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.

Recently, bla_CTX-Ms have become the dominant ESBLs for E. coli strains worldwide. We aim to provide a systematic study on the relationships between sequence types (STs), clinical origins, and the bla_CTX-Ms genotypes of E. coli strains. Totally, 1005 complete sequences of clinical E. coli were collected from NCBI. Multilocus sequence typing (MLST) and antibiotic resistance genes screening were performed. Faeces (26.27%), urine (16.02%), and blood (8.26%) were shown to be the main sources of clinical E. coli isolates. The isolates belong to 153 STs and 26 clonal complexes (CCs). The most prevalent STs were ST2 (11.3%), ST43 (8.6%), and ST8 (5.7%). The positive rate for bla_CTX-Ms was 34.7%. Different samples showed significantly different bla_CTX-Ms positive rates (P<0.05). The main genotypes were bla_{CTX-M-55-like} (47.6%), bla_CTX-M-1-like (31.8%), and bla_CTX-M-2-like (22.1%). The majority of ST2 strains had bla_{CTX-M-55-like} genes. In ST8 strains, there was a homogeneous distribution of bla_CTX-M-9, bla_CTX-M-65, bla_CTX-M-55, bla_CTX-M-2, and bla_CTX-M-1. Only ST43 strains exhibited the presence of bla_CTX-M-79. The bla_CTX-Ms showed a pattern of cross-continental transmission with intra-regional spread. Among the 349 bla_CTX-Ms-producing E. coli strains, 148 strains also carried carbapenem resistance genes, including bla_NDM (119, 34.1%), bla_KPC (16, 4.6%), bla_OXA-48 (9, 2.6%) and bla_IMP (4, 1.1%). Also, 81 strains carried the mcr gene (23.2%). E. coli has become increasingly rich in bla_CTX-Ms genotypes. Our findings about the connection between E. coli STs and bla_CTX-Ms can be utilized to identify E. coli strains with high potential to spread drug resistance in the future.

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.

Vibrio alginolyticus, a halophilic bacterium in the genus of Vibrio, is widely distributed in marine and estuarine habitats such as seawaters, estuaries, ocean sediment, and aquaculture settings[1]...

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.