A Sustainable Future
To highlight the vital role microbiology plays in delivering on the UN Sustainable Development Goals (SDGs), we have created a collection of must-read research on three critical aspects of the SDGs: antimicrobial resistance, soil health, and the circular economy.
Collection Contents
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In vitro activity of imipenem-relebactam against resistant phenotypes of Enterobacteriaceae and Pseudomonas aeruginosa isolated from intraabdominal and urinary tract infection samples – SMART Surveillance Europe 2015–2017
Introduction. Infections attributable to carbapenem-resistant Gram-negative bacilli are increasing globally. New antimicrobial agents are urgently needed to treat patients with these infections.
Aim. To describe susceptibility to the novel carbapenem-β-lactamase inhibitor combination imipenem-relebactam and comparators of clinical isolates of non-Proteeae Enterobacteriaceae (NPE) and Pseudomonas aeruginosa from intraabdominal infections (IAIs) and urinary tract infections (UTIs).
Methods. Broth microdilution MICs were determined for isolates collected in 22 European countries in 2015–2017 and interpreted using EUCAST breakpoints; imipenem-relebactam MICs were interpreted using imipenem breakpoints.
Results. For NPE, 98.4 % of isolates from IAIs (n=10,465) and 98.5 % of UTI isolates (n=7,446) were susceptible to imipenem-relebactam, as were 42.4 % of imipenem-nonsusceptible (n=474), 98.6 % of Klebsiella pneumoniae carbapenemase (KPC)-positive (n=138), and 93.9 % of multidrug-resistant (MDR) isolates (n=4,424) from IAIs and UTIs combined. Molecular analysis demonstrated that two-thirds of imipenem-nonsusceptible isolates rendered susceptible by relebactam carried KPCs; 96 % (261/271) of imipenem-nonsusceptible isolates of NPE that remained nonsusceptible in the presence of relebactam carried metallo-β-lactamase (MBL)-type and/or OXA-48-like carbapenemases. Among P. aeruginosa , 94.4 % of IAI (n=1,245) and 93.0 % of UTI isolates (n=714) were susceptible to imipenem-relebactam, as were 74.4 % of imipenem-nonsusceptible (n=469) and 79.8 % of MDR isolates (n=595) from IAIs and UTIs combined. Among the 120 isolates of P. aeruginosa that remained nonsusceptible to imipenem upon addition of relebactam, 72 % carried MBLs. The distribution of NPE and P. aeruginosa carrying carbapenemases varied substantially across Europe, as did resistance to imipenem and imipenem-relebactam.
Conclusions. Continued surveillance of antimicrobial resistance and resistance mechanisms, including the study of imipenem-relebactam as it approaches regulatory approval, appears warranted.
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In vivo acquisition and risk of inter-species spread of bla KPC-3-plasmid from Klebsiella pneumoniae to Serratia marcescens in the lower respiratory tract
In recent years, Serratia marcescens has emerged as an important agent of hospital-acquired infections, such as pneumonia, urinary tract infection, septicaemia and meningitis, particularly in vulnerable patients. Compared to Klebsiella pneumoniae and Escherichia coli , S. marcescens is less commonly associated with bla KPC genes, yet few cases of plasmid transmission at the gastrointestinal level from K. pneumoniae carbapenemase (KPC)-producing Enterobacterales to S. marcescens have been described. Here we report a case of in vivo acquisition, during a 3-month period of hospitalization in the intensive care unit, of a bla KPC-3 gene carried by a pKpQIL-IT plasmid, and its probable transmission at the bronchial level among different species of Enterobacterales , including K. pneumoniae and S. marcescens . By using whole genome sequence analyses we were able provide insight into the dynamics of carbapenem-resistance determinants acquisition in the lower respiratory tract, a novel anatomical region for such plasmid transmission events, that usually involve the gastrointestinal tract. The co-presence at the same time of both wild-type and resistant Enterobacterales could have been the critical factor leading to the spread of plasmids harbouring carbapenem-resistance genes, of particular importance during surveillance screenings. The possibility of such an event may have significant consequences in terms of antimicrobial treatment, with a potential limitation of therapeutic options, thereby further complicating the clinical management of high-risk critically ill patients.
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Isolation and characterization of novel soil- and plant-associated bacteria with multiple phytohormone-degrading activities using a targeted methodology
More LessEthylene (ET), salicylic acid (SA) and indole-3-acetic acid (IAA) are important phytohormones regulating plant growth and development, as well as plant-microbe interactions. Plant growth-promoting bacteria (PGPB) naturally associate with plants and facilitate plant growth through a variety of mechanisms, including the ability to modulate the concentrations of these phytohormones in planta. Importantly, the wide presence of phytohormone degradation mechanisms amongst symbiotic and other soil- and plant-associated bacteria indicates that the ability to modulate phytohormone concentrations plays an important role in bacterial colonization and plant-growth promotion abilities. Obtaining phytohormone-degrading bacteria is therefore key for the development of novel solutions aiming to increase plant growth and protection. In this paper, we report an optimized targeted methodology and the consequent isolation of novel soil- and plant-associated bacteria, including rhizospheric, endophytic and phyllospheric strains, with the ability to degrade the phytohormones, SA and IAA, as well as the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). By using an optimized targeted methodology, we rapidly isolated diverse soil- and plant-associated bacteria presenting phytohormone-degrading abilities from several plants, plant tissues and environments, without the need for prior extensive and laborious isolation and maintenance of large numbers of isolates. The developed methodology facilitates PGPB research, especially in developing countries. Here, we also report, for the first time, the isolation of bacterial strains able to concomitantly catabolize three phytohormones (SA, IAA and ACC). Ultimately, the described targeted methodology and the novel phytohormone-degrading bacteria obtained in this work may be useful tools for future plant-microbe interaction studies, and in the development of new inoculant formulations for agriculture and biotechnology.
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In vitro activities of six antifungal agents and their combinations against Chaetomium spp.
Lingyue Sun, Zhe Wan, Ruoyu Li and Jin YuPurpose. To assess in vitro activities of six antifungal agents (amphotericin B, itraconazole, voriconazole, posaconazole, caspofungin and terbinafine) and the combined effects of eight pairs of them (caspofungin or terbinafine with amphotericin B, itraconazole, voriconazole or posaconazole) against 22 isolates of Chaetomium spp.
Methodology. The broth microdilution method drafted by the Clinical and Laboratory Standards Institute and the checkerboard method were used in this study to evaluate in vitro activities of antifungal drugs both alone and in combination against Chaetomium spp.
Results. Amphotericin B and triazoles exhibited lower geometric mean, MIC50 and MIC90 than caspofungin and terbinafine. Besides, all the paired drugs displayed varying degrees of synergism, with the interactions between caspofungin and itraconazole ranking first (86.36 %).
Conclusion. Our study illustrated varying degrees of synergism between caspofungin or terbinafine and itraconazole, voriconazole, posaconazole or amphotericin B towards Chaetomium spp., which could be a reference for the clinical treatment of Chaetomium spp. infections.
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In vitro activity of mecillinam and nitroxoline against Neisseria gonorrhoeae – re-purposing old antibiotics in the multi-drug resistance era
In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l−1 and MICs of cefotaxime ranged from < 0.016 to 1 mg l−1 . Five isolates were penicillinase-producers. MICs of MCM ranged from 16 to > 128 mg l−1 whereas MICs of NIT ranged from 0.125 to 2 mg l−1 . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.
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In vitro efficacy of sodium selenite in reducing toxin production, spore outgrowth and antibiotic resistance in hypervirulent Clostridium difficile
Purpose. This study investigated the efficacy of the essential mineral, selenium (sodium selenite), in reducing the toxin production, spore outgrowth and antibiotic resistance of Clostridium difficile in vitro.
Methodology. Two hypervirulent C. difficile isolates were cultured in brain heart infusion broth with and without a sub-minimum inhibitory concentration (sub-MIC) of sodium selenite, and the supernatant and bacterial pellet were harvested for total toxin quantitation and RT-qPCR analysis of toxin-encoding genes, respectively. Additionally, C. difficile isolates were cultured in brain heart infusion broth containing 0.5 or 1× the minimum inhibitory concentration (MIC) of either ciprofloxacin or vancomycin with or without sub-MICs of sodium selenite. Further, the effect of sodium selenite on C. difficile germination and spore outgrowth was also determined by exposing C. difficile spores to a sub-MIC of sodium selenite in a germination medium and measuring the germination and outgrowth by measuring the optical density at 600 nm.
Results. Sodium selenite significantly reduced C. difficile toxin synthesis, cytotoxicity and spore outgrowth. Further, the expression of the toxin production genes, tcdA and tcdB, was downregulated in the presence of sodium selenite, while sodium selenite significantly increased the sensitivity of C. difficile to ciprofloxacin , but not vancomycin, as revealed by decreased bacterial growth in samples containing ciprofloxacin+selenium compared to the antibiotic control. Although the sub-MIC of sodium selenite did not inhibit spore germination, it was capable of completely inhibiting spore outgrowth.
Conclusion. Our results suggest that sodium selenite could potentially be used to control C. difficile and indicate that future in vivo studies are warranted.
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Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China
Purpose. The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China.
Methodology. The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing.
Results. A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3′)-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3′)-IIa]. The oqxAB–aph(3′)-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin.
Conclusion. This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB–aph(3′)-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.
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An investigation of antifungal stewardship programmes in England
Purpose. We sought to explore the current status of antifungal stewardship (AFS) initiatives across National Health Service (NHS) Trusts within England, the challenges and barriers, as well as ways to improve current AFS programmes.
Methodology. An electronic survey was sent to all 155 acute NHS Trusts in England. A total of 47 Trusts, corresponding to 30 % of English acute Trusts, responded to the the survey; 46 Trusts (98 %) had an antimicrobial stewardship (AMS) programme but only 5 (11 %) had a dedicated AFS programme. Overall, 20 (43 %) Trusts said they included AFS as part of their AMS programmes. From those conducting AFS programmes, 7 (28 %) have an AFS/management team, 16 (64 %) monitor and report on antifungal usage, 5 (20 %) have dedicated AFS ward rounds and 12 (48 %) are directly involved in the management of invasive fungal infections.
Results/Key findings. Altogether, 13 acute Trusts (52 %) started their AFS programme to manage costs, whilst 12 (48 %) commenced the programme due to clinical need; 27 (73 %) declared that they would increase their AFS initiatives if they could. Of those without an AFS programme, 14 (67 %) responded that this was due to lack of resources/staff time. Overall, 12 Trusts (57 %) responded that the availability of rapid diagnostics and clinical support would enable them to conduct AFS activities.
Conclusion. Although a minority of Trusts conduct dedicated AFS programmes, nearly half include AFS as part of routine AMS activities. Cost issues are the main driver for AFS, followed by clinical need. The availability of rapid diagnostics and clinical support could help increase AFS initiatives.
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In vitro antifungal susceptibilities of Candida species to liposomal amphotericin B, determined using CLSI broth microdilution, and amphotericin B deoxycholate, measured using the Etest
The antifungal susceptibilities of 598 isolates of Candida spp. (bloodstream and other sterile sites) to liposomal amphotericin B (L-AmB) versus amphotericin B (AmB) were determined. MICs were calculated using the Clinical and Laboratory Standards Institute broth microdilution (M27-A3) method for L-AmB and the Etest method for AmB. The MIC50/MIC90 (µg ml−1) values for L-AmB broth microdilution and AmB Etest were 0.25/1 and 0.19/0.5, respectively. The overall essential agreement (±2 dilutions) was 91.5 %, ranging from 37.5 % (Candida lusitaniae) to 100 % (Candida glabrata and Candida krusei). Categorical agreement between the two methods was categorized based on a previously published breakpoint (susceptible/resistant MIC cut-off of 1 µg ml−1). The overall categorical agreement at the 48 h reading was 97.3 %, ranging from 72.7 % (C. krusei) to 100 % (Candida albicans). Major and very major discrepancies occurred in 2.3 and 0.3 %, respectively. Spearman’s ρ was 0.48 (P<0.0001). These results demonstrate the utility of the AmB Etest as a surrogate marker to predict the sensibility and resistance of Candida spp. to L-AmB and thus to support its use in antifungal treatment.
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Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil
Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. ‘atroviride type B’ morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (l-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. ‘atroviride type B’ growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.
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Influence of root exudates on the extracellular proteome of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42
More LessProteins secreted by Bacillus amyloliquefaciens FZB42, a root-associated plant growth-promoting rhizobacterium, are thought to play an important role in the establishment of beneficial interactions with plants. To investigate the possible role of proteins in this process, extracellular proteome maps of B. amyloliquefaciens FZB42 during the late exponential and stationary growth phases were generated using 2D gel electrophoresis. Out of the 121 proteins identified by MALDI-TOF MS, 61 were predicted to contain secretion signals. A few of the others, bearing no signal peptide, have been described as elicitors of plant innate immunity, including flagellin proteins, cold-shock proteins and the elongation factor Tu, suggesting that B. amyloliquefaciens FZB42 protects plants against disease by eliciting innate immunity. Our reference maps were used to monitor bacterial responses to maize root exudates. Approximately 34 proteins were differentially secreted in response to root exudates during either the late exponential or stationary phase. These were mainly involved in nutrient utilization and transport. The protein with the highest fold change in the presence of maize root exudates during the late exponential growth phase was acetolactate synthase (AlsS), an enzyme involved in the synthesis of the volatile acetoin, known as an inducer of systemic resistance against plant pathogens and as a trigger of plant growth.
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