Diversity of Clinical and Environmental Isolates of Vibrio cholerae in Natural Transformation and Contact-Dependent Bacterial Killing Indicative of Type VI Secretion System Activity.

The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS.

Competence and natural transformation in vibrios.

Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors: (i) nutrient limitation, (ii) availability of extracellular nucleosides, (iii) high cell density and (iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by co-ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.

Natural competence in Vibrio cholerae is controlled by a nucleoside scavenging response that requires CytR-dependent anti-activation.

Competence for genetic transformation in Vibrio cholerae is triggered by chitin-induced transcription factor TfoX and quorum sensing (QS) regulator HapR. Transformation requires expression of ComEA, described as a DNA receptor in other competent bacteria. A screen for mutants that poorly expressed a comEA-luciferase fusion identified cytR, encoding the nucleoside scavenging cytidine repressor, previously shown in V. cholerae to be a biofilm repressor and positively regulated by TfoX, but not linked to transformation. A ΔcytR mutant was non-transformable and defective in expression of comEA and additional TfoX-induced genes, including pilA (transformation pseudopilus) and chiA-1 (chitinase). In Escherichia coli, 'anti-activation' of nucleoside metabolism genes, via protein-protein interactions between critical residues in CytR and CRP (cAMP receptor protein), is disrupted by exogenous cytidine. Amino acid substitutions of the corresponding V. cholerae CytR residues impaired expression of comEA, pilA and chiA-1, and halted DNA uptake; while exogenous cytidine hampered comEA expression levels and prevented transformation. Our results support a speculative model that when V. cholerae reaches high density on chitin, CytR-CRP interactions 'anti-activate' multiple genes, including a possible factor that negatively controls DNA uptake. Thus, a nucleoside scavenging mechanism couples nutrient stress and cell-cell signalling to natural transformation in V. cholerae as described in other bacterial pathogens.

Staphylococcus aureus and Pseudomonas aeruginosa Isolates from the Same Cystic Fibrosis Respiratory Sample Coexist in Coculture.

Respiratory infections with bacterial pathogens remain the major cause of morbidity in individuals with the genetic disease cystic fibrosis (CF). Some studies have shown that CF patients that harbor both Staphylococcus aureus and Pseudomonas aeruginosa in their lungs are at even greater risk for more severe and complicated respiratory infections and earlier death. However, the drivers for this worse clinical condition are not well understood. To investigate the interactions between these two microbes that might be responsible for their increased pathogenic potential, we obtained 28 pairs of S. aureus and P. aeruginosa from the same respiratory samples from 18 individuals with CF. We compared the survival of each S. aureus CF isolate cocultured with its corresponding coinfecting CF P. aeruginosa to when it was cocultured with non-CF laboratory strains of P. aeruginosa. We found that the S. aureus survival was significantly higher in the presence of the coinfecting P. aeruginosa compared to laboratory P. aeruginosa strains, regardless of whether the coinfecting isolate was mucoid or nonmucoid. We also tested how a non-CF S. aureus strain, JE2, behaved with each P. aeruginosa CF isolate and found that its interaction was similar to how the CF S. aureus isolate interacted with its coinfecting P. aeruginosa. Altogether, our work suggests that interactions between S. aureus and P. aeruginosa that promote coexistence in the CF lung are isolate-dependent and that this interaction appears to be driven mainly by P. aeruginosa. IMPORTANCE Previous studies have shown that in laboratory settings, Pseudomonas aeruginosa generally kills Staphylococcus aureus. However, these bacteria are often found coinfecting the lungs of cystic fibrosis (CF) patients, which has been associated with worse patient outcomes. To investigate the interactions between these two bacteria, we competed 28 coinfection pairs obtained from the same lung samples of 18 different CF patients. We compared these results to those we previously reported of each CF S. aureus isolate against a non-CF laboratory strain of P. aeruginosa. We found that S. aureus survival against its corresponding coinfection P. aeruginosa was higher than its survival against the laboratory strain of P. aeruginosa. These results suggest that there may be selection for coexistence of these microbes in the CF lung environment. Further understanding of the interactions between P. aeruginosa and S. aureus will provide insights into the drivers of coexistence and their impact on the host.

Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill Staphylococcus aureus.

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes. Staphylococcus aureus (S. aureus) is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill S. aureus, it remains largely unknown why PMNs fail to eliminate S. aureus in CF. The goal of this study was to observe how the CF airway environment affects S. aureus killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of S. aureus were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of S. aureus tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum, S. aureus killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing S. aureus. Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of S. aureus was not diminished. The S. aureus-induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of S. aureus by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

Genotypic and Phenotypic Diversity of Staphylococcus aureus Isolates from Cystic Fibrosis Patient Lung Infections and Their Interactions with Pseudomonas aeruginosa.

Staphylococcus aureus has recently overtaken Pseudomonas aeruginosa as the most commonly recognized bacterial pathogen that infects the respiratory tracts of individuals with the genetic disease cystic fibrosis (CF) in the United States. Most studies of S. aureus in CF patient lung infections have focused on a few isolates, often exclusively laboratory-adapted strains, and how they are killed by P. aeruginosa Less is known about the diversity of S. aureus CF patient lung isolates in terms of both their virulence and their interaction with P. aeruginosa To begin to address this gap, we recently sequenced 64 clinical S. aureus isolates and a reference isolate, JE2. Here, we analyzed the antibiotic resistance genotypes, sequence types, clonal complexes, spa types, agr types, and presence/absence of other known virulence factor genes of these isolates. We hypothesized that virulence phenotypes of S. aureus, namely, toxin production and the mucoid phenotype, would be lost in these isolates due to adaptation in the CF patient lung. In contrast to these expectations, we found that most isolates can lyse both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), suggesting that these phenotypes were not lost during adaptation to the CF lung. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus isolates from CF patients, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.IMPORTANCEStaphylococcus aureus is now the most frequently detected recognized pathogen in the lungs of individuals who have cystic fibrosis (CF) in the United States, followed closely by Pseudomonas aeruginosa When these pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus Here, we present an in-depth study of 64 S. aureus clinical isolates from CF patients, for which we investigated genetic diversity utilizing whole-genome sequencing, virulence phenotypes, and interactions with P. aeruginosa We found that S. aureus isolated from CF lungs are phylogenetically diverse; most retain known virulence factors and vary in their interactions with P. aeruginosa (i.e., they range from being highly sensitive to P. aeruginosa to completely tolerant to it). Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

Whole-Genome Sequences of Staphylococcus aureus Isolates from Cystic Fibrosis Lung Infections.

Staphylococcus aureus is an early colonizer in the lungs of individuals with cystic fibrosis (CF), but surprisingly, only a limited number of genomes from CF-associated S. aureus isolates have been sequenced. Here, we present the whole-genome sequences of 65 S. aureus isolates obtained from 50 individuals with CF.

Vibrio cholerae: Measuring Natural Transformation Frequency.

Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation.