Clinical Implications of Cefazolin Inoculum Effect and β-Lactamase Type on Methicillin-Susceptible Staphylococcus aureus Bacteremia

Abstract

Background: Cefazolin is a common antibiotic for methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. Type A or C β-lactamase-producing MSSA frequently shows the cefazolin inoculum effect (CIE). However, the clinical implication of the CIE for MSSA bacteremia is obscure. Methods: MSSA bacteremic patients treated with cefazolin were included in a retrospective cohort study. The blaZ gene of the isolates was sequenced to identify the type of β-lactamase. The patients whose isolates showed a ≥4-fold increase in cefazolin, the minimal inhibitory concentration (MIC) at the high inoculum (∼5×10⁷ CFU/ml), were assigned to the CIE-positive group and the remainder to the CIE-negative group. Treatment failure was assessed at 12 weeks after cefazolin was initiated. Results: A total of 113 MSSA bacteremic patients were included. Among the 113 isolates, 57.5% showed the CIE and 77.9% carried the blaZ gene; type A β-lactamase was 15.0% and type C was 40.7%. Persistent bacteremia was more common in the CIE-positive group (9% vs. 0%, p=0.04). Treatment failure rates were higher in the CIE-positive group with high bacterial burden infection, but the difference was not significant (48% vs. 25%, p=0.13). There was no significant difference of failure between groups with high-inoculum MIC ≥16 and ≤1 μg/ml (13% vs. 5%, p=0.31). In the multivariable analysis, underlying cardiovascular diseases, pneumonia, osteoarticular infections, and endocarditis were significant risk factors for treatment failure and the CIE was not significantly associated with treatment failure. Conclusion: The CIE might be associated with persistent bacteremia if cefazolin is used for MSSA bacteremia with a high burden of infections. However, the sites of infections are more important factors for the clinical outcome than the CIE.

Introduction

The β-lactamase-producing Staphylococcus aureus has disseminated worldwide since the widespread use of penicillin. Approximately 90% of the clinical S. aureus isolates are β-lactamase producers.²⁸ The β-lactamases have four variants (A, B, C, and D) based upon the amino acids at residues 128 and 216.^13,14,26,31 Each of the four variants has different hydrolytic kinetics to other β-lactam antibiotics.³² Type A β-lactamase efficiently hydrolyzes cefazolin, and the type C β-lactamase also hydrolyzes cefazolin.³³ Cefazolin is a common antibiotic for methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. However, there is concern that cefazolin use for severe MSSA infections is associated with treatment failure because it can be hydrolyzed by β-lactamase produced by S. aureus.^{3,21,22,25,27} Despite concerns over the risk of treatment failure, cefazolin is widely used for MSSA infections and is recommended as an alternative agent to antistaphylococcal penicillins for MSSA endocarditis because of its convenient dosing and tolerability.^2,17

Nannini et al.²³ demonstrated that a pronounced inoculum effect against cefazolin was present in one-fifth of the clinical MSSA strains, the strains that produced the type A or C β-lactamases, and suggested that these strains have significant clinical implications in infections with a high bacterial burden such as endocarditis, osteomyelitis, septic arthritis, pneumonia, and large abscesses. However, previous studies were small, making it difficult to clearly discern the clinical implications of the cefazolin inoculum effect (CIE).²³

The purpose of this study was to evaluate the clinical impact of the CIE on outcomes of MSSA bacteremia treated with cefazolin.

Materials and Methods

Study design and patients

A retrospective cohort study and molecular analysis of the blaZ gene with clinical isolates were performed. From January 2004 to June 2009 in Seoul National University Hospital, and from March 2008 to March 2013 in Kyungpook National University Hospital and Daegu Fatima Hospital, all patients who had MSSA bacteremia and received cefazolin as definite antibiotics and whose isolates were preserved in repositories were included. Demographic and clinical data were reviewed, retrospectively. The Institutional Review Board at Daegu Fatima Hospital approved the study protocol and informed consent was waived.

Definitions

The sites of infection were defined as follows: catheter-associated infection was considered the source of bacteremia if the catheter had been in place for ≥72 hr, if the culture of a specimen of purulent drainage from the insertion site showed S. aureus, or if clinical signs improved after the catheter was removed and there was no other source of bacteremia.¹⁵ Pneumonia was considered the source of S. aureus bacteremia if the patient had clinical symptoms and signs of a lower respiratory tract infection and if there was radiological evidence of pulmonary infiltrates not attributable to other causes. Soft tissue infection was considered the source of S. aureus bacteremia when patients had an S. aureus culture from a tissue, or a drainage specimen from the affected site, as well as signs of infection.¹⁷ Surgical wound infection followed the definition of the US Centers for Disease Control and Prevention.⁷ The McCabe classification, performed by a clinician (Shinwon Lee), designated the severity of the underlying illness.¹⁹ High bacterial burden was defined as MSSA bacteremia accompanying endocarditis, osteoarticular infections, pneumonia, deep-seated abscess, or metastatic infection.²³ High-dose cefazolin treatment was considered if patients with glomerular filtration rate (GFR) >50 ml/min/1.73 m² received ≥6 g/day, patients with GFR 50–10 ml/min/1.73 m² received ≥4 g/day, and patients with GFR <10 ml/min/1.73 m² received ≥2 g/day of cefazolin. Persistent bacteremia was defined as persisting MSSA bacteremia by ≥72 hr after cefazolin treatment. Treatment failure was defined as (i) switching of antibiotics due to the clinician's decision of the treatment failure (i.e., a lack of improvement of the clinical symptoms and signs, decided by the clinicians involved in the patient's care), (ii) recurrence of MSSA infection (i.e., resolution of clinical signs of infection during therapy, but recurrent MSSA infection), or (iii) MSSA bacteremia-associated mortality.¹⁷ All outcomes, except persistent bacteremia, were assessed at 12 weeks after MSSA bacteremia initiated.

Susceptibility tests

The minimal inhibitory concentrations (MICs) of MSSA strains against cefazolin were determined by a broth microdilution method using the cation-adjusted Mueller-Hinton II broth (Becton, Dickinson and Company, Sparks, MD), according to the Clinical and Laboratory Standards Institute (CLSI) guidelines,⁶ except the inoculum size of the strains. Cefazolin was obtained from Sigma-Aldrich Chemicals (St. Louis, MO). MICs of high inoculum (∼5×10⁷ CFU/ml) were compared with standard inoculum (∼5×10⁵ CFU/ml) to identify the stains with the CIE. The inoculum was estimated by optical density measurements. For the high inocula, dilute inoculums were matched with a 0.5 McFarland standard, concentrated by centrifugation, and resuspended in one-fifth of the original volume of the broth.¹⁸ The MICs were read by one observer who was blinded to the clinical outcomes. We defined the CIE as a more than fourfold increase in the MIC from the standard inoculum to the high inoculum and marked the CIE as an increased MIC ≥16 μg/ml at the high inoculum. The S. aureus strain TX 0117 (high-level producer of type A β-lactamase),²² S. aureus ATCC 29213 (known to produce small amounts of type A β-lactamase),¹³ and S. aureus ATCC 25923 (β-lactamase-negative strain) were used as controls.

Sequence analysis

All isolates underwent extraction of genomic DNA by the spin-column-based extraction method using commercially available kits (Solgent, Daejeon, South Korea). PCRs were performed using the following primers designed to amplify a 355-bp region within the structural blaZ gene: 5′-CAAAGATGATATAG TTGCTT ATTC-3′ and 5′-CAT ATGTTATTGCTTGCACCAC-3′.²³ Identification of the blaZ gene and verification of the PCR product size were performed by gel electrophoresis. PCR products with a positive blaZ gene were analyzed by DNA sequencing. Sequence analysis was performed using the NCBI BLAST network service (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The classification of the β-lactamase type of each strain was based on the amino acids at residues 128 and 216 encoded by the blaZ gene. For β-lactamase type A, threonine is found at position 128 and serine is at position 216; for type B, lysine at position 128 and asparagine at position 216; for type C, threonine at position 128 and asparagine at position 216; and for type D, alanine at position 128 and serine at position 216.³¹

Statistical analyses

The SPSS software, version 18.0 (SPSS, Inc., an IBM Company, Chicago, IL), was used for all statistical analyses. Fisher's exact test or Pearson χ² test was used as appropriate to compare categorical variables, and continuous variables were compared using Student's t-test. Subgroup analyses of those with high burden infections were performed by the Mantel–Haenszel χ² test. To evaluate the clinical impact of the CIE and type A β-lactamase while adjusting for potential confounders, the multivariable logistic regression analysis was used. The variables, for which p-value was≤0.2 in a bivariable analysis, were included in a multivariable logistic regression model and stepwise backward elimination was performed to create a final model. To estimate the clinical implications of the positive CIE—marked CIE—and type A blaZ, the variables were separately forced into the final model of multivariable analysis. All tests of significance were two-tailed; p≤0.05 was considered to be statistically significant.

Results

One hundred thirteen patients were included: 90 patients from Seoul National University Hospital, 15 patients from Daegu Fatima Hospital, and 8 patients from Kyungpook National Hospital. Of the 113 patients with MSSA bacteremia, 66 (58.4%) were male and 68 (60.2%) were community-acquired infections. Skin and soft tissue infections were most common (34.5%), following osteoarticular infections (22.1%), primary bacteremia (20.4%), catheter-associated infections (17.7%), pneumonia (8%), and endocarditis (3.5%). Eleven patients (9.7%) had metastatic infections. β-lactam antibiotics were empirically administered in 77.9%, glycopeptide in 33.6%, and β-lactam/glycopetide combination in 15.9%.

Among the 113 MSSA isolates from patients, 88 (77.9%) were positive for the blaZ gene—17 (15%) carried type A, 24 (21.2%) type B, 46 (40.7%) type C, and 1 (0.9%) carried the type D blaZ gene. The geometric mean MIC, MIC₅₀, and MIC₉₀ of the isolates classified by the type of β-lactamases are shown in Table 1. Sixty-five isolates (57.5%) showed CIE. All the CIE-positive isolates showed increased MICs to ≥4 μg/ml and 23 (20.4%) showed increased MICs to ≥16 μg/ml at high inoculum (marked CIE). Of the 27 type A blaZ-positive strains, 94.1% showed the CIE and 64.7% showed a marked CIE. Of the 56 type C blaZ-positive strains, 95.7% showed the CIE and 26.1% showed a marked CIE (Table 1).

Table 1.

Mean Cefazolin MICs and CIEs Among the 113 MSSA from Total Patients and Classified by the Type of β-Lactamases

Type of β-lactamase	Total (n=113)	Type A (n=17)	Type B (n=24)	Type C (n=46)	Type D (n=1)	Bla−(n=25)
MICs in standard inocula (μg/ml)
Mean (GM) MIC	1.03	1.13	1	1	1	1
MIC₅₀	1	1	1	1	1	1
MIC₉₀	1	2	1	1	1	1
MICs in high inocula (μg/ml)
Mean (GM) MIC	3.9	18.83	1.46	7.65	1	1.06
MIC₅₀	4	32	1	8	1	1
MIC₉₀	16	64	4	16	1	1
No. (%) of inoculum effect-positive strains
Increase to ≥4-folds	65 (57.5)	16 (94.1)	4 (16.7)	44 (95.7)	0	1 (4.0)
Increase to MIC ≥16 μg/ml^a	23 (20.4)	11 (64.7)	0	12 (26.1)	0	0

MIC ≥16 μg/ml is nonsusceptible range of cefazolin according to the Clinical and Laboratory Standards Institute guidelines.

Bla−, isolates without blaZ gene; CIE, cefazolin inoculum effect; GM, geometric mean; MIC, minimal inhibitory concentration; MSSA, methicillin-susceptible Staphylococcus aureus.

Sixty-five patients (57.5%) whose isolates were positive for the CIE were assigned to the CIE-positive group and 48 patients whose isolates were negative for the CIE were assigned to the CIE-negative group. The clinical characteristics, demographic data of the patients, usage of empirical antibiotics, and the doses of cefazolin in both groups were comparable, and details are shown in Table 2. However, type A and C β-lactamase were predominant in the CIE-positive group and type B was predominant in the CIE-negative group (Table 2).

Table 2.

Demographics and Clinical Characteristics of 113 Patients with MSSA Bacteremia and Type of β-Lactamase of Their Isolates in the CIE-Positive and -Negative Groups

Characteristic	CIE-P (n=65)	CIE-N (n=48)	p-Value
Mean age (years, mean±SD)	56.5±18.9	57.2±16.8	0.83
Male	41 (63.1)	25 (52.1)	0.24
Community-acquired infection	36 (55.4)	32 (66.7)	0.23
McCabe classification of
Nonfatal	30 (46.2)	20 (41.7)	0.64
Ultimately fatal	28 (43.1)	22 (45.8)	0.77
Rapidly fatal	7 (10.8)	6 (12.5)	0.78
Underlying disease
Hematologic malignancy	9 (13.8)	6 (12.5)	0.84
Solid tumor	17 (26.2)	15 (31.3)	0.55
Cardiovascular disease	10 (15.4)	6 (12.5)	0.66
Liver cirrhosis	10 (15.4)	9 (18.8)	0.64
End-stage renal disease	5 (7.7)	2 (4.2)	0.70
Neutropenia	10 (15.4)	3 (6.3)	0.13
Site of infection
Catheter associated	12 (18.5)	7 (12.5)	0.59
Osteoarticular infections	13 (20.0)	12 (25.0)	0.53
Skin and soft tissue	24 (36.9)	17 (35.4)	0.87
Endocarditis	1 (1.5)	3 (6.3)	0.31
Pneumonia	8 (12.3)	1 (2.1)	0.08
Surgical site	2 (3.1)	6 (12.5)	0.07
Primary bacteremia	12 (18.5)	11 (22.9)	0.56
Eradicated foci of infection	20 (32.3)	17 (33.3)	0.60
With deep-seated abscess	6 (9.2)	4 (8.3)	0.87
Metastatic infection	6 (9.2)	5 (10.4)	>0.99
β-Lactamase producers	64 (98.5)	23 (47.9)	<0.01
Type A	16 (24.6)	1 (2.1)	<0.01
Type B	4 (6.2)	20 (41.7)	<0.01
Type C	44 (67.7)	2 (4.2)	<0.01
Type D	0 (0)	1 (2.1)	0.43
Empirical antibiotics
Glycopeptide only	14 (21.5)	6 (12.5)	0.213
β-Lactam only	38 (58.5)	32 (66.7)	0.375
Glycopeptide+β-lactam	11 (16.9)	7 (14.6)	0.737
Cefazolin	19 (29.2)	18 (37.5)	0.354
Mean time to cefazolin (day, mean±SD)	3.3±2.4	3.4±3.2	0.807
High-dose cefazolin treatment	44 (66.7)	27 (56.3)	0.213

Data were presented as the number (%) of patients except indicated variables.

CIE-N, cefazolin inoculum effect-negative group; CIE-P, cefazolin inoculum effect-positive group.

Persistent MSSA bacteremia was more common in the CIE-positive group (9% vs. 0%, p=0.04) and 67% of persistent MSSA bacteremia occurred in infections with high bacterial burden. There was no persistent bacteremia in the CIE-negative group with high bacterial burden. However, the overall treatment failure rates and mortality rates of both groups were not significantly different (Table 3). In the subgroup analysis of subjects with high bacterial burden, the CIE-positive group had a higher treatment failure rate (48%) than the CIE-negative group (25%), although the difference was not statistically significant (Table 3). In comparison between groups with MIC ≥16 μg/ml (n=23) and ≤1 μg/ml (n=42), the rate of treatment failure (13% vs. 5%, p=0.31), mortality (22% vs. 12%, p=0.34), and MSSA bacteremia-related mortality (4% vs. 2%, p>0.99) were not significantly different.

Table 3.

Comparison of Outcomes of MSSA Bacteremia Between CIE-Positive and -Negative Groups: Overall and Stratified Analysis by High Bacterial Burden Infection and MIC at High Inoculum

	Overall analysis				Stratified analysis ^a
Characteristics	CIE-P (n=65)	CIE-N (n=48)	OR (95% CI)	p	CIE-P (n=25)	CIE-N (n=20)	OR (95% CI)	p
Median time to defervescence (IQR)	4 (2–8)	5 (2–8)		0.76	8 (4–17)	6 (2–10)		0.33
Persistent bacteremia (>72 hr)	6 (9%)	0 (0%)	NC	0.04	4 (16%)	0 (0%)	NC	NC
Overall mortality	5 (8%)	2 (4%)	1.92 (0.36–10.33)	0.70	3 (12%)	0 (0%)	1.88 (0.36–9.78)	0.46
Treatment failure	14 (22%)	6 (13%)	1.92 (0.68–5.44)	0.21	12 (48%)	5 (25%)	2.39 (0.77–7.45)	0.13
SAB-related deaths	3 (5%)	1 (2%)	2.27 (0.23–22.56)	0.64	3 (12%)	0 (0%)	2.27 (0.25–20.61)	0.47
Antibiotics change due to clinical failure	9 (14%)	5 (10%)	1.38 (0.43–4.42)	0.58	8 (32%)	5 (25%)	1.63 (0.45–5.86)	0.45
Relapse	2 (3%)	0 (0%)	NC	0.51	1 (4%)	0 (0%)	NC	NC

High bacterial burden: MSSA bacteremia that accompanied endocarditis, irremovable vascular graft infection, osteomyelitis, pneumonia, deep-seated abscess, or metastatic infection.

Stratified analysis performed by Mantel–Haenszel χ² test.

IQR, interquartile range; NC, not calculable; OR, odds ratio; SAB, Staphylococcus aureus bacteremia.

All the variables regarding demographics, clinical characteristics of infections, empirical antibiotics, doses of cefazolin, types of β-lactamase, and the CIE were included in a bivariable analysis of risk factors of treatment failure, and the variables for which p-value was≤0.2 in the bivariable analysis were included in a multivariable analysis. In multivariable risk factor analysis of treatment failure, underlying cardiovascular diseases (adjusted odds ratio [aOR] 4.57; 95% confidence interval [CI], 1.10–18.02; p=0.04), pneumonia (aOR 9.66; 95% CI, 1.93–48.43; p=0.01), osteoarticular infections (aOR 6.39; 95% CI, 1.81–22.54; p=0.01), and endocarditis (aOR 9.86; 95% CI, 1.00–101.38; p=0.05) were significantly associated with treatment failure, and the type A β-lactamase, the CIE, and the marked CIE were not (Table 4).

Table 4.

Multivariable Logistic Regression Analysis for Risk Factors of Treatment Failure

	Bivariable analysis		Multivariable analysis
Risk factors	OR (95% CI)	p	aOR (95% CI)	p
Solid tumor	0.39 (0.11–1.43)	0.15
Cardiovascular diseases	2.49 (0.76–8.18)	0.16	4.57 (1.10–18.02)	0.04
Site of infections
Endocarditis	5.06 (0.67–38.27)	0.14	9.86 (1.00–101.38)	0.05
Pneumonia	7.42 (1.79–30.80)	0.01	9.661 (1.93–48.43)	0.01
Osteoarticular infections	2.25 (0.69–7.32)	0.18	6.39 (1.81–22.54)	0.01
Primary bacteremia	0.09 (0.01–0.71)	0.01
Eradicated foci of infection	0.31 (0.08–1.12)	0.06
Deep-seated abscess	3.63 (0.92–14.31)	0.07
High-dose cefazolin treatment	2.76 (0.86–8.91)	0.08
Type A β-lactamase^a	0.58 (0.12–2.76)	0.73	0.46 (0.08–2.87)	0.41
CIE-P^a	1.92 (0.68–5.44)	0.21	2.07 (0.61–7.01)	0.24
Marked CIE-P^a	1.39 (0.45–4.32)	0.38	1.30 (0.35–4.88)	0.69

Marked CIE: cefazolin MIC ≥16 μg/ml at the high inoculum (nonsusceptible).

Separately forced into the final model of multivariable analysis.

aOR, adjusted odds ratio; CI, confidence interval.

Discussion

Clinical isolates of MSSA are usually β-lactamase producers.²⁸ Among them, type A or C β-lactamase-producing strains can exhibit the CIE.²³ In animal studies, mortality rates were higher when infections of β-lactamase-producing S. aureus were treated with cefazolin.^4,9 An association between types of β-lactamase and the CIE has been demonstrated,^18,23 and some investigators suggested that cefazolin might be associated with treatment failure in serious infections by S. aureus with the CIE.^22,23

This study found that the CIE was associated with higher rates of persistent bacteremia when MSSA bacteremia was treated with cefazolin. All the CIE-negative MSSA bacteremia, even with high bacterial burden did not persist beyond 72 hr. These findings suggest that CIE is important in the early stage of infection. The bacterial burden is usually highest just before initiating antibiotics, but the concentration of antibiotics could be suboptimal until the concentrations attain a steady state. During this period, cefazolin treatment could be compromised by a high bacterial burden and/or the infecting MSSA strains with CIE.

However, the CIE was not associated with overall treatment failure. In the subgroup analysis of subjects with high bacterial burden, treatment failure was more common in the CIE-positive group than the CIE-negative group although the difference was not statistically significant.

CIE is a dynamic phenomenon where other factors such as the infection site, the cefazolin dose, and the degree of debridement (debulking) are also important factors impacting the patient's clinical outcome. Infection sites were actually predictive of the overall treatment failure rather than the CIE, especially in pneumonia, osteoarticular infections, and endocarditis in this study. This finding supports findings that site and severity of MSSA infection predict poor outcome of MSSA bacteremia.^17,30 Pneumonia and endocarditis caused by S. aureus result in high mortality rates despite appropriate antibiotic treatment.^8,15,17,20

Two prior studies of CIE prevalence used different definitions: Nannini et al.²³ defined the CIE as an MIC of ≥16 μg/ml at the high inoculum and Livorsi et al.¹⁸ ≥4-fold increasing in MIC from the standard inoculum to the high inoculum. We used Livorsi's definition, but also presented data according to Nannini's definition because the clinical relevance of the two definitions was obscure.

This study also provided some epidemiologic information because it was the first survey of the type of β-lactamase and the CIE in South Korea. It demonstrated that more than 50% of MSSA presented with the CIE and 20% were nonsusceptible to cefazolin at the high inoculum. Type C β-lactamase-producing S. aureus was the predominant strain and more than 90% of type A or C β-lactamase-producing isolates exhibited the CIE in this study. The rate of CIE was similar to Nannini et al.,²³ but higher than Livorsi et al.¹⁸ However, mean MIC and MIC₅₀ in type A or C β-lactamase-producing strains of this study were higher than previous studies,^18,23 suggesting that most type A or C blaZ gene-carried MSSA isolates were hyperproducers of β-lactamase in South Korea. We hypothesized that this results from overuse of oral antibiotics such as amoxicillin–clavulanate. It is proven that the use of amoxicillin–clavulanate can cause herd selection of S. aureus strains producing higher levels of β-lactamases.¹⁰

This study has limitations. First, our study has limited external validity because we enrolled only MSSA bacteremic patients treated with cefazolin and evaluated clinical characteristics retrospectively. The mortality rate of our study (6.2%) is lower than previous epidemiologic studies of MSSA bacteremia (11%,⁵ 24%¹⁶), suggesting that fewer severe cases among the total MSSA bacteremic cases were included in this study. Physicians tend to prescribe antistaphylococcal penicillin for serious MSSA infections rather than cefazolin.¹⁷

Second, few cases of endocarditis were included in our study because cefazolin poorly penetrates the blood–brain barrier and concerns regarding metastatic infection of the brain could hamper the use of cefazolin in the case of endocarditis. Therefore, these results cannot be generalized for MSSA bacteremia associated with endocarditis.

Third, virulence factors other than an inoculum effect, which can affect the clinical outcomes, were not considered in this study. These include the ability to form biofilms,²⁴ the small colony variant phenotype,²⁹ and high vancomycin MIC.^1,11,12 However, this study evaluated only the CIE among the various characteristics of S. aureus.

In conclusion, the severity of infection is a more important factor for overall treatment failure than the CIE. Although the CIE was not significantly implicated in the overall treatment failure, it was associated with persistent bacteremia when MSSA bacteremia was treated by cefazolin. Therefore, cefazolin should be used with caution for MSSA bacteremia, especially in the high bacterial burden such as pneumonia, osteoarticular infections, or endocarditis.

Footnotes

Acknowledgments

The authors especially thank B.E. Murray and her colleagues of the University of Texas Medical School for providing the strain TX 0117, high-level producer of type A β-lactamase, and gracious advice about this study.

Disclosure Statement

There are no conflicts of interest.

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