Repetitive Extragenic Palindromic Sequences Research Articles

Sodium Fluoride Exposure Leads to ATP Depletion and Altered RNA Decay in Escherichia coli under Anaerobic Conditions.

Although fluoride-containing compounds are widely used to inhibit bacterial growth, the reprogramming of gene expression underlying cellular responses to fluoride, especially under anaerobic conditions, is still poorly understood. Here, we compare the genome-wide transcriptomic profiles of E. coli grown in the absence (control) or presence (20 and 70 mM) of sodium fluoride (NaF) under anaerobic conditions and assess the impact of fluoride-dependent ATP depletion on RNA turnover. Tiling array analysis revealed transcripts displaying altered abundance in response to NaF treatments. Quantile-based K-means clustering uncovered a subset of genes that were highly upregulated and then downregulated in response to increased and subsequently decreased fluoride concentrations, many of which (~40%) contained repetitive extragenic palindromic (REP) sequences. Northern blot analysis of some of these highly upregulated REP-containing transcripts (i.e., osmC, proP, efeO and yghA) confirmed their considerably enhanced abundance in response to NaF treatment. An mRNA stability analysis of osmC and yghA transcripts demonstrated that fluoride treatment slows down RNA degradation, thereby enhancing RNA stability and steady-state mRNA levels. Moreover, we demonstrate that turnover of these transcripts depends on RNase E activity and RNA degradosome. Thus, we show that NaF exerts significant effects at the whole-transcriptome level under hypoxic growth (i.e., mimicking the host environment), and fluoride can impact gene expression posttranscriptionally by slowing down ATP-dependent degradation of structured RNAs. IMPORTANCE Gram-negative Escherichia coli is a rod-shaped facultative anaerobic bacterium commonly found in microaerobic/anaerobic environments, including the dental plaques of warm-blooded organisms. These latter can be treated efficiently with fluoride-rich compounds that act as anticaries agents to prevent tooth decay. Although fluoride inhibits microbial growth by affecting metabolic pathways, the molecular mechanisms underlying its activity under anaerobic conditions remain poorly defined. Here, using genome-wide transcriptomics, we explore the impact of fluoride treatments on E. coli gene expression under anaerobic conditions. We reveal key gene clusters associated with cellular responses to fluoride and define its ATP-dependent stabilizing effects on transcripts containing repetitive extragenic palindromic sequences. We demonstrate the mechanisms controlling the RNA stability of these REP-containing mRNAs. Thus, fluoride can affect gene expression posttranscriptionally by stabilizing structured RNAs.

First Report of Bacterial Blight on arugula (Eruca vesicaria subsp. sativa) caused by Pseudomonas cannabina pv. alisalensis in New York.

Bacterial blight of arugula (Eruca vesicaria subsp. sativa cv. Standard) was observed in a commercial crop being grown in a high tunnel under overhead irrigation in Argyle, NY in January 2021. Approximately 80-100% of the plants were affected. Symptoms started as small, angular, water-soaked lesions visible on both sides of the leaves, then expanded, coalesced and later dried and turned tan. Fluorescent pseudomonads from five different plants were isolated on King's medium B agar amended with boric acid, chloramphenicol, and cycloheximide (Schaad et al. 2001) from surface disinfested symptomatic leaf tissues macerated in phosphate buffer (10mM, pH 7.0). Five representative isolates from each of the five plants produced levan and were negative for arginine dihydrolase and oxidase. They did not rot potatoes but were able to induce a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv Glurk) within 24 h, thus demonstrating that the isolates belonged to LOPAT group 1 (Lelliott et al. 1966). DNA fragment banding patterns of these isolates generated by repetitive extragenic palindromic sequence PCR (repPCR) with BOXA1R primers were compared to the pathotype strains of Pseudomonas cannabina pv. alisalensis and Pseudomonas syringae pv. maculicola, known fluorescent pathogens of the Brassicaceae. The repPCR banding pattern of all isolates matched the pattern of Pseudomonas cannabina pv. alisalensis. Bacterial inoculum for pathogenicity experiments was prepared from 48 h KBBC agar cultures suspended in phosphate buffer (10mM, pH 7.0) and adjusted to 0.6 optical density at 600nm yielding approximately 108 CFU/ml. Five-week-old arugula plants were sprayed until run-off with one of the three isolates from arugula, a negative control (sterile buffer), or a positive control (P. cannabina pv. alisalensis) that is pathogenic to crucifers and also reported to cause disease on arugula in California (Bull et al. 2004). Experiments consisted of three replications of each treatment and two independent experiments were conducted. Small water-soaked spots resembling the original symptoms developed on all plants inoculated with the three representative isolates and P. cannabina pv. alisalensis. Moreover, reisolates from the symptomatic tissues were fluorescent on KBBC and had identical repPCR banding pattern as inoculated strains, demonstrating Koch's postulates. To our knowledge, this is the first report of bacterial blight on arugula caused by Pseudomonas cannabina pv. alisalensis in the Northeastern US. It was previously reported in California, Nevada, and Minnesota (Bull and du Toit 2009; Bull et al. 2004). This report may have significance for all brassica leafy green growers in the Northeast as P. cannabina pv. alisalensis has a broad host range including members of the Brassicaceae and oats which are commonly used as cover crops in mixed vegetable production.

Repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence‐based typing of Shiga toxin‐producing Escherichia coli from bovine samples

AbstractThe strain typing of Shiga toxin‐producing Escherichia coli (STEC) is challenging due to its high genetic variability. In the present study, two polymerase chain reaction (PCR)‐based typing methods—repetitive extragenic palindromic sequences PCR (REP‐PCR) and enterobacterial repetitive intergenic consensus sequences PCR (ERIC‐PCR)—are evaluated for their efficacy in differentiating the strains. The present study used 100 strains of STEC isolated from bovine meat, bovine feces, and swabs from the chopping boards. The REP‐PCR profiles showed 21 different banding patterns, whereas ERIC‐PCR profiles obtained 18 patterns among the tested strains. The dendrogram prepared by the neighbor‐joining method formed eight and seven clusters for REP and ERIC‐PCR, respectively. The discriminatory indices for the typing methods were 87.83 and 83.03% for REP and ERIC‐PCR, respectively. Thus, the clusters based on REP and ERIC‐PCR fingerprints were unambiguous and thus could be useful for phylogenetic clustering and epidemiological surveillance. The nonparametric multidimensional scaling plots showed more than 75 and 60% similarities within populations of different REP and ERIC clusters, respectively, regarding its source of isolation, serogroup, and distribution virulence markers. The REP and the ERIC sequences are good molecular markers that offer rapid, cost‐effective, and easy molecular typing for STEC strains.

Mechanisms Affecting the Biosynthesis and Incorporation Rate of Selenocysteine.

Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure–activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.

A small RNA is functional in Escherichia fergusonii despite containing a large insertion.

Bacterial small RNAs (sRNAs) are important regulators of gene expression; however, the impact of natural mutations on sRNA functions has not been studied extensively. Here we show that the sRNA MgrR contains a unique 53 bp insertion in Escherichia fergusonii, a close relative of Escherichia coli and Salmonella enterica. The insertion is a repetitive extragenic palindromic (REP) sequence that could block transcription, but full-length MgrR is produced in E. fergusonii, showing that the insertion has not affected sRNA production. Additionally, despite containing the large insertion, the sRNA appears to be functional because deletion of mgrR made E. fergusonii more susceptible to H2O2. The molecular details of MgrR's roles in H2O2defence are yet to be defined, but our results suggest that having an alternative function allowed the sRNA to be retained in E. fergusonii despite it sustaining a large, potentially disruptive mutation.

Expression of the ISPpu9 transposase of Pseudomonas putida KT2440 is regulated by two small RNAs and the secondary structure of the mRNA 5'-untranslated region.

Insertion sequences (ISs) are mobile genetic elements that only carry the information required for their own transposition. Pseudomonas putida KT2440, a model bacterium, has seven copies of an IS called ISPpu9 inserted into repetitive extragenic palindromic sequences. This work shows that the gene for ISPpu9 transposase, tnp, is regulated by two small RNAs (sRNAs) named Asr9 and Ssr9, which are encoded upstream and downstream of tnp, respectively. The tnp mRNA has a long 5′-untranslated region (5′-UTR) that can fold into a secondary structure that likely includes the ribosome-binding site (RBS). Mutations weakening this structure increased tnp mRNA translation. Asr9, an antisense sRNA complementary to the 5′-UTR, was shown to be very stable. Eliminating Asr9 considerably reduced tnp mRNA translation, suggesting that it helps to unfold this secondary structure, exposing the RBS. Ectopic overproduction of Asr9 increased the transposition frequency of a new ISPpu9 entering the cell by conjugation, suggesting improved tnp expression. Ssr9 has significant complementarity to Asr9 and annealed to it in vitro forming an RNA duplex; this would sequester it and possibly facilitate its degradation. Thus, the antisense Asr9 sRNA likely facilitates tnp expression, improving transposition, while Ssr9 might counteract Asr9, keeping tnp expression low.

Detection of β-Lactamase–Producing Proteus mirabilis Strains of Animal Origin in Andhra Pradesh, India and Their Genetic Diversity

Proteus mirabilis is abundant in soil and water. Although this bacterium is part of the normal human intestinal flora, it can cause serious infections in humans, including complicated urinary tract infections. This pathogen is also commonly associated with multidrug resistance. In the present study, analysis of 1,093 samples from foods of animal origin and animal intestinal samples recovered 232 P. mirabilis isolates identified by PCR assay. Of these 232 isolates, 72 produced β-lactamase (determined by both phenotypic and genotypic methods), with the highest prevalence in poultry cloacal swabs (11.82%) followed by mutton (9.18%), khoa (6.32%), pork (5.63%), pig rectal swabs (5.52%), beef (5.45%), and chicken (5.13%) but none from sheep rectal swabs and bovine rectal swabs. Among β-lactamase genes, blaTEM was the predominant gene detected (59 isolates) followed by blaOXA (11 isolates), blaSHV (5 isolates), blaFOX (5 isolates), blaCIT (4 isolates), blaCTX-M1 and blaCTX-M9 (2 isolates each) and blaCTX-M2, blaDHA, and blaEBC (1 isolate each). None of the isolates carried blaACC, blaMOX, or carbapenemase genes (blaVIM, blaIMP, blaKPC, and blaNDM-1). Dendrogram analysis of enterobacterial repetitive intergenic consensus sequences and repetitive extragenic palindromic sequences obtained with PCR analysis of β-lactamase-producing isolates revealed 63 isolates, but 9 isolates did not yield bands. The analysis revealed that 6.58% of the samples had β-lactamase-producing P. mirabilis isolates that may affect food safety and contaminate the environment. Further genotyping revealed the genetic relationships between isolates of different origin. These findings emphasize the need for careful use of antibiotics to control the spread of β-lactamase-producing bacteria.

Detecting and molecular typing of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a prevalent food-borne pathogen in many Asian countries and has been spreading rapidly following the appearance of pandemic strains. This article reviews conventional and molecular methods for culturing, identifying, enumerating and rapidly detecting V. parahaemolyticus and its virulence factors, such as thermostable direct hemolysin (TDH), TDH-related hemolysin and thermolabile hemolysin. It also discusses various molecular typing methods, namely, pulsed-field gel electrophoresis, ribotyping, random amplified polymorphic DNA and three other polymerase chain reaction methods based on specific conserved nucleotide sequences, such as ribosomal gene spacer, repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequences, for use in epidemiological investigations of this pathogen.

Molecular characterization of Bacillus thuringiensis strains to control Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) population

Abstract The main objective of this study was to characterize the toxicity and genetic divergence of 18 Bacillus thuringiensis strains in the biological control of Spodoptera eridania. Bacterial suspensions were added to the S. eridania diet. Half of the selected B. thuringiensis strains caused high mortality seven days after infection. The genetic divergence of B. thuringiensis strains was assessed based on Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromic (REP) sequences, and five phylogenetic groups were formed. Despite their genetic diversity B. thuringiensis strains did not show any correlation between the collection sites and toxicity to larvae. Some B. thuringiensis strains are highly toxic to S. eridania, thus highlighting the potential of their endotoxins as biopesticides.

Molecular Characterization of Escherichia Coli Isolates from Food Animals

The present study was carried out for isolation and molecular characterization of Escherichia coli from faecal and meat samples of food animals viz. cattle, pigs and poultry. A total of 66 E. coli isolates were recovered from 420 samples of different food animals and further confirmed by PCR targeting E. coli specific uidA gene. These isolates were sensitive to chloramphenicol and ciprofloxacin and resistant to ampicillin and cloxacillin. Out of 66 isolates, 42 were typed into 13 different ‘O’ serogroups, 13 untypable and remaining 11 were identified as rough. Serogroup O84, O101, O118, O120 and O147 were predominant and serogroup O118 was found to be common in the samples of all 3 species of food animals. Five (7.57%) and 3 (4.54%) of E. coli isolates were found to harbor virulent genes, stx2 and est, respectively. Twenty representative E. coli isolates selected randomly from 20 different locations were subjected to molecular typing by PCR targeting Repetitive Extragenic Palindromic (REP) sequences. The region specific molecular types of E. coli could not be detected by using REP-PCR based discrimination.

Virulence and DNA fingerprinting analysis of Xanthomonas oryzae pv. oryzae identify a new pathotype in Guangxi, South China.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice worldwide. However, little is known about the population structure of this organism in Guangxi Zhuang Autonomous Region, South China. Here, pathotypic and DNA fingerprint analyses were conducted to characterize the isolates of Xoo collected from rice leaves in five districts of the region from 2013 to 2016. Their pathogenicity was tested by leaf clipping, and the DNA fingerprints were analyzed by repetitive sequence-based polymerase chain reaction and endogenous insertion sequence element-based polymerase chain reaction assays using the repetitive extragenic palindromic sequence and enterobacterial repetitive intergenic consensus primers, respectively. Pathogenicity assays of 70 representative isolates were conducted using a series of near-isogenic lines and two new pathotypes were identified. All the pathotypes were found to be incompatible with xa5 and Xa7. One pathotype was virulent to Xa14, Xa21, and Xa23, whereas another virulent to Xa21 and Xa23, but incompatible with Xa14. A dendrogram generated for the data sets obtained from DNA fingerprinting suggested the prevalence of high genetic diversity of Xoo throughout Guangxi, and no association between the molecular haplotypes and pathotypes was identified.

Genomic polymorphism of Trifolium repens root nodule symbionts from heavy metal-abundant 100-year-old waste heap in southern Poland

In total, 77 rhizobial strains isolated from the root nodules of T. repens, inhabiting heavy metal-contaminated waste heap (36 isolates) and control grassland (41 ones) in southern Poland, were analyzed for genome polymorphism and strength of the heavy metals’ (mainly Zn, Pb, Cd) selective pressure on bacterial genome polymorphism using two PCR-based techniques, ERIC- (enterobacterial repetitive intergenic consensus) and REP-PCR (repetitive extragenic palindromic) sequences. Both methods of different discriminatory power index (D) (ERIC-PCR D = 0.9737; REP-PCR D = 0.9826) allowed to distinguish 47 and 44 rhizobial strains, respectively. Combined analysis of ERIC-PCR and REP-PCR DNA amplicons differentiated all tested isolates. Both ERIC- and REP-PCR DNA fingerprinting techniques showed significant decline of the genome polymorphism (h) in rhizobial population from metalliferous waste heap (h = 0.89 ± 0.03; h = 0.90 ± 0.02, respectively) compared to rhizobia from control non-metalliferous area (h = 0.99 ± 0.01; h = 0.98 ± 0.02, respectively) as well as substantial differences in the genomic polymorphism between both these populations (FST = 0.162, p = 0.008; FST = 0.170, p = 0.000, respectively).

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

Abstract The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

First Report of Bacterial Leaf Spot Caused by Pseudomonas syringae pv. tomato on Kale in California

HomePlant DiseaseVol. 101, No. 3First Report of Bacterial Leaf Spot Caused by Pseudomonas syringae pv. tomato on Kale in California PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Bacterial Leaf Spot Caused by Pseudomonas syringae pv. tomato on Kale in CaliforniaS. T. Koike, E. I. Alger, L. Ramos Sepulveda, and C. T. BullS. T. KoikeSearch for more papers by this author, E. I. AlgerSearch for more papers by this author, L. Ramos SepulvedaSearch for more papers by this author, and C. T. BullSearch for more papers by this authorAffiliationsAuthors and Affiliations S. T. Koike , University of California Cooperative Extension, Salinas 93901 E. I. Alger , Undergraduate Research Opportunities Center, California State University Monterey Bay, Seaside 93955 L. Ramos Sepulveda , The Pennsylvania State University, University Park 16802 C. T. Bull , The Pennsylvania State University, University Park 16802, and USDA/ARS, Salinas, CA 93901. Published Online:20 Dec 2016https://doi.org/10.1094/PDIS-10-16-1460-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In 2014, bacterial leaf spot was seen on commercial kale (Brassica oleracea var. acephala) grown in Monterey County, CA. Initial symptoms were small (2 to 4 mm in diameter), angular, water soaked lesions observable on both adaxial and abaxial surfaces. Lesions enlarged, became tan to light brown, and were often surrounded by chlorotic haloes. Incidence was as high as 10% and due to the impracticality of sorting healthy and diseased leaves, large portions of fields were unharvested. From macerated spots, blue-green fluorescent pseudomonads were consistently isolated on King’s Medium B agar. Six retained isolates were gram-negative, oxidase negative, arginine dihydrolase negative, produced levan, but did not nucleate ice. Isolates did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Samsun). These isolates therefore belong to Lelliott’s LOPAT group 1a Pseudomonas syringae (Lelliott 1966). Multilocus sequence analysis using the concatenated gene fragments of the loci gap1, gltA, gyrB, and rpoD placed the strains in P. syringae genomospecies 3, and sequences were identical to those of the pathotype of P. syringae pv. tomato. For Koch’s postulates, potted kale (cvs. Lacinato, Red Russian, Siberian) at the six-leaf stage were inoculated with bacteria grown on sucrose peptone agar for 48 h and suspended in sterile distilled water (SDW) to a concentration of 107 CFU/ml. Each of six isolates was sprayed until runoff onto 12 replicate plants per cultivar. Plants were enclosed in plastic bags for 24 h and then maintained in a greenhouse (23 to 25°C). After 4 to 5 days, water soaked lesions developed on all inoculated plants; lesions turned tan and were similar to symptoms seen in the field. Control plants sprayed with SDW developed no symptoms. Fluorescent pseudomonads were reisolated from all inoculated cultivars. The experiment was conducted twice and results were the same. The reisolates were identical to the original strains according to repetitive extragenic palindromic sequence PCR with the BOXA1R primer, confirming Koch’s postulates. Using methods described above, kale strain BS3736, P. syringae pv. tomato (CFBP2122PT), and P. syringae pv. maculicola (CFBP1657PT) were inoculated onto the three kales and tomato (cv. Super Beefsteak). In three experiments, all kale and tomato inoculated with the kale strain and the pathotype of P. syringae pv. tomato developed necrotic spots and yellow halos; distinctive pepper spot symptoms were only observed on kale cv. Lacinato inoculated with P. syringae pv. maculicola; pepper spot symptoms consist of small (3 to 4 mm in diameter), tan to brown spots visible from both sides of leaves. In this study, P. syringae pv. maculicola and P. syringae pv. tomato (including the kale strains) had overlapping but distinct experimental host ranges. P. syringae strains from kale were identified previously as P. syringae pv. maculicola though genetically identical to the pathotype strain of P. syringae pv. tomato (Zhao et al. 2000). While P. syringae has been reported on kale previously (Bull et al. 2003), this is the first report of bacterial leaf spot on kale caused by P. syringae pv. tomato in California and one of the few reports of P. syringae pv. tomato causing a natural disease outbreak on crucifers.

Repetitive extragenic palindromic DNA sequences from Brucella melitensis stimulate Toll-like receptor 9 signaling in macrophages

Brucella DNA activates the host innate immune system via the intracellular Toll-like receptor 9 (TLR9). However, the Brucella DNA sequences which are responsible for these immunostimulatory effects remain to be elucidated. The present study demonstrated that repetitive extragenic palindromic (REPs) sequences present in Brucella DNA were able to stimulate macrophages through TLR9. The induction of interferon-α (IFN-α) production by Brucella REPs was detected in cultured RAW264.7 mouse macrophages as well as in Wistar rats. Knockdown of TLR9 expression by siRNA in macrophages led to a reduction in IFN-α production following REPs stimulation. In addition, it was confirmed that the activating capacity of Brucella REPs is CpG dependent. Induction of IFN-α by Brucella REPs was completely abrogated when REP sequences were transformed into non-CpG sequences or by C-methylated modifications. Furthermore, it was observed that REPs-initiated TLR9/NF-κB and TLR9/MAPK signaling pathways contributed to the production of IFN-α. The identification of Brucella REPs as natural TLR9 agonists may be useful for the development of novel therapeutic applications.

Molecular Epidemiology of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Strains Isolated from Children with Urinary Tract Infections

Background: Uropathogenic Escherichia coli (UPEC) and Klebsiella pneumoniae (K. pneumoniae) are major pathogens which cause urinary tract infections (UTI) in pediatric patients. The presence of extended-spectrum β-lactamases (ESBLs) in these pathogens may further exacerbate infections and hamper successful treatment. Objectives: We undertook a study to investigate the prevalence of ESBL genetic indicators among K. pneumoniae strains isolated from pediatric patients in Tehran, Iran. Moreover, genotyping of blaCTX-M-15-positive isolates was determined through repetitive extragenic palindromic sequence polymerase chain reactions (REP-PCR). Methods: A total of 76 non-duplicate K. pneumoniae isolates were collected from outpatients admitted with UTIs at the pediatric nephrology wards of two hospitals in Tehran, Iran. The antibacterial susceptibility of K. pneumoniae isolates was determined by the disk diffusion method. The isolates were examined phenotypically and genotypically for ESBL production using the combined-disk method and PCR, respectively. The blaCTX-M-positive isolates were subjected to minimal inhibitory concentration (MIC) testing for ceftazidime and cefotaxime. The clonal relationships of blaCTX-M-15-positive isolates were determined through REP-PCR. Results: The highest rates of antibiotic resistance were obtained for ampicillin (92.1%), followed by ceftazidime (40.8%), cefotaxime (40.8%), and aztreonam (39.5%). However, only one isolate (1.3%) was resistant to imipenem. Among the ESBL-positive isolates, blaCTX-M(64.5%) was the most prevalent gene, followed by blaSHV (54.8%) and blaTEM (41.9%). Of 20 blaCTX-M-carrying isolates, 14 isolates showed MICs of 256 μg/mL against cefotaxime. The other six isolates had MICs of 512 μg/mL. However, 16 out of 20 blaCTX-M-carrying isolates exhibited MICs of 128 μg/mL against ceftazidime. The other four K. pneumoniae isolates showed MICs of 256 μg/mL. Of 17 blaCTX-M-15-positive K. pneumoniae isolates, 16 distinct REP-PCR patterns (genotypes) were obtained. Conclusions: The frequency of blaCTX-Mamong K. pneumoniae isolates was at an alarming rate, indicating that more efforts should be undertaken to track and monitor the spread of K. pneumoniae that produce CTX-M β-lactamases.

Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus Planktothrix.

BackgroundBloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes.ResultsMutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5–6.9 %), when compared with the neutral mutations (7.5–20.6 %). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2–19.4 %) and/or mcyHA (0–7.3 %) exceeded the abundance of neutral mutations.ConclusionsIt is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0639-1) contains supplementary material, which is available to authorized users.

Molecular Epidemiology and Drug Resistance of Acinetobacter baumannii Isolated from Hospitals in Southern Poland: ICU as a Risk Factor for XDR Strains.

The objectives of the present study were to investigate the carbapenemase and metallo-beta-lactamase genes of Acinetobacter baumannii clinical isolates by polymerase chain reaction (PCR) and real time PCR and to determine the molecular epidemiology of the strains using the DiversiLab tool. From these data, correlations between drug resistance, resistance genes, and epidemiological clones may be revealed. The study was conducted on 125 A. baumannii collected over the 2013 year. The majority of the isolates from both intensive care unit (ICU) and non-ICU cases originated from pneumonia infections (79.2%), isolates from blood infections accounted for 17.6% and 3.2% were from meningitis infections. In the ICU cases compared with the non-ICU cases, bloodstream infections were more frequently diagnosed (19.2% vs. 11.5%). Sixty percent of A. baumannii strains were resistant to all the antimicrobials tested with the exception of colistin. All strains were susceptible to colistin and polymyxin B. Extensively drug-resistant (XDR) strains accounted for 80.8% of the isolates tested and these XDR strains were more frequently isolated from ICU cases than from non-ICU cases (93.9% vs. 30.8%). Among the 101 isolates of A. baumannii exhibiting the XDR pattern of resistance, 80 possessed the blaOXA-24 gene and 29 had the blaOXA-23 gene. Only two isolates possessed the blaVIM gene. The presence of the ISAba1element was confirmed among 10 strains from patients hospitalized in the ICU. Using repetitive extragenic palindromic sequence PCR (DiversiLab typing), six clones and 12 unique strains were identified, of which two clones dominated. Most isolates belonging to clone 1 (66.7%) and clone 2 (85.5%) were susceptible only to colistin. In summary, it is clear from our findings and those of other studies that carbapenem resistance among A. baumannii strains presents a serious clinical problem worldwide. Furthermore, the presence of XDR international clone II in ICUs poses a potential risk for future outbreaks of A. baumannii infection and controlling A. baumannii infections in hospitals presents a serious challenge.

The effects of repetitive extragenic palindromic sequences from Brucella melitensis DNA on the toll-like receptor 9-mediated interferon-α production

To screen the repetitive extragenic palindromic sequences with activation of toll-like receptor 9 (TLR9) activity from Brucella melitensis DNA, providing new ideas and new targets for prevention and treatment of brucellosis. Bioinformatics methods were used to detect repetitive extragenic palindromic (REP) sequences from Brucella melitensis DNA. The studied REPs were selected and synthesized. RAW264.7 was cultured and transfected with REPs mediated by lipofectamine 3000. Additionally, TLR9-siRNA was used to downregulate TLR9 expression. The content of interferon-α (IFN-α) in the supernatant was then measured by ELISA. A total of 2 200 REP sequences in Brucella melitensis DNA were identified. Twelve REP sequences were synthesized for further detecting of the TLR9 agonistic activity. IFN-α expression in RAW264.7 treated with M2, M3, M4, M5, M6, M7, M9, M12 were (26.944 ± 1.868), (46.461 ± 2.562), (34.980 ± 2.055), (43.016 ± 2.162), (62.533 ± 4.031), (67.125 ± 5.069), (18.908 ± 1.633), (39.572 ± 2.465) pg/ml respectively, which significantly increased when compared with the negative control group [(12.594 ± 1.338) pg/ml, t=10.817, 20.295, 15.812, 20.724, 20.365, 18.016, 5.180, 16.660, all P<0.05]. Additionally, TLR9-siRNA can significantly decrease the levels of IFN-α in RAW264.7 treated with M6. REP sequences presented in Brucella melitensis DNA are able to induce IFN-α expression through TLR9, which can be helpful for the understanding of pathogenesis and immunity of Brucella melitensis.