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Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity
Jiménez, Kenia Barrantes;McCoy, Clyde B.;Achí, Rosario;
Brazilian Journal of Microbiology , 2010, DOI: 10.1590/S1517-83822010000400018
Abstract: a multiplex polymerase chain reaction (pcr) assay to be used as an alternative to the conventional culture method in detecting shigella and enteroinvasive escherichia coli (eiec) virulence genes ipah and ial in lettuce was developed. efficacy and rapidity of the molecular method were determined as compared to the conventional culture. lettuce samples were inoculated with different shigella flexneri concentrations (from 10 cfu/ml to 107 cfu/ml). dna was extracted directly from lettuce after inoculation (direct-pcr) and after an enrichment step (enrichment pcr). multiplex pcr detection limit was 104 cfu/ml, diagnostic sensitivity and specificity were 100% accurate. an internal amplification control (iac) of 100 bp was used in order to avoid false negative results. this method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. also, the culture method detection limit was 106 cfu/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. in this study a multiplex pcr method for detection of virulence genes in shigella and eiec was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to shigella conventional culture.
A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium
Marie Bugarel, Sophie A Granier, Fran?ois-Xavier Weill, Patrick Fach, Anne Brisabois
BMC Microbiology , 2011, DOI: 10.1186/1471-2180-11-151
Abstract: A total of 538 unrelated S. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, intI1, sul1 and blaTEM determinants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources.The GeneDisc? assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.Non-typhoid salmonellosis is one of the most frequently-reported bacterial foodborne diseases and is a major economic and public health issue worldwide. European data show that Salmonella is the second most predominant bacterial pathogen, causing around 132,000 human cases in 2008 [1]. In the United States, Salmonella serotypes cause an estimated 1.4 million cases of foodborne disease each year [2]. The primary reservoirs of Salmonella are food-producing animals, the three main sources being poultry, cattle and pigs. Of the numerous different serotypes, only a few are frequently isolated from human and animal sources. Serotypes Enteritidis and Typhimurium are the most frequently encountered in human and animal sources. Together, they represent 80% of confirmed human salmonellosis cases in Europe, with a marked decrease in serotype Enteritidis cases but an increase in S. Typhimurium cases [1]. Serotype Typhimurium was implicated in 47% of the notified foodborne outbreaks in France in 2
The Effect of the Potential PhoQ Histidine Kinase Inhibitors on Shigella flexneri Virulence  [PDF]
Xia Cai, Jian Zhang, Mingliang Chen, Yang Wu, Xueqing Wang, Jiayu Chen, Junqin Zhang, Xu Shen, Di Qu, Hualiang Jiang
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023100
Abstract: PhoQ/PhoP is an important two-component system that regulates Shigella virulence. We explored whether the PhoQ/PhoP system is a promising target for new antibiotics against S. flexneri infection. By using a high-throughput screen and enzymatic activity coupled assay, four compounds were found as potential PhoQ inhibitors. These compounds not only inhibited the activity of SF-PhoQc autophosphorylation but also displayed high binding affinities to the SF-PhoQc protein in the Surface Plasmon Resonance response. A S. flexneri cell invasion assay showed that three of these potential PhoQ inhibitors inhibit the invasion of HeLa cells by S. flexneri 9380. In a Mouse Sereny test, mice inoculated with S. flexneri 9380 pre-treated with the potential PhoQ inhibitors 1, 2, 3 or 4 displayed no inflammation, whereas mice inoculated with S. flexneri 9380 alone displayed severe keratoconjunctival inflammation. All four potential PhoQ inhibitors showed no significant cytotoxicity or hemolytic activity. These data suggest that the four potential PhoQ inhibitors inhibited the virulence of S. flexneri and that PhoQ/PhoP is a promising target for the development of drugs against S. flexneri infection.
Evaluation of new primers for detecting toxigenic vibrio cholerae by multiplex PCR  [cached]
Jalil F. Mehrabadi,Parisa Morsali,Hamideh Rohani nejad,Abbas Ali Imani Fooladi
Microbiology Research , 2011, DOI: 10.4081/mr.2011.e1
Abstract: Vibrio cholerae is the etiological agent of cholera that has emerged as an endemic disease in different regions of the world in recent years. Traditional microbial culture and microscopy methods are considered to be the best standard for diagnosing V. cholerae infection. These methods, however, delay any available confirmatory answer by days. Molecular methods have the potential to provide sensitive, accurate, and rapid analysis of V. cholerae infection. We have developed a multiplex PCR assay to detect virulence and toxigenic-associated (VTA) genes (ctxA, tcpA, and ompW). To evaluate PCR specificity, additional bacteria from the enterobacteriaceae family (Salmonella typhi, Shigella dysantry, and entrotoxigenic E. coli) and Aeromonas hidrophyla were examined in this study. Specificity tests were evaluated using the genome dilution method. Importantly, the results show that our PCR specificity method represents the best tool for the rapid detection of VTA genes because of its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used for examining the existence of VTA genes in patient samples, and therefore will distinguish V. cholerae from other vibrios and bacteria. This method is able to detect 10-100 colony forming units (CFUs) of V.Cholerae and 8.5-85 picograms (pg) of genomic DNA. The multiplex PCR method is also more specific and sensitive than other methods, validating it as an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.
Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes
Sousa, Mireille ?ngela Bernardes;Mendes, Edilberto Nogueira;Collares, Guilherme Birchal;Péret-Filho, Luciano Amedée;Penna, Francisco José;Magalh?es, Paula Prazeres;
Memórias do Instituto Oswaldo Cruz , 2013, DOI: 10.1590/S0074-02762013000100005
Abstract: diarrhoeal disease is still considered a major cause of morbidity and mortality among children. among diarrhoeagenic agents, shigella should be highlighted due to its prevalence and the severity of the associated disease. here, we assessed shigella prevalence, drug susceptibility and virulence factors. faeces from 157 children with diarrhoea who sought treatment at the children's hospital jo?o paulo ii, a reference children′s hospital in belo horizonte, state of minas gerais, brazil, were cultured and drug susceptibility of the shigella isolates was determined by the disk diffusion technique. shigella virulence markers were identified by polymerase chain reaction. the bacterium was recovered from 10.8% of the children (88.2% shigella sonnei). the ipah, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1a was only detected in shigella flexneri. additionally, patients were infected by shigella strains of different ial, sat, sen and set1a genotypes. compared to previous studies, we observed a marked shift in the distribution of species from s. flexneri to s. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.
Virulence of Environmental Stenotrophomonas maltophilia Serologically Cross-reacting with Shigella-specific Antisera  [PDF]
T.S. Bonny,N. Azmuda,S.I. Khan,N.K. Birkeland
Pakistan Journal of Biological Sciences , 2010,
Abstract: This research involved an environmental strain of Stenotrophomonas maltophilia which has been reported to produce serological cross-reactivity with Shigella dysenteriae type 8 specific antisera. Since clinical diagnosis of shigellosis is largely based on culture and serology, the investigation was aimed at in vivo and in vitro virulence comparison between the culturally similar environmental S. maltophilia isolate and the reference S. dysenteriae strains. The findings of this study revealed the absence of virulent genes of Shigella sp. like ipaH, virA and stx1 and characteristic invasive large plasmid in the test isolate. The Western blot analysis revealed that serological cross-reactivity of Stenotrophomonas maltophilia was due to certain protein component(s) in its outer membrane. The isolate was capable of producing extracellular protease, exhibited alpha hemolysis and was negative for hemagglutinating assay. The isolate gave negative reaction with rabbit ileal loop and Sereny tests. The S. maltophilia isolate did not possess any enterotoxic or invasive property as that of virulent S. dysenteriae strains. Further characterizations and adequate genetic manipulations of this environmental isolate may contribute to the development of a potential vaccine candidate for shigellosis.
Sequential multiplex PCR assay for determining capsular serotypes of colonizing S. pneumoniae
Sarah Jourdain, Pierre-Alexandre Drèze, Jozef Vandeven, Jan Verhaegen, Laurence Van Melderen, Pierre R Smeesters
BMC Infectious Diseases , 2011, DOI: 10.1186/1471-2334-11-100
Abstract: We designed a novel multiplex PCR assay for specific detection of the 30 classical colonizing S. pneumoniae serogroups/types. This multiplex assay is composed of 7 consecutive PCR reactions and was validated on a large and recent collection of Streptococcus pneumoniae isolated during a prospective study conducted in Belgium at the time of PCV7 adoption.The multiplex PCR assay allowed the typing of more than 94% of the isolates of a collection of pneumococci isolated from Belgian preschool attendees (n = 332). Seventy-five percent of the isolates were typed after 3 subsequent PCR reactions. Results were in agreement with the Quellung identification.Our novel multiplex assay is an accurate and reliable method which can be used in place of the conventional method for S. pneumoniae carriage studies.S. pneumoniae is a major cause of morbidity and mortality worldwide. The World Health Organisation estimates that 1.6 million people among which 0.7 to 1 million children under 5 die of pneumococcal diseases each year [1]. The nasopharynx of young children represents the ecological niche of S. pneumoniae as well as the reservoir for pneumococcal transmission within the community. Colonisation is also recognized as the first step of disease development [2]. S. pneumoniae major virulence factor is the capsular polysaccharide upon which S. pneumoniae classification is based. Today, 93 different serotypes (including the recently discovered 6D and 11E [3,4]), belonging to 46 serogroups, have been described [5,6]. Serotypes differ by geographical prevalence [7], attack rate [8], age distribution [7] tendency to cause outbreaks [9,10] and propensity to acquire antimicrobial resistance genes [11-13]. The capsular polysaccharide is also the target of pneumococcal vaccine. The currently available protein conjugate vaccine, PCV7, protects against 7 clinically-relevant serotypes in young children (4, 6B, 9V, 14, 18C, 19F, 23F). Because of its coverage limitation, it has induced replaceme
PathogenMip Assay: A Multiplex Pathogen Detection Assay  [PDF]
Michael S. Akhras, Sreedevi Thiyagarajan, Andrea C. Villablanca, Ronald W. Davis, P?l Nyrén, Nader Pourmand
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0000223
Abstract: The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.
Multiplex PCR (Polymerase Chain Reaction) Assay for Detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in Spiked Shrimps (Penaeus monodon)
M.D. Fakruddin,Mahmuda Sultana,Monzur Morshed Ahmed,Abhijit Chowdhury
Pakistan Journal of Biological Sciences , 2013,
Abstract: The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.
Survivality and Virulence of Shigella sonnei and Shigella boydii in Different Physico-Chemical Stress Conditions  [PDF]
Ishrat Sultana,Rahman Md. Mizanur,Shakhawat Hossain Bhuiyan,Md. Majibur Rahman
Journal of Biological Sciences , 2002,
Abstract: Acids and salts are important environmental conditions encountered by Shigella spp. during its survivality and pathogenesis. These studies have shown that S. sonnei and S. boydii could survive longer than 120 min to acidic environment at pH 3 and approximately 32% survivality for both the species was recorded. The acid tolerance was found to be dependent on the growth phase and pH of the growth medium. Shigella spp. previously grown in low-salt broth at pH 7.2, produced organisms which were markedly more acid sensitive when subsequently cultured in the same broth supplemented with 200 mM or more salt at 37 °C. A differential survival pattern was recorded with salt-treated Shigella spp. in a number of aquatic samples. Fluorescent microscopic examination revealed that Shigella spp. treated with 500 mM salt went into non-culturable state but remain viable. However, salt treatment could produce no significant changes to the infective properties of Shigella spp.
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