Search Results (8)

Neisseria gonorrhoeae, the causative agent of gonorrhea, represents a major public health concern due to its increasing antimicrobial resistance. While often asymptomatic—particularly in extragenital infections—untreated cases can lead to severe complications and further transmission. Despite global efforts to monitor antimicrobial resistance, data on the molecular determinants underlying decreased susceptibility in N. gonorrhoeae remain scarce in Peru. This study aimed to detect mutations in the penA and 23S rRNA genes, which confer decreased susceptibility to cephalosporins and azithromycin resistance. We extracted DNA from 124 N. gonorrhoeae-positive clinical rectal specimens collected in Aptima Combo 2 transport tubes from MSM patients. These DNA samples were then screened using the Mismatch Amplification Mutation Assay-based real-time PCR (MAMA-qPCR) to identify mutations in the 23S rRNA and penA genes. Each sample underwent separate reactions to detect A2059G and C2611T mutations in the 23S rRNA gene, and 86 of these samples were further tested in individual qPCR assays for the penA D345 deletion (D345del) or G545S mutations. Sanger sequencing was performed on all DNA samples positive for 23S rRNA mutations by MAMA-qPCR assay, and on 27 DNA samples that yielded sufficient penA amplicons for additional sequencing. Using the MAMA-qPCR assay for the 23S rRNA gene, 64 of 124 samples amplified in the A2059G reaction: 2 (3.1%) carried the mutation, and 62 were classified as wild type. In the C2611T reaction, 42 of 124 samples amplified, and none of them carried the mutation. Using the MAMA-qPCR assay for the penA gene, we only analyzed 86 samples, as the remaining 38 samples had insufficient DNA yield. A total of 44 of the 86 samples amplified in the D345del reaction: 5 (11.4%) carried the D345del, and 39 were classified as wild type. In the G545S reaction, 4 (6.4%) carried the mutation, and 58 were classified as wild type. Finally, sequencing of the penA gene in the 27 samples revealed mutations related to decreased susceptibility to cephalosporins. This study identified genetic mutations conferring resistance to azithromycin and decreased susceptibility to cephalosporins, providing an overview of the circulating mutations conferring resistance in N. gonorrhoeae strains in Peru. Full article

Duck hepatitis A virus type 3 (DHAV-3) is a viral pathogen that causes acute, high-mortality hepatitis in ducklings, and vaccination with attenuated live vaccines is currently the main preventive measure against it. However, differentiating infected from vaccinated animals (DIVA) is crucial for clinical diagnosis and effective disease control. This study aimed to develop a rapid mismatch amplification mutation assay PCR (MAMA-PCR) diagnostic method to simultaneously detect and differentiate between wild-type and vaccine strains. The method was specifically designed to target the critical single-nucleotide polymorphism (SNP) site (T→C at position 1143 in the VP0 gene) unique to the Korean vaccine strain AP04203-P100. MAMA-PCR demonstrated high sensitivity and specificity, with detection limits as low as 10^2.4 ELD₅₀/mL for wild strains and 10^0.5 ELD₅₀/mL for vaccine strains, and showed no cross-reactivity with 11 other common duck pathogens. The clinical sample results were completely consistent with those obtained using nested PCR detection and gold-standard sequencing. In summary, we successfully developed a rapid, one-step MAMA-PCR method that is more suitable for clinical diagnosis than traditional sequencing methods. Full article

Duck hepatitis A virus type 1 (DHAV-1) is the main pathogen causing viral hepatitis in ducks, marked by high contagion and acute mortality. Live attenuated DHAV-1 vaccines are widely used to control the disease. This study aims to develop a mismatch amplification mutation assay (MAMA)-PCR for the rapid detection and differentiation of Korean DHAV-1 wild-type strains from vaccine strains. A MAMA primer was designed to target a single nucleotide polymorphism (SNPs) at position 2276 within the VP1 gene, allowing differentiation in a single PCR reaction. The MAMA-PCR accurately identified both strains, with detection limits of 10^0.5 ELD₅₀/mL and 10^2.3 ELD₅₀/mL, respectively. The MAMA-PCR demonstrated specificity, showing no cross-reactivity with 12 other viral and bacterial pathogens. The MAMA-PCR was applied to 89 farms, yielding results consistent with nested-PCR and sequence determination, identifying four positive farms for DHAV-1 vaccine strains. In conclusion, this study is the first to employ the MAMA-PCR method to distinguish between DHAV-1 wild-type and vaccine strains. The developed method is rapid, simple, specific, and sensitive, thereby serving as an effective tool for clinical diagnostics in identifying and differentiating between Korean DHAV-1 wild-type and vaccine strains. Full article

The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in Campylobacter coli isolates collected from broilers, laying hens and poultry farm environments. One hundred and thirty-nine C. coli isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6)-Ib gene was performed using polymerase chain reaction (PCR) and specific primers. aac(6′)-Ib amplicons were further analyzed by digestion with BtsCI to identify the aac(6′)-Ib-cr variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 C. coli isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the qnrB, qnrS, qepA, and aac(6′)-Ib-cr were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in C. coli isolates in Tunisia and globally. Full article

It has been proven that single-nucleotide polymorphisms (SNPs) in LEP and LEPR genes could predispose individuals to an increased risk of pregnancy adverse outcomes (PAOs) such as recurrent pregnancy loss (RPL) and pre-eclampsia. Preterm birth (PTB) is the leading cause of infant mortality. We decided to investigate the correlation between PTB and LEP and LEPR SNPs. The study cohort included families who underwent spontaneous PTB and control samples of families who had at-term-born (≥37 weeks of gestational age) children. Swabs were performed by rubbing the sticky end for about 30 s on the gum and on the inside of the cheek, allowing us to collect the flaking cells of the oral mucosa. Genotyping of the three SNPs—LEPRA668G, LEPG2548A and A19G—was carried out via an ARMS-MAMA real-time PCR procedure, as previously described. Regarding LEPG2548A, we found that the most expressed genotype in infants both in the preterm and the at-term group was AG; however, we did not discover any statistically significant difference (p = 0.97). Considering LEPA19G, none among the infants and parents were found to carry the AA genotype. No statistically significant differences were found between children, mothers and fathers belonging to preterm and at-term groups. We did not find a statistically significant association in newborns and their mother, but our results show a statistical correlation with the LEPRA668G genotype GG of the father. This fact can contribute to defining genetic risk factors for PTB. Further studies are certainly needed to better clarify the role of genetics in influencing preterm delivery. Full article

To investigate the persistence risk of Campylobacter spp. in poultry farms, and to study the virulence and antimicrobial resistance characteristics in the recovered strains, we collected 362 samples from breeding hen flocks, before and after disinfection. The virulence factors were investigated by targeting the genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB, and ceuE by PCR. Antimicrobial susceptibility was tested and genes encoding antibiotic resistance were investigated by PCR and MAMA-PCR. Among the analyzed samples, 167 (46.13%) were positive for Campylobacter. They were detected in 38.7% (38/98) and 3% (3/98) of environment samples before and after disinfection, respectively, and in 126 (75.9%) out of 166 feces samples. In total, 78 C. jejuni and 89 C. coli isolates were identified and further studied. All isolates were resistant to macrolids, tetracycline, quinolones, and chloramphenicol. However, lower rates were observed for beta-lactams [ampicillin (62.87%), amoxicillin-clavulanic acid (47.3%)] and gentamicin (0.6%). The tet(O) and the cmeB genes were detected in 90% of resistant isolates. The bla_OXA-61 gene and the specific mutations in the 23S rRNA were detected in 87% and 73.5% of isolates, respectively. The A2075G and the Thr-86-Ile mutations were detected in 85% and 73.5% of macrolide and quinolone-resistant isolates, respectively. All isolates carried the flaA, cadF, CiaB, cdtA, cdtB, and cdtC genes. The virB11, pldA, and racR genes were frequent in both C. jejuni (89%, 89%, and 90%, respectively) and C. coli (89%, 84%, and 90%). Our findings highlight the high occurrence of Campylobacter strains exhibiting antimicrobial resistance with potential virulence traits in the avian environment. Thus, the improvement of biosecurity measures in poultry farms is essential to control bacterial infection persistence and to prevent the spread of virulent and resistant strains. Full article

Globally, Campylobacter is a significant contributor to gastroenteritis. Efficient pathogens are qualified by their virulence power, resistance to antibiotics and epidemic spread. However, the correlation between antimicrobial resistance (AR) and the pathogenicity power of pathogens is complex and poorly understood. In this study, we aimed to investigate genes encoding virulence and AR mechanisms in 177 Campylobacter isolates collected from layer hens and eggs in Tunisia and to assess associations between AR and virulence characteristics. Virulotyping was determined by searching 13 virulence genes and AR-encoding genes were investigated by PCR and MAMA-PCR. The following genes were detected in C. jejuni and C. coli isolates: tet(O) (100%/100%), bla_OXA-61 (18.82%/6.25%), and cmeB (100%/100%). All quinolone-resistant isolates harbored the Thr-86-Ile substitution in GyrA. Both the A2074C and A2075G mutations in 23S rRNA were found in all erythromycin-resistant isolates; however, the erm(B) gene was detected in 48.38% and 64.15% of the C. jejuni and C. coli isolates, respectively. The machine learning algorithm Random Forest was used to determine the association of virulence genes with AR phenotypes. This analysis showed that C. jejuni virulotypes with gene clusters encompassing the racR, ceuE, virB11, and pldA genes were strongly associated with the majority of phenotypic resistance. Our findings showed high rates of AR and virulence genes among poultry Campylobacter, which is a cause of concern to human health. In addition, the correlations of specific virulence genes with AR phenotypes were established by statistical analysis. Full article

Background: The aim of this study is to assess the serum values of IL-4, IL-5, IL-10, and IL-13 in a group of infants with non-IgE mediated food allergies treated with a hydrolyzed formula and compare them with a group of healthy peers. Methods: A total of 53 infants aged 1 to 4 months, of which 34 with non-IgE mediated food allergies and 19 healthy infants were enrolled in this study. Infants were eligible if they had gastrointestinal symptoms of food allergy and needed to switch from their initial formula to hydrolyzed formulas with an improvement of symptoms. Controls were fed with either breastmilk or standard formula. Blood samples were taken within one week of a special diet for cases. Interleukinsin in peripheral blood was detected and analyzed using the real-time PCR MAMA method. Fecal calprotectin was evaluated using a quantitative assay. Results: Values of IL-4 and IL-13 were significantly higher in the non-IgE food allergy group compared to the control group (p < 0.05), while IL-5 and IL-10 were significantly lower than the control group (p < 0.05). Fecal calprotectin in the non-IgE food allergy group was significantly higher compared to the control group (p < 0.05). Conclusion: This study provides a theoretical basis that Th2 cytokine expression in infants with a non-IgE mediated food allergy is significantly different than in healthy infants; this finding supports the use of early dietetic treatment with hydrolyzed formulas. Full article