Journal of Applied Microbiology最新文献

Aims: Hygienic monitoring (HM) of germ-free (GF) mouse colonies is exceptionally challenging. The test accuracy of the applied diagnostic methodology has to be outstanding to provide proof of absence of all living microorganisms confirming the GF status. In this context, microscopy of native intestinal content serves as a highly sensitive diagnostic tool for the detection of bacterial contaminants. However, with this method residual microorganisms may be detected. To overcome this risk of false-positive results, we complemented our analyses with a bacterial viability staining of the intestinal content of GF mice.

Methods and results: Intestinal contents of GF mice (n = 13) from five isolators were analyzed by bacterial culture and phase-contrast microscopy. Additionally, 16S rRNA gene PCR analysis and metagenomic sequencing were performed. To distinguish between live and dead bacteria, intestinal content was stained by a Bacterial Viability Kit and analyzed by fluorescence microscopy. While culture medium proved sterility of the sample material, increased amounts of scattered bacterial structures were detected during microscopic analysis, indicating potential contamination. Molecular techniques pointed to a presence of environmental bacteria. However, viability staining revealed the presence of only dead (double-stained) bacteria in all samples. Likewise, non-viable bacteria have been identified in samples obtained from irradiated feed, probably being the source of bacterial structures found in GF mice.

Conclusions: Altogether, detected bacterial structures were proven to be non-viable and therefore should not be interpreted as isolator contaminants. Thus, in our hands, with the herein described report of suspected contamination, we prove that bacterial viability staining served as a highly valuable screening tool, enhancing diagnostic quality of the HM of GF colonies.

Aims: Monascus purpureus is a widely used filamentous fungus in food and medicinal fermentation, yet the co-production of citrinin, a nephrotoxic mycotoxin, poses significant safety concerns. In this study, we established a co-growth system of M. purpureus with Panax ginseng to investigate the regulatory effects of P. ginseng on citrinin biosynthesis and related toxicity.

Methods and results: HPLC analysis revealed that P. ginseng supplementation significantly reduced citrinin production by approximately 68% without impairing fungal biomass accumulation. RT-qPCR results showed that the expression levels of four key citrinin biosynthetic genes (pksCT, ctnA, citB, citD) were markedly downregulated in the P. ginseng-treated group. In vivo toxicity evaluation using mice demonstrated that administration of M. purpureus fermentation extract led to increased serum blood urea nitrogen, creatinine, uric acid levels, and renal inflammation, whereas co-growth with P. ginseng effectively alleviated these nephrotoxic effects and significantly attenuated renal mRNA expression of pro-inflammatory cytokines, including IL-6, TNF-α, IL-1β, and MCP-1 in renal tissue.

Conclusions: These findings indicate that P. ginseng can attenuate citrinin production and associated renal toxicity via transcriptional modulation of fungal secondary metabolism, suggesting a potentially effective strategy for the safe development of Monascus-derived functional foods and traditional medicine fermentation products.

Aims: Methicillin-resistant Staphylococcus aureus (MRSA) remains a major public health concern due to its antibiotic resistance and virulence potential. This study aimed to characterizes tst-positive MRSA strains identified among 150 clinical isolates collected between 2020 and 2023 at Mustapha Bacha Hospital, Algiers, Algeria.

Methods and results: Antimicrobial susceptibility testing was performed according to CLSI guidelines. The methicillin resistance genes (mecA and mecC) and virulence gene tst were detected by PCR. In addition, whole genome sequencing (WGS) was conducted on all tst-positive isolates to determine their multilocus sequence types (MLST), antimicrobial resistance and virulence gene profiles, and single nucleotide polymorphisms (SNPs), in order to determine clonal relatedness. Among the 150 isolates tested, 143 carried the mecA gene, while the remaining seven were negative. The mecC gene was not detected. Eight MRSA (5.3%) carried the tst gene encoding toxic shock syndrome toxin-1 (TSST-1). The predominant clone was ST22-MRSA-IV, detected in seven isolates, while one strain belonged to ST39-MRSA-II.

Conclusions: This study reports the detection of tst-positive ST22-MRSA (Gaza epidemic clone) and a multidrug-resistant ST39-MRSA isolates at Mustapha Bacha Hospital, underscoring the need for ongoing genomic surveillance to monitor their occurrence and potential spread.

Aims: Carbapenem- and colistin-resistant Escherichia coli co-carrying blaNDM-5 and mcr-1 represent a growing therapeutic challenge with limited treatment options. Fusidic acid (FA), a protein synthesis inhibitor, is inactive against Gram-negative bacteria because of its limited ability to permeate their outer membrane. This study aimed to evaluate whether colistin (CST) can restore FA activity against mcr-1-positive E. coli through membrane permeabilization-mediated synergy.

Methods and results: Three E. coli isolates carrying the blaNDM-5 and mcr-1 were subjected to antimicrobial susceptibility testing, checkerboard and time-kill assays, NPN uptake, intracellular FA accumulation analyses, and a murine thigh infection model. All isolates exhibited multidrug resistance but only low-level CST resistance (MIC 4-8 mg l-1). Checkerboard assays revealed strong synergy, with CST reducing FA minimum inhibitory concentrations (MICs) by >1000-fold and restoring apparent susceptibility. Time-kill studies confirmed concentration-dependent synergistic bactericidal activity, achieving complete bacterial eradication when sub-MIC of FA was combined with CST. The tests for NPN uptake evolution and FA accumulation revealed that CST markedly increased membrane permeability and intracellular FA levels (5- to 8-fold). In the mouse thigh infection model, combination therapy with CST and FA at clinically equivalent doses produced a ≥3.0-log10 cfu g-1 reduction relative to the control and a >2.0-log10 cfu g-1 reduction compared with either monotherapy.

Conclusions: CST markedly enhances FA activity against mcr-1-positive E. coli by compromising outer membrane integrity and facilitating intracellular antibiotic accumulation. This synergy highlights a rational combination approach to restore the efficacy of obsolete antibiotics for multidrug-resistant Gram-negative bacteria.

Aims: This study aimed to design and evaluate novel antimicrobial peptides (AMPs) with antibacterial and antibiofilm activity against Pseudomonas aeruginosa. Computational analyses included interactions with quorum sensing (QS) receptors as potential targets involved in bacterial virulence regulation.

Methods and results: AMP sequences were generated using TACaPe, a deep learning model based on transformers, to predict peptides with antibacterial activity. The selected AMPs were assessed in silico for their ability to bind QS receptors (LasR, RhlR, and PqsR) using molecular docking analysis. The five AMPs with the highest binding affinities were chemically synthesized and tested in vitro against P. aeruginosa ATCC® 27853. Two peptides exhibited significant antibacterial effects and dose-dependent inhibition of biofilm formation. Additionally, both peptides showed synergistic activity with meropenem, lowering its minimum inhibitory concentration (MIC). Hemolytic and cytotoxic assays indicated their potential for therapeutic application.

Conclusions: Computationally designed AMPs exhibited antibacterial and antibiofilm activity against P. aeruginosa. Their synergistic effects with meropenem further enhance their therapeutic potential.

Aims: Screenings for biocontrol organisms against fungal and oomycete pathogens are typically performed on mycelium. While this allows for high-throughput screenings, it omits a major actor in pathogenicity, i.e. the spores. This study aims to improve the screening strategy using a spore-based confrontation assay (SBCA), as well as comparing its performance to the traditional mycelium-based confrontation assay (MBCA) and microscopy analyses of spore germination.

Methods and results: The SBCA was used to screen for 38 candidate biocontrol bacteria against two relevant broad-spectrum phytopathogens, Botrytis cinerea and Phytophthora cactorum. The performance of the SBCA was benchmarked to the traditional mycelium-based confrontation assay and microscopy observations for spore germination inhibition. The SBCA demonstrated a higher hit rate and reproducibility than its counterparts. The bacteria tested exhibited diverse traits in vitro such as production of lytic enzymes, biosurfactant, bioactive volatile organic compounds, and cell-free extracts. These characteristics suggest potential biocontrol modes of action, such as antibiosis (via diffusible metabolites and enzymes) or competition for nutrients and space. For two Pseudomonas strains, the biocontrol activity against P. cactorum was confirmed in planta in a detached leaf assay.

Conclusions: This study showcases a versatile and robust spore-based screening that outperforms conventional screening methods. Through the use of the SBCA, two promising biocontrol strains with antagonistic activity against P. cactorum in strawberry were identified.

Aims: This study investigates the individual and synergistic antibacterial and antibiofilm effects of nisin, resveratrol, naringenin, and saponins against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028. The objective is to identify safe, effective compound combinations to combat Salmonella persistence in food processing environments, which is often caused by biofilm formation.

Methods and results: Using broth microdilution, checkerboard synergy testing, and biofilm/motility assays, the antimicrobial effects were evaluated. Molecular docking and qPCR provided mechanistic insights. Naringenin and resveratrol demonstrated significant antibacterial and antibiofilm activities, especially when combined. The resveratrol-naringenin combination produced the strongest synergistic effect (Fractional Inhibitory Concentration Index (FICI) value of 0.1875), effectively inhibiting bacterial growth and biofilm formation. Molecular analyses revealed this combination targets key proteins (e.g. BcsA, CsgD) and significantly downregulates genes for biofilm formation, quorum sensing, and motility.

Conclusions: The combination of resveratrol and naringenin is a promising natural strategy for controlling Salmonella. Its synergistic action impairs bacterial viability and disrupts biofilm by targeting essential proteins and downregulating key virulence genes.

Aim: This study aimed to investigate the intrinsic cold tolerance of Sphingomonas paucimobilis ZJSH1 and to evaluate the role of endogenous salicylic acid (SA) in mediating cold adaptation. The research focused on highlighting the influence of microbial SA over plant osmolyte accumulation, antioxidant activity, and broader metabolic responses under low-temperature stress.

Methods and results: Wild-type S. paucimobilis ZJSH1 and an SA-deficient ΔpchB mutant were subjected to prolonged low-temperature stress. The ΔpchB mutant, which is unable to produce SA, showed impaired growth and lower osmolyte and exopolysaccharide (EPS) accumulation compared to the wild type. Additionally, the mutant exhibited diminished antioxidant enzyme activities (SOD, POD, and CAT) under cold stress but showed partial recovery when supplemented with exogenous SA. In plant inoculation experiments, wild-type ZJSH1 significantly improved rosette diameter (53.1% over control) and fresh weight (approximately 36% over control) in Arabidopsis plants under cold stress, outperforming the ΔpchB mutant (34.67% and 14%, respectively). Transcriptomic analysis revealed that the wild-type strain upregulated genes involved in SA biosynthesis (pchB), redox detoxification (CatA, CatC, sod), membrane stabilization (Omp16), and nutrient cycling (FixK, NifU, PhoU). In contrast, the ΔpchB mutant activated compensatory pathways, including antioxidant enzymes (KatC), nutrient scavenging systems (pstB, cstA), and membrane-modifying proteins (LptF, MreC), suggesting a SA-independent metabolic flexibility.

Conclusion: S. paucimobilis ZJSH1 exhibits intrinsic cold tolerance with both SA-dependent and SA-independent mechanisms contributing to its adaptation to low temperatures. While SA plays a critical role in enhancing osmolyte accumulation, antioxidant activity, and membrane stabilization, the SA-deficient ΔpchB mutant activates alternative metabolic pathways. These findings suggest that ZJSH1 may be useful in improving plant stress tolerance under cold stress conditions.

Aims: This study investigated the presence of viable Mycobacterium bovis in faecal samples collected from 79 free-ranging domestic cattle in rural KwaZulu-Natal, South Africa.

Methods and results: Faecal samples were processed under biosafety level 3 (BSL-3) conditions and analysed using mycobacterial culture followed by molecular speciation, as well as being screened using the GeneXpert® MTB/RIF Ultra (GXU®) assay. Viable M. bovis was isolated from two animals, confirmed through region-of-difference PCR and spoligotyping. These findings provide rare field-based confirmation of natural faecal shedding of viable M. bovis in cattle. The GXU® detected Mycobacterium tuberculosis complex (MTBC) DNA in eight samples (10.1%), including those that were culture positive, supporting its utility as a rapid screening tool. However, its inability to confirm bacterial viability or differentiate MTBC members remains a limitation. Additionally, nontuberculous mycobacteria (NTMs), including M. avium and M. litorale, were isolated, highlighting environmental exposure and diagnostic challenges in endemic regions.

Conclusions: This study reinforces the need to consider faecal shedding and environmental reservoirs in bTB transmission dynamics, particularly in communal grazing systems. It also emphasizes the importance of integrating culture-based and molecular diagnostics for accurate detection and differentiation of mycobacterial species. These findings have important implications for One Health approaches to bTB surveillance, control, and zoonotic risk mitigation.

Aims: Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) infections are increasingly problematic, leading to worse treatment outcomes and higher healthcare expenditure. This study evaluated laboratory workflows for the detection of ESBL-E in healthcare environments. We aimed to optimize workflows for organism yield, detection accuracy and practical feasibility to support future surveillance and transmission studies, which are needed to inform targeted interventions to interrupt the spread of drug-resistant organisms.

Methods and results: We sampled sinks, toilets, shower drains, shower heads, and high-touch surfaces, within six healthcare facilities in Liverpool, UK. We then assessed recovery of ESBL-producing Escherichia coli (ESBL-Ec) and Klebsiella pneumoniae (ESBL-Kp) using two swab types (foam and polyester), two pre-enrichment broths [Buffered Peptone Water (BPW) and Tryptic Soy Broth (TSB)], two incubation times (4 and 18 h) and four selective agars (CHROMagar ESBL, cefotaxime-supplemented MacConkey, Membrane Lactose Glucuronide Agar (MLGA) and Simmons Citrate Agar with Inositol).Pre-enrichment for 18 h significantly increased recovery of third-generation cephalosporin resistant Gram-negatives, compared to 4 h. The choice of selective agar impacted the number of ESBL-Ec and ESBL-Kp detected and the number of samples requiring additional species confirmation. We found CHROMagar ESBL and cefotaxime-supplemented MLGA performed best overall, demonstrating the highest yield and detection accuracy (i.e. colour of colonies matched their confirmed species) of ESBL-Ec and ESBL-Kp.

Conclusions: Pre-enrichment for 18 h in BPW, followed by plating onto cefotaxime-supplemented MLGA, was the most effective screening method in our context, having a high detection efficacy, whilst maintaining a scalable and accessible cost.