title: Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation.
abstract: Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase β chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical School, Naples, Italy SDN-Foundation, Naples, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Colicchio
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical School, Naples, Italy Ceinge Advanced Biotechnologies, Naples, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Pagliuca
authors.affiliation: Department of Sciences and Technologies, University of Sannio, Benevento, Italy.
authors.forename: Gabiria
authors.initials: G
authors.lastname: Pastore
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical School, Naples, Italy.
authors.forename: Annunziata Gaetana
authors.initials: AG
authors.lastname: Cicatiello
authors.affiliation: Department of Sciences and Technologies, University of Sannio, Benevento, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Pagliarulo
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy.
authors.forename: Adelfia
authors.initials: A
authors.lastname: Talà
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical School, Naples, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Scaglione
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical School, Naples, Italy.
authors.forename: Josè Camilla
authors.initials: JC
authors.lastname: Sammartino
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bucci
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy pietro.alifano@unisalento.it psalvato@unina.it.
authors.forename: Pietro
authors.initials: P
authors.lastname: Alifano
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical School, Naples, Italy Ceinge Advanced Biotechnologies, Naples, Italy pietro.alifano@unisalento.it psalvato@unina.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Salvatore
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003470:Culture Media; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D056084:Genetic Fitness; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009000:Monocytes / Q000187:drug effects / Q000382:microbiology; D009154:Mutation; D009345:Neisseria meningitidis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D018272:Porins / Q000235:genetics / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003470:Culture Media; C534217:HrpA protein, Neisseria meningitidis; D018272:Porins; D037521:Virulence Factors; C057699:porin protein, Neisseria; D012321:DNA-Directed RNA Polymerases; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase; D012293:Rifampin