Detection and Quantification of Klebsiella pneumoniae in Fecal Samples Using Digital Droplet PCR in Comparison with Real-Time PCR.

This study aimed to develop a rapid and sensitive droplet digital PCR (ddPCR) assay for the specific detection of Klebsiella pneumoniae in fecal samples, and to evaluate its application in the clinic by comparison with real-time PCR assay and conventional microbial culture. Specific primers and a probe targeting the K. pneumoniae hemolysin (khe) gene were designed. Thirteen other pathogens were used to evaluate the specificity of the primers and probe. A recombinant plasmid containing the khe gene was constructed and used to assess the sensitivity, repeatability, and reproducibility of the ddPCR. Clinical fecal samples (n = 103) were collected and tested by the ddPCR, real-time PCR, and conventional microbial culture methods. The detection limit of ddPCR for K. pneumoniae was 1.1 copies/µL, about a 10-fold increase in sensitivity compared with real-time PCR. The ddPCR was negative for the 13 pathogens other than K. pneumoniae, confirming its high specificity. Clinical fecal samples gave a higher rate of positivity in the K. pneumoniae ddPCR assay than in analysis by real-time PCR or conventional culture. ddPCR also showed less inhibition by the inhibitor in fecal sample than real-time PCR. Thus, we established a sensitive and effective ddPCR-based assay method for K. pneumoniae. It could be a useful tool for K. pneumoniae detection in feces and may serve as a reliable method to identify causal pathogens and help guide treatment decisions. IMPORTANCE Klebsiella pneumoniae can cause a range of illnesses and has a high colonization rate in the human gut, making it crucial to develop an efficient method for detecting K. pneumoniae in fecal samples.

Bacteriophage targeting microbiota alleviates non-alcoholic fatty liver disease induced by high alcohol-producing Klebsiella pneumoniae.

Our previous studies have shown that high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the intestinal microbiome could be one of the causes of non-alcoholic fatty liver disease (NAFLD). Considering antimicrobial resistance of K. pneumoniae and dysbacteriosis caused by antibiotics, phage therapy might have potential in treatment of HiAlc Kpn-induced NAFLD, because of the specificity targeting the bacteria. Here, we clarified the effectiveness of phage therapy in male mice with HiAlc Kpn-induced steatohepatitis. Comprehensive investigations including transcriptomes and metabolomes revealed that treatment with HiAlc Kpn-specific phage was able to alleviate steatohepatitis caused by HiAlc Kpn, including hepatic dysfunction and expression of cytokines and lipogenic genes. In contrast, such treatment did not cause significantly pathological changes, either in functions of liver and kidney, or in components of gut microbiota. In addition to reducing alcohol attack, phage therapy also regulated inflammation, and lipid and carbohydrate metabolism. Our data suggest that phage therapy targeting gut microbiota is an alternative to antibiotics, with potential efficacy and safety, at least in HiAlc Kpn-caused NAFLD.

Three Klebsiella species as potential pathobionts generating endogenous ethanol in a clinical cohort of patients with auto-brewery syndrome: a case control study.

BACKGROUND: Patients with auto-brewery syndrome (ABS) become inebriated after the ingestion of an alcohol-free, high-carbohydrate diet. Our previous work has shown that high-alcohol-producing (HiAlc) Klebsiella pneumoniae can generate excessive endogenous ethanol and cause non-alcoholic fatty liver disease (NAFLD). Therefore, it is reasonable to speculate that such bacteria might play an important role in the pathogenesis of ABS. METHODS: The characteristics and metabolites of the intestinal flora from a clinical cohort of patients with ABS were analysed during different stages of disease and compared to a group of healthy controls. An in vitro culture system of relevant samples was used for screening drug sensitivity and ABS-inducing factors. Rabbit intestinal and murine models were established to verify if the isolated strains could induce ABS in vivo. FINDINGS: We observed intestinal dysbiosis with decreased abundance of Firmicutes and increased of Proteobacteria in patients with ABS compared with healthy controls. The abundance of the genus Klebsiella in Enterobacteriaceae was strongly associated with fluctuations of patient's blood alcohol concentration. We isolated three species of HiAlc Klebsiella from ABS patients, which were able to induce ABS in mice. Monosaccharide content was identified as a potential food-related inducing factor for alcohol production. Treatments with antibiotics, a complex probiotic preparation and a low-carbohydrate diet not only alleviated ABS, but also erased ABS relapse during the follow-up observation of one of the patients. INTERPRETATION: Excessive endogenous alcohol produced by HiAlc Klebsiella species was an underlying cause of bacterial ABS. Combined prescription of appropriate antibiotics, complex probiotic preparation and a controlled diet could be sufficient for treatment of bacteria-caused ABS. FUNDING: The funders are listed in the acknowledgement.

Rapid detection of mpox virus using recombinase aided amplification assay.

A recent, unprecedented outbreak of human mpox virus infection has led to cases in non-African nations, and the number of confirmed or suspected cases outside of Africa has exceeded 1,000 within 5 weeks. Mpox may pose a double threat to public health in the context of the ongoing COVID-19 pandemic. It is difficult to distinguish mpox virus infection from other diseases in the early stages, and patients are contagious from the onset of nonspecific symptoms; therefore, it is crucial to develop rapid and specific diagnostic methods. The diagnosis of mpox relies on real-time polymerase chain reaction, a time-consuming method that requires a highly sophisticated thermal cycler, which makes it unsuitable for widespread use in underdeveloped areas, where the outbreak is still severe. In this study, we developed a recombinase-aided amplification (RAA) assay that can detect mpox virus within 5-10 minutes. The conserved regions of the A27L gene and F3L gene were selected as targets, as they amplify well from different mpox virus clades with no cross-reaction from other pathogens. The sensitivity of this RAA assay is 10 copies/reaction for the A27L gene and 102 copies/reaction for the F3L gene. When applied to simulated clinical samples, both targets showed 100% specificity, and the detection limits were consistent with the sensitivity results. Moreover, through clinical blinded sample detection, RAA exhibits the same detection power as RT-PCR. In summary, the RAA mpox assay described here exhibits rapid detection, high sensitivity and specificity, and low operational difficulty, making it suitable for mpox virus detection in less developed countries and regions.

Recombinase-Aided Amplification Assay for Rapid Detection of Hypervirulent Klebsiella pneumoniae (hvKp) and Characterization of the hvKp Pathotype.

Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes peg344 and rmpA, and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets peg344-set1 and rmpA-set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors iroN and iutA were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. IMPORTANCEKlebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes peg344 and rmpA. Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.

Bacteriophage Effectively Rescues Pneumonia Caused by Prevalent Multidrug-Resistant Klebsiella pneumoniae in the Early Stage.

Pneumonia caused by multidrug-resistant (MDR) Klebsiella pneumoniae of sequence types ST11 and ST383 have highlighted the necessity for new therapies against these prevalent pathogens. Bacteriophages (phages) may be used as alternatives or complements to antibiotics for treating MDR bacteria because they show potential efficacy in mouse models and even individual clinical cases, and they also cause fewer side effects, such as microbiota-imbalance-induced diseases. In the present study, we screened two phages, pKp11 and pKp383, that targeted ST11 and ST383 MDR K. pneumoniae isolates collected from patients with pneumonia, and they exhibited a broad host range, high lytic activity, and high environmental adaptability. Both phages pKp11 and pKp383 provided an effective treatment for the early stage of pneumonia in a murine infection model without promoting obvious side effects, and cocktails consisting of the two phages were more effective for reducing bacterial loads, inflammation, and pathogenic injuries. Our findings support the application of phages as new medications for refractory ST11 and ST383 K. pneumoniae infections and emphasize the potential of enhancing phage therapy modalities through phage screening. These data provided important resources for assessing and optimizing phage therapies for MDR ST11 and ST383 infection treatment. However, substantial amounts of further work are needed before phage therapy can be translated to human therapeutics. IMPORTANCE K. pneumoniae is recognized as the most common pathogen of hospital- and community-acquired pneumonia across the world. The strains of ST11 and ST383 are frequently reported in patients with pneumonia. However, the efficacy of antibiotics toward K. pneumoniae is decreasing dramatically. As a new approach to combat MDR bacteria, phages have exhibited positive clinical effects and efficacy as synergetic or alternative strategies to antibiotics. Thus, we screened two phages that targeted ST11 and ST383 MDR K. pneumoniae, and they exhibited a broad host range, high lytic activity, and high environmental adaptability. Both phages provided an effective treatment for the early stage of pneumonia in mice, and cocktails consisting of the two phages were more effective in reducing bacterial loads, inflammation, and pathogenic injuries. Although these data suggest that phages are effective alternatives or complements to antibiotics, more research is needed before they can be translated into therapeutics for humans.

Development of a Loop-Mediated Isothermal Amplification Method for Rapid and Visual Detection of Monkeypox Virus.

Monkeypox virus (MPXV) is a human pathogenic virus that belongs to the genus Orthopoxvirus. In 2022, MPXV caused an unprecedented number of infections in many countries. As it is difficult to distinguish MPXV from other pathogens by its symptoms in the early stage of infection, a rapid and reliable assay for MPXV detection is needed. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of MPXV and evaluated its application in simulated clinical samples. The A27L-1 and F3L-1 primer sets were identified as the optimal primers, and 63°C was the most appropriate reaction temperature for sequence amplification. The detection limits of the LAMP assay using primer sets A27L-1 and F3L-1 were both 20 copies/reaction mixture, which were >100-fold higher in terms of sensitivity, compared with conventional PCR. The LAMP assay findings were negative for all 21 non-MPXV pathogens, confirming the high specificity of our assay. All three types of simulated clinical samples were clearly identified by our LAMP assay, and the detection limits were consistent with the sensitivity results, indicating efficient clinical sample identification. Our rapid and reliable MPXV LAMP assay could be useful for MPXV detection and on-site diagnosis, especially in primary hospitals and rural areas. IMPORTANCE MPXV outbreaks rapidly grew in the first half of 2022, and this virus has been recognized as an increasing public health threat, particularly in the context of the COVID-19 pandemic. Thus, developing reliable and fast detection methods for MPXV is necessary.

Rapid detection of Burkholderia cepacia complex carrying the 16S rRNA gene in clinical specimens by recombinase-aided amplification.

The Burkholderia cepacia complex (BCC) is a group of opportunistic pathogens, including Burkholderia cepacia, Burkholderia multivorans, Burkholderia vietnamiensis and Burkholderia ambifaria, which can cause severe respiratory tract infections and lead to high mortality rates among humans. The early diagnosis and effective treatment of BCC infection are therefore crucial. In this study, a novel and rapid recombinase-aided amplification (RAA) assay targeting the 16S rRNA gene was developed for BCC detection. The protocol for this RAA assay could be completed in 10 min at 39°C, with a sensitivity of 10 copies per reaction and no cross-reactivity with other pathogens. To characterize the effectiveness of the RAA assay, we further collected 269 clinical samples from patients with bacterial pneumonia. The sensitivity and specificity of the RAA assay were 100% and 98.5%, respectively. Seven BCC-infected patients were detected using the RAA assay, and three BCC strains were isolated from the 269 clinical samples. Our data showed that the prevalence of BCC infection was 2.60%, which is higher than the 1.40% reported in previous studies, suggesting that high sensitivity is vital to BCC detection. We also screened a patient with B. vietnamiensis infection using the RAA assay in clinic, allowing for appropriate treatment to be initiated rapidly. Together, these data indicate that the RAA assay targeting the 16S rRNA gene can be applied for the early and rapid detection of BCC pathogens in patients with an uncharacterized infection who are immunocompromised or have underlying diseases, thereby providing guidance for effective treatment.

How Do MinC-D Copolymers Act on Z-Ring Localization Regulation? A New Model of Bacillus subtilis Min System.

Division site selection in rod-shaped bacteria is strictly regulated spatially by the Min system. Although many sophisticated studies, including in vitro recombination, have tried to explain these regulations, the precise mechanisms are still unclear. A previous model suggested that the concentration gradient of MinC, an FtsZ inhibitor, regulates the position of the Z-ring in the cell. In Escherichia coli, the oscillation of MinCDE proteins leads to a gradient of Min proteins with the average concentration being lowest in the middle and highest near the poles. In contrast to the Min system of E. coli, the Min system of Bacillus subtilis lacks MinE and exhibits a stable concentration distribution, which is regulated by the binding of DivIVA to the negative curvature membrane. The Min proteins first accumulate at the poles of the cell and relocalize near the division site when the membrane invagination begins. It is inconsistent with the previous model of high concentrations of MinC inhibiting Z-ring formation. Our preliminary data here using electron microscopy and light scattering technology reported that B. subtilis MinC (BsMinC) and MinD (BsMinD) also assembled into large straight copolymers in the presence of ATP, similar to the Min proteins of E. coli. Their assembly is fast and dominated by MinD concentration. When BsMinD is 5 µM, a clear light scattering signal can be observed even at 0.3 µM BsMinC. Here, we propose a new model based on the MinC-D copolymers. In our hypothesis, it is not the concentration gradient of MinC, but the MinC-D copolymer assembled in the region of high concentration MinD that plays a key role in the regulation of Z-ring positioning. In B. subtilis, the regions with high MinD concentration are initially at both ends of the cell and then appear at midcell when cell division began. MinC-D copolymer will polymerize and form a complex with MinJ and DivIVA. These complexes capture FtsZ protofilaments to prevent their diffusion away from the midcell and narrow the Z-ring in the middle of the cell.

Rapid Detection of Multi-Resistance Strains Carrying mcr-1 Gene Using Recombinase-Aided Amplification Directly on Clinical Samples.

With the increasingly severe problem of bacterial resistance, colistin, as the last line of defense, has attracted attention again. Mobile colistin resistance (mcr-1) gene is involved in the horizontal transmission of colistin resistance in Gram-negative bacteria (GNB), which is a serious threat to human health. Therefore, rapid detection of mcr-1 gene presence in clinical samples is crucial. In this study, a Recombinase-aided amplification(RAA) method for mcr-1 was successfully constructed, with sensitivity of 20 copies/reaction. In addition, amplification signal could only be detected in the strain containing mcr-1 gene among 14 different bacterial species. The method was then used to test a total of 672 clinical samples from a pediatric hospital in Beijing. Five strains harbored mcr-1 genes were isolated from mcr-1-positive clinical samples and identified as Escherichia coli. Multi-locus sequence typing (MLST) analysis showed that the five E. coli belonged to different ST types. Notably, the mcr-1 gene from the isolates could be transferred conjugately to the recipient strain E. coli J53, with highest transfer efficiency up to 57-58%, suggesting that the mcr-1 gene was located on the plasmid. These findings showed that the RAA assay has potential to be a rapid and sensitive mcr-1 gene screening test for clinical samples, and mcr-1 could be transmitted vertically and horizontally between and within bacterial species in a plasmid-mediated manner.

Genetic Diversity and Pathogenic Features in Klebsiella pneumoniae Isolates from Patients with Pyogenic Liver Abscess and Pneumonia.

While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. In this study, we investigated the prevalence and molecular characteristics of K. pneumoniae isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and genomic and phenotypic assays were used to determine the differences among the isolates. A total of 232 K. pneumoniae isolates were subtyped into 74 sequence types (STs). The isolates from different sources had their own STs, and the predominant subtypes in liver abscess and pneumonia patients were ST23 and ST11, respectively. Pangenome analysis also distinguished three phylogroups that were consistent with the isolate sources. The isolates collected from liver abscess patients carried significantly more virulence factors, and those from pneumonia patients harbored significantly more resistance genes and replicons. Almost all isolate STs (93/97 [95.88%]) from liver abscesses strongly correlated with the virulence factor salmochelin, while most pneumonia isolate STs (52/53 [98.11%]) from pneumonia did not correlate with salmochelin. The isolates collected from liver abscesses showed higher virulence in the cytotoxicity and mouse models. These data provide genomic support for the proposal that isolates collected from carriers, liver abscess patients, and pneumonia patients have distinct genomic features. Isolates from the different sources are largely nonoverlapping, suggesting that different patients may be infected via different sources. Further studies on the pathogenic mechanisms of salmochelin and other virulence factors will be required. IMPORTANCE While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. We collected 232 isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and the isolates from different sources had their own sequence types. Pangenome analysis also distinguished three phylogroups that were consistent with the isolate sources. The isolates collected from liver abscess patients carried significantly more virulence factors, and those from pneumonia patients harbored significantly more resistance genes and replicons. Besides, there was a strong link between salmochelin and liver abscess. The isolates collected from liver abscesses also showed higher virulence in the cytotoxicity and mouse models. Isolates collected from different sources have distinct genomic features, suggesting that different patients may be infected via different sources.

High alcohol-producing Klebsiella pneumoniae causes fatty liver disease through 2,3-butanediol fermentation pathway in vivo.

High alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the gut microbiota had been demonstrated to be the causative agent of fatty liver disease (FLD). However, the catabolic pathways for alcohol production in vivo remain unclear. Here, we characterized the genome of HiAlc and medium alcohol-producing (MedAlc) Kpn and constructed an adh (an essential gene encoding alcohol dehydrogenase) knock-out HiAlc Kpn W14 strain (W14Δadh) using CRISPR-Cas9 system. Subsequently, we established the mouse model via gavage administration of HiAlc Kpn W14 and W14 Δadh strains, respectively. Proteome and metabolome analysis showed that 10 proteins and six major metabolites involved in the 2,3-butanediol fermentation pathway exhibited at least a three-fold change or greater during intestinal growth. Compared with HiAlc Kpn W14-fed mice, W14Δadh-fed mice with weak alcohol-producing ability did not show apparent pathological changes at 4 weeks, although some steatotic hepatocytes were observed at 12 weeks. Our data demonstrated that carbohydrate substances are catabolized to produce alcohol and 2,3-butanediol via the 2,3-butanediol fermentation pathway in HiAlc Kpn, which could be a promising clinical diagnostic marker. The production of high amounts of endogenous alcohol is responsible for the observed steatosis effects in hepatocytes in vivo.

A Recombinase Aided Amplification Assay for Rapid Detection of the Klebsiella pneumoniae Carbapenemase Gene and Its Characteristics in Klebsiella pneumoniae.

Klebsiella pneumoniae carbapenemase genes (blaKPC) play an important role in carbapenem-resistant Enterobacteriaceae in China. A rapid detection method for blaKPC genes and investigations into the molecular characteristics of blaKPC positive Klebsiella pneumoniae were necessary. In this study, an easy and rapid recombinase aided amplification assay (RAA) for blaKPC was established. This protocol could be completed at 39°C in 15-20 min. The sensitivity of this assay was determined as 48 copies per reaction, and the specificity was 100%. The blaKPC RAA method could be used for clinical diagnosis and epidemiological investigation. Among 801 fecal samples from inpatients, 34 blaKPC positive isolates were identified from each sample, of which 23 isolates were K. pneumoniae. ST11 with blaKPC-2 was the most prevalent type. All these strains were multidrug resistant and carried various virulence genes. Fecal carriage of blaKPC positive carbapenem-resistant K.pneumoniae poses significant challenges for public health control.

Rapid Detection of New Delhi Metallo-ß-Lactamase Gene Using Recombinase-Aided Amplification Directly on Clinical Samples From Children.

New Delhi metallo-ß-lactamase, a metallo-ß-lactamase carbapenemase type, mediates resistance to most ß-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect bla NDM genes in children's clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5-15 min, was established to target bla NDM genes in children's clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without bla NDM genes did not amplify. This method was used to detect bla NDM genes in 112 children's stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying bla NDM in children, 15 carbapenem-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca, Acinetobacter junii, and Proteus mirabilis) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. bla NDM - 1 and bla NDM - 5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid bla NDM gene screening test for clinical samples. The common existence of bla NDM and multi-drug resistance genes presents major challenges for pediatric treatment.

Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children.

Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/µL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4-100%), specificity 99.0% (95% CI 97.8-99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy.

Assembly properties of bacterial tubulin homolog FtsZ regulated by the positive regulator protein ZipA and ZapA from Pseudomonas aeruginosa.

Bacterial tubulin homolog FtsZ self-assembles into dynamic protofilaments, which forms the scaffold for the contractile ring (Z-ring) to achieve bacterial cell division. Here, we study the biochemical properties of FtsZ from Pseudomonas aeruginosa (PaFtsZ) and the effects of its two positive regulator proteins, ZipA and ZapA. Similar to Escherichia coli FtsZ, PaFtsZ had a strong GTPase activity, ~ 7.8 GTP min-1 FtsZ-1 at pH 7.5, and assembled into mainly short single filaments in vitro. However, PaFtsZ protofilaments were mixtures of straight and "intermediate-curved" (100-300 nm diameter) in pH 7.5 solution and formed some bundles in pH 6.5 solution. The effects of ZipA on PaFtsZ assembly varied with pH. In pH 6.5 buffer ZipA induced PaFtsZ to form large bundles. In pH 7.5 buffer PaFtsZ-ZipA protofilaments were not bundled, but ZipA enhanced PaFtsZ assembly and promoted more curved filaments. Comparable to ZapA from other bacterial species, ZapA from P. aeruginosa induced PaFtsZ protofilaments to associate into long straight loose bundles and/or sheets at both pH 6.5 and pH 7.5, which had little effect on the GTPase activity of PaFtsZ. These results provide us further information that ZipA functions as an enhancer of FtsZ curved filaments, while ZapA works as a stabilizer of FtsZ straight filaments.

Research on calibrating rock mechanical parameters with a statistical method.

Research on the modeling of rock mechanics parameters is of great significance to the exploration of oil and gas. The use of logging data with the Kriging interpolation to study rock mechanics parameters has been proven to be effective in reservoir prediction and other oilfield applications and can provide additional data. However, there will sometimes be a great deviation due to the limited samples and the strong heterogeneity of a layer. To solve this problem, a new approach was proposed to calibrate rock mechanical models through the statistical analysis of logging data. A module was developed to calibrate rock mechanics parameters automatically, which was then applied to the Wangyao area of the Ansai oilfield. This method significantly improved the accuracy of rock mechanics modeling.