Acquisition of a stable and transferable plasmid coharbouring hypervirulence and MDR genes with low fitness cost: Accelerating the dissemination of ST11-KL64 CR-HvKP.

OBJECTIVES: This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism. METHODS: We employed whole-genome sequencing to decipher the genetic architecture of pS130-4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests. RESULTS: pS130-4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both blaKPC-2 and hypervirulence genes. Our conjugation experiments established that pS130-4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10-5 and 10-4. Remarkably, pS130-4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130-4's virulence remained intact postconjugation. CONCLUSION: A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid.

Quantitative real-time PCR and magnetic separation strategy for specific detection of group B streptococcus in perinatal Women's urine.

Introduction: Group B streptococcus(GBS)often causes adverse outcomes such as urinary system infection, intrauterine infection, premature birth, and stillbirth in perinatal women. Perinatal screening of GBS is conducive to guiding clinical scientific intervention and improving delivery outcomes.This study quantitative real-time PCR (RT-qPCR) combined with magnetic separation was used for GBS detection. Materials and methods: Sample pre-treatment in this study involved the utilization of magnetic separation (MS) technology, aiming to expedite the detection process and enhance detection sensitivity, and the cfb gene of group B streptococcus was used as the target gene to establish quantitative real-time PCR (RT-qPCR) to detect group B streptococcus. Results: It was found that penicillin-functionalized magnetic beads had a good ability to enrich and capture group B Streptococcus.The findings revealed an exceptional detection sensitivity, with the ability to detect B streptococcus in urine samples at levels as low as 102 CFU/mL. Conclusions: The utilization of MS technology in conjunction with the RT-qPCR (MS-RT-qPCR) assay, as demonstrated in this study, offers a viable approach for prenatal screening of group B streptococcus among perinatal women.

Horizontal gene transfer via OMVs co-carrying virulence and antimicrobial-resistant genes is a novel way for the dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Introduction: The rapidly increased isolation rate of CR-HvKP worldwide has brought great difficulties in controlling clinical infection. Moreover, it has been demonstrated that the transmission of drug-resistant genes among bacteria can be mediated by outer membrane vesicles (OMVs), which is a new way of horizontal gene transfer (HGT). The transmission of virulence genes among bacteria has also been well studied; however, it remains unclear whether virulence and drug-resistant genes can be co-transmitted simultaneously. Co-transmission of virulence and drug-resistant genes is essential for the formation and prevalence of CR-HvKP. Methods: First, we isolated OMVs from CR-HvKP by cushioned-density gradient ultracentrifugation (C-DGUC). TEM and DLS were used to examine the morphology and size of bacterial OMVs. OMV-mediated gene transfer in liquid cultures and the acquisition of the carbapenem gene and virulence gene was confirmed using colony-PCR. Antimicrobial susceptibility testing, mCIM and eCIM were conducted for the resistance of transformant. Serum killing assay, assessment of the anti-biofilm effect and galleria mellonella infection model, mucoviscosity assay, extraction and quantification of capsules were verified the virulence of transformant. Pulsed-field gel electrophoresis (PFGE), S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting hybridization confirmed the plasmid of transformant. Results: Firstly, OMVs were isolated from CR-HvKP NUHL30457 (K2, ST86). TEM and DLS analyses revealed the spherical morphology of the vesicles. Secondly, our study demonstrated that CR-HvKP delivered genetic material, incorporated DNA within the OMVs, and protected it from degradation by extracellular exonucleases. Thirdly, the vesicular lumen DNA was delivered to the recipient cells after determining the presence of virulence and carbapenem-resistant genes in the CR-HvKP OMVs. Importantly, S1-PFGE and Southern hybridization analysis of the 700603 transformant strain showed that the transformant contained both drug-resistant and virulence plasmids. Discussion: In the present study, we aimed to clarify the role of CRHvKP-OMVs in transmitting CR-HvKP among K. pneumoniae. Collectively, our findings provided valuable insights into the evolution of CR-HvKP.

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10-5 and 8.7× 10-7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

Investigation of VOC characteristics, source analysis, and chemical conversions in a typical petrochemical area through 1-year monitoring and emission inventory.

To effectively investigate the characteristics, source analysis, and chemical conversions of volatile organic compounds (VOCs) pollution in a typical petrochemical area, 81 VOC species from nine sampling sites were collected from 1st January to 31th December 2019 in Jinshan District. Results showed the concentration of VOCs was 51.63 ± 36.05 ppbv, and VOCs were dominated by alkane (40.10%) and alkenes (39.91%). The temporal variations of VOCs showed that the highest average VOC concentration appeared in July, and the lowest concentration of VOCs was in February. The concentration of VOCs was mainly connected with industrial processes and was transported to other areas through the downwind direction. Six PMF-derived sources including petrochemical industry, solvent utilization, vehicle exhaust, fuel evaporation, combustion, and other industry processes, contributing 37.08%, 16.74%, 16.69%, 14.99%, 9.53%, and 4.97%, respectively. Meanwhile, an anthropogenic VOC emission inventory was established by emission factors and the activity statistics for 2019, results indicated that the total emission of VOCs was estimated as 6.22 kt, petrochemical industry was the most important contributor of human-produced VOCs. The LOH concentration was 396.12 ppbv via OH radical loss rate method, and the OFP was 210.44 ppbv based on the MIR factor. Alkenes and aromatics were the important components of O3 formation. This study provides effective information for corresponding governments to establish VOCs contamination control directives.

Spatiotemporal variations of ambient air pollutants and meteorological influences over typical urban agglomerations in China during the COVID-19 lockdown.

To investigate the air quality change during the COVID-19 pandemic, we analyzed spatiotemporal variations of six criteria pollutants in nine typical urban agglomerations in China using ground-based data and examined meteorological influences through correlation analysis and backward trajectory analysis under different responses. Concentrations of PM2.5, PM10, NO2, SO2 and CO in urban agglomerations respectively decreased by 18%-45% (30%-62%), 17%-53% (22%-39%), 47%-64% (14%-41%), 9%-34% (0%-53%) and 16%-52% (23%-56%) during Lockdown (Post-lockdown) period relative to Pre-lockdown period. PM2.5 pollution events occurred during Lockdown in Beijing-Tianjin-Hebe (BTH) and Middle and South Liaoning (MSL), and daily O3 concentration rose to grade Ⅱ standard in Post-lockdown period. Distinct from the nationwide slump of NO2 during Lockdown period, a rebound (∼40%) in Post-lockdown period was observed in Cheng-Yu (CY), Yangtze River Middle-Reach (YRMR), Yangtze River Delta (YRD) and Pearl River Delta (PRD). With slightly higher wind speed compared with 2019, the reduction of PM2.5 (51%-62%) in Post-lockdown period is more than 2019 (15%-46%) in HC (Harbin-Changchun), MSL, BTH, CP (Central Plain) and SP (Shandong-Peninsula), suggesting lockdown measures are effective to PM2.5 alleviation. Although O3 concentrations generally increased during the lockdown, its increment rate declined compared with 2019 under similar sunlight duration and temperature. Additionally, unlike HC, MSL and BTH, which suffered from additional (> 30%) air masses from surrounding areas after the lockdown, the polluted air masses reaching YRD and PRD mostly originated from the long-distance transport, highlighting the importance of joint regional governance.

Atmospheric characteristics and population exposure assessment of black carbon at a regional representative site in the Yangtze River Delta region, China based on the five-year monitoring.

To investigate the multi-year atmospheric characteristic and population exposure level of black carbon (BC) in the Yangtze River Delta (YRD) region, China, about five years of ambient BC measurement was performed at Dianshan Lake (DSL) regional Supersite from February 2014 to February 2019. BC concentration at DSL was 1.39 ± 1.15 µg m-3, which was at low to medium level compared to other areas in the world, and annual BC level was decreased by an average of 45.4% from 2014 to 2018. The absorption Ångström exponent (AAE) value was 1.30 ± 0.173, indicating the predominant contribution of liquid fuel sources such as traffic exhaust to BC. Meanwhile, AAE and BC values both showed the winter-high and summer-low temporal patterns, which highlighted the increasing contribution from solid fuels to BC in winter. Moreover, diurnal characteristics of BC, AAE, carbon monoxide and nitrogen oxide demonstrated the dominance of traffic sources for BC. The average estimated daily intakes (EDIs) of BC through inhalation for 17 population age subgroups were 0.0177-0.0811 µg kg-1 day-1, which the highest EDIs for male and female were both observed in infants (9 months ~ < 1 year). Male generally taken higher exposure level of BC compared to female. This work could improve the insights for atmospheric characteristic and population exposure level of BC, and potentially facilitate the development of abatement policies of BC in YRD region, China.

Ozone pollution mitigation in guangxi (south China) driven by meteorology and anthropogenic emissions during the COVID-19 lockdown.

With the implementation of COVID-19 restrictions and consequent improvement in air quality due to the nationwide lockdown, ozone (O3) pollution was generally amplified in China. However, the O3 levels throughout the Guangxi region of South China showed a clear downward trend during the lockdown. To better understand this unusual phenomenon, we investigated the characteristics of conventional pollutants, the influence of meteorological and anthropogenic factors quantified by a multiple linear regression (MLR) model, and the impact of local sources and long-range transport based on a continuous emission monitoring system (CEMS) and the HYSPLIT model. Results show that in Guangxi, the conventional pollutants generally declined during the COVID-19 lockdown period (January 24 to February 9, 2020) compared with their concentrations during 2016-2019, while O3 gradually increased during the resumption (10 February to April 2020) and full operation periods (May and June 2020). Focusing on Beihai, a typical Guangxi region city, the correlations between the daily O3 concentrations and six meteorological parameters (wind speed, visibility, temperature, humidity, precipitation, and atmospheric pressure) and their corresponding regression coefficients indicate that meteorological conditions were generally conducive to O3 pollution mitigation during the lockdown. A 7.84 µg/m3 drop in O3 concentration was driven by meteorology, with other decreases (4.11 µg/m3) explained by reduced anthropogenic emissions of O3 precursors. Taken together, the lower NO2/SO2 ratios (1.25-2.33) and consistencies between real-time monitored primary emissions and ambient concentrations suggest that, with the closure of small-scale industries, residual industrial emissions have become dominant contributors to local primary pollutants. Backward trajectory cluster analyses show that the slump of O3 concentrations in Southern Guangxi could be partly attributed to clean air mass transfer (24-58%) from the South China Sea. Overall, the synergistic effects of the COVID-19 lockdown and meteorological factors intensified O3 reduction in the Guangxi region of South China.

Improving VOCs control strategies based on source characteristics and chemical reactivity in a typical coastal city of South China through measurement and emission inventory.

In China, the corresponding control directives for volatile organic compounds (VOCs) have been based on primary emissions, rarely considering reactive speciation. To seek more effective VOCs control strategies, we investigated 107 VOC species in a typical coastal city (Beihai) of South China, from August to November 2018. Meanwhile, a high-resolution anthropogenic VOCs monthly emission inventory (EI) was established for 2018. For source apportionments (SAs) reliability, comparisons of source structures derived from positive matrix factorization (PMF) and EI were made mainly in terms of reaction losses, uncertainties and specific ratios. Finally, for the source-end control, a comprehensive reactivity control index (RCI) was established by combing SAs with reactive speciation profiles. Ambient measurements showed that the average concentration of VOCs was 26.38 ppbv, dominated by alkanes (36.7%) and oxygenated volatile organic compounds (OVOCs) (29.4%). VOC reactivity was estimated using ozone formation potential (52.35 ppbv) and propylene-equivalent concentration (4.22 ppbv). EI results displayed that the entire VOC, OFP, and propylene-equivalent emissions were 40.98 Gg, 67.98 Gg, and 105.93 Gg, respectively. Comparisons of source structures indicated that VOC SAs agreed within ±100% between two perspectives. Both PMF and EI results showed that petrochemical industry (24.0% and 33.0%), food processing and associated combustion (19.1% and 29.2%) were the significant contributors of anthropogenic VOCs, followed by other industrial processes (22.2% and 13.3%), transportation (18.9% and 12.0%), and solvent utilization (9.1% and10.5%). Aimed at VOCs abatement according to RCI: for terminal control, fifteen ambient highly reactive species (predominantly alkenes and alkanes) were targeted; for source control, the predominant anthropogenic sources (food industry, solvent usage, petrochemical industry and transportation) and their emitted highly reactive species were determined. Particularly, with low levels of ambient VOC and primary emissions, in this VOC and NOx double-controlled regime, crude disorganized emission from food industry contributed a high RCI.