infections of patients in our burn care hospital in Azerbaijan, Iran.

840. First Report of Pseudomonas aeruginosa Isolates Harboring the CTX-M2 and PER genes in Algeria

BackgroundDespite significant improvements in burn care, multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) remains one of the most common causes of life-threatening infections in patients suffering from thermal injuries. The objective of this study is to investigate the prevalence of MDR PA producing Extended-Spectrum Beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria.MethodsBetween April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR).ResultsAmong the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for five groups of bla genes encoding ESBL-type enzymes, namely CTX-M2, PER, TEM, SHV, VEB, and GES. Out of the 28 ESBL-producing strains, 23 (82.14%) were CTX-M2 positive; 18 (38.29%) were PER positive, and 16 (34.04%) were TEM positive, while 5 (17.9%) were co-harboring CTX-M2, TEM, and PER genes. The SHV, VEB, and GES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the NDM-1, IMP, VIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the OprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 6 distinct PA clones among the CTX-M2 positive strains.Table 1: Occurrence of beta-lactamase genes in relation to the antimicrobial susceptibility profiles of the PA isolates. ConclusionThis is the first report of CTX-M2-producing PA in the North Africa region and the first to detect CTX-M2-positive and PER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains in this part of the world. Identification of genotypic alterations that confer antibiotic resistance is critical in determining effective antimicrobial strategies. Hence, these findings could potentially guide antibiotic choice decisions.World map with countries where PER- and CTX-M2-postive Pseudomonas aeruginosa isolates have been reported. Disclosures All Authors: No reported disclosures

The novel outer membrane protein from OprD/Occ family is associated with hypervirulence of carbapenem resistant Acinetobacter baumannii ST2/KL22

ABSTRACT Acinetobacter baumannii has become a major healthcare threat that causes nosocomial infections, especially in critically ill patients. The spread of carbapenem-resistant A. baumannii (CRAB) strains has long been a clinical concern. It is important to study the epidemiology and virulence characteristics of different CRAB isolates in order to tailor infection prevention and antibiotic prescribing. In this study, a total of 71 CRAB isolates were collected in the hospital, and clinical characteristics of infections were analyzed. The genomic characteristics and phylogenetic relationships were elucidated based on genome sequencing and analysis. The isolates were assigned to three sequence types (STs, Pasteur) and nine capsular polysaccharide (KL) types, among which ST2/KL22 was the most prevalent CRAB in the hospital. Even though all the ST2/KL22 isolates contained the same reported virulence genes, one specific clade of ST2/KL22 showed more pathogenic in mouse infection model. Complete genomic analysis revealed differences at the oprD locus between the low- and high-virulent isolates. More specifically, a premature stop codon in the low-virulence strains resulted in truncated OprD expression. By evaluating pathogenicity in C57BL/6 J mice, knock-out of oprD in high-virulent isolate resulted in virulence attenuation, and complementing the avirulent strain with full-length oprD from high-virulent isolate enhanced virulence of the former. The oprD gene may be associated with the enhanced virulence of the specific ST2/KL22 clone, which provides a potential molecular marker for screening the hypervirulent A. baumannii strains.

Molecular Detection and Quantification Gene Expression of Efflux Pump Antibiotic Resistance Genes in Extensive Drug Resistance Pseudomonas Aeruginosa Isolated from Clinical Infection Patients in Al Diwaniyah City of Iraq

The study was included investigated 16 extensive drug resistance pseudomonas aeruginosa isolateswhich previously isolated from sputum, burns, urine, and ear swab clinical patients samples. The reversetranscription Real Time PCR (RT-qPCR) was performed for molecular detection and quantification of geneexpression levels for efflux pump antibiotic resistance gene (mexA, mexB, mexC, mexE, and mexY) andchromosomal lactamase (AmpC) gene, as well as outer membrane prion protein (OprD) gene in extensivedrug pseudomonas aeruginosa isolates. The relative expression analysis was done by used the 2-??CT (Livak)method, this method was used for normalization of expression levels (mRNA transcript levels) in targetgenes with suitable housekeeping gene. In this study 30S ribosomal protein S12 (rpsL) housekeeping genewas used in normalization of antibiotic resistance genes expression levels in extensive drug pseudomonasaeruginosa isolates. The efflux pump resistance gene expression were by RT-qPCR technique. The relativegene expression analysis were showed highest frequency of overexpression at (100%, 100%, 87.5%,81.25%, 56.25%) for (mexB, mexE, mexA,mexC, and mexY) with fold change ranged (2.16- 184.82) foldchange compared to wild type PA control isolate. The relative gene expression analysis of chromosomallactamase AmpC gene was showed highest frequency of overexpression (100%) in XDR isolates withexpression levels ranged (6.77- 73.1) fold change compared to wild type PA control isolate. Whereas, therelative gene expression analysis of OprD gene was showed (68.75%) frequency of down expression inXDR Pseudomonas aeruginosa isolates with expression levels ranged (0.01-0.16) fold change compared towild type PA control isolate. In conclusion, The XDR Pseudomonas aeruginosa isolates can ability to overexpression production of inducible chromosomal lactamase AmpC gene, efflux pumps protein genes, andlow expression outer membrane prion protein (OprD) gene as important antimicrobial mechanism againstCephalosporins and Carbapenems antibiotic classes.

Evaluation of chromosomally and acquired mechanisms of resistance to carbapenem antibiotics among clinical isolates of Pseudomonas aeruginosa in Kerman, Iran

IntroductionMultidrug-resistant (MDR) of Pseudomonas aeruginosa isolates are increasing around the world which can cause serious problems for antimicrobial therapy in hospital settings. Our aim in present study was to evaluation of intrinsic and acquired mechanisms of resistance to carbapenem antibiotics among 170 clinical isolates of P. aeruginosa in Kerman, Iran. Material and methodsCLSI recommendations were used for determination of antimicrobial susceptibility testing of isolates. Metallo-β-lactamase (MBL) producing isolates were detected by the double-disc synergy test (DDST) using the 2-mercaptopropionic acid and efflux pump over-activity were determined by the microdilution method using PabN. PCR-sequencing technique was used for to detection and sequencing of carbapenemase, blaCTX-M and oprD genes. Transcriptional levels of the mexA gene in carbapenem-resistant isolates was evaluated by qReal-time PCR (qPCR) and ERIC-PCR technique was used for molecular typing of carbapenem-resistant isolates. ResultsAccording to our findings, colistin was the most active agent against P. aeruginosa isolates. All carbapenem-resistant P. aeruginosa were efflux pumps overproducers and 4.92% isolates were MBL producing. The MBL genes including: blaNDM, blaVIM, blaIMP and blaSIM were detected in (2, 3.3%), (1, 1.6%), (1, 1.6%), (1, 1.6%), of the isolates, respectively. The oprD gene was disrupted in 9 isolates by insertion sequences (ISs). The qPCR experiment showed that transcriptional levels of mexA gene in 26 (42.6%) carbapenem-resistant P. aeruginosa have more than 2-7-fold change with comparison to P. aeruginosa PAO1 strain. ERIC-PCR results was showed 10 clusters and 6 singletons in carbapenem-resistant isolates. ConclusionsIn this study, we reported blaNDM-1 and blaSIM positive P. aeruginosa isolates for the first time in Kerman, Iran. Furthermore, our results were showed that intrinsic resistance mechanisms including overexpression of efflux pump and inactivation of oprD have an important role in resistance to carbapenem antibiotics in clinical isolates of P. aeruginosa in Kerman, Iran.

Expression of MexAB-OprM efflux pump and OprD porin in carbapenemase producing Pseudomonas aeruginosa clinical isolates

In recent years, the prevalence of carbapenem resistant of Pseudomonas aeruginosa isolates has become a global concern. The aim of this study was to examine the relationship between the expression of efflux pump and OprD genes in Metallo-beta-lactamase (MBL) and Klebsiella pneumoniae carbapenemase (KPC) producing P. aeruginosa isolates.The study was performed on the 97 P. aeruginosa strains that were isolated from clinical samples in Hamadan hospitals, western Iran in 2017–2018. Antibiotic susceptibility testing was performed using disk diffusion and Minimal Inhibitory Concentration (MIC) methods. Carbapenemase production enzymes was identified by Imipenem-EDTA combined disc (ECD) and Modified Hodge Test (MHT) and Polymerase chain reaction (PCR). Expression levels of efflux pump and OprD genes were mesured by Real-Time PCR (RT-PCR). Finally, the relationship between the presence of carbapenemase enzymes with expression levels of efflux pump and OprD genes was analyzed with chi-square test. The results, showed that the highest resistance was to cefoxitin 92 (94.8%) and the lowest resistance was to piperacillin-tazobactam 38 (39.2%). Also, among of the carbapenem antibiotics, the highest resistant was to imipenem 48 (49.4%) and the lowest resistant was to meropenem 40 (41.2%). Out of 49 (51.51%) Carbapenem resistant isolates, 42 (85.7%), 25 (51.02%) and 26 (53.06%) isolates had positive results for MIC, MHT, and ECD tests, respectively. The highest and lowest presence of the carbapenemase genes in P.aeruginosa isolates was related to the IMP 20 (40/80%) and AmpC 12 (24.4%) genes, respectively. Out of 20 selected isolates, decreased and increased expression level of the OprD, MexA, MexB and OprM genes were observed in 60% (12/20), 55% (11/20), 60% (12/20) and 55% (11/20), of isolates, respectively. This study demonstrated that there was a significant relationship between the presence of carbapenemase enzymes (MBL and KPC) and expression level of efflux pump and OprD genes (P-value <.05).

Distinctive Regulation of Carbapenem Susceptibility in Pseudomonas aeruginosa by Hfq.

Carbapenems are often the antibiotics of choice to combat life threatening infections caused by the opportunistic human pathogen Pseudomonas aeruginosa. The outer membrane porins OprD and OpdP serve as entry ports for carbapenems. Here, we report that the RNA chaperone Hfq governs post-transcriptional regulation of the oprD and opdP genes in a distinctive manner. Hfq together with the recently described small regulatory RNAs (sRNAs) ErsA and Sr0161 is shown to mediate translational repression of oprD, whereas opdP appears not to be regulated by sRNAs. At variance, our data indicate that opdP is translationally repressed by a regulatory complex consisting of Hfq and the catabolite repression protein Crc, an assembly known to be key to carbon catabolite repression in P. aeruginosa. The regulatory RNA CrcZ, which is up-regulated during growth of P. aeruginosa on less preferred carbon sources, is known to sequester Hfq, which relieves Hfq-mediated translational repression of genes. The differential carbapenem susceptibility during growth on different carbon sources can thus be understood in light of Hfq-dependent oprD/opdP regulation and of the antagonizing function of the CrcZ RNA on Hfq regulatory complexes.

Mutational Variation Analysis of oprD Porin Gene in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa.

The present study deals with the outer membrane OprD porin protein in 29 clinical bacterial isolates of multidrug-resistant Pseudomonas aeruginosa. oprD porin gene expression was investigated using real-time reverse transcription-PCR. Amplicons from oprD and its transcriptional regulator mexT gene were sequenced and analyzed for mutations. Hypothetical models of selected mutant OprD-porin proteins were predicted and refined by homology modeling approach. oprD ampliconic sequences were also screened for restriction fragment length polymorphism (RFLP). The oprD gene was found to be downregulated in 89.7% (n = 26) of the isolates in comparison to the transcript levels in the reference strain P. aeruginosa-PAO (MTCC-3541). Interestingly, all these isolates displayed the presence of a conspicuous 8-bp deletion (GGCCAGCC) at nucleotide position 235 of mexT regulatory gene. Based on the mutational patterns observed in oprD gene, the isolates were classified into categories designated as A, B1-2, C1-4, D1-6, E1-2, and F. Our hypothetical models revealed that mutations were predominantly confined to the extracellular loops emanating from the β-barrel porin protein. These protein models also enabled clear visualization of loss of substantial portions of the truncated polypeptide. Incidentally, since most of the oprD amplicons of the clinical isolates were found to display distinct RFLP banding patterns, our results also provide a useful diagnostic tool for detection of P. aeruginosa porin mutants.

Genotypes, carbapenemase carriage, integron diversity and oprD alterations among carbapenem-resistant Pseudomonas aeruginosa from Russia

Pseudomonas aeruginosa is a serious opportunistic pathogen demonstrating a high level of resistance to many groups of antibiotics, including carbapenems. This study aimed to characterise the molecular epidemiology and prevalence of mobile genetic elements associated with resistance to carbapenems among P. aeruginosa (CRPA) clinical isolates. Among 145 carbapenem-resistant P. aeruginosa isolates, 34 different sequence types (STs) were detected; the six most common STs were ST654 (24%), ST235 (24%), ST111 (8%), ST446 (6%), ST357 (5%) and ST2592 (a novel single-locus variant of ST357) (4%). A carbapenemase gene was found in 94 isolates (64.8%). The blaVIM-2 gene was harboured by 64 isolates (44.1%) restricted to ST111, ST235 and ST654, and the blaGES-type and blaOXA-10 group genes were each detected in 15 isolates (10.3%); none of other tested carbapenemase genes, including blaIMP, blaNDM and blaGIM, were detected. Among the blaVIM-2-positive isolates, five types of blaVIM-2-containing integrons were discovered, including In56, In559, In59-like, In59 and In249. The oprD gene was disrupted by an insertion sequence (IS) in 15.9% of isolates. Overall, five types of IS elements were found (ISPsme1, ISPa1328, ISPa26, ISPst2 and ISPa195). Observed rearrangements within variable regions of blaVIM-2-carrying integrons in conjunction with the discovery of a novel type of oprD-disrupting IS element illustrate the ongoing evolution of CRPA a, which warrants further investigation.

EMERGENCE OF PLASMID MEDIATED COLISTIN RESISTANCE GENE MCR-1 IN CARBAPENEM-RESISTANT PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES FROM ALGERIA: A NEW SUCCESSFUL RESISTANCE COMBINATION TOWARD A THERAPEUTIC IMPASSE?

Ponte Academic JournalApr 2020, Volume 76, Issue 4 EMERGENCE OF PLASMID MEDIATED COLISTIN RESISTANCE GENE MCR-1 IN CARBAPENEM-RESISTANT PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES FROM ALGERIA: A NEW SUCCESSFUL RESISTANCE COMBINATION TOWARD A THERAPEUTIC IMPASSE?Author(s): Addouda Abir ,Ayachi Ammar, Hassaine Hafida, Dhaouadi Sana, Soufi Leila, Mahjoubi Mouna, Benmehidi Messaoud, Grainat Nadia, Elandalousi Ramzi BoubakerJ. Ponte - Apr 2020 - Volume 76 - Issue 4 doi: 10.21506/j.ponte.2020.4.16 Abstract:Objectives: Pseudomonas aeruginosa is one of the main causes of nosocomial infections,\r\nespecially in immunocompromised patients in burn and intensive care units, who have\r\ninherited a number of drug resistance determinants via horizontal mutation. For this reason,\r\nour work has also been conducted to prove the genetic relationship between strains and to\r\ninvestigate the occurrence of genes conferring high levels of carbapenem and colistin\r\nresistance in Pseudomonas aeruginosa isolates from the Algerian hospital.\r\nMethodology: 27 colistin resistant Pseudomonas aeruginosa (CO-RPA) recovered from\r\npatients and hospital environment were analyzed using a combination of phenotypic and\r\nmolecular typing methods for the identification of plasmid mediated colistin resistance genes;\r\ncarbapenem resistant Pseudomonas aeruginosa (CRPA) isolates were tested for the presence\r\nof genes encoding β-lactamases, quinolones and β-lactamases.\r\nPCR-Ribotyping and PCR 16s rDNA were used to establish the phylogeny and clonal\r\nrelationship of strains.\r\nResults: Molecular Screening for mcr encoding genes showed that 02 tested isolates\r\nexamined contained mcr-1, one isolate of which expressed mcr-1 and OprD genes at the same\r\ntime. Interestingly, 62.95 % of the colistin-resistant strains were imipenem-resistant in this\r\nstudy. Actually, loss/mutation in OprD was founded in 29, 41% (05/17) of imipenem-resistant\r\nstrains. PCR- Ribotyping revealed an outbreak of clonal distribution of colistin and\r\ncarbapenem resistant Pseudomonas aeruginosa isolates.\r\nConclusion: The current study highlights the first potential spread of the mcr-1 gene in\r\nPseudomonas aeruginosa strains in the health care setting in Algeria and around the world\r\nthat appears to be linked to the uncontrolled use of antibiotics. Strict precautions will be\r\nexpected to prevent the further spread of these pathogens in hospital settings. Download full text:Check if you have access through your login credentials or your institution Username Password