Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

Abstract Background Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5′-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells. Results We observed that 20.3% (+/− 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner. Conclusions Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis

Resistance to Ceftriaxone and Azithromycin in Neisseria gonorrhoeae Isolates From 7 Countries of South America and the Caribbean: 2010-2011

Fil: Thakur, Sidharath Dev. Department of Microbiology, College of Medicine; Estados Unidos.Fil: Araya, Pamela. Instituto de Salud Publica de Chile, Santiago; Chile.Fil: Borthagaray, Graciela. Faculdad de Quimica, Universidad de la Republica, Montevideo; Uruguay.Fil: Galarza, Patricia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Hernandez, Alina Llop. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Medicina Tropical; Argentina.Fil: Payares, Daisy. Instituto Nacional Higiene "Rafael Rangel", Caracas; Venezuela.Fil: Sanabria Cruz, Olga Marina. Instituto National de Salud, Bogota; Colombia.Fil: Carvallo, Maria Elena Trigoso. Centro Departmental de Vigilancia Information y Referencia, CDVIR La Paz; Bolivia.Fil: Corredor, Aura Helena. Vaccine and infectious Disease Organization-International Vaccine Center, University of Saskatchewan, Saskatchewan; Canada.Fil: Dillon, Jo-Anne R. Vaccine and infectious Disease Organization-International Vaccine Center, University of Saskatchewan, Saskatchewan; Canada.Seven countries in Latin America and the Caribbean report on (2010 and 2011) the susceptibility of 2235 isolates of Neisseria gonorrhoeae to 6 antibiotics. Thirteen isolates had ceftriaxone minimum inhibitory concentrations (MICs) of 0.125 to ≥ 0.25 mg/L. The percentage of resistant isolates to the following antibiotics was: azithromycin, 1.0% to 1.7%; ciprofloxacin, 42.1% to 36.2%; penicillin, 31% to 35%; tetracycline, 21.8% to 22.6%

Quality assurance for antimicrobial susceptibility testing of Neisseria gonorrhoeae in Latin American and Caribbean countries, 2013-2015

Fil: Sawatzky, Pam. Streptococcus and STI Unit, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba; Canada.Fil: Martin, Irene. Streptococcus and STI Unit, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba; Canada.Fil: Galarza, Patricia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Carvallo, Marıa Elena Trigoso. Programa de ITS/VIH/SIDA, Laboratorio del Centro Departamental de Vigilancia, Informacion y Referencia, La Paz; Bolivia.Fil: Araya Rodriguez, Pamela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Cruz, Olga Marina Sanabria. Grupo de Microbiología, Instituto National de Salud, Bogota; Colombia.Fil: Hernandez, Alina Llop. Laboratorio Nacional de Referencia de Microbiología, Instituto de Medicina Tropical 'Pedro Kouri' (IPK), La Habana; Cuba.Fil: Martinez, Mario Fabian. Seccion Antimicrobianos, Laboratorio Central De Salud Pública, Asunción; Paraguay.Fil: Borthagaray, Graciela. Laboratorio de Microbiologia, Departamento de Biociencias, Facultad de Quimica, Universidad de la Republica, Montevideo; Uruguay.Fil: Payares, Daisy. Departamento de Bacteriologia, Instituto Nacional de Higine 'Rafael Rangel', Caracas; Venezuela.Fil: Moreno, José E. Laboratorio Central de Referencia en Salud Publica, Instituto Conmemeorahvo Gorgas de Estudios de la Salud, Panamá; Panamá.Fil: Chiappe, Marina. Facultad de Salud Pública y Administración, Universidad Peruana Cayetano Heredia, Lima; Peru.Fil: Corredor, Aura Helena. Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan; Canada.Fil: Thakur, Sidharath Dev. Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan; Canada.Fil: Dillon, Jo-Anne R. Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan; Canada.A Neisseria gonorrhoeae antimicrobial susceptibility quality control comparison programme was re-established in Latin America and the Caribbean to ensure antimicrobial susceptibility data produced from the region are comparable nationally and internationally