sigA Genes Research Articles

Evaluate the distribution of virulence genes and to investigate antibiotic resistance pattern among Shigella species isolated from children with shigellosis in Iran

The present study was performed to evaluate the frequency of virulence genes, including ipaH, ial, set1A, set1B, sen, virF, invE, sat, sigA, pic and sepA and assess antibiotic resistance pattern among Shigella isolates acquired from pediatric patients in Shiraz, Iran. Three hundred and nine isolates were isolated, from October 2018 to March 2019 from the feces of children with diarrhea; we used standard microbiological and serological tests for Shigella species. Overall, 73 (23.6%) isolates were positive for Shigella spp. The highest rate species of Shigella was S. sonnei (61.64%), followed by S. flexneri (37%) and S. boydii (1.36%). All the isolates were positive for ipaH gene, followed by ial (95.8%), virF (85%), InvE (75.3%), sen (28.7%), set1A (10.9%) and set1B (10.9%). The presence of sigA, sepA, sat and pic genes as SPATE genes was confirmed among 80.8%, 32.8%, 24.6% and 6.8% of Shigella isolates, respectively. Our investigations also indicated a high resistance percentage against trimethoprim-sulfamethoxazole and ampicillin in Shigella isolates. Our results displayed that S. sonnei was the predominant serogroup detected from children with shigellosis in Shiraz, Iran. As demonstrated in this study, most of the isolates showed a high prevalence rate of drug-resistance. S. flexneri carried a higher number of virulence genes. Our results indicated that certain symptoms were found to be related to the presence of specific different virulence factors. In addition, this study provided information that can increase our knowledge of the contribution of virulence genes in disease severity.

Dietary supplement of essential oil from oregano affects growth performance, nutrient utilization, intestinal morphology and antioxidant ability in Pekin ducks.

A study was conducted to investigate the effects of oregano essential oil (EO) on growth performance, nutrients utilization, intestinal morphology, intestinal barrier-related gene expression and antioxidant capability in meat ducks. A total of 360 1-day-old ducks were divided into three groups (12 replicates pens per diet of 10 ducks in each pen): negative control (no essential oil or antibiotic), positive control (antibiotic: 500mg/kg aureomycin of diet) and oregano EO (100mg/kg of diet). The experiment was carried out for 35days. Ducks were given feed and water ad libitum. Ducks fed EO supplement showed similar body weight and feed to gain ratio to antibiotic fed ducks. EO supplementation significantly increased (p<.05) feed intake (day 1-35), jejunal villus height (VH) to crypt depth (CD) ratio, serum superoxide dismutase activities (SOD) and jejunal total antioxidant capacity (T-AOC) of ducks compared to controls. Ducks fed diets supplemented with oregano EO also had decreased (p<.05) jejunal CD, serum and hepatic malondialdehyde (MDA) concentration, and the mRNA expression of jejunal zonula occludens-3 (ZO-3) and secretory immunoglobulin A (sIgA) genes in comparison to the control group. Compared to the antibiotic supplementation group, the mRNA expression of claudin1 (CLND1) and CLND2 significantly increased (p<.05), but the mRNA expression of ZO-3 and mucin 2 markedly decreased (p<.05) in the jejunum of ducks in oregano EO supplementation group. These results suggest that oregano EO improves the antioxidant capacity and intestinal defence and structural measures and may aide in helping to maintain enteric health in production without growth-promoting antibiotics.

Distribution of Genes Encoding Virulence Factors and the Genetic Diversity of Enteroinvasive Escherichia coli (EIEC) Isolates from Patients with Diarrhea in Ahvaz, Iran.

BackgroundEntero-invasive E. coli (EIEC) is one of the causes of bacillary dysentery in adults and children. The ability of EIEC to invade and colonize the surface of epithelial cells is influenced by many virulence factors. This study aimed to investigate the distribution of virulence factor genes in EIEC strains isolated from patients with diarrhea in Ahvaz, Iran, as well as the genetic diversity between these isolates by Multilocus variable-number tandem repeat analysis (MLVA).Materials and MethodsA total of 581 diarrheic stool samples were collected from patients with diarrhea attending two hospitals, in Ahvaz, Iran. The E. coli strains were identified by biochemical methods. Subsequently, all E. coli isolates were identified as EIEC by polymerase chain reaction (PCR) for the ipaH gene. The EIEC isolates evaluated by PCR for the presence of 8 virulence genes (ial, sen, virF, invE, sat, sigA, pic, and sepA). All EIEC strains were genotyped by the MLVA typing method.ResultsA total of 13 EIEC isolates were identified. The presence of ial, virF, invE, sen, sigA, pic, and sat genes was confirmed among 92.3%, 84.6%, 84.6%, 76.9%, 69.2%, and 15.3% of EIEC isolates, respectively. On the other hand, none of the isolates were positive for the sepA gene. The EIEC isolates were divided into 11 MLVA types.ConclusionOur results showed a high distribution of virulence genes among EIEC isolates in our region. This study showed that MLVA is a promising typing technique for epidemiological studies. MLVA can supply data in the form of codes that can be saved in the database and easily shared among laboratories, research institutes, and even hospitals.

First investigation of the presence of SPATE genes in Shigella species isolated from children with diarrhea infection in Ahvaz, southwest Iran.

Background: SPATE (serine protease autotransporters of enterobacteriaceae) genes are considered as a group of the main virulence factors of Shigella species This study aimed to investigate for the first time the distribution of SPATE genes among Shigella spp. isolated from children with diarrhea infection in Ahvaz, Iran.Methodology: In this study, a total of 74 Shigella isolates were collected between August 2016 and June 2017 from feces of children with diarrhea and identified by biochemical and molecular methods for Shigella species. The frequency distribution of the SPATE genes, including pic, pet, sat, sigA and sepA, was evaluated using PCR. The genetic relationship of all isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR.Results: The most common species of Shigella was S. flexneri, followed by S. sonnei and S. boydii. In total, 95.94% of Shigella isolates had at least one of the SPATE genes. The presence of pic, pet, sat, sigA and sepA genes was confirmed among 35.13%, 27%, 47.29%, 58.1% and 39.18% of Shigella isolates, respectively. Of these SPATE genes, the sat and sigA genes were recognized as the most common autotransporters among S. flexneri and S. sonnei isolates, respectively. Also, either S. flexneri or S. sonnei isolates belonging to a same clone type had similar SPATE genes profile.Conclusion: Our results revealed that the high distribution of SPATE genes among Shigella isolates in our region. Hence, this study highlights a need for epidemiological programs to monitor the distribution of SPATE genes locally for prevention from further dissemination of the Shigella isolates harboring them.

Characterization of a serologically atypical Shigella flexneri Z isolated from diarrheal patients in Bangladesh and a proposed serological scheme for Shigella flexneri.

BackgroundAtypical Shigella flexneri Z variant, that agglutinate with E1037 group factor specific monoclonal antisera against Shigella flexneri IV-I but not with other group or type specific antisera, has continuously being isolated in Bangladesh since 1997. Later this serotype has been reported in Indonesia, China and Argentina. Despite being a provisional serotype, continuous isolation of these strains in diverse geographical regions implicated a great necessity to study the overall characteristics of these strains. Therefore, we extensively characterized S. flexneri Z strains using various phenotypic and molecular tools.MethodOf 3569 S. flexneri isolated between 1997 and 2015, 95 strains were identified as S. flexneri Z using a panel of polyvalent absorbed antisera and monoclonal antisera of S. flexneri (MASF). Of them, randomly selected 65 strains were molecular O-serotyped using multiplex PCR and characterized using different phenotypic and molecular techniques (i.e.biotyping, plasmid profile, virulence marker and PFGE) to determine relationship with other subserotypes of S. flexneri.ResultsAll these atypical S. flexneri Z strains were agglutinated with MASF B and IV-I antisera. Concordantly, these strains were positive to opt-gene, responsible for MASF IV-I sero-positive phenotype. However, molecular O-serotyping of all 65 strains could not differentiate between Z and Yb giving similar amplification products (wzx1-5 and opt). Contrarily, MASF based serotypic scheme distinguished among Z and Yb as well as Ya. All these S. flexneri Z showed typical biochemical reaction of S. flexneri, harboured a 140 MDa virulence plasmid and virulence markers namely ipaH, ial, sen, sigA and sepA genes. Along with the virulence plasmid, small plasmids (2.6, 1.8 and 1.6 MDa) were present as core plasmid. Moreover, a middle ranged plasmid and a 4.0 MDa sized plasmid were observed in 65% and 20% strains, respectively. Analysis of PFGE on XbaI-digested chromosomal DNA of Bangladeshi strains showed that S. flexneri Z had a close relatedness with Ya and Yb but completely different than the strains of Xa, Xb, 2a and 2b. This observation was found to be unequivocal while the overall result of biotyping, plasmid profile, and virulence factors was compared. Therefore, we conclude that these atypical serotype Z isolated in Bangladesh had a clonal relationship with Ya and Yb of Bangladesh and the opt gene played an important role in serotypic switching among them. Current serotyping scheme of S. flexneri strains fails to place many such atypical strains (1c, 1c+6, 1d, type 4, and 4c) including S. flexneri Z isolated from different parts of the world. Therefore, an updated serotyping scheme for identification of subserotypes of S. flexneri has been proposed to avoid multiple naming of the same subserotype having similar agglutination pattern.

Fitness-compensatory mutations facilitate the spread of drug-resistant F15/LAM4/KZN and F28 Mycobacterium tuberculosis strains in KwaZulu-Natal, South Africa.

While the acquisition of drug resistance is often accompanied by fitness costs, Mycobacterium tuberculosis has developed mechanisms to overcome these costs in the form of compensatory mutations. In an attempt to dissect strain-specific differences in biological fitness, 10 M. tuberculosis genomes, representing F15/LAM4/KZN, Beijing, F11 and F28 genotypes were sequenced on the Illumina MiSeq platform. Drug-susceptible F15/LAM4/KZN strains differed by 43 SNPs, demonstrating that heterogeneity exists even among closely-related strains. We found unique, nonsynonymous single-nucleotide polymorphisms (SNPs) in the sigA and grcC1 genes of multidrug resistant (MDR) and XDR F15/LAM4/KZN strains, respectively. The F28 MDR strain harboured a novel ubiA mutation in combination with its embB M306I mutation, which may be related to ethambutol resistance. In addition, it possessed a low-frequency rpoC mutation, suggesting that this strain was in the process of developing compensation. In contrast, no compensatory mutations were identified in Beijing and F11 MDR strains, corroborating its low in vitro fitness. Clinical strains also harboured unique SNPs in a number of important genes associated with virulence, highlighting the need for future studies which examine the correlation of genetic variations with phenotypic diversity. In summary, whole-genome sequencing revealed the presence of fitness-compensatory mutations in F15/LAM4/KZN and F28 genotypes which predominate in MDR and/or extensively drug resistant (XDR) forms in KwaZulu-Natal, South Africa.

Molecular and phenotypic characterization of strains of Shigella sonnei isolated over 31 years suggests the circulation of two prevalent subtypes in São Paulo State, Brazil.

Shigella sonnei is an important causative agent of bacillary dysentery worldwide that has recently emerged in developing countries. However, there are few studies that have characterized strains ofS. sonnei isolated in Brazil. The aims of this study were to assess the presence of 12 virulence genes, the antimicrobial resistance profile against 16 drugs and the genotypic diversity of strains of S. sonnei isolated in this country. Seventy-two strains of S. sonnei isolated from human diarrhoeic faeces in São Paulo State, Brazil from 1983-2014 were studied. All of the strains contained the ipaH, iuc and sigA genes. The ipaBCD gene was detected in 19 % of the strains, the ial and virF genes in 18 % and the sen gene in 10 % of the strains. The set1A, set1B, pic,sepA and sat genes were not detected. A total of 42 (58.3 %) strains were resistant to trimethoprim-sulfamethoxazole. Thirty (41.6 %) strains were resistant to tetracycline. The S. sonnei strains were grouped in two clusters called A and B by PFGE and ERIC-PCR, and the majority of the strains comprised in each cluster presented ≥80 % similarity. In conclusion, the pathogenic potential of the strains studied was highlighted by the presence of important virulence genes. The high rates of resistance to trimethoprim-sulfamethoxazole and tetracycline are alarming once those drugs can be used in the treatment of shigellosis. The PFGE and ERIC-PCR results suggest that there are two prevalent subtypes in the studied strains of S. sonnei that differed little over 31 years and have been contaminating humans and causing diseases in São Paulo State, Brazil.

Molecular characterization and phylogeny of Shiga toxin–producing Escherichia coli isolates obtained from two Dutch regions using whole genome sequencing

Shiga toxin–producing Escherichia coli (STEC) is one of the major causes of human gastrointestinal disease and has been implicated in sporadic cases and outbreaks of diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome worldwide. In this study, we determined the molecular characteristics and phylogenetic relationship of STEC isolates, and their genetic diversity was compared to that of other E. coli populations. Whole genome sequencing was performed on 132 clinical STEC isolates obtained from the faeces of 129 Dutch patients with gastrointestinal complaints. STEC isolates of this study belonged to 44 different sequence types (STs), 42 serogenotypes and 14 stx subtype combinations. Antibiotic resistance genes were more frequently present in stx1-positive isolates compared to stx2 and stx1 + stx2–positive isolates. The iha, mchB, mchC, mchF, subA, ireA, senB, saa and sigA genes were significantly more frequently present in eae-negative than in eae-positive STEC isolates. Presence of virulence genes encoding type III secretion proteins and adhesins was associated with isolates obtained from patients with bloody diarrhoea. Core genome phylogenetic analysis showed that isolates clustered according to their ST or serogenotypes irrespective of stx subtypes. Isolates obtained from patients with bloody diarrhoea were from diverse phylogenetic backgrounds. Some STEC isolates shared common ancestors with non-STEC isolates. Whole genome sequencing is a powerful tool for clinical microbiology, allowing high-resolution molecular typing, population structure analysis and detailed molecular characterization of strains. STEC isolates of a substantial genetic diversity and of distinct phylogenetic groups were observed in this study.

Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

Transcriptional analysis of the sigA and sigB genes of Brevibacterium lactofermentum.

Transcription of the sigA and sigB genes of Brevibacterium lactofermentum, encoding the principal sigma factors of RNA polymerase, has been investigated by Northern blot and primer extension analysis. Northern hybridizations revealed that sigA is transcribed as a monocistronic mRNA of 1.7 kb and sigB gives two transcripts of 1.1 and 1.5 kb. Similar transcription patterns of sigA and sigB genes in nutrient-rich medium were observed; transcripts of both genes occurred simultaneously throughout the exponential growth phase and decayed clearly when the stationary phase was reached. Primer extension analysis of B. lactofermentum RNA showed that the sigA and sigB transcription initiation sites are located 17 bp and 24 bp upstream from the first nucleotide of the respective translation initiation codons. Alignment of the sigA and sigB promoter regions provided evidence for highly conserved sequences in both of them.

Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB, from Brevibacterium flavum.

Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomyces aureofaciens, we identified two sigma70-like genes in a library of Brevibacterium flavum. Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum. We designated them similarly sigA and sigB. Transcription of B. flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions. Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively. Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock. Expression of both genes is significantly reduced after heat shock. Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.

Transcriptional analysis of the sigA and sigB genes of Brevibacterium lactofermentum

Transcription of the sigA and sigB genes of Brevibacterium lactofermentum, encoding the principal sigma factors of RNA polymerase, has been investigated by Northern blot and primer extension analysis. Northern hybridizations revealed that sigA is transcribed as a monocistronic mRNA of 1.7 kb and sigB gives two transcripts of 1.1 and 1.5 kb. Similar transcription patterns of sigA and sigB genes in nutrient-rich medium were observed; transcripts of both genes occurred simultaneously throughout the exponential growth phase and decayed clearly when the stationary phase was reached. Primer extension analysis of B. lactofermentum RNA showed that the sigA and sigB transcription initiation sites are located 17 bp and 24 bp upstream from the first nucleotide of the respective translation initiation codons. Alignment of the sigA and sigB promoter regions provided evidence for highly conserved sequences in both of them.

Cloning, sequencing, and transcriptional analysis of the gene coding for the vegetative sigma factor of Agrobacterium tumefaciens

The sigA gene of Agrobacterium tumefaciens was cloned and sequenced. Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors. However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (sigma 32) promoters. It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region of the groE operon in A. tumefaciens. The transcription initiation site of the gene was determined and found to be at the same position during normal growth and under heat shock conditions. Furthermore, no heat shock activation was observed at the transcriptional level.

Localization of a second SigH promoter in the Bacillus subtilis sigA operon and regulation of dnaE expression by the promoter

The presence of a second SigH promoter in the sigA operon of Bacillus subtilis was demonstrated by use of a promoter probe plasmid, a sigH deletion mutant, primer extension studies, and in vitro transcription with E sigma H holoenzyme. Both SigH promoters were expressed at low levels even during the growth phase but were expressed at higher levels during the early stationary phase. Expression from the upstream SigH promoter allowed the expression of both dnaE and sigA genes; however, expression from the downstream SigH promoter, which was located in the ribosome-binding site of the dnaE gene, resulted only in the expression of the sigA gene, since the truncated dnaE ribosome-binding site could not be used for initiating translation. Thus, promoter switching during the early stationary phase resulted not only in expression from SigH promoters but also in differential expression of the genes in the sigA operon.