Microbiology spectrum最新文献

The detection of anti-Aspergillus antibodies is key for diagnosing chronic pulmonary aspergillosis (CPA). Available techniques are limited and pose a considerable challenge in resource-limited settings. The objective of this study was to evaluate the performance of the point-of-care test LDBio ICT IgG/IgM lateral flow assay (LDBio) versus that of the Bordier Elisa assay in a field study in Nigeria. Of the 97 serum samples tested in the CPA patient group, 71 tested positive by the LDBio assay with 69.4% sensitivity. In the non-CPA group, 286 of the 289 sera tested negative by the LDBio assay with 98.7% specificity. The Bordier test was positive for 97 of the 97 CPA serum samples tested, showing 100% sensitivity, significantly different from the LDBio test in detecting Aspergillus antibodies (P < 0.0001). However, there was no significant difference in the specificity between the two tests (P = 0.617). The results were in agreement for 353 of the 386 samples tested (91.5%), with a Cohen's kappa coefficient of 0.75, indicating substantial agreement between the LDBio and Bordier test results. The LDBio lateral flow assay (LFA) test is a simple and rapid point-of-care test that can be used in field studies in which the Elisa test is not available. The lower sensitivity of the LDBio LFA in our field study could have been due to A. fumigatus being the species involved in only 50%-60% of cases in Nigeria, with A. flavus being more frequent than in Northern countries.IMPORTANCEAvailable techniques for the detection of Aspergillus IgG are limited and pose a considerable challenge in resource-limited settings in terms of affordability, skilled personnel, equipment, and a regular power supply. A point-of-care test would address most of these challenges. The LDBio lateral flow assay (LFA) test is a simple and rapid point-of-care test that can be used in field studies in which the Elisa test is not available. When combined with clinical features, the LFA can be used as a screening tool for chronic pulmonary aspergillosis in settings such as ours; however, a lower sensitivity was observed compared to the Elisa.

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection that is challenging to diagnose and treat due to the resistance of Acanthamoeba to standard antimicrobial agents. Current detection methods have limitations. This study aimed to develop and validate a sensitive viability PCR (v-PCR) assay using a photoreactive dye to distinguish viable from non-viable Acanthamoeba for rapid identification of viable Acanthamoeba trophozoites and cysts. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures containing decreasing percentages of viable Acanthamoeba, including reference strains Acanthamoeba polyphaga trophozoites and cysts, Acanthamoeba castellanii trophozoites, and Acanthamoeba castellanii trophozoites from a clinical sample, were prepared. Disinfectant efficacy against Acanthamoeba was also assessed. Samples were divided into PMAxx-treated and non-PMAxx-treated parts, and v-PCR assay was applied to both. The difference in viable Acanthamoeba was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of viable Acanthamoeba trophozoites and cysts showed increasingly lower delta Ct values as the percentage of viable Acanthamoeba decreased, as expected. This relationship was observed across all tested samples. Menicon Progent effectively eliminated A. polyphaga trophozoites and cysts, while propamidine, chlorhexidine, or their combination resulted in approximately 2-log reductions in A. polyphaga trophozoites and cysts. In the current study, a rapid v-PCR assay was developed that can distinguish between viable and non-viable Acanthamoeba, for both trophozoites and cysts, across multiple species. The presence of viable Acanthamoeba, as determined by v-PCR, allows monitoring of treatment response and efficacy in AK.IMPORTANCEThe development of a sensitive viability PCR (v-PCR) assay using propidium monoazide (PMAxx) as a photoreactive dye marks a significant advancement in the diagnosis and treatment of Acanthamoeba keratitis (AK), a severe corneal infection notorious for its resistance to conventional antimicrobials. This innovative assay offers a rapid and accurate method to distinguish viable from non-viable Acanthamoeba trophozoites and cysts, addressing a critical need in the field. By effectively distinguishing between viable and non-viable Acanthamoeba, this test enables monitoring of treatment response and efficacy, essential for guiding clinical interventions in AK cases. The successful validation of this v-PCR assay across various Acanthamoeba species and its ability to assess disinfectant efficacy further underline its potential as a valuable tool for improving diagnostic precision and therapeutic outcomes in the treatment of AK.

Zinc (Zn²⁺) is essential for all bacteria, but excessive Zn²⁺ levels are toxic. Bacteria maintain zinc homeostasis through regulators, such as Zur, AdcR, and ZntR. Riemerella anatipestifer is a significant Flavobacteriales pathogen causing acute serositis in ducks and other birds. In this study, we identified a homolog of ZntR, a regulator for zinc homeostasis, and demonstrated its contribution to the pathogenicity of R. anatipestifer. Deletion of zntR makes the bacteria hypersensitive to excess Zn²⁺ but not to other metals like manganese (Mn²⁺), copper (Cu²⁺), cobalt (Co²⁺), and nickel (Ni²⁺). Deletion of zntR also leads to intracellular zinc accumulation but not of other metals. Additionally, compared to the wild type, the deletion of zntR increases resistance to oxidants hydrogen peroxide (H₂O₂) and sodium hypochlorite (NaOCl), respectively. The deletion of zntR causes significant changes in transcriptional and protein expression levels, revealing 35 genes with potential zinc metabolism functions. Among them, zupT, which is inhibited by ZntR, is required for zinc transport and resistance to oxidative stress. Finally, deletion of zntR leads to attenuation of colonization in ducklings. In summary, ZntR is a crucial regulator for zinc homeostasis and contributes to the pathogenicity of R. anatipestifer.IMPORTANCEZinc homeostasis plays a critical role in the environmental adaptability of bacteria. Riemerella anatipestifer is a significant pathogen in poultry with the potential to encounter zinc-deficient or zinc-excess environment. The mechanism of zinc homeostasis in this bacterium remains largely unexplored. In this study, we showed that the transcriptional regulator ZntR of R. anatipestifer is critical for zinc homeostasis by altering the transcription and expression of a number of genes. Importantly, ZntR inhibits the transcription of zinc transporter ZupT and contributes to colonization in R. anatipestifer. The results are significant for understanding zinc homeostasis and the pathogenic mechanisms in R. anatipestifer.

Clinical studies of vaccines generally require collections of microbial isolates obtained from various body sites over multiple years. Further, large microbe collections are needed for research due to increasing appreciation for the phenotypic and genotypic diversity among a single microbial species. However, large collections are not generally available due to method limitations. We show a new way to create, recover, and manage microbe collections in 96- or 384-well plates using 50% glycerol at -20°C. Fifty percent glycerol remains liquid at -20°C and permits only the chosen isolates to be reliably sampled without first thawing all other isolates in the plate. Consequently, the glycerol sampling allows integration of microbe collection, labeling, recovery, and storage steps. Creating a microbe collection as an array in microplates reduces physical storage space by 6- or 23-fold with 96-well or 384-well plates, respectively. The array permits direct analysis of the collection with high-throughput assay systems. Further, we show that Streptococcus pneumoniae could be stored for 11 years as a microplate array. Standardized microbe arrays created in microplates with the new method could be easily distributed for studies of microbial structures, genetic diversity, antibiotics, and microbe-host interactions. This integrated method suggests how automated microbe management systems can be created.IMPORTANCEEpidemiologic and microbiology studies require large microbial collections, and the use of microplates could facilitate the creation and management of these collections. However, recovering individual isolates from microplates is manual and tedious. In this study, we demonstrate a simple method for recovering a selected individual isolate from a microplate at -20°C using 50% glycerol. Additionally, we found that Streptococcus pneumoniae could be revived for more than 10 years in microplates. This new method of recovering microbes from frozen microplates could greatly streamline many large-scale epidemiologic studies, particularly those related to pneumococcal vaccine studies. This new method may ultimately automate the collection, management, and storage of microbial isolates.

Acinetobacter baumannii is a significant nosocomial pathogen, particularly problematic due to its extensive drug resistance. This study investigates 56 extensively drug-resistant A. baumannii (XDRAB) strains collected from various ICU wards at Jinhua Central Hospital, Zhejiang Province, China. Strains were isolated from diverse clinical samples, including sputum, blood, cerebrospinal fluid, and wound secretions. Identification was confirmed via matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was conducted using the VITEK 2 Compact system, E-test, and Kirby-Bauer methods. All strains were susceptible to polymyxin, with four showing intermediate susceptibility to tigecycline, while resistance rates to other antibiotics were 100%. Molecular typing through pulsed-field gel electrophoresis (PFGE) classified the strains into 10 types, with the dominant type (G) primarily found in ICU3, indicating a potential clonal outbreak. Whole-genome sequencing (WGS) and multi-locus sequence typing (MLST) identified ST208 as the predominant sequence type. Resistance gene screening revealed the presence of blaOXA-23, blaTEM-1D, and aminoglycoside resistance genes in most strains. Phylogenetic analysis confirmed the clonal transmission of ST208 strains across the hospital, with a high degree of genomic similarity among the isolates. These findings highlight the importance of continuous monitoring and effective infection control measures to prevent the spread of XDRAB in healthcare settings.IMPORTANCEExtensively drug-resistant Acinetobacter baumannii is a critical public health threat, particularly in hospital environments where it causes a variety of infections. The global spread of extensively drug-resistant A. baumannii (XDRAB) and its resistance to most antibiotics make treatment options limited, increasing the risk of patient morbidity and mortality. This study provides important insights into the molecular epidemiology of XDRAB in a hospital setting, revealing the clonal transmission of the ST208 sequence type. By utilizing both pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS), we identified genetic links between strains and the presence of key resistance genes. The findings underscore the urgent need for robust infection control protocols, routine surveillance, and judicious use of antibiotics to mitigate the spread of XDRAB and ensure better patient outcomes.

Ralstonia pseudosolanacearum strain OE1-1 secretes methyl 3-hydroxymyristate (3-OH MAME) as a quorum-sensing (QS) signal. Strain OE1-1 senses the chemical by the sensor histidine kinase PhcS, leading to the activation of the LysR family transcriptional regulator PhcA. The activated PhcA controls the expression of QS-dependent genes responsible for QS-regulated phenotypes including virulence. The autophosphorylation of the histidine at amino acid position 230 (H230-PhcS) in PhcS following the 3-OH MAME sensing is required for the PhcA activation. The alternative sensor histidine kinase PhcK is involved in the regulation of phcA, which is independent of 3-OH MAME sensing. Furthermore, the H230Q-PhcS substitution of H230-PhcS with glutamine significantly decreases phcA expression. However, how PhcK and PhcS regulate phcA expression remains unclear. To elucidate the mechanisms of the phcA regulation, we generated a phcK mutant with the H205Q-PhcK substitution of autophosphorylated histidine at amino acid position 205 of PhcK with glutamine. A transcriptome analysis using quantitative real-time polymerase chain reaction assay and RNA sequencing showed that the H230Q-PhcS substitution, but not the H205Q-PhcK substitution, significantly decreased the expression level of phcA. The H230Q-PhcS substitution led to significant changes in the expression levels of QS-dependent genes and a loss of virulence, similar to phcA or phcK deletion. It is thus thought that PhcS participates in not only the 3-OH MAME sensing-independently PhcK-mediated regulation of phcA but also the PhcA activation following 3-OH MAME sensing. Both functions of PhcS are significantly influenced by the autophosphorylation of H230-PhcS.

Importance: The soil-borne Ralstonia solanacearum species complex (RSSC) infects more than 300 plant species in over 50 families, including solanaceous plants, causing the devastating wilt disease that substantially decreases agricultural production worldwide. The cell density-dependent gene regulation system, QS, is required for RSSC virulence and involves two signaling pathways for the induction and activation of PhcA, which is the master transcriptional regulator in QS. In the present study, we describe the contribution of sensor histidine kinase PhcS to the PhcA induction, along with the alternative sensor kinase PhcK, independently of the sensing of QS signal methyl 3-hydroxymyristate in a phylotype I strain of RSSC, R. pseudosolanacearum strain OE1-1. This study further expands our knowledge of multiple networks, suggesting that several PhcS-mediated two-component systems are likely necessary for RSSC QS and virulence.

Nucleic acid amplification tests (NAATs) are the method of choice for Chlamydia trachomatis diagnosis, but these strategies are susceptible to target site mutations. C. trachomatis variants escaping detection with the Aptima Combo 2 (AC2) assay on the Hologic Panther instrument from 23S rRNA mutations have been reported in Nordic countries, England, Japan, and the United States. Given the potential for false negative results, this study investigated whether strains of C. trachomatis with AC2 target site mutations were present in Canada. Surveillance was conducted in Canadian laboratories from 2019 to 2021. Specimens suspected of AC2 target site mutations included those with low-value detections on the AC2 assay, with subsequent high-value detections on the Aptima Chlamydia Trachomatis (ACT) assay used for confirmatory testing. Specimens with AC2/ACT discrepant results were subjected to sequencing of the AC2 target (i.e., 23S rRNA). Sequencing revealed 15 (4.8%) diagnostic escape variants which were carrying either C1514T, G1523A, or G1526A mutations. All specimens with a diagnostic escape mutation were detected with a reformulated version of the AC2 assay. Overall, while the prevalence of C. trachomatis variants was rare, their presence in the Canadian population supports the use of the new AC2 kit formulation and the need for ongoing genetic surveillance for NAAT-based assays.

Importance: Molecular tests are commonly used for the detection of sexually transmitted infections (STIs) like Chlamydia trachomatis. Mutations impacting C. trachomatis molecular target detection on the Hologic Panther AC2 assay have been reported in several countries, raising concerns about potential false negative results. This study showed C. trachomatis target detection failures in specimens submitted for C. trachomatis testing in Canadian laboratories from 2019 to 2021. A reformulated version of the AC2 molecular test is now available that can identify C. trachomatis strains harboring target site mutations that were impacted by the previous test formulation. While target site mutations were rare in Canada, revealing their presence is important to ensure accurate molecular detection of C. trachomatis with existing testing methods. This study supports ongoing genetic monitoring of C. trachomatis molecular test target sites, as well as the use of the reformulated test to avoid false negative results and subsequent transmissions.

Incorporating molecular testing for human papillomavirus (HPV) into the screening of cervical specimens can improve risk stratification and, in turn, patient management. Infection with a high-risk (HR) HPV genotype is associated with greater risk for persistent infection, viral integration, and progression of cervical neoplasia. Current guidelines consider HPV 16 or HPV 18 clinically actionable with referral to colposcopy; however, 12 Other HR HPV genotypes have been associated with cervical cancer risk, suggesting a benefit of extended genotyping. In this multi-center study, we evaluated the performance of the Alinity m HR HPV assay, which reports HPV 16, 18, and 45 individually and aggregates of HPV 31/33/52/58 and HPV 35/39/51/56/59/66, compared with cobas HPV and Aptima HPV assays, across a variety of cytology result categories. A total of 746 de-identified residual cervical specimens, collected as part of routine cervical cancer screening programs, were tested using Alinity m HR HPV and at least one comparator assay. The overall percent agreement was ≥90.7% for results from the Alinity m HR HPV assay and cobas HPV assays and 90.5% for results from the Alinity m HR HPV and Aptima HPV assay. In patients with any abnormal cytology result, Alinity m identified 78 specimens with non-HPV 16/18 results, underscoring the benefit of detecting additional HR HPV genotypes to guide patient management more accurately. Among specimens with normal cytology, Alinity m detected 14 additional specimens with non-HPV 16/18 genotypes. Extended HR HPV testing can provide additional information to triage patients for appropriate testing and follow-up.IMPORTANCEExtended genotyping for high-risk human papillomavirus (HPV) types enhances diagnostic precision by identifying additional oncogenic HPV types beyond 16 and 18 therefore offering a more nuanced risk profile. This more comprehensive detection may aid in identifying persistent infections that are more likely to progress, thereby supporting future risk-based patient management strategies.

Escherichia coli synthesizes aromatic amino acids (AAAs) through the common pathway to produce the precursor, chorismate, and the three terminal pathways to convert chorismate into Phe, Tyr, and Trp. E. coli also imports exogenous AAAs through five transporters. GcvB small RNA post-transcriptionally regulates more than 50 genes involved in amino acid uptake and biosynthesis in E. coli, but the full extent of GcvB regulon is still underestimated. This study examined all genes involved in AAA biosynthesis and transport using translation reporter assay and qRT-PCR analysis. In addition to previously verified targets, aroC, aroP, and trpE, we identified new target genes that were significantly repressed by GcvB primarily via the R1 seed region. Exceptionally, GcvB strongly inhibits the expression of aroG, which encodes the major isozyme of the first reaction in the common pathway, through direct base pairing between the aroG translation initiation region and the GcvB R3 seed sequence. RNase E mediates the degradation of target mRNAs except aroC and aroP via its C-terminal domain. GcvB overexpression prolongs the lag phase and reduces the growth rate in minimal media supplemented with AAAs and confers resistance to an antibiotic compound, azaserine, by repressing AAA transporters.IMPORTANCEE. coli strains have been genetically modified in relevant transcription factors and biosynthetic enzymes for industrial use in the fermentative production of aromatic amino acids (AAAs) and their derivative compounds. This study focuses on GcvB small RNA, a global regulator of amino acid metabolism in E. coli, and identifies new GcvB targets involved in AAA biosynthesis and uptake. GcvB represses the expression of the first and last enzymes of the common pathway and the first enzymes of Trp and Phe terminal pathways. GcvB also limits import of AAAs. This paper documents the impact of RNA-mediated regulation on AAA metabolism in E. coli.