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Construction and characterization of a Salmonella enterica serovar Typhi tolC deletion mutant
226
1st International Conference on Molecular Diagnostic and Biomarker Discovery/Asian Pac J Trop Dis 2014; 4(3): 223-252
identification and detection of a unique gene in Salmonella enterica subsp. enterica Weltevreden using PCR-based approach
The
doi: 10.1016/S2222-1808(14)60516-8
Clarissa Tan , Goay Yuan Xin , Yeoh Chiann Ying , Asma Ismail 1
1
1
襃 2014
2
by the Asian Pacific Journal of Tropical Disease. All rights reserved.
and Phua Kia Kien1.
Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia Kubang Kerian, Kelantan, Malaysia, 2Universiti Sains Islam Malaysia, Negeri Sembilan, Malaysia.
1
ABSTRACT
Introduction: Salmonella enterica are important food-borne pathogens that cause human gastroenteritis, bacteremia and subsequent focal infections. The incidences of food-borne salmonellosis due to non-typhoidal Salmonella serotypes are increasing periodically in Malaysia. Other than the two predominant non-typhoidal Salmonella serotypes, S. Typhimurium and S. Enteritidis, an emerging serotype, S. Weltevreden, is increasingly being isolated from hospitalized-gastroenteritis patients in Malaysia. Serotyping is the conventional method for differentiation of this pathogen from the 2500 other serotypes known to exist. However, this method has a number of disadvantages including low specificity and high cost. Therefore, a molecular method for identification of S. Weltevreden in clinical isolates based on amplification of a specific DNA sequence present in its genome is proposed. Methods: A total of 234 clinical isolates were collected from the Department of Clinical Microbiology and Parasitology, HUSM from year 2010-2012. There were 49 S. Weltevreden isolates which have been confirmed by the Institute of Medical Research (IMR) using conventional serotyping method. Two sets of primers targeting a unique gene in S. Weltevreden (SWE349) and a pan-Salmonella gene (invA284) were designed using Primer3 online software, and a multiplex-PCR (mPCR) assay was developed to detect S. Weltevreden in clinical isolates. The optimized S. Weltevreden mPCR assay was evaluated on a panel of known Salmonella and non-Salmonella spp. to determine the specificity and sensitivity of the mPCR assay. Results & Discussion: The S. Weltevreden serovar specific primer, SWE349, and the internal control primer, invA284, resulted in 2 PCR products of 349bp and 284bp, respectively. The mPCR assay for S. Weltevreden targeting gene SWE349 showed 100% sensitivity (49/49) and specificity (36/36) in concordance with the conventional serotyping method in detecting only S. Weltevreden clinical strains, but not from the panel of 26 non-typhoidal Salmonella and 10 non- Salmonella isolates. Conclusion: A cost effective, sensitive and specific mPCR assay has been developed for the detection and confirmation of Salmonella Weltevreden from clinical isolates of gastroenteritis patients, using genes SWE349 and invA284.
Construction and characterization tolC deletion mutant doi: 10.1016/S2222-1808(14)60517-X
of a Salmonella enterica serovar Typhi 襃 2014
by the Asian Pacific Journal of Tropical Disease. All rights reserved.
Ashraf Hussain, Eugene Boon Beng Ong, Phua K.K., Prabha Balaram and Ismail A Institute for Research in Molecular Medicine (INFORMM), Main Campus, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
ABSTRACT
Introduction: Salmonella enterica serovar Typhi (S.Typhi), the organism responsible for typhoid fever, is a human specific pathogen that is able to survive in the macrophage and form biofilm in the host. TolC belongs to a family of outer membrane proteins that are found in all Gramnegative pathogenic bacteria. Together with two other proteins, AcrA and AcrB, TolC forms a channel through the outer membrane. Besides its role in the efflux of various molecules from the cell, TolC has been hypothesized to also play a key role in bacterial colonisation, adhesion and invasion of macrophages. Objectives: To investigate the role of the outer membrane protein TolC of S.Typhi in macrophage infection and biofilm formation. Methods: The tolC deletion mutant (吤tolC) strain of S.Typhi was constructed using a single-step gene knockout technique. S.Typhi cell invasion and biofilm formation ability of wild-type (WT) and 吤tolC strains were tested using an in vitro macrophage invasion assay and crystal violet biofilm assay, respectively. Results: The 吤tolC strain was compromised in their ability to infect macrophages and promote biofilm formation. The 吤tolC strain invaded THP-1 macrophage cells very poorly compared with the WT (counts for wild-type were mean 5.1伊105cfu/ml [n=4] compared with mean 1.6伊105cfu/ml [n=4] for 吤tolC; p<0.0001). Crystal violet biofilm assay revealed that WT strains were able to attach to polystyrene wells and form dense biofilm structures, whereas the 吤tolC strain were unable to do so (OD595 readings mean 3.43, SD 依 0.17 versus 0.30; SD 依 0.08 p<0.0001). Conclusions: Our data demonstrated the role of the S. Typhi TolC protein in biofilm formation and invasion of human macrophage cells.
1st International Conference on Molecular Diagnostic and Biomarker Discovery/Asian Pac J Trop Dis 2014; 4(3): 223-252
identification and detection of a unique gene in Salmonella enterica subsp. enterica Weltevreden using PCR-based approach
The
doi: 10.1016/S2222-1808(14)60516-8
Clarissa Tan , Goay Yuan Xin , Yeoh Chiann Ying , Asma Ismail 1
1
1
襃 2014
2
by the Asian Pacific Journal of Tropical Disease. All rights reserved.
and Phua Kia Kien1.
Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia Kubang Kerian, Kelantan, Malaysia, 2Universiti Sains Islam Malaysia, Negeri Sembilan, Malaysia.
1
ABSTRACT
Introduction: Salmonella enterica are important food-borne pathogens that cause human gastroenteritis, bacteremia and subsequent focal infections. The incidences of food-borne salmonellosis due to non-typhoidal Salmonella serotypes are increasing periodically in Malaysia. Other than the two predominant non-typhoidal Salmonella serotypes, S. Typhimurium and S. Enteritidis, an emerging serotype, S. Weltevreden, is increasingly being isolated from hospitalized-gastroenteritis patients in Malaysia. Serotyping is the conventional method for differentiation of this pathogen from the 2500 other serotypes known to exist. However, this method has a number of disadvantages including low specificity and high cost. Therefore, a molecular method for identification of S. Weltevreden in clinical isolates based on amplification of a specific DNA sequence present in its genome is proposed. Methods: A total of 234 clinical isolates were collected from the Department of Clinical Microbiology and Parasitology, HUSM from year 2010-2012. There were 49 S. Weltevreden isolates which have been confirmed by the Institute of Medical Research (IMR) using conventional serotyping method. Two sets of primers targeting a unique gene in S. Weltevreden (SWE349) and a pan-Salmonella gene (invA284) were designed using Primer3 online software, and a multiplex-PCR (mPCR) assay was developed to detect S. Weltevreden in clinical isolates. The optimized S. Weltevreden mPCR assay was evaluated on a panel of known Salmonella and non-Salmonella spp. to determine the specificity and sensitivity of the mPCR assay. Results & Discussion: The S. Weltevreden serovar specific primer, SWE349, and the internal control primer, invA284, resulted in 2 PCR products of 349bp and 284bp, respectively. The mPCR assay for S. Weltevreden targeting gene SWE349 showed 100% sensitivity (49/49) and specificity (36/36) in concordance with the conventional serotyping method in detecting only S. Weltevreden clinical strains, but not from the panel of 26 non-typhoidal Salmonella and 10 non- Salmonella isolates. Conclusion: A cost effective, sensitive and specific mPCR assay has been developed for the detection and confirmation of Salmonella Weltevreden from clinical isolates of gastroenteritis patients, using genes SWE349 and invA284.
Construction and characterization tolC deletion mutant doi: 10.1016/S2222-1808(14)60517-X
of a Salmonella enterica serovar Typhi 襃 2014
by the Asian Pacific Journal of Tropical Disease. All rights reserved.
Ashraf Hussain, Eugene Boon Beng Ong, Phua K.K., Prabha Balaram and Ismail A Institute for Research in Molecular Medicine (INFORMM), Main Campus, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
ABSTRACT
Introduction: Salmonella enterica serovar Typhi (S.Typhi), the organism responsible for typhoid fever, is a human specific pathogen that is able to survive in the macrophage and form biofilm in the host. TolC belongs to a family of outer membrane proteins that are found in all Gramnegative pathogenic bacteria. Together with two other proteins, AcrA and AcrB, TolC forms a channel through the outer membrane. Besides its role in the efflux of various molecules from the cell, TolC has been hypothesized to also play a key role in bacterial colonisation, adhesion and invasion of macrophages. Objectives: To investigate the role of the outer membrane protein TolC of S.Typhi in macrophage infection and biofilm formation. Methods: The tolC deletion mutant (吤tolC) strain of S.Typhi was constructed using a single-step gene knockout technique. S.Typhi cell invasion and biofilm formation ability of wild-type (WT) and 吤tolC strains were tested using an in vitro macrophage invasion assay and crystal violet biofilm assay, respectively. Results: The 吤tolC strain was compromised in their ability to infect macrophages and promote biofilm formation. The 吤tolC strain invaded THP-1 macrophage cells very poorly compared with the WT (counts for wild-type were mean 5.1伊105cfu/ml [n=4] compared with mean 1.6伊105cfu/ml [n=4] for 吤tolC; p<0.0001). Crystal violet biofilm assay revealed that WT strains were able to attach to polystyrene wells and form dense biofilm structures, whereas the 吤tolC strain were unable to do so (OD595 readings mean 3.43, SD 依 0.17 versus 0.30; SD 依 0.08 p<0.0001). Conclusions: Our data demonstrated the role of the S. Typhi TolC protein in biofilm formation and invasion of human macrophage cells.