Convergent Ser/Thr ligation has been used to prepare a series of teixobactin analogues (28 in total) to establish a structure-activity relationship of teixobactin. anti-bacterial evaluations of these synthetic analogues have revealed the critical amino acid residues and the sites tolerable of modifications. These studies will shed lights on the further development of teixobactin analogues with improved antibacterial activities.Download high-res image (75KB)Download full-size image

•The study investigated the effects on antibiotic treatment on antibiotic resistant sub-population organisms in animal GI tract.•Antibiotic resistant organisms constituted a varied but significant proportion of the gut flora regardless of the degree of previous exposure to antibiotics.•The proportion of sub-population of resistant organisms varied according to the presence of antibiotic pressure.•Increase of ceftiofur-resistant subpopulation was mainly attributed to transmission of blaCTX-M-borne plasmids.BackgroundAntibiotic resistant sub-population of the gut microbiome in both human and animals may be enriched during antimicrobial treatment.MethodsThis study investigated how the size and antibiotic susceptibility profiles of antibiotic resistant sub-population organisms in the animal gastrointestinal tract changed upon exposure to different levels of ceftiofur and enrofloxacin by plating fecal samples collected from piglets before and after antibiotic treatment onto MacConkey agar containing 0.5 μg/ml cefotaxime and determination of the resistance rate. Genetic characteristics and relatedness of strains collected before and after treatment was tested to determine if resistant Escherichia coli strains that emerged after treatment was due to dissemination of resistance-encoding mobile elements or clonal expansion of specific strains.ResultsAntibiotic resistant organisms constituted a varied but significant proportion of the gut flora regardless of the degree of previous exposure to antibiotics; the proportion of sub-population resistant organisms in the gut flora decreased but remained detectable even 10 days after antibiotic selection pressure had been relieved, and increased again upon renewed exposure to antibiotic regardless of the approach of administration. The increased prevalence of cefotaxime resistant strains in the animal GI tract upon treatment with sub-therapeutic and therapeutic concentrations of ceftiofur/enrofloxacin was respectively attributed to transmission of blaCTX-M-borne plasmids and clonal expansion of pre-existing resistant strains.ConclusionThe gut flora of animals comprises a high baseline level of resistance, among which resistant organisms cannot be eradicated but instead being further enriched in successive episodes of antibiotic treatment.

•Over 90% of CRE strains in China harbored the blaNDM or blaKPC-2 gene.•The predominant types of carbapenem-resistant K. pneumoniae and E. coli were ST11 and ST131/ST167 respectively.•Conservative mobile elements carrying blaNDM and blaKPC-2 contributed to transmission of carbapenemase genes in CRE.Large scale surveillance of CRE was rarely reported worldwide except for a recent survey reported in European countries. A nationwide survey was conducted in this study to depict the epidemiological features of CRE strains in China and the types of resistance elements that they harbored. We showed that the increasing prevalence of CRE strains in China was due to dissemination of major resistant clones that harbor several major types of blaNDM or blaKPC-2 – bearing conjugative plasmids, and that infections caused by such strains remain treatable by various non-β-lactam antibiotics, including colistin.The increasing incidence of carbapenem-resistant Enterobacteriaceae (CRE) - mediated hospital infections in China prompted a need to investigate the genetic basis of emergence of such strains. A nationwide survey was conducted in China covering a total of 1105 CRE strains collected from 25 geographical locales with results showing that acquisition of two carbapenemase genes, blaKPC-2 and blaNDM, was responsible for phenotypic resistance in 90% of the CRE strains tested (58% and 32% respectively), among which several major strain types, such as ST11 of K. pneumoniae and ST131/ST167 of E. coli, were identified, suggesting that dissemination of specific resistant clones is mainly responsible for emergence of new CRE strains. Prevalence of the fosA3 gene which mediates fosfomycin resistance, was high, while the colistin resistance determinant mcr-1 was rarely present in these isolates. Consistently, the majority of the blaNDM-bearing plasmids recoverable from the test strains belonged to IncX3, which contained a common core structure, blaNDM-blaMBL-trpF. Likewise, the core structure of ISKpn27-blaKPC-2-ISKpn2 was observed among plasmids harboring the blaKPC-2 gene, although they were genetically more divergent. In conclusion, the increasing prevalence of CRE strains in China is attributed to dissemination of conservative mobile elements carrying blaNDM or blaKPC-2 on conjugative and non-conjugative plasmids.

We report the discovery of a promising NDM-1 inhibitor, ebselen, through a cell-based screening approach. Enzymatic kinetic study and ESI-MS analysis suggested that ebselen could bind to NDM-1 by forming a S–Se bond with the Cys221 residue at the active site, thereby exhibiting a new inhibition mechanism with broad spectrum inhibitory potential.

Botulinum neurotoxins are among the most potent toxic bacterial proteins for humans and there is a great need to develop simple, rapid and sensitive methods for toxin detection and protease activity quantification in field deployment. In this paper, a nanoporous membrane based impedance sensor was developed to monitor the activity of the BoNT serotype A light chain protease (LcA). Synaptosomal-associated protein 25 (SNAP-25) was first immobilized inside nanopore walls via silane linkers. BoNT LcA was then injected over the nanoporous membrane substrate sensor and specifically cleaved SNAP-25. The cleavage activity could be monitored by measuring impedance signals across nanoporous membranes which represented the nanopore blockage degree. This initial device could achieve a 500 pM LcA detection limit within 25 minutes.

•H2S negative Salmonella are prevalent in meat products in particular chicken.•H2S negative Salmonella are seen in all clinical important serotypes in particular S. Enteriditis.•H2S negative Salmonella possess virulent plasmid as common as H2S positive Salmonella.Hydrogen sulfide (H2S) production is considered a typical characteristic of Salmonella and an important marker for Salmonella isolation. In this study, a total of 82 (26%) Salmonella strains were isolated from 113 chicken and 204 pork samples, within which 49 Salmonella strains were H2S positive and 33 were H2S negative. Salmonella enterica serovar Derby was most prevalent in both pork and chicken followed by S. Typhimurium in pork and S. Heidelberg in chicken. Salmonella isolated from pork exhibited a much higher H2S positive rate than those from chicken (68% versus 31%). The most prevalent H2S negative serotypes were S. Derby (40%) and S. Heidelberg (30%) in chicken, and S. Typhimurium (23%) and S. Enteritidis (23%) in pork. spvC, a plasmid-encoded virulence marker, was detected in 51% and 42% of the H2S positive and negative Salmonella respectively. The presence of the two most important serotypes, S. Enteritidis and S. Typhimurium, as well as a virulence plasmid in H2S negative Salmonella suggested that H2S negative Salmonella is also a significant public health concern. Such finding warrants the development of an improved method for effective coverage of H2S negative Salmonella.

•Clostridia Neurotoxins BoNT/B and TeNT cleave substrate VAMP2 at the same scissile bond, but with different mechanisms.•Their difference on substrate recognition is due to the different composition of S1 and S1′ pocket residues.•Optimization of these residues increases LC/B and LC/T activities.•More active Clostridia Neurotoxins were engineered.BoNT/B and TeNT cleave substrate VAMP2 at the same scissile bond, yet these two toxins showed different efficiency on substrate hydrolysis and had different requirements for the recognition of P2′ site of VAMP2, E78. These differences may be due to their different composition of their substrate recognition pockets in the active site. Swapping of LC/T S1′ pocket residue, L230, with the corresponding isoleucine in LC/B increased LC/T activity by ∼25 fold, while swapping of LC/B S1′ pocket residue, S201, with the corresponding proline in LC/T increased LC/B activity by ∼10 fold. Optimization of both S1 and S1′ pocket residues of LC/T, LC/T (K168E, L230I) elevated LC/T activity by more than 100-fold. The highly active LC/T derivative engineered in this study has the potential to be used as a more effective tool to study mechanisms of exocytosis in central neuron. The LC/B derivative with elevated activity has the potential to be developed into novel therapy to minimize the impact of immunoresistance during BoNT/B therapy.Download full-size image

•Carbapenem-resistant Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas otitidis were isolated from retail meats.•No acquired carbapenemase genes were detectable in these isolates.•Overexpression of efflux pumps was responsible for carbapenem resistance.•Loss of porins also played a role in carbapenem resistance.•The intrinsic metallo-β-lactamase blaPOM also contributed to the decreased carbapenem susceptibility in P. otitidis.Pseudomonas spp. are ubiquitous in nature. Carbapenem resistance in environmental isolates of members of this genus is thought to be rare but the exact resistance rate is unknown. In this study, carbapenem-resistant Pseudomonas spp. were isolated from chicken and pork samples and the mechanisms underlying the carbapenem resistance in these strains were investigated. A total of 16 carbapenem-resistant Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas otitidis isolates were recovered from eight samples of chicken and pork. The isolates exhibited meropenem minimum inhibitory concentrations (MICs) of 8 to ≥32 mg/L and imipenem MICs of <0.5–16 mg/L yet did not harbour any acquired carbapenemase genes. Meropenem resistance in various strains was found to be mediated by efflux systems only, whereas overexpression of MexAB–OprM efflux pump and lack of OprD porin were responsible for carbapenem resistance in P. aeruginosa. The intrinsic metallo-β-lactamase gene blaPOM in P. otitidis and overexpression of the TtgABC efflux system in P. putida were also responsible for carbapenem resistance in these organisms. In conclusion, this study reports for the first time the isolation of carbapenem-resistant P. aeruginosa, P. otitidis and P. putida strains from food. The resistance mechanisms of these strains are rarely due to production of carbapenemases. Further selection of such carbapenem-resistant Pseudomonas spp. in the environment and the risk by which they are transmitted to clinical settings are of great public health concern.

Vibrio parahaemolyticus is a major causative agent of gastroenteritis and is the leading cause of food-borne illness in Hong Kong. Recent studies of resistance to extended-spectrum β-lactams and fluoroquinolones in V. parahaemolyticus have caused huge concern. This work reports the characterisation of a multidrug resistance conjugative plasmid in V. parahaemolyticus isolated from shrimp samples from Hong Kong. The plasmid is ca. 200 kb and carries multidrug resistance genes, including a novel plasmid-mediated quinolone resistance gene qnrVC6 surrounded by several known and novel insertion sequence (IS) elements, an extended-spectrum β-lactamase gene blaPER-1 mediated by ISCR1, and a ca. 3-kb four-gene cassette (aacA3, catB2, dfrA1 and aadA1) class 1 integron. Transmission of this multidrug resistance conjugative plasmid among Vibrio spp. would compromise the effectiveness of Vibrio infection control and pose a huge threat to public health.

•BoNTs exhibited variable activities on their recombinant and endogenous substrates.•Combinations of LC/A and LC/B, as well as LC/A and LC/F, could enhance the activity of each.•Provides insights into development of new BoNT serotypes for clinical application.•Provide solution to minimize the immuno-resistance issues of BoNT based therapies.Botulinum neurotoxins are the most potent protein neurotoxin known to human. The dual roles of BoNTs as both the causative agent of human botulism and a widely used protein-based therapeutic agent for treatment of numerous neuromuscular disorders/cosmetic uses make it an extremely hot topic of research. Biochemical characterization of these toxins was mainly confined to the recombinant light chains and substrate and little is known about their efficiency on the cleavage of endogenous substrates. In the present study, we showed that BoNTs exhibited variable activities on their endogenous substrates and that their efficiency to cleave recombinant and endogenous substrate was not consistent, presumably due to the differential recognition of their respective substrates in the natural SNARE complex format. Through testing the combinatorial effects of different BoNTs on cleavage of endogenous substrates, we showed that the combinations of LC/A and LC/B, as well as LC/A and LC/F, could enhance the activity of each individual BoNT. This finding may shed light on the future development of new BoNT serotypes for clinical application, and formulation of combinatorial uses of different BoNTs to minimize the development of immuno-resistance by using a lower amount of individual type.

Botulinum neurotoxins are among the most potent toxic bacterial proteins for humans and there is a great need to develop simple, rapid and sensitive methods for toxin detection and protease activity quantification in field deployment. In this paper, a nanoporous membrane based impedance sensor was developed to monitor the activity of the BoNT serotype A light chain protease (LcA). Synaptosomal-associated protein 25 (SNAP-25) was first immobilized inside nanopore walls via silane linkers. BoNT LcA was then injected over the nanoporous membrane substrate sensor and specifically cleaved SNAP-25. The cleavage activity could be monitored by measuring impedance signals across nanoporous membranes which represented the nanopore blockage degree. This initial device could achieve a 500 pM LcA detection limit within 25 minutes.

We report the discovery of a promising NDM-1 inhibitor, ebselen, through a cell-based screening approach. Enzymatic kinetic study and ESI-MS analysis suggested that ebselen could bind to NDM-1 by forming a S–Se bond with the Cys221 residue at the active site, thereby exhibiting a new inhibition mechanism with broad spectrum inhibitory potential.