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Abstract This study evaluated the antimicrobial capacity of BlueM® mouthwash against the bacterium Streptococcus mutans and its influence on gbpA gene expression as well as its cytotoxic effect on fibroblast cells. BlueM® showed antimicrobial activity, with MIC and MBC values of 0.005% and 0.01%, respectively. The MBIC was 6.25% for S. mutans. CFU count and confocal microscopy revealed significant effect of BlueM® on S. mutans biofilm pre-formed on dentin surfaces. Interestingly, the analysis of gbpA gene expression indicated a decrease in gene expression after 15 min of treatment with BlueM® at a concentration of 25%. Moreover, BlueM® exhibited low levels of cytotoxicity. In conclusion, our results showed the antimicrobial effectiveness of BlueM® against S. mutans, its ability to modulate the expression of the gbpA gene and its low cytotoxicity. This study supports the therapeutic potential of BlueM® as an alternative agent for the control of oral biofilm.
Resumo Este estudo avaliou a capacidade antimicrobiana do enxaguatório BlueM® contra a bactéria Streptococcus mutans e sua influência na expressão do gene gbpA, bem como seu efeito citotóxico em células de fibroblastos. BlueM® apresentou atividade antimicrobiana, com valores de CIM e CBM de 0,005% e 0,01%, respectivamente. O MBIC foi de 6,25% para S. mutans. A contagem de UFC e a microscopia confocal revelaram efeito significativo do BlueM® no biofilme de S. mutans pré-formado nas superfícies de dentinas. Curiosamente, a análise da expressão do gene gbpA, indicou uma diminuição na expressão do gene após 15 min de tratamento com BlueM® na concentração de 25%. Além disso, BlueM® exibiu baixos níveis de citotoxicidade. Em conclusão, nossos resultados mostraram a eficácia antimicrobiana do BlueM® contra S. mutans, sua capacidade de modular a expressão do gene gbpA e sua baixa citotoxicidade. Este estudo apoia o potencial terapêutico do BlueM® como agente alternativo para o controle do biofilme oral.
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@#Objective To isolate and identify the pathogenic bacteria in Tongliao area,Inner Mongolia Autonomous Region,China in 2019 and determine the virulence gene distribution and pathogenicity.Methods Three Gram positive isolates,FL1,FL2 and FL3,from the clinical specimens in Tongliao area were subjected to biochemical test,drug sensitivity test,16S rDNA sequencing,virulence gene test and pathogenicity test.Results Bio-chemical test proved the three isolates as Bacillus cereus. The strains FL1 and FL2 from bovine origin were resistant to penicillin and carbamazillin,while the strain FL3 from horse origin to Cefradine. Strains FL1 and FL2 showed the closest relationship to the isolate(KR063181. 1)from Hunan,China,while strain FL3 to the isolate(HM104658. 1)from Shandong,China. However,strain FL3 showed relatively far relationship to strains FL1 and FL2. All the distributions of eight virulence genes of B.cereus were observed in the three strains,while the distributions of four virulence genes of B.anthracoides were different. All the three isolates showed pathogenicity.Conclusion All the three isolates were pathogenic B.cereus,which showed a certain drug resistance. However,there were differences in virulence gene distri-bution between bovine and equine B.cereus.
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Objective:To study the molecular phylogeny and virulence gene profile of Francisella salimarina. Methods:Phylogenetic analysis of Francisella salimarina was performed based on the global genome data of related Francisella species on GenBank database. The consistency in phylogenetic analysis based on single marker genes (such as 16S rRNA gene, rpoB gene and mdh gene) and the core genome as compared. Virulence genes and antibiotic resistance genes were annotated using the virulence factor database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD), respectively. The virulence of Francisella salimarina was analyzed with a Galleria mellonella (greater wax moth) infection model using Francisella philomiragia ATCC 25015 T as reference strain. Results:The phylogenetic analysis revealed that Francisella salimarina was closely related to Francisella philomiragia. The phylogenetic tree based on mdh gene was highly similar to that based on the core genome. Francisella salimarina could be differentiated from other related species by 16S rRNA gene or mdh gene, with the latter being more accurate. Eight Francisella salimarina strains carried multiple virulence genes, mainly involved in secretion, adhesion, immune regulation, motility and stress survival. Moreover, beta-lactam resistance gene blaFPH was identified in all eight strains. Francisella salimarina showed high lethality in the Galleria mellonella infection model, which was similar to Francisella philomiragia ATCC 25015 T. Conclusions:Francisella salimarina was a rare pathogen with similar pathogenicity to Francisella philomiragia. The mdh gene could be used as a molecular target for rapid identification of Francisella salimarina.
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Objective:To analyze the molecular epidemiological characteristics of Campylobacter fetus subsp. testudinum ( Cft). Methods:Fifteen strains of Cft collected in our laboratory from 2010 to 2022 were subjected to whole-genome sequencing. Their epidemiological characteristics were analyzed based on the global genome data of Cft on GenBank database. MLST-GrapeTree software was used to obtain the genetic structure of Cft strains. A phylogenetic tree was constructed using core-genome single nucleotide polymorphism (cgSNP) analysis, and the sequence clusters were identified using rhierBAPS. Virulence genes and drug resistance genes of Cft strains were annotated using CARD, ResFinder and VFDB database. Their susceptibility to antibiotics was tested using E-test method and the results were analyzed using the CLSI-M45 sensitivity standard for Campylobacter jejuni/ Campylobacter coli. Results:Based on average nucleotide identity (ANI) analysis, the genome data of 41 Cft strains including 24 isolated from human, 13 from animals and four of unknown sources were collected from GenBank database. Among the 24 human-derived strains, 20 were linked to Asian descent and only one was linked to Caucasian descent (spouse of Asian descent), showing statistically significant differences in human ethnicity. All of the 13 animal-derived strains were originated from reptilian sources, including six from turtles, four from snakes and three from lizards. MLST revealed that ST46 was the predominant ST in China, while ST15 was the major sequence type in the United States. Grapetree analysis also demonstrated that the genetic diversity in China was greater than that in the United States. The phylogenetic tree constructed based on cgSNP and BAPS identified six distinct sequence clusters. The Chinese isolates were scattered in diverse sequence clusters and closely related to animal-derived strains, while the American isolates mainly belonged to ST15. The genes encoding virulence factors such as flagella, glycosylation systems and adhesins were carried by all of the 41 Cft strains (100.00%). The invasion-related virulence genes, such as the genes encoding the IV type secretion system ( virB4, virB9, virD4) and the resistance-related tetO efflux pump gene were specifically identified in the emerging ST74 clones. In vitro drug susceptibility testing of 15 Chinese isolates revealed 46.67% of the Cft strains were resistant to ciprofloxacin and 100.00% were sensitive to erythromycin. Conclusions:The global sequence clusters of Cft isolates showed a great genetic diversity. Most of the people with Cft infection had basic immune diseases and might have eaten or had contact with reptiles. Notably, the Chinese domestic infection of ST46 and the emerging ST74 should arouse our more attention.
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@#Abstract: Objective To establish a method for detecting sdaB virulence gene of Streptococcus pyogenes with loop mediated isothermal amplification (LAMP). Methods According to the conserved sequence of Streptococcus pyogenes sdaB gene published in GenBank (GenBank: 69901515), LAMP primers were designed with Primer Explorer V5.0 software. Main components of LAMP reaction system were optimized including of fluorescent dye, MgSO4, betaine, deoxyribonucleosidetriphosphate (dNTP), and Bst DNA polymerase, with the concentration of MgSO4 from 0 mmol/L to 12 mmol/L, betaine from 0 mol/L to 2.4 mol/L, dNTP from 0.2 µmol/L to 2 µmol/L, forward inner primer (FIP) and backward inner primer (BIP) from 0.2 µmol/L to 2 µmol/L respetively, forward outer primer (F3) and backward outer primer (B3) from 0.2 µmol/L to 0.4 µmol/L, Bst DNA polymerase from 0.16 U/µL to 0.96 U/µL, fluorescent dye from 0.2 µmol/L to 2 µmol/L. With the optimized system, the methodological specificity and the minimum detection limit were evaluated on the ABI7500 real-time fluorescent quantitative PCR analyzer, and 13 standard strains including Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus viridis, Enterococcus faecalis, Enterococcus faecium, Neisseria gonorrhoeae, Lactobacillus acidophilus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were detected. Finally, 103 clinical samples were tested. Results The optimized reaction system contained 25 µL reaction mixture, including 0.8 µL of 25 µmol/L fluorescent dye, 1 µL of 100 mmol/L MgSO4, 6 µL of 5 mol/L betaine, 1.4 µL of 25 mmol/L dNTP, 2 µL of 20 µmol/L FIP and BIP, 0.5 µL of 20 µmol/L F3 and B3, 1 µL of 8 U/µL Bst DNA polymerase, and 2 µL of template. After adding deionized water, the mixture was incubated at 63°C for 45 min to complete the reaction. The limit of detection (LOD) was 500 pg/µL. All 12 non-S. pyogenes strains tested were negative. Compared with the culture method, the clinical sensitivity and specificity were 100.0% (16/16) and 96.6% (84/87), respectively, for 103 clinical samples. Conclusions This LAMP assay is reliable for the detection of Streptococcus pyogenes in clinic and is suitable for field detection with good specificity and sensitivity, as well as simply operation.
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@#Objective To detect the virulence gene of E. coli from calves with diarrhea in Tongliao City，Inner Mongolia Autonomous Region，China and analyze its antimicrobial resistance as well as the distribution of antimicrobial resistant genes. Methods The sensitivities of 82 E. coli isolates from the fecal samples of calves with diarrhea to thirteen kinds of antibiotics were determined by disk diffusion test. The carrying statuses of thirteen virulence genes and twelve antimicrobial resistant genes of the E. coli isolates were determined by PCR，based on which the phylogenetic background was investigated. Results Of the 82 pathogenic E. coli isolates，48. 78%（40／82）、31. 71%（26／82）、14. 63%（12／82）and 4. 88%（4／82）belonged to phylogenic groups A，B1，B2 and D respectively，indicating that the prominent one was group A. A total of 11 virulence genes were detected in 82 isolates. The detection rates of irp2，fyuA，eaeA and STb genes were 79. 27%（65／82），63. 41%（52／82），53. 66%（44／82）and 50%（41／82）respectively，while those of other virulence genes were less than 50%，and no tsh or LT1 was detected. The 82 isolates were significantly resistant to 13 kinds of antibiotics，in which the resistant rates to tetracycline，doxycycline and amoxicillin were 100%（82／82），97. 56%（80／82）and 90. 24%（74／82）respectively. All the isolates were mutidrug resistant，most of which were resistant to eight kinds of antibiotics（16／82，19. 51%）. A total of twelve antimicrobial resistant genes were detected in the 82 isolates，in which the positive rates of genes resistant to β ‑ lactams（blaTEM），sulfonamide（sul1 and sul2），tetracycline（tetB and tetD）and aminoglycosides（aadB）were more than 70%. Conclusion The 82 pathogenic E. coli isolates mainly belonged to group A，with high detection rates of virulence gene and antimicrobial resistant gene as well as high and multiple drug resistance. The study provided a reference for the prevention and treatment of and clinical medication of E. coli‑associated diseases in calves in Tongliao Region.
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@#Abstract: Objective To investigate the molecular characteristics and drug resistance of non-O1/non-O139 Vibrio cholerae in Zhongshan City, and to provide laboratory basis for cholera prevention and control. Methods The strains of non-O1/non-O139 Vibrio cholerae isolated from sporadic patients and aquatic products from 2015 to 2021 in Zhongshan city were collected. The identification and cluster analysis of the strains were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the ctxA virulence gene of strains were detected by real-time fluorescence quantitative PCR, the cluster analysis of the strains was analyzed by pulsed-field gel electrophoresis (PFGE), and the drug resistance of the strains were analyzed by microbroth dilution method. Results From 2015 to 2021, 33 strains of non-O1/non-O139 Vibrio cholerae were isolated from Zhongshan City, including 28 strains from sporadic patients and 5 strains from aquatic products. Through MALDI-TOF-MS identification, 33 strains of non-O1/non-O139 Vibrio cholera can be identified to the level of species, and the identification results were all Vibrio cholerae. Among 33 non-O1/non-O139 Vibrio cholerae strains, 1 strain carried the ctxA virulence gene. The drug-resistant strains accounted for 69.7% (23/33), and the multidrug resistant strains accounted for 18.2% (6/33). A total of 7 kinds of drug resistance spectrum were produced, including 3 kinds of multidrug resistant spectrum, and showed drug resistance to 8 antibiotics, among which the resistance rates to streptomycin, cefazolin and compound sulfamethoxazole were above 30%. The 33 strains of non-O1/non-O139 Vibrio cholerae were divided into 32 PFGE fingerprints with a similarity ranging from 61.7% to 100%. MALDI-TOF-MS cluster analysis divided 33 non-O1/non-O139 Vibrio cholerae strains into two clusters. Conclusions The results of molecular typing of non-O1/non-O139 Vibrio cholerae in Zhongshan City presented diversity, and no significant correlation was found between PFGE and MALDI-TOF-MS cluster analysis. The strains demonstrated various degrees of resistance to certain antibiotics, and there were multidrug-resistant and toxigenic strains. Therefore, it is necessary to alert to the harmfulness of non-O1/non-O139 Vibrio cholerae and enhance monitoring.
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Objective To investigate the etiological characteristics of food poisoning isolates of Vibrio parahaemolyticus (VP) from 2019 to 2021 in Zhongshan City. Methods A total of 37 strains of Vibrio parahaemolyticus isolated from 8 food poisoning incidents in Zhongshan City from 2019 to 2021 were collected, including 1 residual food isolate and 36 human isolates. The genetic correlation of Vibrio parahaemolyticus food poisoning isolates in this region was analyzed by serological typing, virulence gene detection (TLH, TDH, and TRH), drug sensitivity test, pulsed field gel electrophoresis (PFGE) and multipoint sequence typing (MLST). Results The 37 strains of Vibrio parahaemolyticus were divided into 4 serotypes: O3:K6, O10:K4, O4:K8, and O4:KUT. The tdh+ and trh- were the main virulence genotypes, accounting for 97.30% (36/37). The drug resistance rate of cefazolin was 40.54% (15 strains R, 22 strains I), and no multidrug-resistant strains were found. The 37 VP strains were divided into 23 PFGE types and 6 cluster groups, with correlation coefficients ranging from 60.4%-100%. The multipoint sequencing typing showed that the 37 VP strains were divided into 9 ST types and 3 complex groups, of which ST3 type was the main type (23 strains, 62.1%). Conclusion This study has found that the dominant virulence types of Vibrio parahaemolyticus food poisoning isolates in Zhongshan City from 2019 to 2021 are tdh+ and trh-, and 37 representative strains can be divided into 6 PFGE clusters and 9 ST types with MLST type being mainly ST3. This study has identified the rare serotype O10:K4 which has caused an increase in the proportion of food poisoning events, suggesting that we should strengthen detection and be alert to the risk of continued local epidemics of new rare serotype strains.
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Objective:To study the effect of the siderophore virulence gene entB on the virulence of carbapenem-resistant Klebsiella pneumonia (CRKP). Methods:CRKP-27 was selected as the experimental strain from 30 CRKP strains collected from the First Affiliated Hospital of Kunming Medical University. The knockdown strain (Δ entB) and complementing strain (C-Δ entB) were constructed by the clustered regularly interspaced short palindromic repeat-Cas9 technology, and verified by polymerase chain reaction (PCR). In order to initially understand the effect of entB on CRKP colony morphology and virulence phenotype, the colony morphology of CRKP-27, Δ entB, and C-Δ entB strains were observed and string test were tested. Draw the growth curve of the strains and determine the effect of entB on the growth of the CRKP strains. The siderophores production ability of the strains were detected quantitatively using chrome azurol S (CAS) detection solution. Mice model of inflammation was established to observe the survival rate of mice and intuitively understand the effect of entB on CRKP virulence. Results:The PCR results showed that the Δ entB strain and C-Δ entB stranin were constructed successfully. The entB has no significant effect on the colony morphology, capsule and virulence phenotype of CRKP. The growth rate of Δ entB was significantly faster than that of CRKP-27( P=0.008) and C-Δ entB ( P=0.001), which showed that entB weakened the growth ability of CRKP. Compared with CRKP-27( P=0.001) and C-Δ entB( P=0.001), the siderophore production of Δ entB was significantly decreased by 11.739 3% and 11.964 2%, indicating that entB gene increased the capacity of CRKP to produce siderophpres. In animal experiments, compared with CRKP-27( P=0.023) and C-Δ entB( P=0.024), the survival rate of mice in the Δ entB group was significantly increased, indicating that the entB increased the virulence of the CRKP. Conclusion:The siderophore virulence gene entB significantly weakened the growth ability of the strain, but clearly enhanced the siderophore production capacity and virulence of CRKP.
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Objective:To investigate the molecular epidemiological characteristics and antibiotic resistance of Clostridioides difficile ( Cd) in hospitalized diarrhea patients in a tertiary hospital in Shaanxi Province. Methods:This study collected 425 stool samples of hospitalized diarrhea patients from October 2018 to December 2021 for isolation and identification of Cd. Toxin genes carried by the isolates were detected. Multilocus sequence typing (MLST) was performed to analyze the phylogenetic profile. Antibiotic susceptibility was analyzed by E-test. Results:Forty-nine strains of Cd were isolated from the 425 samples, including 37 strains of toxigenic Cd (75.5%, 37/49). The detection rate of Cd was 14.0% (25/179) in diarrhea patients aged ≥65 years old and 36.4% (4/11) in Nephrology Department. In the 37 toxigenic Cd strains, A -B + CDT -Cd, A + B + CDT -Cd and A + B + CDT +Cd accounted for 18.9% (7/37), 78.4% (29/37) and 2.7% (1/37), respectively. There were 24 ST types, among which ST2, ST3 and ST54 were the predominant types, each accounting for 12.2% (6/49). All strains were sensitive to metronidazole and vancomycin, with a high resistance rate of 93.9% (46/49) to ciprofloxacin and a low resistance rate of 12.2% (6/49) and 10.2% (5/49) to rifampicin and meropenem, respectively. Conclusions:The main type of toxigenic strains was A + B + CDT -. ST2, ST3 and ST54 were the predominant types and the distribution of ST types was scattered. All isolates were sensitive to metronidazole and vancomycin and most of them were resistant to ciprofloxacin.
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Objective:To identify a pathogenic strain JM-1 isolated from the pus of a patient stabbed by a sea shrimp and to analyze its antibiotic susceptibility and virulence genes, aiming to provide reference for screening clinically related infections caused by Cysteiniphilum litorale as a rare pathogen and improving prognosis. Methods:Biochemical phenotype identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S rRNA gene sequencing, analysis of average nucleotide identity (ANI) and average amino acid identity (AAI) based on the whole genome and phylogenetic analysis of the 16S rRNA gene and the whole genome were performed to accurately determine the taxonomic status of the strain JM-1. E-test was used to detect antibiotic susceptibility, and the results were interpreted according to the interpretation standards of Francisella tularensis in CLSI M45-A3. The virulence factor database (VFDB) was used for genome-wide annotation and analysis of virulence genes. Results:After culturing the strain JM-1 on the Columbia blood plate for 3 d, some grey-white, medium-sized, smooth, round and convex hemolytic colonies were observed. Gram staining result showed lightly colored Gram-negative Coccobacillus. API NH identification results suggested that the isolate JM-1 was Moraxella catarrhalis (biochemical code: 3010), while there was no identification result in Vitek2 system NH card (biochemical code: 0211002121). The EXS3000 mass spectrometer self-built database identified the isolate JM-1 as Cysteiniphilum litorale. The phylogenetic analysis based on the 16S rRNA gene and the whole genome showed that the isolate JM-1 and Cysteiniphilum litorale DSM 101832 T clustered into the same branch, and the ANI and AAI values between the two strains were 95.07% and 95.65%, respectively. The biochemical phenotype identification indicated the isolate JM-1 producing β-lactamase and penicillinase. Antibiotic susceptibility test results showed the strain was resistant to penicillin and sensitive to gentamicin, streptomycin, doxycycline, tetracycline, ciprofloxacin, levofloxacin, and chloramphenicol. Genome annotation suggested the virulence genes of the isolate JM-1 were similar to those of Francisella, including Francisella pathogenicity island (FPI), type Ⅳ fimbriae, capsule and lipopolysaccharide. Conclusions:Cysteiniphilum litorale was a rare pathogen with virulence genes similar to those of Francisella, and its antibiotic susceptibility was also similar to that of Francisella. This study confirmed a case of clinical infection caused by Cysteiniphilum litorale. The self-built MALDI-TOF MS system could be used for its rapid identification.
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Objective To study the biological characteristics of clinical isolates of human infected Streptococcus suis type 2 in Zhongshan City from 2016 to 2019 and classify the strains using pulse-field gel electrophoresis (PFGE), and to provide a scientific basis for clinical prevention and treatment of porcine streptococcus suis disease. Methods Twelve strains of Streptococcus suis type 2 were collected from 2016 to 2019 and identified by automatic bacterial identification instrument. The carrying status of five major virulence genes of Streptococcus suis was detected by nucleic acid and protein analyzer, including capsular polysaccharide (cps2J), lysozyme-releasing protein (mrp), hemolysin (sly), glutamate dehydrogenase (gdh), and extracellular factor (ef). The susceptibility of Streptococcus suis to 12 kinds of commonly used antibiotics was determined by the broth microdilution method, and the homology analysis was carried out by PFGE method. Results Twelve strains of Streptococcus suis type 2 were divided into four virulence genotypes, mainly mrp-/sly+/ef+/cps2J+/gdh+ (6strains) and mrp-/sly-/ef+/cps2J+/gdh+(4strains). Drug susceptibility test results showed that 12 strains of Streptococcus suis type 2 were resistant to erythromycin, tetracycline and clindamycin, and they all were multi-resistant strains. According to the classification results of PFGE, the 12 strains were classified into 7 PFGE types based on 100% similarity coefficient. The PFGE band types of Streptococcus suis in the same year had high homology. Conclusion The virulence genotypes of 12 clinical isolates of human infected type 2 Streptococcus suis in Zhongshan from 2016 to 2019 are diverse, and the strains are resistant to multiple antibiotics. Most strains in the same year are the same clone strains. PFGE genotypes are not correlated with virulence genotypes and drug resistance spectrum.
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To investigate the relationship of biofilm-forming ability of (PA) with swimming motility, twitching motility and virulence gene distribution. A total of 192 clinical isolates of PA were collected consecutively. Microtiter plate method was used to evaluate the ability to form biofilm. The swimming and twitching motilities were detected by plate method. Polymerase chain reaction (PCR) was used to detect virulence genes. Of the 192 PA clinical isolates, 186 (96.9%) showed biofilm-forming ability. Among them, 36 isolates showed weak biofilm-forming ability, 84 exhibited moderate biofilm-forming ability and 66 showed strong biofilm-forming ability. The diameters of the swimming ring for PA with none biofilm-forming ability, weak biofilm-forming ability, moderate biofilm-forming ability, strong biofilm-forming ability were (9.12±6.76), (18.42±7.51), (19.10±4.77) and respectively. The diameters of the twitching ring for PA in above groups were (8.38±1.50), (17.21±7.42), (18.49±5.62) and respectively. The swimming motility and twitching motility of none biofilm-forming ability group were weaker than biofilm-forming ability groups (all <0.05). Among 192 PA strains, 163 were positive (84.9%), 40 were positive (20.8%), 183 were positive (95.3%), and 189 were positive (98.4%). The positive rate of PA virulence gene , and were different in strains with different biofilm-forming abilities (<0.05). The rate of in the strong biofilm-forming ability group was lower than that in the moderate biofilm-forming ability group (=9.293, <0.01) and the weak biofilm-forming ability group (=9.997, <0.01). The rate of in the strong biofilm-forming ability group was higher than that in the weak biofilm-forming ability group (=10.803, <0.01). Most clinical isolates of PA can form biofilm. Swimming and twitching motilities are related to the formation of biofilm, but not significantly related to strength of biofilm-forming ability. The virulence genes of type Ⅲ secretion system for PA may be related to the biofilm-forming ability.
Subject(s)
Humans , Biofilms , Swimming , Virulence/geneticsABSTRACT
Objective@#This study was performed to compare the genetic diversity, virulence, and antimicrobial resistance of @*Methods@#A total of 38 clinical strains and 19 strains from healthy individuals were isolated from the samples collected in Ma'anshan City, Anhui Province. Their taxonomy was investigated using concatenated @*Results@#The 57 @*Conclusions@#The taxonomy, virulence properties, and antibiotic resistance of
Subject(s)
Humans , Aeromonas/pathogenicity , Case-Control Studies , Drug Resistance, Bacterial/genetics , Genetic Variation , Virulence Factors/geneticsABSTRACT
Objective:To determine the distribution and epidemic characteristics of Vibrio parahaemolyticus in Jinshan District of Shanghai from 2016 through 2018. Methods:Serotype analysis，examination of virulence genes， including thermolabilehemolysin（TLH），thermostable direct hemolysin（TDH），and TDH related hemolysin（TRH），and antimicrobial susceptibility test and pulsed field gel electrophoresis （PFGE） molecular typing were performed on 218 Vibrio parahaemolyticus isolated from diarrhea patients. Results:A total of 218 strains were divided into 8 groups and 21 serotypes. Of them， 147 strains were serotyped，with O3：K6 as the most common serotype （57.3%）. All tested strains were divided into 25 clusters based on the similarity of 85% and above，in which the dominant cluster was JSVP02，and the total similarity was 56.3%-100.0%. Two hundred and one strains （92.2%） carried tdh gene，13 strains （6.0%） carried trh gene，and 7 strains were negative for both tdh and trh. A total of 35 strains were completely sensitive to 17 kinds of antibiotics，while the remaining 183 strains showed different drug resistance. Conclusion:Vibrio parahaemolyticus isolated from diarrhea patients in Jinshan District from 2016 through 2018 is diverse. Majority of the strains have TDH gene and are resistant to the first generation cephalosporins such as cefazolin and penicillin ampicillin. Construction of regional PFGE molecular typing database will facilitate screening，identification and early warning of high-risk strains.
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Objective:To understand the contamination and antimicrobial resistance of Vibrio parahaemolyticus in seafood sold in a place of Zhejiang Province， and to provide basic data for the prevention and control of food-borne diseases caused by V. parahaemolyticus. Methods:V. parahaemolyticus was isolated and identified from seafood according to GB 4789.7—2013 method. The virulence genes were identified by real-time fluorescent PCR， and the antimicrobial sensitivity test was performed by Kirby-Bauer method. Results:V. parahaemolyticus was isolated from 55 of 210 seafood samples. The detection rate was 26.19%， and the detection rates of different species were quite different. None of the 55 isolates contained virulence genes tdh and trh， while all isolates have species-specific genes tlh and toxR. Most isolates were resistant to ampicillin and amoxicillin， with the same antimicrobial resistance rate of 96.36%. All isolates were fully sensitive to ampicillin/sulbactam， tetracycline， ciprofloxacin， levofloxacin， loxacin and chloramphenicol. Conclusion:V. parahaemolyticus contamination is common in sold seafood. The antimicrobial resistance of the isolates has reached a certain level. Monitoring of the pollution status of V. parahaemolyticus in seafood should be strengthened and the aquaculture and clinical use of antibiotics should be standardized.
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Objective@#This study aimed to evaluate the genetic diversity, virulence, and antimicrobial resistance of isolates from clinical patients, tap water systems, and food.@*Methods@#Ninety isolates were obtained from Ma'anshan, Anhui province, China, and subjected to multi-locus sequence typing (MLST) with six housekeeping genes. Their taxonomy was investigated using concatenated sequences, while their resistance to 12 antibiotics was evaluated. Ten putative virulence factors and several resistance genes were identified by PCR and sequencing.@*Results@#The 90 isolates were divided into 84 sequence types, 80 of which were novel, indicating high genetic diversity. The isolates were classified into eight different species. PCR assays identified virulence genes in the isolates, with the enterotoxin and hemolysin genes , , , and found in 47 (52.2%), 13 (14.4%), 22 (24.4%), and 12 (13.3%) of the isolates, respectively. The majority of the isolates (≥ 90%) were susceptible to aztreonam, imipenem, cefepime, chloramphenicol, gentamicin, tetracycline, and ciprofloxacin. However, several resistance genes were detected in the isolates, as well as a new variant.@*Conclusions@#Sequence type, virulence properties, and antibiotic resistance vary in isolates from clinical patients, tap water systems, and food.
Subject(s)
Aeromonas , Genetics , Virulence , Anti-Bacterial Agents , Pharmacology , China , Drinking Water , Microbiology , Drug Resistance, Bacterial , Food Microbiology , Genetic Variation , Gram-Negative Bacterial Infections , Microbiology , Species Specificity , VirulenceABSTRACT
Objective: To investigate the relationship of the clustered regularly interspaced short palindromic repeats (CRISPR) with resistance genes and virulence genes. Methods: The Shigella genome sequences, resistance genes and virulence genes sequences were obtained from GenBank Database. Then we obtained the distribution of CRISPR1, CRISPR2 and CRISPR-Q1 and the information of repeat and spacers in Shigella by multiple sequences alignment. Meanwhile, the distribution of resistance genes and virulence genes in Shigella were obtained by BLAST and the relationship of the CRISPR with resistance genes and virulence genes was computed by SPSS 17.0. Results: The spacers of CRISPR1 and CRISPR2 showed obvious differences in different Shigella strains, and the CRISPR-Q1 was more widely distributed (over 99%). The virulence genes were widely distributed in Shigella (more than 56%); the distribution of resistance genes (blaOXA-1, camr, Sul2, and tetB) was also wide (over 50%). There were significant differences between the CRISPR1/CRISPR2 and 9 kinds of virulence genes and 4 kinds of resistance genes (P<0.05). There were significant differences between the CRISPR-Q1 and 3 kinds of virulence genes and 4 kinds of resistance genes (P<0.05). There were significant differences between the CRISPR1/CRISPR2, CRISPR-Q1 and the number of virulence genes and resistance genes (P<0.05). There were significant differences between the CRISPR1/CRISPR2 and the number of spacers of CRISPR-Q1 (χ2=61.6, P<0.001). Conclusion: The distribution of CRISPR was quite different in Shigella, and the number of spacers was also different. There were negative correlation of CRISPR of Shigella with some resistance genes and virulence genes. The CRISPR1/CRISPR2 had a positive relationship with the number of the spacers.
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Objective@#To investigate the antimicrobial resistance, macrolide-resistance genes, virulence genes and emm types in Streptococcus pyogenes isolates.@*Methods@#A total of 247 oropharyngeal swab specimens were collected from pediatric outpatients (aged 2-11 years) who were clinically diagnosed as scarlet fever in Children′s Hospital of Fudan University from January to December, 2018. These specimens were timely sent to the Microbiology Laboratory for isolation and identification of Streptococcus pyogenes strains were isolate after culturing and identified with bacitracin susceptibility test. Moreover, the diameter of bacitracin inhibition zone was measured by vernier caliper. Their susceptibility to seven antibiotics, including erythromycin, clarithromycin, clindamycin, ampicillin, ceftriaxone, norfloxacin and chloramphenicol, were measured using KB method. Macrolide-resistance genes (mefA, ermA, ermB and Tn916 transposon) and virulence genes (speA, speB, speC, speG, speH, speI, speJ and speK) were detected by PCR. Amplification and sequencing of emm gene were conducted according to the protocol in the website of Centre for Disease Control and Prevention (CDC).@*Results@#A total of 86 strains of Streptococcus pyogenes were isolated from the 247 specimens. Their resistance rates to erythromycin, clarithromycin and clindamycin were 89.5%, 95.3% and 96.5%, respectively. However, these isolates showed high susceptibility to ampicillin (100.0%), ceftriaxone (100.0%), norfloxacin (90.7%) and chloramphenicol (95.3%). The positive rates of mefA, ermA, ermB and Tn916 genes were 20.9%, 24.4%, 98.8% and 97.7%. There was significant difference in the mefA-carrying rates between patients with scarlet fever and angina. The positive rates of virulence genes of speA, speB, speC, speG, speH, speI, speJ, speK, speL and speM were 8.1%, 100.0%, 95.3%, 100.0%, 80.2%, 90.7%, 10.5%, 100.0%, 5.8% and 5.8%. Seven emm types were identified and the predominant types were emm12.0 (75.6%), emm12.19 (9.3%) and emm1.0 (8.1%). The diameter of bacitracin inhibition zone was smaller in isolates of emm1.0 type than in emm12.0 type strains. The profile of virulence genes varied in the strains of different emm types. All types of strains carried speB, speG and speK genes. The isolates of emm1.0 type carried speA virulence gene, while speB, speC, speG, speH, speI and speK genes were more often identified in emm12.0 type isolates.@*Conclusions@#This study showed that emm types were associated with the profile of virulence genes and the diameter of bacitracin inhibition zone. It was recommended that the diameter of bacitracin inhibition zone should be measured in bacitracin inhibition susceptibility test apart from only observing the formation of inhibition zone.
ABSTRACT
Objective@#To analyze the molecular typing characteristics by pulsed field gel electrophoresis (PFGE), drug resistance and virulence genes of Salmonella typhimurium in Longyan city in order to provide reference for the prevention and control.@*Methods@#A total of 79 Salmonella typhimurium strains were isolated from sporadic cases of diarrhea and food poisoning and raw poultry meat samples during 2010 to 2017. PFGE was performed to measure the minimum inhibitory concentrations (MIC) of 15 commonly used drugs against them. PCR was used to detect nine virulence genes (sopB, invA, sifA, sscA, sseE, spvB, pefA, spvR, spvC) in 55 strains.@*Results@#The 79 Salmonella typhimurium strains belonged to 61 PFGE types. There were 10, three and four strains of P1, P3 and P21 types, respectively. Seven P1 type strains were isolate from one food poisoning event. According to the 85% classification standard, 79 Salmonella typhimurium strains could be divided into five predominant gene clusters (G1-G5). Drug susceptibility test showed that the 79 strains had the highest resistance rate to ampicillin (88.61%), followed by that to tetracycline (87.34%) and streptomycin (73.41%). Multidrug resistant bacteria resistant to three or more antibacterial drugs accounted for 84.81% (67/79). All of the 55 strains carried invA, sopB, sseE and sscA genes. The other five genes, sifA, spvC, spvB, spvR and pefA, were detected in 54, 31. 10, 11 and 12 strains, respectively. There were 76.4% (42/55) of the strains carrying five or six virulence genes and all were positive for invA, sopB, sseE, sscA and sifA, and negative for spvB, spvR and pefA. The strains carrying all of the nine virulence genes accounted for 18.2% (10/55).@*Conclusions@#Salmonella typhimurium strains isolated in Longyan city had a diverse PFGE type. P1, P3 and P21 types were the three predominant PFGE types. In the food poisoning event, PFGE molecular typing could quickly alert the outbreak and traceability of Salmonella typhimurium. Attention should be paid to the multidrug resistance in Salmonella typhimurium. Monitoring of multidrug-resistant strains and supervision on antibacterial drug usage should be strengthened. Salmonella typhimurium had high virulence as it carried many virulence genes.