SARS-CoV-2 nucleocapsid protein, rather than spike protein, triggers a cytokine storm originating from lung epithelial cells in patients with COVID-19.

PURPOSE: The aim of this study was to elucidate the factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may initiate cytokine cascades and correlate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with their serum cytokine profiles. METHODS: Recombinant baculoviruses displaying SARS-CoV-2 spike or nucleocapsid protein were constructed and transfected into A549 cells and THP-1-derived macrophages, to determine which protein initiate cytokine release. SARS-CoV-2-specific antibody titers and cytokine profiles of patients with COVID-19 were determined, and the results were associated with their clinical characteristics, such as development of pneumonia or length of hospital stay. RESULTS: The SARS-CoV-2 nucleocapsid protein, rather than the spike protein, triggers lung epithelial A549 cells to express IP-10, RANTES, IL-16, MIP-1α, basic FGF, eotaxin, IL-15, PDGF-BB, TRAIL, VEGF-A, and IL-5. Additionally, serum CTACK, basic FGF, GRO-α, IL-1α, IL-1RA, IL-2Rα, IL-9, IL-15, IL-16, IL-18, IP-10, M-CSF, MIF, MIG, RANTES, SCGF-ß, SDF-1α, TNF-α, TNF-ß, VEGF, PDGF-BB, TRAIL, ß-NGF, eotaxin, GM-CSF, IFN-α2, INF-γ, and MCP-1 levels were considerably increased in patients with COVID-19. Among them, patients with pneumonia had higher serum IP-10 and M-CSF levels than patients without. Patients requiring less than 3 weeks to show negative COVID-19 tests after contracting COVID-19 had higher serum IP-10 levels than the remaining patients. CONCLUSION: Our study revealed that nucleocapsid protein, lung epithelial cells, and IP-10 may be potential targets for the development of new strategies to prevent, or control, severe COVID-19.

Development of an EBOV MiniG plus system as an advanced tool for anti-Ebola virus drug screening.

The incidence of zoonotic diseases, such as coronavirus disease 2019 and Ebola virus disease, is increasing worldwide. However, drug and vaccine development for zoonotic diseases has been hampered because the experiments involving live viruses are limited to high-containment laboratories. The Ebola virus minigenome system enables researchers to study the Ebola virus under BSL-2 conditions. Here, we found that the addition of the nucleocapsid protein of human coronaviruses, such as severe acute respiratory syndrome coronavirus 2, can increase the ratio of green fluorescent protein-positive cells by 1.5-2 folds in the Ebola virus minigenome system. Further analysis showed that the nucleocapsid protein acts as an activator of the Ebola virus minigenome system. Here, we developed an EBOV MiniG Plus system based on the Ebola virus minigenome system by adding the SARS-CoV-2 nucleocapsid protein. By evaluating the antiviral effect of remdesivir and rupintrivir, we demonstrated that compared to that of the traditional Ebola virus minigenome system, significant concentration-dependent activity was observed in the EBOV MiniG Plus system. Taken together, these results demonstrate the utility of adding nucleocapsid protein to the Ebola virus minigenome system to create a powerful platform for screening antiviral drugs against the Ebola virus.

Modifications of lipid pathways restrict SARS-CoV-2 propagation in human induced pluripotent stem cell-derived 3D airway organoids.

BACKGROUND: Modifications of lipid metabolism were closely associated with the manifestations and prognosis of coronavirus disease of 2019 (COVID-19). Pre-existing metabolic conditions exacerbated the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection while modulations of aberrant lipid metabolisms alleviated the manifestations. To elucidate the underlying mechanisms, an experimental platform that reproduces human respiratory physiology is required. METHODS: Here we generated induced pluripotent stem cell-derived airway organoids (iPSC-AOs) that resemble the human native airway. Single-cell sequencing (ScRNAseq) and microscopic examination verified the cellular heterogeneity and microstructures of iPSC-AOs, respectively. We subjected iPSC-AOs to SARS-CoV-2 infection and investigated the treatment effect of lipid modifiers statin drugs on viral pathogenesis, gene expression, and the intracellular trafficking of the SARS-CoV-2 entry receptor angiotensin-converting enzyme-2 (ACE-2). RESULTS: In SARS-CoV-2-infected iPSC-AOs, immunofluorescence staining detected the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins and bioinformatics analysis further showed the aberrant enrichment of lipid-associated pathways. In addition, SARS-CoV-2 hijacked the host RNA replication machinery and generated the new isoforms of a high-density lipoprotein constituent apolipoprotein A1 (APOA1) and the virus-scavenging protein deleted in malignant brain tumors 1 (DMBT1). Manipulating lipid homeostasis using cholesterol-lowering drugs (e.g. Statins) relocated the viral entry receptor angiotensin-converting enzyme-2 (ACE-2) and decreased N protein expression, leading to the reduction of SARS-CoV-2 entry and replication. The same lipid modifications suppressed the entry of luciferase-expressing SARS-CoV-2 pseudoviruses containing the S proteins derived from different SARS-CoV-2 variants, i.e. wild-type, alpha, delta, and omicron. CONCLUSIONS: Together, our data demonstrated that modifications of lipid pathways restrict SARS-CoV-2 propagation in the iPSC-AOs, which the inhibition is speculated through the translocation of ACE2 from the cell membrane to the cytosol. Considering the highly frequent mutation and generation of SARS-CoV-2 variants, targeting host metabolisms of cholesterol or other lipids may represent an alternative approach against SARS-CoV-2 infection.

Biofilm formation is not an independent risk factor for mortality in patients with Acinetobacter baumannii bacteremia.

In the past decades, due to the high prevalence of the antibiotic-resistant isolates of Acinetobacter baumannii, it has emerged as one of the most troublesome pathogens threatening the global healthcare system. Furthermore, this pathogen has the ability to form biofilms, which is another effective mechanism by which it survives in the presence of antibiotics. However, the clinical impact of biofilm-forming A. baumannii isolates on patients with bacteremia is largely unknown. This retrospective study was conducted at five medical centers in Taiwan over a 9-year period. A total of 252 and 459 patients with bacteremia caused by biofilm- and non-biofilm-forming isolates of A. baumannii, respectively, were enrolled. The clinical demographics, antimicrobial susceptibility, biofilm-forming ability, and patient clinical outcomes were analyzed. The biofilm-forming ability of the isolates was assessed using a microtiter plate assay. Multivariate analysis revealed the higher APACHE II score, shock status, lack of appropriate antimicrobial therapy, and carbapenem resistance of the infected strain were independent risk factors of 28-day mortality in the patients with A. baumannii bacteremia. However, there was no significant difference between the 28-day survival and non-survival groups, in terms of the biofilm forming ability. Compared to the patients infected with non-biofilm-forming isolates, those infected with biofilm-forming isolates had a lower in-hospital mortality rate. Patients with either congestive heart failure, underlying hematological malignancy, or chemotherapy recipients were more likely to become infected with the biofilm-forming isolates. Multivariate analysis showed congestive heart failure was an independent risk factor of infection with biofilm-forming isolates, while those with arterial lines tended to be infected with non-biofilm-forming isolates. There were no significant differences in the sources of infection between the biofilm-forming and non-biofilm-forming isolate groups. Carbapenem susceptibility was also similar between these groups. In conclusion, the patients infected with the biofilm-forming isolates of the A. baumannii exhibited different clinical features than those infected with non-biofilm-forming isolates. The biofilm-forming ability of A. baumannii may also influence the antibiotic susceptibility of its isolates. However, it was not an independent risk factor for a 28-day mortality in the patients with bacteremia.

Interleukin-6 Test Strip Combined With a Spectrum-Based Optical Reader for Early Recognition of COVID-19 Patients With Risk of Respiratory Failure.

The COVID-19 pandemic has had a globally devastating impact. This highly contagious virus has significantly overburdened and undermined medical systems. While most infected patients experience only mild symptoms, those who are severely affect require urgent medical interventions and some develop acute respiratory failure and require mechanical ventilation. The broad and potentially deadly impact of infection underscores the critical need for early recognition, especially for those at risk for respiratory failure. Those who are severely impacted and at high risk for respiratory failure have been found to present high levels of serum cytokines, such as interleukin-6 (IL-6). Timely diagnosis and management of those at risk for respiratory failure is crucial. Measurement of IL-6 may provide a means for distinguishing such patients. Currently, most serum IL-6 detection relies on the use of laboratory-based conventional enzyme-linked immunosorbent assays. Although some rapid assays have been developed recently, they need to be conducted by specific technicians in central laboratory settings with advanced and expensive equipment. In this study, we propose an IL-6 test strip combined with a spectrum-based optical reader for early recognition of COVID-19-infected patients at imminent risk of acute respiratory failure requiring mechanical ventilator support. For our analyses, clinical demographic data and sera samples were obtained from three medical centers, and test strip specificity and detection performance were analyzed. This would help healthcare personnel stratify the risk of respiratory failure and provide prompt, and suitable management.

A Lateral Flow Immunoassay Coupled with a Spectrum-Based Reader for SARS-CoV-2 Neutralizing Antibody Detection.

As of August 2021, there have been over 200 million confirmed case of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus and more than 4 million COVID-19-related deaths globally. Although real-time polymerase chain reaction is considered to be the primary method of detection for SARS-CoV-2 infection, the use of serological assays for detecting COVID-19 antibodies has been shown to be effective in aiding with diagnosis, particularly in patients who have recovered from the disease and those in later stages of infection. Since it has a high detection rate and few limitations compared to conventional enzyme-linked immunosorbent assay protocols, we used a lateral flow immunoassay as our diagnostic tool of choice. Since lateral flow immunoassay results interpreted by the naked eye may lead to erroneous diagnoses, we developed an innovative, portable device with the capacity to capture a high-resolution reflectance spectrum as a means of promoting diagnostic accuracy. We combined this spectrum-based device with commercial lateral flow immunoassays to detect the neutralizing antibody in serum samples collected from 30 COVID-19-infected patients (26 mild cases and four severe cases). The results of our approach, lateral flow immunoassays coupled with a spectrum-based reader, demonstrated a 0.989 area under the ROC curve, 100% sensitivity, 95.7% positive predictive value, 87.5% specificity, and 100% negative predictive value. As a result, our approach exhibited great value for neutralizing antibody detection. In addition to the above tests, we also tested plasma samples from 16 AstraZeneca-vaccinated (ChAdOx1nCoV-19) patients and compared our approach and enzyme-linked immunosorbent assay results to see whether our approach could be applied to vaccinated patients. The results showed a high correlation between these two approaches, indicating that the lateral flow immunoassay coupled with a spectrum-based reader is a feasible approach for diagnosing the presence of a neutralizing antibody in both COVID-19-infected and vaccinated patients.

Dynamic Changes of the Blood Chemistry in Syrian Hamsters Post-Acute COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that causes coronavirus disease 2019 (COVID-19). However, the long-term health consequences of COVID-19 are not fully understood. We aimed to determine the long-term lung pathology and blood chemistry changes in Syrian hamsters infected with SARS-CoV-2. Syrian hamsters (Mesocricetus auratus) were inoculated with 105 PFU of SARS-CoV-2, and changes post-infection (pi) were observed for 20 days. On days 5 and 20 pi, the lungs were harvested and processed for pathology and viral load count. Multiple blood samples were collected every 3 to 5 days to observe dynamic changes in blood chemistry. Infected hamsters showed consistent weight loss until day 7 pi At day 5 pi, histopathology of the lungs showed moderate to severe inflammation and the virus could be detected. These results indicate that SARS-CoV-2 has an acute onset and recovery course in the hamster infection model. During the acute onset, blood triglyceride levels increased significantly at day 3 pi During the recovery course, uric acid and low-density lipoprotein levels increased significantly, but the total protein and albumin levels decreased. Together, our study suggests that SARS-CoV-2 infection in hamsters not only causes lung damage but also causes long-term changes in blood biochemistry during the recovery process. IMPORTANCE COVID-19 is now considered a multiorgan disease with a wide range of manifestations. There are increasing reports of persistent and long-term effects after acute COVID-19, but the long-term health consequences of COVID-19 are not fully understood. This study reported for the first time the use of blood samples collected continuously in a SARS-CoV-2-infected hamster model, which provides more information about the dynamic changes in blood biochemistry during the acute and recovery phases of SARS-CoV-2 infection. Our study suggests that SARS-CoV-2 infection in hamsters not only causes lung damage but also causes long-term changes in blood biochemistry during the recovery process. The study may be used by several researchers and clinicians, especially those who are studying potential treatments for patients with post-acute COVID-19 syndrome.

Tumor Necrosis Factor-Alpha Exacerbates Viral Entry in SARS-CoV2-Infected iPSC-Derived Cardiomyocytes.

The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.

Ser253Leu substitution in PmrB contributes to colistin resistance in clinical Acinetobacter nosocomialis.

Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis.

Rapid typing of carbapenem-resistant Acinetobacter baumannii and Acinetobacter nosocomialis by multiplex Pan- and OXA-PCR assays.

Introduction. Outbreaks of carbapenem-resistant A. baumannii and A. nosocomialis have occurred worldwide in healthcare settings. Rapid and reliable molecular typing of bacterial isolates is vital for the effective surveillance of institutional outbreaks. The Pan-PCR and OXA-PCR assays are two multiplex PCR-based assays for the molecular typing of Acinetobacter species.Gap statement. However, few studies have investigated the discriminatory power of two multiplex PCR assays in in the genotyping of Acinetobacter species.Aim. We aimed to evaluate the efficacies of the Pan-PCR and OXA-PCR assays for molecular typing of A. baumannii and A. nosocomialis.Methodology. A total of 105 carbapenem-resistant A. baumannii isolates (CRABs) and 93 carbapenem-resistant A. nosocomialis isolates (CRANs) obtained from blood cultures were used for molecular typing by the Pan-PCR and OXA-PCR assays and two multilocus sequence typing (MLST) schemes.Results. The isolates were individually divided into 12 and 21 different sequence types via the Pasteur and Oxford MLST schemes, respectively. Additionally, these isolates were distinguished into 18 different types by the Pan-PCR and OXA-PCR assays. The results of the Pan-PCR and OXA-PCR assays distinguished CRABs and CRANs with a sensitivity of 98.13 % and a specificity of 100 %.Conclusion. The Pan-PCR and OXA-PCR assays are promising alternative methods for rapid molecular typing of CRABs and CRANs in a routine laboratory setting.

Highlight of severe acute respiratory syndrome coronavirus-2 vaccine development against COVID-19 pandemic.

The pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has brought an unprecedented impact upon the global economy and public health. Although the SARS-CoV-2 virology has been gradually investigated, measures to combat this new threat in public health are still absent. To date, no certificated drug or vaccine has been developed for the treatment or prevention of coronavirus disease Extensive researches and international coordination has been conducted to rapidly develop novel vaccines against SARS-CoV-2 pandemic. Several major breakthroughs have been made through the identification of the genetic sequence and structural/non-structural proteins of SARS-CoV-2, which enabled the development of RNA-, DNA-based vaccines, subunit vaccines, and attenuated viral vaccines. In this review article, we present an overview of the recent advances of SARS-CoV-2 vaccines and the challenges that may be encountered in the development process, highlighting the advantages and disadvantages of these approaches that may help in effectively countering COVID-19.

Clinical and molecular characterization of Acinetobacter seifertii in Taiwan.

OBJECTIVES: Acinetobacter seifertii, a new member of the Acinetobacter baumannii group, has emerged as a cause of severe infections in humans. We investigated the clinical and molecular characteristics of A. seifertii. PATIENTS AND METHODS: This retrospective study enrolled 80 adults with A. seifertii bloodstream infection (BSI) at four medical centres over an 8 year period. Species identification was confirmed by MALDI-TOF MS, rpoB sequencing and WGS. Molecular typing was performed by MLST. Clinical information, antimicrobial susceptibility and the mechanisms of carbapenem and colistin resistance were analysed. Transmissibility of the carbapenem-resistance determinants was examined by conjugation experiments. RESULTS: The main source of A. seifertii BSI was the respiratory tract (46.3%). The 28 day and in-hospital mortality rates of A. seifertii BSI were 18.8% and 30.0%, respectively. High APACHE II scores and immunosuppressant therapy were independent risk factors for 28 day mortality. The most common MLST type was ST553 (58.8%). Most A. seifertii isolates were susceptible to levofloxacin (86.2%), and only 37.5% were susceptible to colistin. Carbapenem resistance was observed in 16.3% of isolates, mostly caused by the plasmid-borne ISAba1-blaOXA-51-like genetic structure. A. seifertii could transfer various carbapenem-resistance determinants to A. baumannii, Acinetobacter nosocomialis and other A. seifertii isolates. Variations of pmrCAB and lpxCAD genes were not associated with colistin resistance of A. seifertii. CONCLUSIONS: Levofloxacin and carbapenems, but not colistin, have the potential to be the drug of choice for A. seifertii infections. A. seifertii can transfer carbapenem-resistance determinants to other species of the A. baumannii group and warrants close monitoring.

AdeABC Efflux Pump Controlled by AdeRS Two Component System Conferring Resistance to Tigecycline, Omadacycline and Eravacycline in Clinical Carbapenem Resistant Acinetobacter nosocomialis.

Carbapenem-resistant Acinetobacter nosocomialis (CRAn) is a significant public health concern. Tigecycline non-susceptible CRAn (Tn-CRAn) isolates have emerged worldwide. Tigecycline resistance is mainly related to the overexpression of AdeABC efflux pump controlled by AdeRS two-component system (TCS). Two novel tetracycline derivatives, omadacycline and eravacycline, may present a treatment option for CRAn. This study investigated the in vitro antimicrobial activity of tigecycline, omadacycline and eravacycline against clinical CRAn isolates and the contribution of efflux pumps in their resistance. Eighty-nine clinical CRAn isolates, including 57 Tn-CRAn isolates were evaluated for minimum inhibitory concentrations (MICs) by the broth microdilution. The relationship between the antimicrobial resistance and efflux pump expression was assessed by their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). The contribution of the AdeABC efflux pump in their resistance was determined by the complementation of the AdeRS two-component system in wild-type, adeRS operon and adeB gene knockout strains. Among the 89 isolates, omadacycline and eravacycline MICs were correlated closely with those of tigecycline. They demonstrated improved potency, based on MIC90 values, by showing a 4 to 8-fold greater potency than tigecycline. The synergetic effects of tigecycline, omadacycline and eravacycline with NMP were observed in 57 (100%), 13 (22.8%), and 51 (89.5%) of Tn-CRAn isolates, respectively. Further analysis showed that the laboratory strain carrying the Type 1 adeRS operon increased the tigecycline, omadacycline and eravacycline MICs by 4-8-folds, respectively. Eravacycline demonstrated improved potency over tigecycline against populations of CRAn, including Tn-CRAn isolates. The over-expression of AdeABC efflux pumps was directly activated by the AdeRS two-component system and simultaneously reduced the susceptibilities of tigecycline, eravacycline, and omadacycline. Omadacycline and eravacycline MICs were correlated closely with those of eravacycline.

Current diagnostic tools for coronaviruses-From laboratory diagnosis to POC diagnosis for COVID-19.

The Coronavirus-2019 (COVID-19) pandemic has put tremendous strain on healthcare systems worldwide. It is challenging for clinicians to differentiate COVID-19 from other acute respiratory tract infections via clinical symptoms because those who are infected display a wide range of symptoms. An effective, point-of-care (POC) diagnostic tool could mitigate healthcare system strain, protect healthcare professionals, and support quarantine efforts. We believe that a POC tool can be developed that would be rapid, easy to use, and inexpensive. It could be used in the home, in resource-limited areas, and even in clinical settings. In this article, we summarize the current state of COVID-19 diagnostic methods and make a case for an all-in-one, highly sensitive POC assay that integrates antibody detection, protein detection, and serum cytokine detection to diagnose COVID-19 infection. We believe this article will provide insights into the current state of diagnostics for COVID-19, and promote additional research and tool development that could be exceptionally impactful.

Confronting Tigecycline-Resistant Acinetobacter baumannii via Immunization Against Conserved Resistance Determinants.

Antimicrobial-resistant (AMR) bacterial infections, including those caused by Acinetobacter baumannii, have emerged as a clinical crisis worldwide. Immunization with AMR determinants has been suggested as a novel approach to combat AMR bacteria, but has not been validated. The present study targeted tigecycline (TGC) resistance determinants in A. baumannii to test the feasibility of this approach. Using bioinformatic tools, four candidates, AdeA, AdeI, AdeK, and TolC, belonging to the resistance-nodulation-division (RND) efflux pump were identified as highly conserved and exposed antigens from 15 A. baumannii genomes. Antisera generated from recombinant proteins showed the capability to reserve Hoechst 33342, a substrate of the efflux pump, in bacterial cells. The rTolC antisera had the highest complement-dependent killing and opsonophagocytosis effect compared to the sera from phosphate-buffered saline immunized mice. Among the antisera, anti-rAdeK-specific antisera decreased the minimal inhibitory concentration of TGC in 26.7% of the tested isolates. Immunization with rAdeK significantly potentiated TGC efficacy in treating TGC-resistant A. baumannii pneumonia in the murine model. The bacterial load (7.5 × 105 vs. 3.8 × 107, p < 0.01) and neutrophil infiltration in the peri-bronchial vasculature region of immunized mice was significantly lower compared to the PBS-immunized mice when TGC was administrated concomitantly. Collectively, these results suggest that active immunization against resistance determinants might be a feasible approach to combat multidrug-resistant pathogens in high risk population.

Multicentre study of risk factors for mortality in patients with Acinetobacter bacteraemia receiving colistin treatment.

Colistin remains a last-line antibiotic for the treatment of infections by multidrug-resistant Acinetobacter species. However, mortality rates are high in patients with Acinetobacter infection receiving colistin treatment. This multicentre study evaluated whether colistin susceptibility, additional antimicrobial agents or other prognostic factors influenced the clinical outcomes of patients receiving colistin treatment for Acinetobacter bacteraemia. This retrospective study enrolled 122 adults receiving colistin for monomicrobial Acinetobacter bacteraemia at six medical centres in the ACTION Study Group over an 8-year period. Clinical information, antimicrobial susceptibility and colistin resistance determinants were analysed. The primary outcome measure was 14-day mortality. Among 122 patients, 18 and 104 were infected with colistin-resistant (ColR) isolates [minimum inhibitory concentration (MIC) ≥4 mg/L] and colistin-susceptible (ColS) isolates (MIC ≤2 mg/L), respectively. Patients infected with ColR and ColS isolates did not differ significantly with regard to Charlson comorbidity index, invasive procedures, sources of bacteraemia, disease severity and 14-day mortality rate (44.4% vs. 34.6%; P = 0.592). No specific additional antimicrobial agent was independently associated with higher or lower mortality. Coronary artery disease, higher Acute Physiology and Chronic Health Evaluation (APACHE) II score and bacteraemia caused Acinetobacter baumannii were independent risk factors associated with 14-day mortality. Mechanisms of colistin resistance were associated with amino acid variants in the pmrCAB operon. Finally, previously unreported Acinetobacter nosocomialis amino acid variants related to colistin resistance were identified. In conclusion, colistin susceptibility and colistin combination antimicrobial treatment were not associated with decreased 14-day mortality in patients with Acinetobacter bacteraemia receiving colistin treatment.

In vitro activities of imipenem, vancomycin, and rifampicin against clinical Elizabethkingia species producing BlaB and GOB metallo-beta-lactamases.

Elizabethkingia genus is emerging in hospitals and resistant to multiple antibiotics. The intrinsic imipenem resistance of Elizabethkingia genus is related to two chromosome-encoded metallo-beta-lactamases (MBLs), BlaB and GOB. This study was aimed to investigate the in vitro activity of imipenem, vancomycin, and rifampicin in clinical Elizabethkingia species. The distribution and heterogeneity of MBLs responsible for imipenem resistance were also evaluated. A total of 167 Elizabethkingia isolates from different patients were collected, including E. anophelis (142), E. meningoseptica (11), and E. miricola (14). All isolates were evaluated by the broth microdilution assay, ethylenediaminetetraacetic acid (EDTA) combination disk test, and EDTA-based microdilution test. The characteristics of BlaB and GOB were evaluated in phylogenetic analysis and heterologous expression experiments. Most of the isolates were susceptible to rifampin (94%), whereas none of the isolates were susceptible to imipenem. Vancomycin showed intermediate effectiveness. EDTA could reduce 4 folds or more minimum inhibitory concentrations (MICs) of imipenem in 105 isolates (62.9%). Of the isolates, the amino acid sequences of BlaB and GOB were divided into 22 and 25 different types, respectively. Phylogenetic analysis showed BlaB and GOB are species-specific proteins. Furthermore, GOB and BlaB from E. anophelis showed higher imipenem hydrolysis efficiency than those from the other two species. Rifampicin remained the most active agent in the current study. The mechanism of Elizabethkingia resistance to imipenem primarily stemmed from MBLs but other mechanisms could also exist, which requires further investigation.

Multicentre MDR Elizabethkingia anophelis isolates: Novel random amplified polymorphic DNA with capillary electrophoresis systems to rapid molecular typing compared to genomic epidemiology analysis.

Elizabethkingia species are ubiquitous bacteria that uncommonly cause human infection. Elizabethkingia anophelis was first identified in 2011 from the mosquito Anopheles gambiae. The currently available bacterial typing systems vary greatly with respect to labour, cost, reliability, and ability to discriminate among bacterial strains. Polymerase chain reaction (PCR)-based fingerprinting using random amplified polymorphic DNA (RAPD) is commonly used to identify genetic markers. To our knowledge, no system coupling RAPD-PCR and capillary gel electrophoresis (CGE) has been utilized for the epidemiological typing of E. anophelis. Thus, the aim of the present study was to establish a reliable and reproducible molecular typing technique for E. anophelis isolates based on a multi-centre assessment of bacteraemia patients. Here, we used a rapid CGE-light-emitting diode-induced fluorescence (LEDIF)-based method in conjunction with RAPD-PCR to genotype E. anophelis with a high level of discrimination. All clinical isolates of E. anophelis were found to be typeable, and isolates from two hospitals formed two distinct clusters. The results demonstrated the potential of coupling RAPD and CGE as a rapid and efficient molecular typing tool, providing a reliable method for surveillance and epidemiological investigations of bacterial infections. The proposed method shows promise as a novel, cost-effective, high-throughput, first-pass typing method.