The Mechanism of Tigecycline Resistance in Acinetobacter baumannii under Sub-Minimal Inhibitory Concentrations of Tigecycline.

The presence of sub-minimal inhibitory concentration (sub-MIC) antibiotics in our environment is widespread, and their ability to induce antibiotic resistance is inevitable. Acinetobacter baumannii, a pathogen known for its strong ability to acquire antibiotic resistance, has recently shown clinical resistance to the last-line antibiotic tigecycline. To unravel the complex mechanism of A. baumannii drug resistance, we subjected tigecycline-susceptible, -intermediate, and -mildly-resistant strains to successive increases in sub-MIC tigecycline and ultimately obtained tigecycline-resistant strains. The proteome of both key intermediate and final strains during the selection process was analyzed using nanoLC-MS/MS. Among the more than 2600 proteins detected in all strains, we found that RND efflux pump AdeABC was associated with the adaptability of A. baumannii to tigecycline under sub-MIC pressure. qRT-PCR analysis also revealed higher expression of AdeAB in strains that can quickly acquire tigecycline resistance compared with strains that displayed lower adaptability. To validate our findings, we added an efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), to the medium and observed its ability to inhibit tigecycline resistance in A. baumannii strains with quick adaptability. This study contributes to a better understanding of the mechanisms underlying tigecycline resistance in A. baumannii under sub-MIC pressure.

Molecular characterization and biofilm formation ability of Enterococcus faecium and Enterococcus faecalis bloodstream isolates from a Chinese tertiary hospital in Beijing.

To investigate the molecular characteristics and biofilm-forming ability of 116 Enterococcus faecium (Efm) and 72 Enterococcus faecalis (Efs) isolates obtained from patients with bloodstream infections (BSI) at a Chinese hospital between July 2011 and March 2018. The presence of glycopeptide resistance genes and five virulence genes (esp, gelE, asa1, hyl, and cylA) was screened using two multiplex PCR. MLST was used to assess the clonality. Crystal violet staining was used to detect biofilms. Vancomycin resistance was detected in 30.1% of Efm and 2.8% of Efs isolates, respectively. All VRE strains carried the vanA gene. The esp, gelE, asa1, and cylA genes in 72 Efs strains were detected at 62.5%, 84.7%, 84.7%, and 69.4%, respectively. Among the 116 Efm isolates, 74.1% and 25.8% carried esp and hyl, respectively. The esp gene was significantly associated with vancomycin-resistant Efm (VREfm) compared to vancomycin-susceptible Efm (VSEfm). In total, 91.7% of Efs and 20.0% of Efm produced biofilms. Twenty-six STs were identified among the 72 Efs isolates, with ST4 (29.2%) being the predominant. In total, 116 Efm strains were grouped into 26 STs, with ST78 (46.6%) being the predominant. Both VREfm (41.7%) and VSEfm (48.8%) were dominant in ST78. There is no clear evidence suggesting that some STs are associated with vancomycin resistance or biofilm formation. Both Efm and Efs BSI isolates showed a polyclonal pattern with a dominant clone and many unique types, implying the coexistence of clonal dissemination and an influx of new clones. The horizontal transmission of resistance genes may play a more important role in VREfm prevalence than clonal expansion.

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii Revealed by Proteomic and Genomic Analysis.

The mechanism of tigecycline resistance in A. baumannii remains largely unclear. In this study, we selected a tigecycline-resistant and a tigecycline-susceptible strain from a tigecycline-susceptible and a resistant strain, respectively. Proteomic and genomic analyses were performed to elucidate the variations associated with tigecycline resistance. Our study showed proteins associated with efflux pump, biofilm formation, iron acquisition, stress response, and metabolic ability are upregulated in tigecycline resistant strains, and efflux pump should be the key mechanism for tigecycline resistance. By genomic analysis, we found several changes in the genome that can explain the increased level of efflux pump, including the loss of the global negative regulator hns in the plasmid and the disruption of the hns gene and acrR gene on the chromosome by the insertion of IS5. Collectively, we not only revealed the phenomenon that the efflux pump is mainly responsible for tigecycline resistance, but also highlighted the mechanism at the genomic level, which will help in understanding the resistance mechanism in detail and provide clues for the treatment of clinical multiple drug-resistant A. baumannii.

The type VI secretion system in Acinetobacter baumannii clinical isolates and its roles in antimicrobial resistance acquisition.

Acinetobacter baumannii is a successful pathogen that can acquire various antibiotic resistance in a short time. However, little is known about how it can evolve from an antibiotic sensitive to a resistant phenotype. In this study, we investigated the roles of the type VI secretion system (T6SS) in the acquisition of antibiotic resistance of A. baumannii. T6SS gene cluster was found to be present in 51 of 77 A. baumannii clinical isolates, of which, it was found in 62% (8/13) of the multiple drug resistant (MDR) isolates, 90% (36/40) of the extensively drug-resistant (XDR) isolates and 26% (6/23) of the antibiotic sensitive isolates. There is a close relationship between the antimicrobial resistance and the presence of T6SS. Besides, T6SS + isolates showed lower biofilm formation activity and higher survival ability in the presence of normal human serum than T6SS- isolates. A. baumannii A152 with complete T6SS can outcompete E.coli effectively and can acquire the antibiotic resistance plasmids released by E.coli. In contrast, the T6SS core gene mutant A152Δhcp showed significantly decreased ability to acquire antimicrobial resistance plasmids from the prey bacteria. These results suggest that T6SS mediated bacterial competition plays important roles in the antimicrobial resistance of A. baumannii, which points out a new direction for us to study the antimicrobial resistance of A. baumannii.

Complete genome sequence and virulence characterization of a neonatal meningitis Escherichia coli isolate.

Neonatal bacterial meningitis is a life-threatening disease in newborns, and neonatal meningitis Escherichia coli (NMEC) is the second most frequent bacteria causing this disease worldwide. In order to further understand the characteristics of this pathogen, an E. coli isolate W224 N from newborns with meningitis was sequenced for detailed genetic characterization and the virulence was tested by a series of phenotypic experiments. W224 N has a circular chromosome and three plasmids. It belongs to ST95 and the serotype is O18:H7. Comparative genomic analysis showed that W224 N was closely related to E. coli neonatal meningitis isolates RS218 and NMEC O18. There are 11 genomic islands in W224 N and most of the GIs are specific to W224 N. W224 N has most of the virulence factors other neonatal meningitis isolates have. The virulence genes located both on the genome and plasmid. At the same time, we found a virulence factor cdiA only present in W224 N but absent in the other five genomes analyzed. In vitro experiment showed that W224 N has strong serum resistance ability, low biofilm formation ability and high flagellar motility. It also has a very strong toxicity to mice and amoeba. The whole genome as well as in vitro and in vivo experiments showed that W224 N is a high virulent strain. The results can help us better learn about the pathogenicity of neonatal meningitis E. coli.

[Contamination and dietary exposure assessment of fatty acid esters of chloropropanediols in vegetable oil and fat-rich food in Shaanxi Province].

OBJECTIVE: To investigate the contamination of chloropropanediols in fat-rich food in Shaanxi Province, and to assess the dietary chloropropanediols exposure and the health risk of intaking chloropropanediols from fat-rich food for Shaanxi residents. METHODS: In 2015-2016, three kinds of food including vegetable oil, fried food and baked food were collected randomly from ten cities of Shaanxi Province, and determined by gas chromatography. Dietary intake assessment of human exposure to 3-monochloropropane-1, 2-diol ester(3-MCPD ester) was carried out in combination of food consumption data with concentration of chloropropanediols. RESULTS: A total of 203 samples were detected, the total detection rates of 3-MCPD ester and 2-monochloropropane-1, 3-diol ester(2-MCPD ester) were 31.53% and 12.30%, respectively, with the mean values of 0.22 and 0.02 mg/kg.Among all food samples, baked food were more seriously polluted than other kinds of foods with 3-MCPD ester, its detection rate was 47.06%. The highest detection rate of 2-MCPD ester was fried food with 20.00% detection rate. Getting rid of rapeseed oil, olive oil, pastries and biscuits, and 2-MCPD esters were detected in the rest. The ratio of the average exposure and high-end exposure to the maximum daily tolerance of 3-MCPD ester in the three fatty foods were less than 100%. CONCLUSION: The overall level of 3-MCPD esters contamination in fat-rich food is common in Shaanxi Province. The risk assessment result suggests that the current dietary intake of 3-MCPD esters from fried food in Shaanxi Province has acceptable effect on health.

[Investigation on the level of dioxin-like compounds in different animal-derived foods from Shaanxi Province].

OBJECTIVE: To investigate the pollution level of dioxin-like compounds in animal-derived foods from 10 cities of Shaanxi Province. METHODS: Collect four representative food samples( pork, beef, fish and eggs) to determine the concentrations of29 types of dioxin-like compounds by high-resolution gas chromatographs/high-resolution mass spectrometers( HRGC-HRMS). RESULTS: The result showed that the toxic equivalent quantity( TEQ) value of dioxin-like compounds in fish was 0. 085 pg TEQ/g, which was the biggest among the 4 food samples. The TEQ values for pork, beef and eggs were 0. 049, 0. 034 and 0. 040 pg TEQ/g, respectively. The TEQ values of dioxin-like compounds were higher in the fish meat of Ankang and the pork of Hanzhoung, but were lower than the max limit of commission regulation European Union. The total dietary intake of the above four foods was 2. 01 pg/( kg·BW·month), which was much lower than the provisional tolerable monthly intake( PTMI) of Joint FAO/WHO Expert Committee on Food Additives 70 pg/( kg·BW·month). CONCLUSION: The residents in the 10 cities of Shaanxi Province are at a low health risk caused by the intake of dioxin-like compounds.

Fitness Costs of Tigecycline Resistance in Acinetobacter baumannii and the Resistance Mechanism Revealed by a Transposon Mutation Library.

Acinetobacter baumannii is one of the main pathogens causing nosocomial and community-acquired infections. Tigecycline is an important antibiotic for the treatment of multidrug-resistant A. baumannii infections, but strains resistant to tigecycline have also emerged. There are still many unclear questions concerning the mechanism of tigecycline resistance in A. baumannii. In this study, tigecycline-susceptible and tigecycline-intermediate strains were gradually cultured with sub-minimum inhibitory concentrations of tigecycline to select for tigecycline-resistant mutants, and a tigecycline-resistant strain was cultured under 42 °C to select for tigecycline-susceptible mutants. We found that the acquisition of tigecycline resistance affected the susceptibility of the strains to other antibiotics. Resistance to ampicillin-sulbactam is negatively correlated with tigecycline resistance. The strains will experience fitness costs along with the acquisition of tigecycline resistance. Tigecycline resistance in the strains was not related to 16S rRNA target variation or outer membrane integrity alteration. By constructing a transposon mutation library, we found that transposon insertion of the adeL gene reduced the sensitivity of A. baumannii to tigecycline. This study provides important clues for understanding the mechanism of tigecycline resistance in A. baumannii.

Visual chiral recognition of tryptophan enantiomers using unmodified gold nanoparticles as colorimetric probes.

A simple protocol to distinguish enantiomers is extremely intriguing and useful. In this study, we propose a low-cost, facile, sensitive method for visual chiral recognition of enantimers. It is based on the inherent chirality of gold nanoparticles (AuNPs), and the unmodified AuNPs are used as chiral selector for D- and L-Tryptophan (Trp). In the presence of D-Trp, an appreciable red-to-blue color change of AuNPs solution can be observed, whereas no color change is found in the presence of L-Trp. The method can be used to detect D-Trp in the range of 0.2-10 µM, and the limit of detection is 0.1 µM. The chiral assay described in this work is easily readout with the naked eye or using a UV-vis spectrometer. Furthermore, the AuNPs can selectively adsorb D-Trp, and simple centrifugation can allow the precipitation of D-Trp with AuNPs and leave a net excess of the other enantiomer in solution, thus resulting in enantioseparation. In this method, AuNPs do not need any labeling or modifying with chiral molecules. The method is more attractive because of its high sensitivity, low cost, ready availability and simple manipulation.