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Abstract Background Shewanella xiamenensis, widely distributed in natural environments, has long been considered as opportunistic pathogen. Recently, significant changes in the resistance spectrum have been observed in S. xiamenensis, due to acquired antibiotic resistance genes. Therefore, a pan-genome analysis was conducted to illuminate the genomic changes in S. xiamenensis. Results Phylogenetic analysis revealed three major clusters and three singletons, among which close relationship between several strains was discovered, regardless of their host and niches. The “open” genomes with diversity of accessory and strain-specific genomes took advantage towards diversity environments. The purifying selection pressure was the main force on genome evolution, especially in conservative genes. Only 53 gene families were under positive selection pressure. Phenotypic resistance analysis revealed 21 strains were classified as multi-drug resistance (MDR). Ten types of antibiotic resistance genes and two heavy metal resistance operons were discovered in S. xiamenensis. Mobile genetic elements and horizontal gene transfer increased genome diversity and were closely related to MDR strains. S. xiamenensis carried a variety of virulence genes and macromolecular secretion systems, indicating their important roles in pathogenicity and adaptability. Type IV secretion system was discovered in 15 genomes with various sequence structures, indicating it was originated from different donors through horizontal gene transfer. Conclusions This study provided with a detailed insight into the changes in the pan-genome of S. xiamenensis, highlighting its capability to acquire new mobile genetic elements and resistance genes for its adaptation to environment and pathogenicity to human and animals.

Abstract Background Recently, extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) isolates have been increasingly detected and posed great challenges to clinical anti-infection treatments. However, little is known about extensively resistant hypervirulent P. aeruginosa (XDR-hvPA). In this study, we investigate its epidemiological characteristics and provide important basis for preventing its dissemination. Methods Clinical XDR-PA isolates were collected from January 2018 to January 2023 and identified using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry; antibiotic susceptibility testing was performed by broth microdilution method, and minimum inhibitory concentrations (MICs) were evaluated. Virulence was evaluated using the Galleria mellonella infection model; molecular characteristics, including resistance genes, virulence genes, and homology, were determined using whole-genome sequencing. Results A total of 77 XDR-PA strains were collected; 47/77 strains were XDR-hvPA. Patients aged > 60 years showed a significantly higher detection rate of XDR-hvPA than of XDR-non-hvPA. Among the 47 XDR-hvPA strains, 24 strains carried a carbapenemase gene, including bla GES−1 (10/47), bla VIM−2 (6/47), bla GES−14 (4/47), bla IMP−45 (2/47), bla KPC−2 (1/47), and bla NDM−14 (1/47). ExoU, exoT, exoY, and exoS, important virulence factors of PA, were found in 31/47, 47/47, 46/47, and 29/47 strains, respectively. Notably, two XDR-hvPA simultaneously co-carried exoU and exoS. Six serotypes (O1, O4–O7, and O11) were detected; O11 (19/47), O7 (13/47), and O4 (9/47) were the most prevalent. In 2018–2020, O4 and O7 were the most prevalent serotypes; 2021 onward, O11 (16/26) was the most prevalent serotype. Fourteen types of ST were detected, mainly ST235 (14/47), ST1158 (13/47), and ST1800 (7/47). Five global epidemic ST235 XDR-hvPA carried bla GES and showed the MIC value of ceftazidime/avibactam reached the susceptibility breakpoint (8/4 mg/L). Conclusions The clinical detection rate of XDR-hvPA is unexpectedly high, particularly in patients aged > 60 years, who are seemingly more susceptible to contracting this infection. Clonal transmission of XDR-hvPA carrying bla GES, which belongs to the global epidemic ST235, was noted. Therefore, the monitoring of XDR-hvPA should be strengthened, particularly for elderly hospitalized patients, to prevent its spread.

Abstract Background The prevalence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP) has gradually increased. It poses a severe threat to human health. However, polymyxin-resistant hvKP is rare. Here, we collected eight polymyxin B-resistant K. pneumoniae isolates from a Chinese teaching hospital as a suspected outbreak. Results The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. HvKP was identified by detecting virulence-related genes and using a Galleria mellonella infection model. Their resistance to serum, growth, biofilm formation, and plasmid conjugation were analyzed in this study. Molecular characteristics were analyzed using whole-genome sequencing (WGS) and mutations of chromosome-mediated two-component systems pmrAB and phoPQ, and the negative phoPQ regulator mgrB to cause polymyxin B (PB) resistance were screened. All isolates were resistant to polymyxin B and sensitive to tigecycline; four were resistant to ceftazidime/avibactam. Except for KP16 (a newly discovered ST5254), all were of the K64 capsular serotype and belonged to ST11. Four strains co-harbored bla KPC-2, bla NDM-1, and the virulence-related genes p rmpA, p rmpA2, iucA, and peg344, and were confirmed to be hypervirulent by the G. mellonella infection model. According to WGS analysis, three hvKP strains showed evidence of clonal transmission (8–20 single nucleotide polymorphisms) and had a highly transferable pKOX_NDM1-like plasmid. KP25 had multiple plasmids carrying bla KPC-2, bla NDM-1, bla SHV-12, bla LAP-2, tet(A), fosA5, and a pLVPK-like virulence plasmid. Tn1722 and multiple additional insert sequence-mediated transpositions were observed. Mutations in chromosomal genes phoQ and pmrB, and insertion mutations in mgrB were major causes of PB resistance. Conclusions Polymyxin-resistant hvKP has become an essential new superbug prevalent in China, posing a serious challenge to public health. Its epidemic transmission characteristics and mechanisms of resistance and virulence deserve attention.

Abstract Background The heteroresistance of polymyxin B, a last-resort antibiotic used to treat many serious bacterial infections, may lead to antibiotic treatment failure. However, polymyxin B-heteroresistant isolates are rare in individuals living in the community. We report a polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae (hvKP) isolate from an individual in the community with asymptomatic bacteriuria. Results The NYTJ35 isolate had multiple virulence genes that encoded a mucoid phenotype regulator (rmpA), aerobactin (iucABCD-iutA), salmochelin (iroBCDN), yersiniabactin (irp1–2 and ybtAEPQSTUX), and a truncated rmpA2. Infection of galleria mellonella larvae indicated the isolate was hypervirulent. Antimicrobial susceptibility testing showed it was susceptible to all tested antibiotics except polymyxin B. The proportion of surviving bacteria was 1.2 × 10− 7 based on the population analysis profile (PAP) method, suggesting the presence of polymyxin B heteroresistance. The isolate was not hypermucoviscous, but it was a strong biofilm producer. It had capsular serotype K1 and belonged to sequence type 23 (ST23). The isolate also had the D150G substitution in phoQ, which is known to confer polymyxin B resistance. Conclusions We identified the co-occurrence of hypervirulence and polymyxin B heteroresistance in a K. pneumoniae isolate from an individual with asymptomatic bacteriuria. We suggest the use of increased screening for hvKP in individuals living in the community.

Abstract Background Asymptomatic bacteriuria (ASB) frequently occurs among all ages and may develop into urinary tract infections (UTIs). Hypervirulent Klebsiella pneumoniae (hvKP) has become a new threat to human health. In our study, we aimed to investigate the epidemiological characteristics of hvKP in population with ASB. Results A total of 61 K. pneumoniae isolates were collected from 7530 urine samples between October and December 2020. The strains were sensitive to most of the antimicrobial agents tested, but a polymyxin resistant strain was found (MIC>16 μg/mL). Three serotypes were detected, including K1 (16.4%, 10/61), K5 (1.6%, 1/61) and K57 (3.2%, 2/61). Four strains (KPNY9, KPNY31, KPNY40, and KPNY42) carried a combination of two or more hypervirulent markers (peg-344, iroB, iucA, p rmpA, and p rmpA2), and their survival rates after Galleria mellonella infection were lower than those of the other strains (40.0 vs. 70.0%), suggesting that they were hvKP. These hvKP strains with lower biofilm forming ability than classical K. pneumoniae (0.2625 ± 0.0579 vs. 0.6686 ± 0.0661, P = 0.033) were identified as belonging to K2-ST65, K2-ST86, K57-ST592, and K2-ST5559 (a new ST type). KPNY31 (ST5559) shared a close genetic relationship with KPNY42 (ST86) and other ST86 isolates, which have been detected in both nosocomial and community-acquired infections. Conclusions The hvKP with relatively weak biofilm formation was detected in a population with ASB, which was more likely to cause bacteremia and serious consequences. A novel sequence type (ST5559) hvKP derived from ST86 was found. Therefore, hvKP should be monitored in the population with ASB.

Abstract Background Fosfomycin has been proven to be a vital choice to treat infection caused by multidrug resistance bacteria, especially carbapenem-resistant Klebsiella pneumoniae (CRKP). However, fosfomycin resistant cases has been reported gradually. In this study, we reported the fosfomycin-resistant rate in CRKP strains and further revealed the molecular mechanisms in resistance gene dissemination. Results A total of 294 non-duplicated CRKP strains were collected. And 55 fosfomyin-resistant strains were detected, 94.5% of which were clustered to sequence type (ST) 11 by PCR followed up sequencing. PFGE further revealed two major groups and four singletons. The positive rates of genes responsible to fosfomycin and carbapenem resistance were 81.8% (fosA3), 12.7% (fosA5) and 94.5% (bla KPC-2), respectively. Genomic analysis confirmed insertion sequence (IS) 26 was the predominant structure surrounding fosA3. The fosA3 genes in six isolates were located on plasmids which were able to transfer to E. coli J53 recipient cells by means of conjugation. Conclusions Although the resistant rate of CRKP to fosfomycin is relatively low in our area, considering its gene is located on transferrable plasmid and inserted in IS structure, continuous monitoring is still needed.

Abstract Background Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, “M2-like” macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4). Methods To enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing. Results We observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain. Conclusion The results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.