Category Archives: Journal of Applied Microbiology

Transcriptome reveals BCAAs biosynthesis pathway is influenced by lovastatin and can act as a potential control target in Phytophthora sojae

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Abstract

Aims

Lovastatin has been indicated to impair growth and development of Phytophthora sojae. Therefore, this study was performed to understand the inhibitory mechanism of lovastatin and investigate the metabolic pathway potentially served as a new control target for this plant pathogen.

Methods and Results

Whole transcriptome analysis of lovastatin-treated P. sojae was performed by RNA-sequencing. The results revealed that 84 genes were upregulated and 58 were downregulated with more than fourfold changes under treatment. Kyoto Encyclopaedia of Genes and Genomes analysis indicated that the branched-chain amino acids (BCAAs) biosynthesis pathway was abundantly enriched. All enzymes in the BCAAs biosynthesis pathway were identified in the P. sojae genome. Moreover, the study found that the herbicide flumetsulam targeting acetohydroxyacid synthase (AHAS) of the BCAAs biosynthesis pathway could effectively inhibit mycelial growth of P. sojae.

Conclusions

Lovastatin treatment significantly influences the BCAAs biosynthesis pathway in P. sojae. Moreover, the herbicide flumetsulam targets AHAS and inhibits growth of P. sojae.

Significance and Impact of the Study

The present study revealed that BCAAs biosynthesis pathway was influenced by lovastatin treatment and its key enzyme AHAS was identified as a potential new control target, which provides clues for exploring more oomycetes to control plant diseases caused by P. sojae.

Dynamics of the fermentation quality and microbiotsa in Ephedra sinica treated native grass silage

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Abstract

Aims

This study aimed to evaluate the effects of Ephedra sinica on physicochemical characteristics and bacterial community of ensiled native grass by multiple physicochemical analyses combined with high-throughput sequencing.

Methods and Results

Treatments were a control treatment with no additive (CON), E. sinica was added at 1% (CEa1), 3% (CEa2), and 5% of the fresh materials (CEa3). Compared to the CON group, the dry matter and water-soluble carbohydrate contents were significantly (p < 0.05) decreased in the CEa1 group. Compared to the CON group, the pH was significantly (p < 0.05) decreased in E. sinica treated silages, and a higher lactic acid content was observed in E. sinica treated silages. At the genus level, the abundance of Enterococcus, Lactobacillus, Pediococcus, and Weissella were the predominant member in the CON, CEa1, CEa2, and CEa3 groups, respectively. The abundance of Lactobacillus was significantly (p < 0.05) increased in the CEa1 group and Pediococcus was significantly (p < 0.05) increased in the CEa2 group. According to the 16S rRNA gene-predicted functional profiles, the inoculation of E. sinica accelerated the carbohydrate metabolism.

Conclusions

In summary, the addition of E. sinica could improve the silage quality of native grass by regulating the bacterial community, and the addition of a 1% percentage of fresh materials exhibited the potential possibility of responding to get high-quality native grass silages.

Significance and Impact of the Study

The utilization of herbal additives on fermentation quality combined with 16S rRNA gene-predicted functional analyses will contribute to the direction of future research in improving silage quality.

Identification of spoilage microflora in draught beer using culture‐dependent methods

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Abstract

Aims

To determine whether the culture-dependent spoilage microflora found in draught beer are influenced by beer style.

Methods and Results

Four beer styles—lager, ale, stout and cask ale – were sampled twice from five different public houses (accounts) in four different locations. The microbiological quality of the dispensed beers was determined by a culture-dependent method (‘forcing’), measuring the increase in turbidity after incubation at 30°C. The quality of draught beer varied from ‘excellent’ to ‘poor’ with cask beer samples having a higher Quality Index (90%) with keg ale the lowest (67.5%). With PCR amplified DNA (ITS1, ITS4, 16S rRNA primers) and blast identification of microflora, 386 colonies from agar plates were identified with 28 different micro-organisms from five genera of yeast and six of bacteria. Seven micro-organisms were found in all beer styles with Brettanomyces bruxellensis, B. anomalus and Acetobacter fabarum representing 53% of the identified micro-organisms. A subsequent, limited study using PALL multiplex PCR GeneDisc technology on forced samples (without selection on plates) suggests that draught beer microflora is qualitatively broader. It is noteworthy that the microflora of spoilt draught beer resembles that involved in the production of Belgian Lambic sour beers.

Conclusions

Draught beer was of variable quality. Culture-dependent analysis suggests that species of Brettanomyces and Acetobacter are core microflora with some micro-organisms being associated with beer style.

Significance and Impact of the Study

The microbiological quality of draught beer is important both commercially and to the consumer. Here, we report the core and diverse microflora found in different styles of draught beer using culture-dependent methods.

A 16S rRNA amplicon approach to the structural and functional diversity of bacterial communities associated with horse gram crop for drought mitigation and sustainable productivity

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Abstract

Aim

In this study, 16S rRNA amplicon sequencing analyses were performed to determine the diversity of the bacterial community present in the soil, rhizosphere region, root nodules and seeds of the horse gram plant.

Methods and Results

We observed the dominance of Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria, Bacteroidetes, Planctomycetes and Gemmatimonadetes across all four domains of the horse gram plant. For community analyses, the significance of the alpha diversity was estimated using the Shannon index, Simpson index and Chao1 index, which revealed no significant difference among the samples. However, the estimation of the beta diversity indicated a significant difference among the samples, with p < 0.001 and R2 = 1. A strong positive correlation was found between the rhizosphere and root nodule samples. Comparative genomics of the 16S rRNA gene showed that ammonium-oxidizing metabolism (amoA), nitrite-reducing metabolism (nirK) and nitrogen-fixing metabolism (nifH) were prominent mechanisms in all samples. The genes involved in the biosynthesis of amino acids, purine metabolism and nitrogen metabolism were identified as the key genes associated with the functional traits of microbial domains in horse gram.

Conclusion

The culturable microbes associated with horse gram can be used as a substitute for synthetic fertilizers to maintain soil fertility and ecological health in agricultural practices.

Significance and Impact of the study

Determining the survival strategies of bacterial communities that positively respond to multiple gate selection helps in understanding the structural diversity and functional traits primarily focused on the development of beneficial microbial consortium for promoting plant growth.

Transfer of Phi6 bacteriophage between human skin and surfaces common to consumer‐facing environments

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Abstract

Aims

This study aimed to determine the extent of Phi6 (Φ6) transfer between skin and surfaces relevant to consumer-facing environments based on inoculum matrix, surface type and contact time.

Methods and Results

Φ6 transfer rates were determined from skin-to-fomite and fomite-to-skin influenced by inoculum matrix (artificial saliva and tripartite), surface type (aluminium, plastic, stainless steel, touchscreen, vinyl and wood) and contact time (5 and 10 s). Significant differences in estimated means were observed based on surface type (both transfer directions), inoculum matrix (skin-to-fomite) and contact time (both transfer directions). During a sequential transfer experiment from fomite-to-skin, the maximum number of consecutive transfer events observed was 3.33 ± 1.19, 2.33 ± 1.20 and 1.67 ± 1.21 for plastic, touchscreen and vinyl, respectively.

Conclusions

Contact time significantly impacted Φ6 transfer rates, which may be attributed to skin absorption dynamics. Surface type should be considered for assessing Φ6 transfer rates.

Significance and Impact of the Study

Although the persistence of Φ6 on fomites has been characterized, limited data are available regarding the transfer of Φ6 among skin and fomites. Determining Φ6 transfer rates for surfaces in consumer-facing environments based on these factors is needed to better inform future virus transmission mitigation strategies.

Flexible multiplex PCR to detect SARS‐CoV‐2, coronavirus OC43 and influenza A virus in nasopharyngeal swab samples

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Abstract

Introduction

Quantitative reverse transcription PCR (RT-qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits.

Methods and results

In this study, combinations of four primers/probe sets were used to construct a flexible RT-qPCR assay which is capable of discriminating between SARS-CoV-2 and the seasonal human coronavirus HCoV-OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed.

Conclusions

This study provides an efficient method for the simultaneous detection of SARS-CoV-2, HCoV-OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results.

Significance and impact of the study

The multiplex RT-qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.

Ultrasound‐assisted facile synthesis of Boron‐Heck‐coupled sclareol analogues as potential antibacterial agents against Staphylococcus aureus

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Abstract

Aim

To evaluate the antimicrobial capability of sclareol and its derivatives against Staphylococcus aureus and its Methicillin-resistant strain (MRSA).

Methods and Results.

A new series of Boron-Heck-coupled sclareol analogues were prepared by structural modifications at the C-15 terminal double bond of sclareol using ultrasonication. The structural modifications were designed to keep the stereochemistry of all the five chiral centres of sclareol intact. A two-step reaction scheme consisting of Boron-Heck coupling of sclareol followed by Wittig reaction was carried out to produce novel sclareol congeners for antimicrobial evaluation. Three compounds SAJ-1, SAJ-2 and SB-11 exhibited strong antibacterial activity against S. aureus and Methicillin-resistant strain (MRSA) with MIC values between 3 and 11 μM. Among all the screened compounds, SAJ-1 and SAJ-2 showed the best antibiofilm profiles against both strains. Moreover, SAJ-1 and SAJ-2 acted synergistically with streptomycin against S. aureus while creating varying outcomes in combination with ciprofloxacin, penicillin and ampicillin. SAJ-1 also acted synergistically with ampicillin against S. aureus, while SB-11 showed synergism with ciprofloxacin against both pathogens. Moreover, SAJ-1 and SAJ-2 also inhibited staphyloxanthin production in S. aureus and MRSA and induced postantibiotic effects against both pathogens.

Conclusions

It can be inferred that SAJ-1, SAJ-2 and SB-11 may act as potential chemical entities for the development of antibacterial substances. The study revealed that SAJ-1 and SAJ-2 are the most suitable sclareol analogues for further studies towards the development of antibacterial substances.

Significance and Impact of the Study

SAJ-1, SAJ-2 and SB-11 show promising antibacterial properties against Staphylococcus aureus. Efforts should be made and more research should be done utilizing in vivo models to determine their efficacy as antibiotics.

Effects of Bacillus subtilis natto JLCC513 on gut microbiota and intestinal barrier function in obese rats

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Abstract

Aims

This study aimed to investigate the effects of Bacillus subtilis natto JLCC513 (JLCC513) on gut microbiota, inflammation and intestinal barrier function in high-fat-diet (HFD) rats.

Methods and Results

Sprague–Dawley (SD) rats were fed HFD for 16 weeks, and treated with JLCC513 in 9th week. The oral administration of JLCC513 decreased body weight and reduced the inflammation level in HFD rats. Pathologically, JLCC513 prevented the detachment of ileal villus and increased the villus height in rats. Mechanistically, western blot analysis showed that the protein levels of tight junction (TJ) proteins involved in intestinal barrier function, including zonula occludens-1 (ZO-1), occludin and claudin-1, were increased after JLCC513 treatment. Meanwhile, JLCC513 treatment also decreased the protein levels of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB) and NOD-like receptor protein 3 (NLRP3), indicating inhibition of the TLR4/NF-κB/NLRP3 pathway. Furthermore, faecal analysis showed that JLCC513 increased the abundance of Lactobacillus and Oscillospira and the ratio of Firmicutes/Bacteroidetes (F/B), and decreased the levels of Blautia and C_Clostridium.

Conclusions

JLCC513 alleviated intestinal barrier dysfunction by inhibiting TLR4/NF-κB/NLRP3 pathway and regulating gut microbiota disorders.

Significance and Impact of Study

Our study might provide new treatment strategies for obesity and metabolic diseases.

Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification‐lateral flow assay combined with immunomagnetic separation

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Abstract

Aims

The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk.

Methods and results

Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction.

Conclusions

The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min.

Significance and Impact of the Study

The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

Altered molecular attributes and antimicrobial resistance patterns of Vibrio cholerae O1 El Tor strains isolated from the cholera endemic regions of India

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Abstract

Aims

The present study aimed to document the comparative analysis of differential hypervirulent features of Vibrio cholerae O1 strains isolated during 2018 from cholera endemic regions in Gujarat and Maharashtra (Western India) and West Bengal (Eastern India).

Methods and Results

A total of 87 V. cholerae O1 clinical strains from Western India and 48 from Eastern India were analysed for a number of biotypic and genotypic features followed by antimicrobial resistance (AMR) profile. A novel polymerase chain reaction was designed to detect a large fragment deletion in the Vibrio seventh pandemic island II (VSP-II) genomic region, which is a significant genetic feature of the V. cholerae strains that have caused Yemen cholera outbreak. All the strains from Western India belong to the Ogawa serotype, polymyxin B-sensitive, hemolytic, had a deletion in VSP-II (VSP-IIC) region and carried Haitian genetic alleles of ctxB, tcpA and rtxA. Conversely, 14.6% (7/48) of the strains from Eastern India belonged to the Inaba serotype, polymyxin B-resistant, nonhemolytic, harboured VSP-II other than VSP-IIC type, classical ctxB, Haitian tcpA and El Tor rtxA alleles. Resistance to tetracycline and chloramphenicol has been observed in strains from both regions.

Conclusions

This study showed hypervirulent, polymyxin B-sensitive epidemic causing strains in India along with the strains with polymyxin B-resistant and nonhemolytic traits that may spread and cause serious disease outcomes in future.

Significance and impact of the study

The outcomes of this study can help to improve the understanding of the hyperpathogenic property of recently circulating pandemic Vibrio cholerae strains in India. Special attention is also needed for the monitoring of AMR surveillance because V. cholerae strains are losing susceptibility to many antibiotics used as a second line of defence in the treatment of cholera.