Microbiology spectrum最新文献

{"title":"Exploring phage-host interactions in <i>Burkholderia cepacia</i> complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics.","authors":"Ben Diaz, Rohan Krishna, Joseph S Schoeniger, Catherine M Mageeney","doi":"10.1128/spectrum.01936-25","DOIUrl":"https://doi.org/10.1128/spectrum.01936-25","url":null,"abstract":"<p><p>Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in <i>Burkholderia cenocepacia</i> K56-2. <i>Burkholderia cepacia</i> complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of <i>B. cenocepacia</i> K56-2 with Bcep176, a temperate phage isolated from <i>Burkholderia multivorans</i>. A genome-wide dCas9 knockdown library targeting <i>B. cenocepacia</i> K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of <i>B. cenocepacia K56-2</i> during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of <i>B. cenocepacia</i> and its phages<i>,</i> and for designing phage therapies.IMPORTANCE<i>Burkholderia cepacia</i> complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. <i>Burkholderia</i> bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic <i>Burkholderia</i> bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in <i>Burkholderia cenocepacia</i> K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing <i>Burkholderia</i> bacteria.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0193625"},"PeriodicalIF":3.8,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206917","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A universal, high-quality, and high-yield DNA purification method for mycobacteria, including <i>Mycobacterium tuberculosis</i>: large-scale assessment of the chloroform-bead method.","authors":"Yoshiro Murase, Makiko Hosoya, Yuta Morishige, Yoshiko Shimomura, Miori Nagai, Aki Tamaru, Akiko Takaki, Satoshi Mitarai","doi":"10.1128/spectrum.00765-25","DOIUrl":"https://doi.org/10.1128/spectrum.00765-25","url":null,"abstract":"<p><p>Genomic analysis of mycobacteria has become increasingly crucial for understanding drug-resistance mechanisms, molecular epidemiology, and pathogenesis. However, efficient extraction of high-molecular-weight genomic DNA from these organisms remains challenging because of their thick mycolic acid-rich cell walls. In this study, we report the chloroform-bead method, a universal DNA extraction protocol that combines chemical and mechanical disruptions to overcome these challenges. Multi-laboratory evaluation (16 sites) demonstrated the chloroform-bead method's superiority over conventional methods for <i>Mycobacterium tuberculosis</i> (DNA yield: 17.9 vs 1.9 µg, purity A260/A230: 1.86 vs 1.22, both <i>P</i> < 0.001). Single-facility assessment extended these findings to >32 nontuberculous mycobacterial species (<i>n</i> = 1,058), showing performance comparable to <i>M. tuberculosis</i> (<i>n</i> = 1,000), with both achieving median yields of 22.2 µg DNA and consistent quality metrics. The chloroform-bead method significantly reduced the processing time from 2 to 3 days to 2 h while ensuring complete sample sterilization, eliminating the need for species-specific optimization. This streamlined and universally applicable protocol represents a practical advancement in mycobacterial DNA extraction methodology, ideal for high-throughput genomic studies and routine clinical diagnostics.</p><p><strong>Importance: </strong>Mycobacterial genomics is crucial for understanding pathogenesis and drug resistance; however, DNA extraction remains a significant challenge because of its unique cell wall. Traditional methods rely on enzymatic treatments, resulting in complex and time-consuming protocols with variable results. The chloroform-bead method introduces a paradigm shift by chemically and mechanically disrupting the mycolic acid layer and eliminating the need for enzymatic treatment. This standardized approach ensures consistent, high-quality DNA extraction across diverse mycobacterial species, thereby enhancing research capabilities and clinical applications.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0076525"},"PeriodicalIF":3.8,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic diversity, virulence genes, antimicrobial resistance, and biofilm formation of <i>Klebsiella pneumoniae</i> isolated from bovine mastitis milk in South Korea.","authors":"Hye Jeong Kang, Ju-Yeon You, Seung Hoe Kim, Jin-San Moon, Ha-Young Kim, Jae-Myung Kim, Hyun-Mi Kang","doi":"10.1128/spectrum.01343-25","DOIUrl":"https://doi.org/10.1128/spectrum.01343-25","url":null,"abstract":"<p><p><i>Klebsiella pneumoniae</i>, a zoonotic agent, is a causative pathogen of bovine mastitis. Despite its clinical relevance in dairy farms, studies on <i>K. pneumoniae</i> in bovine mastitis remain limited. Additionally, studies on <i>K. pneumoniae</i>'s genetic diversity and virulence characteristics in South Korea remain limited. Therefore, in this study, we aimed to elucidate the genetic diversity, antimicrobial resistance, virulence genes, and biofilm-forming capacity of 29 <i>K</i>. <i>pneumoniae</i> strains isolated from bovine mastitis milk samples in South Korea between 2017 and 2023. Multilocus sequence typing revealed 23 sequence types, four of which were novel, indicating substantial genetic heterogeneity and the absence of a dominant clonal lineage. Excluding intrinsic resistance, the highest resistance rates were observed for tetracycline (34.5%) and sulfisoxazole (31.0%), whereas resistance to the other antibiotics tested ranged from 0% to 20.7%. In addition, multidrug resistance (MDR) was noted in 20.7% of isolates. Virulence gene analysis revealed that most isolates carried the <i>ureA</i>, <i>uge</i>, <i>wabG</i>, and <i>fimH</i> genes, whereas <i>allS</i>, <i>rmpA</i>, <i>iucB</i>, and <i>iroNB</i> were not detected. Two isolates exhibited a hypermucoviscous phenotype, and one belonged to the capsular serotype K2. All isolates demonstrated biofilm-forming ability, with moderate-to-strong production observed in over 89.0% of cases, indicating potential for persistence and treatment challenges. In conclusion, <i>K. pneumoniae</i> isolates from mastitis milk carried multiple virulence genes and showed MDR as well as robust biofilm formation. Therefore, continued surveillance and further characterization of <i>K. pneumoniae</i> are needed to support mastitis control and protect public health.IMPORTANCE<i>Klebsiella pneumoniae</i> is an emerging environmental pathogen associated with clinical mastitis in dairy cows, raising concerns regarding antimicrobial resistance and public health. To the best of our knowledge, this study provides the first comprehensive characterization of <i>K. pneumoniae</i> isolates from mastitis milk in South Korea, including analyses of genetic diversity, antimicrobial resistance, virulence factors, and biofilm formation. The findings advance our current understanding of <i>K. pneumoniae</i> associated with bovine mastitis and highlight the need for continued surveillance that will contribute to mastitis control efforts and safeguard public health.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0134325"},"PeriodicalIF":3.8,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pangenome analysis of <i>Lactobacillus mulieris</i> strains reveals distinct subspecies clusters with defined ecological adaptations.","authors":"Jake Adolf V Montecillo, Heon Jong Yoo, Yoo-Young Lee, Chul Min Park, Angela Cho, Hyunsu Lee, Hee Yeon Yoon, Jong Mi Kim, Nan Young Lee, Sun-Hyun Park, Nora Jee-Young Park, Hyung Soo Han, Incheol Seo, Gun Oh Chong","doi":"10.1128/spectrum.02011-25","DOIUrl":"https://doi.org/10.1128/spectrum.02011-25","url":null,"abstract":"<p><p><i>Lactobacillus mulieris</i> is a recently described species, reportedly isolated from human urine, vagina, and gut. Previous genomic studies of <i>L. mulieris</i> highlighted significant genetic diversity among its strains. To gain a deeper understanding of this genomic diversity, we conducted a comprehensive genomic comparison of 70 <i>L</i>. <i>mulieris</i> strains from diverse sources. Phylogenomic and genome relatedness analysis identified three distinct clades, each representing a potential subspecies cluster. Pangenome analysis revealed distinct gene clusters shaping the functional characteristics and unique ecological adaptations of each clade. Clade 1 demonstrated a generalist lifestyle, with strains isolated from diverse sources and enriched in serine/threonine protein kinases, suggesting adaptive versatility. Clade 2, predominantly composed of urinary isolates, displayed enrichment in genes facilitating nutrient acquisition and osmotic regulation, enabling survival in the nutrient-limited and high osmolarity conditions of the urinary tract. Clade 3, exclusively composed of vaginal isolates, exhibited significant enrichment in genes supporting glycogen metabolism, carbohydrate transport, and capsular polysaccharide biosynthesis-features indicative of adaptation to the vaginal environment. Collectively, our findings provide essential genomic insights into the ecological specialization of <i>L. mulieris</i>, shedding light on their genetic variability and adaptive traits within their respective ecological niches.IMPORTANCERecognizing the genomic diversity within <i>Lactobacillus mulieris</i> is essential for understanding its ecological specialization and adaptation strategies across distinct human-associated environments. By identifying three distinct clades with unique functional traits, our study highlights the critical role of niche-specific genetic adaptations in microbial survival. The presence of specialized gene functions within each clade underscores how evolutionary pressures shape bacterial resilience in different environments. Despite their coexistence in overlapping environments, these clades exhibit distinct genomic profiles that may influence their colonization potential and interactions with the host and within the host-associated microbiota. Our findings emphasize the need for a classification framework that accounts for these genetic and functional differences and the necessity for further investigation to understand their distinct roles and impact on human health.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0201125"},"PeriodicalIF":3.8,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel, rapid, and reliable typing of vancomycin-resistant <i>Enterococcus faecium</i> CC17/ST80 strains using MALDI-TOF MS.","authors":"Silke Huber, Christina Brühwasser, David Eisele, Cornelia Lass-Flörl, Stefan Fuchs, Miriam Govrins","doi":"10.1128/spectrum.02702-25","DOIUrl":"https://doi.org/10.1128/spectrum.02702-25","url":null,"abstract":"<p><p>Vancomycin-resistant <i>Enterococcus faecium</i> (VREfm) is an important nosocomial pathogen. The recent emergence of the highly virulent clonal complex 17 (CC17) is posing a challenge for both therapeutic interventions and hospital infection control measures. Hence, prompt discrimination of CC17 VREfm from unrelated and less-virulent VREfm strains is essential for preventing its spread in hospitals and beyond. Between January 2022 and November 2024, 340 VREfm primary isolates have been identified in our lab and underwent genotyping by pulsed-field gel electrophoresis (PFGE) to survey a potential outbreak in the Tyrol region. In addition, whole-genome sequencing (WGS) was performed on a selected subset (<i>n</i> = 40). To curtail the lengthy time-to-result (TTR) of these methods, a novel typing protocol using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was established, validated, and optimized for rapid sample processing. PFGE and WGS showed that 61.2% of isolates (<i>n</i> = 208) belonged to a specific VREfm cluster identified as CC17 sequence type (ST) 80 <i>vanA</i> VREfm. A comprehensive MALDI-TOF MS analysis identified a distinct peak pattern specific to this lineage. This phenotypic characterization was used as a novel typing method with excellent performance (sensitivity: 1.00 [0.98-1.00], specificity: 0.89 [0.70-0.97]) and demonstrated a short TTR of 1 day after the cultural growth of VREfm. A rapid and novel MALDI-TOF MS-based typing approach for a specific CC17/ST80 <i>vanA</i> VREfm cluster was developed and enabled real-life application in routine diagnostics to assure accurate infection prevention and control measures. Future outbreak investigations may benefit from adopting this cost- and labor-efficient approach.IMPORTANCEThis study addresses the urgent need for faster ways to detect problematic hospital bacteria. A highly transmissible strain of <i>Enterococcus faecium</i> (CC17) has been spreading in healthcare settings, making infections harder to treat and control. Traditional methods to identify and track outbreaks are accurate but slow and resource-intensive, delaying critical infection control actions. By developing and validating a new method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the researchers demonstrated that this strain can be identified quickly, reliably, and at lower cost. Importantly, the new approach delivers results within a day, compared to the lengthy turnaround times of existing methods. This rapid detection tool provides hospitals with a practical solution to respond to outbreaks more effectively, prevent further spread, and protect vulnerable patients. The findings highlight a valuable step forward in strengthening hospital infection control and improving patient safety.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0270225"},"PeriodicalIF":3.8,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Polydextrose reduces the infection of <i>Klebsiella pneumoniae</i> in mice by downregulating the expression of TamA.","authors":"Lin Su, Huajie Zhao, Hafiz Muhammad Ishaq, Ningning Liu, Yalan Yang, Duan Li, Liang Liu, Chuansheng Wang, Fan Yang","doi":"10.1128/spectrum.01017-25","DOIUrl":"https://doi.org/10.1128/spectrum.01017-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Polydextrose (PDX), as a prebiotic, is an extensively branched glucose polymer that can promote the growth of beneficial bacteria in the gut. Recent research indicates that PDX regulates intestinal function and supports immune balance, which helps to protect the gut from pathogenic bacteria. However, scarce research has been found that PDX prevents the host infection through the direct effects on the pathogen. In this study, we developed a mouse model infected with &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; by pretreating with PDX, assessed the effect of PDX on &lt;i&gt;K. pneumoniae&lt;/i&gt; acute infection in mice, and explored its potential mechanisms. We developed a mouse model that is infected with &lt;i&gt;K. pneumoniae&lt;/i&gt; by pretreating with PDX. Colony counting quantified the &lt;i&gt;K. pneumoniae&lt;/i&gt; bacterial load in the parenchymal organs of mice. A scanning electron microscope was used to investigate the morphological characteristics of &lt;i&gt;K. pneumoniae&lt;/i&gt;. The expression level of TamA (translocation and assembly module A) was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The CRISPR-Cas9 technique was applied to construct the &lt;i&gt;tamA&lt;/i&gt; mutant strains (Δ&lt;i&gt;tamA&lt;/i&gt;) and the &lt;i&gt;tamA&lt;/i&gt; complement strain (&lt;i&gt;C-&lt;/i&gt;Δ&lt;i&gt;tamA&lt;/i&gt;). The biofilm formation capacity was evaluated by the crystal violet assay. The capsule production was quantified by measuring uronic acid content. In the PDX pretreated model, PDX did not alter the growth characteristics and morphological structure of &lt;i&gt;K. pneumoniae&lt;/i&gt;. However, it significantly reduces the load of &lt;i&gt;K. pneumoniae&lt;/i&gt; in the lung, liver, spleen, and intestinal tract of mice, which is related to inhibiting the expression of the outer membrane TamA protein by PDX. In an &lt;i&gt;in vitro&lt;/i&gt; study, the results indicated that deletion of &lt;i&gt;tamA&lt;/i&gt; significantly inhibited capsule production and biofilm formation of &lt;i&gt;K. pneumoniae&lt;/i&gt;, weakened interspecific and intraspecific competitive abilities with other members of the &lt;i&gt;Enterobacteriaceae&lt;/i&gt; family, and reduced the adhesion ability to Caco-2 and murine lung epithelial (MLE) cells. Compared with the wild strain, PDX treatment and the deletion of &lt;i&gt;tamA&lt;/i&gt; inhibit the expression of adhesion factors (including &lt;i&gt;FimH&lt;/i&gt;, &lt;i&gt;FimC&lt;/i&gt;, &lt;i&gt;FimD&lt;/i&gt;, and &lt;i&gt;MrkD&lt;/i&gt;) and the capsule synthesis genes (including &lt;i&gt;galF&lt;/i&gt;, &lt;i&gt;wzi&lt;/i&gt;, and &lt;i&gt;manC&lt;/i&gt;) in &lt;i&gt;K. pneumoniae&lt;/i&gt;. PDX can prevent the infection of &lt;i&gt;K. pneumoniae&lt;/i&gt; in mice. The potential mechanism may involve downregulating TamA expression and inhibiting adhesion-related molecules. Therefore, PDX can serve as a potential prebiotic to reduce &lt;i&gt;K. pneumoniae&lt;/i&gt; infections in both humans and animals.IMPORTANCEOur findings revealed that polydextrose (PDX) could significantly reduce the load of &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; in the lung, liver, spleen, and intestinal tract of mice. The potential mechanism is related to inhibiting the expression of the outer membrane Ta","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0101725"},"PeriodicalIF":3.8,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reassessing the role of wild birds in the spread of antibiotic resistance: the white stork as a model species in studying populations from Central European river valley.","authors":"Andżelina Łopińska, Alicja Nowak-Zaleska, Jakub Z Kosicki, Leszek Jerzak, Alicja Węgrzyn, Grzegorz Węgrzyn","doi":"10.1128/spectrum.00990-25","DOIUrl":"https://doi.org/10.1128/spectrum.00990-25","url":null,"abstract":"<p><p>It is commonly accepted that the emergence of multidrug-resistant bacteria in the hospital environment, in food production, and in the wild is the consequence of the overuse of antibiotics. The presence of antibiotic-resistant bacterial strains was reported in both wild and farmed animals. It was suggested previously that wild birds may be carriers of antibiotic-resistant bacteria acquired from agriculture and/or industrial/urban habitats. Here, we used the white stork (<i>Ciconia ciconia</i>) as a model species to assess the presence and origin of antibiotic-resistant bacteria in a breeding population located in river valley landscapes of western Poland. Cloacal swabs from 50 nestlings across 19 nests were collected during the 2019 breeding season, along with 49 environmental samples (soil, water, and food items) from known foraging sites. A total of 147 bacterial isolates representing 49 species were obtained. The majority originated from cloacal samples (58.5%), followed by water (16.3%), food (15.0%), and soil (10.2%). Resistance testing against 19 antibiotics revealed significantly higher resistance in food-derived isolates, while no significant differences were found among soil, water, and cloacal isolates. Correlation analyses based on resistance profiles showed no significant relationship between cloacal isolates and those from soil (rs = 0.11; <i>P</i> = 0.647) or food (rs = 0.25; <i>P</i> = 0.289), but a strong correlation with water isolates (rs = 0.68; <i>P</i> = 0.001). These results might suggest that wild birds' role in transmitting antibiotic-resistant microorganisms might be more limited than predicted previously. Therefore, the role of wild birds, especially the white stork, in the transmission of antibiotic-resistant bacteria appears unclear and requires further consideration and re-examination.IMPORTANCEDue to the overuse of drugs during animal breeding and the use of manures from breeding farms, antibiotic residues are present in the natural environment, especially in soil and water reservoirs. This causes a selection pressure on bacteria, creating a reservoir of antibiotic-resistant bacteria. It was suggested previously that wild birds may be carriers of antibiotic-resistant bacteria acquired from agriculture and/or industrial/urban habitats. Here, using a model of the white stork as a synanthropic bird, we asked whether this species is colonized by antibiotic-resistant bacteria, and if there is any significant correlation between bacteria present in samples of cloacal swabs and environmental samples from their habitats. We found no significant correlations between antibiotic-resistance patterns in bacteria isolated from white stork cloaca and those identified in bacteria isolated from soil and stork's food. Thus, roles of wild birds in transmitting antibiotic-resistant microorganisms might be constrained and less significant than predicted previously.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0099025"},"PeriodicalIF":3.8,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and characterization of strictly lytic bacteriophages against carbapenem-resistant <i>Enterobacter cloacae</i> complex.","authors":"Han-Yueh Kuo, Carl Jay Ballena Bregente, Tran Thi Dieu Thuy, Jazon Harl Hidrosollo, Donna May Dela Cruz-Papa, Tracey Antaeus Gutierrez, Yun-Tsung Huang, Yu-Jui Chuang, Po-Ren Hsueh, Cheng-Yen Kao","doi":"10.1128/spectrum.00835-25","DOIUrl":"https://doi.org/10.1128/spectrum.00835-25","url":null,"abstract":"<p><p>The global surge of carbapenem-resistant <i>Enterobacter cloacae</i> complex (CR-ECC) poses a significant clinical challenge due to limited treatment options. This study aimed to isolate and characterize lytic bacteriophages (phages) targeting CR-ECC. CR-ECC CYEBC080 was used as the bacterial host for isolating lytic phages, and a comprehensive evaluation was conducted on isolated phages, including phage stability under various pH and temperature conditions, host range analysis, killing curves, and therapeutic efficacy in <i>Galleria mellonella</i> larvae and a murine bacteremia model. Twelve lytic phages with distinct random amplified polymorphic DNA patterns were isolated, and transmission electron microscopy confirmed their classification under the <i>Straboviridae</i> family within the Caudoviricetes class. All phages remained stable across pH 3-11 for up to 90 minutes, with an optimal temperature range of 25°C-37°C. Among them, CYPEBC012 exhibited the broadest host range, lysing 93.75% of 80 CR-ECC isolates, while CYPEBC006 displayed the narrowest, lysing only 65%. Whole-genome sequencing revealed 12 phages with linear double-stranded DNA genomes ranging from 177,624 to 180,648 bp. Phage treatment administered at a multiplicity of infection of 10, 1 hour post-infection, significantly improved larval survival at day 7, reaching ≥80% in most groups, except CYPEBC001 (50%) and CYPEBC004 (60%) treatment groups. In CYEBC080-infected mice, CYPEBC012 treatment resulted in 100% survival by day 3 and 80% survival through day 7. Additionally, phage-treated mice exhibited significantly reduced bacterial loads and high phage titers in blood and liver. This study demonstrates the therapeutic potential of CYPEBC012 as a promising strategy against CR-ECC infections, offering an alternative to conventional antimicrobial treatments.</p><p><strong>Importance: </strong>This study identified and characterized lytic bacteriophages targeting carbapenem-resistant <i>Enterobacter cloacae</i> complex, with CYPEBC012 exhibiting the broadest host range and significantly improving survival in a murine bacteremia model. Its stability and efficacy highlight its potential for clinical application. Our findings demonstrate that phage therapy offers a promising alternative to conventional treatments to combat antibiotic-resistant infections.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0083525"},"PeriodicalIF":3.8,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200275","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>In vitro</i> and <i>in vivo</i> efficacy of vancomycin against <i>Elizabethkingia</i> species and the impact of increased vancomycin MICs.","authors":"Tzu-Wen Huang, Teng-Kuang Yeh, Shu-Yuan Hsu, Mei-Chen Tan, Wei-Cheng Huang, Shu-Chen Kuo","doi":"10.1128/spectrum.02371-25","DOIUrl":"https://doi.org/10.1128/spectrum.02371-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;This study aimed to evaluate the concordance of vancomycin susceptibility testing methods, its &lt;i&gt;in vivo&lt;/i&gt; and &lt;i&gt;in vitro&lt;/i&gt; efficacy, and the mechanisms underlying elevated MICs in &lt;i&gt;Elizabethkingia&lt;/i&gt; spp. Vancomycin susceptibilities of 18 &lt;i&gt;E. anophelis&lt;/i&gt; isolates were determined using multiple assays. The efficacy of vancomycin against five clinical isolates and one laboratory-induced mutant with an elevated vancomycin MIC was evaluated using time-kill assays and &lt;i&gt;Galleria mellonella&lt;/i&gt; and murine models. Vancomycin MICs (16-32 mg/L) determined by broth microdilution were consistent with agar dilution, Etest, and MBC assay results. All isolates had zone diameters &lt; 17 mm and were, thus, categorized as non-susceptible according to the CLSI criteria for &lt;i&gt;Enterococcus&lt;/i&gt; spp. Time-kill assays of five clinical isolates demonstrated that vancomycin at a clinically relevant concentration (4 mg/L) exhibited poor bactericidal activity similar to that of teicoplanin. Vancomycin improved &lt;i&gt;Galleria mellonella&lt;/i&gt; survival in a dose-dependent manner, whereas teicoplanin, dalbavancin, oritavancin, and daptomycin were ineffective. Murine models revealed that vancomycin at a human-equivalent dose (25 mg/kg twice daily) prolonged survival in most infections and modestly reduced bacterial load, while teicoplanin remained ineffective. Vancomycin efficacy was significantly reduced in &lt;i&gt;G. mellonella&lt;/i&gt; and mice infected with a mutant strain exhibiting an elevated MIC (128 mg/L), which was attributable to spontaneous mutations in &lt;i&gt;pbp&lt;/i&gt;4. In conclusion, &lt;i&gt;E. anophelis&lt;/i&gt; were consistently non-susceptible to vancomycin as determined by multiple &lt;i&gt;in vitro&lt;/i&gt; assays. However, vancomycin demonstrated unique &lt;i&gt;in vivo&lt;/i&gt; activity among glycopeptides although this effect was abrogated by spontaneous mutations leading to elevated MICs.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;&lt;i&gt;Elizabethkingia anophelis&lt;/i&gt; is a multidrug-resistant pathogen associated with limited treatment options and high mortality. Most commonly considered agents, including fluoroquinolones, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole, are increasingly compromised by resistance, toxicity, or inconsistent efficacy. Although vancomycin is not routinely used for Gram-negative infections due to limited outer membrane permeability, case reports have suggested potential benefit in &lt;i&gt;Elizabethkingia&lt;/i&gt; infections under critical conditions. In this study, we show that &lt;i&gt;E. anophelis&lt;/i&gt; isolates are uniformly non-susceptible to vancomycin &lt;i&gt;in vitro&lt;/i&gt; and exhibit minimal bactericidal activity. However, vancomycin conferred a modest but statistically significant survival benefit in two independent animal models. Importantly, this effect was lost in strains with vancomycin-induced MIC elevation, and genome analysis identified &lt;i&gt;pbp4&lt;/i&gt; mutations as a potential underlying mechanism. These findings suggest vancomycin may offer therapeutic benefit when no pr","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0237125"},"PeriodicalIF":3.8,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discrepancies in isoniazid susceptibility profiles: Bactec MGIT 960-resistant but GenoType MTBDR<i>plus</i>-susceptible <i>Mycobacterium tuberculosis</i> strains in Hunan, China.","authors":"Zhenhua Chen, Peilei Hu, Jingwei Guo, Jue Wang, Binbin Liu, Yunhong Tan","doi":"10.1128/spectrum.01101-25","DOIUrl":"https://doi.org/10.1128/spectrum.01101-25","url":null,"abstract":"<p><p>Discordant drug susceptibility testing (DST) results between the Bactec MGIT 960 system (MGIT) and the GenoType MTBDR<i>plus</i> assay (MTBDR<i>plus</i>) for isoniazid (INH) complicate clinical decision-making. In this study, we performed minimum inhibitory concentration (MIC) assays and whole-genome sequencing (WGS) on 53 <i>Mycobacterium tuberculosis</i> strains identified as INH-resistant by MGIT but INH-susceptible by MTBDR<i>plus</i>. The variants conferring INH resistance were evaluated by the WHO mutation catalogue. Our results showed that only five strains carried variants classified as \"associated with resistance\" (Group 1/2), including <i>katG</i> Trp39STOP, <i>katG</i> Ser315Asn, <i>inhA</i> -154G>A, and <i>inhA</i> Ser94Ala. In addition, 44 strains carried 70 variants classified as \"Group 3: Uncertain significance\" across nine genes, including <i>katG</i>, <i>ahpC</i>, <i>inhA</i>, <i>Rv0010c</i>, <i>Rv1129c</i>, <i>Rv2752c</i>, <i>mshA</i>, <i>dnaA</i>, and <i>Rv1258c</i>. The remaining four strains carried no variants (Groups 1-3) linked to INH resistance. No significant difference in the prevalence of high-level INH resistance was observed between lineage 2 and lineage 4 strains (<i>χ</i>² = 0.232, <i>P</i> = 0.630). Our findings indicate that the variants classified as \"uncertain significance\" may be the main genetic determinants causing discordant results, highlighting their associations with INH resistance that need to be further investigated.</p><p><strong>Importance: </strong>This study addresses a critical challenge in drug susceptibility testing (DST): the discrepancies in DST results for isoniazid (INH) between the Bactec MGIT 960 system and the GenoType MTBDR<i>plus</i> assay. These discordant results significantly complicate treatment decisions, potentially leading to suboptimal patient outcomes. Using MIC assays and WGS on 53 clinical <i>Mycobacterium tuberculosis</i> strains, we provide valuable insights into the genetic basis of INH resistance. Our findings showed that only a small fraction of strains carried variants definitively linked to INH resistance, while a larger number harbored variants of uncertain significance across multiple genes, underscoring the complexity of INH resistance mechanisms. This study highlights the urgent need to refine our understanding of these \"Group 3: uncertain significance\" variants, as they appear to be a primary driver of the discrepancies. Additionally, this study emphasizes the importance of integrating advanced sequencing tools into DST to improve the accuracy of INH resistance detection.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0110125"},"PeriodicalIF":3.8,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}