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Background: Persistent infections caused by Helicobacter pylori (H. pylori), which are resistant to antibiotic treatment, pose a growing global public health concern. Biofilm formation is known to be associated with persistent infections due to its role in enhancing antimicrobial resistance and the tolerance of many pathogenic bacteria. Objective: This study aims to evaluate the biofilm formation of clinical isolates of H. pylori and its impact on antibiotic eradication. Methods: The thickness, morphology, and structure of biofilms derived from nine H. pylori strains were examined using confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy. Subsequently, the susceptibility of both planktonic and biofilm bacteria was assessed through the determination of minimum inhibitory concentration and minimum biofilm eradication concentration for amoxicillin, clarithromycin, levofloxacin, and tetracycline. Results: The results revealed varying biofilm thicknesses and densities among the strains, characterised by the presence of numerous filaments intertwining and connecting bacterial cells. Additionally, several cases exhibited susceptibility based on MIC measurements but resistance according to MBEC measurements, with MBEC indicating a higher resistance rate. Pearson Correlation analysis demonstrated a positive correlation between biofilm thickness and MBEC results (0 < r < 1), notably significant for amoxicillin (r = 0.801, P = 0.009) and tetracycline (r = 0.696, P = 0.037). Conclusion: Different strains of H. pylori exhibit variations in their capacity to release outer membrane vesicles (OMVs) and form biofilms. Biofilm formation can influence the effectiveness of amoxicillin and tetracycline in eradicating susceptible bacterial strains.
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The following are our views regarding the "letter to the editor" (Helicobacter is preserved in yeast vacuoles! Does Koch's postulates confirm it?) by Alipour and Gaeini, and the response "letter to the editor" (Candida accommodates non-culturable Helicobacter pylori in its vacuole-Koch's postulates aren't applicable) by Siavoshi and Saniee. Alipour and Gaeini rejected the methods, results, discussion, and conclusions summarized in a review article by Siavoshi and Saniee. The present article reviews and discusses evidence on the evolutionary adaptation of Helicobacter pylori (H. pylori) to thrive in Candida cell vacuoles and concludes that Candida could act as a Trojan horse, transporting potentially infectious H. pylori into the stomach of humans.
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Infecciones por Helicobacter , Helicobacter pylori , Helicobacter pylori/patogenicidad , Humanos , Infecciones por Helicobacter/microbiología , Candida/fisiología , Candida/crecimiento & desarrollo , Candida/patogenicidad , Vacuolas/microbiología , Vacuolas/metabolismo , Estómago/microbiología , Mucosa Gástrica/microbiologíaRESUMEN
Dental fluorosis, resulting from long-term environmental exposure to fluoride, is prevalent among diverse populations worldwide. Severe fluorosis not only compromises the aesthetic appeal of teeth but also impairs their functionality. This study aims to investigate the oral microbiome in dental fluorosis and the health individuals of adolescents living in the endemic fluorosis area of Guizhou, China through full-length 16S rDNA sequencing. Fourty-six individuals meet the sampling criteria, and we divided these samples into the following groups: a healthy group (H = 23) and a dental fluorosis group (F = 23), and two subgroups of Miao ethnicity: a healthy Miao group (Hm = 13) and a dental fluorosis Miao group (Fm = 15). A total of 660,389 high-quality sequences were obtained, and 12,007 Amplicon Sequence Variants (ASVs) were identified, revealing significant variations in oral microbiome between Fm and Hm groups. The composition of oral microbiota was similar between the H and F groups. At the genus level, Pseudopropionibacterium and at the species level, Streptococcus oralis_subsp.dentisani_clade_058 were less abundant in group F than in group H (P < 0.05). Further analysis revealed that the abundance of Capnocytophaga gingivalis and Kingella denitrificans was significantly lower in Fm fluorosis patients than in the Hm group (P < 0.05). Based on the LEfSe analysis, the potential core biomarkers in the oral of Fm fluorosis patients were identified at different taxonomic levels, ranging from phylum to species. These include Gammaproteobacteria, Prevotella sp_HMT_304, Gemella sanguinis, and Gracilibacteria_(GN02). Network analysis revealed that the microbiota in the fluorosis group exhibited more complex interactions with each other than the healthy group. Notably, within the Hm group, the potential biomarkers Capnocytophaga gingivalis and Kingella denitrificans exhibited a positive correlation. Finally, we employed PICRUSt2 analysis to explore the abundance clustering of the top 30 functional units in each sample, and we found that the metabolic pathway compositions of the four groups were similar. In summary, our findings suggest that the microbial composition of plaque in Hm patients with dental fluorosis is significantly altered, and we identified the potential marker microorganisms that contribute to these changes.
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BACKGROUND: Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida. METHODS: Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity. RESULTS: A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive. CONCLUSION: In the form of BLBs, H. pylori can internalize into gastric Candida and even vaginal Candida, which might have great significance in its transmission and pathogenicity.
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Candidiasis Vulvovaginal , Infecciones por Helicobacter , Helicobacter pylori , Excreción Vaginal , Femenino , Humanos , Ureasa/genética , Infecciones por Helicobacter/microbiología , Fluoresceína-5-Isotiocianato , Agar , Antígenos Bacterianos/genética , Mucosa Gástrica/microbiología , Candida/genética , Biopsia , ADN Ribosómico , Urea , Proteínas Bacterianas/genéticaRESUMEN
Helicobacter pylori are transmissible from person to person and among family members. Mother-to-child transmission is the main intrafamilial route of H. pylori transmission. However, how it transmits from mother to child is still being determined. Vaginal yeast often transmits to neonates during delivery. Therefore, H. pylori hosted in yeast might follow the same transmission route. This study aimed to detect intracellular H. pylori in vaginal and fecal yeasts isolates and explore the role of yeast in H. pylori transmission. Yeast was isolated from the mothers' vaginal discharge and neonates' feces and identified by internal transcribed spacer (ITS) sequencing. H. pylori 16S rRNA and antigen were detected in yeast isolates by polymerase chain reaction and direct immunofluorescence assay. Genetic relationships of Candida strains isolated from seven mothers and their corresponding neonates were determined by random amplified polymorphic DNA (RAPD) fingerprinting and ITS alignment. The Candida isolates from four mother-neonate pairs had identical RAPD patterns and highly homologous ITS sequences. The current study showed H. pylori could be sheltered within yeast colonizing the vagina, and fecal yeast from neonates is genetically related to the vaginal yeast from their mothers. Thus, vaginal yeast presents a potential reservoir of H. pylori and plays a vital role in the transmission from mother to neonate.
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Infecciones por Helicobacter , Helicobacter pylori , Recién Nacido , Humanos , Femenino , Madres , Helicobacter pylori/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Transmisión Vertical de Enfermedad Infecciosa , Saccharomyces cerevisiae/genética , ARN Ribosómico 16S/genética , Infecciones por Helicobacter/diagnóstico , Candida/genética , HecesRESUMEN
Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.
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Clostridioides difficile , Clostridioides , Humanos , Clostridioides/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia , Antibacterianos/farmacología , Antibacterianos/metabolismoRESUMEN
The alternative σ factor TcdR controls the synthesis of two major enterotoxins: TcdA and TcdB in Clostridioides difficile. Four potential TcdR-dependent promoters in the pathogenicity locus of C. difficile showed different activities. In this study, we constructed a heterologous system in Bacillus subtilis to investigate the molecular basis of TcdR-dependent promoter activity. The promoters of the two major enterotoxins showed strong TcdR-dependent activity, while the two putative TcdR-dependent promoters in the upstream region of the tcdR gene did not show detectable activity, suggesting that the autoregulation of TcdR may need other unknown factors involved. Mutation analysis indicated that the divergent -10 region is the key determinant for different activities of the TcdR-dependent promoters. Analysis of the TcdR model predicted by AlphaFold2 suggested that TcdR should be classified into group 4, i.e., extracytoplasmic function, σ70 factors. The results of this study provide the molecular basis of the TcdR-dependent promoter recognition for toxin production. This study also suggests the feasibility of the heterologous system in analyzing σ factor functions and possibly in drug development targeting these factors.
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Toxinas Bacterianas , Clostridioides difficile , Toxinas Bacterianas/genética , Clostridioides/metabolismo , Factor sigma/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Enterotoxinas/genéticaRESUMEN
Thermophilic group II intron is one type of retrotransposon composed of intron RNA and intron-encoded protein (IEP), which can be utilized in gene targeting by harnessing their novel ribozyme-based DNA integration mechanism termed "retrohoming." It is mediated by a ribonucleoprotein (RNP) complex that contains the excised intron lariat RNA and an IEP with reverse transcriptase (RT) activity. The RNP recognizes targeting sites by exon-binding sequences 2 (EBS2)/intron-binding sequences 2 (IBS2), EBS1/IBS1, and EBS3/IBS3 bases pairing. Previously, we developed the TeI3c/4c intron as a thermophilic gene targeting system-Thermotargetron (TMT). However, we found that the targeting efficiency of TMT varies significantly at different targeting sites, which leads to a relatively low success rate. To further improve the success rate and gene-targeting efficiency of TMT, we constructed a Random Gene-targeting Plasmids Pool (RGPP) to analyze the sequence recognition preference of TMT. A new base pairing, located at the -8 site between EBS2/IBS2 and EBS1/IBS1 (named EBS2b-IBS2b), increased the success rate (2.45- to 5.07-fold) and significantly improved gene-targeting efficiency of TMT. A computer algorithm (TMT 1.0), based on the newly discovered sequence recognition roles, was also developed to facilitate the design of TMT gene-targeting primers. The present work could essentially expand the practicalities of TMT in the genome engineering of heat-tolerance mesophilic and thermophilic bacteria. IMPORTANCE The randomized base pairing in the interval of IBS2 and IBS1 of Tel3c/4c intron (-8 and -7 sites) in Thermotargetron (TMT) results in a low success rate and gene-targeting efficiency in bacteria. In the present work, we constructed a randomized gene-targeting plasmids pool (RGPP) to study whether there is a base preference in target sequences. Among all the successful "retrohoming" targets, we found that a new EBS2b-IBS2b base paring (A-8/T-8) significantly increased TMT's gene-targeting efficiency, and the concept is also applicable to other gene targets in redesigned gene-targeting plasmids pool in E. coli. The improved TMT is a promising tool for the genetic engineering of bacteria and could promote metabolic engineering and synthetic biology research in valuable microbes that recalcitrance for genetic manipulation.
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Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Here, we describe a streamlined CRISPR-Cpf1-based protocol to achieve precise genome editing in C. difficile with high efficiencies. Our work highlighted the first application of CRISPR-Cpf1 for genome editing in C. difficile, which are both crucial for understanding pathogenic mechanism of C. difficile and developing strategies to fight against C. difficile infection (CDI). In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. Approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.
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Clostridioides difficile , Edición Génica , Sistemas CRISPR-Cas/genética , Clostridioides difficile/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Plásmidos/genéticaRESUMEN
Group â ¡ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group â ¡ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg2+ concentration. Finally, we dissected the potential of group â ¡ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group â ¡ introns in biotechnology.
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ARN Catalítico , ADN , Eucariontes , Marcación de Gen , Intrones/genética , ARN Catalítico/genéticaRESUMEN
Background: Reactive oxygen species (ROS)-induced oxidative stress (OS) and hyper-proliferation of gastric epithelial cells (GECs) due to Helicobacter pylori (Hp) infection are important mechanisms that lead to gastric carcinoma. Phycocyanin is a marine functional food additive with antioxidant and anti-inflammatory properties. Methods: The flow cytometry was used to detect the effect of 150 µM phycocyanin intervention on the cell cycle of human gastric adenocarcinoma cell line (AGS) infected with Hp. The transcriptomics of AGS cells intervened by 150 µM phycocyanin for 24 h and infected by Hp were detected. Differential gene expression analysis was performed using a cutoff at the normalized gene expression (log2) of 2 and a P-value of <0.05. Comparisons of the transcriptomes were made between the following groups: (I) AGS cells not infected with Hp and not using phycocyanin action and AGS cells infected with Hp only; (II) AGS cells not infected with Hp and not using phycocyanin action and AGS cells infected with phycocyanin action only; and (III) AGS cells infected with Hp only and phycocyanin treated and infected with Hp cells. c-myc and CyclinD1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting. Phosphorylation and non-phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 Mitogen-activated protein kinase (p38MAPK) were detected by immunoblotting. Intracellular ROS was detected by immunofluorescence. Results: Phycocyanin alleviates the Hp infection-induced increased cell viability and expression of cell cycle regulatory proteins c-myc and CyclinD1. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the differentially expressed genes in phycocyanin-treated Hp-infected AGS cells were most significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway. Phycocyanin could inhibit the Hp infection-induced phosphorylation of JNK, ERK, and p38MAPK and reduce the level of cellular ROS. Conclusions: This study suggests that phycocyanin can regulate the ROS/MAPK signaling pathway and reduce c-myc and CyclinD1 expression to inhibit the hyper-proliferation of AGS cells. Phycocyanin may serve as an inhibitor of malignant progression of Hp infection-induced gastric disease.
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Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.
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Clostridioides difficile , Clostridioides , Antígenos de Superficie , Adhesión Celular , Pared Celular , Clostridioides difficile/genética , Farmacorresistencia Microbiana , Manitol , Octoxinol , Proteínas RecombinantesRESUMEN
BACKGROUND: Environmental conditions affect the biology of mosquito vectors. Aedes albopictus is a major vector of many important diseases including dengue, Zika, and chikungunya in China. Understanding the development, fecundity, and survivorship of Ae. albopictus mosquitoes in different environmental conditions is beneficial for the implementation of effective vector control measures. METHODS: Aedes albopictus larval and adult life-table experiments were conducted under natural conditions in indoor, half-shaded, and fully shaded settings, simulating the three major habitat types in Hainan Province, a tropical island in the South China Sea. Temperature, humidity, and light intensity were recorded daily. Larval rearing used habitat water and tap water, with and without additional artificial food. Development time, survivorship, pupation rate, and adult emergence rates were monitored. Adult mosquito survivorship and fecundity were monitored daily and reproductive rates were determined, and age-dependent survivorship and reproduction were analyzed. RESULTS: The pupation time and male and female emergence times were significantly shorter in indoor conditions than in shaded and half-shaded conditions for both tap water with added food and habitat water with added food groups. For habitat water with added food, the shaded environment had the lowest pupation rate among the settings. For tap water with added food group, the shaded environment had the lowest pupation rate. The mean survival time of females was 27.3 ± 0.8 days in the indoor condition, which was significantly longer than that in the half-shaded (18.4 ± 0.6 days) and shaded (13.8 ± 1.2 days) conditions. Adult mortality was age-dependent, and the rate of change in mortality with age was not significantly different among different environmental conditions. The mean net replacement rate (R0) of female mosquitoes showed no significant difference among the three conditions, whereas the per capita intrinsic growth rate (r) in the shaded condition was 42.0% and 20.4% higher than that in the indoor and half-shaded conditions, respectively. Female daily egg mass was also age-dependent in all the settings, decaying exponentially with age. CONCLUSIONS: Our results imply that half-shaded conditions are likely the best natural condition for adult emergence and female reproduction, and food supply is crucial for larval development and pupation. The results provide new avenues for integrated mosquito management in indoor and outdoor areas, especially in half-shaded areas.
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Aedes/fisiología , Mosquitos Vectores/fisiología , Aedes/crecimiento & desarrollo , Animales , Ecosistema , Conducta Alimentaria , Femenino , Larva/crecimiento & desarrollo , Larva/fisiología , Masculino , Mosquitos Vectores/crecimiento & desarrollo , Reproducción , Estaciones del Año , Temperatura , Clima TropicalRESUMEN
PURPOSE: Chronic Helicobacter pylori infection causes peptic ulcers in a subpopulation of individuals and is a risk factor for the development of gastric cancer. Multiple infections and heteroresistant H. pylori contribute to poor treatment efficacy. Here, we investigated the extent of genetic diversity among H. pylori strains within a given host and its influence on the results of antibiotic (metronidazole, levofloxacin, clarithromycin, amoxicillin, and tetracycline) susceptibility testing. MATERIALS AND METHODS: Gastric mucosa biopsy samples were obtained from patients with gastric disorders, including 48 H. pylori positive patients, who were never previously treated for H. pylori infection. Five potential H. pylori colonies isolated from each sample were subcultured for enrichment. Enriched H. pylori colonies were identified through Gram staining and assays for urease, oxidase, and catalase. For each H. pylori monoclonal colony, the antibiotic susceptibility was assessed, genomic DNA was sequenced, and the cytotoxin-associated gene A (cagA) genotype was verified. Co-infection with multiple H. pylori strains was determined using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR). RESULTS: Thirteen gastric mucosa biopsy samples were positive for H. pylori. Five monoclonal strains isolated from each of these 13 patients were identified as H. pylori. RAPD-PCR indicated that intra-patient monoclonal strains of H. pylori in 10 of the 13 samples exhibited heterogeneity. Among the 13 patients, intra-patient monoclonal strains isolated from 4 patients had identical cagA genotype, whereas intra-patient monoclonal strains isolated from the other 9 patients harbored more than one cagA genotype. The antibiotic susceptibility of five intra-patient monoclonal strains from seven patients was inconsistent. CONCLUSION: The existence of heterogeneous H. pylori strains with resistance to different drugs and virulence were common within the gastric mucosa of an individual patient.
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BACKGROUND: Aedes albopictus is a highly invasive mosquito and has become a potential vector of dengue, chikungunya and Zika viruses. Insecticide-based mosquito interventions are the main tools for vector-borne disease control. However, mosquito resistance to insecticides is a major threat to effective prevention and control. Five Ae. albopictus populations across Hainan Province, China were investigated for susceptibility to multiple insecticide and resistance mechanisms. RESULTS: Larval bioassays indicated that resistance to pyrethroids was common in all larval populations. Adult bioassays revealed all populations were either resistant or highly resistant to at least four of the six synthetic insecticides (deltamethrin, permethrin, cyfluthrin, propoxur, malathion, and DDT) tested. Pre-exposure of mosquitoes to the synergistic agent piperonyl butoxide (PBO) increased mosquito mortality by 2.4-43.3% in bioassays to DDT, malathion, and permethrin and rendered mosquito sensitive to deltamethrin, cyfluthrin, and propoxur. The frequency of knockdown resistance (kdr) mutations (F1534S and F1534C) ranged from 69.8% to 89.3% and from 38.1% to 87.0% in field-resistant and sensitive populations, respectively. F1534S mutation was significantly associated with pyrethroid resistance. No mutation was detected in the acetylcholinesterase (ace-1) gene in the two examined populations. CONCLUSION: This study provides evidence of widespread resistance to multiple insecticides in Ae. albopictus in Hainan Province, China. Both kdr mutations and metabolic detoxification were potential causes of insecticide resistance for Ae. albopictus. Our findings highlight the need for insecticide resistance management and mosquito control measures that do not entirely depend on synthetic insecticides. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Aedes , Dengue , Insecticidas , Piretrinas , Infección por el Virus Zika , Virus Zika , Aedes/genética , Animales , China , Dengue/prevención & control , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Piretrinas/farmacología , Infección por el Virus Zika/prevención & controlRESUMEN
BACKGROUND: Mosquitoes are vectors of many tropical diseases. Understanding the ecology of local mosquito vectors, such as species composition, distributions, population dynamics, and species diversity is important for designing the optimal strategy to control the mosquito-borne diseases. METHODS: Entomological surveillance of adult mosquitoes was conducted in five sites representing different ecological settings across Hainan Island from January to December of 2018 using BG Sentinel (BGS) traps and Centers for Disease Prevention and Control (CDC) light traps. In each site, we selected three areas representing urban, suburban and rural settings. Eighteen trap-days were sampled in each setting at each site, and CDC light traps and BGS traps were setup simultaneously. Mosquito species composition, distribution, population dynamics, and species diversity were analyzed. Mosquito densities were compared between different study sites and between different settings. RESULTS: Nine species of mosquitoes belonging to four genera were identified. Culex quinquefasciatus (80.8%), Armigeres subalbatus (13.0%) and Anopheles sinensis (3.1%) were the top three species collected by CDC light traps; Cx. quinquefasciatus (91.9%), Ae. albopictus (5.1%), and Ar. subalbatus (2.8%) were the top three species collected by BGS traps. Predominant species varied among study sites. The population dynamics of Ae. albopictus, An. sinensis and Cx. quinquefasciatus showed clear seasonal variation regardless of study sites with a varied peak season for different species. Mosquito abundance of all species showed significant differences among different study sites and among urban, suburban and rural areas. Danzhou had the highest mosquito biodiversity, with an α, ß, and Gini-Simpson biodiversity index of 8, 1.13 and 0.42, respectively. BGS traps captured Aedes mosquito at a higher efficiency than CDC light traps, whereas CDC light traps captured significantly more Anopheles and Armigeres mosquitoes than BGS traps. CONCLUSIONS: Mosquitoes were abundant on Hainan Island with clear seasonality and spatial heterogeneity. Population density, species composition, distribution, and species diversity were strongly affected by the natural environment. Different tools are required for the surveillance of different mosquito species.
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Biodiversidad , Culicidae , Densidad de Población , Aedes/clasificación , Animales , Anopheles/clasificación , China , Culex/clasificación , Culicidae/clasificación , Control de Mosquitos , Mosquitos Vectores , Estaciones del Año , Enfermedades Transmitidas por Vectores/transmisiónRESUMEN
To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⺠symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.
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Escherichia coli , Silenciador del Gen , Marcación de Gen , Técnicas Genéticas , Temperatura , Escherichia coli/genética , Flagelos , Marcación de Gen/métodos , Mutación , PlásmidosRESUMEN
Clostridioes difficile is a Gram-positive, spore-forming, obligate anaerobic bacterium, and the main cause of hospital-associated diarrhea. In recent years, with the presence of virulent strains (i.e., ribosome type 027), the prevalence and mortality events have increased. Thus, studies on physiological and biochemical characteristics, and pathogenic mechanisms of C. difficile have been performed. The development of efficient and stable genome-editing methods for C. difficile is urgent for the dissection of its physiological and pathogenic mechanism. For example, ClosTron technology plays a key role in study of the relationship between C. difficile toxins (Toxin A and Toxin B) and its pathogenicity. This article reviews the history, recent progress and future prospects of C. difficile genome-editing technologies.
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Edición Génica , Proteínas Bacterianas , Toxinas Bacterianas , Composición de Base , Clostridioides difficile , Enterotoxinas , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADNRESUMEN
Despite extensive studies on the gastric microbiota, including Helicobacter pylori and non-H. pylori, the bacterial composition in children remains unknown. In this study, we analyzed the culturable gastric bacteria in stomach biopsies from 346 children aged 1-15 years affected by gastric diseases. H. pylori and non-H. pylori were identified by specific PCR and 16S rDNA sequencing, respectively. Antibiotic susceptibilities of H. pylori and non-H. pylori were tested by the E-test and disk diffusion methods, respectively. Rapid diagnosis was also performed by H. pylori-specific PCR. Twenty-two H. pylori strains were obtained from culture, and 92 biopsies were positive by H. pylori-specific PCR. The positive rate was higher in boys (40.3%) than in girls (23.3%) (P = 0.001). Resistance rates of 22 H. pylori strains were as follows: metronidazole, 86.4%; tetracycline, 22.7%; amoxicillin, 22.7%; levofloxacin, 31.8%; clarithromycin, 36.4%. Ten isolates were multidrug-resistant. Additionally, among 366 non-H. pylori strains, 204 exhibited urease activity. Non-H. pylori resistance rates were as follows: metronidazole, 94.8%; tetracycline, 26.2%; amoxicillin, 42.6%; levofloxacin, 15.3%; clarithromycin, 46.7%. Our results showed that children with gastric disorders harbor stomach bacteria with urease activity or nitrate reductase activity. Further studies will determine the effects of non-H. pylori bacteria in gastric diseases.