Simultaneous detection of Escherichia coli, Salmonella enterica, Listeria monocytegenes and Bacillus cereus by oligonucleotide microarray

Background: Traditional laboratory methods to detect pathogenic bacteria are time consuming and laborious. Therefore, it is essential to use powerful and reliable molecular methods for quick and simultaneous detection of microbial pathogens. Objectives: The current study aimed to evaluate the capability and efficiency of 23S rDNA sequence for rapid and simultaneous detection of four important food-borne pathogens by an oligonucleotide microarray technique. Materials and Methods: The 23S rDNA sequences of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus were obtained from GenBank databases and used to design the oligonucleotide probes and primers by Vector NTI software. Oligonucleotide probes were placed on a nylon membrane and hybridization was performed between probes and 23S rDNA digoxigenin-labeled polymerase chain reaction (PCR) products. Hybridization signals were visualized by NBT/BCIP color development. Results: Positive hybridization color was produced for Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus. The oligonucleotide microarray detected all bacterial strains in a single reaction in less than five hours. The sensitivity of the performed microarray assay was 103 cfu/mL for each species of pathogen. No cross reaction was found between the tested bacterial species. Conclusions: The obtained results indicated that amplification of 23S rDNA gene followed by oligonucleotide microarray hybridization is a rapid and reliable method to identify and discriminate foodborne pathogens tested under the study

Detection of eight foodborne bacterial pathogens by oligonucleotide array hybridization

Background: Simultaneous and rapid detection of multiple foodborne bacterial pathogens is important for the prevention of foodborne illnesses. Objective: The aim of this study was to evaluate the use of 16S rDNA and 23S rDNA sequences as targets for simultaneous detection of eight foodborne bacterial pathogens. Methods: Nineteen bacterial oligonucleotide probes were synthesized and applied to nylon membranes. Digoxygenin labeled 16S rDNA and 23S rDNA from bacteria were amplified by PCR using universal primers, and the amplicons were hybridized to the membrane array. Hybridization signals were visualized by NBT/BCIP color development. Results: The eight intestinal bacterial pathogens including Salmonella enterica, Escherichia coli, Bacillus cereus, Vibrio cholerae, Shigella dysenteriae, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis were appropriately detected in a panel of oligonucleotide array hybridization. The experimental results showed that the method could discriminate the bacterial pathogens successfully. The sensitivity of oligonucleotide array was 103 CFU/ml. Conclusion: This study showed that 16S rDNA and 23S rDNA genes had sufficient sequence diversity for species identification and were useful for monitoring the populations of foodborne pathogenic bacteria. Furthermore, results obtained in this study revealed that oligonucleotide array hybridization had a powerful capability to detect and identify the bacterial pathogens simultaneously

Genetic analysis of cagA and vacA genes in helicobacter pylori isolates and their relationship with gastroduodenal diseases in the west of Iran

Background: Helicobacter pylori have different virulence factors which are associated with several gastroduodenal diseases; however, this association is variable in different geographical regions. Data of genotypes of Iranian H. pylori isolates are few. Objectives: The aim of the current study was to investigate the cagA/vacA genotypes of Helicobacter pylori isolates and determine the relationship between these genotypes with respect to different gastric disorders in patients of Chaharmahalo Bakhtiarian. Materials and Methods: In this cross-sectional study, gastric biopsies were taken from 200 patients with gastrodoudenal diseases. Histopathological features were recognized by specialist. The samples were subjected to PCR for detection and identification of ureC, cagA and vacA genes. Results: The frequency of the vacA genotypes, sa1/m1, s1a/m1b, s1a/m2, s1b/m1a, s1b/m1b, s1b/m2, s1c/m1a, s1c/m1b, s1c/m2, s2/m1a, s2/m1b and s2/ m2 were 27(6.6%), 8(4.3%), 45(28.04%), 7(3.7%), 5(2.5%), 10 (6.1%), 12 (7.4%), 4 (2.5%), 18(11%), 6(3.7%), 0 and 22(13.5%) respectively. The cagA gene was detected in 92% of strains. Based on our findings, it seemed that cagPAI and vacA s1 genotypes were associated with some gastric disorders in patients with H. pylori. In this region, the isolates carrying s1a/m2 were the most prevalent. Conclusions: We found considerable relationship between s1a/m1a, s1a/m2, s2/m2 and s1c/m1a and some gastric disorders. Further studies about the role of H. pylori virulence factors and gastric disorders were recommended. © 2013, Iranian Red Crescent Medical Journal

Fecal microRNAs as innovative biomarkers of intestinal diseases and effective players in host-microbiome interactions

Over the past decade, short non-coding microRNAs (miRNAs), including circulating and fecal miRNAs have emerged as important modulators of various cellular processes by regulating the expression of target genes. Recent studies revealed the role of miRNAs as powerful biomarkers in disease diagnosis and for the development of innovative therapeutic applications in several human conditions, including intestinal diseases. In this review, we explored the literature and summarized the role of identified dysregulated fecal miRNAs in intestinal diseases, with particular focus on colorectal cancer (CRC) and celiac disease (CD). The aim of this review is to highlight one fascinating aspect of fecal miRNA function related to gut microbiota shaping and bacterial metabolism influencing. The role of miRNAs as "messenger" molecules for inter kingdom communications will be analyzed to highlight their role in the complex host-bacteria interactions. Moreover, whether fecal miRNAs could open up new perspectives to develop novel suitable biomarkers for disease detection and innovative therapeutic approaches to restore microbiota balance will be discussed

Acinetobacter baumannii. An ancient commensal with weapons of a pathogen

Acinetobacter baumannii is regarded as a life-threatening pathogen associated with community-acquired and nosocomial infections, mainly pneumonia. The rise in the number of A. baumannii antibiotic-resistant strains reduces effective therapies and increases mortality. Bacterial comparative genomic studies have unraveled the innate and acquired virulence factors of A. baumannii. These virulence factors are involved in antibiotic resistance, environmental persistence, host-pathogen interactions, and immune evasion. Studies on host–pathogen interactions revealed that A. baumannii evolved different mechanisms to adhere to in order to invade host respiratory cells as well as evade the host immune system. In this review, we discuss current data on A. baumannii genetic features and virulence factors. An emphasis is given to the players in host–pathogen interaction in the respiratory tract. In addition, we report recent investigations into host defense systems using in vitro and in vivo models, providing new insights into the innate immune response to A. baumannii infections. Increasing our knowledge of A. baumannii pathogenesis may help the development of novel therapeutic strategies based on anti-adhesive, anti-virulence, and anti-cell to cell signaling pathways drugs

Serogroups, virulence genes and antibiotic resistance in Shiga toxin-producing Escherichia coli isolated from diarrheic and non-diarrheic pediatric patients in Iran

Background: From a clinical perspective, it is important to know which serogroups, virulence genes and antibiotic resistance patterns are present in Shiga toxin-producing Escherichia coli strains in pediatric patients suffering from diarrheic and non-diarrheic infections. This is the first study in Iran that has comprehensively investigated the Shiga toxin-producing Escherichia coli -related infection characteristics in diarrheic and non-diarrheic pediatric patients of 0-60 months of age. Methods. Two-hundred and twenty four diarrheic and 84 non-diarrheic stool specimens were collected from the Baqiyatallah hospital of Tehran, Iran. The stool samples were cultured immediately and those that were E. coli-positive were analyzed for the presence of antibiotic resistance genes and bacterial virulence factors using PCR. Antimicrobial susceptibility testing was performed using disk diffusion method. Results: One-hundred and fifty four out of 224 (68.75%) diarrheic stools and 31 out of 84 (36.90%) non-diarrheic stools harbored E. coli. In addition, children in 13-24 month-old age group had the highest incidence of infection with this bacterium (77.63%). A significant difference was found between the frequency of Attaching and Effacing Escherichia coli and Enterohaemorrhagic Escherichia coli (P =0.045). The genes encoding Shiga toxins and intimin were the most commonly detected virulence factors. Among all serogroups studied, O26 (27.04%) and O111 (18.85%) had the highest incidences in the diarrheic and non-diarrheic patients. The incidence of genes encoding resistance against sulfonamide (sul1), gentamicin (aac(3)-IV), trimethoprim (aadA1), cephalothin (blaSHV) and tetracycline (tetA) were 82.78%, 68.03%, 60.65%, 56.55% and 51.63%, respectively. High resistance levels against penicillin (100%), tetracycline (86.88%), gentamicin (62.29%) and streptomycin (54.91%) were observed. Marked seasonality in the serogroup distributions was evident, while STEC infections were more common in summer (P =0.041). Conclusions: Our findings should raise awareness about antibiotic resistance in diarrheic pediatric patients in Iran. Clinicians should exercise caution when prescribing antibiotics, especially during the warmer months of the year

FimH and anti-adhesive therapeutics. A disarming strategy against uropathogens

Chaperone-usher fimbrial adhesins are powerful weapons against the uropathogens that allow the establishment of urinary tract infections (UTIs). As the antibiotic therapeutic strategy has become less effective in the treatment of uropathogen-related UTIs, the anti-adhesive molecules active against fimbrial adhesins, key determinants of urovirulence, are attractive alternatives. The best-characterized bacterial adhesin is FimH, produced by uropathogenic Escherichia coli (UPEC). Hence, a number of high-affinity mono-and polyvalent mannose-based FimH antagonists, characterized by different bioavailabilities, have been reported. Given that antagonist affinities are firmly associated with the functional heterogeneities of different FimH variants, several FimH inhibitors have been developed using ligand-drug discovery strategies to generate high-affinity molecules for successful anti-adhesion therapy. As clinical trials have shown d-mannose’s efficacy in UTIs prevention, it is supposed that mannosides could be a first-in-class strategy not only for UTIs, but also to combat other Gram-negative bacterial infections. Therefore, the current review discusses valuable and effective FimH anti-adhesive molecules active against UTIs, from design and synthesis to in vitro and in vivo evaluations

Acinetobacter baumannii targets human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for invasion of pneumocytes

Multidrug-resistant Acinetobacter baumannii is regarded as a life-threatening pathogen mainly associated with nosocomial and community-acquired pneumonia. Here, we show that A. baumannii can bind the human carcinoembryonic antigen- related cell adhesion molecule (CEACAM) receptors CEACAM1, CEACAM5, and CEACAM6. This specific interaction enhances A. baumannii internalization in membrane- bound vacuoles, promptly decorated with Rab5, Rab7, and lipidated microtubule- associated protein light chain 3 (LC3). Dissecting intracellular signaling pathways revealed that infected pneumocytes trigger interleukin-8 (IL-8) secretion via the extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) signaling pathways for A. baumannii clearance. However, in CEACAM1-L-expressing cells, IL-8 secretion lasts only 24 h, possibly due to an A. baumannii-dependent effect on the CEACAM1-L intracellular domain. Conversely, the glycosylphosphatidylinositol- anchored CEACAM5 and CEACAM6 activate the c-Jun NH2-terminal kinase (JNK)1/ 2-Rubicon-NOX2 pathway, suggestive of LC3-associated phagocytosis. Overall, our data show for the first time novel mechanisms of adhesion to and invasion of pneumocytes by A. baumannii via CEACAM-dependent signaling pathways that eventually lead to bacterial killing. These findings suggest that CEACAM upregulation could put patients at increased risk of lower respiratory tract infection by A. baumannii. IMPORTANCE This work shows for the first time that Acinetobacter baumannii binds to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5, and CEACAM6. This binding significantly enhances A. baumannii internalization within alveolar host cell epithelia. Intracellular trafficking involves typical Rab5 and Rab7 vacuolar proteins as well as light chain 3 (LC3) and slowly progresses to bacterial killing by endosome acidification. CEACAM engagement by A. baumannii leads to distinct and specific downstream signaling pathways. The CEACAM1 pathway finely tunes interleukin- 8 (IL-8) secretion, whereas CEACAM5 and CEACAM6 mediate LC3-associated phagocytosis. The present study provides new insights into A. baumannii-host interactions and could represent a promising therapeutic strategy to reduce pulmonary infections caused by this pathogen

Simultaneous molecular detection of Salmonella enterica serovars typhi, enteritidis, infantis, and typhimurium

Background: Salmonella enterica serovar Typhi, as causative agent of typhoid fever, is one of the most important endemic pathogens. Non-typhoidal Salmonella serovars, including Typhimurium, Infantis, and Enteritidis are amongst the most prevalent serotypes worldwide and in developing areas such as Iran. The aim of this study was to apply a uniplex PCR for rapid detection of Salmonella spp., and a multiplex PCR for the simultaneous detection of the four most common Salmonella serovars in Iran. Methods: Current research was done in 2010 at Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. For detection of Salmonella spp a pair of primers was used to replicate a chromosomal sequence. Four other sets of primers were also designed to amplify the target genes of four Salmonella species including S. typhi, and three non-typhoidal Salmonella spp (S. enteritidis, S. infantis, and S. typhimurium). The assay specificity was investigated by testing 15 different Salmonella serovars and 8 other additional non-Salmonella species. Results: The Salmonella genus-specific PCR yielded the expected DNA band of 404 bp in all Salmonella spp., strains tested. The uniplex and multiplex PCR assays produced also the expected fragments of 489 bp, 304 bp, 224 bp, and 104 bp for serovars Typhi, Enteritidis, Typhimurium, and Infantis, respectively. Each species-specific primer pair set did not show any cross-reactivity when tested on other Salmonella serovars or other non- but related- Salmonella strains. Conclusion: Both uniplex and multiplex PCR protocols had a good specificity. They can provide an important tool for the rapid and simultaneous detection and differentiation of the four most prevalent Salmonella serovars in Iran