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.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology.

Objective: Recombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. Methods: Recombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. Results: The M-RAP method had sensitivity rates of 1, 10, and 1 copies/µL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively ( P < 0.05). Conclusion: An M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

[A case of acute purulent otitis media complicated by atypical mastoiditis on the background of an unverified HIV infection]. / Sluchai ostrogo gnoinogo srednego otita, oslozhnennogo atipichnym mastoiditom, na fone neverifitsirovannoi VICh-infektsii.

According to the literature, acute otitis media is complicated by mastoiditis in 0.15-1% of cases. In turn, mastoiditis can be complicated by meningitis, encephalitis, abscess of temporal lobe of brain and cerebellum, epidural and subdural abscesses, facial nerve paresis, labyrinthitis, phlegmon of soft tissues of neck, as well as subperiosteal abscess, which makes 7% in the structure of mastoiditis complications. Nowadays, when doctors have a wide range of antibacterial preparations at their disposal, a complicated course of acute otitis media and further mastoiditis is caused both by an aggressive atypical infectious agent and immunocompromised status of a patient. The article deals with a clinical case of a prolonged course of acute otitis media complicated by mastoiditis and subperiosteal abscess against the background of outpatient courses of antibacterial therapy. The examination revealed an atypical pathogen of otitis media Pseudomonas aeruginosa and HIV-positive status of the patient, previously unknown. Timely surgical intervention and the right combination of antibacterial drugs, meropenem and ciprofloxacin, prevented the development of intracranial and septic complications, despite the presence of multiple foci of bone destruction of the mastoid process and temporal bone pyramid, bordering the middle fossa and sigmoid sinus, according to multispiral head computed tomography. As a part of additional examination in the Center for AIDS and Infectious Diseases Prevention and Control, the patient was diagnosed with HIV infection, clinical stage 4C, progressing phase on the background of absence of antiretroviral therapy, and the necessary amount of treatment was prescribed.

Implications of deduplication on the detection rates of multidrug-resistant organism (MDRO) in various specimens: insights from the hospital infection surveillance program.

BACKGROUND: Currently, different guidelines recommend using different methods to determine whether deduplication is necessary when determining the detection rates of multidrug-resistant organisms (MDROs). However, few studies have investigated the effect of deduplication on MDRO monitoring data. In this study, we aimed to investigate the influence of deduplication on the detection rates of MDROs in different specimens to assess its impact on infection surveillance outcomes. METHODS: Samples were collected from hospitalized patients admitted between January 2022 and December 2022; four types of specimens were collected from key monitored MDROs, including sputum samples, urine samples, blood samples, and bronchoalveolar lavage fluid (BALF) samples. In this study, we compared and analysed the detection rates of carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRECO), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) under two conditions: with and without deduplication. RESULTS: When all specimens were included, the detection rates of CRKP, CRAB, CRPA, and MRSA without deduplication (33.52%, 77.24%, 44.56%, and 56.58%, respectively) were significantly greater than those with deduplication (24.78%, 66.25%, 36.24%, and 50.83%, respectively) (all P < 0.05). The detection rates in sputum samples were significantly different between samples without duplication (28.39%, 76.19%, 46.95%, and 70.43%) and those with deduplication (19.99%, 63.00%, 38.05%, and 64.50%) (all P < 0.05). When deduplication was not performed, the rate of detection of CRKP in urine samples reached 30.05%, surpassing the rate observed with deduplication (21.56%) (P < 0.05). In BALF specimens, the detection rates of CRKP and CRPA without deduplication (39.78% and 53.23%, respectively) were greater than those with deduplication (31.62% and 42.20%, respectively) (P < 0.05). In blood samples, deduplication did not have a significant impact on the detection rates of MDROs. CONCLUSION: Deduplication had a significant effect on the detection rates of MDROs in sputum, urine, and BALF samples. Based on these data, we call for the Infection Prevention and Control Organization to align its analysis rules with those of the Bacterial Resistance Surveillance Organization when monitoring MDRO detection rates.

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.

Correlating Reiff scores with clinical, functional, and prognostic factors: characterizing noncystic fibrosis bronchiectasis severity: validation from a nationwide multicenter study in Taiwan.

BACKGROUND: Our study aimed to confirm a simplified radiological scoring system, derived from a modified Reiff score, to evaluate its relationship with clinical symptoms and predictive outcomes in Taiwanese patients with noncystic fibrosis bronchiectasis (NCFB). METHODS: This extensive multicenter retrospective study, performed in Taiwan, concentrated on patients diagnosed with NCFB verified through high-resolution computed tomography (HRCT) scans. We not only compared the clinical features of various types of bronchiectasis (cylindrical, varicose, and cystic). Furthermore, we established relationships between the severity of clinical factors, including symptom scores, pulmonary function, pseudomonas aeruginosa colonization, exacerbation and admission rates, and HRCT parameters using modified Reiff scores. RESULTS: Data from 2,753 patients were classified based on HRCT patterns (cylindrical, varicose, and cystic) and severity, assessed by modified Reiff scores (mild, moderate, and severe). With increasing HRCT severity, a significant correlation was found with decreased forced expiratory volume in the first second (FEV1) (p < 0.001), heightened clinical symptoms (p < 0.001), elevated pathogen colonization (pseudomonas aeruginosa) (p < 0.001), and an increased annual hospitalization rate (p < 0.001). In the following multivariate analysis, elderly age, pseudomonas aeruginosa pneumonia, and hospitalizations per year emerged as the only independent predictors of mortality. CONCLUSION: Based on our large cohort study, the simplified CT scoring system (Reiff score) can serve as a useful adjunct to clinical factors in predicting disease severity and prognosis among Taiwanese patients with NCFB.

Carbapenem-Resistant Pseudomonas aeruginosa Infection and Mixed Infections Are Risk Factors for Poor Outcome After Lung Transplant.

OBJECTIVES: In this study, we analyzed the effects of carbapenem-resistant Pseudomonas aeruginosa infection and mixed infection on the perioperative prognosis of lung transplant recipients and studied statistics on antibiotic resistance in P aeruginosa. MATERIALS AND METHODS: This was a retrospective casecontrol study. We collected data on lung transplant recipients with combined lower respiratory tract P aeruginosa infection within 48 hours after lung transplant at the China-Japan Friendship Hospital from August 2018 to April 2022. We grouped recipients according to P aeruginosa resistance to carbapenem antibiotics and summarized the clinical characteristics of carbapenem-resistant P aeruginosa infection. We analyzed the effects of carbapenemresistant P aeruginosa infection and mixed infections on all-cause mortality 30 days after lung transplant by Cox regression. We used the Kaplan-Meier method to plot survival curves. RESULTS: Patients in the carbapenem-resistant P aeruginosa group had a higher all-cause mortality rate than those in the carbapenem-sensitive P aeruginosa group at both 7 days (6 patients [22.3%] vs 2 patients [4.5%]; P = .022) and 30 days (12 patients [44.4%] vs 7 patients [15.9%]; P = .003) after lung transplant. In multivariate analysis, both carbapenemresistant P aeruginosa infection and P aeruginosa combined with bacterial infection were independent risk factors for death 30 days after transplant in lung transplant recipients (P < .05). In subgroup analysis, carbapenem-resistant P aeruginosa combined with bacterial infection increased the risk of death 30 days after transplant in lung transplant recipients compared with carbapenem-sensitive P aeruginosa combined with bacterial infection (12 patients [60%] vs 6 patients [19.4%]; P < .001). CONCLUSIONS: Combined lower respiratory tract carbapenem-resistant P aeruginosa infection and P aeruginosa combined with bacterial infection early after lung transplant increased the risk of 30-day mortality after lung transplant.

Considerations for the use of inhaled antibiotics for Pseudomonas aeruginosa in people with cystic fibrosis receiving CFTR modulator therapy.

The major cause of mortality in people with cystic fibrosis (pwCF) is progressive lung disease characterised by acute and chronic infections, the accumulation of mucus, airway inflammation, structural damage and pulmonary exacerbations. The prevalence of Pseudomonas aeruginosa rises rapidly in the teenage years, and this organism is the most common cause of chronic lung infection in adults with cystic fibrosis (CF). It is associated with an accelerated decline in lung function and premature death. New P. aeruginosa infections are treated with antibiotics to eradicate the organism, while chronic infections require long-term inhaled antibiotic therapy. The prevalence of P. aeruginosa infections has decreased in CF registries since the introduction of CF transmembrane conductance regulator modulators (CFTRm), but clinical observations suggest that chronic P. aeruginosa infections usually persist in patients receiving CFTRm. This indicates that pwCF may still need inhaled antibiotics in the CFTRm era to maintain long-term control of P. aeruginosa infections. Here, we provide an overview of the changing perceptions of P. aeruginosa infection management, including considerations on detection and treatment, the therapy burden associated with inhaled antibiotics and the potential effects of CFTRm on the lung microbiome. We conclude that updated guidance is required on the diagnosis and management of P. aeruginosa infection. In particular, we highlight a need for prospective studies to evaluate the consequences of stopping inhaled antibiotic therapy in pwCF who have chronic P. aeruginosa infection and are receiving CFTRm. This will help inform new guidelines on the use of antibiotics alongside CFTRm.

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.

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.

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.

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.

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.

OriPlex: Origami-enabled multiplexed detection of respiratory pathogens.

Origami biosensors leverage paper foldability to develop total analysis systems integrated in a single piece of paper. This capability can also be utilized to incorporate additional features that would be difficult to achieve with rigid substrates. In this article, we report a new design for 3D origami biosensors called OriPlex, which leverages the foldability of filter paper for the multiplexed detection of bacterial pathogens. OriPlex immunosensors detect pathogens by folding nanoparticle reservoirs containing different types of nanoprobes. This releases antibody-coated nanoparticles in a central channel where targets are captured through physical interactions. The OriPlex concept was demonstrated by detecting the respiratory pathogens Pseudomonas aeruginosa (PA) and Klebsiella pneumoniae (KP) with a limit of detection of 3.4·103 cfu mL-1 and 1.4·102 cfu mL-1, respectively, and with a turn-around time of 25 min. Remarkably, the OriPlex biosensors allowed the multiplexed detection of both pathogens spiked into real bronchial aspirate (BAS) samples at a concentration of 105 cfu mL-1 (clinical infection threshold), thus demonstrating their suitability for diagnosing lower tract respiratory infections. The results shown here pave the way for implementing OriPlex biosensors as a screening test for detecting superbugs requiring personalized antibiotics in suspected cases of nosocomial pneumonia.

Fourth national Japanese antimicrobial susceptibility pattern surveillance program: Bacterial isolates from patients with complicated urinary tract infections.

INTRODUCTION: Antimicrobial susceptibility patterns of bacterial pathogens isolated from patients with complicated urinary tract infections were analyzed using the national surveillance data, comprising 793 bacterial strains from eight clinically relevant species. MATERIALS AND METHODS: Data were collected for the fourth national surveillance project from July 2020 to December 2021 by the Japanese Society of Chemotherapy, the Japanese Association for Infectious Disease, and the Japanese Society of Clinical Microbiology. Surveillance was supervised with the cooperation of 43 medical institutions throughout Japan. RESULTS: Fluoroquinolone required a minimum inhibitory concentration (MIC) of 2-64 mg/L to inhibit the 330 tested Escherichia coli strains. The proportion of levofloxacin-resistant E. coli strains increased from 28.6% in 2008 to 29.6% in 2011, 38.5% in 2015, and 44.5% in 2021. The proportion of levofloxacin-resistant strains of Pseudomonas aeruginosa also increased from previous survey results, showing a continuing downward trend. Conversely, the proportion of levofloxacin-resistant strains of Enterococcus faecalis decreased relative to previous reports. Neither multidrug-resistant P. aeruginosa nor carbapenem-resistant Enterobacteriaceae were detected. For methicillin-resistant Staphylococcus aureus (MRSA), the proportion of vancomycin-susceptible strains (MIC of 2 µg/mL) decreased from 14.7% to 7.7%. DISCUSSION: Bacterial strains that produced extended-spectrum ß-lactamase included E. coli (82/330 strains, 24.8%), Klebsiella pneumoniae (11/68 strains, 16.2%), and Proteus mirabilis (4/26 strains, 15.4%). As compared to previous surveillance reports, these strains showed an increase in proportion over the years.

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.

Secondary metabolite profiling of Pseudomonas aeruginosa isolates reveals rare genomic traits.

Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen with remarkable phylogenetic and phenotypic variabilities. In this work, we applied classical molecular networking analysis to secondary metabolite profiling data from seven Pseudomonas aeruginosa strains, including five clinical isolates from the lung secretions of people with cystic fibrosis (CF). We provide three vignettes illustrating how secondary metabolite profiling aids in the identification of rare genomics traits in P. aeruginosa. First, we describe the identification of a previously unreported class of acyl putrescines produced by isolate mFLRO1. Secondary analysis of publicly available metabolomics data revealed that acyl putrescines are produced by <5% of P. aeruginosa strains. Second, we show that isolate SH3A does not produce di-rhamnolipids. Whole-genome sequencing and comparative genomics revealed that SH3A cannot produce di-rhamnolipids because its genome belongs to clade 5 of the P. aeruginosa phylogenetic tree. Previous phylogenetic analysis of thousands of P. aeruginosa strains concluded that <1% of publicly available genome sequences contribute to this clade. Last, we show that isolate SH1B does not produce the phenazine pyocyanin or rhamnolipids because it has a one-base insertion frameshift mutation (678insC) in the gene rhlR, which disrupts rhl-driven quorum sensing. Secondary analysis of the tens of thousands of publicly available genomes in the National Center for Biotechnology Information (NCBI) and the Pseudomonas Genome Database revealed that this mutation was present in only four P. aeruginosa genomes. Taken together, this study highlights that secondary metabolite profiling combined with genomic analysis can identify rare genetic traits of P. aeruginosa isolates.IMPORTANCESecondary metabolite profiling of five Pseudomonas aeruginosa isolates from cystic fibrosis sputum captured three traits present in <1%-5% of publicly available data, pointing to how our current library of P. aeruginosa strains may not represent the diversity within this species or the genetic variance that occurs in the CF lung.

[Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method]. / Pseudomonas aeruginosa Izolatlarinda Virülans Genlerinin Polimeraz Zincir Reaksiyonuyla Tespiti ve Yüksek Riskli Klonlarin Matriksle Desteklenmis Lazer Desorpsiyon/Iyonizasyon Uçus Zamani Kütle Spektrometresi Yöntemi ile Arastirilmasi.

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.

[Molecular Analysis of Antibiotic Resistance in CarbapenemResistant Pseudomonas aeruginosa Isolates Isolated from Clinical Samples]. / Klinik Örneklerden Izole Edilen Karbapenem Dirençli Pseudomonas aeruginosa Izolatlarindaki Antibiyotik Direncinin Moleküler Analizi.

Pseudomonas aeruginosa is an opportunistic pathogen that causes increased morbidity and mortality in risky patient groups. Nowadays, carbapenem resistance has become a threat and resistance genes are spreading among species through mobile genetic elements. The dissemination of carbapenemases among P.aeruginosa is a serious public health concern due to its limited options for the treatment of bacterial infections. In this study, it was aimed to investigate the molecular epidemiology of 47 carbapenem resistant P.aeruginosa (CRPA) isolates derived from various clinical samples from the Central Laboratory Bacteriology Unit of Kocaeli University Research and Training Hospital between October 2021 and March 2023. The rates of resistance to the antibiotics, some carbapenemase and virulence genes, conjugative resistance plasmids, integron gene cassette contents and the clonal similarity of the isolates were investigated and then epidemiologically evaluated. In the study, identification of the bacterial isolates and their susceptibility to some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were determined by the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of the isolates was demonstrated by the imipenem/meropenem-EDTA (IMP/MEM-EDTA) combined disc method. Conjugation experiments were performed by the broth mating method. Alkali lysis method was used in plasmid DNA isolations. Co-transferred antibiotic resistances in transconjugants were detected by disc diffusion method. Carbapenemase genes (blaIMP , blaVIM , blaNDM , blaKPC and blaOXA-48 ), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were screened by specific polymerase chain reactions (PCRs). Clonal relationships of the CRPA isolates were investigated by evaluating the DNA f ingerprintings obtained from the ERIC (enterobacterial repetitive intergenic consensus)-PCR assay. The highest resistance rate of the isolates were to levofloxacin, while the lowest resistance rates were observed against tobramycin, gentamicin and amikacin. MBL production was detected in 25 (53.2%) isolates. In conjugation experiments, 12 (25.5%) isolates were detected to harbour conjugative resistance plasmids. In 90% of the CRPA isolates, lasR and rhlR biofilm genes (encoding for the transcriptional activator protein) were detected by PCR. The blaVIM gene was detected in six (12.8%) isolates. The blaNDM gene was detected in five (10.6%) isolates and the blaOXA-48 gene was detected in three (6.4%) isolates. The blaKPC and blaIMP genes were not detected in CRPA isolates. It was determined that two (16.6%) of the isolates that carried the blaVIM gene, one (8.3%) carried the blaNDM gene and one (8.3%) carried the blaOXA-48 gene contained conjugative plasmids.In integron-specific PCRs, intI1 gene was positive in 39 (82.9%) isolates, while class 1 integron gene cassettes were detected in 24 isolates (51%). IntI1 positive six isolates were found to harbour class 1 integron gene cassettes-bearing conjugative plasmids. Class 2 integrons were not found in the CRPA isolates. Dendrogram analysis of ERIC-PCR patterns showed that there was no clonal similarity between the CRPA isolates and the isolates did not spread by cross-contamination. As a result, it has been observed that most of the CRPA isolates which have the potential to form biofilms, are highly resistant to other antibiotic groups other than carbapenems and can co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains which have the potential to cause epidemics in the clinical arena.