Current and novel therapies for management of Acinetobacter baumannii-associated pneumonia.

Acinetobacter baumannii is a common pathogen associated with hospital-acquired pneumonia showing increased resistance to carbapenem and colistin antibiotics nowadays. Infections with A. baumannii cause high patient fatalities due to their capability to evade current antimicrobial therapies, emphasizing the urgency of developing viable therapeutics to treat A. baumannii-associated pneumonia. In this review, we explore current and novel therapeutic options for overcoming therapeutic failure when dealing with A. baumannii-associated pneumonia. Among them, antibiotic combination therapy administering several drugs simultaneously or alternately, is one promising approach for optimizing therapeutic success. However, it has been associated with inconsistent and inconclusive therapeutic outcomes across different studies. Therefore, it is critical to undertake additional clinical trials to ascertain the clinical effectiveness of different antibiotic combinations. We also discuss the prospective roles of novel antimicrobial therapies including antimicrobial peptides, bacteriophage-based therapy, repurposed drugs, naturally-occurring compounds, nanoparticle-based therapy, anti-virulence strategies, immunotherapy, photodynamic and sonodynamic therapy, for utilizing them as additional alternative therapy while tackling A. baumannii-associated pneumonia. Importantly, these innovative therapies further require pharmacokinetic and pharmacodynamic evaluation for safety, stability, immunogenicity, toxicity, and tolerability before they can be clinically approved as an alternative rescue therapy for A. baumannii-associated pulmonary infections.

Distinct skin microbiome modulation following different topical acne treatments in mild acne vulgaris patients: A randomized, investigator-blinded exploratory study.

The effects of topical non-antibiotic acne treatment on skin microbiota have rarely been demonstrated. In the study, we randomized 45 mild acne vulgaris participants into three treatment groups, including a cream-gel dermocosmetic containing Aqua Posae Filiformis, lipohydroxy acid, salicylic acid, linoleic acid, niacinamide and piroctone olamine (DC), retinoic acid 0.025% cream (VAA) and benzoyl peroxide 2.5% gel (BP). At months 0, 1 and 3, skin specimens were swabbed from the cheek and forehead and sequenced by targeting V3-V4 regions of the 16 S rRNA gene. QIIME2 was used to characterize bacterial communities. Acne severity, sebum level and tolerability were assessed concomitantly in each visit. We found that both VAA and BP could significantly reduce the bacterial diversity at month 1 (p-value = 0.010 and 0.004 respectively), while no significant reduction was observed in DC group. The microbiota compositions also significantly altered for beta diversity in all treatments (all p-value = 0.001). An increased Cutibacterium with decreased Staphylococcus relative abundance was observed at months 1 and 3 in DC group, while an opposite trend was demonstrated in VAA and BP groups. These findings suggest a potential impact of DC, VAA and BP on the diversity and composition profiles of the skin microbiota in mild acne participants.

An Extracorporeal Plasma Filtration Column with Specific Binding to Dengue Virions.

INTRODUCTION: Dengue infection is a significant public health concern that no specific treatment is available. Extracorporeal plasmapheresis or plasma filtration is a treatment option for severe cases with complications. However, the commercial adsorption devices mainly contained size-exclusive porous beads to adsorb the plasma proteins nonselectively. METHODS: We developed a 1:50 simulated circuit for dengue virus-specific adsorption using a flavivirus-specific (4G2) antibody entrapped into the alginate bead. RESULTS: The reduction ratios of the viral titer after 3 h of continuous run were 63.00 ± 1.21%, and 93.97 ± 1.27% measured by reverse transcription qPCR, and plaque titration, respectively. No specific adsorption was observed with Enterovirus A71 or Escherichia coli bacteria. CONCLUSION: This study is a proof-of-concept for the potential use of a dengue virus-specific adsorption column in the 1:50 simulated circuit. The system could be applied to various clinical platforms by substituting target-specific antibodies.

Bacterial contamination and organic residue after reprocessing in duodenoscopes with disposable distal caps compared with duodenoscopes with fixed distal caps: a randomized trial.

BACKGROUND AND AIMS: Newly designed duodenoscopes with disposable distal caps have been developed for better cleaning and preprocessing to reduce the risk of bacterial contamination (BC). We compared BC and organic residue of duodenoscopes with disposable distal caps and duodenoscopes with fixed distal caps after manual cleaning and high-level disinfection (HLD). METHODS: Four hundred duodenoscopes were randomized into group A (fixed distal caps, n = 200) and group B (disposable distal caps, n = 200). After manual cleaning, samples from the elevator were submitted for culture. An adenosine triphosphate (ATP) test was performed for organic residue evaluation. Based on our previous data, ATP < 40 relative light units (RLUs) had 100% sensitivity with 100% negative predictive value to confirm no BC after reprocessing. RESULTS: After manual cleaning, group A had a higher BC rate (14% vs 7%, P = .02), a higher proportion of duodenoscopes with ATP ≥ 40 RLUs (73.5% vs 57%, P = .001), and a higher mean of ATP level (226.6 vs 82.0 RLUs, P < .001) compared with group B. After HLD, the proportion of potential BC (ATP ≥ 40 RLUs) in group A was 2.7 times higher than group B (4% vs 1.5%, P = .13). Mean ATP level after HLD in the 2 groups was significantly lower than before the HLD procedure (group A, 24.2 vs 226.6 RLUs [P < .001]; group B, 20.4 vs 82.0 RLUs [P < .001], respectively). CONCLUSIONS: After manual cleaning, duodenoscopes with disposable distal caps had significantly lower BC and organic residue than duodenoscopes with fixed distal caps. Only a few duodenoscopes from each group did not pass the ATP threshold after HLD.

Effect of vaginal estrogen in postmenopausal women using vaginal pessary for pelvic organ prolapse treatment: a randomized controlled trial.

INTRODUCTION AND HYPOTHESIS: Evidence and recommendations for the use of intravaginal estrogen for prevention of bacterial vaginosis and pessary-related complications are limited and controversial. We hypothesized that adding intravaginal estrogen to pessary use would decrease the incidence of bacterial vaginosis and other pessary-related complications. METHODS: A single-center, open-label, randomized, parallel study was conducted between April 2018 and August 2020. Participants were randomized to either receive intravaginal estriol 0.03 mg plus Lactobacillus acidophilus 100 million viable cell vaginal tablets or have no treatment. The Amsel criteria, normal flora index, visual analog scale, Thai version of the ICIQ-VS (International Consultation on Incontinence Questionnaire-Vaginal symptoms) questionnaire, vaginal abrasions and vaginal bleeding were evaluated at entry and at 2- and 14-week follow-up. RESULTS: Seventy-eight women were included and randomized to two groups (39 women per group). At 2-week follow-up, one participant in the intervention group and two participants in the control group were diagnosed with bacterial vaginosis (2.7% vs. 5.7%, p = 0.609). At 14-week follow-up, two participants in the intervention group and two participants in the control group were diagnosed with bacterial vaginosis (5.7% vs. 6.2%, p = 0.926). Normal flora index was significantly different at 2-week follow-up [8 (6.3) vs. 5 (6.0), p = 0.032]. There was no significant difference in the visual analog scale, Thai version of the ICIQ-VS, vaginal abrasions and vaginal bleeding between the 2- and 14-week follow-ups. CONCLUSIONS: This study shows no benefit of intravaginal estrogen in reducing bacterial vaginosis, vaginal abrasions, vaginal bleeding and pain in postmenopausal women using a vaginal pessary for pelvic organ prolapse treatment.

Rapid Synergistic Biofilm Production of Pseudomonas and Candida on the Pulmonary Cell Surface and in Mice, a Possible Cause of Chronic Mixed Organismal Lung Lesions.

Due to the possible co-presence of Pseudomonas aeruginosa and Candida albicans (the most common nosocomial pathogens) in lungs, rapid interkingdom biofilm production is possible. As such, PA+CA produced more dominant biofilms on the pulmonary epithelial surface (NCI-H292) (confocal fluorescent extracellular matrix staining) with dominant psl upregulation, as demonstrated by polymerase chain reaction (PCR), after 8 h of experiments than PA alone. With a proteomic analysis, rhamnosyltransferase RhlB protein (Psl-associated quorum-sensing protein) was found to be among the high-abundance proteins in PA+CA than in PA biofilms, supporting psl-mediated biofilms in PA+CA on the cell surface. Additionally, PA+CA increased supernatant cytokines (IL-8 and IL-13, but not TNF-α, IL-6, and IL-10) with a similar upregulation of TLR-4, TLR-5, and TLR-9 (by PCR) compared with PA-stimulated cells. The intratracheal administration of PA+CA induced a greater severity of sepsis (serum creatinine, alanine transaminase, serum cytokines, and histology score) and prominent biofilms (fluorescent staining) with psl upregulation (PCR). In comparison with PA+CA biofilms on glass slides, PA+CA biofilms on biotic surfaces were more prominent (fluorescent staining). In conclusion, PA+CA induced Psl-predominant biofilms on the pulmonary cell surface and in mice with acute pneumonia, and these biofilms were more prominent than those induced by PA alone, highlighting the impact of Candida on rapid interkingdom biofilm production.

Chlorhexidine Promotes Psl Expression in Pseudomonas aeruginosa That Enhances Cell Aggregation with Preserved Pathogenicity Demonstrates an Adaptation against Antiseptic.

Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid-solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca2+ in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca2+ in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.

Nationwide Surveillance and Molecular Characterization of Critically Drug-Resistant Gram-Negative Bacteria: Results of the Research University Network Thailand Study.

A large-scale surveillance is an important measure to monitor the regional spread of antimicrobial resistance. We prospectively studied the prevalence and molecular characteristics of clinically important Gram-negative bacilli, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii complex (ABC), and Pseudomonas aeruginosa, from blood, respiratory tract, urine, and sterile sites at 47 hospitals across Thailand. Among 187,619 isolates, 93,810 isolates (50.0%) were critically drug resistant, of which 12,915 isolates (13.8%) were randomly selected for molecular characterization. E. coli was most commonly isolated from all specimens, except the respiratory tract, in which ABC was predominant. Prevalence of extended-spectrum cephalosporin resistance (ESCR) was higher in E. coli (42.5%) than K. pneumoniae (32.0%), but carbapenem-resistant (CR)-K. pneumoniae (17.2%) was 4.5-fold higher than CR-E. coli (3.8%). The majority of ESCR/CR-E. coli and K. pneumoniae isolates carried blaCTX-M (64.6% to 82.1%). blaNDM and blaOXA-48-like were the most prevalent carbapenemase genes in CR-E. coli/CR-K. pneumoniae (74.9%/52.9% and 22.4%/54.1%, respectively). In addition, 12.9%/23.0% of CR-E. coli/CR-K. pneumoniae cocarried blaNDM and blaOXA-48-like. Among ABC isolates, 41.9% were extensively drug resistant (XDR) and 35.7% were multidrug resistant (MDR), while P. aeruginosa showed XDR/MDR at 6.3%/16.5%. A. baumannii was the most common species among ABC isolates. The major carbapenemase gene in MDR-A. baumannii/XDR-A. baumannii was blaOXA-23-like (85.8%/93.0%), which had much higher rates than other ABC species. blaIMP, blaVIM, blaOXA-40-like, and blaOXA-58-like were also detected in ABC at lower rates. The most common carbapenemase gene in MDR/XDR-P. aeruginosa was blaIMP (29.0%/30.6%), followed by blaVIM (9.5%/25.3%). The findings reiterate an alarming situation of drug resistance that requires serious control measures.

A newly designed duodenoscope with detachable distal cap significantly reduces organic residue contamination after reprocessing.

BACKGROUND: A newly designed duodenoscope with detachable distal cap may reduce bacterial contamination by allowing better access to the elevator. We compared bacterial contamination and organic residue evaluated by rapid adenosine triphosphate (ATP) test and culture from duodenoscopes with detachable vs. fixed distal caps after high-level disinfection (HLD). METHODS: During December 2018-April 2019, 108 used newly designed duodenoscopes were enrolled. In group A (n = 54), the distal cap of the duodenoscope was detached before manual cleaning. In group B (n = 54), the distal cap was not detached. After HLD, samples were collected from the elevator, submitted for culture, and evaluated using the ATP test, using the cutoff value of 40 relative light units (RLUs). RESULTS: After HLD, the proportion of potential bacterial contamination and organic residue in group A was significantly lower than in group B (37.0 % vs. 75.9 %; P  < 0.001; relative risk 0.49, 95 % confidence interval 0.33-0.71), and also confirmed by median ATP values (45.2 vs. 141.0 RLU; P  < 0.001). In group B, one sample culture was positive for nonpathogenic bacteria. Pathogenic bacteria were not found in any culture from either group. CONCLUSIONS: The detachable distal cap was more effective at eliminating bacterial contamination and reducing organic residue than a fixed cap. Nonpathogenic bacteria were detected in the fixed cap group after reprocessing. The ATP test with 40 RLU cutoff is a practical method to ensure the cleanliness of duodenoscope reprocessing without the need to wait for bacterial culture results.

Genistein attenuated gastric inflammation and apoptosis in Helicobacter pylori-induced gastropathy in rats.

BACKGROUND: Helicobacter pylori (H. pylori) infection is a major cause of chronic gastritis, peptic ulcer diseases and cancer. Genistein (4',5,7-trihydroxyisoflavone), a tyrosine-specific-protein kinase inhibitor, has been shown to exert an anti-inflammatory property. The aim of this study was to examine the treatment effects of genistein and its mechanisms in rats with H. pylori infection. METHODS: Eighteen male Sprague-Dawley rats were divided into three groups (6 rats per group): (1) control group (Con); (2) H. pylori infected group (HP): the rats were inoculated with H. pylori (108- 1010 CFU/mL; 1 mL/rat.) for 3 consecutive days; and (3) HP + genistein group (HP + Gen): the rats were inoculated with H. pylori as above. Then, they were gavaged with genistein (16 mg/kg BW) for 14 days. Gastric tissue was used for the determination of nuclear factor (NF)-κB expression by immunohistochemistry (IHC), degree of apoptosis by the terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) reaction, and histopathology. Serum samples were used to measure the levels of tumor necrosis factor-alpha (TNF-α) and cytokine-induced neutrophil chemoattractant-1 (CINC-1). RESULTS: Rats in the HP group had significantly higher levels of pro-inflammatory mediators, NF-κB expression and apoptotic cells when compared with the Con group, and these markers significantly decreased in HP + Gen group when compared with the HP group. The histopathology of HP group showed moderate gastric inflammation and many HP colonization. Gastric pathology in HP + Gen group demonstrated the attenuation of inflammatory cell infiltration and H. pylori colonization. CONCLUSION: Genistein exerted its gastroprotective effects through the reduction of pro-inflammatory mediators, nuclear receptor NF-κB expression and gastric mucosal apoptosis in rats with H. pylori-induced gastropathy.

A rapid and simple method for routine determination of antibiotic sensitivity to biofilm populations of Pseudomonas aeruginosa.

Treatment of infections by Pseudomonas aeruginosa forming biofilms after antimicrobial testing on planktonic bacteria can result in substantial failure. Therefore, we offer a robust and simple experimental platform to test the impact of antimicrobials on biofilms. Antibiotic response patterns varied uniquely within biofilm formation capacity and minimal biofilm eradication concentrations (MBECs) has a significantly better discriminatory power than minimum inhibitory concentrations (MICs) to differentiate the overall efficiency of antibiotics to eradicate biofilm. Our resazurin-based 96-well-plate platform is able to emulate bacterial responses to antibiotics under biofilm conditions in a fast, simple, and cost-effective screening method adaptable to automation, and warrants trials in the clinic.

Efficacy of chlorhexidine patches on central line-associated bloodstream infections in children.

BACKGROUND: Central line-associated bloodstream infections (CLABSIs) are important hospital-acquired infections. Chlorhexidine-impregnated dressings (also known as chlorhexidine patches, CHG patches) are reported to decrease CLABSIs in adults. This study aims to determine the efficacy of CHG patches in reducing CLABSIs in children. METHODS: An open-label randomized controlled trial was conducted in children aged 2 months to 18 years, requiring a short-term catheter. Patients were randomized into two groups, allocated to receive CHG patches or standard transparent dressings. Care of the catheter was in accordance with Asia Pacific Society of Infection Control (APSIC) recommendations. Central-line-associated bloodstream infections were defined using National Healthcare Safety Network surveillance criteria. RESULTS: From April 2017 to April 2018, 192 children were enrolled. There were 108 CHG patch catheters and 101 standard dressing catheters, contributing to 3,113 catheter days. The median duration of catheter dwelling was 13 days, with an interquartile range (IQR) of 8-20 days. Half were placed at the jugular vein and 22% at the femoral vein. There were 23 CLABSI events. Incidence rates for CHG patches and standard dressings were 7.98 (95% confidence interval (CI), 4.25-13.65) and 6.74 (95% CI, 3.23-12.39) per 1,000 catheter days, respectively (incidence rate ratio 1.18; 95% CI, 0.52-2.70). The CLABSI pathogens were 15 Gram-negative bacteria, six Gram-positive bacteria, and two Candida organisms. Catheter colonization of CHG patches and standard dressings were 2.02 (95% CI, 0.42-5.91) and 3.07 (95% CI, 1.00-7.16) per 1,000 catheter days, respectively. Only local adverse effects occurred in 6.8% of the participants. CONCLUSIONS: In our setting, there was no difference in CLABSI rates when the chlorhexidine patch dressings were compared with the standard transparent dressings. Strengthening of CLABSI prevention bundles is mandatory.

The cooperation of pharmacologic-dose ascorbate with ceftriaxone against Staphylococcus aureus through bactericidal synergy and enhanced macrophage killing activity.

BACKGROUND: Ascorbate is a low-cost compound with a known bactericidal-synergy to antibitics. However, the synergy depends on concentrations and organisms. Thus, the synergy test by time-kill assay might be appropriate for the screening of the synergy. OBJECTIVE: We aimed to test the adjuvant property of ascorbate with ceftriaxone, a frequently prescribed ß-lactam antibiotic. METHOD: Ascorbate was tested with several bacteria from the American Type Culture Collection (ATCC) including Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli for i) bactericidal property of ascorbate, alone or with ceftriaxone-combination, by time-kill assay, ii) an influence on the killing-activity of bone -marrow-derived macrophage and iii) the attenuation of myositis mouse model. RESULT: The bactericidal synergy (determined with time-kill assay at 24 h) against S. aureus, but not other selected bacteria, was demonstrated in ascorbate (10 and 40 mM) plus ceftriaxone at the minimal inhibitory concentration (1x MIC). Ascorbate alone, without antibiotic, enhanced macrophage killing-activity and directly eliminated bacteria at the concentration 10-40mM and 250mM, respectively (both properties presented against S. aureus and P. aeruginosa, but not other bacteria). Ascorbate with ceftriaxone also reduced bacterial burdens in muscle and serum cytokines of S. aureus -myositis mouse model. Moreover, the synergy against the clinical isolated methicillin resistant S. aureus (MRSA) by time-kill assay and myositis model also presented. CONCLUSION: Ascorbate-ceftriaxone synergy against S. aureus was demonstrated by time-kill assay and myositis model. Time-kill assy might be valuable as a screening test to select the patients that potentially benefit from ascorbate- ceftriaxone adjuvant therapy.

High prevalence of ceftriaxone resistance among invasive Salmonella enterica serotype Choleraesuis isolates in Thailand: The emergence and increase of CTX-M-55 in ciprofloxacin-resistant S. Choleraesuis isolates.

S. Choleraesuis is a highly invasive zoonotic pathogen that causes a serious systemic infection in humans. The emergence and increase of resistance to ceftriaxone and ciprofloxacin among S. Choleraesuis has become a serious therapeutic problem. The present study demonstrated high frequency of antimicrobial resistance in Salmonella Choleraesuis among 414 nontyphoidal Salmonella isolates from bacteremic patients in Thailand. High rates of ceftriaxone (58.3%) and ciprofloxacin (19.6%) resistances were observed in S. Choleraesuis isolates. The dissemination of the self-transferable blaCTX-M-14-carrying IncFIIs, IncFII, and IncI1 plasmids and blaCMY-2-carrying IncA/C plasmid along with the clonal spread of blaCMY-2-harbouring S. Choleraesuis isolates contributed to the high frequency of resistance to extended-spectrum cephalosporins (ESCs; third- and fourth-generation cephalosporins) during 2005-2007. We reported the first occurrence of ceftazidime-hydrolysing CTX-M-55 in S. Choleraesuis isolates which dramatically increased and became the most abundant CTX-M variant among ESC-resistant S. Choleraesuis isolates during 2012-2016. The spread of clone pulsotype B3 was due to the dissemination of IncA/C plasmids carrying both blaCTX-M-55 and qnrS1 among ciprofloxacin-resistant S. Choleraesuis isolates harbouring D87G in GyrA. These isolates were apparently responsible for the high rates of co-resistance to ESCs and ciprofloxacin (51.3%) during 2012-2016. This study emphasizes the importance to have an action plan to control the dissemination of antimicrobial resistance in S. Choleraesuis since this poses a threat to global health due to travel and trade in animal food products.

Comparison of methods for identifying causative bacterial microorganisms in presumed acute endophthalmitis: conventional culture, blood culture, and PCR.

BACKGROUND: Identification of bacterial pathogens in endophthalmitis is important to inform antibiotic selection and treatment decisions. Hemoculture bottles and polymerase chain reaction (PCR) analysis have been proposed to offer good detection sensitivity. This study compared the sensitivity and accuracy of a blood culture system, a PCR approach, and conventional culture methods for identification of causative bacteria in cases of acute endophthalmitis. METHODS: Twenty-nine patients with a diagnosis of presumed acute bacterial endophthalmitis who underwent vitreous specimen collection at King Chulalongkorn Memorial Hospital were enrolled in this study. Forty-one specimens were collected. Each specimen was divided into three parts, and each part was analyzed using one of three microbial identification techniques: conventional plate culture, blood culture, and polymerase chain reaction and sequencing. The results of the three methods were then compared. RESULTS: Bacteria were identified in 15 of the 41 specimens (36.5%). Five (12.2%) specimens were positive by conventional culture methods, 11 (26.8%) were positive by hemoculture, and 11 (26.8%) were positive by PCR. Cohen's kappa analysis revealed p-values for conventional methods vs. hemoculture, conventional methods vs. PCR, and hemoculture vs. PCR of 0.057, 0.33, and 0.009, respectively. Higher detection rates of Enterococcus faecalis were observed for hemoculture and PCR than for conventional methods. CONCLUSIONS: Blood culture bottles and PCR detection may facilitate bacterial identification in cases of presumed acute endophthalmitis. These techniques should be used in addition to conventional plate culture methods because they provide a greater degree of sensitivity than conventional plate culture alone for the detection of specific microorganisms such as E. faecalis. TRIAL REGISTRATION: Thai Clinical Trial Register No. TCTR20110000024 .

Mortality factors and antibiotic options in carbapenem-resistant Enterobacterales bloodstream infections: Insights from a high-prevalence setting with co-occurring NDM-1 and OXA-48.

Bloodstream infections (BSI) caused by carbapenem-resistant Enterobacterales (CRE) are associated with a high mortality rate. This study aimed to investigate factors associated with 14-day mortality and identify a potential treatment option. A retrospective cohort study was conducted on patients with CRE-BSI in Thailand from 2015 to 2020. The multivariate Cox proportional-hazards model was employed to identify factors influencing 14-day mortality. Out of 134 diagnosed cases of CRE-BSI, the all-cause 14-day mortality rate was 35.1%. The most prevalent organism isolated was Klebsiella pneumoniae (85.8%), followed by Escherichia coli (11.9%). Among the 60 isolates tested for carbapenemase genes, the majority exhibited co-occurring blaNDM-1 and blaOXA-48 (51.7%), followed by blaOXA-48 (31.7%) and blaNDM-1 (15.0%). In the multivariate analysis, neutropenia (adjusted hazard ratio [aHR] 2.55; 95% confidence interval [95%CI] 1.28-5.06; p = 0.008), sepsis/septic shock (aHR 3.02; 95%CI 1.33-6.86; p = 0.008), and previous metronidazole exposures (aHR 3.58; 95%CI 1.89-6.71; p < 0.001) were identified as independent factors for 14-day mortality. The fosfomycin-based regimen was found to be protective (aHR 0.37; 95%CI 0.15-0.92; p = 0.032). In patients with CRE-BSI, particularly in regions with a high occurrence of co-occurring blaNDM-1 and blaOXA-48, neutropenia, sepsis/septic shock, and previous metronidazole exposures emerged as independent risk factors for mortality. Moreover, the fosfomycin-based regimen showed an improvement in the survival rate.

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand.

Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017-2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.

CRISPR Interference-Mediated Silencing of the mmpL3 Gene in Mycobacterium smegmatis and Its Impact on Antimicrobial Susceptibility.

BACKGROUND: The discovery of novel therapeutic agents, especially those targeting mycobacterial membrane protein large 3 (mmpL3), has shown promise. In this study, the CRISPR interference-Streptococcus thermophilus nuclease-deactivated Cas9 (CRISPRi-dCas9Sth1) system was utilized to suppress mmpL3 expression in Mycobacterium smegmatis, and its impacts on susceptibility to antimicrobial agents were evaluated. METHODS: The repression of the mmpL3 gene was confirmed by RT-qPCR. The essentiality, growth curve, viability, and antimicrobial susceptibility of the mmpL3 knockdown strain were investigated. RESULTS: mmpL3 silencing was achieved by utilizing 0.5 and 1 ng/mL anhydrotetracycline (ATc), resulting in reductions in the expression of 60.4% and 74.4%, respectively. mmpL3 silencing led to a significant decrease in bacterial viability when combined with one-half of the minimal inhibitory concentrations (MICs) of rifampicin, rifabutin, ceftriaxone, or isoniazid, along with 0.1 or 0.5 ng/mL ATc (p < 0.05). However, no significant difference was observed for clarithromycin or amikacin. CONCLUSIONS: The downregulation of the mmpL3 gene in mycobacteria was achieved through the use of CRISPRi-dCas9Sth1, resulting in growth deficiencies and resensitization to certain antimicrobial agents. The impact was dependent upon the level of gene expression.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Introduction: Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Methods: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. Results and discussion: All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

Early treatment with fluvoxamine, bromhexine, cyproheptadine, and niclosamide to prevent clinical deterioration in patients with symptomatic COVID-19: a randomized clinical trial.

Background: Repurposed drugs with host-directed antiviral and immunomodulatory properties have shown promise in the treatment of COVID-19, but few trials have studied combinations of these agents. The aim of this trial was to assess the effectiveness of affordable, widely available, repurposed drugs used in combination for treatment of COVID-19, which may be particularly relevant to low-resource countries. Methods: We conducted an open-label, randomized, outpatient, controlled trial in Thailand from October 1, 2021, to June 21, 2022, to assess whether early treatment within 48-h of symptoms onset with combinations of fluvoxamine, bromhexine, cyproheptadine, and niclosamide, given to adults with confirmed mild SARS-CoV-2 infection, can prevent 28-day clinical deterioration compared to standard care. Participants were randomly assigned to receive treatment with fluvoxamine alone, fluvoxamine + bromhexine, fluvoxamine + cyproheptadine, niclosamide + bromhexine, or standard care. The primary outcome measured was clinical deterioration within 9, 14, or 28 days using a 6-point ordinal scale. This trial is registered with ClinicalTrials.gov (NCT05087381). Findings: Among 1900 recruited, a total of 995 participants completed the trial. No participants had clinical deterioration by day 9, 14, or 28 days among those treated with fluvoxamine plus bromhexine (0%), fluvoxamine plus cyproheptadine (0%), or niclosamide plus bromhexine (0%). Nine participants (5.6%) in the fluvoxamine arm had clinical deterioration by day 28, requiring low-flow oxygen. In contrast, most standard care arm participants had clinical deterioration by 9, 14, and 28 days. By day 9, 32.7% (110) of patients in the standard care arm had been hospitalized without requiring supplemental oxygen but needing ongoing medical care. By day 28, this percentage increased to 37.5% (21). Additionally, 20.8% (70) of patients in the standard care arm required low-flow oxygen by day 9, and 12.5% (16) needed non-invasive or mechanical ventilation by day 28. All treated groups significantly differed from the standard care group by days 9, 14, and 28 (p < 0.0001). Also, by day 28, the three 2-drug treatments were significantly better than the fluvoxamine arm (p < 0.0001). No deaths occurred in any study group. Compared to standard care, participants treated with the combination agents had significantly decreased viral loads as early as day 3 of treatment (p < 0.0001), decreased levels of serum cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) as early as day 5 of treatment, and interleukin-8 (IL-8) by day 7 of treatment (p < 0.0001) and lower incidence of post-acute sequelae of COVID-19 (PASC) symptoms (p < 0.0001). 23 serious adverse events occurred in the standard care arm, while only 1 serious adverse event was reported in the fluvoxamine arm, and zero serious adverse events occurred in the other arms. Interpretation: Early treatment with these combinations among outpatients diagnosed with COVID-19 was associated with lower likelihood of clinical deterioration, and with significant and rapid reduction in the viral load and serum cytokines, and with lower burden of PASC symptoms. When started very soon after symptom onset, these repurposed drugs have high potential to prevent clinical deterioration and death in vaccinated and unvaccinated COVID-19 patients. Funding: Ped Thai Su Phai (Thai Ducks Fighting Danger) social giver group.