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Jundishapur J Microbiol. 2019 June; 12(6):e85092. doi: 10.5812/jjm.85092.

Published online 2019 June 2. Research Article

In Vitro Determination of Hydrolytic Enzymes and Echinocandin

Susceptibility in Mexican Clinical Isolates of Candida glabrata Sensu
Stricto
Rogelio de J. Treviño-Rangel 1 , José F. Espinosa-Pérez 1 , Hiram Villanueva-Lozano 1 , Laura A.
Soto-Quintana 1 , Alexandra M. Montoya 1 , Carolina E. Luna-Rodríguez 1 and Gloria M. González 1, *
1
Department of Microbiology, Faculty of Medicine, Universidad Autónoma de Nuevo León, Monterrey, Nuevo Leon, Mexico
*
Corresponding author: Department of Microbiology, Faculty of Medicine, Universidad Autónoma de Nuevo León, Monterrey, Nuevo Leon, Mexico. Email:
[email protected]

Received 2018 October 08; Revised 2019 May 17; Accepted 2019 May 19.

Abstract

Background: Candida glabrata is an opportunistic yeast that has emerged as a cause of human fungal disease, and is a complex of
three closely related species. Until now, there is not enough information about its virulence attributes. Moreover, its resistance to
echinocandins has been documented, which is a cause of clinical concern.
Objectives: The objective of this study was to evaluate the in vitro production of aspartyl proteinase, phospholipase, esterase,
hemolysin, DNase, and coagulase in a subset of 107 Mexican clinical isolates of C. glabrata sensu stricto, as well as to determinate
its echinocandin susceptibility.
Methods: The enzymatic determinations were carried out in plate assays using specific substrates, excepting coagulase, which was
determined by the classic tube test. Antifungal susceptibility testing was determined in accordance with the CLSI broth microdilu-
tion method.
Results: Aspartyl proteinase, hemolysin, and phospholipase were detected in 100%, 95%, and 79% of isolates, respectively. Blood
isolates were associated with a very strong activity of aspartyl proteinase and hemolysin, and those recovered from vaginal swabs
were associated with very strong production of aspartyl proteinase, phospholipase, and hemolysin. All isolates were susceptible to
echinocandins, except one bloodstream isolate, which was resistant to echinocandins.
Conclusions: A very strong activity of aspartyl proteinase and hemolysin was particularly associated with both, bloodstream, and
vaginal swab isolates. Echinocandin resistance was rare.

Keywords: Candida glabrata, Phospholipases, Aspartyl Proteinases, Esterases, Hemolysins, Echinocandins

1. Background Candida species, such as C. glabrata.

Treatment of C. glabrata infections represents an au-
Candida glabrata is an emerging human fungal thentic challenge due to the scarce knowledge of C.
pathogen, ranking as the second or third common glabrata pathogenicity, the increased probability of the or-
cause of clinical forms of candidosis (1-3), highlighting ganism to develop resistance to azole derivatives, as well
bloodstream infections due to its unacceptable high as a limited antifungal repertoire (8). The reduced suscep-
mortality rate ranging from 58% to 61% (4). Furthermore, tibility of C. glabrata to fluconazole has increased the clin-
C. glabrata is a species complex comprised of C. glabrata ical value of echinocandins, leading ESCMID and IDSA to
sensu stricto, C. nivariensis, and C. bracarensis (5, 6). On the propose them as the first-line therapy against this yeast (9).
other hand, the adherence to host tissues, rapid response However, resistance to echinocandins has also emerged,
to changes in the microenvironment, and the ability to alarmingly increasing the number of case reports of C.
secrete hydrolytic enzymes are all considered virulence at- glabrata resistant isolates following echinocandin ther-
tributes that enable Candida species to cause disseminated apy (10). Thus, local and global antifungal susceptibility
infections in susceptible hosts (7). Particularly, hydrolytic surveillance of this threatening pathogen becomes critical
enzymes have been exhaustively studied in C. albicans, yet in order to document epidemiological shifts and trends.
there is limited information with respect to non-albicans Overall, the aim of this study was to assess the production

Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
(http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly
cited.
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Treviño-Rangel RJ et al.

of lytic enzymes, such as aspartyl proteinase (AP), phospho- 10 µL was spotted for the hemolytic assay. Plates were in-
lipase (PP), esterase (ES), hemolysin (HM), DNase (DA), and cubated for two, five, seven, and ten days at 37ºC for the de-
coagulase (CO) in a subset of C. glabrata sensu stricto clini- termination of HM, PP, AP, and ES, respectively. Enzymatic
cal isolates, as well as to determine its antifungal suscepti- activities were evaluated using the Pz index and the clas-
bility to echinocandins. sification criteria were as follows: very strong, Pz < 0.69;
strong, Pz = 0.70 - 0.79; mild, Pz = 0.80 - 0.89; weak, Pz =
0.90 to 0.99; and negative, Pz = 1 (17).
2. Objectives Additionally, DA and CO activities were qualitatively
determined employing a medium with methylene green
The present study evaluated the production of some (Difco, USA) and a commercial rabbit plasma (Difco, USA),
hydrolytic enzymes in a subset of 107 Mexican clinical iso- respectively (18). Both determinations were conducted ac-
lates of C. glabrata sensu stricto. Moreover, its antifun- cording to the manufacture’s recommendations.
gal susceptibility to caspofungin, anidulafungin, and mi- All assays were done in duplicates. The quality con-
cafungin was determinated. trols used for AP, PP, ES, and HM determinations were
C. albicans ATCC 90028 and C. tropicalis ATCC 750, while
Staphylococcus aureus ATCC 29213 was utilized for DA and
3. Methods CO assays. Complementarily, in order to compare the re-
sults with other published studies, a comprehensive liter-
3.1. Isolates ature review was performed using the platform PubMed
One hundred and seven isolates of C. glabrata were of the National Center for Biotechnology Information
recollected between 2005 to 2015 at the Department of (www.ncbi.nlm.nih.gov/pubmed/) under the criteria of
Microbiology, UANL in Mexico. These isolates were col- search: ‘Candida glabrata hydrolytic enzymes’.
lected from different patient samples and incubated at
3.3. Antifungal Susceptibility Testing
37ºC on Sabouraud Dextrose Agar (SDA) (Difco, USA) for
24 hours. Isolates were identified as C. glabrata employ- The antifungal susceptibilities of the isolates to the
ing API-20C-AUX strips (bioMérieux, Mexico). In order to echinocandins caspofungin, anidulafungin, and micafun-
confirm the identity of the isolates, DNA extraction by gin were in accordance with the most recent approved
the organic (phenol-chloroform) method was performed broth microdilution method of CLSI, M27-A4 protocol (19).
(11) and the non-coding ITS region of the rDNA was am- In brief, two-fold serial dilutions were prepared for each
plified in a T100 Thermal Cycler (BIO-RAD; Hercules, USA), antifungal, and further dilutions were made in RPMI 1640
using the primers IT5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) with MOPS (Hardy Diagnostics, USA). The drug’s working
and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (12). Once ampli- concentrations were 0.015 to 8 µg/mL. Plates were incu-
cons were purified (Promega; Madison, USA), they were se- bated at 35ºC and then read after 24 hours of incubation.
quenced by Sanger and the obtained sequences were then Furthermore, C. krusei ATCC 6258 and C. parapsilosis ATCC
compared by BLAST with sequence deposits available from 22019 were used as quality control organisms. The MIC
the NCBI/GenBank and ISHAM ITS databases. The samples breakpoints for the interpretation of results were those es-
from which the isolates were collected were as follows: 43 tablished by CLSI. Isolates with MICs of ≤ 0.12 µg/mL for
(40.2%) from blood, 28 (26.2%) from vaginal swab of Vulvo- anidulafungin and caspofungin and of ≤ 0.06 µg/mL for
vaginal Candidiasis (VVC) cases, 23 (21.5%) from urine, and micafungin were considered susceptible; in counterpart,
13 (12.1%) from diverse sites, such as peritoneal fluid (8), isolates with MICs of ≥ 0.5 µg/mL for anidulafungin and
intra-abdominal abscess (2), cerebrospinal fluid (1), hep- caspofungin and of ≥ 0.25 µg/mL for micafungin were
atic cyst (1), and bronchial secretion (1). All isolates were considered resistant.
preserved on agar slants at -20ºC.
3.4. Statistics
The enzymatic profiles of the isolates were correlated
3.2. Enzymatic Activity Assays
based on their clinical origin with chi-square and Fisher’s
The activity of AP, PP, ES, and HM were quantitatively tests using SPSS version 17.0 for Windows (SPSS; Chicago,
assessed using plate assays with specific test media, such USA). P values ≤ 0.05 were considered significant.
as YCB-BSA medium (Difco & Bio Basic, USA) (13), egg-yolk
agar (Difco, USA) (14), tween 80 opacity test medium (Difco 4. Results
& Sigma-Aldrich, USA) (15), and SDA (Difco, USA) with 7%
blood (16), respectively. Five microliters of a 107 CFU/mL ad- All analyzed isolates were identified as C. glabrata sensu
justed suspension was inoculated in specific media, while stricto, according to the sequence analysis of the ITS inter-

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Treviño-Rangel RJ et al.

genic region; sequences are available in GenBank (acces- Table 1. Enzymatic Activities of the Clinical Isolates Studied in This Work
sion numbers: MF187218 to MF187325). Origin (N)
Enzyme
The enzymatic profiles of the C. glabrata sensu stricto Evaluated
Blood (43) Urine (23) Vaginal Others
isolates evaluated in this study are summarized in Table 1. Swab (28) (13)
The AP was secreted by all strains with a very strong activ-
Aspartyl
ity, excepting for two bloodstream isolates and one strain proteinase
from vaginal swab. On the other hand, 84 isolates (78.5%) Very 41 23 27 13
were PP producers with different activity levels, highlight- strong
ing the finding that all vaginal isolates were very good pro- Strong 1 0 1 0
ducers of this enzyme. In counterpart, ES was expressed
Mild 1 0 0 0
only by 13 isolates (12.1%), none from urine. Regarding HM,
Weak 0 0 0 0
102 isolates (95%) were hemolytic, exhibiting a very strong
activity, especially among isolates recovered from blood, Negative 0 0 0 0

vaginal swab, and diverse other origins. The DA and CO Phospholipase

were all negative for the subset of isolates studied. The Very 18 8 27 5
bloodstream isolates were statistically associated with a strong
very strong activity of AP and HM (P < 0.001), while the Strong 2 11 1 0
vaginal specimens were particularly associated with a very
Mild 3 2 0 5
strong production of AP and PP (P < 0.001), as well as HM (P
Weak 0 0 0 2
= 0.022). Moreover, the literature review is presented in Ta-
ble 2, and includes 14 reports; AP and PP were the enzymes Negative 20 2 0 1

more extensively examined. Esterase

Regarding the antifungal susceptibilities of the iso-
Very 5 0 5 3
lates, the MIC90 for the three drugs was 0.0625 µg/mL with strong
ranges of 0.0156 to 1 µg/mL for anidulafungin, 0.03125 to Strong 0 0 0 0
0.5 µg/mL for caspofungin, and 0.03125 to 0.25 µg/mL for
Mild 0 0 0 0
micafungin. A very low incidence of echinocandin resis-
tance was detected with only one bloodstream isolate re- Weak 0 0 0 0

sistant to the three echinocandins, exhibiting a MIC of 1 Negative 38 23 23 10

µg/mL for anidulafungin and caspofungin, and a MIC of 0.5 Hemolysin
µg/mL for micafungin. Very 38 11 27 12
strong

5. Discussion Strong 2 4 0 0

Mild 0 6 0 0
The pathogenicity of Candida spp. is determined by
Weak 0 2 0 0
the expression of several virulence attributes, such as ad-
herence to host cells, phenotypic switching, the ability to Negative 3 0 1 1

form biofilms, and the capability to produce and secrete

hydrolytic enzymes (34, 35). These play a major role in ad-
herence, penetration, invasion, and destruction of host tis- controls several steps in innate immune evasion degrad-
sue (35), contributing significantly to the pathogenicity of ing structural and immunological defense proteins (34).
Candida. Some of the most commonly described extracel- In the present study, the researchers found that all strains
lular hydrolases are AP, lipolytic enzymes, and HM. Impor- showed proteolytic activity, with an elevated percentage
tantly, production of these enzymes varies between species of isolates with very strong activity of AP (97.2%). As de-
and also depends on the source or site of infection (36). In picted in Table 2, Figueiredo-Carvalho et al. (33) recently
this sense, for example, the current study found an associ- reported similar findings in a collection of 91 strains of C.
ation between C. glabrata sensu stricto isolates from vagi- glabrata from diverse clinical sources. However, in the lit-
nal swabs with a very strong production of AP, PP and H; erature, there are several reports with lower variable inci-
findings also reported on a strain of C. bracarensis isolated dence of proteolytic activity (24, 26, 28, 29, 31) and some au-
from the same site of infection in a Mexican woman with thors did not detect such enzymatic activity, probably due
VVC (18). to the very limited number of C. glabrata strains they tested
Aspartyl proteinase enables Candida invasion and tis- (21, 30, 32). On the other hand, PP and ES are extracellular
sue colonization by disruption of host membranes, while lipolytic enzymes that contributed to the virulence of Can-

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Table 2. Comprehensive Literature Review

No. C. glabrata Enzymatic Activities: No. Isolates with Activity (%)

Method (s) of
Reference Country Isolates (Clinical
Identification
Origin) AP PP ES HM CO DA

Aktas et al. (20) Turkey 12 (NS) API 20C AUX - - 0 (0) - - -

Kantarcioglu and Turkey 4 (NS) Fermentation and 0 (0) 1 (25) - - - -

Yucel (21) assimilation tests

Rodrigues et al. (22) Portugal 25 (NS) API ID 32C - - - - 5 (20) -

Gokce et al. (23) Turkey 6 (blood) API ID32C ND 0 (0) - - - -

Oksuz et al. (24) Turkey 9 (RS = 3, GU = 1, OM = API ID 32C 1 (11.1) 0 (0) - - - -

1, fecal = 1, skin = 3)

Yigit et al. (25) Turkey 14 (NS) API 32C AUX - - - - 6 (42.8) -

D’Eca Junior et al. Brazil 10 (urine = 7, TS = 3) Vitek 6 (60) 7 (70) - - - -

(26)

Chin et al. (27) Malaysia 4 (NS) CHROM agar - 0 (0) - 4 (100) - -

Candida/ITS
sequencing

Tellapragada et al. India 13 (VS = 11, blood = 2) HiChrome Candida 2 (15.3) 0 (0) 0 (0) - - -
(28) agar/Vitek 2/PCR
multiplex (ITS)

Deorukhkar et al. India 93 (VS = 41, urine = 24, Assimilation 56 61 - 62 45 -

(29) OM = 11, blood = 8, CSF tests/HiChrome (60.2) (65.5) (66.6) (48.3)
= 4, miscellaneous = 5) Candida agar

Mutlu Sariguzel et Turkey 2 (blood) API 20C AUX/ITS 0 (0) 0 (0) 0 (0) - - -
al. (30) sequencing

Atalay et al. (31) Turkey 14 (blood) CHROM agar 4 (28.5) 5 (35.7) 1 (7.1) - - -
Candida/API 20C AUX

Riceto et al. (32) Brazil 5 (NS) ND 0 (0) 0 (0) - 5 (100) - 0 (0)

Figueiredo-Carvalho Brazil 91 (blood = 25, urine = API 20C AUX/CHROM 87 0 (0) 51 (56) 90 - -
et al. (33) 13, RS = 10, feces = 9, VS agar Candida/Vitek (95.6) (98.9)
= 7, miscellaneous = 2/ITS sequencing
27)

This study Mexico 107 (blood = 43, VS = API 20C AUX/ITS 107 84 13 (12.1) 102 0 (0) 0 (0)
28, urine = 23, sequencing (100) (78.5) (95.3)
miscellaneous = 13)

Abbreviations: AP, aspartyl proteinase; CO, coagulase; CSF, cerebrospinal fluid; DA, DNase; ES, esterase; GU, genitourinary tract; HM, hemolysin; ITS, internal transcribed
spacer region; ND, not defined; NS, not stated; OM, oral mucosa; PCR, polymerase chain reaction; PP, phospholipase; RS, respiratory samples; VS, vaginal swab.

dida spp., possibly through damage to the host cell mem- lated.
brane by digestion of lipids, facilitating tissue invasion as
Hemolysin is important for elemental iron uptake
well as nutrient acquisition (35)
from hemoglobin through lysis of red blood cells. Thus,
In this work, the researchers detected 84 isolates this putative virulence factor enables pathogen survival
(78.5%) with PP activity at different production levels, with and persistence in the host (37). In the present study only
these observations agreeing closely with certain studies five isolates were unable to produce HM, these findings are
(26, 29) and more distantly with some additional reports in agreement with previous publications (27, 32, 33), re-
(21, 31); as opposed, other authors did not find PP activ- flecting the importance of this key virulence attribute for
ity (23, 24, 27, 28, 30, 32), even in collections of numerous C. glabrata. In fact, the hemolytic activity has been demon-
strains of C. glabrata (33). Otherwise, as expected, a lim- strated as necessary for virulence in this yeast (38). On the
ited number of isolates (12.1%) exhibited ES activity, agree- other hand, although less studied, other extracellular lytic
ing with some studies (20, 28, 30, 31) and in contrast to the enzymes that also contribute to the pathogenic fitness of
work of Figueiredo-Carvalho et al. (33), who reported an in- Candida spp. are CO and DA. In this study, these enzymes
cidence of 56%, and further suggested that the production were not produced among the studied isolates, in contrast
of this enzyme may vary according to the clinical source with scarce works (22, 25, 29) and in agreement with the
or the geographic region, from which the strains were iso- findings communicated by Riceto et al. (32).

4 Jundishapur J Microbiol. 2019; 12(6):e85092.

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Treviño-Rangel RJ et al.

Echinocandins are the front-line antifungals for the (http://www.medicina.uanl.mx/investigacion/sometimiento_-

treatment of candidemia and other forms of invasive can- protocolo/).
didiasis due to C. glabrata (9). Although case reports were Financial Disclosure: The authors declare that they
initially infrequent, echinocandin resistance in Candida had no financial interests related to the material in the
spp. is emerging, particularly in C. glabrata. This phe- manuscript.
nomenon mostly occurs in patients with long periods of Funding/Support: This study was supported by internal
echinocandin treatment or prophylaxis, and it is princi- resources of Departamento de Microbiologia, Facultad de
pally related to mutations in hot-spot regions of FKS gene, Medicina, UANL.
which participate in the production of 1,3-β -D-glucan syn-
thase (39), manifesting phenotypically with magnitudes of
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