Category Archives: ORIGINAL ARTICLE

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.

Antibacterial and antibiofilm activities of thiazolidine‐2,4‐dione and 4‐thioxo‐thiazolidin‐2‐one derivatives against multidrug‐resistant Staphylococcus aureus clinical isolates

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Abstract

Aims

Antimicrobial resistance is one of the highest priorities in global public health with Staphylococcus aureus among the most important microorganisms due to its rapidly evolving antimicrobial resistance. Despite all the efforts of antimicrobial stewardship, research and development of new antimicrobials are still imperative. The thiazolidine ring is considered a privileged structure for the development of new antimicrobials. This study aimed to compare the antibacterial effects of two analogue series of thiazolidine-2,4-dione and 4-thioxo-thiazolidin-2-one against multidrug-resistant Staph. aureus clinical isolates.

Methods and Results

The derivatives 1a, 2a and 2b exhibited MIC between 1–32 μg ml−1, with time-to-kill curves showing a bactericidal effect up to 24 h. In the antibiofilm assay, the most active derivatives were able to inhibit about 90% of biofilm formation. The 4-thioxo-thiazolidine-2-one derivatives were more active against planktonic cells, while the thiazolidine-2,4-dione derivatives were able to disrupt about 50% of the preformed biofilm. In the in vivo infection model using Caenorhabditis elegans as a host, the derivatives 1a, 2a and 2b increased nematode survival with a concentration-dependent effect. Exposure of Staph. aureus to the derivatives 2a and 2b induced surface changes and decrease cell size. None of the derivatives was cytotoxic for human peripheral blood mononuclear cells (PBMC) but showed moderate cytotoxicity for L929 fibroblasts.

Conclusion

The 5-(3,4-dichlorobenzylidene)-4-thioxothiazolidin-2-one (2b) was the most active derivative against Staph. aureus and showed higher selective indices.

Significance and Impact of the Study

4-thioxo-thiazolidin-2-one is a promising scaffold for the research and development of new antimicrobial drugs against multidrug-resistant Staph. aureus.

Prevalence, genetic diversity, antibiotic resistance and biofilm formation of Acinetobacter baumannii isolated from urban environments

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Abstract

Aim

Acinetobacter baumannii is a well-known nosocomial pathogen that has been isolated from different clinical sources. This pathogen also causes community-acquired infections, with mortality rates as high as 64%. The exact natural habitat of this bacterium is still unknown. In this study, we investigated the prevalence of A. baumannii in diverse soil and high-touch surface samples collected from a university campus, malls, parks, hypermarkets and produce markets, roundabout playground slides and bank ATMs.

Methods and Results

All obtained isolates were characterized for their antibiotic susceptibility, biofilm formation capacities, and were typed by multi-locus sequence analysis. A total of 63 A. baumannii isolates were recovered, along with 46 Acinetobacter pittii and 8 Acinetobacter nosocomialis isolates. Sequence typing revealed that 25 A. baumannii isolates are novel strains. Toilets and sink washing basins were the most contaminated surfaces, accounting for almost 50% of the isolates. A number of A. baumannii (n = 10), A. pittii (n = 19) and A. nosocomialis (n = 5) isolates were recovered from handles of shopping carts and baskets. The majority of isolates were strong biofilm formers and 4 isolates exhibited a multi-drug resistant phenotype.

Conclusions

Our study is the first to highlight community restrooms and shopping carts as potential reservoirs for pathogenic Acinetobacter species. Further studies are required to identify the reasons associated with the occurrence of A. baumannii inside restrooms. Proper disinfection of community environmental surfaces and spreading awareness about the importance of hand hygiene may prevent the dissemination of pathogenic bacteria within the community.

Significance and Impact of the study

Serious gaps remain in our knowledge of how A. baumannii spreads to cause disease. This study will advance our understanding of how this pathogen spreads between healthcare and community environments. In addition, our findings will help healthcare decision-makers implement better measures to control and limit further transmission of A. baumannii.

Antimicrobial effects of automobile screen washes against Legionella pneumophila

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Abstract

Aims

Legionella pneumophila (Lp), a human pathogen, has been detected in windscreen wiper fluid reservoirs (WWFRs) where commercial screen washes (CSWs) are commonly added. Limited information is available on CSWs against planktonic Lp; however, responses of sessile Lp and planktonic Lp pre-acclimated in nutrient-limited water to CSWs remain unknown. This study thus investigates the antibacterial effects of CSWs on sessile and starved planktonic Lp, in comparison with unstarved Lp.

Methods and Results

Lp biofilms were produced on glass and WWFR materials of high-density polyethylene (HDPE) and polypropylene (PP). Planktonic Lp with and without acclimation in tap water were prepared. Log reductions in cell counts averaged 0.4–5.0 for 10 brands of CSWs against sessile Lp and 1.0–3.9 and 0.9–4.9, respectively, against starved and unstarved planktonic Lp for five CSWs. Both biofilm formation and acclimation in tap water enhanced Lp resistance to CSWs. Significantly different log-reduction values among CSW brands were observed for sessile Lp on HDPE and planktonic Lp regardless of acclimation (p < 0.05).

Conclusions

Biofilm formation, starvation acclimation and CSW brand are crucial factors influencing Lp response to CSWs.

Significance and Impact of Study

This study advances the knowledge of Lp reaction in anthropogenic water systems with CSWs.

Dose–response analysis of Bacillus thuringiensis HD‐1 cry‐ spore reduction on surfaces using formaldehyde with pre‐germination

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Abstract

Aim

To establish a basis for rapid remediation of large areas contaminated with Bacillus anthracis spores.

Methods and Results

Representative surfaces of wood, steel and cement were coated by nebulization with B. thuringiensis HD-1 cry- (a simulant for B. anthracis) at 5.9 ± 0.2, 6.3 ± 0.2 and 5.8 ± 0.2 log10 CFU per cm2, respectively. These were sprayed with formaldehyde, either with or without pre-germination. Low volume (equivalent to ≤2500 L ha−1) applications of formaldehyde at 30 g l−1 to steel or cement surfaces resulted in ≥4 or ≤2 log10 CFU per cm2 reductions respectively, after 2 h exposure. Pre-germinating spores (500 mmol l−1 l-alanine and 25 mmol l−1 inosine, pH 7) followed by formaldehyde application showed higher levels of spore inactivation than formaldehyde alone with gains of up to 3.4 log10 CFU per cm2 for a given dose. No loss in B. thuringiensis cry- viability was measured after the 2 h germination period, however, a pre-heat shock log10 reduction was seen for B. anthracis strains: LSU149 (1.7 log10), Vollum and LSU465 (both 0.9 log10), LSU442 (0.2 log10), Sterne (0.8 log10) and Ames (0.6 log10).

Conclusions

A methodology was developed to produce representative spore contamination of surfaces along with a laboratory-based technique to measure the efficacy of decontamination. Dose–response analysis was used to optimize decontamination. Pre-germinating spores was found to increase effectiveness of decontamination but requires careful consideration of total volume used (germinant and decontaminant) by surface type.

Significance and Impact of the Study

To be practically achievable, decontamination of a wide area contaminated with B. anthracis spores must be effective, timely and minimize the amount of materials required. This study uses systematic dose–response methodology to demonstrate that such an approach is feasible.

Laccase production from Bacillus aestuarii KSK using Borassus flabellifer empty fruit bunch waste as a substrate and assessing their malachite green dye degradation

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Abstract

Aims

The lignocellulosic waste, Borassus flabellifer empty fruit bunch waste (BFEFBW), was employed to produce laccase using Bacillus aestuarii KSK under solid-state fermentation (SSF) conditions and to assess the efficiency of malachite green (MG) dye decolourization.

Methods and Results

Abiotic factors such as pH (5.0–9.0), temperature (25–45°C) and incubation time (24–96 h) were optimized using Response surface methodology-Box-Behenan Design (RSM-BBD) to exploit the laccase production. The anticipated model revealed that the highest laccase activity of 437 U/ml shows after 60 h of incubation at 35°C at pH 7.0. The bacterial laccase was used to remove 89% of the MG dye in less time.

Conclusion

The laccase from B. aestuarii KSK decolorizes the MG and thereby making it a suitable choice for wastewater treatment from industrial effluents.

Significance and Impact of the Study

This study is the first report on the production of laccase from B. flabellifer empty fruit bunch waste as a substrate. Bacillus aestuarii KSK was isolated from the soil sample and used to produce laccase under SSF conditions. The bacterial laccase has the potential for industrial application in textile waste dye treatment.

A novel dibenzofuran from endophytic fungus Mycosphaerella nawae preferentially inhibits CD4+ T cell activation and proliferation

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Abstract

Aim

To obtain promising immunosuppressants from endophytic fungus.

Methods and Results

The endophytic fungus Mycosphaerella nawae (ZJLQ129) was isolated from the plant Smilax china L. and its secondary metabolites extracted and fractionated through column chromatography. The metabolites were further modified by a derivatization reaction with ammonium hydroxide. After isolation and derivatization, a new dibenzofuran named as (+)isomycousnine enamine (iME) was obtained. The structures of the derivatives were determined based on chemical evidences and extensive spectroscopic methods including 2D-NMR, DEPT and HRESI-MS spectra. The immune activities of iME were first evaluated on the proliferation and cytokines (IL-2 and IFN-γ) production of T and B cells by using MTT and ELISA methods respectively. Then, its effects on the proliferation of T-cell subsets (CD4+ and CD8+ T cells), as well as CD25 and CD69 expressions were also determined by flow cytometry. Finally, by using Cytometric Bead Array (CBA), the impacts of iME on the secretion of Th1/Th2/Th17 cytokines from purified CD4+ T cells were assayed. The results showed that iME not only selectively suppressed the immune responses of T cells, but also preferentially inhibited the activation and proliferation of CD4+ T cells.

Conclusion

A novel dibenzofuran derived from endophytic fungus Mycosphaerella nawae preferentially inhibits CD4+ T-cell activation and proliferation.

Significance and Impact of the Study

This work obtained iME, a new dibenzofuran derived from endophytic fungus. iME has the capacity to inhibit CD4+ T-cell activation and therefore is a novel potential immunosuppressant for development in the future.

Simple and field amenable loop‐mediated isothermal amplification‐lateral flow dipstick assay for detection of west Nile virus in human clinical samples

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Abstract

Aim

West Nile encephalitis caused by infection with the West Nile virus (WNV) is endemic in many regions of the world and is a global public health threat. The aim of this report was to develop a method using colorimetry-based reverse-transcription loop-mediated isothermal amplification (cRT-LAMP) and RT-LAMP combined with lateral-flow dipstick (LFD) for rapidly detecting WNV in low-infrastructure settings.

Methods and Results

The primers for the cRT-LAMP and RT-LAMP-LFD assays were designed based on env gene of the WNV. Primers concentration, temperature and time were optimized for cRT-LAMP and RT-LAMP-LFD. The diagnostic performance of the cRT-LAMP and RT-LAMP-LFD assays was evaluated using human serum samples from 110 patients who were clinically suspected to be infected with WNV. The RT-LAMP was performed in a heating block at 63°C for 40 min. The LAMP amplicons were visible in the lateral-flow dipstick within 5 min. The detection limit of the developed cRT-LAMP and RT-LAMP-LFD assays was 10 copies and this assay showed a high degree of specificity for WNV. Compared with quantitative real-time RT-PCR assay, the kappa value of cRT-LAMP and RT-LAMP-LFD were 0.970.

Conclusions

These results showed that the newly developed WNV-specific cRT-LAMP and RT-LAMP-LFD assays can be employed as an alternative method for screening of WN-suspected human samples. The results revealed that the assay could potentially identify the virus without interference from human serum samples. Collectively, all results revealed that cRT-LAMP and RT-LAMP-LFD assays offer a suitable field-based diagnosis of WNV.

Significance and Impact of the Study

The cRT-LAMP and LAMP-LFD platform for the detection of WNV is rapid, accurate and simple-to-perform. Our present method has not only a short turnaround time but also avoided cross-contamination problem. Moreover, the use of simple lateral flow dipsticks broadens its application potential for the point-of-care use in resource-limited settings during outbreak situations. To the best of our knowledge, this is the first report for the development of cRT-LAMP and LAMP-LFD assays for rapid, simple, specific and sensitive detection of WNV using human clinical samples and EvaGreen dye.

Beneficial effects of fermented barley extracts on inflammatory status and gut microbiota in high‐fat diet‐induced obese rats

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Abstract

Aims

To explore how fermented barley extracts could affect obesity-associated inflammatory responses to ameliorate high-fat diet (HFD)-induced obesity, and investigate whether their anti-inflammatory properties were affected by modulating the gut microbiota.

Methods and Results

Twenty-four male rats were assigned randomly to three groups for 8 weeks. Inflammatory status and gut microbiota in HFD-induced obese rats were measured by enzyme linked immunosorbent assay and 16sRNA sequencing technology. The dietary supplementation of Extract of fermented barley with L. plantarum JDM1 (LFBE) reduced HFD-induced obesity and improved insulin sensitivity. LFBE significantly decreased the levels of lipopolysaccharide (LPS) and pro-inflammatory cytokines (tumour necrosis factor-α, interleukin [IL]-6, IL-1β, monocyte chemotactic protein-1), and increased anti-inflammatory cytokines (IL-10) in serum. In addition, LFBE suppressed the activation of nuclear factor-κB (NF-κB) by inhibiting the inhibitor of NF-κB alpha degradation and phosphorylation of JNK/p38 mitogen-activated protein kinases in adipose tissue. Combined with changes in gut microbiota, these results illustrated that LFBE treatment markedly decreased the proportion of the LPS-producing opportunistic pathogens and increased the proportion of Bifidobacterium.

Conclusions

Administration of LFBE has beneficial effects on ameliorating HFD-induced obesity and insulin resistance, lessening HFD-induced gut microbiota dysbiosis and pro-inflammatory cytokines secretion.

Significance and Impact of this Study

The results suggest that fermented barley extracts may be a useful functional compound and beneficial to improve inflammatory status and gut microflora.