A novel PCR-based genotyping method for Proteus mirabilis - Intergenic region polymorphism analysis.

Proteus mirabilis is a predominant species in cases of food poisoning associated with meat products and is also an opportunistic pathogen causing numerous infections in humans. This study aimed to differentiate P. mirabilis isolates using intergenic region polymorphism analysis (IRPA). The IRPA typing scheme was developed to amplify polymorphic fragments in intergenic regions (IGRs). The presence, absence, or size change of amplified products were identified and utilized as genetic markers for rapid differentiation of strains. A total of 75 P. mirabilis isolates were isolated from 63 fresh poultry and pork samples were subtyped using the IRPA and ERIC-PCR methods, and their antibiotic resistance profiles were tested. The majority of P. mirabilis isolates showed resistance to tetracycline (85.3%), doxycycline (93.3%), chloramphenicol (82.7%), streptomycin (92.0%), spectinomycin (80.0%), trimethoprim (97.3%); trimethoprim-sulfalleth (82.7%), and erythromycin (100.0%). In contrast, resistance rates to ceftriaxon, cefoxitin, cefepime, and cefotaxim were lower at only 17.3%, 5.3%, 6.7%, and 13.3%, respectively, among P. mirabilis isolates. Eleven loci were selected for analysis of the genetic diversity of 75 P. mirabilis isolates. A combination of 4 loci was determined as the optimal combination. The results compared to those obtained using ERIC-PCR for the same isolates. The Simpson's index of diversity was 0.999 for IRPA and 0.923 for ERIC-PCR, indicating that IRPA has a higher discriminatory power than ERIC-PCR. The concordance between IRPA and ERIC-PCR methods was low, primarily because IRPA classified isolates from the same ERIC cluster into separate clusters due to its high resolution. The IRPA method presented in this study offers a rapid, simple, reproducible, and economical approach for genotyping P. mirabilis.

Recent Advances in Oral Vaccines for Animals.

Compared to traditional injected vaccines, oral vaccines offer significant advantages for the immunization of livestock and wildlife due to their ease of use, high compliance, improved safety, and potential to stimulate mucosal immune responses and induce systemic immunity against pathogens. This review provides an overview of the delivery methods for oral vaccines, and the factors that influence their immunogenicity. We also highlight the global progress and achievements in the development and use of oral vaccines for animals, shedding light on potential future applications in this field.

Correlation between Antibiotics-Resistance, Virulence Genes and Genotypes among Klebsiella pneumoniae Clinical Strains Isolated in Guangzhou, China.

Klebsiella pneumoniae is an important opportunistic pathogen causing community-acquired and hospital-acquired infections. This aim of this study was to analysis the antibiotic-resistance phenotypes, carbapenemase genes, virulence genes, and genotypes the 62 K. pneumoniae clinical isolates, and to explore the correlations between these isolates. The antimicrobial susceptibility profiles were determined using the BD Phoenix-100 system. Carbapenemase and virulence genes were detected using multiplex PCR. Out of the 62 K. pneumoniae clinical isolates, 79.0% were exhibited resistance to antibiotics, with 69.4% displaying multi-drug resistance. The rate of antibiotic-resistance was highest for penicillin (71.0%), followed by cephalosporins (66.1%), and lowest for carbapenems (29.0%). The detection rates of carbapenemase genes were as follows: KPC (56.5%), VIM (35.5%), and NDM (1.61%). Additionally, seven virulence genes were detected with the highest prevalence rates, of which entB and mrkD were at the top of the carrier rates with 95.2% each. The study classified 62 isolates into 13 clusters and 46 genotypes using ERIC-PCR. Cluster A6 exhibited the highest genetic diversity, comprising 20 strains and 13 genotypes. The statistical analysis revealed a strong correlation between MDR and resistance to penicillin and cephalosporin. Furthermore, genes related to siderophores were closely associated with mrkD. Genotypes identified by ERIC-PCR showed a negative correlation with allS. The study revealed a negative correlation between antibiotic resistance and genes kfu, ybtS, iutA, rmpA, and allS. Conversely, a positive correlation was observed between antibiotic resistance and genes entB and mrkD. The correlations identified in this study provide insights into the occurrence of hospital-acquired infections. The findings of this study may guide the prevention and control of K. pneumoniae outbreaks by utilizing appropriate medication.

Rapid Genotyping of Campylobacter coli Strains from Poultry Meat by PFGE, Sau-PCR, and fla-DGGE.

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains.

Intergenic region polymorphism analysis: a novel genotyping method for Klebsiella pneumoniae.

AIMS: The ability to distinguish between Klebsiella pneumoniae strains is critical for outbreak investigations. A new typing method, intergenic region polymorphism analysis (IRPA), was developed, validated, and the discriminatory power was determined by comparison with multiple-locus variable-number tandem repeat analysis (MLVA) in this study. METHODS AND RESULTS: This method is based on the idea that every IRPA locus (polymorphic fragment of intergenic regions present in one strain but not in other strains or different fragment sizes in other strains) could divide strains into different genotypes. A 9-loci IRPA scheme was designed to type 64 K. pneumoniae isolates. Five IRPA loci were identified that conferred the same level of discrimination as the 9-loci initially examined. Among these K. pneumoniae isolates, 7.81% (5/64), 6.25% (4/64), 4.96% (3/64), 9.38% (6/64), and 1.56% (1/64) were capsular serotypes K1, K2, K5, K20, and K54, respectively. The discriminatory power of the IRPA method was better than that of MLVA expressed in Simpson's index of diversity (SI) at 0.997 and 0.988, respectively. The congruent analysis of the IRPA method and MLVA showed moderate congruence between the two methods (AR = 0.378). The AW indicated that if IRPA data are availabl, one can accurately predict the MLVA cluster. CONCLUSION: The IRPA method was found to have higher discriminatory power than MLVA and allowed for simpler band profile interpretation. The IRPA method is a rapid, simple, and high-resolution technique for molecular typing of K. pneumoniae.

Comparison of pulsed-field gel electrophoresis and a novel amplified intergenic locus polymorphism method for molecular typing of Campylobacter jejuni.

Campylobacter is regarded as the leading cause of zoonotic diseases and Campylobacter jejuni (C. jejuni) is one of the predominant pathogenic species. To track C. jejuni infections, various genotyping methods have been used. In this study, amplified intergenic locus polymorphism (AILP) was used to type C. jejuni for the first time. To confirm its feasibility, pulsed-field gel electrophoresis (PFGE) was performed as a control, and the results obtained by the AILP and PFGE methods were compared. Fifty-one isolates were resolved into 34 and 29 different genotypes with Simpson's indices of 0.976 and 0.967 using the AILP and PFGE methods, respectively. The adjusted Rand coefficient of the two approaches was as high as 0.845. In summary, the data showed that the two genotyping methods were similar for discriminating isolates and were both appropriate methods to distinguish whether two isolates were indistinguishable, but the AILP was faster and less costly than PFGE. Therefore, the AILP is a reliable, rapid, and highly discriminative method to genotype C. jejuni collected from poultry meat, which is helpful to effectively monitor C. jejuni.

Subtyping of Campylobacter coli isolated from raw poultry meat in retail markets using amplified intergenic locus polymorphism - A novel rapid subtyping method.

In order to provide more phylogenetic information of Campylobacter coli in large-scale epidemiological investigation, this work was undertaken to develop a novel genotyping method based on amplified intergenic locus polymorphism (AILP), by using pulsed-field gel electrophoresis (PFGE; using SmaI enzymes) as control. Eleven pairs of primers were selected to type C. coli strains for this purpose. A total of 68 C. coli isolates recovered from 51 retail raw chicken and 37 retail raw duck were subtyped. The Simpson's index of diversity (SID) of AILP and PFGE, as well as the adjusted Rand index (AR) and the adjusted Wallace coefficient (AW) between AILP and PFGE, were calculated. The new AILP method differentiated 68 C. coli isolates into 55 different subtypes (SID = 0.993), compared with 46 different profiles obtained from PFGE (SID = 0.980). The SID value of the AILP method was improved with the increasing number of primers, and a combination of 7 loci was selected as the optimal combination. The congruent analysis of the AILP method and PFGE showed moderate congruence between the two methods (AR = 0.462). The AW indicated that if AILP data is the available one can confidently predict the PFGE cluster. The results of this study showed that the AILP method had higher discrimination than PFGE and also allowed for significant reduction in time and cost.

Mapping of Pesticide Transmission on Biological Tissues by Surface Enhanced Raman Microscopy with a Gold Nanoparticle Mirror.

We presented an improved surface-enhanced Raman scattering (SERS) mapping technique for the imaging of pesticides on biological samples including tomato leaves, fruits, and mouse skin using a gold nanoparticle mirror as the SERS substrate. The gold nanoparticle mirror was fabricated using 50 nm commercial citrate-capped gold nanoparticles upon the interface of water and a mediating solvent that was prepared using acetonitrile and hexane. The properties of the gold nanoparticle mirror were compared with gold nanoparticles, and the mirror displayed higher sensitivity with a limit of detection of 0.07 µg/cm2 and better reproducibility with a relative standard deviation of 5.48% for the SERS mapping of pesticide (ferbam) on biological samples. The gold mirror-based SERS mapping technique was also used to investigate pesticide transmission from tomato fruit surfaces to mouse skin after 1 mg/cm2 of pesticides was administered upon the fruit, and the results showed that about 23% of the pesticide was transmitted from the fruit to the mouse skin. We also found that pesticides on the contaminated hand could not be completely removed by routine rinsing with tap water for 2 min. This study provides an effective approach for the imaging of pesticides on biological tissues that would facilitate research on pesticide behaviors both on and in biological systems.

Genome-Wide Transcriptional Profiling Reveals Two Distinct Outcomes in Central Nervous System Infections of Rabies Virus.

Rabies remains a major public health concern in many developing countries. The precise neuropathogenesis of rabies is unknown, though it is hypothesized to be due to neuronal death or dysfunction. Mice that received intranasal inoculation of an attenuated rabies virus (RABV) strain HEP-Flury exhibited subtle clinical signs, and eventually recovered, which is different from the fatal encephalitis caused by the virulent RABV strain CVS-11. To understand the neuropathogenesis of rabies and the mechanisms of viral clearance, we applied RNA sequencing (RNA-Seq) to compare the brain transcriptomes of normal mice vs. HEP-Flury or CVS-11 intranasally inoculated mice. Our results revealed that both RABV strains altered positively and negatively the expression levels of many host genes, including genes associated with innate and adaptive immunity, inflammation and cell death. It is found that HEP-Flury infection can activate the innate immunity earlier through the RIG-I/MDA-5 signaling, and the innate immunity pre-activated by HEP-Flury or Newcastle disease virus (NDV) infection can effectively prevent the CVS-11 to invade central nervous system (CNS), but fails to clear the CVS-11 after its entry into the CNS. In addition, following CVS-11 infection, genes implicated in cell adhesion, blood vessel morphogenesis and coagulation were mainly up-regulated, while the genes involved in synaptic transmission and ion transport were significantly down-regulated. On the other hand, several genes involved in the MHC class II-mediated antigen presentation pathway were activated to a greater extent after the HEP-Flury infection as compared with the CVS-11 infection suggesting that the collaboration of CD4(+) T cells and MHC class II-mediated antigen presentation is critical for the clearance of attenuated RABV from the CNS. The differentially regulated genes reported here are likely to include potential therapeutic targets for expanding the post-exposure treatment window for RABV infection.