Table_2_Epidemiological characteristics of SHV, cmlv, and FosA6-producing carbapenem-resistant Klebsiella pneumoniae based on whole genome sequences in Jiangsu, China.xlsx

Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly those with high virulence, cause invasive disease in clinical settings. An epidemiological investigation was conducted on the evolution, virulence, and antimicrobial resistance of CRKP isolates in two tertiary teaching hospitals in Jiangsu, China from November 2020 to December 2021. There were 31 different CRKP strains discovered. We performed whole genome sequencing (WGS) on 13 SHV, cmlv, and FosA6-producing CRKP to reveal molecular characteristics. Five ST15/ST11 isolates had CRISPR-Cas systems. By conjugation tests, KPC-2 can be transmitted horizontally to E. coil. A conjugative pHN7A8-related multi-resistance plasmid (KPC-2, blaCTX-M-65, blaTEM-1, fosA3, catII, and rmtB) was first discovered in CRKP clinical isolates. Using bacteriological testing, a serum killing assay, and an infection model with Galleria mellonella, three ST11-K64 KPC-2 generating carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) were identified. These strains harbored a virulent plasmid and an IncFII-family pKPC/pHN7A8 conjugative plasmid, which led to hypervirulence and resistance. One of these CR-hvKPs, which co-harbored KPC-2, NDM-6, SHV-182, SHV-64, and blaCTX-M-122 genes, was first discovered. Importantly, this CR-hvKP strain also produced biofilm and had non-inferior fitness. The widespread use of ceftazidime/avibactam might provide this CR-hvKP with a selective advantage; hence, immediate action is required to stop its dissemination. Another important finding is the novel ST6136 in K. pneumoniae. Finally, the sterilization efficiency rates of Fe2C nanoparticles in CRKP were more than 98%. Furthermore, our novel antibacterial Fe2C nanoparticles may also provide a therapeutic strategy for infections.</p

Table_3_Epidemiological characteristics of SHV, cmlv, and FosA6-producing carbapenem-resistant Klebsiella pneumoniae based on whole genome sequences in Jiangsu, China.xlsx

Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly those with high virulence, cause invasive disease in clinical settings. An epidemiological investigation was conducted on the evolution, virulence, and antimicrobial resistance of CRKP isolates in two tertiary teaching hospitals in Jiangsu, China from November 2020 to December 2021. There were 31 different CRKP strains discovered. We performed whole genome sequencing (WGS) on 13 SHV, cmlv, and FosA6-producing CRKP to reveal molecular characteristics. Five ST15/ST11 isolates had CRISPR-Cas systems. By conjugation tests, KPC-2 can be transmitted horizontally to E. coil. A conjugative pHN7A8-related multi-resistance plasmid (KPC-2, blaCTX-M-65, blaTEM-1, fosA3, catII, and rmtB) was first discovered in CRKP clinical isolates. Using bacteriological testing, a serum killing assay, and an infection model with Galleria mellonella, three ST11-K64 KPC-2 generating carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) were identified. These strains harbored a virulent plasmid and an IncFII-family pKPC/pHN7A8 conjugative plasmid, which led to hypervirulence and resistance. One of these CR-hvKPs, which co-harbored KPC-2, NDM-6, SHV-182, SHV-64, and blaCTX-M-122 genes, was first discovered. Importantly, this CR-hvKP strain also produced biofilm and had non-inferior fitness. The widespread use of ceftazidime/avibactam might provide this CR-hvKP with a selective advantage; hence, immediate action is required to stop its dissemination. Another important finding is the novel ST6136 in K. pneumoniae. Finally, the sterilization efficiency rates of Fe2C nanoparticles in CRKP were more than 98%. Furthermore, our novel antibacterial Fe2C nanoparticles may also provide a therapeutic strategy for infections.</p

Table_1_Epidemiological characteristics of SHV, cmlv, and FosA6-producing carbapenem-resistant Klebsiella pneumoniae based on whole genome sequences in Jiangsu, China.xlsx

Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly those with high virulence, cause invasive disease in clinical settings. An epidemiological investigation was conducted on the evolution, virulence, and antimicrobial resistance of CRKP isolates in two tertiary teaching hospitals in Jiangsu, China from November 2020 to December 2021. There were 31 different CRKP strains discovered. We performed whole genome sequencing (WGS) on 13 SHV, cmlv, and FosA6-producing CRKP to reveal molecular characteristics. Five ST15/ST11 isolates had CRISPR-Cas systems. By conjugation tests, KPC-2 can be transmitted horizontally to E. coil. A conjugative pHN7A8-related multi-resistance plasmid (KPC-2, blaCTX-M-65, blaTEM-1, fosA3, catII, and rmtB) was first discovered in CRKP clinical isolates. Using bacteriological testing, a serum killing assay, and an infection model with Galleria mellonella, three ST11-K64 KPC-2 generating carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) were identified. These strains harbored a virulent plasmid and an IncFII-family pKPC/pHN7A8 conjugative plasmid, which led to hypervirulence and resistance. One of these CR-hvKPs, which co-harbored KPC-2, NDM-6, SHV-182, SHV-64, and blaCTX-M-122 genes, was first discovered. Importantly, this CR-hvKP strain also produced biofilm and had non-inferior fitness. The widespread use of ceftazidime/avibactam might provide this CR-hvKP with a selective advantage; hence, immediate action is required to stop its dissemination. Another important finding is the novel ST6136 in K. pneumoniae. Finally, the sterilization efficiency rates of Fe2C nanoparticles in CRKP were more than 98%. Furthermore, our novel antibacterial Fe2C nanoparticles may also provide a therapeutic strategy for infections.</p

Additional file 3: Figure S2. of Iron-regulated gene ireA in avian pathogenic Escherichia coli participates in adhesion and stress-resistance

Technical routes of construction of the ireA complementary strain. (TIF 102 kb

Additional file 1: Table S1. of Iron-regulated gene ireA in avian pathogenic Escherichia coli participates in adhesion and stress-resistance

Detailed information about the distribution of the ireA gene in APEC strains. (DOCX 21 kb

Table_3_A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages.docx

<p>The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the P_hlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.</p

Image_3_A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages.JPEG

<p>The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the P_hlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.</p

Image_4_A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages.TIF

<p>The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the P_hlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.</p