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Abstract The increasing occurrence of hospital-associated infections, particularly bacteremia, caused by extensively drug-resistant (XDR) carbapenemase-producing colistin-resistant Klebsiella pneumoniae highlights a critical requirement to discover new therapeutic alternatives. Bacteriophages having host-specific bacteriolytic effects are promising alternatives for combating these pathogens. Among 12 phages isolated from public wastewater in Thailand, two phages-vB_kpnM_05 (myovirus) and vB_kpnP_08 (podovirus) showed broad-host range, producing bacteriolytic activities against 81.3% (n = 26) and 78.1% (n = 25) of 32 XDR carbapenemase-producing colistin-resistant K. pneumoniae, with capsular types—K15, K17, K50, K51, K52/wzi-50 and K2/wzi-2. Both phages showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages from Myoviridae and Podoviridae family, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. These characteristics highlighted their promising potential for utilizing in phage therapy for combating XDR K. pneumoniae. Although phage cocktail combining vB_kpnM_05 and vB_kpnP_08 provided significant bacteriolysis for longer duration (8 h) than its monophage (6 h), bacterial regrowth was observed which suggested an evitable development of phage resistance under phages’ selection pressures. Future study will be undertaken to elucidate the precise mechanisms by which these XDR K. pneumoniae developed phage resistance and their associated fitness cost. Remarkably, combining phage cocktail with amikacin at their sub-inhibitory concentrations produced potent synergy by completely suppressing bacterial regrowth in vitro. Our study demonstrated the significant therapeutic and prophylactic effectiveness of a phage cocktail-amikacin combination as a promising alternative strategy for overcoming bacteremia associated with XDR K. pneumoniae having carbapenemase and colistin resistance in vivo.

Objectives: To evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. Methods: DNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). Results: We processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (

Summary: Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

Abstract The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia.

Abstract Development of an effective therapy to overcome colistin resistance in Klebsiella pneumoniae, a common pathogen causing catheter-related biofilm infections in vascular catheters, has become a serious therapeutic challenge that must be addressed urgently. Although colistin and EDTA have successful roles for eradicating biofilms, no in vitro and in vivo studies have investigated their efficacy in catheter-related biofilm infections of colistin-resistant K. pneumoniae. In this study, colistin resistance was significantly reversed in both planktonic and mature biofilms of colistin-resistant K. pneumoniae by a combination of colistin (0.25–1 µg/ml) with EDTA (12 mg/ml). This novel colistin-EDTA combination was also demonstrated to have potent efficacy in eradicating colistin-resistant K. pneumoniae catheter-related biofilm infections, and eliminating the risk of recurrence in vivo. Furthermore, this study revealed significant therapeutic efficacy of colistin-EDTA combination in reducing bacterial load in internal organs, lowering serum creatinine, and protecting treated mice from mortality. Altered in vivo expression of different virulence genes indicate bacterial adaptive responses to survive in hostile environments under different treatments. According to these data discovered in this study, a novel colistin-EDTA combination provides favorable efficacy and safety for successful eradication of colistin-resistant K. pneumonia catheter-related biofilm infections.

Candida albicans is abundant in the human gut mycobiota but this species does not colonize the mouse gastrointestinal tract. C. albicans administration in dextran-sulfate solution (DSS) induced-colitis mouse model (DSS+Candida) might resemble more to human condition, therefore, a DSS colitis model with Candida administration was studied; first, to test the influence of fungi in DSS model and second, to test the efficacy of Lactobacillus rhamnosus L34. We demonstrated serum (1→3)-β-D-glucan (BG) elevation in patients with IBD and endoscopic moderate colitis in clinical remission, supporting the possible influence of gut fungi toward IBD in human. Then, in mouse model, Candida gavage was found to worsen the DSS model indicated by higher mortality rate, more severe colon histology and enhanced gut-leakage (FITC-dextran assay, endotoxemia, serum BG and blood bacterial burdens) but did not affect weight loss and diarrhea. DSS+Candida induced higher pro-inflammatory cytokines both in blood and in intestinal tissue. Worsened systemic pro-inflammatory cytokine responses in DSS+Candida compared with DSS alone was possibly due to the more severe translocation of LPS, BG and bacteria (not fungemia) from gut into systemic circulation. Interestingly, bacteremia from Pseudomonas aeruginosa was more frequently isolated from DSS+Candida than DSS alone. In parallel, P. aeruginosa was also isolated from fecal culture in most of the mice in DSS+Candida group supported by prominent Gammaproteobacteria in fecal microbioata analysis. However, L. rhamnosus L34 attenuated both DSS+Candida and DSS model through the attenuation of gut local inflammation (cytokines and histology), gut-leakage severity, fecal dysbiosis (culture method and microbiome analysis) and systemic inflammation (serum cytokines). In conclusion, gut Candida in DSS model induced fecal bacterial dysbiosis and enhanced leaky-gut induced bacteremia. Probiotic treatment strategy aiming to reduce gut-fungi and fecal dysbiosis could attenuate disease severity. Investigation on gut fungi in patients with IBD is highly interesting.

Gastrointestinal (GI) leakage in Clostridium difficile-associated diarrhea (CDAD) is well known but is not routinely assessed in clinical practice. Serum (1→3)-β-D-glucan (BG), a fungal cell wall component used as a biomarker for invasive fungal disease, was tested in a CDAD mouse model with and without probiotics. Higher serum fluorescein isothiocyanate-dextran (FITC-dextran) and spontaneous gram-negative bacteremia, GI leakage indicators, were frequently found in CDAD mice, which died compared with those which survived. BG, serum macrophage inflammatory protein-2 and FITC-dextran but not quantitative blood bacterial count differentiated the clinical severity. Interestingly, a specific dose of Lactobacillus rhamnosus L34 attenuated CDAD and decreased serum BG and FITC-dextran, but not other parameters. BG also showed a higher area under the receiver operating characteristic curve for 7-day mortality than FITC-dextran. Fifty-five percent of CDAD mice with BG ≥ 60 pg/ml (the human negative cut-off value for invasive fungal disease) at 1 day after C. difficile gavage died within 7 days. In conclusion, serum BG was elevated in mice with severe CDAD, an established model of GI leakage with a strong association with mortality rate. BG monitoring in patients with CDAD is of interest as both a potential prognostic tool and a therapeutic efficacy indicator. [ABSTRACT FROM AUTHOR]