scholarly journals The detection efficiency of digital PCR for the virulence genes of waterborne pathogenic bacteria

Author(s):  
Xiaojie Shi ◽  
Gang Liu ◽  
Liangliang Shi ◽  
Menghao Chen ◽  
Xiaojin Wu ◽  
...  

Abstract Waterborne pathogens are the primary concern for the safe reuse of wastewater. Although digital PCR (dPCR) is considered promising for absolutely quantitating genes, the detection efficiency of dPCR is affected by many factors. This study tested 8 virulence genes of pathogenic bacteria on a control plasmid and reclaimed water samples with reported primer-probe sets and designed ones on quantitative PCR (qPCR) and dPCR. Probe efficiency, data analysis, and PCR inhibition, were found to affect the detection efficiency of dPCR. Firstly, poor probe quality, which is determined by probe quenching and activation efficiencies, was the main cause of PCR failure. Secondly, even if the PCR was successful, the probe quality and signal intensity could still affect the quantitative process. Manual analysis of dPCR data on the weak signal intensity would significantly reduce errors. And lastly, the sensitivity of PCR inhibition was lower in dPCR than qPCR, but inhibition still existed. The dPCR produced various detection efficiencies for different targets in one sample indicating inconstant inhibitory effects. Dilution was still the proper approach to overcome inhibition, but decreased detection limit. More studies are required to ensure accurate waterborne pathogen quantitation by dPCR.

Antibiotics ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 98 ◽  
Author(s):  
Eunice Mgbeahuruike ◽  
Pia Fyhrquist ◽  
Heikki Vuorela ◽  
Riitta Julkunen-Tiitto ◽  
Yvonne Holm

Piper guineense is a food and medicinal plant commonly used to treat infectious diseases in West-African traditional medicine. In a bid to identify new antibacterial compounds due to bacterial resistance to antibiotics, twelve extracts of P. guineense fruits and leaves, obtained by sequential extraction, as well as the piperine and piperlongumine commercial compounds were evaluated for antibacterial activity against human pathogenic bacteria. HPLC-DAD and UHPLC/Q-TOF MS analysis were conducted to characterize and identify the compounds present in the extracts with promising antibacterial activity. The extracts, with the exception of the hot water decoctions and macerations, contained piperamide alkaloids as their main constituents. Piperine, dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine and derivatives of piperine and piperidine were identified in a hexane extract of the leaf. In addition, some new piperamide alkaloids were identified, such as a piperine and a piperidine alkaloid derivative and two unknown piperamide alkaloids. To the best of our knowledge, there are no piperamides reported in the literature with similar UVλ absorption maxima and masses. A piperamide alkaloid-rich hexane leaf extract recorded the lowest MIC of 19 µg/mL against Sarcina sp. and gave promising growth inhibitory effects against S. aureus and E. aerogenes as well, inhibiting the growth of both bacteria with a MIC of 78 µg/mL. Moreover, this is the first report of the antibacterial activity of P. guineense extracts against Sarcina sp. and E. aerogenes. Marked growth inhibition was also obtained for chloroform extracts of the leaves and fruits against P. aeruginosa with a MIC value of 78 µg/mL. Piperine and piperlongumine were active against E. aerogenes, S. aureus, E. coli, S. enterica, P. mirabilis and B. cereus with MIC values ranging from 39–1250 µg/mL. Notably, the water extracts, which were almost devoid of piperamide alkaloids, were not active against the bacterial strains. Our results demonstrate that P. guineense contains antibacterial alkaloids that could be relevant for the discovery of new natural antibiotics.


Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2401
Author(s):  
Jakkrit Aisara ◽  
Pairote Wongputtisin ◽  
Somkid Deejing ◽  
Chutamas Maneewong ◽  
Kridsada Unban ◽  
...  

Red onion is a popular ingredient in many Thai dishes and has recently been promoted for commercial cultivation. In this study, inulin-fructooligosaccharides (inulin-FOSs) were extracted from red onions in a simplified extraction method. The extract contained 24.00 ± 0.38 g/L free glucose, fructose and sucrose, while the level of FOSs was recorded at 74.0 ± 2.80 g/L with a degree of polymerization of 4.1. The extract was resistant to simulated gastrointestinal conditions, while selectively promoting probiotic lactobacilli. These outcomes resulted in inhibitory effects against various pathogenic bacteria. The in vitro batch culture fermentation of the extract by natural mixed culture indicated that an unknown sugar identified as neokestose was more rapidly fermented than 1-kestose and other longer-chain inulin-FOSs. Notably, neokestose selectively encouraged a bifidogenic effect, specifically in terms of the growth of Bifidobacteirum breve, which is an infant-type probiotic bacterium. This is the first report to state that neokestose could selectively enhance the bifidogenic effect. In summary, inulin-FOSs extract should be recognized as a multifunctional ingredient that can offer benefits in food and pharmaceutical applications.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Melanie Radloff ◽  
Isam Elamri ◽  
Tamara N. Grund ◽  
Luca F. Witte ◽  
Katharina F. Hohmann ◽  
...  

AbstractCytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure–activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.


2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Emily Moraes Roges ◽  
Verônica Dias Gonçalves ◽  
Maira Duarte Cardoso ◽  
Marcia Lima Festivo ◽  
Salvatore Siciliano ◽  
...  

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.


2012 ◽  
Vol 503 ◽  
pp. 188-193 ◽  
Author(s):  
Kun Wang Niu ◽  
Ting Liang ◽  
Fang Yang ◽  
Qing Meng ◽  
Kai Wang ◽  
...  

The quality of designing structure and properties of sensor photoionization room,directly determines its weak signal detection sensitivity detection and effecting ionization chamber detection efficiency .The paper experiments on four factors of affect the sensitivity of sensor. The results show that: the signal intensity proportional to the area of the ionization chamber; signal intensity goes up with the increase of bias voltage, and with bias voltage into a square the relationship; signal strength was first increased and then decreased with the growth of the distance between electrodes; signal strength was first increased and then decreased. With the growth of light-driven power.


2009 ◽  
Vol 64 (5-6) ◽  
pp. 355-360 ◽  
Author(s):  
Taicia P. Fill ◽  
Regina M. Geris dos Santos ◽  
Anderson Barisson ◽  
Edson Rodrigues-Filho ◽  
Antonia Q. L. Souza

A fungus, isolated from the root bark of Melia azedarach (Meliaceae), from which a series of meroterpenes have been reported, was identified as Penicillium brasilianum based on analysis of the ITS region of ribosomal DNA. From a rice culture of this fungus, the known phenylpropanoid amides brasiliamide A and B were obtained together with and a new, slightly modified congener, along with the meroterpenoids preaustinoid A1, preaustinoid B2 and austinolide. The compounds were isolated by the use of combined chromatographic procedures and identified by physical methods, mainly 1D and 2D NMR experiments, with distinction for 1H{15N} HMBC applied to brasiliamide A. The amides were tested for their antimicrobial activity and showed only weak inhibitory effects, against a set of pathogenic bacteria


2014 ◽  
Vol 8 (11) ◽  
pp. 1407-1414 ◽  
Author(s):  
Laila Nimri ◽  
Fatina Abu AL- Dahab ◽  
Raymond Batchoun

Introduction: The purpose of the study was to isolate, identify, and determine the antimicrobial resistance of the bacterial pathogens recovered from shawarma (donair) sandwiches served to the public in Jordan. Methodology: Bacterial contamination of 100 shawarma sandwiches with pathogenic bacteria was studied by culture on selective media, serology, PCR assay, and antimicrobial susceptibility testing. Results: One hundred and forty-five bacterial isolates were identified. The predominant species was Escherichia coli (28.3%), with six isolates of serotype O157:H7, followed by Salmonella spp. (25.5%). Higher contamination rates were found in chicken sandwiches. The majority of these bacteria expressed high resistance to several antimicrobials, especially tetracycline and streptomycin. Citrobacter freundii was isolated from 15.9% and Staphylococcus aureus was isolated from 8.3% of the sandwiches. The presence of these pathogens is of primary concern because some strains are capable of producing a heat-stable enterotoxin that causes food poisoning in humans, and should therefore be taken into account in risk assessment. Conclusions: Results signify the importance of sustained surveillance of foodborne pathogens in shawarma sandwiches to minimize the risk of contamination. Availability of data on the isolated pathogens and modes of transmission in food from different countries would provide a common ground for reaching international agreement on food safety regulations.


2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Zafer Asım Kaplancıklı ◽  
Mehlika Dilek Altıntop ◽  
Belgin Sever ◽  
Zerrin Cantürk ◽  
Ahmet Özdemir

In an effort to develop potent antimicrobial agents, new thiosemicarbazone derivatives were synthesizedviathe reaction of 4-[4-(trifluoromethyl)phenyl]thiosemicarbazide with aromatic aldehydes. The compounds were evaluated for their inhibitory effects on pathogenic bacteria and yeasts using the CLSI broth microdilution method. Microplate Alamar Blue Assay was also carried out to determine the antimycobacterial activities of the compounds againstMycobacterium tuberculosisH37Rv. Among these derivatives, compounds5and11were more effective againstEnterococcus faecalis(ATCC 29212) than chloramphenicol, whereas compounds1,2, and12and chloramphenicol showed the same level of antibacterial activity againstE. faecalis. Moreover, compound2and chloramphenicol exhibited the same level of antibacterial activity againstStaphylococcus aureus. On the other hand, the most potent anticandidal derivatives were found as compounds2and5. These derivatives and ketoconazole exhibited the same level of antifungal activity againstCandida glabrata. According to the Microplate Alamar Blue Assay, the tested compounds showed weak to moderate antitubercular activity.


2019 ◽  
Author(s):  
Jacek Piatek ◽  
Henning Sommermeyer ◽  
Arleta Ciechelska-Rybarczyk ◽  
Malgorzata Bernatek

AbstractSupplementation with probiotics is considered as alternative treatment or adjuvant therapy for a number of bacterial infections for which the use of antibiotics is either not recommended or emerging antibiotic resistance is a major concern. Inhibition of the growth of pathogenic bacteria has been related to a number of different activities of probiotic bacteria or yeasts, some of which are very specific for particular strains of probiotics. As the different inhibition activities might act additively or even synergistically, probiotic multistrain products are discussed as potentially being more effective in pathogen inhibition than products containing one or a small number of probiotic strains. The present study investigated the in vitro inhibition of Escherichia (E.) coli, Shigella spp., Salmonella (S.) typhimurium and Clostridum (Cl.) difficile, all being human pathogens of significant worldwide healthcare concerns. The probiotic containing the yeast Sacharomyces (S.) boulardii inhibited all four pathogens. Similar inhibitions were observed with a bacterial probiotic containing three different strains (Pen, E/N and Oxy) of Lactobacillus (Lc.) rhamnosus. Compared to the inhibition found for these probiotics, the inhibitory effects of a complex multistrain synbiotic, containing nine different probiotic strains (6 Lactobacilli and 3 Bifidobacteria) and the prebiotic fructooligosaccharide (FOS), were significantly stronger. The stronger inhibition by the complex multistrain synbiotic was observed for all four tested pathogens. Our findings support a hypothesis that complex synbiotic products containing a larger number of different strains combined with a prebiotic component might be more attractive candidates for further clinical characterization than simpler probiotics containing one or only few probiotic strains.


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