Leonardo Neves Andrade

Purpose Tolerance and persistence occur “silently” in bacteria categorized as susceptible by antimicrobial susceptibility testing in clinical microbiology laboratories. They are different from resistance phenomena, not well-studied, and often remain unnoticeable. We aimed to investigate and characterize ceftazidime-avibactam (CZA) tolerance/persistence in 80 Klebsiella pneumoniae isolates from bloodstream infections. Methods We used the Tolerance Disk Test (TDtest) to detect CZA tolerance/persistence and investigate the avibactam (AVI) influence on them, and time-kill assays with minimal duration for killing (MDK) determination to characterize/differentiate CZA tolerance from persistence, for selected isolates. Whole genome sequencing was performed for 49/80 selected isolates to investigate genes related to beta-lactam tolerance/persistence and resistance as well as phylogeny studies. Results Tolerance/persistence to CZA was detected in 48/80 (60%) isolates, all extensively drug-resistant (XDR) or multidrug-resistant, carbapenem-resistant K. pneumoniae (CRKp), KPC producers, and previously categorized as susceptible (not resistant) to CZA. No heteroresistance was detected. CZA tolerance/persistence occurred due to ceftazidime tolerance/persistence and was not related to AVI in the CZA combination. 5/11 isolates were characterized as CZA-tolerant and 5/11 as CZA-persistent. The single (1/11) XDR and CRKp non-KPC producer was truly susceptible. All the CZA-tolerant/persistent isolates (ST11, ST258, ST340, ST437, ST16, ST17, and ST307) harbored the carbapenemase-encoding gene bla_KPC−2. Mutation in only two genes (rpoS and degQ) related to beta-lactam tolerance/persistence was found in only 7/49 CZA-tolerant/persistent isolates, suggesting the presence of yet unknown beta-lactam tolerance/persistence genes. Conclusion Among the K. pneumoniae bloodstream isolates studied, 60%, previously categorized as susceptible to CZA, were, actually, tolerant/persistent to this antibiotic, all these KPC producers.

We investigated the in vitro antibacterial activity of the combination rifampicin (RIF) + polymyxin B (PB) against extensively drug-resistant (XDR) Klebsiella pneumoniae isolates. We evaluated clinical isolates co-resistant to PB (non-mcr carriers; eptB, mgrB, pmr operon, and ramA mutations) and to carbapenems (KPC, CTX-M, and SHV producers; including KPC + NDM co-producer), belonging to sequence types (ST) ST16, ST11, ST258, ST340, and ST437. We used the standard broth microdilution method to determine RIF and PB minimum inhibitory concentration (MIC) and the checkerboard assay to evaluate the fractional inhibitory concentration index (FICI) of RIF + PB as well as to investigate the lowest concentrations of RIF and PB that combined (RIF + PB) had antibacterial activity. Time-kill assays were performed to evaluate the synergistic effect of the combination against selected isolates. PB MIC (32–256 µg/mL) and RIF MIC (32–1024 µg/mL) were determined. FICI (K. pneumoniae and could be further studied as a potential combination therapy, with cost-effectiveness and promising efficacy.

Introduction: Fungi usually are resistant to disinfection and has pathogenic potential; however, they are not legally controlled in hemodialysis water (HW). Objective: Filamentous fungi (FF) were evaluated in HW from a tertiary hospital in southeast of São Paulo state (Brazil) and is a reference medical center providing service to millions of people every year. Method: A total of 84 water samples were collected in seven hydraulic system points during a period of one year and FF was identified by macroscopic and microscopic features. Results: FF were detected in all points ranging from 1 to 334 CFU/100 mL. Nine genera along the hydraulic system were identified, including Acremonium, Alternaria, Aspergillus, Beltrania, Bipolaris, Cladosporium, Exophiala, Fusarium, and Penicillium. Conclusions: Most fungi are ubiquitous and some cause human infection and mycotoxins producers. The various genera found reveal possible failures in treatment and distribution system, either through inadequate hygiene practices or infusion of these microorganisms into the pipes. FF detection is needed bearing mind immunological vulnerability of patients under hemodialysis treatment requiring continuous health surveillance actions.

Treatments that attenuate the effects of hypoestrogenism in menopausal women have been gaining visibility. This study investigated the skin response to a phytoestrogen-enriched cosmetic formulation created by incorporating a biotransformed soybean extract (BE) into a cream-like matrix. Collagen-I expression was analyzed both in vitro (fibroblast cells) and ex vivo (skin explants). The results revealed an increased amount of collagen-I both in fibroblasts and human skin when treated with BE and BE-incorporated cream. Also, this collagen-I overexpression was inhibited by PHTPP, indicating a dependence on estrogen hormone receptor beta (ERβ) signaling. Moreover, BE was not harmful to skin microbiota, showing a promising nutricosmetic potential. Thus, this work presented a fully functional cream-like formulation that was shown to be safe and effectively increase collagen-I levels both in vitro and ex vivo.

We correlated clinical, epidemiological, microbiological, and genomic data of an outbreak with polymyxin B (PB)- and carbapenem-resistant Klebsiella pneumoniae during the COVID-19 pandemic. Twenty-six PB- and carbapenem-resistant K. pneumoniae were isolated from patients in the COVID-19 ICU (Intensive Care Unit), non-COVID-19 ICU (Intensive Care Unit), clinical, or surgical ward. Bacterial identification, drug susceptibility tests, and DNA sequencing were performed, followed by in silico resistance genes identification. All isolates showed extensively drug-resistant (XDR) phenotypes. Four different sequence types (ST) were detected: ST16, ST11, ST258, and ST437. Nineteen isolates were responsible for an outbreak in the ICU in September 2020. They belong to ST258 and harbored the 42Kb IncX3plasmid (pKP98M3N42) with the same genomic pattern of two K. pneumoniae identified in 2018. Twenty-four isolates carried bla_-KPC-2 gene. No plasmid-mediated colistin (mcr) resistance genes were found. Eight isolates presented mgrB gene mutation. The clonal isolates responsible for the outbreak came from patients submitted to pronation, with high mortality rates in one month. XDR-K. pneumoniae detected during the outbreak presented chromosomal resistance to PB and plasmid-acquired carbapenem resistance due to KPC production in most isolates and 42Kb IncX3(pKP98M3N42) plasmid carrying bla_KPC-2 was associated with ST258 isolates. The outbreak followed the collapse of the local healthcare system with high mortality rates.

This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD + polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Time-kill assays results showed that the combination CBD + PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD + PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. pneumoniae, is particularly promising.