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Simple Summary: Duck hepatitis A virus (DHAV) is an infectious pathogen that causes fatal viral hepatitis in ducklings. Duck hepatitis A virus-1 (DHAV-1) is the most virulent and widely distributed serotype, inflicting severe damage on the global duck farming industry. The main approach to preventing DHAV-1 involves using attenuated live vaccines. However, these vaccine strains may persist in ducks and pose a risk of reverting to virulence. Therefore, distinguishing between infected and vaccinated animals is vital for clinical diagnosis and vaccine safety monitoring. Currently, only time-consuming and costly sequencing methods serve this purpose. In this study, a Mismatch Amplification Mutation Assay (MAMA)-PCR method was developed based on single nucleotide polymorphism (SNPs) in the VP1 gene. The MAMA-PCR method offers a highly specific and sensitive clinical tool for rapidly distinguishing DHAV-1 wild-type from vaccine strains, significantly contributing to accurate clinical diagnosis and epidemiological understanding. 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 100.5 ELD50/mL and 102.3 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. [ABSTRACT FROM AUTHOR]

Background: Quinolone-resistant Escherichia coli may easily be transmitted from poultry to humans and animals. Quinolone misuse in veterinary medicine, therefore, poses a significant public health risk. This study aimed to assess the prevalence of type II topoisomerase mutations commonly associated with resistance to quinolones in E. coli; in Dar es Salaam, Tanzania. Methods: One hundred sixty E. coli samples isolated from broiler cloacal swabs in Dar es Salaam between March and August 2022 were evaluated and analyzed for quinolone resistance by disc diffusion and genetic methods. The study used two Polymerase Chain Reaction (PCR) techniques, one a Mismatch Amplification Mutation Assay (MAMA-PCR) and the other employing enzyme digestion of amplified products (PCR-RFLP). Both approaches targeted regions in type II topoisomerases (gyrA and parC) to determine quinolone resistance (QRDR). Results: Results showed considerable levels of quinolone resistance among E. coli isolated from broiler chickens, where 42.5% of the strains demonstrated non-susceptibility to both ciprofloxacin and norfloxacin. Moreover, a significant minority of isolates (5%) were non-susceptible only to ciprofloxacin. All isolates non-susceptible to both drugs harboured a substitution mutation (S83L) at the gyrA Ser-83 target site (a resistance marker) as revealed by PCR-RFLP. The genotypic-to-phenotypic agreement ratio for norfloxacin was 100%, in contrast to that of ciprofloxacin, which was 89.47%. However, as revealed by MAMA-PCR results, none of the isolates under study harboured parC80 (S80I) substitution mutation (a resistance marker) on the topoisomerase IV gene. Conclusion: Type II topoisomerase mutations other than the ones assayed in this study (gyrA Ser-83 or parC80) or other mechanisms of resistance might be contributing to the resistance against quinolones in E. coli strains circulating in broiler chickens in Dar es Salaam. The level of quinolone resistance revealed by this study calls for immediate intervention to mitigate its spread. [ABSTRACT FROM AUTHOR]

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Background: Campylobacter species are among foodborne pathogens that are known as the main cause of inflammatory diarrhea in humans. In this cross-sectional study, we investigated the prevalence of Campylobacter spp. and their drug resistance status in fecal samples of patients with community-acquired gastroenteritis in Tehran. Materials and Methods: In this survey, infection with Campylobacter spp. was evaluated in 400 stool samples of patients with diarrhea. Accordingly, microscopic examination to show the presence of exudative diarrhea, rejection of fungi and parasitic infections, enrichment and culture in a specific medium under microaerophilic conditions, determination of biochemical identity, and molecular confirmation at genus and species levels for C. jejuni, C. coli, C. lari, and C. upsaliensis were performed. Antibiotic resistance patterns were determined by E-test and disk diffusion methods, and the presence of the dominant gyrA gene mutations in the quinolone-resistance domain of C. jejuni isolates was determined by using Mismatch Amplification Mutation Assay-polymerase chain reaction (MAMA-PCR)i 1. Results: A total of 28 strains of Campylobacter were isolated from the samples obtained from the patients with diarrhea. The most common species were C. jejuni (6%), C. coli (0.5%), C. lari (0.25%), and other unknown species (0.25%). The highest antibiotic resistance rate was observed against tetracycline and ampicillin (53.5% and 50%, respectively), and the lowest rate was detected for nalidixic acid and ciprofloxacin (28.5% and 28.5%). Multiple drug resistance (MDR) phenotype was detected among 51.8% of the strains. Conclusion: Results of this study indicated C. jejuni as the main Campylobacter species responsible for community-acquired diarrhea among the studied patients. The high rate of resistance to antibiotics and MDR phenotype in these strains compared with other countries is considered a risk, especially in the treatment of invasive infections. [ABSTRACT FROM AUTHOR]

Fluoroquinolones are the drug of choice for most of the infections caused by Escherichia coli, and their indiscriminate use has resulted in increased selective pressure for antibiotic resistance. At present, sequencing is the only reliable and direct technique to detect mutations in the quinolone resistance determining region (QRDR). In this study, a rapid and reliable mismatch amplification mutation assay (MAMA) PCR to detect mutations in the QRDR was evaluated and compared to PCR-restriction fragment length polymorphism (PCR-RFLP). One hundred one clinical isolates of E. coli were subjected to MAMA-PCR and PCR-RFLP to detect QRDR mutations. Overall, 92 (91.08%) resistant isolates harbored a point mutation of S83L in gyrA. Double mutations in gyrA were also detected in 45 (44.55%) isolates. Similarly, 41 (40.59%) isolates possessed a point mutation at parC 80, and 25 (24.75%) isolates possessed a point mutation at parC 84. Additionally, MAMA-PCR—the first of its kind—was also standardized to detect mutations in regions gyrB 447 and parE 416, although no mutations were detected in these regions. The rapid and sensitive MAMA-PCR method evaluated in this study would be helpful in exploring the underlying mechanism of fluoroquinolone resistance to enhance control strategies. [ABSTRACT FROM AUTHOR]

A mismatch amplification mutation assay (MAMA)-PCR, which detects a single-nucleotide polymorphism contributed to serological difference between Streptococcus suis serotypes 2 and 1/2, is used to discriminate between these serotypes. The present study reports unusual serotype 1/2 isolates untypable by the MAMA-PCR and improvement of the MAMA-PCR for typing such isolates. • S. suis serotype 1/2 untypable by the original MAMA-PCR were isolated. • The MAMA-PCR was modified to correctly typed these unusual isolates. • Modified MAMA-PCR will help discrimination between S. suis serotypes 1/2 and 2. [ABSTRACT FROM AUTHOR]

Vitamin D has potent antimicrobial and anti-inflammatory properties. Vitamin D deficiency has been shown to be associated with the risk of vulnerability to different infectious diseases, such as neonatal sepsis. Polymorphisms in vitamin D receptor ( VDR ) gene can influence the expression of vitamin D in individuals. Hence, it is essential to study the vitamin D status and VDR gene polymorphisms for assessing neonatal sepsis risk. In this study, we assessed the serum 25(OH)D, the main circulating form of vitamin D and VDR polymorphism on 120 subjects in a case-control approach, recruiting 60 subjects in each category. We genotyped Fok 1, Bsm 1, Apa 1 and Taq 1 gene polymorphisms in VDR by developing a unique mismatch amplification mutation assay (MAMA) and studied their association in both populations. VDR -MAMA primers were designed by addition of dual mismatches (DM) near the 3′ end and were selected based on high ΔCt values in comparison to single mismatch (SM) primers using SYBR-Green RT-PCR, which were eventually used for VDR genotyping. Genotyping was also performed using PCR-RFLP for further confirmation. Serum 25(OH)D ELISA revealed that cases were vitamin D insufficient (Median = 12.16 ng/ml, 95% CI: 3.84–22.22) and controls were vitamin D sufficient (Median = 30.22 ng/ml, 95% CI: 20.08–46.78; p < 0.0001) respectively, which indicated that vitamin D insufficiency was mostly prevalent in cases. We found no evidence of association between genotypes of the Apa 1 polymorphism and neonatal sepsis or 25(OH)D serum levels. The distributions of the Fok 1, Bsm 1, and Taq 1 genotypes were not consistent with Hardy-Weinberg equilibrium in the control group. Future studies in larger populations are required to establish whether the VDR polymorphisms can be potentially used as genetic markers for early screening towards predisposition to neonatal sepsis risk. In this study, we describe a simple, inexpensive and rapid screening of VDR gene polymorphisms using VDR MAMA-PCR, which can be used in both clinical and research laboratories. [ABSTRACT FROM AUTHOR]

Here we describe the international scenario of Vibrio cholerae with a comparative analysis of different aspects of typing. Representative V. cholerae strains (n = 108) associated with endemic cholera regions from 29 states of India and worldwide were subjected to microbiological, molecular and phylogenetic study. All of the strains were V. cholerae serogroup O1 biotype El Tor and were typed according to both the new phage (NP) type and Basu & Mukherjee (BM) typing schemes. The predominant phage type was T-27 (NP)/T-4 (BM) (65.7%; n = 71), followed by phage type T-27 (NP)/T-2 (BM) (14.8%; n = 16), T-26 (NP)/T4 (BM) (12.0%; n = 13), T-13 (NP)/T-4 (BM) (2.8%; n = 3), T-20 (NP)/T-4 (BM) (1.9%; n = 2), T-3 (NP)/T-4 (BM) (0.9%; n = 1), T-23 (NP)/T-4 (BM) (0.9%; n = 1) and T-24 (NP)/T-2 (BM) (0.9%; n = 1). Mismatch amplification mutation assay PCR (MAMA-PCR) findings showed the dominance of ctxB El Tor genotype (77.1%; 54/70) from 1961-1991, whilst the next two epochs showed the supremacy of ctxB classical genotype. Multidrug-resistant strains showed resistance to erythromycin, streptomycin, trimethoprim/sulfamethoxazole, norfloxacin and ampicillin. The regional resistance of epidemic clones in India draws a layout of the rapid dissemination of resistance in the past 30 years and the necessity of proper treatment to protect populations at risk. [ABSTRACT FROM AUTHOR]

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP) + 874 ∗ T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor’s blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p < 0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject ( p = 0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard. [ABSTRACT FROM AUTHOR]

Summary: Cholera epidemics with moderately high case fatality rates in Assam, northeast India were investigated in 2007, 2008 and 2010. Based on mismatch amplification mutation assay PCR for detection of ctxB allele, 40 isolates of Vibrio cholerae O1 El Tor collected from the epidemics were found to harbour the classical ctxB gene allele of cholera toxin (CT). DNA sequencing of ctxB gene confirmed the isolates to be genotype 1 of ctxB. Antimicrobial susceptibility tests reveal that 100% of the isolates were resistant to trimethoprim and 40% were resistant to tetracycline. The recent V. cholerae O1 strains circulating in Assam, India are due to the El Tor variant carrying classical type CT. Emergence of tetracycline and trimethoprim resistant strains necessitates the review of antibiotic use for severe cholera. [ABSTRACT FROM AUTHOR]

The RB gene from wild potato Solanum bulbocastanum imparts broad-spectrum late blight resistance to cultivated potato. To explore marker associations and haplotype frequencies near RB, we developed, optimized, validated, and employed a set of markers specific to the haplotype associated with the RB resistance allele. Our markers, developed using a mismatch amplification mutation assay (MAMA)-PCR approach, have single nucleotide polymorphism-level sensitivity. MAMA markers were validated via linkage mapping and applied to a collection of 60 S. bulbocastanum genotypes representing the geographic distribution of the species. Considerable marker disassociation within a 100 kb region encompassing RB was revealed. Physical distance between markers was not an effective predictor of marker disassociation. Applied to a collection of four S. bulbocastanum genotypes of known RB allelic status, a set of six MAMA markers accurately predicted the presence of the RB allele. Implications for marker aided genotype selection and association mapping in wild potato species are explored. [ABSTRACT FROM AUTHOR]

Abstract: Synopsis: Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A2074C and A2075G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A2075G, whereas no isolate carried the A2074C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains. [Copyright &y& Elsevier]

The bacteriophage lambda (λ) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective λ prophage. Here we describe the generation of a mini-λ DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E. coli strain, including the DH10B-carrying BACs or PACs. The mini-λ DNA integrates into the bacterial chromosome as a defective prophage. In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA. We describe here the use of the mini-λ recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone. In addition, using the mini-λ, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker. The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-λ as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering. [Copyright &y& Elsevier]

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity. Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates. Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5 ) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D ) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method. Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB , tcpA and rtxA gene , 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex , att-prfC , setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml -1 to 2 µg ml -1 . Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.

Introduction: Tuberculosis (TB), commonly caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide. The gold standard, microbiological culture for detection and differentiation of mycobacteria are time-consuming and laborious. The use of fast, easy and sensitive nucleic acid amplification tests (NAATs) for diagnosis of TB remains challenging because there is a high degree of homology within Mtb complex (MTBC) members and absence of target genes in the genome of some strains. This study aimed to identify new candidate genetic marker and to design specific primers to detect Mtb using in silico methods. Methods: Using Basic Local Alignment Search Tool (BLAST) program, Mtb H37Rv chromosome reference genome sequence was mapped with other MTBC members and a single nucleotide polymorphism (SNP) at Rv1970 was found to be specific only for Mtb strains. Mismatch amplification mutation assay (MAMA) combine with polymerase chain reaction (PCR) was used as an alternative method to detect the point mutation. MAMA primers targeting the SNP were designed using Primer-BLAST and the PCR assay was optimized via Taguchi method. Results: The assay amplified a 112 bp gene fragment and was able to detect all Mtb strains, but not the other MTBC members and non-tuberculous Mycobacteria. The detection limit of the assay was 60 pg/µl. Conclusion: Bioinformatics has provided predictive identification of many new target markers. The designed primers were found to be highly specific at single-gene target resolution for detection of Mtb. [ABSTRACT FROM AUTHOR]

Purpose: This study was aimed at investigating the occurrence and genetic mechanisms of resistance to ciprofloxacin, tetracycline and erythromycin in clinical isolates of Campylobacter jejuni recovered from human cases of acute gastroenteritis in Turkey., Methodology: MIC values of each antibiotic were determined with the epsilometer test (E-test). Resistance genes/mutations were first screened by PCR and analysed by subsequent DNA sequencing., Results: From a total of 152 C. jejuni isolates tested, 113 (74.3%), 38 (25%) and 9 (5.9%) were found to be resistant to ciprofloxacin, tetracycline and erythromycin, respectively. Sequence analysis of ciprofloxacin-resistant isolates showed that all resistant strains (n=113) carried Thr-86-Ile substition in the gyrA gene, which is the most frequently observed mutation in fluoroquinolone-resistant Campylobacter. All of the tetracycline-resistant isolates (n=38) carried the tetO gene. All of the erythromycin-resistant isolates (n=9) harboured the point mutation A2075G in the 23S rRNA gene, which is the most common mutation conferring macrolide resistance in C. jejuni., Conclusion: The phenotypic susceptibility testing results were found to agree well with those obtained by genetic detection methods for the C. jejuni isolates tested. The findings of this study showed a very high level of resistance to ciprofloxacin and to a lesser extent to tetracycline while resistance to erythromycin remained at a low level. Thus, erythromycin may be considered as the first choice for treatment of Campylobacter infections in this geographical region when indicated.

ABSTRACT: Campylobacter spp. is a bacterial agent that causes gastroenteritis in humans and may trigger Guillain-Barré Syndrome (GBS) and is also considered one of the main foodborne diseases in developed countries. Poultry and pigs are considered reservoirs of these microorganisms, as well as raw or undercooked by-products are often incriminated as a source of human infection. Treatment in human cases is with macrolide, such erythromycin, that inhibits the protein synthesis of the microorganism. This study aimed to isolate Campylobacter jejuni and Campylobacter coli from intestinal content samples of broiler chickens (n=20) and swine (n=30) to characterize the erythromycin resistance profile of the strains and to detect molecular mechanisms involved in this resistance. The minimum inhibitory concentration was determined by agar dilution. The Mismatch Amplification Mutation Assay-Polymerase Chain Reaction (MAMA-PCR) was performed to detect mutations at positions 2074 and 2075 of 23S rRNA region, in addition to PCR test to detect the erm(B) gene. From the intestinal content of broiler chickens, 18 strains of C. jejuni and two strains of C. coli were isolated, whereas, from swine samples, no C. jejuni strain and 14 strains of C. coli were isolated. All C. coli strains were resistant, and three C. jejuni strains from broilers chickens were characterized with intermediate resistance to erythromycin. The MIC of the strains ranged from ≤0.5mg/μL to ≥128mg/μL. All resistant strains had the A2075G mutation, and one strain with intermediate resistance had the A2075G mutation. However, the A2074C mutation and the erm(B) gene were not detected. High resistance levels were detected in C. coli strains isolated from swine. The MAMA-PCR is a practical tool for detecting the erythromycin resistance in Campylobacter strains.