# Activity of Ceftaroline against Aerobic Gram-Positive and Gram-Negative Pathogens: Effect of Test Method Variability

Authors: Diane M. Citron; Yumi A. Warren; Kerin L. Tyrrell; Ellie J. C. Goldstein
Journal: ISRN Microbiology
 (2011)

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## Abstract

Ceftaroline is a new cephalosporin with bactericidal activity against methicillin-resistantS. aureus (MRSA) as well as gram-negative pathogens. Variations of in vitro test conditions were found to affect ceftaroline activity, with 5% NaCl inhibiting growth and/or reducing the minimum inhibitory concentrations (MICs) for E. coli, K. pneumoniae, M. catarrhalis, H. influenzae, and streptococci, while an inoculum of 106 CFU/mL raised MICs of some E. coli, K. pneumoniae, and M. catarrhalis strains.


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## Body

## 1. Introduction

The emergence of MRSA has spurred the development of alternative therapies such as daptomycin, linezolid, and quinupristin-dalfopristin, which are not active against gram-negative pathogens and require combination therapy. Ceftaroline is a new, parenteral, broad-spectrum cephalosporin with bactericidal activity against MRSA, including vancomycin-intermediate (VISA) strains, and multidrug-resistantStreptococcus pneumoniae (MDRSP); it is also active against common gram-negative pathogens and can therefore be used as monotherapy for mixed infections [1–6]. Since alterations of in vitro test conditions can potentially affect susceptibility results, we  evaluated the effects of 15 variations to the standard test conditions as specified by the Clinical and Laboratory Standards Institute (CLSI) guidelines [7, 8] on the minimum inhibitory concentrations (MICs) of ceftaroline against 30 isolates representing 10 species of clinically important, commonly encountered organisms.


## 2. Materials and Methods

### 2.1. Standard Method

The CLSI reference broth microdilution method (CLSI 2006, 2009) uses cation-adjusted Mueller Hinton broth (CAMHB) (Difco, BD; Sparks, Md, USA), which has a calcium concentration of 25 mg/L, a magnesium concentration of 12.5 mg/L,  and a pH of 7.3+ 0.1. The standard inoculum is 5 × 105 colony-forming units (CFUs)/mL for broth microdilution testing and 104 CFU/spot for agar dilution tests.


### 2.2. Test Variables

Modifications of standard test conditions included adjusting the Ca++ content of CAMHB to 50 mg/L Ca, addition of  NaCl to 5%, adjusting the broth to pH 6 and pH 8, and using inocula of  104 and 106 colony-forming units (CFUs)/mL. Other variations to the standard medium were the addition of 10% and 50% pooled human serum (Sigma; St. Louis, Mo, USA), the addition of  lysed horse blood to 2.5% (LHB) (Hardy Diagnostics, Inc. Santa Maria, Calif, USA), and using Haemophilus test medium (HTM) broth. While MIC panels were incubated at 35°C in ambient conditions, for comparative purposes, additional tests in CAMHB were incubated in the anaerobic chamber or in 5% CO2.


### 2.3. MIC Test Panel Preparation

Ninety-six-well panels were prepared with twice the final concentration of ceftaroline (50μL/well) using the Quick-Spense IIe apparatus (Sandy Springs Instruments; Germantown, Md, USA) and stored at −70°C until used. Addition of 50 μL of the organism inocula to the wells reduced the final ceftaroline concentration to the desired level of 0.008 to 8 μg/mL. Some of the organisms did not achieve a ceftaroline MIC endpoint, and further dilutions were prepared to 0.001 μg/mL for retesting some of those isolates.


### 2.4. Agar Dilution Test Media

Agar dilution MICs were determined on unsupplemented Mueller Hinton agar (MHA) (Difco), with 5% LHB, and on HTM with 1.5% agar (HTMA). Serial twofold dilutions of ceftaroline were added to molten agar deeps to prepare the plates for use on the same day. Concentrations of ceftaroline ranged from 0.008 to 8μg/mL. Drug-free growth control plates were included (CLSI, 2006).


### 2.5. Test Organisms

All 30 strains tested were recent clinical isolates and American Type Culture Collection (ATCC) quality control (QC) strains, which includedEscherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Enterococcus faecalisATCC 29212, Streptococcus pneumoniaeATCC 49619, and Haemophilus influenzaeATCC 49247. Details about the clinical isolates are listed in Table 1. Clinical isolates were selected based on previously demonstrated resistance patterns. The isolates were stored in 20% skim milk at −70°C and were taken from frozen stock and transferred twice on blood or chocolate agar (Hardy Diagnostics Inc.) before testing.Table 1
List of organisms used in the study.
OrganismRMA numberSpecimen sourceDate isolatedCommentsE. coliATCC 2592219089Blood3/7/2007Ampicillin = 4μg/mL19090Primary infection site3/28/2007Ampicillin ≥32μg/mLK. pneumoniae19091Primary infection site3/1/2007Ampicillin = 32μg/mL19092Blood6/6/2007Ampicillin = 16μg/mL19093Blood6/29/2007Ampicillin = 32μg/mLH. influenzaeATCC 4924716081Respiratory12/31/2003β-Lactamase-negative18520Respiratory-sinus12/23/2005β-Lactamase-positiveM. catarrhalis11940Respiratory-sinus6/14/200014032Respiratory-sinus5/22/200218861Respiratory-sputum1/31/2007S. aureusATCC 2921318488Chest infection site2/11/2005Methicillin-S18401Blood8/16/2005Methicillin-S18483Head abscess10/15/2005Methicillin-R18504Primary infection site11/18/2005Methicillin-R18526Blood10/24/2005Methicillin-RE. faecalisATCC 2921218284Foot infection site3/24/200518877Blood4/10/2007S. pyogenes17018Diabetic foot infection site1/22/200317019Diabetic foot infection site10/22/200219047Abdominal lesion10/26/2007Clindamycin-RS. pneumoniaeATCC 4961919094Ear10/29/2007Penicillin-S19095Eye1/9/2007Penicillin-S13345Nasopharynx11/14/2001Penicillin = 8μg/mL13385Nasopharynx12/4/2001Penicillin = 8μg/mL18876Eye1/2/2007Penicillin = 8μg/mLRMA: R.M. Alden (culture collection).ATCC: American Type Culture Collection.


### 2.6. Inoculum Preparation for Microbroth Dilution Tests

Standard inocula were prepared by suspending colonies from overnight cultures in 0.85% saline to equal the turbidity of the 0.5 McFarland standard and diluting it in CAMHB with the various additives at twice their final concentration, which upon addition of 50μL of inoculum to the test panel were diluted 1 : 2. The 104 and 106 cfu/mL inocula were prepared by diluting the saline suspension either 10-fold more (for 104 cfu/mL) or 10-fold less (for 106 cfu/mL). The trays were inoculated with 50 μL of cell suspension for a final inoculum of ~5 × 105 CFU/mL which was validated by quantitative subculture from the growth control well. Inoculum preparation and all testing were performed in duplicate.


### 2.7. Agar Dilution Testing

For agar dilution tests, the cell suspensions prepared as above were diluted 1 : 10 in CAMHB and applied to the agar plates using a Steers replicator device that delivered a final inoculum of 104 CFU/spot.


### 2.8. MIC Determinations

After overnight incubation, the broth microdilution trays were examined for growth. The MIC was the lowest drug concentration that completely inhibited growth [7]. For agar dilution, the plates were incubated at 35°C overnight. The MIC was the lowest concentration that completely inhibited growth or resulted in a marked reduction of growth as compared with the drug-free control [7].


## 2.1. Standard Method

The CLSI reference broth microdilution method (CLSI 2006, 2009) uses cation-adjusted Mueller Hinton broth (CAMHB) (Difco, BD; Sparks, Md, USA), which has a calcium concentration of 25 mg/L, a magnesium concentration of 12.5 mg/L,  and a pH of 7.3+ 0.1. The standard inoculum is 5 × 105 colony-forming units (CFUs)/mL for broth microdilution testing and 104 CFU/spot for agar dilution tests.


## 2.2. Test Variables

Modifications of standard test conditions included adjusting the Ca++ content of CAMHB to 50 mg/L Ca, addition of  NaCl to 5%, adjusting the broth to pH 6 and pH 8, and using inocula of  104 and 106 colony-forming units (CFUs)/mL. Other variations to the standard medium were the addition of 10% and 50% pooled human serum (Sigma; St. Louis, Mo, USA), the addition of  lysed horse blood to 2.5% (LHB) (Hardy Diagnostics, Inc. Santa Maria, Calif, USA), and using Haemophilus test medium (HTM) broth. While MIC panels were incubated at 35°C in ambient conditions, for comparative purposes, additional tests in CAMHB were incubated in the anaerobic chamber or in 5% CO2.


## 2.3. MIC Test Panel Preparation

Ninety-six-well panels were prepared with twice the final concentration of ceftaroline (50μL/well) using the Quick-Spense IIe apparatus (Sandy Springs Instruments; Germantown, Md, USA) and stored at −70°C until used. Addition of 50 μL of the organism inocula to the wells reduced the final ceftaroline concentration to the desired level of 0.008 to 8 μg/mL. Some of the organisms did not achieve a ceftaroline MIC endpoint, and further dilutions were prepared to 0.001 μg/mL for retesting some of those isolates.


## 2.4. Agar Dilution Test Media

Agar dilution MICs were determined on unsupplemented Mueller Hinton agar (MHA) (Difco), with 5% LHB, and on HTM with 1.5% agar (HTMA). Serial twofold dilutions of ceftaroline were added to molten agar deeps to prepare the plates for use on the same day. Concentrations of ceftaroline ranged from 0.008 to 8μg/mL. Drug-free growth control plates were included (CLSI, 2006).


## 2.5. Test Organisms

All 30 strains tested were recent clinical isolates and American Type Culture Collection (ATCC) quality control (QC) strains, which includedEscherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Enterococcus faecalisATCC 29212, Streptococcus pneumoniaeATCC 49619, and Haemophilus influenzaeATCC 49247. Details about the clinical isolates are listed in Table 1. Clinical isolates were selected based on previously demonstrated resistance patterns. The isolates were stored in 20% skim milk at −70°C and were taken from frozen stock and transferred twice on blood or chocolate agar (Hardy Diagnostics Inc.) before testing.Table 1
List of organisms used in the study.
OrganismRMA numberSpecimen sourceDate isolatedCommentsE. coliATCC 2592219089Blood3/7/2007Ampicillin = 4μg/mL19090Primary infection site3/28/2007Ampicillin ≥32μg/mLK. pneumoniae19091Primary infection site3/1/2007Ampicillin = 32μg/mL19092Blood6/6/2007Ampicillin = 16μg/mL19093Blood6/29/2007Ampicillin = 32μg/mLH. influenzaeATCC 4924716081Respiratory12/31/2003β-Lactamase-negative18520Respiratory-sinus12/23/2005β-Lactamase-positiveM. catarrhalis11940Respiratory-sinus6/14/200014032Respiratory-sinus5/22/200218861Respiratory-sputum1/31/2007S. aureusATCC 2921318488Chest infection site2/11/2005Methicillin-S18401Blood8/16/2005Methicillin-S18483Head abscess10/15/2005Methicillin-R18504Primary infection site11/18/2005Methicillin-R18526Blood10/24/2005Methicillin-RE. faecalisATCC 2921218284Foot infection site3/24/200518877Blood4/10/2007S. pyogenes17018Diabetic foot infection site1/22/200317019Diabetic foot infection site10/22/200219047Abdominal lesion10/26/2007Clindamycin-RS. pneumoniaeATCC 4961919094Ear10/29/2007Penicillin-S19095Eye1/9/2007Penicillin-S13345Nasopharynx11/14/2001Penicillin = 8μg/mL13385Nasopharynx12/4/2001Penicillin = 8μg/mL18876Eye1/2/2007Penicillin = 8μg/mLRMA: R.M. Alden (culture collection).ATCC: American Type Culture Collection.


## 2.6. Inoculum Preparation for Microbroth Dilution Tests

Standard inocula were prepared by suspending colonies from overnight cultures in 0.85% saline to equal the turbidity of the 0.5 McFarland standard and diluting it in CAMHB with the various additives at twice their final concentration, which upon addition of 50μL of inoculum to the test panel were diluted 1 : 2. The 104 and 106 cfu/mL inocula were prepared by diluting the saline suspension either 10-fold more (for 104 cfu/mL) or 10-fold less (for 106 cfu/mL). The trays were inoculated with 50 μL of cell suspension for a final inoculum of ~5 × 105 CFU/mL which was validated by quantitative subculture from the growth control well. Inoculum preparation and all testing were performed in duplicate.


## 2.7. Agar Dilution Testing

For agar dilution tests, the cell suspensions prepared as above were diluted 1 : 10 in CAMHB and applied to the agar plates using a Steers replicator device that delivered a final inoculum of 104 CFU/spot.


## 2.8. MIC Determinations

After overnight incubation, the broth microdilution trays were examined for growth. The MIC was the lowest drug concentration that completely inhibited growth [7]. For agar dilution, the plates were incubated at 35°C overnight. The MIC was the lowest concentration that completely inhibited growth or resulted in a marked reduction of growth as compared with the drug-free control [7].


## 3. Results

We obtained MICs from duplicate tests under the variations shown in Table2. In cases of discrepancy, the higher value was recorded.  The ceftaroline MICs for the QC isolates (tested with the reference microbroth methods according to CLSI guidelines) were all within their acceptable ranges. Effects of variables in testing were noted where  5% NaCl inhibited growth and/or reduced MICs for E. coliand K. pneumoniae and completely inhibited the growth of M. catarrhalis,  H. influenzae, and all streptococci. Using an increased inoculum of  106 cfu/mL  increased the MIC 5-fold for 1 of 3 E. coli strains that was also resistant to ampicillin (MIC > 32 μg/mL) and 1 of 3 K. pneumoniaestrains that did not appear to have any unusual resistance pattern (ampicillin MIC 32 μg/mL, ceftriaxone 0.25 μg/mL). This K. pneumoniae isolate produced the same result when retested. The higher inoculum also increased MICs 3- to 5-fold for M. catarrhalis.  The addition of blood or serum to the medium enhanced M. catarrhalis growth without changing the MICs. Testing on agar, especially HTMA, produced MICs that were 1–3 dilutions higher.   All other variables showed minimal effect, and the MICs were generally within one dilution of the reference method.Table 2
Ceftaroline MICs (μg/mL) in the cation-adjusted Mueller-Hinton broth with test variables and agar dilution MICs.
Ceftaroline MICs (μg/mL) in the cation-adjusted Mueller Hinton broth with test variablesAgar dilutionCa++% NaClpHInoculum cfu/mL% serumIncubationMHAMHA/ LHBHTMAOrganismRMA numberREF50 mg/L5681041061050LHBHTMCO2AnaerobicATCCE. coli259220.060.06≤0.0080.1250.060.060.1250.060.1250.1250.1250.1250.030.060.250.25190890.060.1250.0150.060.060.030.1250.060.060.1250.060.1250.1250.1250.250.25190900.030.125≤0.0080.060.030.0320.060.030.1250.1250.1250.1250.030.250.25K. pneumoniae190910.250.250.0150.50.250.25>80.250.1250.250.50.50.250.1250.250.5190920.030.1250.0150.060.060.030.1250.060.060.1250.060.1250.060.060.1250.25190930.060.1250.0150.060.060.030.250.030.060.1250.060.1250.060.060.1250.25ATCCH. influenzae492470.0150.015ngng0.06ng0.060.060.060.06n/t0.060.03ng0.060.12516081≤0.008≤0.008ngng≤0.008ng≤0.008≤0.008≤0.008≤0.008n/t≤0.008≤0.008ng0.060.015185200.0150.015ngng0.030.0150.060.060.030.03n/t0.0150.03ng0.0150.06M. catarrhalis119400.1250.06ngng0.1250.0320.060.1250.1250.1250.06ng0.060.03n.g.140320.060.06ngng0.250.0310.1250.250.1250.1250.25ng0.060.030.03188610.030.03ng0.030.060.01520.250.250.1250.060.25ng0.030.0150.06ATCCS. aureus-MSSA292130.1250.1250.1250.250.250.1250.250.1250.1250.250.250.1250.1250.250.250.5184880.1250.1250.250.250.250.1250.250.1250.1250.250.250.1250.1250.250.250.5184010.1250.1250.1250.250.1250.1250.250.1250.1250.250.250.250.1250.250.250.5S.aureus-MRSA184830.250.50.250.50.50.250.50.250.50.50.50.250.250.50.51185040.250.50.250.250.50.250.50.250.50.50.250.250.250.50.51185260.250.50.250.250.250.250.50.250.50.50.50.250.250.50.51ATCCE. faecalis292120.50.510.510.51110.510.50.510.5218284112221212221122418877110.060.521222120.50.520.54S. pyogenes170180.0020.002ng0.0020.0020.0020.0040.0020.002n/t0.0040.0040.004≤0.008≤0.0080.015170190.0040.004ng0.0040.0040.0040.0040.0020.002n/t0.0040.0040.004≤0.008≤0.0080.015190470.0040.004ng0.0040.0040.0040.0040.0040.004n/t0.0080.0040.004≤0.008≤0.0080.015ATCCS. pneumoniae496190.0150.015ng0.0150.0080.0080.0150.0150.008n/t0.0150.0150.015≤0.0080.015≤0.008190940.0040.004ng0.0040.0040.0040.0040.0040.004n/t0.0080.0040.004≤0.008≤0.008≤0.008190950.0040.004ng0.0040.0040.0040.0040.0040.004n/t0.0040.0040.004≤0.008≤0.008≤0.008133450.1250.125ng0.1250.060.060.1250.1250.06n/t0.060.060.1250.060.1250.125133850.1250.125ng0.1250.060.1250.1250.1250.125n/t0.1250.1250.1250.060.1250.125188760.250.25ng0.1250.250.250.250.250.25n/t0.250.250.1250.1250.250.25ATCC: American Type Culture Collection; CFUs: colony-forming units; HTM:Haemophilus test medium; HTMA: HTM with 1.5% agar; LHB: laked horse blood;  MHA: Mueller Hinton agar; MIC: minimum inhibitory concentration; MRSA: methicillin-resistant Staphylococcus aureus; MSSA: methicillin-susceptible Staphylococcus aureus; ng: no growth; nt: not tested; REF: reference method; RMA: R.M. Alden (culture collection).AllH. influenzae were tested in HTM; all streptococci were tested with 2.5% LHB supplementation.


## 4. Discussion

Standardization of test conditions is an important factor in reliably reporting MICs. Yet variations of conditions may occur, and diverse adjustments may be employed by researchers for unusual testing circumstances worldwide. Jones et al. [9] studied the effects of modifying five parameters (11 variations) of in vitro testing of ceftaroline against 15 selected isolates. They found inocula of 5 × 107 raised the MICs from 8 to >32 and 0.5 to >32 μg/mL on P. aeruginosa and E. cloacae, respectively. They also noted that pH 5.0 impaired the growth of 9/12 organisms and consequently lowered their MICs and that increased calcium concentration lowered the MICs of E. faecalis and E. faecium, but that the addition of serum or LHB and variable incubation conditions did not affect ceftaroline MICs. Our study differed from theirs by including a larger number of isolates and adding such variables as 5% NaCl, serum at 10 and 50% concentrations, variations in incubation conditions, and while there was some overlap in species, they did not report on the respiratory pathogens H. influenza and M. catarrhalis, nor did they study S. pyogenes or K. pneumoniae. While some of our test variations were in accord with those of Jones et al., they differed in several respects as well. Jones et al. [9] noted that high inocula did not affect E. coli MICs but did raise E. cloacae and P. aeruginosaMICs, but we found a fourfold increased MIC for one of three E. coli strains tested as well as a fivefold increase with one of three K. pneumonia isolates, suggesting strain variation. Additionally, we found that M. catarrhalis, H. influenza, and S. pyogenes grew poorly with NaCl supplementation and at pH 6.0. An inoculum of 106 CFU/mL also increased the MICs four- to six-fold for all three M. catarrhalis strains tested.


## 5. Conclusion

The in vitro antibacterial activity of ceftaroline was adversely affected by 5% NaCl which inhibited growth and/or reduced MICs forE. coli, K. pneumoniae, M. catarrhalis, H. influenzae,and streptococci, while an inoculum of 106 CFU/mL raised MICs of some E. coli, K. pneumoniae, and M. catarrhalis strains. The other modifications tested did not adversely affect MIC results. Organisms with special growth requirements can be tested for ceftaroline susceptibility with reasonable assurance that test conditions will not affect the MIC results.



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*Source: 787290-2011-11-17.xml*