Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli in Animal Farms in Hunan Province, China.

Multi-drug resistance of bacteria producing extended-spectrum ß-lactamase (ESBL) is a public health challenge. Thus, this study aimed to investigate the antimicrobial susceptibility of ESBL-producing Escherichia coli (ESBL-EC) in Hunan Province, China. A total of 1366 fecal samples were collected from pig, chicken, and cattle farms over a six-year period, which were assessed using strain isolation, 16S rRNA identification, polymerase chain reaction, drug sensitivity testing, whole-genome sequencing, and bioinformatics analysis. The results showed an overall prevalence of 6.66% for ESBL-EC strains, with ESBL positivity extents for pigs, chickens, and cattle isolates at 6.77%, 6.54%, and 12.5%, respectively. Most ESBL-EC isolates were resistant to cefotaxime, tetracycline, and trimethoprim-sulfamethoxazole; however, all the isolates were susceptible to meropenem, with relatively low resistance to amikacin and tigecycline. Various multi-locus sequence types with different origins and similar affinities were identified, with ST155 (n = 16) being the most common subtype. Several types of resistance genes were identified among the 91 positive strains, with beta-lactamase blaCTX-M-55 being the most common ESBL genotype. IncFIB was the predominant plasmid type. Widespread use of antibiotics in animal farming may increase antibiotic resistance, posing a serious threat to the health of farmed animals and, thus, to human food security and health.

Rapid identification of mushroom toxins by direct electrospray probe mass spectrometry for emergency care.

Mushroom poisoning occurs frequently after the ingestion of toxic wild mushrooms misidentified as edible species. The goal of this study is to develop a mass spectrometric platform to bypass the need for morphological recognition of poisonous mushrooms by experts and rapidly identify the toxins in the mushrooms for emergency care. Trace mushroom toxins were collected by penetrating and removing the mushrooms surface for 3 mm with a direct electrospray probe (DEP). The analytes on the DEP were then dissolved in the solution (70% isopropanol containing 0.1% acetic acid) flowing out of a solvent reservoir on the DEP. Electrospray ionization was induced from the sample solution as a high electric field was generated between the DEP and MS inlet. The obtaining mass spectrometric results were further analyzed with principal component analysis (PCA) to classify mushroom toxins. The mass spectrometric platform for detecting mushroom toxins was assessed for its sensitivity, precision, and efficiency by determining its limit-of-detection (LOD), repeatability, and turnaround time, respectively. As a result, the LODs of the mushroom toxins in pure methanol and spiked in human vomitus by DEP/MS were within 0.001-0.5 ng/µL and 0.01-1 ng/µL, respectively. Linear responses of the mushroom toxins in pure methanol with concentrations between 0.01 and 5 ng/µL (R2 between 0.9922 and 0.998) were obtained. The repeatability of the approach (n = 10) was shown in the low relative standard deviation value (<15%) from ten repeat analysis of mushroom toxins standard solution. The corresponding toxic compounds were identified through matching of the obtained mass spectrometric data with those provided by its companion database library of mushroom toxins. Since no time-consuming pretreatment of the samples is required, identification of mushroom toxins with DEP/MS was complete within 1 min. This will be helpful for the emergency physicians to make correct clinical judgment and prescribe appropriate medical treatment in a timely manner.

Characteristics of tet(X4)-Producing Escherichia coli in Chicken and Pig Farms in Hunan Province, China.

BACKGROUND: The plasmid-mediated tigecycline resistance gene tet(X4) confers a high level of resistance to tigecycline. The experiment aims to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from chicken and pig farms in Hunan province, China. METHODS: A total of six tet(X4) positive strains were identified in 257 E. coli derived from chicken samples in Xiangtan city (n = 2), pig samples in Xiangxiang city (n = 1), Chenzhou city (n = 2), and Zhuzhou city (n = 1). The presence of tet(X4) was directly detected by PCR assay, and then the broth dilution method determined the antimicrobial susceptibility profile of the tet(X4)-positive isolates. Genomic locations were identified by whole-genome sequencing (WGS) and bioinformatics. RESULTS: Almost all tet(X4)-positive strains showed high resistance to multidrug, including tigecycline. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to tetracyclines, ß-lactams, phenicols, quinolones, lincosamides chloramphenicol, aminoglycosides and sulfamids. These tet(X4)-bearing strains exhibited six distract STs, such as ST10, 202, ST218, ST362, ST2077, ST7068. The plasmid replicon types carrying tet(X4) were the hybrid plasmid IncFIA(HI1)/IncHIA/IncHIB(R27) (5/6) and IncX1 (1/6). CONCLUSIONS: The presence of similar genetic environments in E. coli from different cities suggests there may be horizontal transmission pathways promoting the broad spread of drug-resistant genes in Hunan Province, putting great pressure on multidrug resistance monitoring.

Direct immersion solid-phase microextraction combined with ambient ionization tandem mass spectrometry to rapidly distinguish pesticides in serum for emergency diagnostics.

Despite the fact that carbamates and organophosphates cause acute poisoning via different mechanisms and require disparate management, they are indistinguishable by current clinical assays. Herein, direct immersion solid-phase microextraction (DI-SPME) plus thermal desorption-electrospray ionization tandem mass spectrometry (TD-ESI/MS/MS) was developed to discern them. Both pesticides spiked in human serum were extracted by SPME and analyzed by TD-ESI/MS/MS. This is a promising emergency care platform as rapid analyses could be done in tiny sample volumes with satisfactory recovery (89.46%-116.32%), precision (covariance <20%), sensitivity (LOD <0.1 µg/mL), turnaround time (<5 minutes), and linearity (R2 = 0.9827-0.9992) within 0.1-100 µg/mL.

Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation.

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice.

Obtaining molecular imagings of pesticide residues on strawberry surfaces with probe sampling followed by ambient ionization mass spectrometric analysis.

Thermal desorption-electrospray ionization tandem mass spectrometry (TD-ESI/MS/MS) was used to rapidly characterize the residual pesticides collected on the surface of a strawberry with a metallic probe. Twelve pesticides, including nine fungicides and three miticides, were detected; the results were validated by comparison with results that used solvent extraction followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry analyses. The distribution of pesticide residues on a strawberry's surface was explored by collecting multiple samples using probes from 40 positions on the strawberry, with the collected samples being analyzed with TD-ESI/MS/MS. The obtained molecular information was used to construct mass spectrometry imaging of the strawberry's pesticide residues.

Rapid identification of organophosphorus pesticides on contaminated skin and confirmation of adequate decontamination by ambient mass spectrometry in emergency settings.

RATIONALE: Dermal exposure to pesticides may cause severe intoxication and even result in a fatal outcome. To expedite rescue in the emergency department, it is mandatory to develop a point-of-care analytical method for immediate identification of pesticides on the skin of exposed personnel, and to perform immediate dermal decontamination to prevent further harm and optimize the chance for full clinical recovery. METHODS: Four of the most commonly used highly toxic pesticides that contaminate the skin were rapidly characterized by thermal desorption electrospray ionization mass spectrometry. The technique was also applied to confirm the completeness of pesticide decontamination from the skin. Pesticide sampling, desorption, ionization, and detection altogether took less than 30 s. In addition, different fabrics of protective garments worn by farmers were assessed with this efficient ambient mass spectrometric technique for their protective capabilities against dermal exposure to pesticides, and scanning electron microscopy was used to observe their different microstructures. The decontaminating efficacies of different cleansing agents for these skin contaminants were also evaluated by this technical platform. RESULTS: The repeatability of this method had a low relative standard deviation (<22%) for the detection of pesticides on the surface of swine skin. The detection limits of the pesticides in solution were found to be in the range of 3-20 ng/mL. Linearity was observed between the signal intensities and the concentrations of the four pesticides in solution within the range of 50 ng/mL to 50 µg/mL (R2 between 0.9921 and 0.9966). In addition, it was found that PVC fabric is optimal in preventing skin contamination by fenthion and detergent had the best efficiency for fenthion decontamination. CONCLUSIONS: Since the whole analytical process is extremely fast, this technique allows early point-of-care identification of contaminating pesticides on the skin of exposed patients in the emergency room, as well as rapid assessment of the adequacy of decontamination.