Adaptive attenuation of virulence in hypervirulent carbapenem-resistant Klebsiella pneumoniae.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

Early C-reactive Protein Kinetics Predict Response to Immune Checkpoint Blockade in Unresectable Hepatocellular Carcinoma.

Purpose: In recent years, a new therapeutic approach, known as immune checkpoint blockade (ICB), has been proposed as approach to improve outcomes in patients with intermediate stage (Barcelona Clinic Liver Cancer, BCLC B) or advanced stage (BCLC C) hepatocellular carcinoma (HCC). Unfortunately, only a select patients can benefit from ICB. Hence, biomarkers that can predict the success and survival of treatment are still necessary. Patients and Methods: Between 2018 to 2021, 132 patients received ICB treatment for intermediate or advanced stage HCC. Based on the early kinetics of C-reactive protein (CRP), the patients were classified into three groups. The study endpoints were progression-free survival (PFS) and overall survival (OS). Results: Our findings support the predictive power of early CRP kinetics in determining immunotherapy response for intermediate or advanced HCC. Objective response rates (ORR) were found in 41.2% of CRP flare-responders, 13.3% of CRP responders, and 3.5% of CRP non-responders (p<0.001). Disease control rates (DCR) in the three groups were substantially different (p<0.001). The improved PFS and OS were strongly correlated with the early kinetics of CRP. Compared to CRP non-responders, CRP responders, especially CRP flare-responders, had significantly longer PFS (median PFS: CRP flare-responders: 11.6 months vs CRP responders: 5.2 months vs CRP non-responders: 2.3 months, p<0.001). Conclusion: The CRP flare response robustly predicts the immunotherapy response and outcomes in patients with HCC. Early CRP kinetics may be an inexpensive, easily implemented and non-invasive biomarker to anticipate response to ICB therapy in intermediate or advanced HCC, with the potential to optimize treatment monitoring.

Evaluation of packaging capacity at the genomic 2C/3A junction region in Porcine enterovirus G.

Porcine enterovirus G (EV-G) is endogenous to most pig farming countries worldwide. Reports that a papain-like protease (PLP) gene has been naturally inserted into the 2C/3A junction region of the EV-G genome, has increased the potential public health threats from this virus. We constructed a full-length infectious cDNA clone of EV-G, CH/17GXQZ/2017, in order to determine the packaging capacity at the 2C/3A insertion site. Subsequently, recombinants viruses containing the coding tags, GFP, iLOV and His at the 2C/3A junction region, were synthesized. The infectious virus was successfully rescued only with the insertion of the His-tag, which displayed similar virological and molecular properties to its parental strain. This study determined the packaging capacity of the 2C/3A insertion site, and it provides a practical tool for studying the functions and pathogenic mechanisms of EV-G in pigs.

Biological Characteristics and Pathogenicity Analysis of a Low Virulence G2a Porcine Epidemic Diarrhea Virus.

Since the outbreak caused by a porcine epidemic diarrhea virus (PEDV) variant in 2010, the current epidemic of PEDV genotype 2 (G2) has caused huge economic losses to the pig industry in China. In order to better understand the biological characteristics and pathogenicity of the current PEDV field strains, 12 PEDV isolates were collected and plaque purified during 2017 to 2018 in Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate genetic variations, and they were compared with the reported G2a and G2b strains. Phylogenetic analysis of the S protein showed that the 12 isolates were clustered into the G2 subgroup (with 5 and 7 strains in G2a and G2b, respectively) and that they shared 97.4 to 99.9% amino acid identities. Among them, one of the G2a strains, CH/GXNN-1/2018, which had a titer of 106.15 PFU/mL, was selected for pathogenicity analysis. Although piglets infected with the CH/GXNN-1/2018 strain exhibited severe clinical signs and the highest level of virus shedding within 24 h postinfection (hpi), recovery and decreased virus shedding were seen after 48 hpi, and no piglets died during the whole process. Thus, the CH/GXNN-1/2018 strain had low virulence in suckling piglets. Virus neutralizing antibody analysis showed that the CH/GXNN-1/2018 strain induced cross-protection against both homologous G2a and heterologous G2b PEDV strains as early as 72 hpi. These results are of great significance for understanding PEDV in Guangxi, China, and they provide a promising naturally occurring low-virulent vaccine candidate for further study. IMPORTANCE The current epidemic of porcine epidemic diarrhea virus (PEDV) G2 has caused huge economic losses to the pig industry. Evaluation for low virulence of the PEDV strains of subgroup G2a would be useful for the future development of effective vaccines. In this study, 12 field strains of PEDV were obtained successfully, and they were characterized from Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate antigenic variations. One of the G2a strains, CH/GXNN-1/2018, was selected for pathogenicity analysis, and it showed that the CH/GXNN-1/2018 strain had low virulence in suckling piglets. These results provide a promising naturally occurring low-virulent vaccine candidate for further study.

Phylogenetic and Spatiotemporal Analyses of Porcine Epidemic Diarrhea Virus in Guangxi, China during 2017-2022.

Since 2010, porcine epidemic diarrhea virus (PEDV) has swept across China and spread throughout the country, causing huge economic losses. In this study, 673 diarrhea samples from 143 pig farms in Guangxi during 2017-2022 were collected and detected for PEDV. Ninety-eight strains were selected for S1 gene analyses and these strains were classified into four subgroups (G1b, G2a, G2b and G2c), accounting for 1.02 (1/98), 75.51 (74/98), 16.33 (16/98) and 7.14% (7/98) of the total, respectively. Importantly, an increased number of strains in the G2c subgroup was found from 2019 onwards. Bayesian analysis revealed that Guigang may have been the epicenter of PEDVs in Guangxi. In addition, Guigang was identified as the primary hub from which PEDVs spread via two routes, namely Guigang-Wuzhou and Guigang-Laibin. Moreover, several coinfections of novel PEDV variants bearing large deletions in the partial S1 protein and PEDVs possessing an intact partial S1 protein were found in pigs. Further recombination analyses indicated that two of the strains, 18-GXNN-6 and 19-GXBH-2, originated from intra-genogroup recombination. Together, our data revealed a new profile of PEDV in Guangxi, China, which enhances our understanding of the distribution, genetic characteristics and evolutionary profile of the circulating PEDV strains in China.

Isolation, Identification, and Evaluation of the Pathogenicity of a Porcine Enterovirus G Isolated From China.

Enterovirus G (EV-G) infects porcine populations worldwide and the infections are generally asymptomatic, with the insertion of the papain-like cysteine protease gene (PLCP) increasing the potential public health threats. However, the genetic and pathogenic characteristics of EV-G itself are not fully understood as yet. In the present study, one EV-G strain, named CH/17GXQZ/2017, was isolated and purified from piglets with diarrheic symptoms from the Guangxi Province, China. This strain produced stable cytopathic effects on Marc-145 cells with a titer of 5 × 106 PFU/mL. The spherical enterovirus particles with diameters of 25-30 nm were observed by using transmission electron microscopy. The whole genome sequence of the CH/17GXQZ/2017 strain consists of 7,364 nucleotides, and the phylogenetic tree based on the amino acid sequences of VP1 indicated this strain was clustered to the G1 genotype. Seven-day-old piglets were inoculated orally with the CH/17GXQZ/2017 strain in order to evaluate its pathogenicity. Although none of the infected piglets died during the experiment, clinical neurological symptoms were observed manifesting as mild hyperemia and Nissl bodies vacuolization in the cerebrum. In addition, the infection with the CH/17GXQZ/2017 strain decelerated the weight gain of suckling piglets significantly. This study demonstrates that CH/17GXQZ/2017 is pathogenic to neonatal piglets and advance knowledge on the biological characteristics, evolution and pathogenicity of EV-G.

Screening of wheat straw biochars for the remediation of soils polluted with Zn (II) and Cd (II).

The immobilization behaviors of Zn(II) and Cd(II) by wheat straw (WS) biochars could vary with the soil conditions. In the acidic environment, WS biochars produced at low temperature were more competent than those produced at high temperature on Zn(II) and Cd(II) immobilization; while WS biochars produced at high temperature were more effective than those produced at low temperature in the alkaline environment. The ions in the porewater could compromise the sorption capacities of Zn(II) and Cd(II) by WS biochars in acidic soils, while could enhance them in alkaline soils. For biochars produced at the same temperature, residence time had little effect on their behaviors of Zn(II) and Cd(II) immobilization. Only a small portion of immobilized Zn(II)/Cd(II) could be released from WS biochar in the simulated acid rain. Compared with Zn(II)/Cd(II) adsorbed on the acidic functional groups, Zn(II)/Cd(II) precipitates were more stable in 0.01 M CaCl2 solution. Most of the Zn(II) and Cd(II) species on biochar could be released in 1 mM citric acid solution. The immobilized Zn(II) and Cd(II) on WS biochar are likely to be released into the soil environment in the long run.