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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.

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.

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.

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.

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.

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.

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.