Effect of Antifibrotic Use on Mortality in Patients with Idiopathic Pulmonary Fibrosis.

RATIONALE: Observational studies report significant protective effect of antifibrotics on mortality among patients with idiopathic pulmonary fibrosis. Many of these studies, however, were subject to immortal time bias due to the mishandling of delayed antifibrotic initiation. OBJECTIVES: To evaluate the antifibrotic effect on mortality among patients with idiopathic pulmonary fibrosis using appropriate statistical methods that avoid immortal time bias. METHODS: Using a large administrative database, we identified 10,289 patients with idiopathic pulmonary fibrosis, of which 2,300 used antifibrotics. Treating delayed antifibrotic initiation as a time-dependent variable, three statistical methods were used to control baseline characteristics and avoid immortal time bias. Stratified analysis was performed for patients who initiated antifibrotics early and those who initiated treatment late. For comparison, methods that mishandle immortal time bias were performed. A simulation study was conducted to demonstrate the performance of these models in a wide range of scenarios. MEASUREMENTS AND MAIN RESULTS: All three statistical methods yielded non-significant results for the antifibrotic effect on mortality, with the stratified analysis for patients with early antifibrotic initiation suggesting evidence for reduced mortality risk: HR=0.89 (95% CI: 0.79-1.01, p=0.08) for all patients and HR=0.85 (95% CI: 0.73-0.98, p=0.03) for patients who were 65 years or older. Methods that mishandle immortal time bias demonstrated significantly lower mortality risk for antifibrotic users. Bias of these methods was evident in the simulation study, where appropriate methods performed well with little to no bias. CONCLUSIONS: Findings in this study did not confirm an association between antifibrotics and mortality, with a stratified analysis showing support for a potential treatment effect with early treatment initiation.

Impact of a novel pre-hospital stroke notification programme on acute stroke care key performance indicators in Hong Kong: a multicentre prospective cohort study with historical controls.

INTRODUCTION: Early identification and initiation of reperfusion therapy is essential for suspected acute ischaemic stroke. A pre-hospital stroke notification (PSN) protocol using FASE (facial drooping, arm weakness, speech difficulties, and eye palsy) was implemented to improve key performance indicators (KPIs) in acute stroke care delivery. We assessed KPIs and clinical outcomes before and after PSN implementation in Hong Kong. METHODS: This prospective cohort study with historical controls was conducted in the Accident and Emergency Departments of four public hospitals in Hong Kong. Patients were screened using the PSN protocol between August 2021 and February 2022. Suspected stroke patients between August 2020 and February 2021 were included as historical controls. Door-to-needle (DTN) and door-to-computed tomography (DTC) times before and after PSN implementation were compared. Clinical outcomes including National Institutes of Health Stroke Scale score at 24 hours and modified Rankin Scale score at 3 months after intravenous recombinant tissue-type plasminogen activator (IV-rtPA) were also assessed. RESULTS: Among the 715 patients (266 PSN and 449 non-PSN) included, 50.8% of PSN patients and 37.7% of non-PSN patients had a DTC time within 25 minutes (P<0.001). For the 58 PSN and 134 non-PSN patients given IV-rtPA, median DTN times were 67 and 75.5 minutes, respectively (P=0.007). The percentage of patients with a DTN time within 60 minutes was higher in the PSN group than in the non-PSN group (37.9% vs 21.6%; P=0.019). No statistically significant differences in clinical outcomes were observed. CONCLUSION: Although the PSN protocol shortened DTC and DTN times, clinical outcomes did not significantly differ.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Telemedicine acceptance by older adults in Hong Kong during a hypothetical severe outbreak and after the COVID-19 pandemic: a cross-sectional cohort survey.

INTRODUCTION: Telemedicine services worldwide have experienced unprecedented growth since the early days of the coronavirus disease 2019 (COVID-19) pandemic. Multiple studies have shown that telemedicine is an effective alternative to conventional in-person patient care. This study explored the public perception of telemedicine in Hong Kong, specifically among older adults who are most vulnerable to COVID-19. METHODS: Medical students from The Chinese University of Hong Kong conducted in-person surveys of older adults aged ≥60 years. Each survey collected socio-demographic information, medical history, and concerns regarding telemedicine use. Univariate and multivariate logistic regression analyses were conducted to identify statistically significant associations. The primary outcomes were acceptance of telemedicine use during a hypothetical severe outbreak and after the COVID-19 pandemic. RESULTS: There were 109 survey respondents. Multivariate logistic regression analyses revealed that the expectation of government subsidies for telemedicine services was the strongest common driver and the only positive independent predictor of telemedicine use during a hypothetical severe outbreak (P=0.016) and after the COVID-19 pandemic (P=0.003). No negative independent predictors of telemedicine use during a hypothetical severe outbreak were identified. Negative independent predictors of telemedicine use after the COVID-19 pandemic included older age and residence in the New Territories (both P=0.001). CONCLUSION: Government support, such as telemedicine-specific subsidies, will be important for efforts to promote telemedicine use in Hong Kong during future severe outbreaks and after the COVID-19 pandemic. Robust dissemination of information regarding the advantages and disadvantages of telemedicine for the public, especially older adults, is needed.

Molecular drivers of resistance to sulbactam-durlobactam in contemporary clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe infections that are difficult to treat due to pre-existing antibiotic resistance. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat serious infections caused by Acinetobacter, including multidrug- and carbapenem-resistant strains. In a recent global surveillance study of 5,032 ABC clinical isolates collected from 2016 to 2021, less than 2% of ABC isolates had SUL-DUR MIC values >4 µg/mL. Molecular characterization of these isolates confirmed the primary drivers of resistance are metallo-ß-lactamases or penicillin-binding protein 3 (PBP3) mutations, as previously described. In addition, this study shows that certain common PBP3 variants, such as A515V, are insufficient to confer sulbactam resistance and that the efflux of durlobactam by AdeIJK is likely to play a role in a subset of strains.

A 20-Year Study of Capsular Polysaccharide Seroepidemiology, Susceptibility Profiles, and Virulence Determinants of Klebsiella pneumoniae from Bacteremia Patients in Taiwan.

In this study, we selected bacteremic Klebsiella pneumoniae isolates from the Taiwan Surveillance of Antimicrobial Resistance program. A total of 521 isolates were collected over a period of 2 decades, including 121 from 1998, 197 from 2008, and 203 from 2018. Seroepidemiology showed that the top five capsular polysaccharide types were serotypes K1, K2, K20, K54, and K62, constituting 48.5% of the total isolates, and the respective ratios at each time point have remained similar over the past 2 decades. The antibacterial susceptibility tests showed that K1, K2, K20, and K54 were susceptible to most antibiotics, while K62 was relatively resistant compared to other typeable and nontypeable strains. In addition, six virulence-associated genes, clbA, entB, iroN, rmpA, iutA, and iucA, were predominant in K1 and K2 isolates of K. pneumoniae. In conclusion, serotypes K1, K2, K20, K54, and K62 of K. pneumoniae are the most prevalent serotypes and carry more virulence determinants in bacteremia patients, which may indicate their invasiveness. If further serotype-specific vaccine development is performed, these five serotypes should be included. Since the antibiotic susceptibility profiles were stable over a long duration, empirical treatment may be predicted according to serotype if rapid diagnosis from direct clinical specimens is available, such as PCR or antigen serotyping for serotype K1 and K2. IMPORTANCE This is the first nationwide study to examine the seroepidemiology of Klebsiella pneumoniae using blood culture isolates collected over a period of 20 years. The study found that the prevalence of serotypes remained consistent over the 20-year period, with high-prevalence serotypes associated with invasive types. Nontypeable isolates had fewer virulence determinants than other serotypes. With the exception of serotype K62, the other high-prevalence serotypes were highly susceptible to antibiotics. If rapid diagnosis using direct clinical specimens, such as PCR or antigen serotyping, is available, empirical treatment can be predicted based on serotype, particularly for K1 and K2. The results of this seroepidemiology study could also help the development of future capsule polysaccharide vaccines.

First-in-class Microbial Ecosystem Therapeutic 4 (MET4) in combination with immune checkpoint inhibitors in patients with advanced solid tumors (MET4-IO trial).

BACKGROUND: The intestinal microbiome has been associated with response to immune checkpoint inhibitors (ICIs) in humans and causally implicated in ICI responsiveness in animal models. Two recent human trials demonstrated that fecal microbiota transplant (FMT) from ICI responders can rescue ICI responses in refractory melanoma, but FMT has specific limitations to scaled use. PATIENTS AND METHODS: We conducted an early-phase clinical trial of a cultivated, orally delivered 30-species microbial consortium (Microbial Ecosystem Therapeutic 4, MET4) designed for co-administration with ICIs as an alternative to FMT and assessed safety, tolerability and ecological responses in patients with advanced solid tumors. RESULTS: The trial achieved its primary safety and tolerability outcomes. There were no statistically significant differences in the primary ecological outcomes; however, differences in MET4 species relative abundance were evident after randomization that varied by patient and species. Increases in the relative abundance of several MET4 taxa, including Enterococcus and Bifidobacterium, taxa previously associated with ICI responsiveness, were observed and MET4 engraftment was associated with decreases in plasma and stool primary bile acids. CONCLUSIONS: This trial is the first report of the use of a microbial consortium as an alternative to FMT in advanced cancer patients receiving ICI and the results justify the further development of microbial consortia as a therapeutic co-intervention for ICI treatment in cancer.

Appropriate antibiotic therapy is a predictor of outcome in patients with Stenotrophomonas maltophilia blood stream infection in the intensive care unit.

BACKGROUND/PURPOSE: The study was to assess the relationship between antibiotic therapy and the outcome in intensive care unit (ICU) patients with Stenotrophomonas maltophilia bloodstream infection (BSI). METHODS: ICU patients with monomicrobial S. maltophilia BSI from January 2004 to December 2019 were included and divided into two groups-those with- and without appropriate antibiotic therapy after BSI-for comparison. The primary outcome was the relationship between appropriate antibiotic therapy and 14-day mortality. The secondary outcome was the influence of different antibiotic therapies: levofloxacin- and trimethoprim-sulfamethoxazole (TMP/SMX)-containing regimens, on 14-day mortality. RESULTS: A total of 214 ICU patients were included. Patients received appropriate antibiotic therapy (n = 133) after BSI had a lower 14-day mortality than those (n = 81) without appropriate antibiotic therapy (10.5% vs. 46.9%, p < 0.001). No difference on 14-day mortality between groups of patients by time of appropriate antibiotic therapy was observed (p > 0.05). After a propensity score matching, the results is consistent that 14-day mortality were lower in patients with appropriate antibiotic therapy than those without appropriate antibiotic therapy (11.5% vs. 39.3%, p < 0.001). Among patients with S. maltophilia BSI receiving appropriate antibiotic therapy, there was a trend levofloxacin-containing regimens is associated with lower mortality than TMP/SMX-containing regimens (HR 0.233, 95% CI 0.050-1.084, p = 0.063). CONCLUSION: Appropriate antibiotic therapy was associated with decreased 14-day mortality in ICU patients with S. maltophilia BSI regardless of time. Levofloxacin-containing regimens may be better choice than TMP/SMX -containing regimens in treating ICU patients with S. maltophilia BSI.

Breast Cancer Screening, Diagnosis, and Surgery during the Pre- and Peri-pandemic: Experience of Patients in a Statewide Health Information Exchange.

BACKGROUND: Measures taken to address the COVID-19 pandemic interrupted routine diagnosis and care for breast cancer. The aim of this study was to characterize the effects of the pandemic on breast cancer care in a statewide cohort. PATIENTS AND METHODS: Using data from a large health information exchange, we retrospectively analyzed the timing of breast cancer screening, and identified a cohort of newly diagnosed patients with any stage of breast cancer to further access the information available about their surgical treatments. We compared data for four subgroups: pre-lockdown (preLD) 25 March to 16 June 2019; lockdown (LD) 23 March to 3 May 2020; reopening (RO) 4 May to 14 June 2020; and post-lockdown (postLD) 22 March to 13 June 2021. RESULTS: During LD and RO, screening mammograms in the cohort decreased by 96.3% and 36.2%, respectively. The overall breast cancer diagnosis and surgery volumes decreased up to 38.7%, and the median time to surgery was prolonged from 1.5 months to 2.4 for LD and 1.8 months for RO. Interestingly, higher mean DCIS diagnosis (5.0 per week vs. 3.1 per week, p < 0.05) and surgery volume (14.8 vs. 10.5, p < 0.05) were found for postLD compared with preLD, while median time to surgery was shorter (1.2 months vs. 1.5 months, p < 0.0001). However, the postLD average weekly screening and diagnostic mammogram did not fully recover to preLD levels (2055.3 vs. 2326.2, p < 0.05; 574.2 vs. 624.1, p < 0.05). CONCLUSIONS: Breast cancer diagnosis and treatment patterns were interrupted during the lockdown and still altered 1 year after. Screening in primary care should be expanded to mitigate possible longer-term effects of these interruptions.

Placental mesenchymal stem cells boost M2 alveolar over M1 bone marrow macrophages via IL-1ß in Klebsiella-mediated acute respiratory distress syndrome.

RATIONALE: Acute respiratory distress syndrome (ARDS) is a lethal complication of severe bacterial pneumonia due to the inability to dampen overexuberant immune responses without compromising pathogen clearance. Both of these processes involve tissue-resident and bone marrow (BM)-recruited macrophage (MΦ) populations which can be polarised to have divergent functions. Surprisingly, despite the known immunomodulatory properties of mesenchymal stem cells (MSCs), simultaneous interactions with tissue-resident and recruited BMMΦ populations are largely unexplored. OBJECTIVES: We assessed the therapeutic use of human placental MSCs (PMSCs) in severe bacterial pneumonia with elucidation of the roles of resident alveolar MΦs (AMΦs) and BMMΦs. METHODS: We developed a lethal, murine pneumonia model using intratracheal infection of a clinically relevant Klebsiella pneumoniae (KP) strain with subsequent intravenous human PMSC treatment. Pulmonary AMΦ and recruited BMMΦ analyses, histological evaluation, bacterial clearance and mice survival were assessed. To elucidate the role of resident AMΦs in improving outcome, we performed AMΦ depletion in the KP-pneumonia model with intratracheal clodronate pretreatment. MEASUREMENTS AND MAIN RESULTS: Human PMSC treatment decreased tissue injury and improved survival of severe KP-pneumonia mice by decreasing the presence and function of recruited M1 BMMΦ while preserving M2 AMΦs and enhancing their antibacterial functions. Interestingly, PMSC therapy failed to rescue AMΦ-depleted mice with KP pneumonia, and PMSC-secreted IL-1ß was identified as critical in increasing AMΦ antibacterial activities to significantly improve pathogen clearance-especially bacteraemia-and survival. CONCLUSIONS: Human PMSC treatment preferentially rescued resident M2 AMΦs over recruited M1 BMMΦs with overall M2 polarisation to improve KP-related ARDS survival.

Pembrolizumab monotherapy versus chemotherapy in platinum-pretreated, recurrent or metastatic nasopharyngeal cancer (KEYNOTE-122): an open-label, randomized, phase III trial.

BACKGROUND: Pembrolizumab previously demonstrated robust antitumor activity and manageable safety in a phase Ib study of patients with heavily pretreated, programmed death ligand 1 (PD-L1)-positive, recurrent or metastatic nasopharyngeal carcinoma (NPC). The phase III KEYNOTE-122 study was conducted to further evaluate pembrolizumab versus chemotherapy in patients with platinum-pretreated, recurrent and/or metastatic NPC. Final analysis results are presented. PATIENTS AND METHODS: KEYNOTE-122 was an open-label, randomized study conducted at 29 sites, globally. Participants with platinum-pretreated recurrent and/or metastatic NPC were randomly assigned (1 : 1) to pembrolizumab or chemotherapy with capecitabine, gemcitabine, or docetaxel. Randomization was stratified by liver metastasis (present versus absent). The primary endpoint was overall survival (OS), analyzed in the intention-to-treat population using the stratified log-rank test (superiority threshold, one-sided P = 0.0187). Safety was assessed in the as-treated population. RESULTS: Between 5 May 2016 and 28 May 2018, 233 participants were randomly assigned to treatment (pembrolizumab, n = 117; chemotherapy, n = 116); Most participants (86.7%) received study treatment in the second-line or later setting. Median time from randomization to data cut-off (30 November 2020) was 45.1 months (interquartile range, 39.0-48.8 months). Median OS was 17.2 months [95% confidence interval (CI) 11.7-22.9 months] with pembrolizumab and 15.3 months (95% CI 10.9-18.1 months) with chemotherapy [hazard ratio, 0.90 (95% CI 0.67-1.19; P = 0.2262)]. Grade 3-5 treatment-related adverse events occurred in 12 of 116 participants (10.3%) with pembrolizumab and 49 of 112 participants (43.8%) with chemotherapy. Three treatment-related deaths occurred: 1 participant (0.9%) with pembrolizumab (pneumonitis) and 2 (1.8%) with chemotherapy (pneumonia, intracranial hemorrhage). CONCLUSION: Pembrolizumab did not significantly improve OS compared with chemotherapy in participants with platinum-pretreated recurrent and/or metastatic NPC but did have manageable safety and a lower incidence of treatment-related adverse events.

Oncology phase I trial design and conduct: time for a change - MDICT Guidelines 2022.

In 2021, the Food and Drug Administration Oncology Center of Excellence announced Project Optimus focusing on dose optimization for oncology drugs. The Methodology for the Development of Innovative Cancer Therapies (MDICT) Taskforce met to review and discuss the optimization of dosage for oncology trials and to develop a practical guide for oncology phase I trials. Defining a single recommended phase II dose based on toxicity may define doses that are neither the most effective nor the best tolerated. MDICT recommendations address the need for robust non-clinical data which are needed to inform trial design, as well as an expert team including statisticians and pharmacologists. The protocol must be flexible and adaptive, with clear definition of all endpoints. Health authorities should be consulted early and regularly. Strategies such as randomization, intrapatient dose escalation, and real-world eligibility criteria are encouraged whereas serial tumor sampling is discouraged in the absence of a strong rationale and appropriately validated assay. Endpoints should include consideration of all longitudinal toxicity. The phase I dose escalation trial should define the recommended dose range for later testing in randomized phase II trials, rather than a single recommended phase II dose, and consider scenarios where different populations may require different dosages. The adoption of these recommendations will improve dosage selection in early clinical trials of new anticancer treatments and ultimately, outcomes for patients.

Phase I, first-in-human study of MSC-1 (AZD0171), a humanized anti-leukemia inhibitory factor monoclonal antibody, for advanced solid tumors.

BACKGROUND: Activation of leukemia inhibitory factor (LIF) is linked to an immunosuppressive tumor microenvironment (TME), with a strong association between LIF expression and tumor-associated macrophages (TAMs). MSC-1 (AZD0171) is a humanized monoclonal antibody that binds with high affinity to LIF, promoting antitumor inflammation through TAM modulation and cancer stem cell inhibition, slowing tumor growth. In this phase I, first-in-human, open-label, dose-escalation study, MSC-1 monotherapy was assessed in patients with advanced, unresectable solid tumors. MATERIALS AND METHODS: Using accelerated-titration dose escalation followed by a 3 + 3 design, MSC-1 doses of 75-1500 mg were administered intravenously every 3 weeks (Q3W) until progression or unmanageable toxicity. Additional patients were enrolled in selected cohorts to further evaluate safety, pharmacokinetics (PK), and pharmacodynamics after escalation to the next dose had been approved. The primary objective was characterizing safety and determining the recommended phase II dose (RP2D). Evaluating antitumor activity and progression-free survival (PFS) by RECIST v1.1, PK and immunogenicity were secondary objectives. Exploratory objectives included pharmacodynamic effects on circulating LIF and TME immune markers. RESULTS: Forty-one patients received treatment. MSC-1 monotherapy was safe and well tolerated at all doses, with no dose-limiting toxicities. The maximum tolerated dose was not reached and the RP2D was determined to be 1500 mg Q3W. Almost half of the patients had treatment-related adverse events (TRAEs), with no apparent trends across doses; no patients withdrew due to TRAEs. There were no objective responses; 23.7% had stable disease for ≥2 consecutive tumor assessments. Median PFS was 5.9 weeks; 23.7% had PFS >16 weeks. On-treatment changes in circulating LIF and TME signal transducers and activators of transcription 3 signaling, M1:M2 macrophage populations, and CD8+ T-cell infiltration were consistent with the hypothesized mechanism of action. CONCLUSIONS: MSC-1 was very well tolerated across doses, with prolonged PFS in some patients. Biomarker and preclinical data suggest potential synergy with checkpoint inhibitors.

Cross-protection induced by highly conserved outer membrane proteins (Omps) in mice immunized with OmpC of Salmonella Typhi or OmpK36 of Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Outer membrane proteins (Omps) are a family of proteins that are highly conserved throughout the evolution of Enterobacteriaceae. Previous studies using sequence comparisons have found a high degree of sequence homology between OmpK36 of Klebsiella pneumoniae and OmpC of Salmonella enterica serovar Typhi. Whether highly conserved OmpC can be directly extrapolated as a common vaccine candidate against K. pneumoniae or other Enterobacteriaceae remains to be verified. METHODS: OmpK36 and OmpC were purified and used to immunize BALB/c mice. After immunization, five mice from each group were injected intraperitoneally with a cell suspension of K. pneumoniae or S. Typhi, and the mice were monitored daily for 14 days to measure the severity of illness and assess their survival. RESULTS: Cross-reacting OmpK36 and OmpC antibodies were identified in the mice immunized with OmpK36 or OmpC. No cross-protection was observed in the mice immunized with OmpC in the presence of K. pneumoniae infection. CONCLUSION: Although a high degree of similarity was observed for the amino acid sequences between OmpK36 and OmpC, our results suggested that no cross-protection occurred in the mice challenged with other species.

A Resistance Mechanism in Non-mcr Colistin-Resistant Escherichia coli in Taiwan: R81H Substitution in PmrA Is an Independent Factor Contributing to Colistin Resistance.

Colistin resistance due to the mcr-type genes in Escherichia coli is well characterized. In order to study the resistance mechanism in mcr-negative colistin-resistant E. coli, strains were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. A total of 11 mcr-negative colistin-resistant isolates among 7,942 (0.1%) clinical E. coli isolates were identified between 2008 and 2018. Their prevalence was low and remained stable during the study period. Since 2012, ST131 and ST1193 clones with multiple drug-resistant phenotypes have emerged. All resistant strains displayed higher expression levels of the operons pmrHFIJKLM and pmrCAB than the control MG1655 strain. Although several amino acid substitutions were identified in PmrA or PmrB, only R81H in PmrA was associated with overexpression of pmrHFIJKLM and colistin resistance. The effect of substitution R81H in PmrA in colistin resistance was confirmed by complementation experiments. Although some strains harbored substitutions in PmrB, the identified mutations in pmrB did not contribute to colistin resistance. In conclusion, the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. IMPORTANCE The molecular epidemiology and resistance mechanisms of mcr-negative colistin-resistant E. coli are not well described. In this study, a total of 11 mcr-negative colistin-resistant E. coli isolates were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. We determined the resistance mechanism of non-mcr colistin-resistant strains using gene knockout and complementation experiments. We observed the occurrence of the global multiple-drug-resistant E. coli clones ST131 and ST1193 starting in 2012. Moreover, for the first time, we proved that the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. The study results helped to gain an insight into the diversity and complexity of chromosome-encoded colistin resistance in E. coli.

Virulence among different types of hypervirulent Klebsiella pneumoniae with multi-locus sequence type (MLST)-11, Serotype K1 or K2 strains.

BACKGROUND: Two different types of hypervirulent K. pneumoniae (HvKp), the MLST-11 and serotype K1/K2 strains, have been frequently described in recent studies. Although these two types of strains were described to be HvKp, their virulence was not compared. In this study, in vitro and in vivo approaches were used to assess differences in virulence. MATERIALS AND METHODS: A total of twenty-nine isolates, including 6 strains of each of serotype K1 and K2 isolates and 17 strains of ST11 isolates, were selected for this study. Phenotypic tests of virulence were performed by the string test and analysis of the virulent associated genes was detected by PCR. In vitro models of serum resistance and phagocytosis were used as the parameters to assess the virulence. In-frame deletion of virulence-associated genes was performed to study their contributions to virulence. The median lethal dose, i.e., the LD50, in mice was determined following IP injection. RESULTS: Although serotype K1 and K2 strains and ST11 isolates had similar virulence gene profiles, the ST11 isolates showed less serum and phagocytic resistance than the serotype K1/K2 isolates. The mouse lethality test revealed that all ST11 isolates were unable to cause lethality, even at > 107 CFU, while serotypes K1 and K2 showed an LD50 at ≤ 103 CFU. Aerobactin or capsule knockout mutants exhibited a lower LD50 than the parental strain, while capsule mutants showed a more significant decrease in LD50. CONCLUSION: Since there was a significant difference in virulence levels between the two types of HvKp when assessed in in vitro and in vivo models, it may be better to use the designation "HvKp" for some strains based on animal studies to avoid confusion. Virulence and non-virulence could be analysed in a relative manner, especially in comparison studies.