Journal of Applied Microbiology最新文献

Aims: To evaluate the diversity, prevalence, and phenotypic and genotypic characteristics of carbapenem-resistant Gram-negative bacteria (CR-GNB) causing bloodstream infections, and assess the mechanisms driving their dissemination through a multi-center study in nine hospitals of Ecuador.

Methods and results: Between November 2021 and May 2022, 297 Gram-negative bacteria (GNB) were isolated from 273 patients across nine hospitals in Ecuador. Genotypic characterization of carbapenem-resistant GNB from blood cultures was performed by whole genome sequencing (WGS). CR-GNB accounted for 18.8% (56/297), predominantly Klebsiella pneumoniae (41.1%), followed by Enterobacter cloacae complex (16.1%), Acinetobacter baumannii (12.5%), and Pseudomonas aeruginosa (7.1%). CR-GNB showed high resistance to cephalosporins (80%-95%), piperacillin-tazobactam (85.7%), ampicillin-sulbactam (91.1%), and ciprofloxacin (78.6%). Genomic analysis revealed carbapenemase genes blaKPC-2 (most frequent), blaNDM-1, and blaOXA-181 across high-risk clones (e.g. K. pneumoniae ST307, ST258, ST147; A. baumannii ST1187). Carbapenemase genes were plasmid-borne (IncA/C, IncM, IncN, IncF, IncHI2, IncX3, and non-typeable) and associated with transposons (Tn4401, Tn125, and Tn3). Also, blaVIM-2 in Pseudomonas spp. was plasmid- and chromosomally encoded.

Conclusions: Our findings demonstrate a high burden of CR-GNB, primarily due to K. pneumoniae and E. cloacae complex. Furthermore, the widespread distribution of blaKPC-2, blaNDM-1, and blaOXA-181 in high-risk clones, coupled with the frequent plasmid- and transposon-mediated mobilization of these genes, highlights the crucial role of horizontal gene transfer in the dissemination of resistance.

Aims: This study aimed to characterize a colistin- and carbapenem-resistant Pseudomonas aeruginosa ST1560 strain isolated from Guanabara Bay, Brazil, and to investigate the molecular mechanisms underlying its resistance phenotype.

Methods and results: Six surface water samples from Guanabara Bay were collected, yielding 71 P. aeruginosa subjected to antimicrobial susceptibility testing. Three isolates exhibited elevated minimal inhibitory concentrations (MICs) to colistin (≥512, 64, and 8 mg/l) in the absence of mcr genes (1-10). Among these, only strain CCVSU 5861 demonstrated carbapenemase confirmed by Blue Carba test. This strain was selected for whole-genome sequencing (Illumina). Genomic analysis identified the presence of blaKPC-2 and blaOXA-395, along with additional resistance determinants associated with aminoglycosides and fosfomycin. Genes involved in lipopolysaccharide modification, (arnA, arnT, and basS) were also detected, likely contributing to colistin resistance. The blaKPC-2 gene was located adjacent to the mobile genetic element ISKpn6, suggesting potential horizontal gene transfer.

Conclusions: The P. aeruginosa ST1560 displays a complex multidrug resistance profile, including resistance to both colistin and carbapenems. This phenotype appears to be mediated by a combination of acquired resistance genes and chromosomal mechanisms. The localization of blaKPC-2 within a mobile genetic element underscores the risk of dissemination in aquatic environments.

Aims: Vaginal health is crucial to a woman's overall well-being. Bacterial vaginosis, a common gynecological condition resulting from dysbiosis, remains a significant clinical challenge. This study aims to investigate whether postbiotics derived from vaginal Lactobacillus strains exhibit therapeutic effects against bacterial vaginitis.

Methods and results: Postbiotics, consisting of inanimate microorganisms and/or their components, were analyzed and found to contain lactic acid and acetic acid as the primary acidic constituents. In a model of Gardnerella vaginalis-induced bacterial vaginosis, postbiotics demonstrated enhanced antibacterial and antioxidant activities. They significantly alleviated clinical symptoms, modulated the composition of the vaginal microbiota, and increased microbial diversity. Specifically, postbiotics reduced the abundance of endotoxin-producing Escherichia-Shigella and Enterobacteriaceae, while promoting beneficial bacteria such as Muribaculaceae, Lachnospiraceae, and Streptococcus. Additionally, postbiotic treatment restored the balance between Th17 and Treg cells and regulated associated inflammatory factors.

Conclusions: These findings indicate that postbiotics improve bacterial vaginitis through multiple mechanisms, including antibacterial and antioxidant effects, immune regulation, and restoration of vaginal flora structure and metabolic balance. This study highlights the potential clinical value of postbiotics in the treatment of bacterial vaginosis.

Aims: Targeting bacterial virulence is a promising alternative to traditional antibiotics. In this study, we aimed to identify microbiome-derived factors capable of suppressing virulence traits in pathogenic bacteria.

Methods and results: Eighty-two cell-free extracts (CFEs) applied at 10% (v/v), from human (n = 56) and soil (n = 26) bacterial isolates were screened for effects on planktonic growth, biofilm formation, and virulence using a Galleria mellonella infection model. Growth inhibition was rare, observed only for Staphylococcus aureus exposed to a Bacillus flexus extract. Anti-biofilm activity was frequent against Pseudomonas aeruginosa (65/82 CFEs, 79%), less so for S. aureus (20/82, 24%), and absent or reversed for Escherichia coli. Eight CFEs improved larval survival in P. aeruginosa infections; one partly protected against S. aureus, while (47/82, 57%) increased mortality in E. coli-infected larvae. Thirty-eight of 65 anti-biofilm CFEs (58%) for P. aeruginosa tested positive (i.e. observed) in a violacein-based quorum-sensing inhibition assay. One extract (Streptococcus australis) also protected keratinocytes from P. aeruginosa-induced cell loss. Proteomic analysis of P. aeruginosa exposed to S. australis CFE indicated modulation of proteins associated with biofilm regulation and quorum-sensing pathways (e.g. the RsmA/CsrA family), consistent with anti-virulence activity.

Conclusions: Of 82 microbiome-derived CFEs, (65/82, 79%) partially inhibited P. aeruginosa biofilm formation, (20/82, 24%) affected S. aureus, and none inhibited E. coli biofilms. Ten percent improved in vivo survival in P. aeruginosa infections, while (47/82, 57%) increased mortality in E. coli-infected larvae. Anti-virulence effects were pathogen-specific and occurred without measurable impacts on planktonic growth under the conditions tested.

Aims: Most chemically defined media for Bacillus are developed with a focus on an individual species. To broaden the applicability, this study aimed to formulate a chemically defined medium that supports the growth of multiple food-relevant Bacillus species. Specifically, it was the aim to support growth of both food fermentation strains from the Bacillus subtilis clade as well as pathogenic strains from the Bacillus cereus clade.

Methods and results: We developed a new chemically defined medium, named Pafoba, using thirteen Bacillus strains: two from the Bacillus cereus clade and eleven strains from the Bacillus subtilis clade, representing seven species. Medium optimisation involved substituting ammonium chloride and sodium chloride with ammonium sulphate and trisodium citrate, enriching glucose, iron, and phosphate concentrations, and applying nutrientomission assays to identify growth requirements. All strains exhibited growth on Pafoba. Ten strains reached a comparable or higher maximum optical density (OD600) on Pafoba medium compared to Brain Heart Infusion broth. Strain-specific nutrient requirements were identified, including a biotin dependency for Bacillus subtilis strain PRO64, and essential amino acid requirements in Bacillus mycoides and Bacillus cereus strains.

Conclusions: The Pafoba medium supports consistent growth across diverse Bacillus species, making it suitable for both fundamental studies and practical applications such as detection and isolation of Bacillus spp. in food-related contexts.

Aims: Copper-rich shape memory alloys (Cu-SMAs) combine unique mechanical properties and catalytic redox activity, supporting the development of advanced endodontic files to improve patient treatments. This study evaluated the bactericidal activity of CuAlBe and CuAlNi wires combined with H2O2-containing solutions against Enterococcus faecalis, a resilient bacterial species frequently associated with persistent root canal infections.

Methods and results: Activity was assessed on planktonic bacteria through CFU counts and on a 2-week-old monospecies biofilm grown on hydroxyapatite discs using SYTO9/propidium iodide staining and fluorescence confocal microscopy. Both Cu-SMAs combined with H2O2 and ascorbic acid (AA) reduced bacterial viability of planktonic cells by 6 log₁₀ after 1-min exposure. In biofilms, CuAlNi/H₂O₂/AA caused cell permeabilization and lysis within 15 min at the wire-biofilm interface. Prolonged exposure led to a time-dependent spatial expansion of bactericidal effects. Diffusing H₂O₂ reacted with Cu²⁺ from the alloy, sustaining a Fenton-like reaction. Gas bubbles formed along the wire generated a convective flow dispersing the reactive mixture millimetres away from the wire surface.

Conclusions: CuAlNi/H₂O₂/AA combination couples radical generation and convection, enabling deep biofilm eradication beyond the wire/solution interface.

Gram-negative bacterial infections are increasingly becoming resistant to available antibiotic treatment options. The World Health Organization attributed over 1 million deaths to bacterial antimicrobial resistance (AMR) in 2021. While there is a crisis in terms of the available effective antibiotic repertoire, there is also a simultaneous decline in novel drug discovery. In this scenario, the search for alternative or complementary therapeutic options is not only relevant, but also urgently needed. Bacterial virulence factors have been proposed as alternative therapeutic targets since there is lesser propensity for emergence of resistance to these effector molecules. Type 1 fimbriae or FimH of Enterobacteriaceae constitute such a potential target, as these structures are crucial for the initial adhesion and colonization by binding mannose-rich host cell-surface receptors. Additionally, FimH has been associated with multiple diseases, including urinary tract infections (UTIs) and Crohn's disease (CD). The elucidation of Escherichia coli FimH crystal structure has opened the possibility for structure-based drug design to combat these diseases. Many mannose-based compounds are being tried as alternative therapeutics against UTIs and CD with a few molecules showing promise. In this review, we discuss the role of FimH in different diseases, its potential and scope for structure-based development of different mannose-based compounds, and other advanced FimH-blocking therapeutics in preventing these infections.

Aims: Multidrug-resistant (MDR) foodborne pathogens pose an urgent global threat. We surveyed retail foods in Ismailia Governorate, Egypt, to identify highly resistant strains and develop a phage-based biocontrol strategy.

Methods and results: Staphylococcus aureus was detected in 45% of samples (70% meat) and Escherichia coli in 52.5% (80% in arugula), often exceeding safety standards. From 40 representative isolates, the most resistant strains: S. aureus SL4 from lettuce (GenBank OR646818; MAR index 0.44) and E. coli ER2 from arugula (GenBank OR646817; MAR index 0.72), were selected for phage targeting. Two sewage-derived lytic phages, STB (Siphoviridae; host SL4) and ECB (Podoviridae; host ER2), were isolated. Both had ideal biocontrol traits: latent periods ∼10 min, burst sizes ∼2 × 10¹¹ PFU cell⁻¹, and stability at 28-55°C and pH 5-9. In simulated decontamination trials, phage soaking on meat (CFU g⁻¹), arugula, and cutting boards (CFU cm⁻²) reduced bacterial counts by 3.4-6.4 log₁₀ after 1 h, 4.7-6.4 log₁₀ after 3 h, and near-complete eradication by 6 h (9.20-8.58 log₁₀ for SL4, 9.41-7.86 log₁₀ for ER2). Soaking, spraying, and the phage cocktail all outperformed 5% vinegar by 0.8-3.8 log₁₀; the cocktail broadened host range but had slightly slower kill kinetics.

Conclusions: Optimized phage application enables rapid, chemical-free eradication of MDR pathogens from foods and food-contact surfaces.

Aims: To achieve optimal application of antimicrobials to poultry processing requires an understanding of the potential for resistance by foodborne pathogens such as Salmonella. The objective of this study was to use transposon sequencing (Tn-seq) to identify genetic factors required for Salmonella Typhimurium's tolerance to PAA.

Methods and results: A genome-saturated Tn5 mutant library (input pool) was inoculated in two replicates into either 6% chicken meat extract (CME) or 11% diluted Luria-Bertani (LB) broth, both supplemented with 15 ppm PAA. Cultures were incubated for 90 minutes at 37°C. Viable Tn5 mutant cells recovered on LB agar plates were combined to form four output pools (two CME and two LB). Genomic DNA extracted from these pools were deep sequenced (Tn5-junction reads). Conditionally essential genes required for fitness in 6% CME and 11% LB were identified and subjected to pathway enrichment analysis (ShinyGO graphical gene-set enrichment tool). We identified two overlapping sets of conditionally essential genes (276 common genes) required for survival in the presence of PAA. In CME, 362 conditionally essential genes were identified, while LB media revealed 536 genes. Pathway enrichment analysis showed that these genes were significantly enriched in pathways such as pyruvate metabolism, the tricarboxylic acid cycle, fumarate reductase/succinate dehydrogenase (transmembrane subunit and 2Fe-2S iron-sulfur cluster binding domain), stress response, and oxidoreductase activity. Notably, genes previously shown to increase sensitivity to PAA upon inactivation (sdhC, zwf, pta, and icdA) were identified as conditionally essential in this study, further validating the Tn-seq data.

Aims: Biofilms formed by Salmonella are a significant concern in the poultry industry due to their role in pathogen persistence. However, there is a lack of data observing the expression of biofilm related genes in different Salmonella serovars. The aim of this study was to investigate the expression patterns of key biofilm-associated genes across three Salmonella serovars, namely Salmonella Typhimurium, Kentucky, and Reading, throughout their biofilm growth cycles.

Methods and results: The expressions of csgD, bapA, bcsA, adrA, and luxS were analyzed in cultures representing different biofilm growth phases: 12 h and 24 h planktonic cells, 4-day old biofilms, and 5-day old biofilms under nutrient deprivation. The findings from this study revealed that only S. Reading exhibited upregulation of these genes at the 24 h planktonic stage at a maximum of 9.58-fold. In contrast, a downregulation of all five genes was noted in the 4-day old biofilms for all serovars. Most notably, bapA was downregulated by 3 765-fold in S. Typhimurium. Upon subjecting the biofilms to nutrient deprivation, there was a notable recovery in the activity of these genes across all serovars with the exception of csgD in S. Typhimurium.

Conclusion: These results suggest that expression of biofilm-associated genes is stimulated by nutrient availability even at biofilm maturity and may vary among different serovars.