Rev Panam Infectol 2007;9(2):18-22
artículo original/artigo original
Spread of a methicillin-resistant Staphylococcus
aureus clone isolated from bacteremia in elderly
patients in a Brazilian general hospital
Disseminação do clone de Staphylococcus aureus resistente à meticilina
isolado de bacteremia em pacientes idosos em um hospital geral brasileiro
Rosineide Marques Ribas1
Ana Lúcia da Costa Darini2
Joseane Cristina Ferreira3
Claudete Freitas4
Paulo Pinto Gontijo Filho5
Adjunct Professor, Hospital de Clínicas, Laboratório de Microbiologia da Universidade
Federal de Uberlândia, Uberlândia, MG, Brasil.
2
Professor, Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de
Ciências Farmacêuticas, Ribeirão Preto – USP,
SP, Brasil. 3
Technician, Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de
Ciências Farmacêuticas, Ribeirão Preto – USP,
SP, Brasil. 4
Technician, Hospital de Clínicas, Laboratório
de Microbiologia da Universidade Federal de
Uberlândia, Uberlândia, MG, Brasil.
5
Professor, Hospital de Clínicas, Laboratório
de Microbiologia da Universidade Federal de
Uberlândia, Uberlândia, MG, Brasil.
1
Rev Panam Infectol 2007;9(2):18-22
Conflicto de intereses: ninguno
Recibido en 5/12/2006.
Aceptado para publicación en 31/5/2007.
18
Abstract
A study of the genomic diversity of MRSA strains isolated from
elderly patients with infection/colonization in three repeated prevalence, cross sectional studies was performed in the 1999-2000
period. In this study, 13 MRSA isolates from blood cultures and 5
from rectal and nare cultures were obtained from 18 patients (13
elderly and 5 adults). Most of the patients were being treated with
two or more antimicrobials (83.3%), had insertion of invasive devices (88.9%) and were managed in ICU (Intensive Care Unit) and/
or surgical units (66.7%). MIC (Minimum Inhibitory Concentration)
data showed that 88.9% of the MRSA strains were resistant to high
concentrations of oxacillin (MIC > 256 μg/mL), 94.5% of the MRSA
carried the mecA gene in their genome, and most (65.0%) of the
isolates were indistinguishable according to their DNA finger­printing
generated by PFGE (Pulsed-field gel electrophoresis). Although
PFGE typing was performed with a few MRSA isolates, our results
demonstrate that one MRSA clone was associated with infection/
colonization in patients with an obvious connection among five out
of eleven patients who stayed in the same clinic and ICU during the
same period. Hospital acquired infection, a major “silent epidemy”,
is associated with prolonged hospital stay and high mortality rate
and its cause must be better evaluated.
Key words: MRSA, epidemic clone, elderly patients.
Resumo
Nós estudamos a diversidade genética de amostras de MRSA
isoladas de pacientes idosos com colonização/infecção, através de
três inquéritos repetidos de prevalência realizados entre o período
de 1999-2000. Nesse estudo, foram analisados 13 isolados de
sangue e cinco de culturas de narina e intestino de 18 pacientes
(13 idosos e cinco adultos). A maioria dos pacientes estava sendo
tratada com dois ou mais antibióticos (83,3%), estavam com
dispositivos invasivos (88,9%) e internados na UTI (Unidade de
Ribas RM et al • Spread of a methicillin-resistant Staphylococcus aureus...
Terapia Intensiva) e/ou unidades cirúrgicas (66,7%).
A CIM (Concentração Inibitória Mínima) demonstrou
que 88,9% das amostras de MRSA foram resistentes
a altas concentrações de oxacilina (MIC > 256 μg/mL)
e 94,5% carreavam o gene mecA no seu genoma e
a maioria (65%) dos isolados foram indistinguíveis
de acordo com o DNA “fingerprinting” gerado pelo
PFGE (“Pulsed-field gel electrophoresis”). Embora a
tipagem pelo PFGE tenha sido feita para um pequeno
número de isolados, nossos resultados demonstram
que um clone de MRSA foi associado com infecção/
colonização em pacientes idosos, com uma conexão
entre cinco dos 11 pacientes que estavam na mesma
enfermaria ou UTI e hospitalizados durante o mesmo
período. As infecções adquiridas no hospital são associadas com hospitalização prolongada e um aumento
na mortalidade, e essa epidemia silenciosa causada por
esse microrganismo precisa ser melhor avaliada.
Palavras-chave: MRSA, clone epidêmico, paciente
idoso.
Introduction Elderly population will grow rapidly over the next
25 years(1). There is little information about hospital
infections in this age group in Brazil(2). In a situation
similar to Mexico, most Brazilian medical care relies on
government institutions, and infection control polices
have to face two crucial problems: limited resources
for health care and low awareness of the importance
of preventing nosocomial infections(3).
Studies on methicillin-resistant Staphylococcus
aureus (MRSA) in the elderly have been performed with
hospitalized patients in nursing homes or long term
care facilities(4). Introduction of MRSA into a health
care setting may occur mainly as a result of an infected
or colonized patient being admitted(4). Risk factors for
colonization or infection have been identified and they
include advanced age, previous hospitalization, length
of stay, admission to either a burn unit or ICU, previous
antibiotic therapy, exposure to a colonized or infected
patient, and the use of invasive procedures(5).
This study aimed to identify cloned profiles of
MRSA isolates from elderly patients and the proba­
bility of intra-hospital dissemination of one or more
clones.
Methods
Patients: Staphylococci isolated from 13 hospitalized elderly patients (older than 65 years) were studied;
5 hospitalized adults (age range 14-64) were used as
controls of the PFGE test. The patients were admitted
to the University Hospital of the Uberlandia Faculty of
Medicine, in Minas Gerais, Brazil, a 503-bed teaching,
tertiary hospital.
Study design: During the study three repeated
prevalence, cross-sectional studies were carried out
in January 1999, February 2000 and October 2000
in different units of the hospital. Clinical and demographic patient information, including intrinsic (age
and length of hospitalization) and extrinsic risk factors
(surgery, invasive devices and use of antimicrobial
agents) were obtained from clinical records. Microbiological techniques: We evaluated a total of 50
blood cultures and 16 rectal and nare cultures from the
Microbiology laboratory. Eighteen isolates were selected
for PFGE. Mannitol salt agar containing 6μg/mL of oxacillin was used for MRSA isolation. Staphylococcus
aureus was identified in accordance with its growth
characteristics on mannitol salt agar, coagulase reaction and the presence of the coa gene. Multiresistant
isolates were assessed by the disk diffusion method,
according to CLSI recommendations (6), to the following antimicrobial agents: oxacillin, cephalotin,
ceftazidime, ceftriaxone, erythromycin, ciprofloxacin,
levofloxacin, imipenen, gentamicin, clindamicin, tetracycline, vancomycin, trimethoprim-sulfamethoxazole,
chloramphenicol, rifampin and amikacin.
Oxacillin agar screening method: Agar screen tests
for susceptibility to oxacillin were performed as indicated by CLSI guidelines(6). Inoculum suspensions
were prepared from 18-24h cultures on blood agar
and adjusted to 0.5 on the MacFarland scale (Densimat, bioMerieux, l´Etoile, France). For each isolate,
an area 10 to 15 mm in diameter was spotted with a
1 μL disposable loop on Mueller- Hinton agar (Oxoid)
supplemented with 4% NaCl and 6 μg mL of oxacillin. The plates were then incubated for 24h at 35°C.
Control strains: Staphylococcus aureus American Type
Culture Collection (ATCC) 29213 and Staphylococcus
aureus ATCC 43300 were used as negative and positive
control, respectively.
Determination of minimum inhibitory concentration
(MIC): The oxacillin MICs were determined by Brain
Heart Infusion (BHI) broth dilution. The broth dilution
was performed as previously described in the CLSI approved standard(6). Colonies were taken from overnight
blood agar inoculums, cultivated at 35°C, and read
after 24 h incubation.
Amplification by PCR for mecA, coa and 16SrRNA
genes: The PCR method was carried out to detect the
presence of the mecA, coa and 16SrRNA genes. Chromosomal DNA of the strains was purified after lyses
of cells in the presence of lysozyme (100 mg/mL) and
lysosthaphin (1 mg/mL), using standard protocols(7).
The reported primers (table 1) were used for amplification of the genes by PCR. PCR was carried out for
30 cycles of 60s at 94°C, 30s at 52°C, and 30s at
72°C, with a final extension step at 72°C for 10 min.
19
Rev Panam Infectol 2007;9(2):18-22
Table 1. Primers used in this study
Primer
Gene
Sequence (5’-3’)
MecA1
mecA
CTCAGGTACTGCTATCCACC
Accession
Position
GeneBank
4506-4525 Y 14051
MecA2
mecA
CACTTGGTATATCTTCACC
4954-4936 Y 14051
16S1
16S rRNA CGAAAGCCTGACGGAGCAAC
378-397
AJ 536434
16S2
16S rRNA AACCTTGCGGTCGTACTCCC
520-501
AY 144447
Coa1
coa
GAACAAAGCGGCCCCATCATTA
1301-1322 X 17679
Coa2
coa
TAAGAAATATGCTCCGATTGTCG
2153-2131 X 17679
a
a
refers to the positions on the gene sequence.
Table 2. Characteristics of 18 hospitalized patients
(13 elderly and 5 adults) at Uberlandia Federal
University hospital
Characteristic
Age (years) median
Hospitalization (days) median
Antibiotics
Yes
N>2
Invasive devices
N>2
Indwelling urinary catheter
Mechanical ventilation
Intravascular catheter
Peripheral
Central
Surgery
Patients N = 18 n (%)
62.3
41.3
16 (88.9)
15 (83.3)
16 (88.9)
14 (77.8)
6 (33.3)
15 (83.3)
11 (61.1)
10 (55.6)
Table 3. Distribution of patients with hospital acquired
infection/colonization by units
Units
Internal medicine units
Surgical units
Intensive care units
Emergency units
Total
Number (%)
5 (27.8)
7 (38.9)
5 (27.8)
1 (5.6)
18 (100.0)
The same conditions were used for amplification of the
16SrRNA gene, except for the annealing temperature
which was 62°C. The amplified products were analyzed
by 1.5% agarose gel electrophoresis and the size of the
amplification products was estimated by comparison
with the molecular size standard 123 bp ladder. Control
strain: Staphylococcus aureus ATCC 27923 was used
as control strain for the PCR amplification method. Pulsed Field Gel Electrophoresis (PFGE): Clone
identity between MRSA isolates was confirmed by
PFGE. Bacterial lysis, SmaI digestion of chromosomal
DNA and analysis of DNA fragments by PFGE were
carried out as previously described(8). Electrophoresis
was briefly performed in a Gene Navigator apparatus at
20
200v for 25 h, at 14°C. The equipment was adjusted
for a pulse of 25s for 20 h, 5s for 4 h and 0.5s for 1 h.
Banding patterns were visualized by ethidium bromide
staining and UV transillumination. Previously described
criteria(9) were used, from visual comparisons of ban­
ding patterns of samples run together in the same gel.
Dendrogram was constructed by computer-assisted
(MVSP 3.0) comparison.
Ethical approval: Ethical approval to conduct the
study was obtained from the Institutional Ethics Committees of the participating hospital.
Results In the present study, 18 MRSA isolates from clinical
speciments were obtained: 13 blood samples and 5
nasal and gut colonizers. Median age was 70.2 among
the 13 elderly patients. Most of the patients were
treated with two or more antibiotics (83.3%), insertion of invasive devices (88.9%) and were managed
in surgical wards (38.9%) (table 2).
Twenty-eight percent of patients colonized and infected with MRSA were managed in an ICU, and most
of them (72%) were elderly. A prolonged hospital stay
(median 14.3 days) and high proportion of antimicrobial therapy (89%) were also found (tables 2 and 3).
MIC data showed that 88.9% of the MRSA strains
were resistant to high concentrations of oxacillin
(MIC > 256 μg/mL) and all isolates showed resistance to
1 μg/mL oxacillin by disk diffusion test. Resistance to
vancomycin was not observed. 94.5% of the MRSA
isolates carried mecA gene in their genome resulting
in a 154 pb fragment after PCR amplification. There
was concordance (72.2%) in our study between PCR
results and those of the agar plate screen.
The identical PFGE patterns in lanes (1-3, 6-7,
11, 13-17) and nearly identical (80%) patterns (4,
9-10 and 18) from elderly (A) and adults (A-1) patients isolates, respectively, suggests the presence of
a predominant MRSA clone, responsible for most of
infection/colonization in these patients (figure 1).
Besides the involvement of a single clone with
infection/colonization in 11 elderly patients, a connection was found between the length of stay in a private
ward and half of patients (table 4).
Discussion From a hospital perspective, patients with MRSA
tend to be older, have more chronic illnesses, a recent
history of hospitalization, some type of surgery and a
longer hospital stay(10). The present study has detected
and considered such factors.
Susceptibility to oxacillin was initially determined by
disk diffusion methods and the agar plate screening test
supplemented with 4% sodium chloride and containing
Ribas RM et al • Spread of a methicillin-resistant Staphylococcus aureus...
Table 4. Properties of the strains of MRSA
Strains
(patient)
32 (1)
35 (14)
41 (13)
43 (4)
44 (5)
46 (6)
47 (2)
48 (18)
49 (3)
50 (16)
57 (7)
58 (11)
59 (8)
60 (9)
61 (10)
64 (15)
75 (17)
76 (12)
Age
(years)
73
45
40
71
65
66
72
75
66
69
68
69
70
71
78
34
41
47
Site (infection/
colonization)
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Nare
Nare
Nare
Nare
Gut
Blood
Blood
Blood
Data of isolation
(month/year)
10/2000
7/2000
9/2000
4/2000
3/2000
5/2000
6/2002
7/2002
6/2002
4/2002
10/2000
1/1999
10/2000
1/1999
11/2000
10/2002
5/2002
7/2002
MIC oxacillin
(µg/mL)
512
512
512
512
256
512
256
1.0
512
16
512
512
512
512
512
1024
256
1024
Ward
Sur2
ICU
Sur2
ICU
ICU
Med
Sur2
Sur1
Sur2
Med
Sur1
Med
Med
Med
Emer
Med
Sur2
Med
PFGE
group
A1
A2
A1
A
A
A
A1
B
A1
C
A
A
A
A
A
A1
B
A1
Sur1, surgical ward 1; Sur2, surgical ward 2; Med, medicine ward; Emer, emergency; ICU, intensive care unit.
oxacillin (6 μg/mL) was used as the confirmatory test(11).
Ninety-five percent of the methicillin-resistant strains
of Staphylococcus aureus carry the mecA gene, which
is responsible for phenotypic expression of methici­llin
resistance(12). Thus, detection of the mecA gene by
PCR has been found as potentially sensitive methods
to detect methicillin-resistant strains(12). This research
has involved the use of PCR with primers specific for
mecA gene and all MRSA isolates presented mecA
gene. From a clinical perspective, it is important to
differentiate isolates that have mec-positive resistance,
which is the classic type of oxacillin resistance, because they are usually resistant to other agents(11).
Monitoring and investigation of MRSA isolates
requires a precise typing method. DNA fingerprin­
ting by macro restriction and PFGE have been consi­
dered the gold standard technique for MRSA typing
because of their high discriminative power and their
good correlation with epidemiological data(13). Our
results showed that the majority (11/18, 65.0%) of
the isolates were similar, as their DNA fingerprinting
a)
b)
Figure1. PFGE profiles of SmaI-digested
genomic DNA of 18 Staphylococcus aureus strains.
a) Lanes M represent the concatamer of DNA lambda; lanes 1
to 3, 6 and 7, 13 to 17 contain the digested DNA with identical
PFGE patterns and nearly identical patterns (4, 9-10 and 18)
from elderly (A) and adults (A-1) patients isolates, respectively.
b) Dendrogram resulting from the computer-assisted
analysis of the PFGE profiles.
21
Rev Panam Infectol 2007;9(2):18-22
were all mecA gene.
The predominance of specific MRSA clones over
large geographic areas, especially the Brazilian endemic clone (BEC), widespread in Brazilian hospitals,
has been reported elsewhere(14-16). In this study, we
were not able to verify if our MRSA isolates belonged
to the BEC, since we did not include a representative of this epidemic clone for comparison purposes
at PFGE. However, our results indicated that eleven
strains (65.0%) had indistinguishable PFGE genotypes. Although PFGE typing was performed with a
reduced number of MRSA isolates, our results demonstrate the presence of a MRSA clone associated
with infection/colonization of elderly patients. This
clone may have spread among colonized and infected
patients, since there was an obvious connection between five out the eleven patients who were on the
same medicine ward (patients 9, 11), surgical ward
(patients 2, 3) and ICU (patients 4,5) all hospitalized
during same period, with all MRSA isolates from the
same clone (profile A).
In developing countries the proportion of elderly
people in the general population and among those
hospitalized has been increasing and they represent a
large proportion in hospitals, where they stay longer than
younger patients. Thus, along with other factors of risk
(ICU, antibiotics and surgery), they become more susceptible to infection by MRSA as the findings show.
Acknowledgements
We thank I. C. V. Palazzo for technical assistance
during the molecular experiments. This work was partially supported by Pro-Reitoria de Pesquisa–University
of São Paulo – Brazil and CAPES.
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Correspondence:
Profa. Dra. Rosineide Marques Ribas
R. Nordau Gonçalves Melo, 1.245, B - Santa Mônica
CEP 38408-218 - Uberlândia - MG - Brazil.
e-mail: [email protected]