Novel resistance ICEs carrying the blaFIM-1 metallo-ß-lactamase gene from an ST235 Pseudomonas aeruginosa sublineage.

FIM-1 is an acquired metallo-ß-lactamase identified in a multidrug-resistant Pseudomonas aeruginosa (index strain FI-14/157) of clinical origin isolated in 2007 in Florence, Italy. Here we report on a second case of infection by FIM-1-positive P. aeruginosa (FI-17645), which occurred in 2020 in the same hospital. Both FIM-1-positive strains exhibited resistance to all anti-Pseudomonas antibiotics except colistin and cefiderocol. Comparative genomic characterization revealed that the two FIM-positive strains were closely related [core genome difference, 16 single nucleotide polymorphisms (SNPs)], suggesting a local circulation of similar strains. In the FI-14/157 index strain, the blaFIM-1 gene was associated with an ISCR19-like element that likely contributed to its capture downstream an integron platform inserted aboard a Tn21-like transposon, named Tn7703.1, which was associated with a large integrative and conjugative element (ICE) named ICE7705.1, integrated into an att site located within the 3'-end of tRNAGly CCC gene of the P. aeruginosa chromosome. In strain FI-17645, blaFIM-1 was associated with a closely related ICE, named ICE7705.2, integrated in the same chromosomal site. Similar ICE platforms, lacking the blaFIM-1-containing region, were detected in other ST235 P. aeruginosa strains from different geographic areas, suggesting a common ancestry and underscoring the role of these elements in the dissemination of resistance genes in P. aeruginosa. Sequence database mining revealed two draft P. aeruginosa genomes, one from Italy and one from the USA (both isolated in 2012), including a contig with blaFIM-1, suggesting that this resistance gene could have a broader distribution than originally anticipated.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Neonatal sepsis due to NDM-1 and VIM-2 co-producing Pseudomonas aeruginosa in Morocco.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco. MATERIALS AND METHODS: P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types. RESULTS: P. aeruginosa O82J1 co-expressed two metallo-ß-lactamases, blaNDM-1 and blaVIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The blaVIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the blaNDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a blaPER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB. CONCLUSIONS: The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults.

In vitro activity of cefiderocol in Pseudomonas aeruginosa isolates from people with cystic fibrosis recovered during three multicentre studies in Spain.

OBJECTIVES: Despite the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, Pseudomonas aeruginosa is still a major pathogen in people with cystic fibrosis (pwCF). We determine the activity of cefiderocol and comparators in a collection of 154 P. aeruginosa isolates recovered from pwCF during three multicentre studies performed in 17 Spanish hospitals in 2013, 2017 and 2021. METHODS: ISO broth microdilution was performed and MICs were interpreted with CLSI and EUCAST criteria. Mutation frequency and WGS were also performed. RESULTS: Overall, 21.4% were MDR, 20.8% XDR and 1.3% pandrug-resistant (PDR). Up to 17% of the isolates showed a hypermutator phenotype. Cefiderocol demonstrated excellent activity; only 13 isolates (8.4%) were cefiderocol resistant by EUCAST (none using CLSI). A high proportion of the isolates resistant to ceftolozane/tazobactam (71.4%), meropenem/vaborbactam (70.0%), imipenem/relebactam (68.0%) and ceftazidime/avibactam (55.6%) were susceptible to cefiderocol. Nine out of 13 cefiderocol-resistant isolates were hypermutators (P < 0.001). Eighty-three STs were detected, with ST98 being the most frequent. Only one isolate belonging to the ST175 high-risk clone carried blaVIM-2. Exclusive mutations affecting genes involved in membrane permeability, AmpC overexpression (L320P-AmpC) and efflux pump up-regulation were found in cefiderocol-resistant isolates (MIC = 4-8 mg/L). Cefiderocol resistance could also be associated with mutations in genes related to iron uptake (tonB-dependent receptors and pyochelin/pyoverdine biosynthesis). CONCLUSIONS: Our results position cefiderocol as a therapeutic option in pwCF infected with P. aeruginosa resistant to most recent ß-lactam/ß-lactamase inhibitor combinations.

A comparison of strategies for identifying patients at risk for carbapenem-resistant or extended ß-lactam-resistant Pseudomonas aeruginosa.

OBJECTIVES: To assess risk factors for carbapenem-resistant Pseudomonas aeruginosa (CR) and extended-ß-lactam-resistant P. aeruginosa (EBR) infection/colonization, and to develop and compare tools for predicting isolation of CR and EBR from clinical cultures. METHODS: This retrospective study analysed hospitalized patients with positive P. aeruginosa cultures between 2015 and 2021. Two case-control analyses were performed to identify risk factors and develop scoring tools for distinguishing patients with CR versus carbapenem-susceptible (CS) P. aeruginosa and EBR versus CS P. aeruginosa. The performance of institutionally derived scores, externally derived scores and the presence/absence of key risk factors to predict CR and EBR were then compared. RESULTS: A total of 2379 patients were included. Of these, 8.3% had a positive culture for CR, 5.0% for EBR and 86.7% for CS P. aeruginosa. There was substantial overlap in risk factors for CR and EBR. Institutional risk scores demonstrated modestly higher area under the ROC curve values than external scores for predicting CR (0.67 versus 0.58) and EBR (0.76 versus 0.70). Assessing the presence/absence of ≥1 of the two strongest predictors (prior carbapenem use or CR isolation within 90 days) was slightly inferior to scoring tools for predicting CR, and comparable for predicting EBR. CONCLUSIONS: Clinicians concerned about CR in P. aeruginosa should consider the likelihood of EBR when making treatment decisions. A simple approach of assessing recent history of CR isolation or carbapenem usage performed similarly to more complex scoring tools and offers a more pragmatic way of identifying patients who require coverage for resistant P. aeruginosa.

Evaluation of fortimicin antibiotic combinations against MDR Pseudomonas aeruginosa and resistome analysis of a whole genome sequenced pan-drug resistant isolate.

BACKGROUND: Multidrug-resistant (MDR) P. aeruginosa is a rising public health concern, challenging the treatment of such a ubiquitous pathogen with monotherapeutic anti-pseudomonal agents. Worryingly, its genome plasticity contributes to the emergence of P. aeruginosa expressing different resistant phenotypes and is now responsible for notable epidemics within hospital settings. Considering this, we aimed to evaluate the synergistic combination of fortimicin with other traditional anti-pseudomonal agents and to analyze the resistome of pan-drug resistant (PDR) isolate. METHODS: Standard methods were used for analyzing the antimicrobial susceptibility tests. The checkerboard technique was used for the in vitro assessment of fortimicin antibiotic combinations against 51 MDR P. aeruginosa and whole genome sequencing was used to determine the resistome of PDR isolate. RESULTS: Out of 51 MDR P. aeruginosa, the highest synergistic effect was recorded for a combination of fortimicin with ß-lactam group as meropenem, ceftazidime, and aztreonam at 71%, 59% and 43%, respectively. Of note, 56.8%, 39.2%, and 37.2% of the tested MDR isolates that had synergistic effects were also resistant to meropenem, ceftazidime, and aztreonam, respectively. The highest additive effects were recorded for combining fortimicin with amikacin (69%) and cefepime (44%) against MDR P. aeruginosa. Resistome analysis of the PDR isolate reflected its association with the antibiotic resistance phenotype. It ensured the presence of a wide variety of antibiotic-resistant genes (ß-lactamases, aminoglycosides modifying enzymes, and efflux pump), rendering the isolate resistant to all clinically relevant anti-pseudomonal agents. CONCLUSION: Fortimicin in combination with classical anti-pseudomonal agents had shown promising synergistic activity against MDR P. aeruginosa. Resistome profiling of PDR P. aeruginosa enhanced the rapid identification of antibiotic resistance genes that are likely linked to the appearance of this resistant phenotype and may pave the way to tackle antimicrobial resistance issues shortly.

Coexistence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes among clinical Pseudomonas aeruginosa isolates in Egypt.

BACKGROUND: Data about the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) production in P. aeruginosa compared to the Enterobacteriaceae family is limited. The availability of limited therapeutic options raises alarming concerns about the treatment of multidrug-resistant P. aeruginosa. This study aimed to assess the presence of PMQR and ESBL genes among P. aeruginosa strains. METHODS: Fifty-six P. aeruginosa strains were isolated from 330 patients with different clinical infections. Phenotypically fluoroquinolone-resistant isolates were tested by PCR for the presence of six PMQR genes. Then, blaTEM, blaSHV, and blaCTX-M type ESBL genes were screened to study the co-existence of different resistance determinants. RESULTS: Overall, 22/56 (39.3%) of the studied P. aeruginosa isolates were phenotypically resistant to fluoroquinolones. PMQR-producing P. aeruginosa isolates were identified in 20 isolates (90.9%). The acc(6')-Ib-cr was the most prevalent PMQR gene (77.3%). The qnr genes occurred in 72.7%, with the predominance of the qnrA gene at 54.5%, followed by the qnrS gene at 27.3%, then qnrB and qnrC at 22.7%. The qepA was not detected in any isolate. The acc(6')-Ib-cr was associated with qnr genes in 65% of positive PMQR isolates. Significant differences between the fluoroquinolone-resistant and fluoroquinolone-susceptible isolates in terms of the antibiotic resistance rates of amikacin, imipenem, and cefepime (P value < 0.0001) were found. The ESBL genes were detected in 52% of cephalosporin-resistant P. aeruginosa isolates. The most frequent ESBL gene was blaCTX-M (76.9%), followed by blaTEM (46.2%). No isolates carried the blaSHV gene. The acc(6')-Ib-cr gene showed the highest association with ESBL genes, followed by the qnrA gene. The correlation matrix of the detected PMQR and ESBL genes indicated overall positive correlations. The strongest and most highly significant correlation was between qnrA and acc(6')-Ib-cr (r = 0.602) and between qnrA and blaCTX-M (r = 0.519). CONCLUSION: A high prevalence of PMQR genes among the phenotypic fluoroquinolone-resistant P. aeruginosa isolates was detected, with the co-carriage of different PMQR genes. The most frequent PMQR was the acc(6')-Ib-cr gene. Co-existence between PMQR and ESBL genes was found, with 75% of PMQR-positive isolates carrying at least one ESBL gene. A high and significant correlation between the ESBL and PMQR genes was detected.

Microbiological and molecular studies on a multidrug-resistant Pseudomonas aeruginosa from a liver transplant patient with urinary tract infection in Egypt.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen responsible for complicated UTIs and exhibits high antibiotic resistance, leading to increased mortality rates, especially in cases of multidrug-resistant strains. This study aimed to investigate the antibiotic susceptibility patterns and genomic characterization of XDR strains identified in end-stage liver disease patients who underwent liver transplants. METHODS: In this study, a number of 30 individuals who underwent liver transplants were registered. Ninety urine and 60 wound site swab samples were collected and processed for culturing, identification, and antimicrobial sensitivity. Extensively drug-resistant strain EMARA01 was confirmed through Sanger sequencing and was then processed for whole genome sequencing to characterize the genomic pattern. Sequencing data were processed for de novo assembly using various tools and databases, including genome annotation, serotype identification, virulence factor genes, and antimicrobial resistance gene. Pangenome analysis of randomly selected 147 reference strains and EMAR01 sequenced strain was performed using the Bacterial Pan Genome Analysis (BPGA) software. RESULTS: Of these total examined samples, nosocomial infection due to P. aeruginosa was detected in twelve patients' samples. AST analysis showed that P. aeruginosa strains exhibit resistance to tobramycin, erythromycin, and gentamicin, followed by piperacillin and ofloxacin, and no strains exhibit resistance to meropenem and imipenem. The CARD database identified 59 AMR genes similar to the EMAR01 strain genome and mostly belong to the family involved in the resistance-nodulation-cell division (RND) antibiotic efflux pump. Five genes; nalC, nalD, MexR, MexA, and MexB, exhibit resistance to 14 classes of antibiotics, while two AMR; CpxR, and OprM, exhibit resistance to 15 classes of drugs. Pangenome analysis revealed that the pan-genome remained open, suggesting the potential for acquiring accessory and unique genes. Notably, the genes predominantly involved in amino acid transport metabolism were identified using the KEGG database. CONCLUSIONS: This study provides valuable insights into the antimicrobial resistance profile, genetic features, and genomic evolution of P. aeruginosa strains causing UTIs in liver transplant patients. The findings emphasize the significance of comprehending AMR mechanisms and genetic diversity in P. aeruginosa for developing effective treatment strategies and infection control measures.

Prevalence and molecular characterization of colistin resistance in Pseudomonas aeruginosa isolates: insights from a study in Ardabil hospitals.

BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa infections. While polymyxins have been the mainstay for treatment, there is a global increase in resistance to these antibiotics. Therefore, our study aimed to determine the prevalence and molecular details of colistin resistance in P. aeruginosa clinical isolates collected between June 2019 and May 2023, as well as the genetic linkage of colistin-resistant P. aeruginosa isolates. RESULTS: The resistance rate to colistin was 9% (n = 18) among P. aeruginosa isolates. All 18 colistin-resistant isolates were biofilm producers and carried genes associated with biofilm formation. Furthermore, the presence of genes encoding efflux pumps, TCSs, and outer membrane porin was observed in all colistin-resistant P. aeruginosa strains, while the mcr-1 gene was not detected. Amino acid substitutions were identified only in the PmrB protein of multidrug- and colistin-resistant strains. The expression levels of mexA, mexC, mexE, mexY, phoP, and pmrA genes in the 18 colistin-resistant P. aeruginosa strains were as follows: 88.8%, 94.4%, 11.1%, 83.3%, 83.3%, and 38.8%, respectively. Additionally, down-regulation of the oprD gene was observed in 44.4% of colistin-resistant P. aeruginosa strains. CONCLUSION: This study reports the emergence of colistin resistance with various mechanisms among P. aeruginosa strains in Ardabil hospitals. We recommend avoiding unnecessary use of colistin to prevent potential future increases in colistin resistance.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Characterization and genetic analysis of extensively drug-resistant hospital acquired Pseudomonas aeruginosa isolates.

BACKGROUND: The incidence of hospital-acquired infections in extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) has been increasing worldwide and is frequently associated with an increase in mortality and morbidity rates. The aim of this study was to characterize clinical XDR-PA isolates recovered during six months at three different hospitals in Egypt. RESULTS: Seventy hospital-acquired clinical isolates of P. aeruginosa were classified into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR), according to their antimicrobial resistance profile. In addition, the possession of genes associated with mobile genetic elements and genes encoding antimicrobial resistance determinants among isolates were detected using polymerase chain reaction. As a result, a significant percentage of the isolates (75.7%) were XDR, while 18.5% were MDR, however only 5.7% of the isolates were non-MDR. The phenotypic detection of carbapenemases, extended-spectrum ß-lactamases (ESBLs) and metallo ß-lactamase (MBL) enzymes showed that 73.6% of XDR-PA isolates were carbapenemases producers, whereas 75.5% and 88.7% of XDR-PA isolates produced ESBLs and MBL respectively. In addition, PCR screening showed that oxa gene was the most frequently detected gene of carbapenemases (91.4%), while aac(6')-lb gene was mostly detected (84.3%) among the screened aminoglycosides-resistance genes. Furthermore, the molecular detection of the colistin resistance gene showed that 12.9% of isolates harbored mcr-1 gene. Concerning mobile genetic element markers (intI, traA, tnp513, and merA), intI was the highest detected gene as it was amplified in 67 isolates (95.7%). Finally, phylogenetic and molecular typing of the isolates via ERIC-PCR analysis revealed 10 different ERIC fingerprints. CONCLUSION: The present study revealed a high prevalence of XDR-PA in hospital settings which were resistant to a variety of antibiotics due to several mechanisms. In addition, 98% of the XDR-PA clinical isolates contained at least one gene associated with movable genetic elements, which could have aided the evolution of these XDR-PA strains. To reduce spread of drug resistance, judicious use of antimicrobial agents and strict infection control measures are therefore essential.

Whole-genome sequencing, multilocus sequence typing, and resistance mechanism of the carbapenem-resistant Pseudomonas aeruginosa in China.

Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed carbapenem-resistant Pseudomonas aeruginosa (CRPA) at a tertiary hospital in Shandong, China, using whole-genome sequencing (WGS). The objective was to explore the mechanisms and molecular characteristics of carbapenem resistance. A retrospective analysis of 91 isolates from January 2022 to March 2023 was performed, which included strain identification and antimicrobial susceptibility testing. WGS was utilized to determine the genome sequences of these CRPA strains, and the species were precisely identified using average nucleotide identification (ANI), with further analysis on multilocus sequence typing and strain relatedness. Some strains were found to carry the ampD and oprD genes, while only a few harbored carbapenemase genes or related genes. Notably, all strains possessed the mexA, mexE, and mexX genes. The major lineage identified was ST244, followed by ST235. The study revealed a diverse array of carbapenem resistance mechanisms among hospital isolates, differing from previous studies in mainland China. It highlighted that carbapenem resistance is not due to a single mechanism but rather a combination of enzyme-mediated resistance, AmpC overexpression, OprD dysfunction, and efflux pump overexpression. This research provides valuable insights into the evolutionary mechanisms and molecular features of CRPA resistance in this region, aiding in the national prevention and control of CRPA, and offering references for targeting and developing new drugs.

Systemic antibiotics for Pseudomonas aeruginosa infection in outpatients with non-hospitalised exacerbations of pre-existing lung diseases: a randomised clinical trial.

BACKGROUND: The effect of dual systemic antibiotic therapy against Pseudomonas aeruginosa in patients with pre-existing lung disease is unknown. To assess whether dual systemic antibiotics against P. aeruginosa in outpatients with COPD, non-cystic fibrosis (non-CF) bronchiectasis, or asthma can improve outcomes. METHODS: Multicenter, randomised, open-label trial conducted at seven respiratory outpatient clinics in Denmark. Outpatients with COPD, non-CF bronchiectasis, or asthma with a current P. aeruginosa-positive lower respiratory tract culture (clinical routine samples obtained based on symptoms of exacerbation not requiring hospitalisation), regardless of prior P. aeruginosa-status, no current need for hospitalisation, and at least two moderate or one hospitalisation-requiring exacerbation within the last year were eligible. Patients were assigned 1:1 to 14 days of dual systemic anti-pseudomonal antibiotics or no antibiotic treatment. Primary outcome was time to prednisolone or antibiotic-requiring exacerbation or death from day 20 to day 365. RESULTS: The trial was stopped prematurely based in lack of recruitment during the COVID-19 pandemic, this decision was endorsed by the Data and Safety Monitoring Board. Forty-nine outpatients were included in the study. There was a reduction in risk of the primary outcome in the antibiotic group compared to the control group (HR 0.51 (95%CI 0.27-0.96), p = 0.037). The incidence of admissions with exacerbation within one year was 1.1 (95%CI 0.6-1.7) in the dual antibiotic group vs. 2.9 (95%CI 1.3-4.5) in the control group, p = 0.037. CONCLUSIONS: Use of dual systemic antibiotics for 14 days against P. aeruginosa in outpatients with chronic lung diseases and no judged need for hospitalisation, improved clinical outcomes markedly. The main limitation was the premature closure of the trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03262142, registration date 2017-08-25.

Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention. METHODS: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples). RESULTS: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 100 copies/µL for fluorescence assay and 101 copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements. CONCLUSIONS: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.

Carbapenem-resistant gram-negative bacterial infections and risk factors for acquisition in a Kenyan intensive care unit.

BACKGROUND: Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a critical public health threat globally; however, there are inadequate surveillance data, especially in intensive care units (ICU), to inform infection prevention and control in many resource-constrained settings. Here, we assessed the prevalence of CR-GNB infections and risk factors for acquisition in a Kenyan ICU. METHODS: A hospital-based cross-sectional study design was adopted, recruiting 162 patients clinically presenting with bacterial infection after 48 h of ICU admission, from January to October 2022 at the Nairobi West Hospital, Kenya. Demographics and clinical data were collected by case report form. The type of sample collected, including blood, tracheal aspirate, ascitic tap, urine, stool, and sputum depended on the patient's clinical presentation and were transported to the hospital Microbiology laboratory in a cool box for processing within 2 h. The samples were analyzed by cultured and BD Phoenix system used for isolates' identity and antimicrobial susceptibility. RESULTS: CR-GNB infections prevalence was 25.9% (42/162), with Klebsiella pneumoniae (35.7%, 15/42) and Pseudomonas aeruginosa (26.2%, 11/42) predominating. All isolates were multidrug-resistant (MDR). P. aeruginosa and A. baumannii were 100% colistin-resistant, while K. pneumoniae (33.3%) was tigecycline-resistant. History of antibiotics (aOR = 3.40, p = 0.005) and nasogastric tube (NGT) use (aOR = 5.84, p = < 0.001) were the risk factors for infection. CONCLUSION: Our study highlights high MDR- and CR-GNB infections in ICU, with prior antibiotic exposure and NGT use as risk factors, and diminishing clinical value of colistin and tigecycline. In this study setting and beyond, strict implementation of antimicrobial stewardship programs and adherence to infection prevention and control through monitoring, evaluation and feedback are warranted to curb CR-GNB infections, especially among the risk groups.

In search of the best method to detect carriage of carbapenem-resistant Pseudomonas aeruginosa in humans: a systematic review.

BACKGROUND: Detection of carbapenem-resistant Pseudomonas aeruginosa (CR-PA) in humans is important to prevent transmission. However, the most optimal culture method to detect CR-PA is unknown. This systematic review aims to determine which culture method is most sensitive and which culture methods are used to detect CR-PA in humans. Second, to establish the most feasible culture method taking into account the turnaround time (TAT), and third, to provide an overview of the sampling sites used to detect carriage. METHODS: We systematically searched the electronic databases Embase, Medline Ovid, Cochrane, Scopus, CINAHL, and Web of Science until January 27, 2023. All diagnostic accuracy studies comparing two or more culture methods to detect CR-PA and recent outbreak or surveillance reports on CR-PA carriage or infection in humans, which describe culture methods and their results, were eligible for inclusion. We used QUADAS-2 guideline for diagnostic accuracy studies and the STROBE or ORION guideline for outbreak-surveillance studies to assess the risk of bias. RESULTS: Six diagnostic accuracy studies were included. An enrichment broth was found to increase the detection of CR-PA. Using an enrichment broth extended the TAT by 18-24 h, yet selective media could reduce the TAT by 24 h compared to routine media. In total, 124 outbreak-surveillance studies were included, of which 17 studies with surveillance samples and 116 studies with clinical samples. In outbreak-surveillance studies with surveillance samples, perianal, rectal swabs or stools were the most common sampling site/specimen (13/17, 76%). A large variety was observed in whether and which kind of enrichment broth and selective media were used. CONCLUSIONS: We found a benefit of using an enrichment step prior to inoculation of the material onto selective media for the detection of CR-PA. More research is needed to determine the most sensitive sampling site and culture method. TRAIL REGISTRATION: This study was registered in the PROSPERO International prospective register of systematic reviews (registration number: CRD42020207390, http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42020207390 ).

Distribution of sequence types and antimicrobial resistance of clinical Pseudomonas aeruginosa isolates from dogs and cats visiting a veterinary teaching hospital in Thailand.

BACKGROUND: Pseudomonas aeruginosa is an important opportunistic pathogen in dogs and cats and is resistant to several antimicrobial drugs; however, data on the clonal distribution of P. aeruginosa in veterinary hospital are limited. This study aimed to investigate the clonal dissemination and antimicrobial resistance of clinical P. aeruginosa in a veterinary teaching hospital in Thailand within a 1-year period. Minimum inhibitory concentration determination and whole genome sequencing were used for antimicrobial susceptibility analysis and genetic determination, respectively. RESULTS: Forty-nine P. aeruginosa were isolated mostly from the skin, urinary tract, and ear canal of 39 dogs and 10 cats. These isolates belonged to 39 sequence types (STs) that included 9 strains of high-risk clones of ST235 (n = 2), ST244 (n = 2), ST274 (n = 2), ST277 (n = 1), ST308 (n = 1), and ST357 (n = 1). Overall antimicrobial resistance rate was low (< 25%), and no colistin-resistant strains were found. Two carbapenem-resistant strains belonging to ST235 and ST3405 were identified. CONCLUSIONS: Clinical P. aeruginosa in dogs and cats represent STs diversity. High-risk clones and carbapenem-resistant strains are a public health concern. Nevertheless, this study was limited by a small number of isolates. Continuous monitoring is needed, particularly in large-scale settings with high numbers of P. aeruginosa, to restrict bacterial transfer from companion animal to humans in a veterinary hospital.

Bronchoscopy-guided antimicrobial therapy for cystic fibrosis.

BACKGROUND: Early diagnosis and treatment of lower respiratory tract infections is the mainstay of management of lung disease in cystic fibrosis (CF). When sputum samples are unavailable, diagnosis relies mainly on cultures from oropharyngeal specimens; however, there are concerns about whether this approach is sensitive enough to identify lower respiratory organisms. Bronchoscopy and related procedures such as bronchoalveolar lavage (BAL) are invasive but allow the collection of lower respiratory specimens from non-sputum producers. Cultures of bronchoscopic specimens provide a higher yield of organisms compared to those from oropharyngeal specimens. Regular use of bronchoscopy and related procedures may increase the accuracy of diagnosis of lower respiratory tract infections and improve the selection of antimicrobials, which may lead to clinical benefits. This is an update of a previous review that was first published in 2013 and was updated in 2016 and in 2018. OBJECTIVES: To evaluate the use of bronchoscopy-guided (also known as bronchoscopy-directed) antimicrobial therapy in the management of lung infection in adults and children with cystic fibrosis. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched three registries of ongoing studies and the reference lists of relevant articles and reviews. The date of the most recent searches was 1 November 2023. SELECTION CRITERIA: We included randomised controlled studies involving people of any age with CF that compared the outcomes of antimicrobial therapies guided by the results of bronchoscopy (and related procedures) versus those guided by any other type of sampling (e.g. cultures from sputum, throat swab and cough swab). DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies, assessed their risk of bias and extracted data. We contacted study investigators for further information when required. We assessed the certainty of the evidence using the GRADE criteria. MAIN RESULTS: We included two studies in this updated review. One study enrolled 170 infants under six months of age who had been diagnosed with CF through newborn screening. Participants were followed until they were five years old, and data were available for 157 children. The study compared outcomes for pulmonary exacerbations following treatment directed by BAL versus standard treatment based on clinical features and oropharyngeal cultures. The second study enrolled 30 children with CF aged between five and 18 years and randomised participants to receive treatment based on microbiological results of BAL triggered by an increase in lung clearance index (LCI) of at least one unit above baseline or to receive standard treatment based on microbiological results of oropharyngeal samples collected when participants were symptomatic. We judged both studies to have a low risk of bias across most domains, although the risk of bias for allocation concealment and selective reporting was unclear in the smaller study. In the larger study, the statistical power to detect a significant difference in the prevalence of Pseudomonas aeruginosa was low because Pseudomonas aeruginosa isolation in BAL samples at five years of age in both groups were much lower than the expected rate that was used for the power calculation. We graded the certainty of evidence for the key outcomes as low, other than for high-resolution computed tomography scoring and cost-of-care analysis, which we graded as moderate certainty. Both studies reported similar outcomes, but meta-analysis was not possible due to different ways of measuring the outcomes and different indications for the use of BAL. Whether antimicrobial therapy is directed by the use of BAL or standard care may make little or no difference in lung function z scores after two years (n = 29) as measured by the change from baseline in LCI and forced expiratory volume in one second (FEV1) (low-certainty evidence). At five years, the larger study found little or no difference between groups in absolute FEV1 z score or forced vital capacity (FVC) (low-certainty evidence). BAL-directed therapy probably makes little or no difference to any measure of chest scores assessed by computed tomography (CT) scan at either two or five years (different measures used in the two studies; moderate-certainty evidence). BAL-directed therapy may make little or no difference in nutritional parameters or in the number of positive isolates of P aeruginosa per participant per year, but may lead to more hospitalisations per year (1 study, 157 participants; low-certainty evidence). There is probably no difference in average cost of care per participant (either for hospitalisations or total costs) at five years between BAL-directed therapy and standard care (1 study, 157 participants; moderate-certainty evidence). We found no difference in health-related quality of life between BAL-directed therapy and standard care at either two or five years, and the larger study found no difference in the number of isolates of Pseudomonas aeruginosa per child per year. The eradication rate following one or two courses of eradication treatment and the number of pulmonary exacerbations were comparable in the two groups. Mild adverse events, when reported, were generally well tolerated. The most common adverse event reported was transient worsening of cough after 29% of procedures. Significant clinical deterioration was documented during or within 24 hours of BAL in 4.8% of procedures. AUTHORS' CONCLUSIONS: This review, limited to two well-designed randomised controlled studies, shows no evidence to support the routine use of BAL for the diagnosis and management of pulmonary infection in preschool children with CF compared to the standard practice of providing treatment based on results of oropharyngeal culture and clinical symptoms. No evidence is available for adults.

Frequency and prognosis of peritoneal dialysis-associated peritonitis in children.

BACKGROUND: Peritonitis is the leading cause of peritoneal dialysis (PD) discontinuation. However, few data concern risk factors of peritonitis development and catheter removal caused by treatment failure in pediatric patients. METHODS: This single-center, retrospective study analyzed data from pediatric patients who underwent chronic PD between March 2002 and June 2022. The incidence rates of peritonitis by the person-year method were calculated, and they were stratified by patient age groups. Risk factors for peritonitis development and catheter removal were also analyzed by multivariate analysis using logistic regression model. RESULTS: Ninety patients were enrolled, and 62 peritonitis episodes were observed in 41 (46%) patients. The incidence rate of peritonitis was 0.21 episodes per patient-year, which was the highest in children aged under 2 years old (0.26 episodes per patient-year). Moreover, 44 (71%) cases were successfully cured by antibiotics alone, although 17 (27%) cases required catheter removal, and 4 (6%) cases transitioned to chronic hemodialysis because of peritoneal dysfunction. One patient died. The risk factor for peritonitis development and catheter removal caused by treatment failure was PD insertion at under 2 years old (odds ratio = 2.5; P = 0.04) and Pseudomonas aeruginosa (odds ratio = 11.0; P = 0.04) in the multivariate analysis. P. aeruginosa was also a risk factor for difficulty in re-initiating PD (P = 0.004). CONCLUSIONS: The incidence rate of peritonitis was the highest in children under 2 years old. P. aeruginosa peritonitis is a risk factor for catheter removal and peritoneal dysfunction.

Ceftazidime-avibactam in the treatment of bacteremia due to carbapenem-resistant gram-negative bacteria in hematological patients: Experience in a single center.

INTRODUCTION: Limited experience exists with ceftazidime-avibactam (CAZ-AVI) in treating bacteremia caused by carbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa (CRPA) in hematological patients. METHODS: We performed a single-center, retrospective, observational study including patients who received CAZ-AVI for bacteremia due to CRE or CRPA between 2018 and 2022. The primary outcome was 30-day survival. We conducted a multivariable analysis to identify predictors of survival. RESULTS: 56 patients were included and 57 (41 CRE and 16 CRPA) strains were isolated. 35 strains produced carbapenemase, including 25 metallo-beta-lactamase (MBL) and 10 serine-beta-lactamase. 48 patients (85.7 %) received combination therapy. All patients with MBL-CRE bacteremia (n = 24) received combination therapy with aztreonam (AZT). The susceptibility rates to CAZ-AVI were only 26.8 % (11/41) in CRE and 80.0 % (8/10) in CRPA. The 30-day survival rates were 85.0 % (34/40) in the CRE group and 81.3 % (13/16) in the CRPA group. In patients with MBL-CRE bacteremia, the 30-day survival was as high as 91.7 % (22/24) due to combination with AZT. Ceftazidime did not influence the activity of aztreonam-avibactam against MBL-CRE in-vitro. Multivariable cox analysis revealed neutropenia >14 days (P = 0.002, HR: 34.483, 95%CI: 3.846-333.333) and a higher Pitt bacteremia score (P = 0.005, HR: 2.074, 95%CI: 1.253-3.436) were risk factors for 30-day survival. CONCLUSIONS: CAZ-AVI is highly effective in treating bacteremia due to CRPA and serine-beta-lactamase CRE. The combination of avibactam with AZT is highly effective in treating bacteremia due to AZT-resistant MBL producers.