Ya-Hong Liu

Heteroresistance has seriously affected the evaluation of antibiotic efficacy against pathogenic bacteria, causing misjudgment of antibiotics’ sensitivity in clinical therapy, leading to treatment failure, and posing a serious threat to current medical health. However, the mechanism of Staphylococcus aureus heteroresistance to ciprofloxacin remains unclear. In this study, heteroresistance to ciprofloxacin in S. aureus strain 529 was confirmed by antimicrobial susceptibility testing and population analysis profiling (PAP), with the resistance of subclonal 529_HR based on MIC being 8-fold that of the original bacteria. A 7-day serial MIC evaluation and growth curves demonstrate that their phenotype was stable, with 529_HR growing more slowly than 529, but reaching a plateau in a similar proportion. WGS analysis showed that there were 11 nonsynonymous mutations and one deletion gene between the two bacteria, but none of these SNPs were directly associated with ciprofloxacin resistance. Transcriptome data analysis showed that the expression of membrane potential related genes (qoxA, qoxB, qoxC, qoxD, mprF) was downregulated, and the expression of multidrug resistance efflux pump gene mepA was upregulated. The combination of ciprofloxacin and limonene restored the 529_HR MIC from 1 mg/L to 0.125 mg/L. Measurement of the membrane potential found that 529_HR had a lower potential, which may enable it to withstand the ciprofloxacin-induced decrease in membrane potential. In summary, we demonstrated that upregulation of mepA gene expression and a reduction in membrane potential are the main heteroresistance mechanisms of S. aureus to ciprofloxacin. Additionally, limonene may be a potentially effective agent to inhibit ciprofloxacin heteroresistance phenotypes.

Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009–2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance. IMPORTANCE: Salmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

Prebiotics and postbiotics have gained attention as functional food additives due to their substantial influence on the gut microbiome and potential implications for human health on a broader scale. In addition, the number of patents for these additives has also increased, yet their functional classification has been problematic. In this study, we classified 2215 patents granted from 2001 to 2020 by functionality to enable predictions of future development directions. These patents encompassed subjects as diverse as feed supplementation, regulation of intestinal homeostasis, prevention of gastrointestinal ailments, targeted drug administration and augmentation of drug potency. The progression of patents issued during this time frame could be divided into three phases: occasional accounts prior to 2001, a period from 2001 to 2013 during which an average of 42 patents were issued annually, followed by a surge exceeding 140 patents annually after 2013. The latter increase has indicated that pre- and post-biotics have been recognized as biologically relevant. Patent mining therefore can enable forecasts of the future trajectory of these biologics and provide insights to evaluate their advancement. Moreover, this research is the first attempt to generalize and predict the directions of prebiotics and postbiotics using patent information and offers a comprehensive perspective for the potential utilization of prebiotics and postbiotics across a wide variety of fields.

The emergence of the wide variety of novel tigecycline resistance tet (X) variants including tet (X3), tet (X4), tet (X5) and tet (X6) has posed a significant challenge to the clinical treatment of multidrug-resistant bacterial infections and represents a serious threat to global public health. The purpose of this study was to evaluate the synergism of tigecycline combined with other antibiotics as a means of overcoming tet (X)-mediated resistance in Acinetobacter spp . We found that the combinations of tigecycline with apramycin or amikacin exhibited synergistic activity against tet (X)-harboring Acinetobacter spp . with FICI values of 0.088 and 0.625, respectively. The MIC _TGC decreased >5-fold decrease in the presence of subinhibitory levels of apramycin. This combination was shown to be a therapeutically effective synergism using both in vitro and in vivo (mouse thigh infection model) assays and delayed the increase of MIC values over time. This study highlights the synergism of tigecycline in combination with apramycin which offers a viable therapeutic alternative for infections caused by tet (X)-harboring Acinetobacter .

S. suis is a serious zoonotic pathogen. In this study, we investigated the epidemiological and molecular characteristics of 96 S. suis isolates from 10 different provinces of China from 2008 to 2019.

This study was designed to investigate the occurrence and dissemination of extended-spectrum β-lactamase (ESBL) harboring Klebsiella pneumoniae in various ecological niches under the one health approach. A total of 793 samples were collected from animals, humans, and the environment. The findings of the study revealed the occurrence of K. pneumoniae as follows: animals (11.6%), humans (8.4%), and associated environments (7.0%), respectively. A high occurrence rate of ESBL genes was found in animals compared to human and environmental isolates. A total of 18 distinct sequence types (STs) and 12 clonal complexes of K. pneumoniae were observed. Overall, six STs of K. pneumoniae were identified in commercial chickens, and three were found in rural poultry. The majority of K. pneumoniae STs found in this study were positive for blaSHV, while the positivity of other ESBL-encoding genes combinations was different in different STs. The high occurrence rate of ESBL-harboring K. pneumoniae found in animals as compared to other sources is alarming and has the potential to be disseminated to the associated environment and community.

Objectives: Multidrug-resistant (MDR) Gram-negative bacterial infections have limited treatment options due to the impermeability of the outer membrane. New therapeutic strategies or agents are urgently needed, and combination therapies using existing antibiotics are a potentially effective means to treat these infections. In this study, we examined whether phentolamine can enhance the antibacterial activity of macrolide antibiotics against Gram-negative bacteria and investigated its mechanism of action. Methods: Synergistic effects between phentolamine and macrolide antibiotics were evaluated by checkerboard and time–kill assays and in vivo using a Galleria mellonella infection model. We utilized a combination of biochemical tests (outer membrane permeability, ATP synthesis, ΔpH gradient measurements, and EtBr accumulation assays) with scanning electron microscopy to clarify the mechanism of phentolamine enhancement of macrolide antibacterial activity against Escherichia coli. Results: In vitro tests of phentolamine combined with the macrolide antibiotics erythromycin, clarithromycin, and azithromycin indicated a synergistic action against E. coli test strains. The fractional concentration inhibitory indices (FICI) of 0.375 and 0.5 indicated a synergic effect that was consistent with kinetic time–kill assays. This synergy was also seen for Salmonella typhimurium, Klebsiella pneumoniae, and Actinobacter baumannii but not Pseudomonas aeruginosa. Similarly, a phentolamine/erythromycin combination displayed significant synergistic effects in vivo in the G. mellonella model. Phentolamine added singly to bacterial cells also resulted in direct outer membrane damage and was able to dissipate and uncouple membrane proton motive force from ATP synthesis that, resulted in enhanced cytoplasmic antibiotic accumulation via reduced efflux pump activity. Conclusions: Phentolamine potentiates macrolide antibiotic activity via reducing efflux pump activity and direct damage to the outer membrane leaflet of Gram-negative bacteria both in vitro and in vivo.

The spread of antibiotic resistance genes (ARGs), especially those on plasmids, poses a major risk to global health. However, the extent and frequency of ARG transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a new fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. The tool integrates a fluorescent tag immediately downstream ARGs, allowing for gene transfer tracking without affecting host cell antibiotic susceptibility, fitness, conjugation, or transposition. Our experiments show that sfGFP-tagged plasmid-borne mcr-1 can transfer across different bacteria species in feces, including Escherichia, Shigella, Lactobacillus, Staphylococcus, Enterococcus, and Bacillus. CRISPR-AMRtracker offers a powerful tool for monitoring ARG transfer in microbiota communities and can inform strategies to combat the threat of antibiotic resistance.

Colistin is one of the last-line antibiotics for the clinical treatment of Enterobacteriaceae . However, the emergence of the mobilized colistin resistance ( mcr-1 ) gene has spread throughout the entire human health system and largely threatens the usage of colistin in the clinical setting.

We determined the longitudinal persistence of ceftiofur-resistant Escherichia coli from a chicken breeding farm in China. A total of 150 samples were collected from 5 breeding periods in a flock of layer hens, and the prevalence of ceftiofur-resistant E. coli fluctuated across the 5 chicken breeding stages: eggs, 3.33%; growing period, 100%; early laying period, 36.7%; peak laying period, 66.7% and late laying period, 80%. The most prevalent ceftiofur resistance genes were bla_CTX-M-55, bla_CMY and bla_NDM, and ST101 was the most prevalent and persistent sequence type across the breeding periods. Our results indicated that this breeder flock was heavily contaminated by ST101 ceftiofur-resistant E. coli and that its presence should be intensively monitored on chicken farms.