In vitro passage alters virulence, immune activation and proteomic profiles of Burkholderia pseudomallei

Abstract

Serial passage is a problem among many bacterial species, especially those where strains have been stored (banked) for several decades. Prior to banking with an organization such as ATCC, many bacterial strains were passaged for many years, so the characteristics of each strain may be extremely different. This is in addition to any differences in the original host environment. For Burkholderia pseudomallei, the number of serial passages should be carefully defined for each experiment because it undergoes adaptation during the course of serial passages. In the present study, we found that passaged B. pseudomallei fresh clinical isolates and reference strain in Luria-Bertani broth exhibited increased plaque formation, invasion, intracellular replication, Galleria mellonella killing abilities, and cytokine production of host cells. These bacteria also modulated proteomic profiles during in vitro passage. We presume that the modulation of protein expression during in vitro passage caused changes in virulence and immunogenicity phenotypes. Therefore, we emphasize the need for caution regarding the use of data from passaged B. pseudomallei. These findings of phenotypic adaptation during in vitro serial passage can help researchers working on B. pseudomallei and on other species to better understand disparate findings among strains that have been reported for many years. Burkholderia pseudomallei is a Gram-negative facultative intracellular bacterium and the causative agent of melioidosis, a life threatening disease in humans and animals 1-3. Melioidosis is an important health problem due to poor understanding of the disease process, frequency of misdiagnosis, and lack of effective vaccination 4,5. Therefore, B. pseudomallei clinical and laboratory isolates have been extensively used to investigate pathogenic mechanisms of bacteria contributing to the disease. Serial passages are routinely performed to maintain an active culture or expand the bacterial number. Serial passages may influence bacterial adaptation through genotypic and phenotypic alterations. In a closely related organism, Burkholderia mallei, genome alterations (e.g. gene insertions, deletions, and repetitive sequences) were observed during in vitro passage 6. However, minimal information has been published regarding B. pseudomallei adaptation during serial passage. U' Ren et al. examined genomic mutations in B. pseudomallei that was serially passaged in vitro for 10 days; they found that tandem repeat genome mutations occurred, and suggested that these tandem repeat regions may contribute to generation and maintenance of adaptive genomic variation in B. pseudomallei 7. It was suggested that these tandem repeat regions may play role in generating and maintaining adaptive genomic variation in B. pseudomallei 7. It is likely that B. pseudomallei has an unstable genome 7 , which could be beneficial for bacterial adaptation and evolution. In addition, Price et al. characterized tandem repeat genome mutations in B. pseudomallei isolates from multiple body sites...

Long-Term In Vitro Passaging Had a Negligible Effect on Extracellular Vesicles Released by Leishmania amazonensis and Induced Protective Immune Response in BALB/c Mice

“…Similar studies with L. infantum and L. amazonensis kept for long periods in culture also reduced parasite infectivity [31,32]. Although the loss of infectivity has been observed in our study and by [32], attenuation of L. amazonensis using the in vitro passage was not as evident as demonstrated for other pathogens [33][34][35]. However, this model offers interesting tools for understanding some mechanisms involved in Leishmania infection and studying unknown virulence factors.…”

Galleria mellonella–intracellular bacteria pathogen infection models: the ins and outs

“…Serial passage of bacterial cultures is a regular practice amongst researchers to maintain an active culture or to harvest large numbers of bacteria. Prolonged passage is known to impact bacterial genotype or phenotype, resulting in alteration to virulence (Duangurai et al 2020 ). Sequencing of B. mallei cultures serially passaged in vitro over 10 days identified the introduction of tandem repeat genome mutations (Romero et al 2006 ).…”

Changes and composition of microbial community during aerobic composting of household food waste

“…Enterobacteriaceae family can contaminate fresh products at any stage of production either at pre-harvest or post-harvest stages (34). Many species of Burkholderia are opportunistic pathogens, such as Burkholderia pseudomallei that causes melioidosis (35) and Burkholderia cepacia that causes cystic fibrosis in human lungs (36,37). The relatively abundant bacterial genera in C1 is Ochrobactrum (84.61%), and the proportion of added bacteria is: Bacillus (1.89%), Pseudomonas (0.24%), Paenibacillus (0.01%).Similarly, Ochrobactrum also belongs to the Proteobacteria , and several Ochrobactrum spp.…”