What are Achromobacter spp. and how do they infect the lungs of people with cystic fibrosis?

Achromobacter spp. (Phylum: Pseudomonadota, Class: Betaproteobacteria, Order: Burkholderiales, Family: Alcaligenaceae) are Gram-negative, strictly aerobic, non-spore forming, rod-shaped bacteria with peritrichous flagella, and are commonly found in soil and aquatic environments. The type species Achromobacter xylosoxidans was first described in 1971, as an isolate from human ear discharge, and the genus was validly published in 1981, with several species of the closely related Alcaligenes subsequently reclassified as Achromobacter [1–3]. These bacteria are increasingly isolated from people who are immunocompromised or individuals with cystic fibrosis (CF), raising globally from ~2%–3% between the 1990s and early 2000s to ~7%–10% by the early 2020s [4–8]. In CF patients, Achromobacter prevalence and infection varies based on age, and adults (≥18 years old) are more likely to be colonized [9]. Infections with Achromobacter spp. are frequently difficult to treat because these bacteria harbor numerous antibiotic resistance genes [10]. For example, A. xylosoxidans is frequently resistant to aminoglycosides, aztreonam, and cephalosporins, due to the presence of multidrug resistance pumps from the resistance-nodulation-cell division superfamily [11].

In CF, mutations in the CF transmembrane conductance regulator gene lead to accumulation of thick and viscous mucus in the lungs which serves as a substrate for colonization by Achromobacter and other microbes from the environment [12]. The sinuses or other body sites may be potential reservoirs for Achromobacter, as patients have been reported to become reinfected with the same strain after a bilateral lung transplant. However, it may also be possible that these individuals became reinfected from the same environmental source [5]. Furthermore, patient-to-patient transmission has been observed, as patients in close contact during prolonged periods can harbor strains of A. xylosoxidans with the same genotype [13]. Similar cases of transmission between CF patients have also been identified for other Achromobacter species using genetic distance measurements and epidemiological data [14].

Which species of Achromobacter have been found to infect the lungs of people with CF?

The genus Achromobacter consists of 22 characterized and validly published species, among which 10 have been clinically isolated. Though the primary species associated with infection is A. xylosoxidans, many initial studies did not discriminate between this and other Achromobacter species. Identification of Achromobacter to the species level requires sequencing of the nrdA gene [15] or whole genome sequencing [14]. Other species that have been clinically isolated from CF patients are Achromobacter aegrifaciens, Achromobacter animicus, Achromobacter dolens, Achromobacter insolitus, Achromobacter insuavis, Achromobacter marplatensis, Achromobacter mucicolens, Achromobacter pulmonis, Achromobacter ruhlandii, and Achromobacter spanius. The prevalence of different Achromobacter species in CF patients varies based on geographical location. For example, A. insuavis has been isolated mostly from patients in Denmark and France, compared with the UK, Italy, and Argentina where the rate of its occurrence is low. In the USA, A. dolens is the most prevalent clinical isolate after A. xylosoxidans. In contrast, the occurrence of A. dolens is lower in the UK, Italy, and Denmark [9]. The higher prevalence of some Achromobacter species over others may signify variable environmental distribution [16].

What are the clinical effects of Achromobacter on the lungs of people with CF?

Multiple studies from CF clinics globally have reported associations between infection with Achromobacter spp. and lung function decline (Table 1). Notably, initial case-control studies by Hansen and colleagues revealed that chronic Achromobacter infection leads to significant loss in respiratory function over time, as measured with spirometry using forced expiratory volume in 1 s (FEV₁) [17,18]. Though these initial studies were limited with respect to sample size, an association between Achromobacter infection and FEV₁ decline either compared with pre-infection or measured over the course of a chronic infection has been repeatedly reported in clinical analyses [7,19–22], including from an analysis of >2,000 people with CF and Achromobacter infection in Europe [22]. Furthermore, these analyses have found that patients with Achromobacter infection tend to have increased numbers of pulmonary exacerbations (PEx) [7,19–21,23], leading to more hospitalizations [21,23,24], and are more likely to be prescribed antibiotics [7,22,23].

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Table 1. Associative studies supporting the role of Achromobacter spp. in disease in patients with CF.

https://doi.org/10.1371/journal.ppat.1013067.t001

What is the effect of Achromobacter infection on inflammation?

Achromobacter infections are associated with a strong inflammatory response. People with CF that are infected by Achromobacter spp. exhibit higher serum levels of the proinflammatory cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNFα) [18]. Accordingly, these individuals are also more likely to be prescribed steroids [7,9,10]. Consistent with these findings, mice infected with A. xylosoxidans have elevated levels of the above factors and the proinflammatory interleukins 1α (IL-1α) and 1β (IL-1β) in bronchoalveolar lavage fluid [25,26]. Moreover, the lungs of infected mice show signs of a strong inflammatory response, including alveolar wall thickening, edema, and accumulation of infiltrating neutrophils and other polymorphonuclear cells [25,26]. The degree of lung tissue damage is correlated with levels of TNFα and the anti-inflammatory interleukin 10 (IL-10) [25].

Genes encoding type III secretion systems (T3SS) are common among Achromobacter spp. These needle-like systems are used by Gram-negative bacteria to inject toxic effectors directly into eukaryotic cells in a contact-dependent manner. The most-well characterization of these effectors in Achromobacter is AxoU, an effector with phospholipase activity [27]. In in vitro infection models, A. xylosoxidans adheres to macrophage and macrophage-like cells using a large RTX adhesin named ArtA and is internalized via phagocytosis [28]. Once internalized, A. xylosoxidans activates expression of axoU and other T3SS-associated genes, which are required for its cytotoxicity [27]. Achromobacter insuavis and A. xylosoxidans both use the T3SS to kill macrophages by inducing inflammosome-dependent pyroptosis, which promotes IL-6 production and inflammatory lung damage in vivo [29]. Pyroptosis occurs even in the absence of AxoU, suggesting that the T3SS also delivers other effectors. In mice, A. xylosoxidans spreads from the lungs into the bloodstream, but mutants that are unable to produce a functional T3SS apparatus fail to disseminate [26] and do not induce lung inflammation [29]. In addition to pyroptosis-mediated inflammation, Achromobacter spp. likely produce additional inflammatory factors. For instance, culture supernatants from A. dolens, A. ruhlandii, and A. xylosoxidans upregulate CD11b in monocytes and neutrophils, which induces IL-6 and interleukin-8 (IL-8) release from CF bronchial epithelial cells [30]. The specific identities and underlying mechanisms of the potential contact-independent inflammatory factors produced by Achromobacter spp. are currently unknown.

It is possible that some of these inflammatory factors are secondary metabolites called siderophores. Bacteria and other microbes use siderophores to scavenge ferric iron and other metal ions from their external environment [31]. Siderophores can also contribute to inflammation and virulence. For example, Pseudomonas aeruginosa produces the siderophore pyoverdine, which kills macrophages in vitro, and whose production levels are positively correlated with mortality in murine and worm infection models [32,33]. A recent survey has indicated that 85% and 90% of clinical isolates of Achromobacter spp. from CF and non-CF patients, respectively, produce siderophores. Clinical isolates also produce higher siderophore levels compared with environmental strains [34]. However, it is not currently known how siderophore production is related to Achromobacter spp. virulence in vivo.

Do Achromobacter spp. interact with other CF pathogens during infection?

The lung microbiota in people with CF often comprises multiple bacterial and fungal species, including other pathogenic microbes. Individuals with chronic Achromobacter infection are frequently also co-infected with P. aeruginosa [7,10,19,22,35], Staphylococcus aureus [7,19,22–24], members of the Stenotrophomonas maltophilia complex [22], or nontuberculosis mycobacteria [22]. These co-infections can exacerbate lung function decline. For instance, people with CF that are co-infected with Achromobacter spp. and P. aeruginosa can have worse annual FEV₁ decline when compared with people with mono-infections of either pathogen [20,22]. These effects could be due to an additive effect of infection with two pathogens or may be due specifically to a synergistic effect of co-infection. It is possible that interactions between Achromobacter spp. and P. aeruginosa modulate production of virulence factors by one or both pathogens. For instance, a recent study determined co-culture of one A. xylosoxidans strain decreases P. aeruginosa biofilm formation, motility, siderophore production, and mortality in a zebrafish embryo infection model, while another strain had little effect [36]. It is important to note that in some studies FEV₁ decline in CF patients infected with Achromobacter spp. occurs independently of P. aeruginosa infection [19] and other studies have not found any specific effect on respiratory function associated with Achromobacter infection [6,10].

Achromobacter spp. engage in competitive and cooperative interactions with other CF lung microbes. Co-culture assays with clinical A. xylosoxidans and P. aeruginosa isolates revealed extensive interspecies interactions, with nearly half of the combinations resulting in alterations to growth, motility, or pigment production in P. aeruginosa, both positively and negatively [37]. The specific mechanisms used by Achromobacter spp. to mediate these interactions are unknown but may involve production of secondary metabolites or the type VI secretion system (T6SS). We highlighted siderophores as secondary metabolites that may act as virulence factors, but these same metabolites are frequently used by bacteria to sequester metals from their competitors [31]. Furthermore, a CF sputum isolate of A. xylosoxidans produces the secondary metabolite lasso peptide achromodin-1 [38]. Achromodin-1 is an RNA polymerase inhibitor that has a narrow activity spectrum, affecting only A. pulmonis and an Escherichia coli mutant with increased outer membrane permeability. This limited activity spectrum suggests that achromodin-1 may facilitate competition between closely related Achromobacter spp. co-infecting the lung [38]. In addition, most isolates of Achromobacter spp. encode components for a T6SS, designated as TAX-1 [39]. The T6SS apparatus has components with structural homology to bacteriophage tail proteins and is used by Gram-negative bacteria to deliver toxic proteins into adjacent cells [31]. One of the TAX-1 genes is a predicted effector with phospholipase D (PLD) activity. Strains of A. xylosoxidans can kill E. coli and P. aeruginosa in a T6SS-dependent manner, presumably using the PLD effector or another effector protein [39]. Proteomic analysis of the secretome of A. xylosoxidans revealed that T6SS components are produced in chemically defined medium that mimics the nutrient-limiting conditions of the CF lung and a T6SS component has been detected directly in sputa from people with CF, suggesting a role for this system in vivo [39].

Future directions

Achromobacter spp. are recently emerging and relatively understudied pathogens in the lungs of people with CF, where they are associated with lung function decline, increased PEx, and increased use of antibiotics and steroids. There is a diverse spectrum of Achromobacter species and knowledge regarding their clinical relevance is still limited. Some of these species are more prevalent in clinical settings, though the mechanisms underlying this phenomenon remain unknown. These bacteria provoke inflammation through mechanisms such as T3SS and other virulence factors, potentially including siderophores. Furthermore, Achromobacter spp. interact with other members of the CF lung microbiota, potentially influencing disease progression. However, the mechanisms underlying these interactions remain poorly understood, as does their impact on the composition of the microbiota and disease dynamics in the lungs of people with CF. Achromobacter spp. are also implicated in other chronic infections, including non-CF bronchiectasis [40], and have been recovered alongside P. aeruginosa in green-nail syndrome [41] and S. aureus in osteomyelitis [42], though whether their interactions in these environments parallel those observed in the CF lung is unknown. Investigating interactions between Achromobacter spp. and other microbes may lead to better understanding of the complex interactions among chronic infection microbiomes and lead to the identification of novel therapeutics.