Low Ciprofloxacin concentrations select Multidrug-Resistant Mutants overproducing efflux pumps in clinical isolates of Pseudomonas Aeruginosa

Low antibiotic concentrations present in natural environments are a severe and often neglected threat to public health. Even if they are present below their MICs, they may select for antibiotic-resistant pathogens. Notably, the minimal subinhibitory concentrations that select resistant bacteria, and define the respective sub-MIC selective windows, differ between antibiotics. The establishment of these selective concentrations is needed for risk-assessment studies regarding the presence of antibiotics in different habitats. Using short-term evolution experiments in a set of 12 Pseudomonas aeruginosa clinical isolates (including high-risk clones with ubiquitous distribution), we have determined that ciprofloxacin sub-MIC selective windows are strain specific and resistome dependent. Nonetheless, in all cases, clinically relevant multidrug-resistant (MDR) mutants emerged upon exposure to low ciprofloxacin concentrations, with these concentrations being below the levels reported in ciprofloxacin-polluted natural habitats where P. aeruginosa can be present. This feature expands the conditions and habitats where clinically relevant quinolone-resistant mutants can emerge. In addition, we established the lowest concentration threshold beyond which P. aeruginosa, regardless of the strain, becomes resistant to ciprofloxacin. Three days of exposure under this sub-MIC "risk concentration" led to the selection of MDR mutants that displayed resistance mechanisms usually ascribed to high selective pressures, i.e., the overproduction of the efflux pumps MexCD-OprJ and MexEF-OprN. From a One-Health viewpoint, these data stress the transcendent role of low drug concentrations, which can be encountered in natural ecosystems, in aggravating the antibiotic resistance problem, especially when it comes to pathogens of environmental origin. IMPORTANCE It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic. Herein, we have determined the ciprofloxacin sub-MIC selective windows of a set of Pseudomonas aeruginosa clinical isolates (including high-risk clones with worldwide distribution) and established the lowest concentration threshold, notably an amount reported to be present in natural ecosystems, beyond which this pathogen acquires resistance. Importantly, our results show that this ciprofloxacin sub-MIC selects for multidrug-resistant mutants overproducing clinically relevant efflux pumps. From a One-Health angle, this information supports that low antimicrobial concentrations, present in natural environments, may have a relevant role in worsening the antibiotic resistance crisis, particularly regarding pathogens with environmental niches, such as P. aeruginosa. It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic

Mutation Analysis in Regulator DNA-Binding Regions for Antimicrobial Efflux Pumps in 17,000 Pseudomonas aeruginosa Genomes

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance.This research was funded by Acción Estratégica en Salud from the ISCIII, grant MPY 509/19. This research was also supported by Personalized and Precision Medicine grant from the Instituto de Salud Carlos III (MePRAM Project, PMP22/00092), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.S

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis

COVID-19; SARS-CoV-2; Electronic health recordsCOVID-19; SARS-CoV-2; Registros médicos electrónicosCOVID 19; SARS-CoV-2; Registres mèdics electrònicsBackground Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance.This work was supported by MSD and by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU through grants PI21/00017 and Personalized and precision medicine grant (MePRAM Project, PMP22/00092)

Using the Electronic Nose to Identify Airway Infection During COPD Exacerbations

Background The electronic nose (e-nose) detects volatile organic compounds (VOCs) in exhaled air. We hypothesized that the exhaled VOCs print is different in stable vs. exacerbated patients with chronic obstructive pulmonary disease (COPD), particularly if the latter is associated with airway bacterial infection, and that the e-nose can distinguish them. Methods Smell-prints of the bacteria most commonly involved in exacerbations of COPD (ECOPD) were identified in vitro. Subsequently, we tested our hypothesis in 93 patients with ECOPD, 19 of them with pneumonia, 50 with stable COPD and 30 healthy controls in a cross-sectional case-controlled study. Secondly, ECOPD patients were re-studied after 2 months if clinically stable. Exhaled air was collected within a Tedlar bag and processed by a Cynarose 320 e-nose. Breath-prints were analyzed by Linear Discriminant Analysis (LDA) with "One Out" technique and Sensor logic Relations (SLR). Sputum samples were collected for culture. Results ECOPD with evidence of infection were significantly distinguishable from non-infected ECOPD (p = 0.018), with better accuracy when ECOPD was associated to pneumonia. The same patients with ECOPD were significantly distinguishable from stable COPD during follow-up (p = 0.018), unless the patient was colonized. Additionally, breath-prints from COPD patients were significantly distinguished from healthy controls. Various bacteria species were identified in culture but the e-nose was unable to identify accurately the bacteria smell-print in infected patients. Conclusion E-nose can identify ECOPD, especially if associated with airway bacterial infection or pneumonia

Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone

AbstractEmergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in &gt;100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up β-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.</jats:p

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis

Background: Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods: A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings: A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation: While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance.This work was supported by MSD and by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea —NextGenerationEU through grants PI21/00017 and Personalized and precision medicine grant (MePRAM Project, PMP22/00092).S

Association between Pseudomonas aeruginosa O-antigen serotypes, resistance profiles and high-risk clones: results from a Spanish nationwide survey

Objectives: To evaluate the correlation of O-antigen serotypes with resistance profiles and high-risk clones in a Spanish nationwide survey. Methods: Up to 30 consecutive healthcare-associated Pseudomonas aeruginosa isolates were collected during October 2017 from each of 51 hospitals (covering all Spanish regions) with a total of 1445 isolates studied. MICs of 13 antipseudomonal agents and MDR/XDR profiles had been previously determined, as well as whole-genome sequences of 185 representative XDR isolates. O-antigen serotypes (O1–O16) were determined by agglutination using serotype-specific antisera (BioRad). The Pseudomonas aeruginosa serotyper (PAst) program was used for in silico serotyping. Results: The most frequent serotypes were O6 (17.8%), O1 (15.4%) and O11 (13.3%). In contrast, the most frequent serotype among XDR isolates (17.3%) was O4 (34.1%), distantly followed by O11 (15.9%). Within serotypes, XDR phenotypes were more frequent for O12 (60.0%) and O4 (57.3%). The most frequent clone among the XDR isolates was ST175 (40.9%), followed by CC235 (10.7%), ST308 (5.2%) and CC111 (3.6%). Up to 81.6%of XDR ST175 isolates typed O4, whereas 18.4% were non-typeable. O4 genotype was detected in all sequenced (n=55) ST175 isolates. On the other hand, CC235 and ST308 were associated with O11, whereas CC111 was linked to serotype O12. Conclusions: O4 serotype is linked to the MDR/XDR profile of widespread ST175 (typically only susceptible to colistin, amikacin and the novel combinations ceftolozane/tazobactam and ceftazidime/avibactam) and therefore, after local validation, its detection in the microbiology laboratory might be useful for guiding semi-empirical antipseudomonal therapies and infection control measures in Spanish hospitals

Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network’s technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain

Genomics And Susceptibility Profiles Of Extensively Drug-resistant Pseudomonas Aeruginosa Isolates From Spain

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (> 95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the beta-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in beta-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance

A Large Multicenter Prospective Study of Community-Onset Healthcare Associated Bacteremic Urinary Tract Infections in the Era of Multidrug Resistance: Even Worse than Hospital Acquired Infections?

Introduction: Healthcare-associated (HCA) infections represent a growing public health problem. The aim of this study was to compare community-onset healthcare associated (CO-HCA) bacteremic urinary tract infections (BUTI) and hospital-acquired (HA)-BUTI with special focus on multidrug resistances (MDR) and outcomes. Methods: ITUBRAS-project is a prospective multicenter cohort study of patients with HCA-BUTI. All consecutive hospitalized adult patients with CO-HCA-BUTI or HA-BUTI episode were included in the study. Exclusion criteria were: patients < 18 years old, non-hospitalized patients, bacteremia from another source or primary bacteremia, non-healthcare-related infections and infections caused by unusual pathogens of the urinary tract. The main outcome variable was 30-day all-cause mortality with day 1 as the first day of positive blood culture. Logistic regression was used to analyze factors associated with clinical cure at hospital discharge and with receiving inappropriate initial antibiotic treatment. Cox regression was used to evaluate 30-day all-cause mortality. Results: Four hundred forty-three episodes were included, 223 CO-HCA-BUTI. Patients with CO-HCA-BUTI were older (p < 0.001) and had more underlying diseases (p = 0.029) than those with HA-BUTI. The severity of the acute illness (Pitt score) was also higher in CO-HCA-BUTI (p = 0.026). Overall, a very high rate of MDR profiles (271/443, 61.2%) was observed, with no statistical differences between groups. In multivariable analysis, inadequate empirical treatment was associated with MDR profile (aOR 3.35; 95% CI 1.77–6.35), Pseudomonas aeruginosa (aOR 2.86; 95% CI 1.27–6.44) and Charlson index (aOR 1.11; 95% CI 1.01–1.23). Mortality was not associated with the site of acquisition of the infection or the presence of MDR profile. However, in the logistic regression analyses patients with CO-HCA-BUTI (aOR 0.61; 95% CI 0.40–0.93) were less likely to present clinical cure. Conclusion: The rate of MDR infections was worryingly high in our study. No differences in MDR rates were found between CO-HCA-BUTI and HA-BUTI, in the probability of receiving inappropriate empirical treatment or in 30-day mortality. However, CO-HCA-BUTIs were associated with worse clinical cure. © 2021, The Author(s)