(2021) 20:12
Teixeira et al. Ann Clin Microbiol Antimicrob
https://doi.org/10.1186/s12941-020-00401-y
Annals of Clinical Microbiology
and Antimicrobials
Open Access
RESEARCH
Molecular characterization
of methicillin-resistant Staphylococcus aureus
among insulin-dependent diabetic individuals
in Brazil
Nathalia Bibiana Teixeira1,2,4* , Carlos Magno Castelo Branco Fortaleza1, Matheus Cristovam de Souza2,
Thais Aline Monteiro Pereira2, Bibiana Prada de Camargo Colenci3 and
Maria de Lourdes Ribeiro de Souza da Cunha2
Abstract
Background: People with diabetes mellitus, especially insulin-dependent diabetic patients, are a risk group for
staphylococcal infections. Asymptomatic infection with Staphylococcus aureus is common and favors dissemination of
the microorganism, rendering these individuals a source of infection. This study aimed to characterize the resistance
profile, clonal profile and sequence type, as well as to analyze the prevalence and risk factors for nasal and oropharyngeal carriage of methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from insulin-dependent diabetic individuals in the city of Botucatu, SP, Brazil.
Methods: Staphylococcus aureus was collected from the nasopharynx and oropharynx of 312 community-dwelling
insulin-dependent diabetic individuals over a period of 3 years (October 2015 to December 2018). The isolates were
characterized by susceptibility profiling, detection of the mecA gene, SCCmec typing, and molecular typing by PFGE
and MLST. The risk factors associated with S. aureus and MRSA carriage were determined by logistic regression analysis.
Results: The overall prevalence of colonization with S. aureus and MRSA was 30.4% and 4.8%, respectively. Fifteen of
the 112 S. aureus isolates carried the mecA gene; SCCmec type IV was identified in 10 isolates, SCCmec type I in three,
and SCCmec type II in two. Among the 15 resistant isolates (MRSA), four were susceptible to oxacillin/cefoxitin by the
disc diffusion method and one MSSA isolate was resistant to sulfamethoxazole/trimethoprim. The analysis of risk factors revealed a protective effect of age and lung disease, while lower-extremity ulcers were a risk factor for S. aureus.
For MRSA, only male gender was significantly associated as a risk factor in multivariate analysis. Clonal profile analysis
demonstrated the formation of clusters among MRSA isolates from different patients, with the identification of ST5-IV,
ST5-I, and ST8-IV. Isolates carrying ST398 were identified among MSSA and MRSA (ST398-IV).
Conclusion: Our findings reinforce the importance of epidemiological studies of S. aureus carriage, especially in
populations at high risk of infections such as diabetics. The data suggest widespread dissemination of MRSA in the
*Correspondence: na_tx0402@yahoo.com.br
4
Departamento de Ciências Químicas e Biológicas – Setor Microbiologia
e Imunologia, Instituto de Biociências de Botucatu (IBB)-Laboratório de
Bacteriologia. Rua Plínio Silva, CEP: 18618-970 – Distrito de Rubião Júnior,
Botucatu, SP, Brasil
Full list of author information is available at the end of the article
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Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
Page 2 of 12
population of insulin-dependent diabetic patients studied, as well as the emergence of important lineages among
these individuals.
Keywords: Methicillin-resistant Staphylococcus aureus (MRSA), Diabetes mellitus, Insulin, Resistance, Nasal or
oropharyngeal carriage, Molecular typing
Background
Diabetes mellitus is a progressive chronic disease characterized by high blood glucose levels, which is one of the
most prevalent diseases in modern societies. Its treatment is often inadequate or absent [1]. It is estimated
that more than 370 million people worldwide have diabetes and more than 5 million deaths were attributed to the
disease and its complications in 2017 [2, 3].
People with diabetes are known to be more susceptible
to infections because of their elevated blood glucose levels and suppression of the immune response. In addition,
neuropathy and reduced blood flow to the extremities
are common in these individuals. Consequently, wounds
tend to heal more slowly, increasing the risk of amputations and death [4, 5].
Staphylococcus aureus is one of the leading causes of
infections in diabetic individuals. This microorganism
plays a particularly important role in this scenario by
causing infections that range from superficial to severe
and potentially fatal systemic infections, in addition to
its ability to acquire resistance to multiple drugs. In the
past, the pathogen was mainly found in hospital settings;
however, we are now witnessing an increase particularly
of resistant isolates (MRSA) acquired in the community
that are genetically different from traditional nosocomial
strains [6–8]. Infections caused by methicillin-resistant
S. aureus (MRSA) are associated with a higher mortality rate compared to infections caused by methicillinsusceptible S. aureus (MSSA) [2]. Furthermore, studies
suggest that staphylococcal infections are preceded by
colonization with these microorganisms [2, 9–13] and at
least one third of colonized healthy adults are at risk of
developing subsequent invasive infections [6, 7].
Bacterial resistance has become a major global threat.
In the study of Onanuga and Temedie [14], the prevalence of multidrug-resistant MRSA isolated from the
anterior nares of diabetic patients was 52.5%. This prevalence was 40% among diabetic outpatients in the study of
Kutlu et al. [15]. High antimicrobial resistance of MRSA
isolated from diabetic patients has also been reported
by Alizargar et al. [16], with rates of erythromycin, ciprofloxacin and clarithromycin resistance of 81.9%, 71.3%
and 65.5%, respectively, and a multidrug resistance rate
of 59%. Two vancomycin-resistant Staphylococcus aureus
strains were isolated in that study [16].
Diabetic individuals are considered a risk group for
skin infections such as those caused by S. aureus and are
also more prone to developing severe systemic infections
[13]. Studies have shown that the prevalence of nasal colonization with S. aureus (27–56.6%) and MRSA (1–7.3%)
varies according to geographic location and that the use
of insulin is a risk factor for nasal MRSA carriage in diabetic individuals [16]. However, little is known about the
role of throat colonization in this population.
Staphylococcus aureus is characterized by a high adaptation potential demonstrated by its success in surviving
in different environments and the constant emergence
of new lineages that cause different clinical manifestations and exhibit rapid epidemiological dissemination,
causing a significant impact on health [17]. Globally disseminated MRSA clones included healthcare-associated
[HA]-MRSA. The prevalence of this microorganism has
also been increasing among community-associated [CA]MRSA and livestock-associated [LA]-MRSA infections
[17]. However, studies have demonstrated an exchange
of these lineages with CA-MRSA, causing outbreaks of
healthcare-associated infections in the community, and
HA-MRSA being isolated in community settings [18, 19].
LA-MRSA have also been reported in individuals without any previous contact with animals [17].
Staphylococcal cassette chromosome (SCCmec) typing is a useful epidemiological tool since different types
are more prevalent in hospital or community settings.
Unlike HA-MRSA that harbor large SCCmec (SCCmec
types I, II and III) and are frequently multidrug-resistant,
CA-MRSA are associated with SCCmec types IV and
V, which are smaller and only carry the mecA gene of
methicillin resistance [20–23].
In view of their complex epidemiology, S. aureus and
MRSA lineages have been identified by techniques such
as pulsed-field gel electrophoresis (PFGE), an adequate
tool for the study of local outbreaks. Studies investigating
the clonality of S. aureus are frequently complemented by
multilocus sequence typing (MLST), which permits comparisons between S. aureus sequences described in different parts of the world. MLST and SCCmec typing have
been used for the identification of global MRSA clones,
which resulted in the detection of five large MRSA clonal
complexes (CC) including CC5, CC8, CC30, CC45, and
CC398 [24, 25].
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
Considering their impact on the health of diabetic
individuals, a better understanding of the epidemiology
of and risk factors for colonization with S. aureus and
MRSA is necessary. The aim of this study was to characterize the resistance profile, clonal profile and sequence
type, as well as to analyze the prevalence and risk factors for nasal and oropharyngeal carriage of MSSA and
MRSA isolated from insulin-dependent diabetic individuals in the city of Botucatu, SP, Brazil.
Materials and methods
Study design
This was a cross-sectional study conducted in the city of
Botucatu, São Paulo, Brazil, whose estimated population
is 146,497 inhabitants [26]. We calculated the sample size
from a population of 1631 individuals using an expected
proportion of 50% (the typical value used in situations in
which the proportion is unknown), considering a power
of 80% (i.e., a beta error of 20%) and an alpha error of
5%, with a design effect of 1.0 (since no subsampling was
performed). The formula used for sample size calculation is given in Additional file 1: Appendix S1. Using this
calculation for proportions, we obtained a suggested n of
312 subjects. The subjects were selected randomly from
the database of the Municipal Health Department over
a period of 3 years (October 2015 to December 2018).
If possible, the subjects were recruited during home visits (n = 204); however, in view of the difficulty in locating these individuals at the addresses obtained, many of
them were invited by telephone to the events promoted
at Basic Health Units (n = 70). Some subjects were
recruited at the doctor’s offices (n = 23) and at the headquarter of the Botucatu Association for Diabetes Support
(ABAD in the Portuguese acronym) (n = 15). A questionnaire including the following data was applied to subjects
who agreed to participate in the study: demographic data
(gender and age); type of diabetes (1 or 2); time since
diagnosis (years); time of insulin use (years); clinical data
(comorbidities); presence of ulcers or amputations; tattoos; hospitalizations or medical procedures in the last
year; and use of antimicrobials in the last year. All data
were obtained by interview with the patient and/or legal
representative following ethical standards. The questionnaires were extensively reviewed for inconsistencies.
Inclusion and exclusion criteria
All insulin-dependent diabetic patients living in the city
of Botucatu, who agreed to participate by signing the free
informed consent form (Additional file 1: Table S1), were
included in the study. Patients who did not consent to
participate in the study, patients who did not use insulin
at the time of data collection, patients who had died, and
Page 3 of 12
patients who could not be found based on their personal
data were excluded (Additional file 1: Table S2).
Collection of microbiological specimens
Nasal and oral mucosa samples were collected from
312 insulin-dependent diabetic individuals living in the
city of Botucatu, São Paulo, Brazil, using sterile swabs
in transport medium. Samples from the anterior nares
and oropharynx were obtained using one swab for each
site. For the collection of nasal samples, the swab was
immersed in 0.9% sterile saline and inserted into both
nares, rotating it and gently pressing its end against the
mucosa. The technique for oropharyngeal sampling consisted of immersion of the swab and passing it gently over
the surface of the throat, avoiding contact of the examiner with the tongue.
The swabs were transported in Stuart medium to the
Laboratory of Bacteriology, Department of Microbiology and Immunology, Institute of Biosciences, UNESP,
and seeded onto plates containing Baird-Parker agar,
a selective medium for Staphylococcus. After incubation for 48 h at 37 °C, the isolated microorganisms were
identified.
Identification of Staphylococcus aureus
The microorganisms were submitted to Gram staining
for observation of their morphology and specific staining. After confirmation of these features, catalase and
coagulase tube tests and biochemical tests (maltose, trehalose, and mannitol) were carried out to differentiate S.
aureus from other Staphylococcus species [27, 28].
After DNA extraction with the Illustra Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK), the S.
aureus isolates were confirmed genotypically by detection of the 16S rRNA gene [29] and the DNA SA442
fragment specific for S. aureus [30]. Thus, 112 S. aureus
isolated obtained from 312 patients included in the study
were identified.
Antimicrobial susceptibility testing
All 112 isolates obtained were subjected to antimicrobial susceptibility testing by the disc diffusion method
using impregnated discs according to the criteria of the
Clinical Laboratory Standards Institute (CLSI) [31]. The
inoculum was adjusted to a 0.5 McFarland standard and
seeded onto Mueller–Hinton agar and the plates were
incubated for 24 h at 35 ºC. The following drugs were
used: oxacillin (1 µg), cefoxitin (30 µg), linezolid (30 µg),
quinupristin/dalfopristin (15 μg), and sulfamethoxazole/trimethoprim (25 µg). Antimicrobial activity was
evaluated by determining the diameter of the inhibition
zone, which was interpreted according to the CLSI [31].
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
International reference strains were used as controls for
MSSA (S. aureus ATCC 25923) and MRSA (S. aureus
ATCC 33591).
Determination of the minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of vancomycin was determined by the E-test in all 112 S. aureus
isolates. This quantitative test uses inert and transparent
plastic strips (60 mm long × 5.5 mm wide) with a predefined gradient of concentrations of the antimicrobial to
be tested. The MIC results were classified as susceptible,
intermediate, or resistant according to the definitions of
the CLSI [31]. An international reference strain (S. aureus
ATCC 29213) was included as control.
Detection of the mecA gene of methicillin resistance
All S. aureus isolates were analyzed regarding the presence of the mecA gene by PCR following the parameters
described by Murakami et al. [32]. International reference strains were included as positive (S. aureus ATCC
33591) and negative (S. aureus ATCC 25923) controls
in all reactions. All isolates in which the mecA gene was
detected were classified as MRSA, regardless of the result
of the oxacillin and cefoxitin disc diffusion tests.
Determination of staphylococcal cassette chromosome
mec (SCCmec) type
SCCmec typing was performed by multiplex PCR as
described by Oliveira and de Lencastre [33] and updated
by Milheiriço et al. [34] on all mecA gene-positive S.
aureus isolates. The following strains were used as controls: COL for SCCmec type I; N315 for SCCmec type IA;
PER34 for SCCmec type II; AN546 for SCCmec type III;
HU25 for SCCmec type IIIA, and MW2 for SCCmec type
IV.
Visualization of amplified products
The efficiency of the amplifications was confirmed by
electrophoresis on 2% agarose gel prepared in 0.5 M
Tris–borate-EDTA (TBE) buffer. A 100-bp marker
was used as molecular weight standard. The gel was
stained with SYBR® Safe and photographed under UV
transillumination.
Pulsed field gel electrophoresis (PFGE)
The modified protocol of McDougal et al. [35] was used
to type all 112 S. aureus isolates by PFGE. The isolates
were grown in BHI broth for 24 h. Next, 400 µL of the
sample was added to a microtube and centrifuged at
12,000 rpm for 50 s. The supernatant was discarded and
300 µL TE solution (10 mM Tris, 1 mM EDTA, pH 8.0)
was added. The samples were kept in a water bath for
10 min at 37 °C. After vortexing, 5 µL lysostaphin (1 mg/
Page 4 of 12
mL in 20 mM sodium acetate, pH 4.5) and 300 μL lowmelting agarose were added.
The samples were transferred to plug molds. The plugs
were allowed to solidify and then placed in a 24-well
plate with 2 mL EC solution (6 mM Tris–HCl, 1 M NaCl,
100 mM EDTA, 0.5% Brij-58, 0.2% sodium deoxycholate,
0.5% sodium lauroyl sarcosine) and incubated at 37 °C for
at least 4 h. The EC solution was removed and the plugs
were washed four times with 2 mL TE at room temperature at intervals of 30 min.
The SmaI enzyme (Fast Digest SmaI, Life Science,
Canada) was used for restriction of genomic DNA. Electrophoresis was carried out in a CHEF-DR III System
(BioRad Laboratories, USA) on 1% agarose gel prepared
with 0.5 M TBE (Pulsed Field Certified Agarose, BioRad
Laboratories, USA) under the following conditions: pulse
switch time of 5 to 40 s for 21 h using a linear ramp; 6 V/
cm; angle of 120°; 14 o C; 0.5 M TBE as running buffer.
The Lambda PFG Ladder (New England BioLabs) was
used as molecular marker. The gel was stained with GelRed (10,000× in water; Biotium, USA) for 1 h and photographed under UV transillumination.
Similarity was analyzed with the BioNumerics 7.6 software (Applied Maths, Belgium). The dendrogram was
constructed by the UPGMA method (Unweighted Pair
Group Method with Arithmetic Mean), with band position tolerance and optimization adjusted to 1.2% and 1%,
respectively.
Forty-five isolates, including one MRSA, could not be
typed with the SmaI enzyme and were therefore digested
with the ApaI restriction enzyme.
A similarity coefficient ≥ 80% was chosen for the determination of clusters. A cluster was defined as a group of
three or more isolates showing ≥ 80% similarity.
Multilocus sequence typing (MLST)
Lineages representative of the clusters obtained by PFGE
were selected for MLST. Five MRSA isolates obtained
PFGE-SmaI and four MSSA isolates obtained by PFGEApaI were chosen.
MLST was performed according to the protocol of
Enright et al. [36] by amplification and sequencing of
seven housekeeping genes (arcC, aroE, glpF, gmk, pta,
tpi, and yqiL). The PCR products were purified using
the HiYield™ Gel/PCR Fragments Extraction Kit and
the sequencing reactions were performed in an ABI3500
8-capillary sequencer (50 cm) using POP7 as polymer
(Applied Biosystems). The BioNumerics 7.6 program
(Applied Maths, Belgium) was used for visualization of
the sequences (electropherogram). The sequences were
analyzed and compared to an online database (http://
www.mlst.net) (2004).
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
Statistical analysis
The Epi-Info for Windows software (version 7.26; ©Centers for Disease Control and Prevention, Atlanta, USA)
was used for univariate analysis. Dichotomous variables
were compared using nonparametric tests of proportion,
X2 test, and Fisher’s exact test. Numerical variables were
compared by the Mann–Whitney U test.
Multivariate analysis was performed with the SPSS 20.0
software (©SPSS, Inc.) using a logistic regression model.
The outcomes of interest were the overall presence of S.
aureus or the presence of MRSA irrespective of sampling
site. The variables were selected using a backward stepwise strategy. The criterion for entry and permanence of
the variables in the models was p < 0.1. Final statistical
significance was set at p < 0.05.
Results
Prevalence of Staphylococcus aureus and MRSA carriage
Ninety-five of the 312 subjects included in the study
were colonized with S. aureus, corresponding to an overall prevalence of 30.4% (95%CI 25.6–35.8%). Fifteen of
the 95 subjects were colonized with MRSA either in the
nose and/or oropharynx, corresponding to a prevalence
of 4.8% (95%CI 2.9–7.8%). Regarding the sampling site,
S. aureus was isolated exclusively from the nose in 44
(14.1%) individuals, exclusively from the oropharynx in
34 (10.9%), and from both sites in 17 (5.4%). Analysis of
MRSA demonstrated that eight of the 15 colonized individuals carried the microorganism exclusively in the nose
and six exclusively in the oropharynx. Interestingly, one
Page 5 of 12
subject carried MSSA in the oral mucosa and MRSA in
the nasal mucosa (Fig. 1).
Determination of in vitro antimicrobial susceptibility
All 112 isolates identified as S. aureus were submitted to
in vitro antimicrobial susceptibility testing as previously
described. The disc diffusion method revealed 11 strains
that were resistant to oxacillin and cefoxitin, including
seven resistant to both drugs and four that were resistant only to cefoxitin (inhibition zone ≤ 21). It should be
noted that four of the isolates that were susceptible to
both drugs exhibited resistance by the genotypic method.
Among all isolates, only one was resistant to sulfamethoxazole/trimethoprim and there was no case of resistance to quinupristin/dalfopristin or linezolid. In addition,
the MIC50 and MIC90 for vancomycin were 0.50 and
1.0 μg/mL, respectively, and all isolates were susceptible
(Supplementary Material Table S3).
Detection of the mecA gene and characterization
of SCCmec
Fifteen of the 112 S. aureus isolates carried the mecA
gene of methicillin resistance. There was a predominance
of the SCCmec type IV among isolates (n = 10), but three
isolates harboring SCCmec type I and two harboring
SCCmec type II were also identified. Most individuals
harboring MRSA isolates reported no contact with the
hospital environment in the last year, except for one subject with SCCmec type II and one with SCCmec type IV.
Fig. 1 Flow chart of the total number of individuals colonized with Staphylococcus aureus and number of individuals colonized with MSSA and
MRSA according to sampling site. MSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus. *Overall prevalence of S. aureus. **
Prevalence of MRSA
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
Page 6 of 12
Risk factors for S. aureus and MRSA carriage
All patients in the study used insulin and 78 (82.1%) had
type 2 diabetes. A total of 198 (63.4%) also used other
hypoglycemic drugs. The incidence of complications was
high: 132 (42.3%) patients had retinopathy and 31 (9.9%)
renal impairment. Thirty-one (9.9%) patients reported
lower-extremity ulcers and 10 of them [3.2%] had undergone amputation.
The results of univariate and multivariate (logistic
regression model) analysis to identify risk factors for S.
aureus and MRSA carriage are shown in Tables 1 and 2.
With respect to risk factors for S. aureus carriage, univariate analysis showed a protective effect (median 61 years,
p = 0.04), heart disease (OR = 0.51, 95%CI 0.28–0.95,
p = 0.03) and lung disease (OR = 0.28, 95%CI 0.10–0.83,
p = 0.02), while only the presence of lower-extremity
ulcers was a risk factor (OR = 2.36, 95%CI 1.11–5.01,
p = 0.02). However, the only factors that remained independently associated with S. aureus carriage in insulindependent diabetic individuals were age (OR = 0.98, 95%
CI 0.93–0.99, p = 0.02) and lung disease (OR = 0.31, 95%
CI 0.10–0.92, p = 0.03), which were protective. The only
risk factor was leg ulcer (OR = 2.44, 95%CI 1.11–5.34,
p = 0.03).
The study of risk factors for MRSA carriage revealed
only male gender as a risk factor in uni- and multivariate
analysis (OR = 3.64, 95% CI 1.12–11.78, p = 0.03).
Determination of the clonal profile of S. aureus and MRSA
by pulsed‑field gel electrophoresis (PFGE)
A total of 112 S. aureus isolates were analyzed by PFGE.
Forty-five of these isolates could not be typed repeatedly
with SmaI, including one MRSA isolate. However, molecular typing of all 45 isolates was possible using the ApaI
enzyme.
For clonal profile analysis, one dendrogram was constructed for susceptible S. aureus isolates (MSSA) and
one for resistant isolates (MRSA) using SmaI and ApaI,
which permitted to identify clusters with similarity ≥ 80%
in both groups.
Figure 2 shows the dendrogram of the PFGE-SmaI and
PFGE-ApaI profiles of MRSA isolates, as well as their
in vitro susceptibility profile to oxacillin and cefoxitin,
presence of the mecA gene, SCCmec type and MLST
analysis. Analysis of PFGE-SmaI isolates revealed the
presence of two clusters (A and B). Cluster A contained
five isolates, four of them showing 100% similarity (554O,
555 N, 659 N, and 665 N). All of them were isolated from
Table 1 Uni- and multivariate (logistic regression) analysis of predictors of Staphylococcus aureus carriage in diabetic
individuals
Factor
S. aureus (n = 95)
Negative (n = 217)
Univariate analysis
Logistic regression
(multivariate)
OR (95%CI)
p
0.88
Male gender
42 (44.2)
98 (45.2)
0.96 (0.59–1.56)
Age (median, quartiles)
61 (49–70)
65 (56–71)
–
0.04
Time since diagnosis, years (median, quartiles)
14 (9–20)
16.5 (8–25)
–
0.84
Use of insulin, years (median, quartiles)
8 (4–14)
7 (3–11)
–
0.22
Diabetes type 2
78 (82.1)
184 (84.8)
0.82 (0.43–1.56)
0.35
Heart disease
16 (17.4)
61 (29.0)
0.51 (0.28–0.95)
0.03
Lung disease
4 (4.3)
29 (13.8)
0.28 (0.10–0.83)
0.02
Kidney disease
23 (25.0)
59 (28.1)
0.85 (0.49–1. 49)
0.58
Liver disease
9 (9.8)
18 (8.6)
1.16 (0.50–2.68)
0.73
CNS disease
19 (20.7)
31 (14.8)
1.50 (0.80–2.82)
0.20
Cancer
11 (11.8)
28 (13.1)
0.89 (0.42–1.87)
0.75
Trauma
8 (8.7)
23 (11.0)
0.77 (0.33–1.80)
0.55
Tattoo
4 (4.3)
12 (5.7)
0.75 (0.23–2.39)
0.78
Lower-extremity ulcers
15 (16.3)
16 (7.6)
2.36 (1.11–5. 01)
0.02
Amputation
2 (2.2)
8 (3.8)
0.56 (0.18–2.70)
0.73
Charlson comorbidity index ≥ 1
3 (2–4)
3 (2–4)
-
0.84
Hospitalization in the last year
11 (12.0)
37 (17.6)
0.63 (0.31–1.31)
0.21
Surgery in the last year
10 (10.9)
22 (10.5)
1.04 (0.47–2.30)
0.91
Antimicrobial use in the last year
21 (22.8)
57 (27.3)
0.79 (0.44–1.40)
0.42
All values are reported as number (%) unless otherwise specified. Significant associations are indicated in italic
OR odds ratio, CI confidence interval, CNS central nervous system
OR (95%CI)
p
0.98 (0.93–0.99)
0.02
0.31 (0.10–0.92)
0.03
2.44 (1.11–5.34)
0.03
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
Page 7 of 12
Table 2 Uni- and multivariate (logistic regression) analysis of predictors of MRSA carriage in diabetic individuals
Factor
MRSA (n = 15) Negative (n = 287)
Univariate analysis
OR (95%CI)
Logistic regression
(multivariate)
p
OR (95% CI)
p
3.64 (1.12–11.78)
0.03
Male gender
11 (73.3)
129 (43.3)
3.58 (1.11–11.51)
0.02
Age (median, quartiles)
58 (46–73)
63 (54–71)
–
0.27
Time since diagnosis, years (median, quartiles) 13 (10–19)
15 (9–24)
–
0.56
Use of insulin, years (median, quartiles)
7 (3–12)
–
0.94
8 (3–15)
Diabetes type 2
11 (73.3)
251 (84.5)
0.50 (0.15–1.65)
0.27
Heart disease
3 (20.0)
74 (25.8)
0.72 (0.20–2.62)
0.77
Lung disease
0
33 (11.5)
–
0.39
Kidney disease
4 (26.7)
78 (27.2)
0.97 (0.30–3.15)
1.00
Liver disease
1 (6.7)
26 (9.1)
0.72 (0.09–5.67)
1.00
CNS disease
1 (6.7)
49 (17.1)
0.35 (0.04–2.70)
0.48
Cancer
2 (13.3)
37 (12.7)
1.06 (0.30–4.87)
1.00
Trauma
0
31 (10.8)
–
0.38
Tattoo
1 (6.7)
15 (5.2)
1.29 (0.16–10.52)
0.57
lower-extremity ulcers
3 (20.0)
28 (9.8)
2.31 (0.61–8.69)
0.19
Amputation
1 (6.7)
9 (3.1)
2.21 (0.26–18.65)
0.40
Charlson comorbidity
index ≥ 1
3 (2–4)
3 (2–4)
–
0.41
Hospitalization in the last year
2 (13.3)
46 (16.0)
0.81 (0.18–3.69)
1.00
Surgery in the last year
3 (20.0)
29 (10.1)
2.22 (0.59–8.34)
0.20
Antimicrobial use in the last year
4 (26.7)
74 (25.9)
1.04 (0.32–3.37)
1.00
All values are reported as number (%) unless otherwise specified. Significant associations are indicated in italic
OR odds ratio, CI confidence interval, CNS central nervous system
different individuals and harbored SCCmec type IV. In
cluster B, it is possible to observe two isolates carrying
SCCmec type I (615 N and 637O) and one isolate carrying SCCmec type IV (72O). The MRSA isolate typed with
ApaI is shown in Fig. 2b. None of the isolates grouped
with international clones.
Two clusters (A and C) containing four strains and
two clusters (B and D) containing strains were obtained
for the MSSA isolates (Supplementary Material Figure
S1). Cluster A contained two isolates from the nasal and
oropharyngeal mucosa of the same subject, demonstrating colonization of different sites with the same isolate.
Similarly, in cluster B, two isolates from the nasal and
oropharyngeal mucosa of the same subject were grouped
with one nasal isolate of another subject. On the other
hand, the four isolates of cluster C were obtained from
unrelated individuals and in cluster D all isolates were
from the oral mucosa of different subjects. These findings suggest widespread dissemination of S. aureus in the
community.
Nine (52.9%) of the 17 subjects colonized at both sampling sites (nose/oropharynx) carried the same isolate in
the nasal and oral mucosa, while eight (47.0%) carried
different S. aureus strains in the nose and throat. Interestingly, two of the 17 subjects concomitantly colonized
with S. aureus in the nose and throat had their strains
(nasal and oropharyngeal) typed after digestion with
different restriction enzymes. Isolates 691O and 747 N
were typed with SmaI, while 691 N and 747O could only
be typed with ApaI, confirming that they are different
S. aureus strains. There was also one patient colonized
with MRSA (735 N) in the nasal mucosa and with MSSA
(735O) in the oropharyngeal mucosa, both typed with
ApaI.
Analysis of typable isolates with ApaI revealed three
major clusters (A, C, and D) and three minor clusters
of 3 isolates (B, E, and F). In cluster A which contained
14 isolates with 85.6% similarity, two isolates were from
the nasal and oropharyngeal mucosa of the same patient
and the remaining from different subjects. The same
was observed for clusters C and D. In cluster D, the isolates were grouped with a strain previously identified in
another study as ST398 (strain 76 N). These findings are
shown in Supplementary Material Figure S2.
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
Page 8 of 12
Fig. 2 Dendrogram of the PFGE-SmaI and PFGE-ApaI profiles of MRSA isolated from insulin-dependent diabetic individuals generated by Dice
analysis/UPGMA (BioNumerics, Applied Maths) and their molecular characterization by SCCmec typing and MLST. a. Isolates showing > 80%
similarity (clusters A and B) after digestion with SmaI. b. Band pattern of strain 735 N obtained by digestion with ApaI. N, nasal mucosa; O,
oropharyngeal mucosa; S, susceptible; R, resistant. * International clones used as controls
Molecular typing of S. aureus and MRSA by multilocus
sequence typing (MLST)
Based on the clusters obtained by PFGE, nine S. aureus
isolates (four MSSA and five MRSA) were selected for
molecular typing by the MLST technique.
Typing of the MRSA isolates revealed a predominance
of sequence type ST5 in three of the four isolates analyzed and one isolate with ST8. Regarding SCCmec type,
isolates of the following lineages were obtained: ST5-IV
(n = 2), ST5-I (n = 1), and ST8-IV (n = 1). In addition,
the strain typed with ApaI by PFGE was characterized as
ST398-IV (Fig. 2).
Among the clusters of the MSSA isolates obtained by
PFGE that could not be typed with SmaI and that were
digested with ApaI, four lineages were selected for MLST.
There was a predominance of ST398 (n = 3). However,
one isolate exhibited divergence in the allele of the arcC
gene and was sent to the curator of the MLST database
(https://pubmlst.org/) for identification of the ST. This
isolate was identified as ST 6133 (Fig. 3).
Discussion
The prevalence of colonization with S. aureus and MRSA
among insulin-dependent diabetic individuals was 30.4%
and 4.8%, respectively. Similar data have been reported
by Hart et al. [37] who analyzed 258 patients and found
a prevalence of 39.1% for S. aureus and 3.1% for MRSA
among diabetic individuals. In a population-based survey
Fig. 3 Dendrogram of the PFGE-ApaI profiles of MSSA isolates generated by Dice analysis/UPGMA (BioNumerics, Applied Maths) and sequence
types obtained by MLST. Clustering of isolates digested with ApaI that were analyzed by MLST. All isolates except for 735 N were susceptible to
methicillin (MSSA). N, nasal mucosa; O, oropharyngeal mucosa; arcC, carbamate kinase; aroE, shikimate dehydrogenase; glpF, glycerol kinase;
gmk, guanylate kinase; pta, phosphate acetyltransferase; tpi, triosephosphate isomerase; yqiL, acetyl coenzyme A; ST, sequence type. Isolate 76 N
was identified as ST398 in a previous study from our group. Isolate 700O was sent to the curator of the MLST database (https://pubmlst.org/) for
identification of the new ST. This isolate was identified as ST 6133
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
conducted in the same city, Pires et al. [38] found a similar prevalence among community-dwelling individuals,
with an overall prevalence of S. aureus of 32.7%. However,
the prevalence of MRSA was higher in our study (4.8%
vs 0.9%). In a recent study, Lin et al. [2] found a lower
prevalence of S. aureus and MRSA than that obtained in
the present study (16.4% S. aureus and 2.8% MRSA). The
authors suggested that the presence of different microorganisms in the microbiota of these individuals causes
competition for the same site, which could explain the
variation found in prevalence studies. None of the studies
included oral mucosa as a potential site of colonization.
It should be noted that 10.9% (n = 34) of the subjects
were colonized exclusively in the oral mucosa, six of
them with MRSA. This finding reinforces the suggestion
of Partida et al. [39] that colonization of the oral mucosa
can compromise control measures of pathogen dissemination since the throat is not part of routine screening.
The MRSA isolates were identified by phenotypic
methods (disc diffusion) and by PCR for detection of the
mecA gene. We found four isolates carrying the mecA
gene that did not exhibit phenotypic resistance to cefoxitin or oxacillin. Although mecA gene resistance is present
in all cells of a population with intrinsic resistance, it may
only be expressed by a small proportion of these cells,
a fact that results in the so-called heteroresistance [40].
Isolates carrying the mecA gene but that are susceptible
to oxacillin/cefoxitin have been reported worldwide and
are called oxacillin-susceptible MRSA (OS-MRSA) [41–
45]. According to Andrade-Figueiredo & Leal-Balbino
[46], this phenomenon may be due to partial excision of
SCCmec in multidrug-resistant MRSA isolates or chromosomal integration of the cassette chromosome, resulting in MSSA isolates that contain SCCmec segments.
High antimicrobial resistance of MRSA isolated from
diabetic patients has been reported in different studies
[14–16]. In our study, there were no multidrug-resistant
isolates and none of the isolates was resistant to vancomycin, although one MRSA isolate had a MIC of 1.5 μg/
mL, indicating a potential therapeutic risk [47–49]. Two
other MSSA isolates had a vancomycin MIC of 1.5 μg/
mL.
In the present study, the analysis of risk factors revealed
an association only with lower-extremity ulcers, which is
consistent with literature findings showing that the same
isolate colonizing the nares was present in foot ulcers and
wounds [50, 51].
Age was associated with a lower risk of S. aureus colonization, with a 2% decrease in the risk of colonization
for each additional year of age. Similar data have been
reported by Pereira-Franchi et al. [52]. It is believed that,
with increasing age, individuals are exposed to factors
(not analyzed in our study) that can prevent colonization
Page 9 of 12
with S. aureus, in addition to increased ecological competition with other microorganisms.
Lung disease was also a protective factor against the
acquisition of S. aureus, a fact that might be related to
colonization of the respiratory tract with other microorganisms that are competing with S. aureus. The nasopharyngeal microbiota changes over time; the level of
bacterial colonization is higher during upper respiratory
infection [53] and other species such as Streptococcus
pneumoniae and Haemophilus influenzae may thus interfere with the capacity of S. aureus to persist in the nasal
mucosa. Mueller et al. [54] also found a protective effect
of age but, in contrast to our findings, lung disease was a
risk factor for S. aureus colonization.
Multivariate analysis identified male gender as a risk
factor for colonization with MRSA. Similar results
were verified in a study of the prevalence of S. aureus
and MRSA in bedridden individuals and residents of
Long-Term Care Institutions for the Elderly (ILPIs) in
the same city [55]. In this study developed by Silva [55],
the male gender was associated with increased risk for
the carrying of S. aureus and was also the only variable
that showed to be a risk factor for carrying MRSA. Nillius et al. [56] reported that male ILPI residents had an
almost double risk for carrying MRSA when compared
to women, probably because they had more risk factors
than they did.
With respect to clonality of the MRSA isolates, lineages
belonging to the most widespread clonal complexes were
identified, including CC5-ST5-IV (639O) and CC8-ST8IV (72O) isolated from individuals with colonized oropharyngeal mucosa. This fact reinforces the importance
of throat colonization, which could be a route of transmission within the population examined. Other studies
involving individuals from the same city and region also
found CC5-ST5-IV and CC8-ST8-IV, suggesting that
these strains are prevalent in the region [57–62].
Studies suggest a high clonal diversity among S. aureus
isolates, particularly among MSSA [63]. Among the isolates that could not be typed with SmaI, four were typed
by MLST and were characterized as ST398. This fact was
also observed by de Souza [61]. The ST398 clonal lineage
has been associated with infection and colonization of
humans and domestic animals, such as dogs, horses and
pigs, in many countries around the world [8]. This lineage
is called livestock-associated S. aureus and was described
for the first time among both MSSA and MRSA on pig
farms in France [64, 65]. Since then, ST398 has spread
rapidly to other animals and has been increasingly related
to infections not only in rural workers but also in people and animals without risk factors [31, 66]. Although
susceptible to oxacillin, this S. aureus lineage is associated with severe infections, as reported by Bonesso et al.
Teixeira et al. Ann Clin Microbiol Antimicrob
(2021) 20:12
[67] in patients with ventilator-associated pneumonia in
whom the infection was fatal in most cases.
Our finding demonstrated a predominance of SCCmec
type IV among isolates, in agreement with the findings
of other prevalence studies on non-diabetic individuals
conducted in the State of São Paulo [57, 61]. However,
SCCmec types I and II were also detected, which are
commonly found circulating in health services. This fact
has also been reported by Pereira-Franchi et al. [62] and
Silveira et al. [68] who found a higher prevalence of isolates harboring SCCmec type II, which was attributed to
a history of hospitalization.
It is worth mentioning that patients in hospital-community settings, such as bedridden or institutionalized
older adults with chronic infections, have a higher prevalence of SCCmec types I and II [62, 68]; in addition, hospitalized patients frequently carry isolates that harbor
SCCmec type III. These SCCmec are larger and carry
plasmids and transposons with other resistance genes,
often multidrug-resistant genes. On the other hand,
community-dwelling patients are associated with SCCmec types IV and V, which are smaller and carry only the
mecA gene of methicillin resistance [38].
One limitation of the present study is the small number of
MRSA isolates (n = 15), which may result in a low statistical
power of the analyses. In addition, the patients studied are
not typically community-dwelling since they often need to
seek health services (primary care) because of their diabetes.
The present study provides important data about the epidemiology of S. aureus and MRSA in a population of insulin-dependent diabetic individuals. The isolates analyzed
had a low rate of resistance to the tested drugs, with only
one isolate being resistant to sulfamethoxazole-trimethoprim; however, the prevalence of MRSA was higher than
that found in a population-based study conducted in the
same city on healthy individuals [38]. Within this context,
screening for oral colonization is extremely important since
some individuals were colonized only at this body site. In
the population studied here, clones were detected among
the MSSA and MRSA isolates and an important clonal lineage (ST398) was identified. These data suggest widespread
dissemination of MRSA in the population of insulindependent diabetic patients studied, as well as the emergence of important S. aureus lineages in these individuals.
Supplementary information
Supplementary information accompanies this paper at https://doi.
org/10.1186/s12941-020-00401-y.
Additional file 1. They were included as tables of the characteristics of
the individuals included and excluded from the study. In addition to the
dendrograms of the PFGE-SmaI and PFGE-ApaI profiles of MSSA isolated
from insulin-dependent diabetic individuals.
Page 10 of 12
Acknowledgements
This work was funded by the São Paulo State Research Foundation (FAPESP—
Grant 2017/21396–0 and Grant 2020/15118-0), by the Coordination for
Improvement of Higher Education Personnel (CAPES) through a Master/
Doctorate Grant program, and by the National Council for Scientific and
Technological Development (CNPq; Grant 304051/2017–9).
Authors’ contributions
NBT and MLRSC designed the study and wrote the manuscript. NBT. and MCS
performed the experiments. NBT, TAMP and BPCC collected the samples.
CMCBF contributed to the study design and performed the statistical analysis.
All authors read and approved the final version of the manuscript.
Availability of data and materials
The datasets used and analyzed during the current study are available from
the corresponding author on reasonable request.
Ethical approval and consent to participate
The study was approved by the Ethics Committee of the Botucatu Medical
School, Brazil (Approval No. 1.107.685). All data were obtained by interview
with the patient and/or legal representative following ethical standards, who
agreed to participate by signing the consent form.
Consent for publication
Not applicable.
Competing interests
The author(s) declare no competing interests.
Author details
1
Departamento de Infectologia, Dermatologia, Diagnóstico Por Imagem
e Radioterapia, Faculdade de Medicina de Botucatu, UNESP - Universidade
Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil. 2 Departamento
de Ciências Químicas e Biológicas, Instituto de Biociências de Botucatu, UNESP
- Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil.
3
Departamento de Clínica Médica – Endocrinologia, UNESP - Universidade
Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil. 4 Departamento
de Ciências Químicas e Biológicas – Setor Microbiologia e Imunologia, Instituto de Biociências de Botucatu (IBB)-Laboratório de Bacteriologia. Rua Plínio
Silva, CEP: 18618-970 – Distrito de Rubião Júnior, Botucatu, SP, Brasil.
Received: 27 August 2020 Accepted: 19 November 2020
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