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Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the main Gram-negative bacterium causes of infections in hospital settings, and the spread of them is a significant challenge to public health. Methods: A total of 30 non-duplicate isolates of CRPA were collected. Antibacterial susceptibility of isolates to antibiotic agents, AmpC β-lactamase production, and biofilm formation were determined. Minimum biofilm inhibitory concentrations (MBIC) of isolates to cefepime (FEP), imipenem (IPM), ceftazidime (CAZ), and meropenem (MEM) were evaluated with/without cloxacillin (CLX). The carbapenemase and 16 S rRNA methylase genes were identified by PCR, and the transcription levels of oprD, ampC, and mexA genes were determined by quantitative real-time PCR (qPCR). ERIC-PCR was used to detect genetic relationships among isolates. Results: All isolates were multidrug resistant (MDR) and strong biofilm producers. The resistance genes including blaNDM, blaIMP, blaVIM, blaSIM, blaGES, and armA were detected in 21 (70%), 6 (20%), 3 (10%), 2 (6.6%), 1 (3.3%), and 17 (56.6%) of the isolates, respectively. CLX at 500 and 1000 µg/mL significantly reduced the level of MIC to MEM, IPM, CAZ, and FEP, also at 2000 µg/mL significantly reduced the level of MBIC to MEM, IPM, CAZ, and FEP. In all isolates, the transcription levels of oprD were significantly downregulated as well as significantly increased for ampC and mexA. ERIC-PCR typing results divided 30 isolates into four clusters A to D. Conclusion: In this study, we reported the spread of different clones of CRPA harboring co-existence of various carbapenemase genes with armA 16 S rRNA methylase for the first time in Kerman, Iran. Also, our isolates had several mechanisms of resistance to carbapenems as well as ability biofilm formation along with resistance to aminoglycosides, the further spread of which could cause serious challenges in our hospital settings. Therefore, serious monitoring is necessary to reduce their prevalence. [ABSTRACT FROM AUTHOR]

Background: Efflux pumps are crucial bacterial defense mechanisms that reduce antibiotic susceptibility by expelling antibiotics from cells. In Acinetobacter baumannii , this significantly contributes to multidrug resistance. In addition, porin alterations, essential for carbapenem uptake, further enhance resistance by limiting antibiotic entry. This study examines these mechanisms to improve treatment strategies. Methods: A cross-sectional descriptive study was conducted on 300 clinical samples collected from Hilla Teaching Hospitals between January and June 2024. Samples were obtained from wounds (40%), burns (40%), blood (12%), and urine (8%). Specimens were collected from burn wards and the intensive care unit. Patients ranged in age from 10 to 40 years. Specimens included 200 females and 100 males. Specimens were collected during hospitalization. Bacteria were cultured and identified by Gram stain and biochemical tests and confirmed to be A. baumannii (8.33%) using the Vitek-2 system. Antibiotic susceptibility testing was performed against 16 antibiotics. Molecular detection of efflux pump genes (adeB and adeJ) was performed using polymerase chain reaction (PCR), and gene expression was measured by reverse transcriptase PCR. Results: A. baumannii isolates showed high resistance to antibiotics, especially β-lactams, carbapenems, and aminoglycosides, whereas colistin remained effective. All isolates carried the adeB gene, whereas adeJ was not detected. Overexpression of OperD (80%) and adeB (44%) was observed, along with consistent downregulation of the CarO gene (100%). Conclusion: A. baumannii isolates exhibited significant antibiotic resistance, particularly to β-lactams, carbapenems, and aminoglycosides, complicating infection treatment. Colistin remained the only effective option. Molecular analysis confirmed the presence of the adeB gene in all isolates, with no detection of adeJ. Overexpression of OperD (80%) and adeB (44%) emphasized efflux pumps' role in resistance, whereas CarO gene downregulation (100%) likely contributed to carbapenem resistance. These results highlight the urgent need for continuous monitoring and new treatment approaches for multidrug-resistant A. baumannii. [ABSTRACT FROM AUTHOR]

Objectives: Pseudomonas aeruginosa is a major opportunistic pathogen responsible for a wide range of infections. The emergence of antibiotic resistance in this pathogen poses a significant public health challenge. This study aims to conduct a comprehensive meta-analysis of studies conducted in Iran to determine the frequency of key antibiotic resistance mechanisms in Pseudomonas aeruginosa and their association with multidrug-resistant and extensively drug-resistant strains or pandrug-resistant strains. Methods: Systematic database searches encompassing literature up to June 2023 were undertaken. The selected studies centered on OprD downregulation, efflux pump (mexAB-OprM, mexXY-OprM) expression, and AmpC overproduction. Extracted data were synthesised in a meta-analysis for pooled frequency determination of each resistance mechanism. Results: In total, 24 studies were included. OprD downregulation exhibited a pooled frequency of 61%. Efflux pump component frequency ranged from 48% to 77.5%. AmpC overproduction was identified in 29.1% of isolates. Polymyxin B and colistin demonstrated lower antibiotic resistance rates, with pooled frequency of 1% and 1.6%, respectively. Conversely, resistance to other antibiotics ranged widely, with pooled frequency spanning 38.4% to 98.2%. Conclusions: This study underscores the concerning frequency of diverse antibiotic resistance mechanisms in Pseudomonas aeruginosa strains from Iran. Concurrent OprD downregulation, mexAB, mexXY, OprM expression, and AmpC overproduction highlight the urgent need for stringent infection control and prudent antibiotic usage to curb the dissemination of these resistant strains. PROSPERO: CRD42022379311

Abstract Background Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the main Gram-negative bacterium causes of infections in hospital settings, and the spread of them is a significant challenge to public health. Methods A total of 30 non-duplicate isolates of CRPA were collected. Antibacterial susceptibility of isolates to antibiotic agents, AmpC β-lactamase production, and biofilm formation were determined. Minimum biofilm inhibitory concentrations (MBIC) of isolates to cefepime (FEP), imipenem (IPM), ceftazidime (CAZ), and meropenem (MEM) were evaluated with/without cloxacillin (CLX). The carbapenemase and 16 S rRNA methylase genes were identified by PCR, and the transcription levels of oprD, ampC, and mexA genes were determined by quantitative real-time PCR (qPCR). ERIC-PCR was used to detect genetic relationships among isolates. Results All isolates were multidrug resistant (MDR) and strong biofilm producers. The resistance genes including bla NDM, blaIMP, bla VIM, bla SIM, bla GES, and armA were detected in 21 (70%), 6 (20%), 3 (10%), 2 (6.6%), 1 (3.3%), and 17 (56.6%) of the isolates, respectively. CLX at 500 and 1000 µg/mL significantly reduced the level of MIC to MEM, IPM, CAZ, and FEP, also at 2000 µg/mL significantly reduced the level of MBIC to MEM, IPM, CAZ, and FEP. In all isolates, the transcription levels of oprD were significantly downregulated as well as significantly increased for ampC and mexA. ERIC-PCR typing results divided 30 isolates into four clusters A to D. Conclusion In this study, we reported the spread of different clones of CRPA harboring co-existence of various carbapenemase genes with armA 16 S rRNA methylase for the first time in Kerman, Iran. Also, our isolates had several mechanisms of resistance to carbapenems as well as ability biofilm formation along with resistance to aminoglycosides, the further spread of which could cause serious challenges in our hospital settings. Therefore, serious monitoring is necessary to reduce their prevalence.

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2(N=3), and blaNDM-1(N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones. [ABSTRACT FROM AUTHOR]

ABSTRACT In the United States, carbapenem resistance in Pseudomonas aeruginosa is linked to the regulation of chromosomal resistance determinants, AmpC and OprD. The β-lactamase AmpC requires overexpression and genetic modifications to be capable of inhibiting imipenem activity. The outer membrane porin OprD can be downregulated or undergo genetic modifications that strongly correlate with imipenem non-susceptibility. Co-administration of imipenem and the β-lactamase inhibitor, relebactam, can lower imipenem minimal inhibitory concentrations and restore susceptibility. However, it is not understood how this occurs in P. aeruginosa isolates that do not overproduce AmpC or produce a functional OprD for imipenem entry. Therefore, we investigated whether imipenem could enter P. aeruginosa in the absence of OprD and whether any of the chromosomal β-lactamases (AmpC, OXA-50, and PIB-1) contributed to imipenem and/or imipenem/relebactam non-susceptibility. This investigation evaluated 17 imipenem non-susceptible clinical isolates and three laboratory strains of PAO1, two of which were porin transposon mutants for either oprD or opdP. Expression of OXA-50 and PIB-1 RNA was similar to PAO1. However, all 20 isolates exhibited blaampC induction under sublethal exposure to imipenem. This occurred in the absence of detectable OprD protein in 18 isolates. Collectively, our data identify that (i) OprD was not the only channel required for imipenem entry and (ii) imipenem susceptibility can be restored by imipenem/relebactam due to the interaction between relebactam and blaampC overexpression due to imipenem induction.IMPORTANCEInfections caused by Pseudomonas aeruginosa are associated with high mortality and worsened clinical outcomes. The carbapenem, imipenem, has been combined with the β-lactamase inhibitor relebactam to treat carbapenem-resistant P. aeruginosa. Downregulation of the outer membrane porin, OprD is the major mechanism of imipenem resistance; however, relebactam inhibits the chromosomally encoded AmpC β-lactamase. We investigated how relebactam was able to reduce P. aeruginosa imipenem minimal inhibitory concentrations (MICs) in isolates in which OprD was downregulated. Our data show that imipenem is capable of entering the cell in the absence of OprD and capable of inducing the AmpC β-lactamase. The induction of AmpC provides a substrate for relebactam, impacting the imipenem MIC. The data presented support the use of an alternative porin(s) for entry of imipenem. This study provides the basis for further investigation into modifications of imipenem or similar molecules that would increase the affinity for other porins in isolates resistant to imipenem.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Introduction: Pseudomonas aeruginosa is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.) pathogen and one of the leading etiologies in multiple nosocomial infections. Treatment of P. aeruginosa is becoming increasingly difficult due to its ever-increasing antibiotic resistance trends. This study investigated clinical multidrug resistance (MDR) P. aeruginosa (MDR-PA), their intrinsic resistance determinants, including the presence of chromosomal AmpC ß-lactamase (Ampicillinase), decreased expression of outer membrane porin protein OprD and selected acquired ß-lactamase resistance genes. Methods: Out of 238 clinical specimens, including urines from urinary tract-infected patients, wound swabs, burn swabs, and catheter aspirates, were collected from two major hospitals in Savar, Dhaka, Bangladesh. Samples were inoculated with Cetrimide agar to isolate presumptive P. aeruginosa. Bacteria were identified by cultural, biochemical characterization, 16S rDNA sequencing, and phylogenetic analysis. Virulence-associated genes of P. aeruginosa, namely, toxA, lasB, and plcH, were identified by polymerase chain reaction (PCR). Antibiotic susceptibilities of the isolates were investigated against ten antibiotics belonging to seven groups by disc-diffusion method followed by a selected minimum inhibitory concentration (MIC) assay. Phenotypic expression of Metallo-ß-lactamases (MBLs) production was checked by the double disc synergistic test selectively among the imipenem-resistant isolates. Acquisition of ß-lactam resistance trait was examined by PCR detection of bla-genes variants. Mutational loss of the OprD was analyzed by PCR to investigate intrinsic resistance determinants. Phenotypic overexpression of chromosomal AmpC was assayed with the identification of the AmpC gene by PCR. The expression level of OprD was assessed by real-time quantitative PCR (RT-qPCR). Results: Fifty-three P. aeruginosa was identified, with an overall isolation of 22.3% (53/238), where urine remains the most prevalent source. Virulence genes toxA, lasB, and plcH were identified in the isolates of 92.4%, 96.2%, and 94.3%. The highest phenotypic antimicrobial resistance was observed against ampicillin and ceftriaxone (100%), followed by cefotaxime (96%), tetracycline (89%), azithromycin (72%), imipenem (31%), ciprofloxacin (29%), levofloxacin (29%), gentamycin (27%) and ceftazidime (14%). The antibiogram pattern revealed 85% of isolates as multidrug-resistant, while 12% were considered extensively drug-resistant (XDR)-P. aeruginosa. The carriage of ß-lactamase genes blaTEM, blaSHV, and blaOXA was detected in 4%, 2%, and 2% cephalosporin-resistant isolates, respectively. Double disc synergistic test revealed 87% of imipenem-resistant isolates expressing MBL-mediated resistance phenomenon. All seven ceftazidime-resistant isolates showed the presence of the AmpC gene with phenotypic overproduction of the AmpC enzyme, indicating AmpC-mediated ceftazidime resistance. Mutational loss of OprD was observed in 12% of phenotypically multidrug-resistant isolates, and RT-qPCR analysis revealed reduced expression of OprD porin protein at various levels in the outer membrane of multidrug-resistant isolates. Conclusions: This study depicts the high prevalence of MDR-PA in clinical specimens in Bangladesh. The identified intrinsic and acquired antimicrobial resistance determinants play synergistic roles in emerging MDR-PA. [ABSTRACT FROM AUTHOR]

ABSTRACT Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a bacterial pathogen that may cause serious drug-resistant infections that are potentially fatal. To investigate the genetic characteristics of these organisms, we tested 416 P. aeruginosa strains recovered from 12 types of clinical samples collected in 29 different hospital wards in 10 hospitals in Guangdong Province, China, from 2017 to 2020. These strains were found to belong to 149 known sequence types (STs) and 72 novel STs, indicating that transmission of these strains involved multiple routes. A high rate of resistance to imipenem (89.4%) and meropenem (79.4%) and a high prevalence of pathogenic serotypes (76.4%) were observed among these strains. Six STs of global high-risk clones (HiRiCs) and a novel HiRiC strains, ST1971, which exhibited extensive drug resistance, were identified. Importantly, ST1971 HiRiC, which was unique in China, also exhibited high virulence, which alarmed the further surveillance on this highly virulent and highly resistant clone. Inactivation of the oprD gene and overexpression of efflux systems were found to be mainly responsible for carbapenem resistance in these strains; carriage of metallo-β-lactamase (MBL)-encoding genes was less common. Interestingly, frameshift mutations (49.0%) and introduction of a stop codon (22.4%) into the oprD genes were the major mechanisms of imipenem resistance. On the other hand, expression of the MexAB-OprM efflux pump and MBL-encoding genes were mechanisms of resistance in >70% of meropenem-resistant strains. The findings presented here provide insights into the development of effective strategies for control of worldwide dissemination of CRPA. IMPORTANCE Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major concern in clinical settings worldwide, yet few genetic and epidemiological studies on CRPA strains have been performed in China. Here, we sequence and analyze the genomes of 416 P. aeruginosa strains from hospitals in China to elucidate the genetic, phenotypic, and transmission characteristics of CRPA strains and to identify the molecular signatures responsible for the observed increase in the prevalence of CRPA infections in China. These findings may provide new insight into the development of effective strategies for worldwide control of CRPA and minimize the occurrence of untreatable infections in clinical settings.

Yue Fang,1,* Zulqarnain Baloch,1,* Wei Zhang,2 Ying Hu,2 Rui Zheng,3 Yuzhu Song,1 Wenlin Tai,2 Xueshan Xia1 1The Affiliated AnNing First Hospital & Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, 650500, People’s Republic of China; 2The 2nd Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, 650101, People’s Republic of China; 3The First Hospital of Yunnan Province & The Affiliated Hospital, Kunming University of Science and Technology, Kunming, Yunnan, 650034, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuzhu Song; Xueshan Xia, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, 650500, People’s Republic of China, Tel +86-871-65920756, Fax +86-871-65920570, Email syzzam@126.com; oliverxia2000@aliyun.comIntroduction: The prevalence of carbapenem-resistant Pseudomonas aeruginosa is increasing persistently, particularly in burn ward isolates. Here, we investigate the prevalence of carbapenem-resistant Pseudomonas aeruginosa in a burn ward of a provincial-level hospital at Kunming, Yunnan province, China.Methods: A total of 118 P. aeruginosa strains were isolated from 57 hospitalized patients, and their MICs were measured. Carbapenem-resistant isolates were selected for multilocus sequence typing (MLST). Carbapenem-resistance mechanisms were identified by examining carbapenemase genes and OprD protein and Carba-NP testing. Representative isolates were further characterized by de novo sequencing for carbapenemase molecular background.Results: Among 118 P. aeruginosa isolates, 54 (54/118,45.8%) were carbapenem-resistant Pseudomonas aeruginosa, and 3 genotypes were found (ST292, ST244, and ST2446). Non-carbapenemase-producing ST292 was the most prevalent ST, followed by ST2446 and ST244. A novel 13-bp oprD deletion was found in the ST292 clone, which formed the truncated outer membrane protein and may cause carbapenem resistance. ST244 and ST2446 harbored blaIMP-45 and blaIMP-87, respectively. blaIMP-45 is located in a megaplasmid, together with aac(6’)-Ib3, blaOXA-1, catB3, qnrVC6, armA, msr(E), mph(E), aph(3’)-Ia, tetC/tetR, aac(6’)-Ib3, floR, mexC-mexD-oprJ, fosA and lead to extensive drug resistance. ST2446 contains a carbapenem-resistant gene blaIMP-87 on the chromosome and is acquired by a novel gene cassette array (blaIMP-87-ant(2”)-Ia-blaOXA-10-aac(6’)-Ib3) of class 1 integron.Discussion: For the first time, ST244, ST292 and ST2446 are reported emerging in burn patients, with distinctive carbapenem-resistance mechanisms, respectively. The obtained results highlight the need to surveillance carbapenem-resistant isolates in burn patients.Keywords: P. aeruginosa, carbapenem-resistance, oprD, blaIMP

The prevalence and genetic mechanism of antibiotic heteroresistance (HR) have attracted significant research attention recently. However, non-genetic mechanism of HR has not been adequately explored. The present study aimed to evaluate the role of quorum sensing (QS), an important mechanism of behavioral coordination in different subpopulations and consequent heteroresistance. First, the prevalence of HR to 7 antibiotics was investigated in 170 clinical isolates of P. aeruginosa using population analysis profiles. The results showed that P. aeruginosa was significantly heteroresistant to meropenem (MEM), amikacin (AMK), ciprofloxacin (CIP), and ceftazidime (CAZ). The observed HR was correlated with down-regulation of QS associated genes lasI and rhlI. Further, loss-of-function analysis results showed that reduced expression of lasI and rhlI enhanced HR of P. aeruginosa to MEM, AMK, CIP, and CAZ. Conversely, overexpression of these genes or treatment with 3-oxo-C12-HSL/C4-HSL lowered HR of P. aeruginosa to the four antibiotics. Additionally, although downregulation of oprD and upregulation of efflux-associated genes was evident in heteroresistant subpopulations, their expression was not regulated by LasI and RhlI. Moreover, fitness cost measurements disclosed higher growth rates of PAO1Î"lasI and PAO1Î"rhlI in the presence of sub-MIC antibiotic as compared with that of wild-type PAO1. Our data suggest that under temporary antibiotic pressure, downregulation of QS might result in less fitness cost and promote HR of P. aeruginosa. [ABSTRACT FROM AUTHOR]

In the United States, carbapenem resistance in Pseudomonas aeruginosa is linked to the regulation of chromosomal resistance determinants, AmpC and OprD. The β-lactamase AmpC requires overexpression and genetic modifications to be capable of inhibiting imipenem activity. The outer membrane porin OprD can be downregulated or undergo genetic modifications that strongly correlate with imipenem non-susceptibility. Co-administration of imipenem and the β-lactamase inhibitor, relebactam, can lower imipenem minimal inhibitory concentrations and restore susceptibility. However, it is not understood how this occurs in P. aeruginosa isolates that do not overproduce AmpC or produce a functional OprD for imipenem entry. Therefore, we investigated whether imipenem could enter P. aeruginosa in the absence of OprD and whether any of the chromosomal β-lactamases (AmpC, OXA-50, and PIB-1) contributed to imipenem and/or imipenem/relebactam non-susceptibility. This investigation evaluated 17 imipenem non-susceptible clinical isolates and three laboratory strains of PAO1, two of which were porin transposon mutants for either oprD or opdP . Expression of OXA-50 and PIB-1 RNA was similar to PAO1. However, all 20 isolates exhibited bla ampC induction under sublethal exposure to imipenem. This occurred in the absence of detectable OprD protein in 18 isolates. Collectively, our data identify that (i) OprD was not the only channel required for imipenem entry and (ii) imipenem susceptibility can be restored by imipenem/relebactam due to the interaction between relebactam and bla ampC overexpression due to imipenem induction.IMPORTANCEInfections caused by Pseudomonas aeruginosa are associated with high mortality and worsened clinical outcomes. The carbapenem, imipenem, has been combined with the β-lactamase inhibitor relebactam to treat carbapenem-resistant P. aeruginosa . Downregulation of the outer membrane porin, OprD is the major mechanism of imipenem resistance; however, relebactam inhibits the chromosomally encoded AmpC β-lactamase. We investigated how relebactam was able to reduce P. aeruginosa imipenem minimal inhibitory concentrations (MICs) in isolates in which OprD was downregulated. Our data show that imipenem is capable of entering the cell in the absence of OprD and capable of inducing the AmpC β-lactamase. The induction of AmpC provides a substrate for relebactam, impacting the imipenem MIC. The data presented support the use of an alternative porin(s) for entry of imipenem. This study provides the basis for further investigation into modifications of imipenem or similar molecules that would increase the affinity for other porins in isolates resistant to imipenem., Competing Interests: The authors declare no conflict of interest.

ABSTRACT Most knowledge about Pseudomonas aeruginosa pathoadaptation is derived from studies on airway colonization in cystic fibrosis; little is known about adaptation in acute settings. P. aeruginosa frequently affects burned patients and the burn wound niche has distinct properties that likely influence pathoadaptation. This study aimed to genetically and phenotypically characterize P. aeruginosa isolates collected during an outbreak of infection in a burn intensive care unit (ICU). Sequencing reads from 58 isolates of ST1076 P. aeruginosa taken from 23 patients were independently mapped to a complete reference genome for the lineage (H25338); genetic differences were identified and were used to define the population structure. Comparative genomic analysis at single-nucleotide resolution identified pathoadaptive genes that evolved multiple, independent mutations. Three key phenotypic assays (growth performance, motility, carbapenem resistance) were performed to complement the genetic analysis for 47 unique isolates. Population structure for the ST1076 lineage revealed 11 evolutionary sublineages. Fifteen pathoadaptive genes evolved mutations in at least two sublineages. The most prominent functional classes affected were transcription/two-component regulatory systems, and chemotaxis/motility and attachment. The most frequently mutated gene was oprD, which codes for outer membrane porin involved in uptake of carbapenems. Reduced growth performance and motility were found to be adaptive phenotypic traits, as was high level of carbapenem resistance, which correlated with higher carbapenem consumption during the outbreak. Multiple prominent linages evolved each of the three traits in parallel providing evidence that they afford a fitness advantage for P. aeruginosa in the context of human burn infection. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative pathogen causing infections in acutely burned patients. The precise mechanisms required for the establishment of infection in the burn setting, and adaptive traits underpinning prolonged outbreaks are not known. We have assessed genotypic data from 58 independent P. aeruginosa isolates taken from a single lineage that was responsible for an outbreak of infection in a burn ICU that lasted for almost 2.5 years and affected 23 patients. We identified a core set of 15 genes that we predict to control pathoadaptive traits in the burn infection based on the frequency with which independent mutations evolved. We combined the genotypic data with phenotypic data (growth performance, motility, antibiotic resistance) and clinical data (antibiotic consumption) to identify adaptive phenotypes that emerged in parallel. High-level carbapenem resistance evolved rapidly, and frequently, in response to high clinical demand for this antibiotic class during the outbreak.

Background & objectives: Carbapenems are the main antibiotics for the treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa). The aims of this study were to determine the prevalence of gene encoding outer membrane porin protein (OprD) in carbapenem-resistant P. aeruginosa strains as well as to assess the role of insertion sequence (IS) elements in the inactivation of OprD porin and the emergence of carbapenem resistance. Methods: In this study, 103 clinical isolates of P. aeruginosa including 58, 42 and 23 strains resistant to imipenem, meropenem, and doripenem were used, respectively. The isolates were collected from patients referred to Ardabil hospitals. The presence of oprD gene and IS elements were investigated using polymerase chain reaction (PCR) and sequencing methods. P. aeruginosa PAO1 standard isolate was used as the positive control strain for oprD gene. Results: The frequency of oprD gene among carbapenem-resistant P. aeruginosa strains isolated from Ardabil hospitals was 96.5%. Furthermore, IS elements were not observed in the investigated isolates. Conclusion: Based on the results of this study, the presence of IS elements did not involve in the inactivation of outer membrane porin OprD and resistance to carbapenems among P. aeruginosa clinical strains in Ardabil. Therefore, an investigation of the role of other mutations in reducing the expression of oprD gene and increasing P. aeruginosa resistance to carbapenems is recommended.

Abstract 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.

Purpose: The aim of this study was to investigate the changes over a ten-year period in the resistance and epidemiological characteristics of Pseudomonas aeruginosa strains isolated from the Department of Respiratory in Southwest Hospital. Methods: Antimicrobial resistance was detected using the plate double dilution method. PCR amplification and sequencing were performed to evaluate the carbapenemase genes and the oprD gene. Bacterial genotypes were analyzed by multilocus sequence typing (MLST). Quantitative real-time PCR experiments were performed to assess the expression of efflux pump (mexA and mexX) and ampC gene. Results: We collected 233 P. aeruginosa isolates in 2006–2007 and 128 isolates in 2016–2017. The resistance rate of P. aeruginosa strains to the tested antibiotics was significantly lower in 2016–2017 than in 2006–2007. The MLST results showed 27 genotypes in 2006–2007 and 18 genotypes in 2016–2017. ST235 was the most prevalent sequence type, and there was no significant change in the genotypes over the ten-year period. Both VIM-2 and IMP-4 genes were found in 2006–2007, whereas only IMP-4 was found in 2016–2017. The oprD mutational inactivation was the main factor responsible for carbapenem resistance, and the overexpression of mexX had a good correlation with aminoglycoside resistance. Conclusion: These results indicated that the antibiotic resistance of P. aeruginosa in our respiratory department decreased. The loss of oprD gene was the main mechanism of carbapenem resistance, and mexX overexpression was the major contributing factor to aminoglycoside resistance.

An epidemiological study uncovered that fluoroquinolone (FQ) neutropenic prophylaxis in hematopoietic cell transplant and hematologic malignancy (HCT/HM) patients was associated with breakthrough Pseudomonas aeruginosa bloodstream infections (BSIs) with isolates non-susceptible to both FQs and meropenem. The molecular epidemiology of the FQ/meropenem-non-susceptible P. aeruginosa isolates causing FQ-breakthrough BSIs in the HCT/HM patients remains unclear. Through whole genome sequencing on 57 P. aeruginosa isolates from 54 patients diagnosed with HM or receiving an HCT, we found that ST111 strains predominated, accounting for 22 (38.6%) of the isolates. 17 of 33 (51.5%) FQ-breakthrough BSIs were caused by ST111 strains, of which 15 (88.2%) were meropenem non-susceptible. ST111 strains, but not other oprD -deficient, meropenem-non-susceptible clinical strains, were found to have a colonization advantage over P. aeruginosa strain PA14 in C. elegans and to outcompete PA14 in in vitro co-culture assays. Together, we found that breakthrough P. aeruginosa BSIs during FQ prophylaxis in HCT/HM patients are dominated by clonally-related FQ/meropenem non-susceptible strains, predominantly ST111 type, and that the dominance of ST111 strains may be explained by a relative fitness advantage over other clinical strains. Additional work is necessary to better understand the factors driving the dominance and persistence of these ST111 strains. [ABSTRACT FROM AUTHOR]

An epidemiological study uncovered that fluoroquinolone (FQ) neutropenic prophylaxis in hematopoietic cell transplant and hematologic malignancy (HCT/HM) patients was associated with breakthrough Pseudomonas aeruginosa bloodstream infections (BSIs) with isolates non-susceptible to both FQs and meropenem. The molecular epidemiology of the FQ/meropenem-non-susceptible P. aeruginosa isolates causing FQ-breakthrough BSIs in the HCT/HM patients remains unclear. Through whole genome sequencing on 57 P. aeruginosa isolates from 54 patients diagnosed with HM or receiving an HCT, we found that ST111 strains predominated, accounting for 22 (38.6%) of the isolates. 17 of 33 (51.5%) FQ-breakthrough BSIs were caused by ST111 strains, of which 15 (88.2%) were meropenem non-susceptible. ST111 strains, but not other oprD-deficient, meropenem-non-susceptible clinical strains, were found to have a colonization advantage over P. aeruginosa strain PA14 in C. elegans and to outcompete PA14 in in vitro co-culture assays. Together, we found that breakthrough P. aeruginosa BSIs during FQ prophylaxis in HCT/HM patients are dominated by clonally-related FQ/meropenem non-susceptible strains, predominantly ST111 type, and that the dominance of ST111 strains may be explained by a relative fitness advantage over other clinical strains. Additional work is necessary to better understand the factors driving the dominance and persistence of these ST111 strains.

• P. aeruginosa strain sub-lineages with specific oprD variants have spread between patients. • The oprD variants are important contributors to carbapenem resistance. • People infected with the strain sub-lineages had a lower baseline lung function. • Infection with the sub-lineages was not associated with a worse prognosis. Antimicrobial resistance in cystic fibrosis (CF) Pseudomonas aeruginosa airway infection is complex and often attributed to chromosomal mutations. How these mutations emerge in specific strains or whether particular gene mutations are clinically informative is unclear. This study focused on oprD, which encodes an outer membrane porin associated with carbapenem resistance when it is downregulated or inactivated. Determine how mutations in oprD emerge in two prevalent Australian shared CF strains of P. aeruginosa and their clinical relevance. The two most common shared CF strains in Queensland were investigated using whole genome sequencing and their oprD sequences and antimicrobial resistance phenotypes were established. P. aeruginosa mutants with the most common oprD variants were constructed and characterised. Clinical variables were compared between people with or without evidence of infection with strains harbouring these variants. Frequently found nonsense mutations arising from a 1-base pair substitution in oprD evolved independently in three sub-lineages, and are likely major contributors to the reduced carbapenem susceptibility observed in the clinical isolates. Lower baseline FEV 1 %predicted was identified as a risk factor for infection with a sub-lineage (odds ratio=0.97; 95% confidence interval 0.96-0.99; p <0.001). However, acquiring these sub-lineage strains did not confer an accelerated decline in FEV 1 nor increase the risk of death/lung transplantation. Sub-lineages harbouring specific mutations in oprD have emerged and persisted in the shared strain populations. Infection with the sub-lineages was more likely in people with lower lung function, but this was not predictive of a worse clinical trajectory. [ABSTRACT FROM AUTHOR]

Ye Xu,1 Xiangkuo Zheng,2 Weiliang Zeng,2 Tao Chen,1 Wenli Liao,1 Jiao Qian,1 Jie Lin,1 Cui Zhou,1 Xuebin Tian,2 Jianming Cao,2 Tieli Zhou1 1Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of China; 2Department of Medical Laboratory Science, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of ChinaCorrespondence: Jianming Cao; Tieli Zhou Tel +86-577-88069595; +86-577-86689885Email wzcjming@163.com; wyztli@163.comBackground: Heteroresistance is a phenomenon that occurs in all bacteria and can cause treatment failure. Yet, the exact mechanisms responsible for heteroresistance still remain unknown. The following study investigated the mechanisms of imipenem-heteroresistance and -resistance in Pseudomonas aeruginosa clinical isolates from Wenzhou, China.Methods: Imipenem resistance was detected by the agar dilution method; heteroresistance was determined by population analysis profiles. Biofilm formation assay and modified carbapenem inactivation methods were also performed. Polymerase chain reaction (PCR) was conducted to detect oprD, and quantitative real-time PCR was used to determine expression levels of oprD, ampC and four efflux pump coding genes (mexB, mexD, mexE and mexY).Results: Six imipenem-heteroresistant and -resistant P. aeruginosa isolates were selected respectively. Deficient oprD was detected in all resistant isolates and two heteroresistant isolates. No strains produced carbapenemases. Expression levels of oprD were down-regulated in heteroresistant isolates. Transcription levels of the mexE and mexY were significantly increased in all heterogeneous subpopulations compared with their respective native ones. Compared with the susceptible group, increased mean relative expression levels of mexE and mexY or the decreased mean relative expression levels of oprD were observed in the resistant group (P < 0.05), whereas transcription levels of the mexB and mexD remained unchanged.Conclusion: Down-regulation of oprD contributed to the resistance and heteroresistance of imipenem in our P. aeruginosa clinical isolates. In addition, the marginal up-regulation of efflux systems may indirectly affect imipenem resistance. Contrarily, defective oprD was less common in our experimental heteroresistant strains than resistant strains.Keywords: Pseudomonas aeruginosa, imipenem, resistance, heteroresistance, molecular mechanism, oprD

Objectives: Pseudomonas aeruginosa is a major opportunistic pathogen responsible for a wide range of infections. The emergence of antibiotic resistance in this pathogen poses a significant public health challenge. This study aims to conduct a comprehensive meta-analysis of studies conducted in Iran to determine the frequency of key antibiotic resistance mechanisms in Pseudomonas aeruginosa and their association with multidrug-resistant and extensively drug-resistant strains or pandrug-resistant strains., Methods: Systematic database searches encompassing literature up to June 2023 were undertaken. The selected studies centered on OprD downregulation, efflux pump (mexAB-OprM, mexXY-OprM) expression, and AmpC overproduction. Extracted data were synthesised in a meta-analysis for pooled frequency determination of each resistance mechanism., Results: In total, 24 studies were included. OprD downregulation exhibited a pooled frequency of 61%. Efflux pump component frequency ranged from 48% to 77.5%. AmpC overproduction was identified in 29.1% of isolates. Polymyxin B and colistin demonstrated lower antibiotic resistance rates, with pooled frequency of 1% and 1.6%, respectively. Conversely, resistance to other antibiotics ranged widely, with pooled frequency spanning 38.4% to 98.2%., Conclusions: This study underscores the concerning frequency of diverse antibiotic resistance mechanisms in Pseudomonas aeruginosa strains from Iran. Concurrent OprD downregulation, mexAB, mexXY, OprM expression, and AmpC overproduction highlight the urgent need for stringent infection control and prudent antibiotic usage to curb the dissemination of these resistant strains., Prospero: CRD42022379311., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa . The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr , in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr , the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhou, Shi, Mu, Tian, Zhou, Lyu and Chen.)

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals., Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC β-lactamase., Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, bla IMP-6 (N=38), bla VIM-2 (N=3), and bla NDM-1 (N=2), were detected. bla IMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried bla NDM-1 and rmtB . Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates., Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa . Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.

ABSTRACT 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.

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. [ABSTRACT FROM AUTHOR]

Of all the ESKAPE pathogens, carbapenem-resistant and multidrug-resistant Acinetobacter baumannii is the leading cause of hospital-acquired and ventilator-associated pneumonia. A. baumannii infections are notoriously hard to eradicate due to its propensity to rapidly acquire multitude of resistance determinants and the virulence factor cornucopia elucidated by the bacterium that help it fend off a wide range of adverse conditions imposed upon by host and environment. One such weapon in the arsenal of A. baumannii is the outer membrane protein (OMP) compendium. OMPs in A. baumannii play distinctive roles in facilitating the bacterial acclimatization to antibiotic- and host-induced stresses, albeit following entirely different mechanisms. OMPs are major immunogenic proteins in bacteria conferring bacteria host-fitness advantages including immune evasion, stress tolerance, and resistance to antibiotics and antibacterials. In this review, we summarize the current knowledge of major A. baumannii OMPs and discuss their versatile role in antibiotic resistance and virulence. Specifically, we explore how OmpA, CarO, and OprD-like porins mediate antibiotic and amino acid shuttle and host virulence.

Infections by Pseudomonas aeruginosa are difficult to cure due to its high intrinsic and acquired antibiotic resistance. Once colonized the human host, and thanks to antibiotic treatment pressure, P. aeruginosa usually acquires genetic mutations which provide bacteria with antibiotic resistance as well as ability to better adapt to the host environment. Deciphering the evolutionary traits may provide important insights into the development of effective combinatory antibiotic therapy to treat P. aeruginosa infections. In this study, we investigated the molecular mechanisms by which a clinical isolate (ISP50) yields a carbapenem-resistant derivative (IRP41). RNAseq and genomic DNA reference mapping were conducted to compare the transcriptional profiles and in vivo evolutionary trajectories between the two isolates. Our results demonstrated that oprD mutation together with ampC hyper-expression contributed to the increased resistance to carbapenem in the isolate IRP41. Furthermore, a ldcA (PA5198) gene, encoding murein tetrapeptide carboxypeptidase, has been demonstrated for the first time to negatively influence the ampC expression in P. aeruginosa.

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.

Huiqin Liu,1 Weina Kong,1 Weina Yang,2 Gukui Chen,1 Haihua Liang,1 Yani Zhang1 1College of Life Sciences, Northwest University, Shaanxi, China; 2Department of Clinical Laboratory, The Children’s Hospital of Xi’an City, Shaanxi, China Objectives: To provide information about the genetic relationships and mechanism underlying carbapenem resistance in Pseudomonas aeruginosa clinical isolates of a hospital in China.Materials and methods: One hundred and sixty P. aeruginosa strains were isolated from a hospital in China. Susceptibility to 14 antimicrobial agents was determined by antimicrobial susceptibility testing. Multilocus sequence typing was used to characterize the genetic backgrounds of these clinical isolates. Forty-five strains were randomly selected for further evaluation of their carbapenem resistance mechanism. Their oprD gene was compared with the PAO1 sequence.Results: Multilocus sequence typing analysis demonstrated that these isolates were highly diverse; 68 sequence types were identified, of which 28 were novel sequence types. Polygenic and eBURST analysis demonstrated genetically similar clones with dissimilar resistance profiles. Among the 45 randomly selected strains associated with carbapenem resistance, 2 were metallo β-lactamase producers; all the 45 strains were not AmpC overproducers. Sequence analysis revealed a high diversity in the oprD sequences among isolates. Strains susceptible to imipenem and meropenem with shortened L7 and L8 loops in oprD were the major strain types observed in this hospital.Conclusion: This study indicated that oprD provided the main mechanism for carbapenem resistance. The shortened L7 and L8 loops are responsible for carbapenem susceptibility. Keywords: Pseudomonas aeruginosa, multilocus sequence typing, carbapenem resistance, L7 and L8 loops in OprD

Of all the ESKAPE pathogens, carbapenem-resistant and multidrug-resistant Acinetobacter baumannii is the leading cause of hospital-acquired and ventilator-associated pneumonia. A. baumannii infections are notoriously hard to eradicate due to its propensity to rapidly acquire multitude of resistance determinants and the virulence factor cornucopia elucidated by the bacterium that help it fend off a wide range of adverse conditions imposed upon by host and environment. One such weapon in the arsenal of A. baumannii is the outer membrane protein (OMP) compendium. OMPs in A. baumannii play distinctive roles in facilitating the bacterial acclimatization to antibiotic- and host-induced stresses, albeit following entirely different mechanisms. OMPs are major immunogenic proteins in bacteria conferring bacteria host-fitness advantages including immune evasion, stress tolerance, and resistance to antibiotics and antibacterials. In this review, we summarize the current knowledge of major A. baumannii OMPs and discuss their versatile role in antibiotic resistance and virulence. Specifically, we explore how OmpA, CarO, and OprD-like porins mediate antibiotic and amino acid shuttle and host virulence. [ABSTRACT FROM AUTHOR]

Infections by Pseudomonas aeruginosa are difficult to cure due to its high intrinsic and acquired antibiotic resistance. Once colonized the human host, and thanks to antibiotic treatment pressure, P. aeruginosa usually acquires genetic mutations which provide bacteria with antibiotic resistance as well as ability to better adapt to the host environment. Deciphering the evolutionary traits may provide important insights into the development of effective combinatory antibiotic therapy to treat P. aeruginosa infections. In this study, we investigated the molecular mechanisms by which a clinical isolate (ISP50) yields a carbapenem-resistant derivative (IRP41). RNAseq and genomic DNA reference mapping were conducted to compare the transcriptional profiles and in vivo evolutionary trajectories between the two isolates. Our results demonstrated that oprD mutation together with ampC hyper-expression contributed to the increased resistance to carbapenem in the isolate IRP41. Furthermore, a ldcA (PA5198) gene, encoding murein tetrapeptide carboxypeptidase, has been demonstrated for the first time to negatively influence the ampC expression in P. aeruginosa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hospital environments constitute the main reservoir of multidrug-resistant bacteria. In this study we aimed to investigate the presence of Gram-negative bacteria in one Northwestern Tunisian hospital environment, and characterize the genes involved in bacterial resistance. A total of 152 environmental isolates were collected from various surfaces and isolated using MacConkey medium supplemented with cefotaxime or imipenem, with 81 fermenter bacteria (27 Escherichia coli, and 54 Enterobacter spp., including 46 Enterobacter cloacae), and 71 non-fermenting bacteria (69 Pseudomonas spp., including 54 Pseudomonas aeruginosa, and 2 Stenotrophomonas maltophilia) being identified by the MALDI-TOF-MS method. Antibiotic susceptibility testing was performed by disk diffusion method and E-Test was used to determine MICs for imipenem. Several genes implicated in beta-lactams resistance were characterized by PCR and sequencing. Carbapenem resistance was detected among 12 isolates; nine E. coli (blaNDM-1 (n = 8); blaNDM-1 + blaVIM-2 (n = 1)) and three P. aeruginosa were carbapenem-resistant by loss of OprD porin. The whole-genome sequencing of P. aeruginosa 97H was determined using Illumina MiSeq sequencer, typed ST285, and harbored blaOXA-494. Other genes were also detected, notably blaTEM (n = 23), blaCTX-M-1 (n = 10) and blaCTX-M-9 (n = 6). These new epidemiological data imposed new surveillance strategies and strict hygiene rules to decrease the spread of multidrug-resistant bacteria in this area.

Abstract Carbapenems are the most important therapeutic options that effect against serious infections caused by multidrug resistant Pseudomonas aeruginosa (MDR-PA) isolates. Carbapenems resistant isolates of P. aeruginosa are increasing worldwide. The aim of this study was to determine the carbapenem resistance mechanisms in clinical P. aeruginosa isolates from burn patients, in Tehran, Iran. A total of 53 nonduplicated isolates of carbapenem-resistant P. aeruginosa were collected from burn patients. The presence of carbapenemase genes were determined by PCR. AmpC overproducer isolates were detected by phenotypic method. The mutation and transcription level of oprD were determined by PCR-sequencing and quantitative Real-time PCR (RT-PCR), respectively. Twenty-seven (50.9%) isolates were positive for carbapenemase (blaVIM = 25 and blaIMP = 2) and showed high-level resistance to imipenem and meropenem. Twenty-eight isolates were AmpC overproducers. All isolates had a mutation in the oprD gene and down-regulation of oprD was found in 56.6% of MDR-PA isolates. Although the presence of carbapenemase is the common mechanism of resistant to carbapenem, but carbapenem resistance was found by oprD mutation-driven and the AmpC overproducing isolates in Tehran, Iran.

Carbapenems are the most important therapeutic options that effect against serious infections caused by multidrug resistant Pseudomonas aeruginosa (MDR-PA) isolates. Carbapenems resistant isolates of P. aeruginosa are increasing worldwide. The aim of this study was to determine the carbapenem resistance mechanisms in clinical P. aeruginosa isolates from burn patients, in Tehran, Iran. A total of 53 non-duplicated isolates of carbapenem-resistant P. aeruginosa were collected from burn patients. The presence of carbapenemase genes were determined by PCR. AmpC overproducer isolates were detected by phenotypic method. The mutation and transcription level of oprD were determined by PCR-sequencing and quantitative Real-time PCR (RT-PCR), respectively. Twenty-seven (50.9%) isolates were positive for carbapenemase (blaVIM = 25 and blaIMP = 2) and showed high-level resistance to imipenem and meropenem. Twenty-eight isolates were AmpC overproducers. All isolates had a mutation in the oprD gene and down-regulation of oprD was found in 56.6% of MDR-PA isolates. Although the presence of carbapenemase is the common mechanism of resistant to carbapenem, but carbapenem resistance was found by oprD mutation-driven and the AmpC overproducing isolates in Tehran, Iran.

Background & Objectives: Pseudomonas aeruginosa is a common cause of nosocomial infection. OprD protein is a specific protein regulating the uptake of carbapenem antibiotic. Loss of OprD is the main mechanism of Pseudomonas Aeruginosa resistance to carbapenem. In this study, the presence of OprD gene is investigated in isolated Pseudomonas Aeruginosa in burn patients of Ghotboddin hospital in Shiraz. Material & Methods: 66 Pseudomonas Aeruginosa were isolated from wound specimens of 250 burn patients. Strain characteristics were confirmed by biochemical tests. Antibiogram was done via disc diffusion method. Finally, OprD gene was investigated by PCR. Results: Isolated Pseudomonas Aeruginosa showed more sensitivity to chloramphenicol and colicitin and more resistance to ciprofloxacin, gentamycin, cefotaxim, ceftazidin, imipenem, meropenem, and erythromycin. 61 percent of isolates were positive for OprD gene by PCR. Conclusion: The findings of this study revealed that Colicitin and chloramphenicol are more effective in treatment of Pseudomonas Aeruginosa infections in burn patients, and deletion and mutation in OprD gene cause bacterium resistance to carbapenem antibiotic.

Resistant Pseudomonas aeruginosa isolates are one of the major causes of both hospital-acquired infections (HAIs) and community-acquired infections (CAIs). However, management of P. aeruginosa infections is difficult as the bacterium is inherently resistant to many antibiotics. In this study, a collection of 75 P. aeruginosa clinical isolates from two tertiary hospitals from Athens and Alexnadroupolis in Greece was studied to assess antimicrobial sensitivity and molecular epidemiology. All P. aeruginosa isolates were tested for susceptibility to 11 commonly used antibiotics, and the newly introduced Double Locus Sequence Typing (DLST) scheme was implemented to elucidate the predominant clones. The tested P. aeruginosa isolates presented various resistant phenotypes, with Verona Integron-Mediated Metallo-β-lactamase (VIM-2) mechanisms being the majority, and a new phenotype, FEPR-CAZS, being reported for the first time in Greek isolates. DLST revealed two predominant types, 32-39 and 8-37, and provided evidence for intra-hospital transmission of the 32-39 clone in one of the hospitals. The results indicate that DLST can be a valuable tool when local outbreaks demand immediate tracking investigation with limited time and financial resources.

Pseudomonas aeruginosa is the leading cause of infection in burn patients. The increasing carbapenem resistance of P. aeruginosa has become a serious challenge to clinicians. The present study investigated the molecular typing and carbapenem resistance mechanisms of 196 P. aeruginosa isolates from the bloodstream and wound surface of patients in our burn center over a period of 6 years. By multilocus sequence typing (MLST), a total of 58 sequence types (STs) were identified. An outbreak of ST111, a type that poses a high international risk, occurred in 2014. The isolates from wound samples of patients without bacteremia were more diverse and more susceptible to antibiotics than strains collected from the bloodstream or the wound surface of patients with bacteremia. Importantly, a large proportion of the patients with multisite infection (46.51%) were simultaneously infected by different STs in the bloodstream and wound surface. Antimicrobial susceptibility testing of these isolates revealed high levels of resistance to carbapenems, with 35.71% susceptibility to imipenem and 32.14% to meropenem. To evaluate mechanisms associated with carbapenem resistance, experiments were conducted to determine the prevalence of carbapenemase genes, detect alterations of the oprD porin gene, and measure expression of the ampC β-lactamase gene and the mexB multidrug efflux gene. The main mechanism associated with carbapenem resistance was mutational inactivation of oprD (88.65%), accompanied by overexpression of ampC (68.09%). In some cases, oprD was inactivated by insertion sequence element IS1411, which has not been found previously in P. aeruginosa. These findings may help control nosocomial P. aeruginosa infections and improve clinical practice.

Background & Objectives: Pseudomonas aeruginosa is a common cause of nosocomial infection. OprD protein is a specific protein regulating the uptake of carbapenem antibiotic. Loss of OprD is the main mechanism of Pseudomonas Aeruginosa resistance to carbapenem. In this study, the presence of OprD gene is investigated in isolated Pseudomonas Aeruginosa in burn patients of Ghotboddin hospital in Shiraz. Material & Methods: 66 Pseudomonas Aeruginosa were isolated from wound specimens of 250 burn patients. Strain characteristics were confirmed by biochemical tests. Antibiogram was done via disc diffusion method. Finally, OprD gene was investigated by PCR. Results: Isolated Pseudomonas Aeruginosa showed more sensitivity to chloramphenicol and colicitin and more resistance to ciprofloxacin, gentamycin, cefotaxim, ceftazidin, imipenem, meropenem, and erythromycin. 61 percent of isolates were positive for OprD gene by PCR. Conclusion: The findings of this study revealed that Colicitin and chloramphenicol are more effective in treatment of Pseudomonas Aeruginosa infections in burn patients, and deletion and mutation in OprD gene cause bacterium resistance to carbapenem antibiotic.

Background: Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. The main mechanism of resistance of this bacterium to fluoroquinolones and carbapenems are the modification of type II topoisomerases (DNA gyrase and topoisomerase IV) and alterations in the OprD porin, respectively. The aim of this study was to examine for the occurrence of mutations related to fluoroquinolone resistance of gyrA and parC genes and mutational inactivation of oprD gene of clinical isolates using DNA sequencing technique. Methods: A total of 60 P. aeruginosa isolates were collected from the hospitalized patients in the Intensive Care Units (ICUs) of Al-Zahra hospital located in Isfahan, Iran. The pattern of sensitivity to antibiotics was determined using CLSI disk diffusion and MIC methods. The assay was based on a DNA sequencing method using polymerase chain reaction (PCR) for amplification and sequencing of the selected genes. Results: The results show that replacement of Ile for Thr-83 in gyrA was the only replacement, while other substitutions not observed. No mutations were found in parC. The most frequent amino acid alterations were E185Q, P186G, and V189T, found in five resistance isolates, However, nucleotide insertions and deletions mutations not observed. Conclusion: Our study suggested that mutation of gyrA and oprD genes may play a minor role in fluoroquinolone and carbapenem resistance and other mechanisms may contribute to the fluoroquinolone and carbapenem resistance of P. aeruginosa.

Carbapenems are the most important therapeutic options that effect against serious infections caused by multidrug resistant Pseudomonas aeruginosa (MDR-PA) isolates. Carbapenems resistant isolates of P. aeruginosa are increasing worldwide. The aim of this study was to determine the carbapenem resistance mechanisms in clinical P. aeruginosa isolates from burn patients, in Tehran, Iran. A total of 53 nonduplicated isolates of carbapenem-resistant P. aeruginosa were collected from burn patients. The presence of carbapenemase genes were determined by PCR. AmpC overproducer isolates were detected by phenotypic method. The mutation and transcription level of oprD were determined by PCR-sequencing and quantitative Real-time PCR (RT-PCR), respectively. Twenty-seven (50.9%) isolates were positive for carbapenemase (blaVIM = 25 and blaIMP = 2) and showed high-level resistance to imipenem and meropenem. Twenty-eight isolates were AmpC overproducers. All isolates had a mutation in the oprD gene and down-regulation of oprD was found in 56.6% of MDR-PA isolates. Although the presence of carbapenemase is the common mechanism of resistant to carbapenem, but carbapenem resistance was found by oprD mutation-driven and the AmpC overproducing isolates in Tehran, Iran. [ABSTRACT FROM AUTHOR]

The recently described double-locus sequence typing (DLST) scheme implemented to deeply characterize the genetic profiles of 52 resistant environmental Pseudomonas aeruginosa isolates deriving from aquatic habitats of Greece. DLST scheme was able not only to assign an already known allelic profile to the majority of the isolates but also to recognize two new ones ( ms217-190, ms217-191) with high discriminatory power. A third locus ( oprD) was also used for the molecular typing, which has been found to be fundamental for the phylogenetic analysis of environmental isolates given the resulted increased discrimination between the isolates. Additionally, the circulation of acquired resistant mechanisms in the aquatic habitats according to their genetic profiles was proved to be more extent. Hereby, we suggest that the combination of the DLST to oprD typing can discriminate phenotypically and genetically related environmental P. aeruginosa isolates providing reliable phylogenetic analysis at a local level. [ABSTRACT FROM AUTHOR]

Background: Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. The main mechanism of resistance of this bacterium to fluoroquinolones and carbapenems are the modification of type II topoisomerases (DNA gyrase and topoisomerase IV) and alterations in the OprD porin, respectively. The aim of this study was to examine for the occurrence of mutations related to fluoroquinolone resistance of gyrA and parC genes and mutational inactivation of oprD gene of clinical isolates using DNA sequencing technique. Methods: A total of 60 P. aeruginosa isolates were collected from the hospitalized patients in the Intensive Care Units (ICUs) of Al-Zahra hospital located in Isfahan, Iran. The pattern of sensitivity to antibiotics was determined using CLSI disk diffusion and MIC methods. The assay was based on a DNA sequencing method using polymerase chain reaction (PCR) for amplification and sequencing of t h e selected genes. Results: The results show that replacement of Ile for Thr-83 in gyrA was the only replacement, while other substitutions not observed. No mutations were found in parC. The most frequent amino acid alterations were E185Q, P186G, and V189T, found in five resistance isolates, However, nucleotide insertions and deletions mutations not observed. Conclusion: Our study suggested that mutation of gyrA and oprD genes may play a minor role in fluoroquinolone and carbapenem resistance and other mechanisms may contribute to the fluoroquinolone and carbapenem resistance of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a bacterial pathogen that may cause serious drug-resistant infections that are potentially fatal. To investigate the genetic characteristics of these organisms, we tested 416 P. aeruginosa strains recovered from 12 types of clinical samples collected in 29 different hospital wards in 10 hospitals in Guangdong Province, China, from 2017 to 2020. These strains were found to belong to 149 known sequence types (STs) and 72 novel STs, indicating that transmission of these strains involved multiple routes. A high rate of resistance to imipenem (89.4%) and meropenem (79.4%) and a high prevalence of pathogenic serotypes (76.4%) were observed among these strains. Six STs of global high-risk clones (HiRiCs) and a novel HiRiC strains, ST1971, which exhibited extensive drug resistance, were identified. Importantly, ST1971 HiRiC, which was unique in China, also exhibited high virulence, which alarmed the further surveillance on this highly virulent and highly resistant clone. Inactivation of the oprD gene and overexpression of efflux systems were found to be mainly responsible for carbapenem resistance in these strains; carriage of metallo-β-lactamase (MBL)-encoding genes was less common. Interestingly, frameshift mutations (49.0%) and introduction of a stop codon (22.4%) into the oprD genes were the major mechanisms of imipenem resistance. On the other hand, expression of the MexAB-OprM efflux pump and MBL-encoding genes were mechanisms of resistance in >70% of meropenem-resistant strains. The findings presented here provide insights into the development of effective strategies for control of worldwide dissemination of CRPA. IMPORTANCE Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major concern in clinical settings worldwide, yet few genetic and epidemiological studies on CRPA strains have been performed in China. Here, we sequence and analyze the genomes of 416 P. aeruginosa strains from hospitals in China to elucidate the genetic, phenotypic, and transmission characteristics of CRPA strains and to identify the molecular signatures responsible for the observed increase in the prevalence of CRPA infections in China. These findings may provide new insight into the development of effective strategies for worldwide control of CRPA and minimize the occurrence of untreatable infections in clinical settings., Competing Interests: The authors declare no conflict of interest.