Staphylococcus spp., especially Staphylococcus aureus (S. aureus), is an important pathogen in hospital-acquired infection and food poisoning. Here, we developed a multienzyme isothermal rapid amplification combined with duplex quantitative PCR (duplex MIRA-qPCR) method, which can simultaneously detect the S. aureus species-specific conserved gene FMN-bgsfp and the Staphylococcus genus-specific conserved gene tuf. This assay enabled the amplification of DNA within 20 min at a constant temperature of 39 °C. Specificity analysis indicated that all nine common Staphylococcus species were positive and non-Staphylococcus spp. were negative for tuf gene, whereas S. aureus was positive, non-aureus Staphylococci species and non-Staphylococcus spp. were negative for FMN-bgsfp gene, suggesting that duplex MIRA-qPCR exhibited high specificity. Meanwhile, the sensitivity was tested and the limit of detection (LoD) was 3 × 10² CFU/mL. The coefficient variation values ranged from 0.13% to 2.09%, indicating that the assay had good repeatability. Furthermore, all the nine common Staphylococcus species (including S. aureus) could be detected from four kinds of simulated samples and the LoD of S. aureus was 8.56 × 10³ CFU/mL. In conclusion, the duplex MIRA-qPCR has advantages of stronger specificity, lower detection threshold, shorter detection time, and simpler operation, which is an effective tool to detect S. aureus and non-aureus Staphylococci spp. infections rapidly.

The nonstandard use of disinfectants can lead to the disinfectant resistance of bacteria and even increase antibiotic resistance. However, compared with the study of antibiotic resistance, studies of bacterial resistance to disinfectants are relatively few in number. In this study, we explored the standard strain screening procedure for the evaluation of disinfection efficacy. Staphylococcus aureus strains with different sources and substrates were selected from the National Pathogen Resource Center of China and screened the standard strains that could evaluate the long-term bacteriostatic effect of the chlorine-containing disinfectants through the determination of the physical properties, genome-based safety evaluation, and disinfection test evaluation. In this process, one S. aureus strain was more resistant to the long-term bacteriostasis of chlorine-containing disinfectants than the other strains. This strain and the standard strain ATCC 6538 were cultured in the medium containing a low concentration of chlorine-containing disinfectant synchronously. Then, comparative transcriptome analysis was carried out to investigate the potential mechanism of a high tolerance to chlorine-containing disinfectants. The pathway of significant differential expression is related to the oxocarboxylic acid metabolic mechanism, amino acid metabolic mechanism, and pyrimidine mechanism, which may be the molecular mechanism of S. aureus evolution to adapt to chlorine-containing disinfectants. Our study established a technical process for screening and evaluating standard strains for disinfection, which also provided a reference for studying the bacterial evolution mechanism toward chlorine tolerance.

A rising incidence of clinical infections has been caused by Kluyvera, a significant opportunistic pathogen. Meanwhile, Kluyvera acts as an important reservoir of bla_CTX-Ms, which are the dominant genes of class A extended-spectrum β-lactamases (ESBLs). In this work, 60 strains of Kluyvera were subjected to phylogenetic relationship reconstruction, antimicrobial susceptibility testing, and antibiotic resistance genes prediction. All mature bla_CTX-Ms were gathered to perform subgroup reclassification. The findings demonstrate that Kluyvera has a large gene pool with significant genetic flexibility. Notably, 25% of strains showed simultaneous detection of ESBLs and carbapenem resistance genes. The genotypes of fourteen novel bla_CTX-Ms were identified. A new subgroup classification approach for bla_CTX-Ms was defined by using 20 amino acid site variants, which could split bla_CTX-Ms into 10 subgroups. The results of the subgroup division were consistent with the phylogenetic clustering. More significantly, we proposed a novel bla_CTX-M subgroup, KLUS, that is chromosomally encoded in K. sichuanensis and the new species put forward in this study, showing amino acid differences from the currently known sequences. Cloning and transformation tests demonstrated that the recipient bacteria had a robust phenotype of cefotaxime resistance. Closely related Kluyvera species had bla_CTX-Ms in the same subgroup. Our research lays the groundwork for a deeper comprehension of Kluyvera and emphasizes how important a bla_CTX-M reservoir it is. We provide an update on bla_CTX-M subgroups reclassification from the aspects of phylogenetic relationship, amino acid differences, and the new subgroup KLUS, which needs to be strengthen monitored due to its strong resistance phenotype to cefotaxime.

The genus Vibrio is ubiquitous in marine environments and uses numerous evolutionary characteristics and survival strategies in order to occupy its niche. Here, a newly identified species, Vibrio fujianensis, was deeply explored to reveal a unique environmental adaptability. V. fujianensis type strain FJ201301^T shared 817 core genes with the Vibrio species in the population genomic analysis, but possessed unique genes of its own. In addition, V. fujianensis FJ201301^T was predicated to carry 106 virulence-related factors, several of which were mostly found in other pathogenic Vibrio species. Moreover, a comparative transcriptome analysis between the low-salt (1% NaCl) and high-salt (8% NaCl) condition was conducted to identify the genes involved in salt tolerance. A total of 913 unigenes were found to be differentially expressed. In a high-salt condition, 577 genes were significantly upregulated, whereas 336 unigenes were significantly downregulated. Notably, differentially expressed genes have a significant association with ribosome structural component and ribosome metabolism, which may play a role in salt tolerance. Transcriptional changes in ribosome genes indicate that V. fujianensis may have gained a predominant advantage in order to adapt to the changing environment. In conclusion, to survive in adversity, V. fujianensis has enhanced its environmental adaptability and developed various strategies to fill its niche.

To understand the characteristics of Vibrio isolates in aquatic environments in China and their public health significance, this study investigated water samples in six cities in China in 2020. A total of 88 sampling locations were included and Vibrio isolates were identified in 81 of them. A total of 143 Vibrio isolates belonging to 16 species were selected for characterization. The population structure of Vibrio species showed great differences among the six cities, indicating regional specificity. The presence of virulence genes was examined for the isolates (n = 78) of five pathogenic Vibrio species. All isolates except one (n = 77) contained at least one virulence gene and isolates belonging to the same species showed very similar virulence gene profiles. Then, 26 isolates from 12 species were examined by multilocus sequence typing and were assigned to 25 STs, of which 24 STs were new. Also, the presence of antibiotic-resistant genes was investigated for all 143 isolates and only three isolates were found to contain genes from aminoglycosides, phenicols, beta-lactams or the tetracycline family. Our results provide valuable insights into the Vibrio community in Chinese aquatic environments and can be applied as guidance for the environmental surveillance of the risk of Vibrio isolates.