Evolution of Staphylococcus aureus under Vancomycin Selective Pressure: the Role of the Small-Colony Variant Phenotype

Abstract

Staphylococcus aureus small-colony variants (SCVs) often persist despite antibiotic therapy. Against a 10 8 -CFU/ml methicillinresistant S. aureus (MRSA) (strain COL) population of which 0%, 1%, 10%, 50%, or 100% was an isogenic hemB knockout (Ia48) subpopulation displaying the SCV phenotype, vancomycin achieved maximal reductions of 4.99, 5.39, 4.50, 3.28, and 1.66 log 10 CFU/ml over 48 h. Vancomycin at >16 mg/liter shifted a population from 50% SCV cells at 0 h to 100% SCV cells at 48 h, which was well characterized by a Hill-type model (R 2 > 0.90). Staphylococcus aureus is a virulent pathogen responsible for a myriad of infections ranging from minor community-acquired skin and soft tissue infections to severe nosocomial infections (1). While the current IDSA guidelines recommend vancomycin as the primary agent for treatment of methicillin-resistant S. aureus (MRSA) infections, the utility of the drug has been brought into question due to increasing reports of heterogeneous resistance, treatment failure, and nephrotoxicity (2-4). Despite the global decrease in vancomycin susceptibility, the exact mechanism by which S. aureus develops resistance is not well understood (5). It has been suggested that S. aureus adapts by utilizing an array of genotypic alterations that arise stepwise during the selective pressure of antimicrobial therapy (6, 7).One pathway that S. aureus may exploit during the evolution of antimicrobial resistance is the development of small-colony variants (SCVs) that grow slowly relative to strains of the normal phenotype (NP) (8-10). In vitro testing and macrophage models have confirmed that the SCV phenotype is less susceptible to vancomycin (11, 12). Studies with other antibiotics also suggest that SCV subpopulations may cooperate with NP S. aureus to attenuate antimicrobial activity (13). At present, it is unknown whether SCVs alter vancomycin pharmacodynamics through interactions with NP S. aureus or how the selection of a vancomycin regimen influences the relationship between the two phenotypes. The objective of the current study was, therefore, to utilize reconstructive population biology to determine how the interplay of both phenotypes alters vancomycin pharmacodynamics.The MRSA strain COL (NP) and its isogenic hemB knockout Ia48 (COL hemB::ermB, a stable SCV phenotype) were utilized. The creation of the mutant strain and its features were previously characterized (14). Prior to each experiment, a solution of vancomycin was prepared using analytical-grade powder (Sigma Chemical, St. Louis, MO) in the following concentrations: 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/liter. Brain heart infusion (BHI) broth supplemented with magnesium (12.5 mg/liter) and calcium (25 mg/liter) was used for every experiment. SCV and NP cell suspensions were volumetrically titrated to achieve 5 different starting compositions, with a total bacterial load of 10 8 CFU/ml. Two experiments were conducted exclusively investigating the NP or the SCV phenotype (0% SCV/100% NP cells and 100% SCV/0% NP cells), an...

Clinical Significance and Pathogenesis of Staphylococcal Small Colony Variants in Persistent Infections

“…However, vancomycin is not bactericidal against SCVs (136). Moreover, Lenhard et al (203) showed recently in mixed-population experiments that vancomycin favorably selects for the growth of the SCV subpopulation. Thus, in cases of persistent severe MRSA infections with proven or possible SCV occurrence, a glycopeptide combination regimen or alternative MRSA-active antibiotics should be considered.…”

Sequential Evolution of Vancomycin-Intermediate Resistance Alters Virulence in Staphylococcus aureus: Pharmacokinetic/Pharmacodynamic Targets for Vancomycin Exposure

“…It has been established that alpha-toxin release activates the NLRP3-inflammasome and induces interleukin-1 and interleukin-6 secretion, while the VISA phenotype has been shown to provoke less tumor necrosis factor alpha and interleukin release than its vancomycin-susceptible counterparts (9,37,38). An investigation of the selective pressure of vancomycin on MRSA also found that vancomycin exposure amplifies the relative abundance of the small colony variant phenotype, which is a slowgrowing phenotype implicated in chronic and recurrent S. aureus infections, as well as intracellular persistence (25,39). The consequence of suboptimal vancomycin exposure is therefore not restricted to proliferating antibiotic resistance but is also seen in the shifting of the population dynamics of S. aureus toward a persis-tent state that is capable of enduring host countermeasures and exogenous antimicrobials.…”

“…3). In both USA300 and USA400, ratios of the AUC for the free, unbound fraction of the drug to the MIC (fAUC 24 /MIC ratios) of approximately 200, 371, 554, and 757 achieved a reduction in the total population's log ratio area of 0.344, 1.00, 2.00, and 3.00, respectively (R 2 ϭ 0.990), as described by a Hill-type function utilized previously (25).…”

“…The fAUC/ MICs needed to confer a reduction in the log ratio areas of 0.344, 1, 2, and 3 (black, blue, green, and purple, respectively) are listed above the corresponding vancomycin exposures. The data were described by a Hill-type function as described previously (25).…”

Comparative pharmacodynamics of four different carbapenems in combination with polymyxin B against carbapenem-resistant Acinetobacter baumannii

“…Time–kill experiments were conducted over 48 h at starting inocula of 10 6 CFU/mL and 10 8 CFU/mL in cation-adjusted Mueller–Hinton broth as detailed previously [10]. Solutions of ertapenem, doripenem, meropenem and imipenem (Sigma Chemical Co., St Louis, MO) were freshly prepared on the day of each experiment.…”