The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes. The level of interest in intrinsic resistance genes has increased recently, and one of reasons is that their mobilization could lead to emergence of resistant pathogens. Insertion sequences (ISs) or plasmids can capture intrinsic resistance genes and disseminate them in bacterial populations. In this study, we identified a novel subclass of aminoglycoside nucleotidyltransferases which are intrinsic in A. baumannii and other Acinetobacter species. The genes encoding the aminoglycoside nucleotidyltransferase were frequently horizontally transferred between different Acinetobacter species by homologous recombination. This work reports a novel mechanism of natural resistance in Acinetobacter and an overlooked pathway for the dissemination of resistance among species in this genus.

The convergence of resistance and hypervirulence in Klebsiella pneumoniae represents a significant public health threat, driven by the horizontal transfer of plasmids. Understanding factors affecting plasmid transfer efficiency is essential to elucidate mechanisms behind emergence of these formidable pathogens. Hypermucoviscous K. pneumoniae strains were serially passaged in LB medium to investigate capsule-deficient phenotypes. Capsule-deficient mutants were analyzed using genetic sequencing to identify the types and insertion sites of insertion sequences (IS). Bioinformatics and statistical analyses based on the NCBI and National Microbiology Data Center (NMDC) database were used to map the origins and locations of IS elements. Conjugation assays were performed to assess plasmid transfer efficiency between encapsulated and capsule-deficient strains. A murine intestinal colonization model was employed to evaluate virulence levels and IS excision-mediated capsule restoration. Our research revealed that a hypervirulent K. pneumoniae (hvKP) strain acquired a blaNDM-1-bearing IncX3 plasmid with IS5 and ISKox3 elements. These IS elements are capable of inserting into capsular polysaccharide synthesis genes, causing a notably high frequency of capsule loss in vitro. The IS-mediated capsular phase variation, whether occurring in the donor or recipient strain, significantly increased the conjugation frequency of both the resistance plasmid and the virulence plasmid. Additionally, capsular phase variation enhanced bacterial adaptability in vitro. Experiments in mouse models demonstrated that capsule-deficient mutants exhibited reduced virulence and colonization capacity. However, during long-term intestinal colonization, IS element excision restored capsule expression, leading to the recovery of hypervirulence and enhanced colonization efficiency. Our findings reveal that IS elements mediate capsular phase variation by toggling gene activity, accelerating the genomic convergence of multidrug resistance and hypervirulence in K. pneumoniae, as well as facilitating adaptive transitions in different environments.

Vibrio parahaemolyticus is an important foodborne pathogen and has recently gained particular notoriety because it causes acute hepatopancreatic necrosis disease (AHPND) in shrimp, which has caused significant economic loss in the shrimp industry. Here, we report a whole-genome analysis of 233 V. parahaemolyticus strains isolated from humans, diseased shrimp, and environmental samples collected between 2008 and 2017, providing unprecedented insight into the historical spread of AHPND. The results show that V. parahaemolyticus is genetically diverse and can be divided into 84 sequence types (STs). However, genomic analysis of three STs of V. parahaemolyticus identified seven transmission routes in Asia since 1996, which promoted the transfer of an AHPND-associated plasmid. Notably, the insertion sequence (ISVal1) from the plasmid subsequently mediated the genetic exchange among V. parahaemolyticus STs and resulted in the deletion of an 11-kb region regulating cell mobility and the production of capsular polysaccharides. Phenotype assays confirmed that this deletion enhanced biofilm formation, providing a novel mechanism for environmental adaptation. We conclude that the transmission mode of AHPND consists of two steps, the transmission of V. parahaemolyticus and the subsequent horizontal transfer of the AHPND-associated plasmid. This plasmid allows ISVal1 to mediate genetic exchange and improve pathogen fitness in shrimp ponds. Current shrimp farming practices promoted such genetic exchanges, which highlighted a risk of the emergence of new virulent populations, with potentially devastating consequences for both aquaculture and human health. This study addressed the basic questions regarding the transmission mechanism of AHPND and provided novel insights into shrimp and human disease management. IMPORTANCE Global outbreaks of shrimp acute hepatopancreatic necrosis disease (AHPND) caused by V. parahaemolyticus represent an urgent issue for the shrimp industry. This study revealed that the transmission mode of AHPND consists of two steps, the transregional dissemination of V. parahaemolyticus and the horizontal transfer of an AHPND-associated plasmid. Surprisingly, the introduction of the AHPND-associated plasmid also offers a novel mechanism of genetic exchange mediated by insertion sequences, and it improved the fitness of V. parahaemolyticus in a harsh environment. The results presented herein suggest that current shrimp farming practices promote genetic mixture between endemic and oceanic V. parahaemolyticus populations, which introduced the plasmid and accelerated bacterial adaptation by the acquisition of ecologically important functions. This entails a risk of the emergence of new virulent populations both for shrimp and humans. This study improves our understanding of the global dissemination of the AHPND-associated plasmid and highlights the urgent need to improve biosecurity for shrimp farming.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause serious infections and has been increasingly reported clinically. However, we still lack the knowledge to what degree hvKp colonize the community. In this study, we investigated colonization of hvKp in healthy human gut and the relationship between gut hvKp and clinically important invasive strains. We compile global genomes of gut K. pneumoniae for in-depth genetic analysis and found most hvKp genomes originated from Chinese datasets; therefore, we collected gut K. pneumoniae isolates from healthy people around China. The results revealed a moderate carriage rate of hvKp in the healthy population (4%–5.19%). Phylogenetic analysis indicated a close relationship between gut hvKp and fatal clinical strains. These results demonstrate that the human gut may serve as a reservoir of hvKp and that gut hvKp can play a role in infection of other body parts.