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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.

Rhodococcus equi is a zoonotic pathogen that can cause fatal disease in patients who are immunocompromised. At present, the epidemiology and pathogenic mechanisms of R. equi infection are not clear. This study characterized the genomes of 53 R. equi strains from different sources. Pan-genome analysis showed that all R. equi strains contained 11481 pan genes, including 3690 core genes and 602 ~ 1079 accessory genes. Functional annotation of pan genome focused on the genes related to basic lifestyle, such as the storage and expression of metabolic and genetic information. Phylogenetic analysis based on pan-genome showed that the R. equi strains were clustered into six clades, which was not directly related to the isolation location and host source. Also, a total of 84 virulence genes were predicted in 53 R. equi strains. These virulence factors can be divided into 20 categories related to substance metabolism, secreted protein and immune escape. Meanwhile, six antibiotic resistance genes (RbpA, tetA (33), erm (46), sul1, qacEdelta 1 and aadA9) were detected, and all strains carried RbpA related to rifamycin resistance. In addition, 28 plasmids were found in the 53 R. equi strains, belonging to Type-A (n = 14), Type-B (n = 8) and Type-N (n = 6), respectively. The genetic structures of the same type of plasmid were highly similar. In conclusion, R. equi strains show different genomic characteristics, virulence-related genes, potential drug resistance and virulence plasmid structures, which may be conducive to the evolution of its pathogenesis.

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.

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-aureusStaphylococci 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-aureusStaphylococci spp. infections rapidly.

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae–Shewanella chilikensis–Shewanella indica and Shewanella seohaensis–Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

Background Members of the genus Proteus are mostly opportunistic pathogens that cause a variety of infections in humans. The molecular evolutionary characteristics and genetic relationships among Proteus species have not been elucidated to date. In this study, we developed a multilocus sequence analysis (MLSA) approach based on five housekeeping genes (HKGs) to delineate phylogenetic relationships of species within the genus Proteus. Results Of all 223 Proteus strains collected in the current study, the phylogenetic tree of five concatenated HKGs (dnaJ, mdh, pyrC, recA and rpoD) divided 223 strains into eleven clusters, which were representative of 11 species of Proteus. Meanwhile, the phylogenetic trees of the five individual HKGs also corresponded to that of the concatenated tree, except for recA, which clustered four strains at an independent cluster. The evaluation of inter- and intraspecies distances of HKG concatenation indicated that all interspecies distances were significantly different from intraspecies distances, which revealed that these HKG concatenations can be used as gene markers to distinguish different Proteus species. Further web-based DNA-DNA hybridization estimated by genome of type strains confirmed the validity of the MLSA, and each of eleven clusters was congruent with the most abundant Proteus species. In addition, we used the established MLSA method to identify the randomly collected Proteus and found that P. mirabilis is the most abundant species. However, the second most abundant species is P. terrae but not P. vulgaris. Combined with the genetic, genomic and phenotypic characteristics, these findings indicate that three species, P. terrae, P. cibarius and Proteus genospecies 5, should be regarded as heterotypic synonyms, and the species should be renamed P. terrae, while Proteus genospecies 5 has not been named to date. Conclusions This study suggested that MLSA is a powerful method for the discrimination and classification of Proteus at the species level. The MLSA scheme provides a rapid and inexpensive means of identifying Proteus strains. The identification of Proteus species determined by the MLSA approach plays an important role in the clinical diagnosis and treatment of Proteus infection.

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 FJ201301T shared 817 core genes with the Vibrio species in the population genomic analysis, but possessed unique genes of its own. In addition, V. fujianensis FJ201301T 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.

BackgroundThe increasing rates of carbapenem-resistant Acinetobacter baumannii (CRAB) caused nosocomial infections generate significant comorbidity and sometime cause death among patients. Current treatment options are limited. These infections pose great difficulties for infection control and clinical treatment. This study identifies the antimicrobial resistance, carbapenemases and genetic relatedness of A. baumannii isolates from cerebrospinal fluid (CSF) and blood in a hospital in Shandong, China. Methods A total of 50 nonrepetitive CSF A. baumannii isolates and 44 blood isolates were collected. The resistance phenotypes were determined according to the Clinical and Laboratory Standards Institute guidelines. We performed Polymerase Chain Reaction (PCR) experiments to detect the carbapenem resistance mechanism. Finally, we conducted Multilocus Sequence Typing (MLST) to depict the genetic relatedness of these isolates. Results We observed that eighty-eight of the 94 isolates collected were resistant to imipenem or meropenem. Among them, the bla OXA-23 gene was the most prevalent carbapenemase gene with a 91.5% (86/94) detection rate, followed by the bla OXA-24 gene that showed a 2.1% (2/94) detection rate isolates. Among all CRAB observations in this study, isolates with the bla OXA-23 gene were resistant to both imipenem and meropenem. However, isolates positive for the bla OXA-24 gene but negative for the bla OXA-23 gene showed an imipenem-sensitive but meropenem-resistant phenotype. The outcome of multilocus sequence typing analysis showed 21 different STs were distinguished, of which ST195 (25.5%), ST540 (12.8%) and ST208 (11.7%) were most frequently observed. Eighty of the 94 isolates (85.1%) were clustered into CC92, and all CC92 isolates showed a carbapenem resistance phenotype (except AB13). Five novel STs were detected, and most of them were CRAB, some of which belonged to CC92. Conclusion A high level of carbapenem resistance was detected in this study. The CC92 and bla OXA-23 gene were predominant. Five novel STs were detected, and these new STs require further investigation to understand the nature of and to prevent outbreaks caused by A. baumannii . Our study provides additional observations and epidemiological data of CSF and blood A. baumannii strains, which may improve future infection control measures and aid in potential clinical treatment in hospitals and other clinical settings.

Two strains of Gram-stain-negative, facultatively anaerobic short-rod bacteria were recovered from two different food samples in Ma’anshan city, Anhui province, China in 2008. The bacteria were characterized in a polyphasic taxonomic study that included phenotypic, phylogenetic and genotypic methodologies. Phylogenetic analysis of the 16S rRNA gene demonstrated that the two strains belonged to the genus Proteus and were most similar to Proteus vulgaris ATCC 29905T with a score of 99.7 %. Phylogenetic analysis of the rpoB gene placed the two strains into a cluster with a distinctly interspecies phylogenetic branch that was clearly separated from six type strains of the genus Proteus , with the most closely related species being Proteus mirabilis ATCC 29906T. In silico genomic comparisons, including in silico DNA–DNA hybridization (isDDH) and average nucleotide identity (ANI) analysis showed that the representative strain, 08MAS0041T, and all six Proteus species share less than 70 % isDDH and have a 95 % ANI cutoff level, supporting the designation of the two strains as a novel species of the genus Proteus . The predominant cellular fatty acids of strain 08MAS0041T were C16 : 0 (24.8 %), C16 : 1ω7c/16 : 1ω6c (16.5 %), C18 : 1ω6c/C18 : 1 ω7c (14.5 %), C17 : 0 cyclo (12.6 %) and C16 : 1iso I/C14 : 0 3-OH (10.6 %). The analysis of biochemical, phylogenetic and genomic data confirmed that the two strains were clearly different from all recognized species of the genus Proteus and represent a novel Proteus species, for which the name Proteus alimentorum sp. nov. is proposed. The type strain is 08MAS0041T (=DSM 104685T=CGMCC 1.15939T).

The emergence of multidrug-resistant Salmonella genomic island 1 (SGI1) and Proteus genomic island (PGI) bearing P. mirabilis present a serious threat to public health. In this study, we screened 288 Proteus isolates recovered from seven provinces in China. Fourteen strains (4.9%) all belonged to P. mirabilis were positive for SGI1/PGI2, including twelve from clinical samples (5.3%) and two from food (3.3%). A Blastn search against GenBank and phylogenetic analyses identified eight different SGI1 variants and one PGI2 variant from the fourteen SGI1/PGI2 variants. All SGI1 variants shared a common backbone and harbored different resistance gene(s), except the sul1 gene at its multidrug-resistant (MDR) region. Among the variants, three novel SGI1 variants, designated as SGI1-PmCA11, SGI1-PmCA14 and SGI1-PmCA46, contained different gene cassettes, which were similar to sequences in plasmids or class 1 integrons of Klebsiella pneumoniae, P. mirabilis, Escherichia coli and Salmonella. Moreover, one novel PGI2, designated as PGI2-PmCA72, had an identical gene cassette to the first class 1 integron from PGI2 (GenBank accession no. MG201402.1) in P. mirabilis, but varied due to missing, replaced, inserted and inverted gene clusters. The four novel SGI1/PGI2 variants contained the cmlA5, dfrA14, blaOXA-10, aadA15, blaOXA-1, catB3 and dfrA16 resistance genes, which have never been reported in SGI1/PGI2 variants. Phenotypically, all fourteen SGI1/PGI2-containing strains showed multidrug resistance. All except four strains were resistant to the first, or the second and/or-third generation cephalosporins. Considering the increasing number and the emergence of new SGI1/PGI2 variants, further surveillance is needed to prevent the spreading of the MDR genomic islands among Proteus isolates from human and food.

A Gram-negative, facultatively anaerobic bacillus, strain 08MAS2615T, was isolated from the flesh of a pigeon specimen collected in Ma’anshan, Anhui province, China. Phylogenetic analysis of 16S rRNA gene sequences confirmed that strain 08MAS2615T belonged to the genus Proteus , and formed an independent branch which was clearly separated from the other six known species of Proteus . Strain 08MAS2615T was more closely related to Proteus vulgaris ATCC 29905T and Proteus penneri NCTC 12737T than other Proteus species. Similar independent phylogenetic results were obtained using rpoB gene sequence analysis, whereas strain 08MAS2615T clustered near the species of Proteus cibarius JS9T and Proteus terrae N5/687T. Furthermore, the genome-wide core-single nucleotide polymorphism-based phylogenetic tree confirmed that strain 08MAS2615T formed a monophyletic and robust clade. Based on whole-genome sequences, the range of in silico DNA–DNA hybridization and average nucleotide identity between strain 08MAS2615T and the six Proteus species were 25.5–48.8 % and 82.8–92.9 %, respectively, less than the proposed cutoff level for species delineation, i.e. 70 and 95 %. In addition, the major cellular fatty acid profile of strain 08MAS2615T was C14 : 0 (12.4 %), C16 : 0 (23.8 %), C17 : 0cyclo (14.4 %), summed feature 2 (C16 : 1iso I/C14 : 0 3-OH) (11.0 %), summed feature 3 (C16 : 1ω7c/16 : 1ω6c) (18.5 %) and summed feature 8 (C18 : 1ω6c) (18.6 %). On the basis of these results, strain 08MAS2615T represents a novel species of the genus Proteus , for which the name Proteus columbae sp. nov. is proposed with strain 08MAS2615T (=DSM 104686T=CGMCC 1.15982T) designated as the species type strain.