Mismatch Amplification Mutation Assay Research Articles

Genotyping of vitamin D receptor gene polymorphisms using mismatched amplification mutation assay in neonatal sepsis patients of Odisha, eastern India

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 Fok1, Bsm1, Apa1 and Taq1 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=12.16ng/ml, 95% CI: 3.84–22.22) and controls were vitamin D sufficient (Median=30.22ng/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 Apa1 polymorphism and neonatal sepsis or 25(OH)D serum levels. The distributions of the Fok1, Bsm1, and Taq1 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.

Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients.

To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes. One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed. Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ (2) = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ (2) = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively. HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.

Prevalence of bla TEM-220 gene in Penicillinase-producing Neisseria gonorrhoeae strains carrying Toronto/Rio plasmid in Argentina, 2002 – 2011

BackgroundPenicillinase-producing Neisseria gonorroheae (PPNG) was first isolated in 1976. PPNG strains carrying blaTEM-1 and blaTEM-135 gene have been described in different countries. Recently, a novel blaTEM-220 allele was detected in PPNG isolates carrying Toronto/Rio plasmid. The prevalence and characteristics of TEM-220 strains worldwide are unknown, and therefore, it needs to be studied. The purpose of this study was to detect blaTEM-220 gene in PPNG strains possessing Toronto/Rio plasmid over a period of ten years in Argentina, and to evaluate the proportion of isolates producing non-TEM-220 containing the T539C substitution in the blaTEM allele.MethodsOne hundred and fifty one PPNG isolates carrying Toronto/Rio plasmid were studied between 2002 and 2011. A mismatch amplification mutation assay (MAMA) PCR was used to identify the T539C substitution in the blaTEM allele and a MAMA-PCR protocol was developed to detect the G547A substitution in the blaTEM-220. The reference agar dilution method of the Clinical and Laboratory Standard Institute (CLSI) was used for susceptibility testing to five β-lactams antibiotics, ciprofloxacin, tetracycline and azithromycin. In all TEM-220-producing isolates, the whole blaTEM gene was sequenced and the isolates were typed using N. gonorroheae multiantigen sequence typing (NG-MAST).ResultsMAMA PCR successfully identified the G547A substitution in the blaTEM-220 allele. The proportion of isolates that possessed the blaTEM-220 allele was 2.6 %, and 93.2 % MAMA TEM-220 PCR-negative isolates showed the T539C substitution in the blaTEM gene. No differences in the susceptibility to five beta-lactam antibiotics tested were observed in PPNG isolates TEM-220-producing and PPNG isolates carrying the T539C substitution in the blaTEM gene. All TEM-220 isolates were indistinguishable by NG-MAST.ConclusionThis is the first study which shows the prevalence of blaTEM-220 in N. gonorrhoeae isolates carrying Toronto/Rio plasmid in Argentina. Although the blaTEM-220 allele does not appear to be associated with an extended spectrum beta-lactamase (ESBL) phenotype of resistance, a single nucleotide polymorphism added to the blaTEM-220 or blaTEM containing the T539C substitution could lead to the emergence of ESBL. Thus, it is imperative to investigate in surveillance programs, not only the plasmid type in PPNG isolates and the blaTEM allele associated, but phenotypical characteristics and geographical distribution of isolates.

Identification of the main quinolone resistance determinant in Campylobacter jejuni and Campylobacter coli by MAMA-DEG PCR

Among zoonotic diseases, campylobacteriosis stands out as the major bacterial infection producing human gastroenteritis. Antimicrobial therapy, only recommended in critical cases, is challenged by resistance mechanisms that should be unambiguously detected for achievement of effective treatments. Quinolone (ciprofloxacin) resistance of Campylobacter jejuni and Campylobacter coli, the 2 main Campylobacter detected in humans, is conferred by the mutation gyrA C-257-T, which can be genotyped by several methods that require a previous identification of the pathogen species to circumvent the sequence polymorphism of the gene. A multiplex PCR, based on degenerated oligonucleotides, has been designed for unambiguous identification of the quinolone resistance determinant in Campylobacter spp. isolates. The method was verified with 249 Campylobacter strains isolated from humans (141 isolates) and from the 3 most important animal sources for this zoonosis: poultry (34 isolates), swine (38 isolates), and cattle (36 isolates). High resistance to ciprofloxacin, MIC above 4μg/mL, linked to the mutated genotype predicted by MAMA-DEG PCR (mismatch amplification mutation assay PCR with degenerated primers) was found frequently among isolates from the different hosts.

Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

Trends in the genomic epidemiology of Vibrio cholerae O1 isolated worldwide since 1961

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.

Penicillinase-producing Neisseria gonorrhoeae and its blaTEM-135 gene variants at several gonococcal antimicrobial surveillance sites in China: an epidemiological study

Objective To determine the prevalence of penicillinase-producing Neisseria gonorrhoeae(PPNG) and the distribution of blaTEM-135 gene variants in PPNG at several gonococcal antimicrobial surveillance sites in China, to compare N. gonorrhoeae multi-antigen sequence typing (NG-MAST) types of PPNG and its blaTEM-135 gene variants, and to assess the difference and association in NG-MAST types of blaTEM-135 gene variants among different regions. Methods A total of 572 N. gonorrhoeae isolates were collected at 6 gonococcal antimicrobial surveillance sites from Jiangsu, Shanghai, Zhejiang, Tianjin, Guangdong and Guangxi in 2012. After isolation, purification, and identification, cefalotin paper discs were used for detection of PPNG. DNA was extracted by QIAxtractor DX kits after cultivation of the PPNG strains. Then, mismatch amplification mutation assay (MAMA) PCR was performed to identify blaTEM-135variants, and NG-MAST analysis to determine N. gonorrhoeae genotypes. Results Among the 572 N. gonorrhoeae strains, 38.1%(218/572) were identified as PPNG, and of the PPNG strains, 52.3%(114/218) were blaTEM-135 variants. The detection rate of PPNG at these surveillance sites from high to low was as follows: 51.7%(45/87, Zhejiang), 45.6%(36/79, Shanghai), 38.0%(78/205, Guangdong), 37.5%(12/32, Guangxi), 31.2%(24/77, Jiangsu) and 25.0%(23/92, Tianjin), and that of blaTEM-135 variants was as follows: 68.9%(31/45, Zhejiang), 58.3%(14/24, Jiangsu), 50.0%(39/78, Guangdong), 47.2%(17/36, Shanghai), 39.1%(9/23, Tianjin) and 33.3%(4/12, Guangxi). NG-MAST analysis showed that the ST2318, ST1768, ST1866, ST1053 and ST8726 types predominated among these blaTEM-135 variants, and a strong correlation was found between blaTEM-135 variants and some NG-MAST types, such as ST1768, ST1053 and ST8726 types. The distribution of NG-MAST types was significantly different between the surveillance site in Tianjin (in the Northern part of China) and the other sites (in the Southern part of China), but highly similar among the surveillance sites in Jiangsu, Zhejiang and Shanghai regions. Conclusions There is a high prevalence of PPNG and its blaTEM-135 variants at several gonococcal antimicrobial surveillance sites in China, with significant differences in NG-MAST genotype distribution of PPNG and its blaTEM-135 variants among different regions. Key words: Neisseria gonorrhoeae; beta-Lactamases; Epidemiology; blaTEM-135 Gene variant; Neisseria gonorrhoeae multi-antigen sequence typing

Detection of specific UL49 sequences of Marek's disease virus CVI988/Rispens strain using loop-mediated isothermal amplification

Marek's disease (MD) is a tumoral disease of chickens that can be controlled by vaccines based on non-pathogenic strains of turkey herpesvirus (HVT), SB-1 strain belonging to serotype 2, or the attenuated CVI988/Rispens strain belonging to serotype 1 of Marek's disease virus (MDV). Currently, the ‘gold standard’ in MD prophylaxis is the Rispens strain-based vaccine which protects against very virulent MDV and disease onset. Previous studies have shown that loop-mediated isothermal amplification (LAMP) is a rapid alternative to polymerase chain reaction (PCR) for detection and differentiation of HVT, SB-1 and virulent MDV strains. The aim of this study was to develop and evaluate a novel LAMP assay for the detection of the UL49 Rispens-specific region. This assay was validated using material from infected chicken embryo fibroblasts (CEFs) and tissue samples from vaccinated chickens. The analytical sensitivity of the assay was 10-times higher than PCR and reliably amplified 0.1 log10 TCID50/ml. The MDV Rispens was also detected at 18h after infection of CEFs. The results showed LAMP to be selective and a sensitive method to detect Rispens as early as 3 d.p.v. in all internal organs of chickens. Furthermore, the method was also capable to detect Rispens in 5 out of 26 chickens originating from different flocks. A mismatch amplification mutation assay (MAMA-PCR) confirmed the presence of Rispens strain in all LAMP-positive chickens. This is the first report of the specific visual detection of Rispens in vitro and in vivo using LAMP. The method may be useful for monitoring of successful chicken vaccination as well as in vitro studies in infected cell cultures.

Evaluation of IFN-γ polymorphism +874 T/A in patients with recurrent tonsillitis by PCR Real Time Mismatch Amplification Mutation Assay (MAMA Real Time PCR)

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.

Phenotypic and genotypic characteristics of Vibrio cholerae O1 isolated from the Sierra Leone cholera outbreak in 2012.

This study describes phenotypic, genotypic and antibiotic susceptibility patterns of the strains isolated from the 2012 Sierra Leone cholera outbreak. Rectal swabs were collected from patients and cultured for Vibrio cholerae O1. The isolates were subjected to multiplex PCR, mismatch amplification mutation assay (MAMA) PCR, pulsed field gel electrophoresis (PFGE), and antibiotic sensitivity tests using disk diffusion and minimum inhibitory concentration (MIC) E-test following standard procedures. Out of 17 rectal swabs tested, 15 yielded V. cholerae O1 biotype El Tor, serotype Ogawa. All the strains belonged to 'altered' variants as MAMA PCR result showed the presence of classical cholera toxin B. PFGE result revealed four pulse types. Using antibiotic disk diffusion, all the isolates were resistant to erythromycin, chloramphenicol, furazolidone, and trimethoprim/sulfamethoxazole (SXT) except SL1 which was sensitive to chloramphenicol and SXT. All the isolates were sensitive to nalidixic acid, tetracycline, doxycycline, azithromycin, and ciprofloxacin except SL2 which was resistant to nalidixic acid. However, variable sensitivity patterns were observed for kanamycin. The ranges of MIC were 0.125-0.50 mg/l, 0.003-0.023 mg/l and 0.38-0.75 mg/l for azithromycin, ciprofloxacin and tetracycline, respectively. This study demonstrates that altered variants of V. cholerae O1 of four clonal types were responsible for the 2012 outbreak of cholera in Sierra Leone.

Detection and differentiation of CVI988 (Rispens vaccine) from other serotype 1 Marek's disease viruses.

The serotype 1 Marek's disease virus (MDV) is the causative agent for Marek's disease (MD), a lymphoproliferative disease of chickens of great concern to the poultry industry. CVI988 (Rispens vaccine), an attenuated serotype 1 MDV, is currently the most efficacious commercially available vaccine for preventing MD. However, it is difficult to detect and differentiate CVI988 when other serotype 1 MDVs are present. To facilitate the detection of CVI988, we developed two sets of primers for a mismatch amplification mutation assay (MAMA) PCR that targeted the single nulceotide polymorphism associated with the H19 epitope of the phosphorylated protein 38 gene. The PCR was very specific. One primer set (oncogenic primers) amplified DNA from 15 different serotype 1 MDVs except CVI988. The other primer set (CVI988 primers) amplified DNA from CVI988 but not from any of the other 15 serotype 1 MDVs. A real-time PCR assay was developed using MAMA primers, and specificity and sensitivity was evaluated in vitro and in vivo. Mixtures of plasmids (CVI988 plasmid and oncogenic plasmid) at various concentrations were used to evaluate the sensitivity/specificity of MAMA primers in vitro. Both primer setswere able to amplify as little as one copy of their respective plasmid. Oncogenic primers were highly specific and only amplified CVI988 plasmid when the concentration of oncogenic plasmid was very low (1 X 10(1)) and CVI988 plasmid was very high (1 X 10(6)). Specificity of CVI988 primers was not as high because they could amplify oncogenic plasmids when the concentration of CVI988 plasmid was 1 x 10(3) and the concentration of oncogenic 1 x 10(2). Validation of MAMA primers in in vivo samples demonstrated that oncogenic primers can be used for both early diagnosis of MD in feather pulp (FP) samples collected at 3 wk of age and confirmation of MD diagnosis in tumors. CVI988 primers could be used to monitor CVI988 vaccination in samples with a low load of oncogenic MDV DNA (latently infected samples or negative) but not in samples with a high load of oncogenic MDV DNA (tumors). Our results suggest that monitoring CVI988 vaccination in FP samples collected at 1 wk of age ensures the specificity of the CVI988 primers.

Association between Motilin Receptor Gene Haplotypes and Growth Traits in Japanese Hinai-dori Crossbred Chickens.

We previously identified quantitative trait loci (QTL) for body weight and average daily gain in a common region between ADL0198 (chr 1: 171.7 Mb) and ABR0287 (chr 1: 173.4 Mb) on chicken chromosome 1 in an F2 resource population produced by crossing low- and high-growth lines of the Hinai-dori breed. Motilin receptor (MLNR) is a candidate gene affecting growth traits in the region. In this study, we genotyped polymorphisms of the MLNR gene and investigated its association with growth traits in a Hinai-dori F2 intercross population. All the exons of the MLNR gene in the parental population were subjected to PCR amplification, nucleotide sequenced and haplotypes identified. To distinguish resultant diplotype individuals in the F2 population, a mismatch amplification mutation assay was performed. Three haplotypes (Haplotypes 1–3) were accordingly identified. Six genotypes produced by the combination of three haplotypes (Haplotype 1, 2, and 3) were examined in order to identify associations between MLNR haplotypes and growth traits. The data showed that Haplotype 1 was superior to Haplotype 2 and 3 in body weight at 10 and 14 weeks of age, average daily gain between 4 and 10 weeks, 10 and 14 weeks, and 0 and 14 weeks of age in female in F2 females. It was concluded that MLNR is a useful marker of growth traits and could be used to develop strategies for improving growth traits in the Hinai-dori breed.

The evidence for clonal spreading of quinolone resistance with a particular clonal complex ofCampylobacter jejuni

Campylobacter is the most prevalent cause of bacterial gastroenteritis worldwide and it represents a significant public health risk of increasing severity due to its escalating resistance to clinically important quinolone and macrolide antibiotics. As a zoonotic pathogen Campylobacter is transmitted along the food chain and naturally cycles from environmental waters, feedstuff, animals and food to humans. We determined antibiotic resistance profiles, as well as multilocus sequence types and flaA-SVR types for 52 C. jejuni isolated in Slovenia from human, animal, raw and cured chicken meat and water samples. Twenty-eight different sequence types, arranged in ten clonal complexes, three new allele types and five new sequence types were identified, indicating the relatively high diversity in a small group of strains. The assignment of strains from different sources to the same clonal complexes indicates their transmission along the food supply chain. The most prevalent clonal complex was CC21, which was also the genetic group with 95% of quinolone-resistant strains. Based on the genetic relatedness of these quinolone-resistant strains identified by polymerase chain reaction with a mismatch amplification mutation assay and sequencing of the quinolone resistance-determining region of the gyrA gene, we conclude that the high resistance prevalence observed indicates the local clonal spread of quinolone resistance with CC21.

Real-time PCR assays for genotyping of Cryptococcus gattii in North America.

BackgroundCryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen.MethodsWe developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human.ResultsValidation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA.ConclusionsThe (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.

Outbreak of cholera caused by Vibrio cholerae O1 El Tor variant strain in Bihar, India.

An outbreak of cholera struck Bihar, an Indian state, in August 2008 following a massive flood. Here we report the phenotypic and genotypic characteristics of Vibrio cholerae strains isolated from patients with diarrhea. Rectal swabs were obtained from patients with diarrhea who were admitted to medical camps or the hospital, and the strains were biochemically and serologically characterized. V. cholerae was isolated from 21 (65.6%) of 32 rectal swabs. Serological studies revealed that all the 21 isolates belonged to V. cholerae O1 Ogawa. Mismatch amplification mutation assay (MAMA)-PCR showed that the isolates belonged to El Tor variant group, and pulsed-field gel electrophoresis (PFGE) proved that these isolates were of a different lineage than the conventional El Tor variant strains. These isolates were resistant to several drugs, including ampicillin, streptomycin, tetracycline, nalidixic acid, and furazolidone. The uniqueness of the current report arises from the fact that records of cholera in Bihar are availiable for the early 1960s but not for the next 4 decades. Moreover, the present study is the first to report a cholera outbreak in Bihar that was caused by an El Tor variant strain.

Molecular analysis of ciprofloxacin resistance mechanisms in Malaysian ESBL-producing Klebsiella pneumoniae isolates and development of mismatch amplification mutation assays (MAMA) for rapid detection of gyrA and parC mutations.

Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6′)-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4– ≥ 32 μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6′)-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6′)-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.

Molecular Characterization and Detection of Mutations Associated with Resistance to Succinate Dehydrogenase-Inhibiting Fungicides in Alternaria solani

Early blight, caused by Alternaria solani, is an economically important foliar disease of potato in several production areas of the United States. Few potato cultivars possess resistance to early blight; therefore, the application of fungicides is the primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase-inhibiting (SDHI) fungicide boscalid in this plant pathogen with a concomitant loss of disease control. Two phenotypes were detected, one in which A. solani isolates were moderately resistant to boscalid, the other in which isolates were highly resistant to the fungicide. Resistance in other fungal plant pathogens to SDHI fungicides is known to occur due to amino acid exchanges in the soluble subunit succinate dehydrogenase B (SdhB), C (SdhC), and D (SdhD) proteins. In this study, the AsSdhB, AsSdhC, and AsSdhD genes were analyzed and compared in sensitive (50% effective concentration [EC50] < 5 μg ml(-1)), moderately resistant (EC50 = 5.1 to 20 μg ml(-1)), highly resistant (EC50 = 20.1 to 100 μg ml(-1)), and very highly resistant (EC50 > 100 μg ml(-1)) A. solani isolates. In total, five mutations were detected, two in each of the AsSdhB and AsSdhD genes and one in the AsSdhC gene. The sequencing of AsSdhB elucidated point mutations cytosine (C) to thymine (T) at nucleotide 990 and adenine (A) to guanine (G) at nucleotide 991, leading to an exchange from histidine to tyrosine (H278Y) or arginine (H278R), respectively, at codon 278. The H278R exchange was detected in 4 of 10 A. solani isolates moderately resistant to boscalid, exhibiting EC50 values of 6 to 8 μg ml(-1). Further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values for boscalid of 28 to 500 μg ml(-1). Subsequent sequencing of AsSdhC and AsSdhD genes confirmed the presence of additional mutations from A to G at nucleotide position 490 in AsSdhC and at nucleotide position 398 in the AsSdhD, conferring H134R and H133R exchanges in AsSdhC and AsSdhD, respectively. The H134R exchange in AsSdhC was observed in A. solani isolates with sensitive, moderate, highly resistant, and very highly resistant boscalid phenotypes, and the AsSdhD H133R exchange was observed in isolates with both moderate and very high EC50 value boscalid phenotypes. Detection and differentiation of point mutations in AsSdhB resulting in H278R and H278Y exchanges in the AsSdhB subunit were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations in boscalid-resistant isolates, in addition to mutations in AsSdhC and AsSdhD resulting in an H134R and H133R exchange, respectively, was achieved by the development of a multiplex polymerase chain reaction to detect and differentiate the sensitive and resistant isolates based on the single-nucleotide polymorphisms present in all three genes. A single A. solani isolate with resistance to boscalid did not contain any of the above-mentioned exchanges but did contain a substitution of aspartate to glutamic acid at amino acid position 123 (D123E) in the AsSdhD subunit. Among A. solani isolates possessing resistance to boscalid, point mutations in AsSdhB were more frequently detected than mutations in genes coding for any other subunit.

Prevalence and Molecular Epidemiological Typing of Penicillinase-Producing Neisseria gonorrhoeae and Their blaTEM-135 Gene Variants in Nanjing, China

This study aimed to investigate the prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) and their blaTEM-135 gene variant in 2007 and 2012 in Nanjing, China. In addition, molecular epidemiological typing of all isolates was performed to elucidate the genetic relationships of the PPNG strains. A total of 199 and 77 N. gonorrhoeae isolates were collected at the National Center for STD Control in 2007 and 2012, respectively. Nitrocefin tests were performed to identify PPNG. Mismatch amplification mutation assay was used to identify blaTEM-135. All isolates were genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST), and additionally, porB-based phylogenetic analysis was performed for the PPNG isolates. The total prevalence of PPNG isolates was 41% (114/276) and 58% (66/114) of these PPNG isolates possessed bla(TEM-135). In 2007, 45% (90/199) produced β-lactamase, and of those PPNG, 58% (52/90) possessed bla(TEM-135). In 2012, 31% (24/77) were PPNG, and 58% (14/24) of those isolates contained bla(TEM-135). There were 162 NG-MAST STs among the 276 isolates, and 89 of those were novel STs. A strong association between specific NG-MAST STs and bla(TEM-135) was found, and the porB-based phylogenetic analysis showed a distant evolutionary relationship between isolates in 2007 and isolates in 2012. A high prevalence of PPNG and blaTEM-135 was found in Nanjing, China. bla(TEM-135) might be a precursor in the evolution into an extended-spectrum β-lactamase that can degrade ceftriaxone, which stresses the need to continuously monitor PPNG, blaTEM-135, and additional evolving blaTEM gene variants.

Vibrio choleraeO1 El Tor and O139 Bengal Strains CarryingctxBET, Bangladesh

<i>Vibrio cholerae</i>O1 El Tor and O139 Bengal Strains Carrying<i>ctxB</i>^ET, Bangladesh

Molecular Characterization of Vibrio cholerae O1 Reveals Continuous Evolution of Its New Variants in India.

Vibrio cholerae, the causing agent of cholera is still a major health challenge in most of the developing countries. In this study, V. cholerae strains collected from different cholera outbreaks in India over a period of past 7years were found to have various toxigenic, pathogenic and regulatory genes viz. ctxAB, zot, tcp, hlyA, ace, ompU, ompW, rfbO1, toxT and toxR. The biotype specific genes rstR and rtxC revealed the El Tor biotype in majority of the isolates. However, variants among the isolates were found having genotype of both the biotypes. Sequencing of ctxB gene revealed the presence of altered ctxB of classical biotype with additional variations in isolates of 2007. Mismatch amplification mutation assay PCR also confirmed the isolates belonging to classical biotype. Antibiogram of the isolates revealed resistance for nalidixic acid, co-trimoxazole, streptomycin, and polymyxin B and susceptibility for tetracycline among most of the isolates from India. However, V. cholerae isolates from a recent outbreak in Eastern India were resistant to tetracycline. The study corroborated the continuous emergence and wide-spread of multidrug resistant El Tor variant strains in the Indian subcontinent.