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Effect of Concentrations Exceeding Minimum Inhibitory Concentrations of Caspofungin on the Selection of Resistant Strains of Candida spp.

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Effect of Concentrations Exceeding Minimum Inhibitory Concentrations of Caspofungin on the Selection of Resistant Strains of Candida spp.
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Arora, Divya
Nguyen, Minh Hong ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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Effect of Concentrations Exceeding Minimum Inhibitory Concentrations
of Caspofungin on the Selection of Resistant Strains of Candida spp.

Arora D, Nguyen MH, Clandy CJ


ABSTRACT


Caspofungin is a new antifungal agent with a novel mechanism of action that is effective in the treatment

of infections caused by diverse Candida spp. The potential for emergence of resistance to caspofungin among

clinical Candida isolates has not been well characterized. The purpose of this study is to evaluate the frequency of

the emergence of resistance to caspofungin in vitro. Four strains each of Candida. albicans, Candida

parapsilosis, Candida lusitaniae, and Candida krusei were tested against caspofungin, and we determined

that minimum inhibitory concentrations (MICs) exhibited by each strain were < 1.0 pg/mL. Breakthrough growth

of the C. albicans strains was noted at frequencies of 10-1 in the presence of 4 pg/mL of caspofungin. To determine

if breakthrough growth was associated with increased MICs, we passed the standard C. albicans laboratory

strain SC5314 in the presence of 4 pg/mL of caspofungin and measured MICs. There was no increase in

MICs observed for breakthrough strains. Taken together, our MIC data suggest that caspofungin should be

effective in the treatment of a broad range of Candida infections. The breakthrough growth of C. albicans

suggests the potential for the emergence of resistance with wide-spread clinical use of the drug. It is likely,

however, that increases in MICs will require the extended growth of isolates in the presence of drug, since a

single passage did not induce step-wise increases in MIC.



INTRODUCTION


Studies have shown Candida albicans to be the most common cause of Candida infection (candidiasis), with

a prevalence of about 50%. Other important species of Candida include Candida parapsilosis, Candida krusei,

Candida lusitaniae and Candida glabrata. Morbidity and mortality rates remain unacceptably high for patients

with candidiasis. The main reason for this is the limited number of antifungal drugs.1 Until recently, the

antifungal drugs in the clinical setting were limited to polyenes like amphoterican B and azoles like fluconazole.

Both of these antifungal drugs target ergosterol, a major sterol in fungal cell membranes.1


More recently, newer antifungal agents with broader anti-Candida activity, fewer harmful effects and

minimal resistance have become available. In the past two years, the newer triazoles, voriconazole,

posaconazole, and ravuconazole have been shown to demonstrate excellent activity against Candida spp. in

vitro, including fluconazole-resistant C. krusei and C. glabrata. In agreement with previous reports, in one study






it was shown that most Candida spp. were susceptible to voriconazole and posaconazole, using

susceptibility breakpoints for these drugs of a minimum inhibitory concentration (MIC) of 1 pg/ml or less.2



Echinocandins are the newest antifungal drug class. They act by inhibiting the synthesis of beta-l1,3-glucan,

an important component of the fungal cell wall. Caspofungin is a water-soluble semisynthetic echinocandin that

has been approved by the United States Food and Drug Administration in the past two years for the treatment

of candidiasis as well as refractory aspergillosis.2 Caspofungin displays favorable pharmacodynamic

and pharmacokinetic characteristics and has an excellent toxicological profile.1 Susceptibility MIC breakpoints

have not yet been established because in vitro susceptibility testing methods are currently not established for

this class. However, caspofungin appears to be active against C. albicans and other Candida spp., including C.

krusei and C. glabrata.2



Lately, increased reports of fluconazole-resistant invasive Candida spp. have emerged. It has been found

that Candida spp. resistant to fluconazole exhibit increased MICs of voriconazole and ravuconazole. Risk factors

for the development of azole cross-resistance were found to be the length of therapy and the total previous

azole dose.2 Many of these strains retain susceptibility to caspofungin, however, which likely reflects

the echinocandins' unique mechanism of action.



Resistance in Candida spp. involves increasing drug efflux and regulation of genes encoding the drug target.

Since the sequencing of the C. albicans genome with complementary DNA microarray analysis has been

completed, new genes involved in antifungal resistance of Candida spp. have been discovered. The importance

of correlating resistance of newer antifungal drugs with unsuccessful therapy is highlighted by previous studies

of molecular mechanisms of azole resistance among isolates associated with clinical treatment failures.2



To date, the frequency of caspofungin resistance and the mechanisms by which it might arise are poorly

understood. In this study, we wish to establish whether clinical isolates of Candida spp. are capable of growing in

the presence of caspofungin at concentrations above the MIC. If so, we will determine whether such

breakthrough growth is associated with increases in MICs. Results from this study might give us a sense of

the likelihood of clinical resistance emerging with the use of caspofungin in the future.



MATERIALS AND METHODS


Candida strains and growth conditions. Four strains from each of four Candida spp. were used in this study.

C. albicans, C. parapsilosis, C. lusitaniae, and C. krusei were recovered from the blood of patients at the

Shands Teaching Hospital at the University of Florida. These strains were identified through standard

biochemical procedures (API 20, BioMerieaux) in the clinical microbiology laboratory. A standard laboratory

strain most commonly used for antifungal susceptibility known as C. albicans SC5314 was obtained from Dr. M.

Hong Nguyen at the University of Florida. All the strains were stored at -700C in 8% DMSO . Before performing





the experiments, strains were streaked to isolation on Sabourad dextrose agar (SDA) plates at 370C

for approximately 48 hours. The other specific growth media and conditions are described below.



Determination of the MIC of caspofungin


Isolates were spread on an SDA plate because freshly grown yeast is needed to perform MIC determinations. A

sterile swab was used to lift each isolate from the SDA plate, which was suspended in normal saline sterile solution

to get an absorbance at 340-588 nm between 0.84 and 0.86. This mixture, named the inoculum, was first

diluted 1:10 in sterile distilled water and then diluted further 1:20 in 1X RPMI to get the final concentration of

yeast needed to perform MICs. The original concentration of caspofungin stock was 5mg/mL. This drug was

diluted with distilled water to get a range of different drug concentrations, 0.15mg/mL to 2.0 mg/mL or 0.5 mg/mL

to 8.0 mg/mL, depending upon the specific experiment. The inoculum was then added to each drug

concentration, and the MIC was determined by visual inspection of the tubes after 24 and 48 hour growth at 370

C. The MIC was defined using the standard definition: the lowest concentration of caspofungin that inhibited

the growth of an isolate by at least 80%.



Determination of breakthrough rate of C. albicans SC5314


C. albicans SC5314 was taken from a fresh, frozen stock, and spread on plates containing SDA medium. An

overnight culture of SC5314 was grown at 370C. A suspension was adjusted to give approximately 108 organisms/

mL in sterile distilled water. Serial dilutions were prepared in sterile distilled water and plated on SDA, RPMI

(without drug), and RPMI plates containing caspofungin (4mg/mL). The number of colony forming units (CFUs)

on SDA or RPMI plates (without drug) was determined by counting the number of colonies on the plates.

Similarly, the number of breakthrough colonies was determined by counting the number of colonies on RPMI

plates containing caspofungin (4mg/mL). The breakthrough rate was determined by dividing the number

of breakthrough colonies on the drug plate by the number of colonies on the control SDA plate. This

percentage, therefore, represents the number of SC5314 that survived caspofungin at 4mg/mL.



Serial passage of C. albicans SC5314 breakthrough strains


After breakthrough colonies were created and counted, serial passages were formed on caspofungin drug plates

(4 pg/mL). Eight breakthrough colonies were selected from the original caspofungin plate with a sterile toothpick

and repassed onto a new caspofungin plate at 4 pg/mL. After growth on new caspofungin plate was noted,

these isolates were passed onto a SD plate and the standard MIC procedure was carried out as described above.



RESULTS


Testing of Candida isolates for susceptibility to caspofungin and frequencies of breakthrough growth.

We collected isolates of C. albicans, C. parapsilosis, C. lusitanie, and C. krusei (4 isolates of each spp.) from

patients with candidemia at the Shands Teaching Hospital. As shown in Table 1, we determined that the






caspofungin MIC exhibited by each isolate was _ 1.0 pg/mL. The C. albicans isolates consistently exhibited the

lowest MICs among the Candida spp. tested.



Next, we were interested in determining whether these susceptible isolates were capable of any growth

at caspofungin concentrations above the MIC, since this would indicate the potential for the eventual emergence

of resistance. The strains were cultured overnight, and washed and re-constituted in normal saline solution at

xl106/mL. Next, 0.1 ml inocula of this suspension and serial ten-fold dilutions were spread onto RPMI1640

plates containing 4 pg/mL caspofungin. The inocula were felt to reflect physiologically relevant concentrations,

since most oral OPC samples from our patients contain 103 to 105 CFU. In addition, the drug concentrations

were chosen to be > 4 x MIC, since this is often the level desired in blood or tissue. The plates were incubated for

2 weeks, and observed daily for breakthrough colonies. If breakthrough colonies were found, they were confirmed

for true resistance by re-growing on caspofungin plates. Breakthrough was notable for selected strains of C.

albicans and C. parapsilosis, but was low for all strains of C. lusitaniae and C. krusei (Table 1). The initial MICs of

C. parapsilosis predicted the emergence of breakthrough resistant mutants: mutants develop only among strains

with MICs > 0.5 mg/ml. For other spp., there was no correlation between initial MICs and breakthrough.



Table 1
Determination of caspofungin MICs and breakthrough frequencies of Candida
isolates

Organism 48 hr caspo MICs (pg/mL) Frequency of caspo breakthrough

C. albicans 0.125 10-1 -10-4

C. parapsilosis 0.25-1.0 10-1 - < 10-5

C. lusitaniae 0.125-0.25 10-4 - < 10-5

C. krusei 0.125-0.5 5 10-5


Breakthrough rate of C. albicans SC5314 against caspofungin (4 mg /mL).


The breakthrough rate of C. albicans SC5314 was determined by plating different concentrations of SC5314

on caspofungin (4 mg/mL) plates. We chose to study SC5314 because it is the standard laboratory strain, for

which molecular genetics and DNA sequence are well-defined. These properties would enable us to study

the molecular mechanisms of resistance in future studies, if applicable. Table 2 shows that the breakthrough rate

of C. albicans SC5314 against caspofungin (4 pg/mL) is significant, despite the fact that SC5314 exhibits a 48

hour caspofungin MIC of 1 pg/mL.



Table 2
Breakthrough rates of C. albicans SC5314 against
caspofungin (4 mg /mL)

Number of cells plated Breakthrough rate (%)

102 37.7

103 37.7






Passage of SC5314 in the presence of caspofungin (4 mg /mL).


We picked 32 colonies of C. albicans SC5314 that were able to grow in the presence of 4 mg /mL of caspofungin,

and passed them again in the presence of caspofungin to determine if breakthrough growth was associated with

an increase in MIC. As shown in Table 3, there was no significant increase in MIC among any isolate after a

single passage.


Table 3
MICs of C. albicans SC5314 after a single passage in the presence
of caspofungin (4 mg /mL)

Isolate Number 24 hour MIC reading 48 hour MIC reading

1A 0.5 1.0

1B 0.5 1.0

2A 0.5 1.0

2B 0.5 1.0

3A 0.5 1.0

3B 0.5 1.0

4A 0.5 1.0

4B 0.5 1.0

5A 0.5 1.0

5B 0.5 1.0

6A 1.0 1.0

6B 1.0 1.0

7A 0.5 1.0

7B 1.0 1.0

8A 0.5 1.0

8B 1.0 1.0

9B 0.5 1.0

10B 1.0 1.0

11B 1.0 1.0

13B 1.0 1.0

14B 1.0 1.0

15B 1.0 1.0

16B 1.0 1.0

25A 1.0 1.0

26A 1.0 1.0

27A 1.0 1.0

28A 1.0 1.0

29A 1.0 1.0






30A 1.0 1.0

31A 1.0 1.0

32A 1.0 1.0

12B 1.0 1.0

12B 1.0 1.0



MATERIALS AND METHODS



In this study we examined diverse species of Candida to measure their resistance to a new drug, caspofungin.

Our measurements of MICs suggested that caspofungin is a potentially effective therapeutic agent to

treat candidiasis. This drug is an alternative to many existing antifungal agents since it has a unique mechanism

of action. As mentioned before in the introduction, caspofungin, an echinocandin, acts by inhibiting the synthesis

of beta-1,3-glucan, a vital component of the fungal cell wall.



Despite the low MICs, C. albicans strains showed significant rates of breakthrough growth. This suggests that

the emergence of resistance might eventually be observed clinically, associated with elevated MICs. Other species,

C. parapsilosis, C. lusitaniae, and C. krusei, showed less breakthrough suggesting that these strains might be

less likely to develop resistance to caspofungin clinically.



Serial passage of C. albicans SC5314 revealed breakthrough growth, but this was not associated with increases

in MICs. There could be multiple reasons for this observation. One explanation could be that the concentration

of caspofungin used for the selection of resistant organisms may not have been high enough. Another reason is

that using a liquid medium for MIC measurement could be more efficient than the same amount of drug put into

a solid agar plate, since drug distribution within solid agar is often uneven.3 An alternate way to determine MICs

on solid agar in future studies could be to use caspofungin disks, which will be made available for research in

the near future.



The most likely reason that we did not observe an increase in MICs, however, is that we did not perform a

sufficient number of serial passages. In order to obtain organisms with increased MICs for caspofungin, it is

likely that we would need to perform more serial passages to allow the organism to accumulate the genetic

mutations necessary for measurable resistance to be detected.4



In interpreting our data, it is important to recognize that caspofungin is a fungistatic drug. Fungistatic

antifungal agents inhibit further growth of fungi, but, unlike fungicidal drugs, do not directly kill fungi. For

this reason, some breakthrough isolates might be able to grow at drug concentrations above the MIC, but the

strain might not exhibit increased MIC measurements. In treating infected patients, fungistatic drugs are

effective because if the Candida isolate cannot grow, the infecting organisms will eventually die off or be

eliminated by the patient's immune cells.






Further studies involving a combination of fungistatic and fungicidal antifungal agents could be helpful in

developing more effective therapeutic strategies against candidiasis.5 Caspofungin is potentially very attractive

in combination with polyene or azole agents, because it works by a completely different mechanism than these

other classes of drugs. This has important implications since it suggests that caspofungin and a polyene or

azole might exhibit synergy, whereby the effects of each drug are greater in combination than if the drugs are

given alone. This could be beneficial because it could increase the efficacy of treatment, allow for the use of

lower drug dosages, decrease overall toxicity, and decrease the emergence of resistance.6



A variety of mechanisms could be responsible for development of resistance to caspofungin.7 Further studies

are needed to investigate the genetic changes in Candida that lead to caspofungin resistance.



In summary, we determined the breakthrough rates of a variety of Candida spp. against a new antifungal

drug, caspofungin. We then used these breakthrough colonies and tested them for true resistance to this drug.

We found that for C. parapsilosis, C. lusitaniae, and C. krusei, breakthrough rates as well as MICs were low.

However, for the laboratory isolate, C. albicans SC4314, breakthrough rates were high and MICs were low.

These findings suggest that caspofungin is an effective antifungal agent that should be useful for treating

candidiasis in patients. Nevertheless, the presence of breakthrough growth suggests that resistance, at least

among C. albicans isolates, might ultimately emerge with widespread use of the drug.






ACKNOWLEDGMENTS


I would to thank my mother, Dr. Shiwani Arora for critical reading of this manuscript and helpful comments.

The authors thank the University of Florida Scholars Program for funding these studies.






REFERENCES


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affecting casponfungin susceptibility in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 2004;8:3871-3876.

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vitro. Antimicrob. Agents Chemother. 1994;38:1284-1291.





5. Marr K. Combination antifungal therapy: where are we now, and where are we going? Oncology. 2004;18(13

Suppl. 7):24-29.

6. Clancy CJ, Yu YC, Lewin A, Nguyen MH. Inhibition of RNA synthesis as a therapeutic strategy against Aspergillus

and Fusarium: demonstration of in vitro synergy between rifabutin and amphotericin B. Antimicrob.

Agents Chemother. 1998;42:509-513.

7. Hernandez S, Lopez-Ribot JL, Najvar LK, McCarthy DI, Bocanegra R, Graybill JR. Casopfungin resistance in

Candida albicans: correlating clinical outcome with laboratory susceptibility testing of three isogenic isolates

serially obtained from a patient with progressive Candida esophagitis. AntiMicrob. Agents Chemother.

2004;48:1382-1383.


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