Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed ⌬galT and ⌬cstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (⌬waaF and ⌬lgtF but not ⌬galT or ⌬cstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (⌬cstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The ⌬waaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

Campylobacter jejuni is a leading cause of foodbourne gastroenteritis, despite fragile behaviour under standard laboratory conditions. In the environment, C. jejuni may survive within biofilms, which can impart resident bacteria with enhanced stress tolerance compared to their planktonic counterparts. While C. jejuni forms biofilms in vitro and in the wild, it had not been confirmed that this lifestyle confers stress tolerance. Moreover, little is understood about molecular mechanisms of biofilm formation in this pathogen. We previously found that a ΔcprS mutant, which carries a deletion in the sensor kinase of the CprRS two-component system, forms enhanced biofilms. Biofilms were also enhanced by the bile salt deoxycholate and contained extracellular DNA. Through more in-depth analysis of ΔcprS and WT under conditions that promote or inhibit biofilms, we sought to further define this lifestyle for C. jejuni. Epistasis experiments with ΔcprS and flagellar mutations (ΔflhA, ΔpflA) suggested that initiation is mediated by flagellum-mediated adherence, a process which was kinetically enhanced by motility. Lysis was also observed, especially under biofilm-enhancing conditions. Microscopy suggested adherence was followed by release of eDNA, which was required for biofilm maturation. Importantly, inhibiting biofilm formation by removal of eDNA with DNase decreased stress tolerance. This work suggests the biofilm lifestyle provides C. jejuni with resilience that has not been apparent from observation of planktonic bacteria during routine laboratory culture, and provides a framework for subsequent molecular studies of C. jejuni biofilms.

Combinations of antibiotics, each individually effective against Mycobacterium abscessus, are routinely coadministered based on the concept that this minimizes the spread of antibiotic resistance. However, our in vitro data contradict this assumption and instead document antagonistic interactions between two antibiotics (clarithromycin and amikacin) used to treat M. abscessus infections. Clinically relevant concentrations of clarithromycin induced increased resistance to both amikacin and itself. The induction of resistance was dependent on whiB7, a transcriptional activator of intrinsic antibiotic resistance that is induced by exposure to many different antibiotics. In M. abscessus, the deletion of whiB7 (MAB_3508c) resulted in increased sensitivity to a broad range of antibiotics. WhiB7 was required for transcriptional activation of genes that confer resistance to three commonly used anti-M. abscessus drugs: clarithromycin, amikacin, and tigecycline. The whiB7-dependent gene that conferred macrolide resistance was identified as erm(41) (MAB_2297), which encodes a ribosomal methyltransferase. The whiB7-dependent gene contributing to amikacin resistance was eis2 (MAB_4532c), which encodes a Gcn5-related N-acetyltransferase (GNAT). Transcription of whiB7 and the resistance genes in its regulon was inducible by subinhibitory concentrations of clarithromycin but not by amikacin. Thus, exposure to clarithromycin, or likely any whiB7-inducing antibiotic, may antagonize the activities of amikacin and other drugs. This has important implications for the management of M. abscessus infections, both in cystic fibrosis (CF) and non-CF patients.

Mycobacterium abscessus is a rapidly emerging mycobacterial pathogen causing dangerous pulmonary infections. Because these bacteria are intrinsically multidrug resistant, treatment options are limited and have questionable efficacy. The current treatment regimen relies on a combination of antibiotics, including clarithromycin paired with amikacin and either imipenem or cefoxitin. Tigecycline may be added when triple therapy is ineffective. We initially screened a library containing the majority of clinically available antibiotics for anti-M. abscessus activity. The screen identified rifabutin, which was then investigated for its interactions with M. abscessus antibiotics used in drug regimens. Combination of rifabutin with either clarithromycin or tigecycline generated synergistic anti-M. abscessus activity, dropping the rifabutin MIC below concentrations found in the lung. Importantly, these combinations generated bactericidal activity. The triple combination of clarithromycin, tigecycline, and rifabutin was also synergistic, and clinically relevant concentrations had a sterilizing effect on M. abscessus cultures. We suggest that combinations including rifabutin should be further investigated for treatment of M. abscessus pulmonary infections.

Summary Campylobacter jejuni is a prevalent enteric pathogen that changes morphology from helical to coccoid under unfavorable conditions. Bacterial peptidoglycan maintains cell shape. As C. jejuni transformed from helical to coccoid, peptidoglycan dipeptides increased and tri‐ and tetrapeptides decreased. The DL‐carboxypeptidase Pgp1 important for C. jejuni helical morphology and putative N‐acetylmuramoyl‐L‐alanyl amidase AmiA were both involved in the coccoid transition. Mutants in pgp1 and amiA showed reduced coccoid formation, with ∆pgp1∆amiA producing minimal coccoids. Both ∆amiA and ∆amiA∆pgp1 lacked flagella and formed unseparated chains of cells consistent with a role for AmiA in cell separation. All strains accumulated peptidoglycan dipeptides over time, but only strains capable of becoming coccoid displayed tripeptide changes. C. jejuni helical shape and corresponding peptidoglycan structure are important for pathogenesis‐related attributes. Concomitantly, changing to a coccoid morphology resulted in differences in pathogenic properties; coccoid C. jejuni were non‐motile and non‐infectious, with minimal adherence and invasion of epithelial cells and an inability to stimulate IL‐8. Coccoid peptidoglycan exhibited reduced activation of innate immune receptors Nod1 and Nod2 versus helical peptidoglycan. C. jejuni also transitioned to coccoid within epithelial cells, so the inability of the immune system to detect coccoid C. jejuni may be significant in its pathogenesis.