Microbiology spectrum最新文献

Accurate bacterial quantification is crucial for studying microbial pathogenesis, host-pathogen interactions, and therapeutic interventions. Two widely used methods include agar plating with colony-forming unit (CFU) enumeration and time-lapsed turbidimetry in liquid broth culture. While agar plating remains the gold standard in both in vitro and in vivo infection models, liquid broth turbidimetry is commonly used to assess growth kinetics, microbial fitness, antibiotic susceptibility, and bacterial genetics. While strain-specific CFU and turbidimetry (OD) calibration studies exist, a comprehensive and systematic comparison of these methods for quantifying a broad array of clinically relevant pathogens remains largely unexplored. Here, we conducted a head-to-head comparison of agar plating and liquid broth turbidimetry to quantify the growth of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus in vitro and evaluated their performance in vivo using a murine model of K. pneumoniae pulmonary infection. Across all pathogens tested, both methods exhibited strong correlation over a broad dynamic range (6-7 log₁₀ dilutions). Liquid broth turbidimetry demonstrated enhanced sensitivity at low bacterial densities, as well as greater precision. In the in vivo murine pneumonia model, this method more accurately distinguished bacterial burdens at the site of infection (lung) and dissemination (spleen) between wild-type and Toll-like receptor 4 knockout mice. Overall, liquid broth turbidimetry is a reliable alternative to agar plating with a high degree of correlation for bacterial quantification and improved precision, highlighting its potential utility in studies of bacteriology and infectious diseases.

Importance: Accurate bacterial quantification is fundamental to microbiology research and clinical diagnostics. While agar plating is a widely used method, our study demonstrates that liquid broth turbidimetry provides accurate bacterial quantification, which is fundamental to microbiology research and clinical diagnostics. While agar plating is a widely used method, our study demonstrates that liquid broth turbidimetry provides a highly correlative and more precise approach for quantifying diverse, clinically relevant bacterial pathogens, including when dealing with low bacterial density. The enhanced sensitivity and precision may be valuable for early infection detection, monitoring treatment efficacy, and understanding microbial dynamics in research settings where accurate quantification of even low-density organisms is essential. The findings support considering liquid broth turbidimetry as a complementary or alternative method for bacterial growth quantification.

New anti-infective therapies are urgently needed for the treatment of drug-resistant bacterial infections in the context of the rapid spread of drug resistance. Phages, the natural enemies of bacteria, have irreplaceable advantages in the treatment of bacterial infections. Here, we report a novel phage, vB_KpnP_Henu1_3, that specifically lyses capsule-type K1 Klebsiella pneumoniae. The phage vB_KpnP_Henu1_3 shows relatively favorable thermal stability (4°C-55°C) and pH tolerance (pH = 4-11). In addition, the optimal multiplicity of infection is 0.01, with a burst size of approximately 253 ± 54 PFU/cell. Genomic analysis reveals that phage vB_KpnP_Henu1_3 contains double-stranded DNA (total length of 49,808 bp) with a G + C content of 50.76%, and the genome comprises 75 open reading frames with no virulence- or antibiotic resistance-related genes. Transmission electron microscopy observations revealed that phage vB_KpnP_Henu1_3 possessed an icosahedral head and siphovirus morphology. Based on the genome sequence, phage vB_KpnP_Henu1_3 could be assigned to a new species in the genus Webervirus of the family Drexlerviridae. Furthermore, phage vB_KpnP_Henu1_3 rapidly inhibits the growth of K. pneumoniae within 4 h, prevents biofilm formation, and disrupts mature biofilms in vitro. In infection animal models, phage vB_KpnP_Henu1_3 significantly increases the survival rate of K. pneumoniae-infected Galleria mellonella larvae and mice while reducing the bacterial loads. These findings demonstrate that phage vB_KpnP_Henu1_3 has promising potential as a safe alternative for controlling and treating multidrug-resistant K1-type K. pneumoniae infections.IMPORTANCEThe widespread use of antibiotics has led to increasing antibiotic resistance, which is a growing global health concern. Therefore, the development of novel antimicrobial therapy that can cure drug-resistant bacteria-induced infections is imperative. Phages are of increasing interest as natural enemies of bacteria with clear advantages in antibacterial.

This study analyzed 570 non-duplicate Klebsiella pneumoniae isolates (n = 570) from outpatient urine cultures collected between 1998 and 2018 through the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program. Serotyping, antimicrobial susceptibility testing, and virulence gene profiling were performed. Results were compared with 521 previously reported bloodstream isolates collected in 1998, 2008, and 2018. The most common urinary tract infection (UTI) serotypes were K2, K64, K62, K1, and K25, with non-typeable isolates comprising 19.3%. Compared to bloodstream isolates, non-typeable strains were more frequent in UTIs, while K1 and K2 were more common in bloodstream infections. Resistance to cephalosporins, carbapenems, and ciprofloxacin increased significantly after 2014. K64 showed over 72.2% resistance to cephalosporins and rising quinolone resistance. In contrast, K1 and K2 exhibited low resistance but carried the highest burden of virulence genes. Non-typeable isolates lacked virulence determinants, suggesting lower pathogenic potential. This 20-year analysis highlights the distinct serotype and resistance patterns in community-onset UTI isolates in Taiwan and underscores the importance of incorporating prevalent, drug-resistant serotypes into future vaccine strategies.

Importance: Klebsiella pneumoniae is an important cause of urinary tract infections in the community. With rising resistance to commonly used antibiotics, preventive strategies, such as vaccination, are urgently needed. This 20-year analysis provides detailed data on the distribution of bacterial types and their resistance patterns in Taiwan. The findings offer valuable insight for selecting vaccine targets that cover the most prevalent and drug-resistant strains, supporting the development of effective preventive tools against multidrug-resistant infections.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has emerged as a major public health threat due to its high virulence, multidrug resistance, and increasing global prevalence. However, the molecular characteristics and adaptive mechanisms underlying the CR-hvKP pathogenesis remain poorly understood. In this study, we collected 217 CRKP isolates from a tertiary hospital and identified 46 as CR-hvKP. Genomic analysis revealed that the majority of CR-hvKP strains belonged to ST11-KL64 (48.7%, 20/46) and ST11-KL25 (46.3%, 19/46), followed by ST11-KL47 (4.8%, 2/46). Interestingly, while iroBCDN was present in various sequence types, it was found in ST11 strains only with the KL47 capsule type, which showed high virulence in Galleria mellonella models. To investigate the biological role of iroBCDN, we constructed an iroBCDN deletion mutant and a complemented strain in an ST11-KL47 background. The lactate dehydrogenase cytotoxicity assays and G. mellonella infection models revealed no significant difference in virulence among the wild-type, knockout, and complemented strains. Remarkably, phenotypic assays showed that the iroBCDN deletion mutant exhibited enhanced growth fitness, competitive advantage, oxidative stress resistance, and survival in human whole blood. Transcriptomic analysis revealed that iroBCDN deletion led to the upregulation of genes involved in oxidative stress response, capsule biosynthesis, and energy metabolism, while genes related to fimbrial assembly and carbon metabolism were downregulated. These findings suggest that the loss of iroBCDN does not attenuate virulence in ST11 CR-hvKP but instead enhances its environmental adaptability, potentially contributing to the persistence and dissemination of epidemic clones.IMPORTANCEThe emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a severe global health threat. This study reveals a critical paradox: deletion of the iroBCDN locus traditionally associated with virulence does not attenuate pathogenicity in ST11-KL47 CR-hvKP. Instead, its loss significantly enhances bacterial fitness by improving growth competitiveness, oxidative stress resistance, and survival in human blood. It demonstrates how loss of specific genetic elements may facilitate the dominance of high-risk clones like ST11-KL64/KL25 by optimizing environmental adaptation and persistence-key factors in hospital transmission. Understanding this fitness trade-off is vital for developing strategies against resilient CR-hvKP epidemics.

African swine fever is a fatal, febrile, infectious disease that affects pigs and wild boars and is caused by infection with African swine fever virus (ASFV). The current pandemic strain, belonging to genotype II, first emerged in Georgia in 2007 and subsequently spread across Russia, Europe, Asia, and the Caribbean. Developing live attenuated viruses represents the most promising vaccine strategy; however, biosafety and biosecurity concerns due to long-term persistence of attenuated viruses in immunized animals may lead to secondary transmission or reversion to virulence. To address this concern, we developed a replication-restricted ASFV by deleting the S273R gene that encodes an essential factor for core shell formation and viral maturation within host cells. The resulting strain, AQSΔS273R, was unable to replicate in natural host cells because these cells lack a gene that complements the function of S273R. To enable propagation of infectious progeny in vitro, we established S273R gene-complemented immortalized porcine kidney macrophages, which are competent to support virus production. In animal trials, pigs immunized with AQSΔS273R exhibited no clinical signs or viremia. In the challenge study, vaccination with replication-restricted ASFV (AQSΔS273R) conferred approximately 30% protection, with partial reduction of clinical signs compared with controls. These findings demonstrate the feasibility of a novel approach for developing replication-restricted ASFV vaccine candidates with enhanced biological safety.IMPORTANCETo date, no reliable African swine fever (ASF) vaccines are available. Although some attenuated African swine fever viruses (ASFVs) have been approved for field applications and have attracted attention as potential vaccine candidates, their long-term persistence in inoculated animals raises concerns about virulence reversion or genetic recombination in field settings. In this study, we developed a novel approach to generate safer vaccines by creating a replication-restricted, S273R gene-deleted virus in combination with genetically modified host cells stably expressing the S273R gene. Not only did this mutant virus fail to induce any clinical signs in immunized pigs, but it also partially protected them against challenge with virulent ASFV. These results demonstrate that this newly developed replication-restricted ASFV strain is expected to be a promising and biologically safe vaccine candidate against ASF.

Pathogen whole-genome sequencing (WGS) has significant potential for improving healthcare-associated infection (HAI) outcomes. However, methods for integrating WGS with epidemiologic data to quantify risks for pathogen spread remain underdeveloped. To identify analytic strategies for conducting WGS-based HAI surveillance in high-burden settings, we modeled patient- and facility-level transmission risks of carbapenem-resistant Klebsiella pneumoniae (CRKP) in a long-term acute care hospital (LTACH). Using rectal surveillance data collected over 1 year, we fit three pairwise regression models with three different metrics of genomic relatedness for pairs of case isolates, a proxy for transmission linkage: (i) single-nucleotide variant genomic distance, (ii) closest genomic donor, and (iii) common genomic cluster. To assess the performance of these approaches under real-world conditions defined by passive surveillance, we conducted a sensitivity study including only cases detected by admission surveillance or clinical symptoms. Genomic relatedness between pairs of isolates was associated with room sharing in two of the three models and overlapping stays on a high-acuity unit in all models, echoing previous findings from LTACH settings. In our sensitivity analysis, qualitative findings were robust to the exclusion of cases that would not have been identified with a passive surveillance strategy; however, uncertainty in all estimates also increased markedly. Taken together, our results demonstrate that pairwise regression models combining relevant genomic and epidemiologic data are useful tools for identifying HAI transmission risks.IMPORTANCEWhole-genome sequencing of healthcare-associated infections (HAIs) is becoming more common, and new methods are necessary to integrate these data with epidemiologic risk factors to quantify transmission drivers. We demonstrate how pairwise regression models, in which the outcome of a regression model represents genomic similarity between a pair of isolates, can identify known transmission risk factors of carbapenem-resistant Klebsiella pneumoniae in a long-term acute care facility. Such pairwise regression models could be used with rich epidemiologic data in other settings to identify important risk factors of endemic HAI transmission.

Under bedaquiline (BDQ) pressure, a temporary persistence period (24-96 h) has been observed, during which H37Rv undergoes metabolic rerouting. However, little is known of transcriptomic changes in BDQ-resistant Mycobacterium tuberculosis (Mtb) isolates during this period. We explored transcriptomic adaptations occurring under inhibitory concentrations of BDQ to delineate pathways supporting drug tolerance and contributing to BDQ resistance. We report overexpression of genes involved in the biosynthesis of L-arginine and L-cysteine in our study isolates. Among stress response genes, genes from the suf operon, involved in Fe-S biogenesis, were upregulated in the study isolates. Differentially expressed amino acid gene clusters likely indicate an under-recognized metabolic pathway contributing to BDQ persistence in the study clinical isolates. Furthermore, Fe-S stress response activated under BDQ pressure may be of particular interest as a mechanism broadly used by Mtb in mitigating different environmental stresses. We propose that these pathways should be explored further as potential drug targets.

Importance: Keeping in mind the complex interplay between mutations, gene expression, and drug resistance, knowledge of pathways induced under bedaquiline (BDQ) stress in BDQ-resistant clinical Mycobacterium tuberculosis (Mtb) isolates is limited. Furthermore, focusing on mechanisms supporting tolerance can help identify potential targets for drugs that act against dormant bacilli or select synergistic drug combinations. Such information may be useful in identifying other alternate mechanisms of resistance and tolerance. Our study explores changes occurring in the transcriptome of BDQ-resistant isolates exposed to inhibitory concentrations of BDQ under a specific tolerance time point. Our study identifies differentially expressed pathways and genes that are: (i) similarly expressed in both H37Rv strain and clinical isolates, (ii) expressed only in clinical isolates, and (iii) reported to be similarly induced by literature in Mtb exposed to other anti-tuberculosis drugs. These genes and pathways present themselves as potential markers that may have diagnostic, prognostic, and therapeutic value that can be explored further.