Burkholderia pseudomallei is an environmental bacterium and opportunistic pathogen found mainly in the soil and surface water of tropical and subtropical regions. It is the etiologic agent of melioidosis, an emerging public health concern and potential biothreat agent (Cheng and Currie, 2005; Cheng et al., 2005; Currie, 2015; Limmathurotsakul et al., 2016; Dance and Limmathurotsakul, 2018; Wiersinga et al., 2018). Infections with B. pseudomallei are most commonly acquired through inhalation or direct cutaneous inoculation of the organism into a human or animal host (Currie, 2015; Limmathurotsakul, et al., 2016; Dance and Limmathurotsakul, 2018; Wiersinga, et al., 2018). It is a major cause of sepsis and mortality in endemic tropical regions such as Southeast Asia and Northern Australia (Currie, 2015; Limmathurotsakul, et al., 2016; Dance and Limmathurotsakul, 2018; Wiersinga, et al., 2018; Kaewrakmuk et al., 2023). Furthermore, recent advances in the environmental isolation of B. pseudomallei and the increased identification of melioidosis cases in locations such as South Asia, Africa, and the Americas, clearly support the expanded ecological range of B. pseudomallei and its clinical impact (Limmathurotsakul, et al., 2016; Dance and Limmathurotsakul, 2018).

Melioidosis is also being detected in the United States. Most of the cases have been related to travel to areas with endemic melioidosis or to exposure to contaminated imported products (Dawson et al., 2021; Gee et al., 2022; Currie et al., 2023). However, three non-travel-associated cases of melioidosis in Mississippi, which were found to be genetically associated with B. pseudomallei isolated locally from soil and water samples, clearly suggest that B. pseudomallei is endemic in the southern region of the USA (CDC, 2022; Currie, et al., 2023). Similar cases in Texas have been reported that were suggestive of local acquisition; but attempts to isolate a comparable strain from environmental samples were unsuccessful (Cossaboom et al., 2020). Burkholderia thailandensis, an opportunistic pathogen which only rarely causes human infections, is closely related to B. pseudomallei, and is similarly found in the environment. B. thailandensis has been isolated from water in Texas and Puerto Rico and from the soil in Mississippi, demonstrating an environment suitable for Burkholderia species colonization in parts of the Southern United States and in outlying territories (Hall et al., 2023). Interestingly, climate change appears to play a role in the increased range and magnitude of infections by these bacteria (Gassiep et al., 2023).

Melioidosis is a complex disease with manifestations ranging from acute and rapidly fatal to protracted chronic infections. Infected individuals usually display symptoms of acute disease (pneumonia, bacteremia, or localized infection) upon medical evaluation. However, ~9-15% manifest a chronic infection, with symptoms lasting over two months, or are subclinically infected (Cheng et al., 2005; Currie et al., 2010; Currie, 2015; CDC, 2022; Seng et al., 2023). Most patients have coexisting risk factors for the disease including diabetes, excessive alcohol use, chronic lung or renal disease, and other immunocompromising conditions (Currie et al., 2010; Currie, 2015; Limmathurotsakul et al., 2016; Dance and Limmathurotsakul, 2018; Wiersinga et al., 2018; Kaewrakmuk et al., 2023). Several animal models have been developed to study the pathogenesis of melioidosis (Leakey et al., 1998; Conejero et al., 2011; Limmathurotsakul et al., 2015; Welkos et al., 2015; Amemiya et al., 2017; Bearss et al., 2017; Trevino et al., 2018; Trevino et al., 2021; Nelson et al., 2023). The BALB/c is a highly susceptible strain of mice and is typically used to model acute melioidosis; in contrast, the C57BL/6 mouse strain is significantly more resistant, representing a more chronic model of disease which is often used for vaccine evaluation (Leakey et al., 1998; Tan et al., 2008; Lever et al., 2009; Srisurat et al., 2010; Conejero et al., 2011; Massey et al., 2014; Amemiya et al., 2017; Bearss et al., 2017; Trevino et al., 2018; Funnell et al., 2019; Nelson et al., 2023). By varying the dose of B. pseudomallei delivered by aerosolization (a relevant route when evaluating countermeasures for public health or biodefense purposes), both acute and more protracted models of disease have been modelled in BALB/c mice (Srisurat et al., 2010; Bearss et al., 2017; Funnell et al., 2019).

Mice infected with B. pseudomallei can display acute or chronic disease which resembles that of human melioidosis (Tan et al., 2008; Srisurat et al., 2010; Conejero et al., 2011; Massey et al., 2014; Bearss et al., 2017; Burtnick et al., 2018; Amemiya et al., 2019; Klimko et al., 2022). Despite their sensitivity, BALB/c mice (which survive the initial phase of infection due to intervention with medical countermeasures) can continue to succumb to disease throughout the course of the experimental infection. This apparent persistence of the infection is observed despite resolution of symptomatic disease and has been reported in naïve animals as well as those immunized with vaccines and treated with antibiotics. This has been demonstrated, in studies in which the animals are monitored for two to three months post exposure, with bacteria often recovered in tissues collected from survivors, e.g., spleen, liver, and lungs, at the study endpoint. (Conejero et al., 2011; Burtnick et al., 2018; Biryukov et al., 2022; Klimko et al., 2022). This chronic infection state could be attributable to the ability of B. pseudomallei to survive intracellularly, e.g., in pyogranulomatous lesions (Valvano et al., 2005; Finlay and McFadden, 2006; Limmathurotsakul et al., 2006; Conejero et al., 2011; Titball et al., 2017; Avraham, 2023; Seng et al., 2023) potentially facilitated by the formation of multi-nucleated giant cells, antibiotic-resistant biofilms and/or the development of a bacterial ‘persister’ state (Velapatino et al., 2012; Lazar Adler et al., 2013; Butt et al., 2014; Austin et al., 2015; Stockton and Torres, 2020). Alternately, the apparent persistence or relapse of infection may be due to re-infection following clearance of the initial infection.

Our objective was to evaluate the basis of protracted infection with delayed mortality in the BALB/c mouse model. Whilst B. pseudomallei is not typically transmissible (person-to-person spread has rarely been documented) and the bacteria are not known to be readily transmitted between animals (Aziz et al., 2020; Rees et al., 2021), we hypothesized that mice exposed to infection and recovered following treatment could potentially become re-infected by chronically-infected mice (Cheng and Currie, 2005; Cheng et al., 2005; Wiersinga et al., 2018; DPIRD, 2018). In the current study, we tested this hypothesis by cohousing naïve mice with mice exposed to B. pseudomallei by the aerosol or parenteral routes of infection and followed the disease progression for several months. Whilst infrequent, we showed that infected mice can infect naïve mice when cohoused. It is impractical to avoid cohousing laboratory rodents, however, the possibility that animals showing clinical signs of infection can infect other animals must be considered when interpreting in vivo data, particularly from long-duration therapeutics studies.

Mouse models of melioidosis have been invaluable to understand B. pseudomallei pathogenesis and for the development and evaluation of novel medical countermeasures to treat melioidosis. Importantly, there are several “waves” of clinical illness that can be observed when performing long-duration laboratory evaluations. We evaluated the hypothesis that clinically ill mice could infect naive mice or theoretically reinfect cage mates that had cleared an initial bacterial infection during medical countermeasure evaluations. In this manuscript, we have demonstrated that mouse-to-mouse transmission of B. pseudomallei is infrequent in the laboratory setting, but possible. Whilst not enough naïve mice became infected for statistical analyses, in both studies, transmission was observed in mice housed in ventilated cages. It is tempting to correlate the ventilated caging system with the possible aerosolization of bacteria and subsequent infection of naïve mice, however, it is impossible to draw that conclusion with our current data set. Additionally, there are reports suggesting that rodents housed in ventilated caging systems are the least-likely to transmit infectious diseases to cohoused mice (Compton et al., 2004). As melioidosis is known to be transmitted via ingestion of contaminated material (Sanchez-Villamil et al., 2020; Nelson et al., 2021), we believe that other routes of transmission are possible, including ingestion or inhaling contaminated fecal material or urine and by fomite transmission via the watering system. Additionally, whilst cannibalism of dead mice was not observed in the cages where transmission was observed, it is another possibility for a route of infection if the tissues consumed by cage mates contain B. pseudomallei (Lane-Petter, 1968; Nicklas et al., 2012). We also demonstrated that several naïve cage mates with no clinical signs of melioidosis had anti-B. pseudomallei antibody titers ( Tables 2 , 3 ). Whilst it is possible that these mice were colonized with B. pseudomallei at concentrations below the level of detection or in an organ not sampled, the mice had no detectable bacteria in their lungs or spleens. This seroconversion of seemingly healthy mice may be reminiscent of what is reported in humans living in highly endemic areas and the debate regarding the prevalence of subclinical infections in healthy, yet seropositive, individuals (Limmathurotsakul and Peacock, 2011; Nithichanon et al., 2018).

It is our interpretation that this low-level of transmission, reported here, is unlikely to impact the results of vaccination experiments performed with C57BL/6 mice due to their increased resistance to B. pseudomallei, but this assumption has not been investigated (Tan et al., 2008; Srisurat et al., 2010; Conejero et al., 2011; Massey et al., 2014; Bearss et al., 2017; Burtnick et al., 2018; Amemiya et al., 2019; Klimko et al., 2022). This low-level of transmission should be considered when analyzing data obtained from lengthy therapeutic studies using more sensitive animal models (i.e. BALB/c mouse or hamster) (Amemiya et al., 2017; Nelson et al., 2023). Whilst it is impractical to singly house mice for numerous reasons (including ethical reasons) in certain circumstances it may be prudent to isolate animals showing signs of disease to reduce the risk of them infecting their cage mates that may have resolved the infection after medical countermeasure intervention (Wurbel, 2001; Kappel et al., 2017). These extra measures may only be warranted for pivotal studies in cases where medical countermeasures are undergoing advanced development, and even then, it would be difficult to implement for mouse studies.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal study was approved by The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) Institutional Animal Care and Use Committee (IACUC). The study was conducted in accordance with the local legislation and institutional requirements.

CK: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing – review & editing. KB: Conceptualization, Writing – review & editing. NR: Investigation, Writing – review & editing. JS: Investigation, Writing – review & editing. JD: Data curation, Formal Analysis, Investigation, Writing – review & editing. MH: Investigation, Writing – review & editing. SW: Writing – original draft. DD: Funding acquisition, Writing – review & editing. SB: Formal Analysis, Investigation, Writing – review & editing. SH: Conceptualization, Writing – review & editing. CC: Conceptualization, Writing – review & editing, Data curation, Formal Analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft.