The Prevalence of <i>Escherichia coli</i> Derived from Bovine Clinical Mastitis and Distribution of Resistance to Antimicrobials in Part of Jiangsu Province, China

Bovine mastitis is often taken as one of the most common diseases in dairy farms, which its pathophysiology leads to a reduction of milk production and its quality. The penetration of pathogenic bacteria into the mammary gland, through either a contagious or environmental approach, has been determined the way of infection. The mastitis derived bacteria have become a challenge in practice, since the increasing exposure of antimicrobial. In order to identify characteristics of the epidemiological regulation and drug resistance of the pathogenic bacteria of bovine mastitis in northern Jiangsu, 156 clinical mastitis milk samples were collected from 3 large-scale farms for the epidemiological investigation and analysis of the drug resistance of E. coli. The bacteria were positively isolated in a total of 143 milk samples. The results showed that 78 strains of E. coli were detected, with a prevalence rate of 26.99%, followed by 67 strains of K. pneumoniae, with a prevalence of 23.19%, and 38 strains of Staphylococcus, with a prevalence of spp. 13.15%. In addition, 78 strains of E. coli isolated from bovine mastitis were tested for susceptibility to 8 kinds of antibiotics. It was shown that gentamicin and tetracycline were the most effective against E. coli, with the susceptibility rate of 83.3%, followed by streptomycin and ciprofloxacin, with 73.1% and 71.8% respectively. However, β-lactams including penicillin, cefothiophene, and amoxicillin showed serious resistance to E. coli isolates. There were 12 drug resistance genes detected by PCR, including β-lactam (blaTEM, blaCTX-M, and blaSHV), aminoglycoside (armA and armB), tetracycline (tetA, tetB, and tetC), and quinolone (qnrS, qepA, oqxA, and oqxB) related genes. Notably, all E. coli isolates carried blaTEM gene (100%). The detection rate of blaCTX-M was 53.8%, followed by the detection of blaSHV (20.5%), armA (9.0%), tetA (26.9%), tetB (2.6%), tetC (20.5%), qnrS (29.5%), oqxA (37.2%) and oqxB (1.3%). The present study provides crucial information on the distribution of bovine mastitis derived bacterial pathogens in Jiangsu province, as well as highlighting the antimicrobial resistance which might help to improve the efficiency of antibiotics treatment on bovine mastitis

Identification of Five Structurally Unrelated Quorum-Sensing Inhibitors of Pseudomonas aeruginosa from a Natural-Derivative Database

Bacteria communicate by means of small signal molecules in a process termed quorum sensing (QS). QS enables bacteria to organize their activities at the population level, including the coordinated secretion of virulence factors. Certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), have been shown to effectively block QS and subsequently attenuate the virulence of Pseudomonas aeruginosa, as well as increasing its susceptibility to both antibiotics and the immune system. In this study, a structure-based virtual screening (SB-VS) approach was used for the discovery of novel QSI candidates. Three-dimensional structures of 3,040 natural compounds and their derivatives were obtained, after which molecular docking was performed using the QS receptor LasR as a target. Based on docking scores and molecular masses, 22 compounds were purchased to determine their efficacies as quorum-sensing inhibitors. Using a live reporter assay for quorum sensing, 5 compounds were found to be able to inhibit QS-regulated gene expression in P. aeruginosa in a dose-dependent manner. The most promising compound, G1, was evaluated by isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis, and it was found to significantly affect the abundance of 46 proteins (19 were upregulated; 27 were downregulated) in P. aeruginosa PAO1. It specifically reduced the expression of several quorum-sensing-regulated virulence factors, such as protease IV, chitinase, and pyoverdine synthetases. G1 was also able to reduce extracellular DNA release and inhibited the secretion of the virulence factor, elastase, whose expression is regulated by LasR. These results demonstrate the utility of SB-VS for the discovery of target-specific QSIs.Published versio

RpoN Regulates Virulence Factors of <i>Pseudomonas aeruginosa</i> via Modulating the PqsR Quorum Sensing Regulator

The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs) quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator

C-di-GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that controls the lifestyles of many bacteria. A high intracellular level of c-di-GMP induces a biofilm lifestyle, whereas a low intracellular level of c-di-GMP stimulates dispersal of biofilms and promotes a planktonic lifestyle. Here, we used the expression of different reporters to show that planktonic cells, biofilm cells, and cells dispersed from biofilms (DCells) had distinct intracellular c-di-GMP levels. Proteomics analysis showed that the low intracellular c-di-GMP level of DCells induced the expression of proteins required for the virulence and development of antimicrobial peptide resistance in Pseudomonas aeruginosa. In accordance with this, P. aeruginosa cells with low c-di-GMP levels were found to be more resistant to colistin than P. aeruginosa cells with high c-di-GMP levels. This finding contradicts the current dogma stating that dispersed cells are inevitably more susceptible to antibiotics than their sessile counterparts.Published versio

Functional Amyloids Keep Quorum-sensing Molecules in Check

The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-L-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-L-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio