Adjunctive Clavulanic Acid Abolishes the Cefazolin Inoculum Effect in an Experimental Rat Model of Methicillin-Sensitive Staphylococcus aureus Endocarditis

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Staphylococcus aureus is a major pathogen in both community and health care settings, with a significant burden of morbidity and mortality (1). In many areas, a resurgence of methicillin-susceptible S. aureus (MSSA) infections has occurred, (2, 3) and despite in vitro susceptibility to a variety of antimicrobials, the treatment of invasive infections remains a challenge. Semisynthetic penicillins, such as nafcillin, have traditionally been used as a first-line therapy owing to their stability to hydrolysis by staphylococcal β-lactamases (4–6). However, recent retrospective clinical data have led some clinicians to favor cefazolin for definitive therapy, due to a favorable side effect profile, advantageous dosing schedule, and, possibly, an improved efficacy (4, 7).

The clinical failures of cefazolin in deep-seated MSSA infections, including infective endocarditis, have been described in association with isolates producing a β-lactamase (most commonly types A and C) (8–10) and showing an inoculum effect with cefazolin (CzIE). In vitro, the CzIE is defined as a significant increase in the MIC to 16 μg/ml or greater of cefazolin at a high inoculum (10⁷ or more CFU/ml compared to the MIC at the standard inoculum [10⁵ CFU/ml]) (11). It is assumed that clinical failures occur as a consequence of strains possessing the CzIE at a high bacterial burden at the site of infection. In a previously described experimental model of endocarditis in rats, an MSSA isolate positive for the CzIE was associated with significantly higher bacterial counts in vegetations when treated with cefazolin compared to those with nafcillin, ceftaroline, or daptomycin (12, 13). Furthermore, prospective observational data on the use of cefazolin in patients with MSSA bacteremia where the presence of the CzIE was assessed demonstrated that CzIE-positive isolates were associated with a significant increase in therapeutic failure and mortality (14, 15). Thus, strategies to preserve the usefulness of cefazolin in the setting of the CzIE are needed. In this study, we sought to examine whether the use of clavulanic acid, a commonly available β-lactamase inhibitor, could restore the efficacy of cefazolin against an MSSA isolate exhibiting the CzIE in vitro and in a rat model of endocarditis. We postulated that the inhibition of the staphylococcal β-lactamase, by impairing the degradation of cefazolin, would prevent the inoculum effect in vivo.

We initially sought to test this hypothesis in vitro by determining broth microdilution MICs at standard and high inocula. Three MSSA strains were tested: (i) ATCC 29213, a BlaZ-producing isolate lacking the CzIE, (ii) TX0117, a prototypical strain exhibiting the CzIE, and (iii) ATCC 25923, an MSSA strain lacking β-lactamase (Table 1). At a standard inoculum, there was minimal difference between the MICs of amoxicillin-clavulanate (AMC) and cefazolin for each of the strains. At a high inoculum, ATCC 25923 (without β-lactamase activity) had no change in cefazolin MIC and ATCC 29213 displayed an increase from 0.5 to 4 μg/ml (below the 16 μg/ml cutoff for the CzIE), while the cefazolin MIC for TX0117 was 128 μg/ml, consistent with prior studies (10). When tested in combination, even low concentrations of AMC restored the cefazolin MIC of TX0117 to below the 16 μg/ml cutoff in a dose-dependent manner (cefazolin MICs were reduced to 1, 0.5, and <0.125 μg/ml in the presence of 0.125, 0.5, and 2.5 μg/ml of the clavulanic acid component, respectively). Of note, for in vitro susceptibilities, AMC was tested together, as clavulanic acid is available clinically only as a coformulation with amoxicillin. The dual β-lactam combination of cefazolin plus ampicillin (at a fixed ampicillin concentration of 10 μg/ml, corresponding to the highest concentration of amoxicillin used with the coformulation) produced only a 1-fold dilution change in the MIC, showing that clavulanic acid and not a synergistic interaction between the two β-lactams was responsible for the observed shift in the MIC. To test whether β-lactamase inhibitors available for the intravenous (i.v.) route were also effective, MICs of cefazolin were performed in the presence of fixed subinhibitory concentrations of both ampicillin-sulbactam (SAM; 0.5/0.25 μg/ml) and piperacillin-tazobactam (TZP; 1/0.125 μg/ml). The sulbactam combination resulted in a 64-fold reduction (128 μg/ml to 2 μg/ml) in cefazolin MIC at a high inoculum, and the combination with tazobactam resulted in a 128-fold reduction (128 μg/ml to 1 μg/ml) (Table 1).

Using time-kill curves, we confirmed that clavulanic acid restored the efficacy of cefazolin at a high inoculum to levels comparable to that for cefazolin with a standard inoculum against TX0117 (Fig. 1). At a standard inoculum, a (3.5 ± 0.8)-log₁₀ decrease in CFU/ml was observed in the presence of 64 μg/ml of cefazolin. At a high inoculum, cefazolin alone at 64 μg/ml resulted in only a slight decrease of CFU/ml at 4 h, with regrowth to 8.2 × 10⁸ CFU/ml by 24 h, similar to that for TX0117 grown without antibiotics. The addition of AMC (1/0.25 μg/ml) to cefazolin restored the killing activity of cefazolin, with a (3.1 ± 0.4)-log₁₀ reduction in CFU/ml at 24 h. AMC alone (1/0.25 μg/ml) showed no inhibition of growth.

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FIG 1

Time-kill curve of S. aureus strain TX0117 at standard and high inocula. At a standard inoculum, growth in the presence of CFZ 64 μg/ml resulted in an approximately (3.5 ± 0.8)-log₁₀ reduction in CFU/ml at 24 h. At a high inoculum, growth in the presence of CFZ 64 μg/ml or AMC 1/0.25 μg/ml showed similar colony counts to the antibiotic-free control. The combination of CFZ 64 μg/ml plus AMC 1/0.25 μg/ml resulted in a reduction of 3 ± 0.4 log₁₀ CFU/ml at 24 h. These results are geometric means from three independent experiments; error bars represent the standard deviations. AMC, amoxicillin-clavulanate; CFZ, cefazolin.

Using the rat endocarditis model (12), we sought to examine whether the combination of cefazolin plus clavulanic acid would be effective in vivo at doses comparable to those achieved in human serum with i.v. administration of cefazolin (6-g total daily dose) and oral administration of clavulanic acid (125-mg dose) (16, 17). This protocol was approved by the Animal Welfare Committee at UTHealth. Rats infected with S. aureus strain TX0117 were treated beginning 30 h after inoculation with intramuscular injection of either cefazolin alone (50 mg/kg every 8 h) or cefazolin plus clavulanic acid (50 mg/kg and 4 mg/kg, respectively, given together every 8 h) for 72 h. The strain TX0117c, a derivative of TX0117 lacking β-lactamase activity (12), was used as a control for the cefazolin-only treatment arm. The differences in CFU between groups were compared using the Mann-Whitney Wilcoxon unpaired test, with significance at a P value of <0.05 using two-tailed significance levels. There was no significant difference in mean (± standard deviation [SD]) bacterial counts between TX0117 (7.3 ± 1.3 log₁₀ CFU/g vegetation) and TX0117c (7.9 ± 0.8 log₁₀ CFU/g) in the infected control animals prior to starting treatment (T = 0). Figure 2A shows that rats infected with TX0117 and treated with cefazolin alone showed a reduction of 2 ± 0.6 log₁₀ CFU/g after 72 h of antibiotic therapy. When cefazolin was given in combination with clavulanic acid, however, there was a (7.1 ± 0.5)-log₁₀ CFU/g reduction in bacterial colony counts (mean difference between arms of 5 ± 0.7 log₁₀ CFU/g, P < 0.0001). Importantly, 6 of the 7 vegetations from the combination treatment group were sterile, while all of the 11 vegetations from the cefazolin monotherapy group were infected. Furthermore, the efficacy of the combination of cefazolin plus clavulanic acid in this work is comparable to that of nafcillin (2.0 ± 2.9 log₁₀ CFU/g) as assessed in a previous study of rat endocarditis using S. aureus strain TX0117 published by our group (12). The effect of combination therapy with cefazolin plus clavulanic acid was comparable to that using cefazolin alone against TX0117 lacking β-lactamase (TX0117c) (Fig. 2B), with a mean reduction of 6.5 ± 0.6 log₁₀ CFU/g compared to that of untreated TX0117c controls (P < 0.0001).

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FIG 2

Clavulanic acid abolishes the cefazolin inoculum effect in a rat endocarditis model. (A) S. aureus TX0117 treated with CFZ plus CLA, CFZ alone, or untreated control animals (T = 0). Animals treated with CFZ plus CLA showed an additional mean reduction of 5 ± 0.7 log₁₀ in CFU/g of vegetation compared to CFZ alone after 72 h of therapy. (B) S. aureus TX0117c without BlaZ activity, treated with cefazolin alone showed a mean reduction of 6.5 ± 0.6 log₁₀ CFU/g compared to that of the untreated Bla-cured infected controls (T = 0), a decrease comparable to the parental strain treated with the cefazolin plus clavulanic acid combination (decrease of 7.1 ± 0.5 CFU/g). CLA, clavulanic acid; CFZ, cefazolin; βla+, β-lactamase positive; βla-cured, no β-lactamase activity; SD, standard deviation.

On the basis of the available clinical data, the use of cefazolin as a primary therapy for the treatment of serious MSSA infections is likely to increase. Clinical isolates of S. aureus possessing the blaZ gene are common (73% to 87% isolates) (11, 18), and studies have shown that the presence of the inoculum effect can range from 3% in low-prevalence settings to as much as 55% in settings where cefazolin is used as the front line agent for MSSA (15, 18). Rapid and efficient diagnostics for the detection of the CzIE in the clinical microbiology laboratory are not currently available, and although the investigation of rapid detection is ongoing, broth microdilution using a high inoculum remains the gold standard. Recent data from a prospective clinical study in Argentina where cephalosporins were used as a first-line therapy showed that patients infected with MSSA isolates exhibiting the CzIE were significantly more likely to have higher 30-day all-cause mortality (risk ratio [RR], 2.65; 95% confidence interval [CI], 1.10 to 6.42; P = 0.03) than those without the effect (15). A study in Korea supported these observations, as patients with CzIE-positive isolates treated with cefazolin were noted to have increased rates of treatment failure (61.5% versus 28.9%, P = 0.049) or increased all-cause mortality at 1 month (15.4% versus none, P = 0.047) compared to those with CzIE-negative isolates (14). This effect was not seen in those patients who received nafcillin, suggesting that the CzIE itself, and not the increased virulence of these strains, drives treatment failure. These observations suggest that effective therapeutic interventions to counter the CzIE are important when cefazolin is used as a first-line therapy. In the present study, we show that the addition of the β-lactamase inhibitor clavulanic acid is sufficient to restore the efficacy of cefazolin against the β-lactamase-producing isolate S. aureus TX0117 in vivo. This provides a proof of concept for the use of β-lactamase inhibitors in situations where the CzIE may be of clinical concern. Our observations, both in vitro and in vivo, suggest that even small concentrations of the β-lactamase inhibitor are sufficient to reverse the CzIE. While we administered clavulanic acid with each cefazolin dose, with the rationale of having the β-lactamase inhibitor at effective levels during the peak exposure to the drug, the ideal dosing regimen and duration of the combination therapy remain to be established. While the established treatment for MSSA suspected of possessing the inoculum effect is still an antistaphylococcal penicillin (i.e., nafcillin) when available, our data lend support to the addition of a β-lactamase inhibitor to cefazolin in certain scenarios (such as the intolerance of nafcillin, a need for less frequent dosing intervals, or in those areas where isoxazolyl penicillins are not readily available).

In summary, the expanding use of cefazolin as a first-line treatment for serious MSSA infections may increase the risk of therapeutic failures associated with the CzIE. We demonstrate that the addition of clavulanic acid to cefazolin abolishes the inoculum effect in a rat model of endocarditis with S. aureus TX0117, and this combination may serve as an interesting therapeutic strategy in the future in select situations.

(Parts of this work were presented at the American Society for Microbiology Microbe 2016 meeting and at IDWeek 2017.)