Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa - PubMed (original) (raw)

Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa

Lucas R Hoffman et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Opportunistic infections are often polymicrobial. Two of the most important bacterial opportunistic pathogens of humans, Pseudomonas aeruginosa and Staphylococcus aureus, frequently are coisolated from infections of catheters, endotracheal tubes, skin, eyes, and the respiratory tract, including the airways of people with cystic fibrosis (CF). Here, we show that suppression of S. aureus respiration by a P. aeruginosa exoproduct, 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), protects S. aureus during coculture from killing by commonly used aminoglycoside antibiotics such as tobramycin. Furthermore, prolonged growth of S. aureus with either P. aeruginosa or with physiological concentrations of pure HQNO selects for typical S. aureus small-colony variants (SCVs), well known for stable aminoglycoside resistance and persistence in chronic infections, including those found in CF. We detected HQNO in the sputum of CF patients infected with P. aeruginosa, but not in uninfected patients, suggesting that this HQNO-mediated interspecies interaction occurs in CF airways. Thus, in all coinfections with P. aeruginosa, S. aureus may be underappreciated as a pathogen because of the formation of antibiotic-resistant and difficult to detect small-colony variants. Interspecies microbial interactions, analogous to those mediated by HQNO, commonly may alter not only the course of disease and the response to therapy, but also the population structure of bacterial communities that promote the health of host animals, plants, and ecosystems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

HQNO produced by P. aeruginosa simultaneously suppresses the growth of S. aureus and protects it from tobramycin killing. (A) Colonies of P. aeruginosa grown on a lawn of S. aureus on LB agar plates with tobramycin added as indicated. WT, wild-type P. aeruginosa PAO1. The pqsA mutant is defective for HQNO production. The MIC of tobramycin for these P. aeruginosa strains is 1 μg/ml. White arrow, zone of S. aureus growth. S. aureus grows slowly in the zone surrounding the wild-type (WT) P. aeruginosa colony, most evident when the S. aureus lawn is suppressed by tobramycin concentrations >0.3 μg/ml, but similar S. aureus densities are present in the zones under each condition (data not shown). (B) A paper disk (containing 15 μg of pure HQNO) and colonies of P. aeruginosa on a lawn of S. aureus cells on agar media containing 0.6 μg/ml tobramycin. WT, wild-type P. aeruginosa PA14. The pqsL mutant is defective for HQNO production. White arrow, zone of S. aureus growth. (C) Experiment performed as in B, except without tobramycin. (D) S. aureus colonies (of cells from a diluted culture) growing on LB agar near disks containing HQNO or methanol solvent alone. For all P. aeruginosa experiments shown, equivalent results were obtained by using P. aeruginosa strains PAO1 and PA14 and their derived pqsA and pqsL mutants.

Fig. 2.

Fig. 2.

HQNO inhibits S. aureus electron transport, ultimately selecting for SCVs. (A) S. aureus grown for 5 days in static cultures alone, with P. aeruginosa PAO1, or with HQNO as indicated, before plating on selective media to distinguish species and morphotypes. Results shown are on sheep's blood agar. Cells from the culture with HQNO were incubated longer to better display SCVs. Results are representative of three separate experiments; equivalent results were obtained with P. aeruginosa strains PAO1 and PA14 and with S. aureus clinical isolates from five separate CF patients. White arrowheads, normal S. aureus. Black arrows, SCV S. aureus. White arrow, P. aeruginosa. (B) Colonies of cells from S. aureus cultures grown overnight in the presence of the indicated tobramycin and/or HQNO concentrations. Results are representative of two separate experiments. (C) Alamar blue, a redox-sensitive dye, was used to quantify reduction potential (as a measure of electron transport) of the S. aureus strains indicated in the presence and absence of HQNO. Results shown are the average of triplicates ± SD and are representative of three separate experiments. (D) Cells of S. aureus SCVs isolated after HQNO exposure form colonies of wild-type size when growing in close proximity to a disk containing 1.5 μg of menadione and with colony size diminishing with distance from the disk. No such colony changes were observed with disks containing thymidine or hemin. Results are representative of three separate experiments.

Fig. 3.

Fig. 3.

Synergy between HQNO and tobramycin for S. aureus SCV formation. Quantitative results of the experiment are described in Fig. 2_B_. The relative proportions of SCVs were determined among 100–300 total colony-forming units in overnight cultures of S. aureus grown in HQNO and tobramycin. Growth in the presence of either 10 μg/ml HQNO (Left) or 12 μg/ml tobramycin alone (Right) yielded ≤5% SCVs, whereas in combination, they yielded nearly 100% SCVs, consistent with synergy. Results are representative of two separate experiments, each with at least two technical replicates.

Fig. 4.

Fig. 4.

HQNO selects for S. aureus SCVs that are resistant to tobramycin. (A) Tobramycin susceptibility of wild-type S. aureus and of an SCV isolated after HQNO exposure. A disk containing 1.5 μg of tobramycin was placed on a S. aureus lawn on LB agar. (B) The MIC of tobramycin for wild-type S. aureus growing in the absence and in the presence of HQNO, and for SCVs derived as indicated. Results shown are averages of technical duplicates ± SD and are representative of three separate experiments. (C) The MIC of tobramycin, with and without menadione, for wild-type S. aureus and SCVs derived as indicated. Menadione restored sensitivity to tobramycin to S. aureus SCVs (but menadione itself was toxic to wild-type S. aureus at 10 μg/ml); results shown are representative of two separate experiments.

Fig. 5.

Fig. 5.

Model for interspecies interactions between P. aeruginosa and S. aureus. After short-term exposure (hours), S. aureus grown in the presence of P. aeruginosa producing HQNO becomes transiently resistant to aminoglycosides and, possibly, other antibiotics (20, 21). After long-term exposure (days), S. aureus becomes stably resistant because of selection for S. aureus SCVs. Such SCVs can persist intracellularly in nonprofessional phagocytes and are difficult to detect (20). The presence of tobramycin also can select for S. aureus SCVs either independently (20) or synergistically with HQNO (Fig. 3). The production of HQNO and, thus, selection pressure for S. aureus SCVs, could be augmented by increased biofilm formation, either due to reversible induction by aminoglycosides (27) or associated with hyperadherent P. aeruginosa SCVs (28).

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