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IntroductionKluyvera is a Gram-negative, flagellated, motile bacillus within the Enterobacteriaceae. The case reports of clinical infections shed light on the importance of this organism as an emerging opportunistic pathogen. The genus Phytobacter, which often be misidentified with Kluyvera, is also an important clinically relevant member of the Enterobacteriaceae. However, the identification of Kluyvera and Phytobacter is problematic, and their phylogenetic relationship remains unclear.MethodsHere, 81 strains of Kluyvera and 16 strains of Phytobacter were collected. A series of comparative genomics approaches were applied to the phylogenetic relationship reconstruction, virulence related genes profiles description, and antibiotic resistance genes prediction.ResultsUsing average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH), we offered reliable species designations of 97 strains, in which 40 (41.24%) strains were incorrectly labeled. A new Phytobacter genomospecies-1 were defined. Phytobacter and Kluyvera show great genome plasticity and inclusiveness, which may be related to their diverse ecological niches. An intergenomic distances threshold of 0.15875 was used for taxonomy reassignments at the phylogenomic-group level. Further principal coordinates analysis (PCoA) revealed 11 core genes of Kluyvera (pelX, mdtL, bglC, pcak-1, uhpB, ddpA-2, pdxY, oppD-1, cptA, yidZ, csbX) that could be served as potential identification targets. Meanwhile, the Phytobacter specific virulence genes clbS, csgA-C, fliS, hsiB1_vipA and hsiC1_vipB, were found to differentiate from Kluyvera. We concluded that the evolution rate of Kluyvera was 5.25E-6, approximately three times higher than that of Phytobacter. Additionally, the co-existence of ESBLs and carbapenem resistance genes were present in approximately 40% strains, suggesting the potential development of extensively drug-resistant or even fully drug-resistant strains.DiscussionThis work provided a better understanding of the differences between closely related species Kluyvera and Phytobacter. Their genomes exhibited great genome plasticity and inclusiveness. They not only possess a potential pathogenicity threat, but also a risk of multi-drug resistance. The emerging pathogens Kluyvera and Phytobacter warrant close attention.

IntroductionShewanella is an important opportunistic pathogen distributed in marine environments that has caused an increasing number of clinical infections. However, there are few reports on the distribution and characteristics of Shewanella in the diarrheal pathogen spectrum. In this study, we have systematically described the prevalence of Shewanella infections in diarrhea patients in Beijing, China 2017–2019, and genome characteristics and antimicrobial susceptibility of Shewanella isolates.MethodsStool samples were collected from diarrhea patients in a surveillance project from 2017 to 2019. Shewanella strains were isolated, and identified using VITEKR 2 COMPACT and MALDI-TOF MS. Average nucleotide identity (ANI) analysis, multi-locus sequence typing (MLST), phylogenetic analysis, virulence-associated genes and antimicrobial resistance genes analysis were used for genome characteristics description. The antibiotic susceptibility test was performed with microbroth dilution method.Results1104 fecal samples were collected, and the Shewanella detection rate was 2.36% (26/1104). The main manifestations of infection caused by Shewanella spp. were diarrhea (100%, 26/26), abdominal pain (65.38%, 17/26), and vomiting (38.46%, 10/26). The 26 isolates were classified into 3 species (S. algae (n = 18), S. indica (n = 5), and S. chilikensis (n = 3)) and 22 sequence types. Core genome single nucleotide polymorphism-based evolutionary tree identified three clone groups corresponding to three infection events in the same months in 2017 and 2019. The putative virulence-associated gene pool consisted of 56 potential virulence genes, including 19 virulence gene factors. The resistance rates of the 26 isolates to 17 antibiotics from high to low were as follows: polymyxin E (76.92%), cefotaxime (57.69%), ampicillin (50%), ampicillin-sulbactam (34.62%), nalidixic acid (15.38%), ciprofloxacin (11.54%), selectrin (3.846%,1/26), and tetracycline (3.846%, 1/26). The rate of multidrug resistance was 38.46% (10/26).DiscussionMonitoring for Shewanella spp. should be added to the routine surveillance of infectious diarrhea during the epidemic season.

IntroductionStenotrophomonas maltophilia complex (Smc) comprises opportunistic Gram-negative bacilli responsible for various nosocomial infections. Limited data exists concerning its evolutionary lineage, global prevalence and pathogenicity.MethodsWe conducted an extensive genomic analysis on 734 Smc genomes, of which 90 were newly sequenced and isolated from different patients. The species composition and evolutionary relationships of Smc were examined using core protein sequence analysis. Pathogenicity evaluation was used by assays for swimming motility, biofilm formation and identification of virulence factors. The broth microdilution method was used to evaluate the drug resistance spectrum of clinical isolates.ResultsPhylogenetic analyses delineated 24 species-level clades, dominated by S. maltophilia (42.8%), S. sepilia (13.6%) and S. geniculata (9.9%). Geographically, strains were primarily distributed in Europe (34.2%), Asia (33.7%) and North America (24.0%), with intricate global distribution patterns. Meanwhile, 154 virulence-associated genes and 46 antimicrobial resistance genes within Smc were identified. These genes encoded span various functions, including motility, adherence, toxin, RND antibiotic efflux pumps, beta-lactamases and aminoglycoside-modifying enzymes. Moreover, significant variations were indicated in swimming motility and biofilm-forming capability across the different species, with S. sepilia exhibiting superior levels of both traits. Additionally, no statistically significant discrepancy was detected among Smc species to other antibiotics, despite the fact that all S. geniculata isolates were resistant to Ceftazidime and much higher than other species.ConclusionOur findings indicate the need to pay increased attention to other mainstream species of Smc besides S. maltophilia in order to better manage Smc-related infections and tailor effective treatment strategies.

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.

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.