Impaired Virulence and Fitness of a Colistin-Resistant Clinical Isolate of Acinetobacter baumannii in a Rat Model of Pneumonia

Abstract

f We compared the fitness and lung pathogenicity of two isogenic clinical isolates of Acinetobacter baumannii, one resistant (ABCR) and the other susceptible (ABCS) to colistin. In vitro, ABCR exhibited slower growth kinetics than ABCS. In a rat model of pneumonia, ABCR was associated with less pronounced signs of infection (lung bacterial count, systemic dissemination, and lung damage) and a better outcome (ABCR and ABCS mortality rates, 20 and 50%, respectively [P ‫؍‬ 0.03]). The emergence of colistin-resistant strains of Acinetobacter baumannii is perceived as a formidable threat in clinical settings. Recent contradictory clinical observations and experimental data have questioned the virulence of such strains (1, 2, 3). Experimental models of surgical infections have previously assessed the virulence of A. baumannii with in vitro-induced colistin resistance (4), but a specific model of pneumonia with parental clinical isolates is required to focus on lung pathogenicity.We compared the fitness and virulence, in vitro and in vivo, of A. baumannii according to its sensitivity to colistin in a rat model of acute pneumonia using two isogenic clinical strains.Two strains of A. baumannii were successively isolated from the respiratory tract of a patient with ventilator-associated pneumonia. The first was colistin susceptible (ABCS), and the second was colistin resistant (ABCR). Whole-genome sequencing confirmed that they were parental strains, with ABCR differing from ABCS by a mutation in the pmrA and rpoB genes and by the loss of a prophage (5, 6).The ABCS strain was obtained from bronchoalveolar lavage fluid (colistin MIC ϭ 0.064 mg/liter) (5). The ABCR strain was isolated from tracheal aspirates after the patient had received intravenous colistin (colistin MIC ϭ 32 mg/liter) (5). Apart from the colistin and rifampin susceptibility of ABCS, both strains were resistant to all of the antibiotics tested, including cefepime and sulbactam.The 24-h growth curves of ABCR, ABCS, and reference strain AYE (7) showed a reduced slope in the exponential phase for ABCR versus ABCS (0.14 Ϯ 0.0038 versus 0.18 Ϯ 0.0053 [P ϭ 0.01]) and versus AYE (0.14 Ϯ 0.0038 versus 0.23 Ϯ 0.0066 [P ϭ 0.003]), indicating slower growth of ABCR bacteria.Both the ABCR and ABCS strains were tested for virulence in an animal model of acute pneumonia. Adult Sprague-Dawley male pathogen-free rats (weighing 300 to 350 g) were used for in vivo experiments after approval was obtained from the departmental ethics committee (study agreement 58-08112012).Sixty animals received 250 l of phosphate-buffered saline (PBS) containing 10 9 CFU/ml of ABCR (n ϭ 30) or ABCS (n ϭ 30) and 250 l of porcine mucin diluted to 10% via the tracheal route. Ten controls received only 250 l of PBS and 250 l of porcine mucin. The follow-up period lasted 48 h with daily body weight recording. After death, blood and spleen samples were cultured to assess bacteremia and the right lung was cultured. A histological severity score (HSS; minimum, 0; maximum, 4) was calculated on the ba...

Transcriptional and Mutational Profiling of an Aminoglycoside-Resistant Pseudomonas aeruginosa Small-Colony Variant

“…Polymyxin resistance due to the activation of the arnBCADTEF operon is associated with an altered LPS structure and thus might contribute to fitness costs and/or growth retardation (41,(49)(50)(51), which may also affect colony morphology (40,52). In some cases, reduced virulence was also observed (51,53).…”

Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia

“…For in vivo experiments [36] , aliquots containing P. aeruginosa PAO1 strain were thawed and cultured on COS (Biomerieux, France) (Columbia with 5% Sheep blood) agar plates. Ten fresh PAO1 colonies were sampled and cultured at 37°C in tryptic soy broth (TSB, Biomerieux, France) with continuous shaking until the OD 600 nm = 1.…”

Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria

“…Such costs have been observed in colistin-resistant isolates both in vitro and in vivo and include growth retardation, impaired virulence, and substantially reduced clinical invasiveness (Fernandez-Reyes et al, 2009 ; Lopez-Rojas et al, 2011 ; Rolain et al, 2011 ; Pournaras et al, 2014 ). Furthermore, Hraiech et al have reported that colistin-resistant A. baumannii possesses a reduced ability to cause infections, including systemic dissemination and lung damage, in a rat model with pneumonia (Hraiech et al, 2013 ). Some of these biological costs, such as the impairment of virulence, have been linked with the reduced expression of metabolic proteins and of the OmpA porin (involved in the virulence of Acinetobacter ) (Lopez-Rojas et al, 2011 ).…”

“…These proteins include outer membrane proteins, chaperones, translation factors, and enzymes involved in metabolism (Fernandez-Reyes et al, 2009 ). Interestingly, some of these isolates incurring biological cost also harbor mutations in the pmrA/pmrB TCS (Fernandez-Reyes et al, 2009 ; Lopez-Rojas et al, 2011 ; Rolain et al, 2011 ; Hraiech et al, 2013 ; Pournaras et al, 2014 ). Beceiro et al have recently demonstrated significant biological costs (in virulence and fitness) in colistin-resistant A. baumannii lpx mutants (lacking LPSs) compared to colistin-resistant A. baumannii pmrA/pmrB mutants (PEtN-modified LPSs) (Beceiro et al, 2014 ).…”