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Brazilian Journal of Infectious Diseases

Print version ISSN 1413-8670On-line version ISSN 1678-4391

Braz J Infect Dis vol.21 no.2 Salvador Mar./Apr. 2017

http://dx.doi.org/10.1016/j.bjid.2016.09.015 

Brief Communications

Complete substitution of the Brazilian endemic clone by other methicillin-resistant Staphylococcus aureus lineages in two public hospitals in Rio de Janeiro, Brazil

Raiane Cardoso Chamona  1

Sthefanie da Silva Ribeiroa  1

Thaina Miranda da Costaa 

Simone Aranha Nouérb 

Katia Regina Netto dos Santosa  * 

aUniversidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Médica, Rio de Janeiro, RJ, Brazil

bUniversidade Federal do Rio de Janeiro, Faculdade de Medicina, Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, RJ, Brazil

Abstract

Staphylococcus aureus is an important cause of bloodstream infections. Therefore, the main purpose of this work was to characterize a collection of 139 S. aureus isolates from bloodstream infections in two public hospitals in relation to their antimicrobial susceptibility profile, staphylococcal cassette chromosome mec types, and clonal relationship. Methicillin resistance and resistance to other 12 agents were accessed by the disk diffusion test. Minimum inhibitory concentration to mupirocin was also determined. The SCCmec types were accessed by multiplex PCR, and the clonal relationship was determined by pulsed field gel electrophoresis method and restriction modification system characterization. Besides, multilocus sequence typing was performed for representative methicillin-resistant S. aureus isolates. The military hospital showed a dissemination of the New York/Japan (USA100/ST5/CC5/SCCmecII) lineage associated to multidrug resistance, including mupirocin resistance, and the teaching hospital presented polyclonal and non-multidrug resistant MRSA isolates. Complete substitution of the Brazilian endemic clone by other lineages was found in both hospitals. These findings can highlight differences in policy control and prevention of infections used in the hospitals and a change in the epidemiological profile of MRSA in Brazilian hospitals, with the replacement of BEC, a previously well-established clone, by other lineages.

Keywords: Staphylococcus aureus; Bloodstream infections; USA100; Mupirocin resistance

Staphylococcus aureus is considered an important cause of bloodstream infections (BSI), which is associated with high rates of mortality and morbidity.1 Analysis of molecular characteristics of S. aureus isolates have indicated a variety of circulating lineages inside hospitals, according to the geographic area. In United States, the New York/Japan clone (USA100/ST5/CC5/SCCmecII) has been replaced by the community-acquired MRSA (USA300/ST8/CC8/SCCmecIV) lineage.1 In China, two pandemic hospital-acquired MRSA (HA-MRSA) clones are disseminated, the Brazilian endemic clone (BEC/ST239/CC8/SCCmecIII) and the USA100.2 In Brazil, the BEC lineage remained prevalent inside hospitals,3 but an increasing presence of the clones USA400 (ST1/CC1/SCCmecIV) and the Pediatric clone (USA800/ST5/CC5/SCCmecIV) have been reported in the last decade.3,4 More recently, SCCmecII carrying isolates associated to the CC5 were detected replacing, almost completely the BEC lineage among BSI isolates at a hospital located in São Paulo city.5

The implementation of a Health Care Associated Prevention and Control Committee (HAIPCC) is mandatory by law in Brazilian hospitals since 1997.6 These measures apply to the whole health care system, such as the public and the private sector. Public hospitals are responsible for the care of about 75% of the Brazilian population, estimated in 192 millions of habitants (2012 data). However, funding for the Unified Health System (Sistema Único de Saúde – SUS) has not been sufficient to ensure adequate financial resources for the public health system, leading to inappropriate control of dissemination of endemic resistant microorganisms.6 The aim of the present study was to characterize S. aureus isolates from BSI at two public hospitals as their antimicrobial resistance and clonal dissemination associated with clinical aspects.

We evaluated 139 S. aureus consecutive isolates from BSI recovered in a 532-bed military hospital (Hospital 1) and in a 490-bed university teaching hospital (Hospital 2), both located in Rio de Janeiro city, between January 2008 and June 2009. This study was approved by the Research Ethics Committee under No. 159/07. Clinical data from patients with S. aureus BSI were retrospectively abstracted from the hospital records. S. aureus isolates were identified by standard methods. BSIs were classified as hospital-acquired (HA) or community-acquired (CA) according to the Centers for Disease Control (CDC) criteria.

In order to characterize methicillin resistance, cefoxitin disk diffusion test was used according to CLSI.7 Isolates identified as MRSA were also submitted to antimicrobial susceptibility test for 12 agents by the disk diffusion method.7 Minimum inhibitory concentration (MIC) to mupirocin was determined by Etest ® (AB-Biodisk, Solna, Sweden). The SCCmec types were assessed by multiplex-PCR for MRSA isolates.8 Clonal relationship was determined by pulsed-field gel electrophoresis (PFGE).9 Restriction modification system characterization (RM test)10 was used to identify the clonal complexes (CC) of methicillin susceptible S. aureus (MSSA) isolates. Besides, multilocus sequence typing (MLST) was performed for representative MRSA isolates.11 The Fisher's exact test and chi-square test were used to compare categorical data. Significance level was established at 5% (p < 0.05).

The distribution of the 139 S. aureus isolates and their SCCmec types and clonal complexes in each hospital is shown in Table 1. Out of 75 isolates of Hospital 1 (H1), 32 (43%) were characterized as MRSA, whereas in Hospital 2 (H2) from 64 isolates 13 (20%) were MRSA isolates (p = 0.006). While at H1 the majority of MRSA isolates carried the SCCmec type II (69%), at H2 the SCCmec type IV (69%) was the most prevalent. Overall, only one isolate from H2 carried the SCCmec type III and was assigned as ST889/CC5. In relation to the CC assignment, the majority of MRSA and MSSA isolates (83%; 62/75) at H1 were related to CC1 and CC5. However, there was a polyclonal distribution of S. aureus isolates causing BSI (CCs 1, 5, 8, 30, 45, 221) at H2 regardless of their methicillin resistance.

Table 1 Distribution of 139 methicillin-susceptible and -resistant Staphylococcus aureus isolates, SCCmec types and clonal complexes from bloodstream infections. 

Hospital/methicillin-resistance (number of isolates) N (%) of isolates
SCCmec type Clonal complexes
II III IV 1 5 8 30 45 221 ND
Hospital 1
MRSA (32) 22 (69) 0 10 (31) 9 (28) 23 (72) 0 0 0 0 0
MSSA (43) - - - 14 (32) 16 (37) 1 (3) 2 (5) 4 (9) 0 6 (14)
Total (75) 23 (31) 39 (52) 1 (1) 2 (3) 4 (5) 0 6 (8)
Hospital 2
MRSA (13) 2 (23) 1a (8) 9 (69) 3 (23) 7 (53) 0 1 (8) 1 (8) 1 (8) 0
MSSA (51) - - - 17 (33) 6 (12) 7 (14) 7 (14) 4 (8) 0 10 (19)
Total (64) 20 (31) 13 (20) 7(11) 8 (12.5) 5 (8) 1 (2) 10 (15.5)

MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; SCCmec, Staphylococcal cassette chromosome mec; ND, not determined; N, number.

aST889/CC5.

Characteristics of 45 MRSA isolates from BSI of patients from the two hospitals evaluated are presented in Table 2. Overall, 93% (42 isolates), 75% (34), and 35% (16) of the MRSA isolates were resistant to ciprofloxacin, clindamycin, and mupirocin, respectively. Among the MRSA isolates from H1, resistance to three or more drug classes (multidrug resistance – MDR) was verified in 59.3% (19/32), while among the MRSA isolates from H2, MDR was found in only 23% (3/13) (p = 0.05). Moreover, 97% of the MRSA isolates from H1 were related to only two disseminated lineages, USA100/ST5/CC5/SCCmecII (69%) and USA400/ST1/CC1/SCCmecIV (28%), all of them causing hospital-acquired BSI. Furthermore, 94% (15/16) of mupirocin-resistant isolates were found at H1 and it was associated to the USA100 lineage. Two USA100 isolates (1238a and 1240a) showed high levels of mupirocin resistance (MIC > 1024 µg/mL) (data not shown). In this hospital, the prevalent USA100/ST5/SCCmecII lineage was found widely disseminated. At H2, 62% of MRSA isolates carried the SCCmec IV and were related to USA800 (38%) or USA400 (23%) lineages. Besides, the polyclonal presence of sporadic lineages (STs 484, 889, 3050, and 221) was identified in this hospital, the ST3050 being described for the first time in this study.

Table 2 General characteristics of 45 methicillin-resistant Staphylococcus aureus isolates from bloodstream infections. 

Hospital/genotype (no of isolates) Isolate number Isolation date (mm/dd/yy) Unit or floor Acquisition mode SCCmec type PFGE subtype Clonality ST/CC Antimicrobial resistance profile
Hospital 1 (32)
A (22) 1223a 01/10/2008 11 HA II A1 USA100 5/5 cip cli ery mup tec
1224a 01/12/2008 ICU HA II A1 USA100 5/5 cip cli ery mup
1255a 09/02/2008 ICU HA II A1 USA100 5/5 cip cli ery mup
1258a 09/16/2008 ICU HA II A1 USA100 5/5 cip cli ery mup
1265a 09/23/2008 9 HA II A1 USA100 5/5 cip cli ery mup
1266a 09/24/2008 9 HA II A1 USA100 5/5 cip cli ery mup
1276a 02/12/2008 9 HA II A1 USA100 5/5 cip cli ery mup
1288a 03/12/2009 ICU HA II A1 USA100 5/5 cip cli ery mup
1289a 03/13/2009 11 HA II A1 USA100 5/5 cip cli ery mup
1309a 03/14/2009 ND HA II A1 USA100 5/5 cip cli ery mup clo rif
1290a 03/19/2009 11 HA II A1 USA100 5/5 cip cli clo ery
1291a 03/31/2009 9 HA II A1 USA100 5/5 cip cli ery mup
1305a 06/04/2009 11 HA II A1 USA100 5/5 cip cli ery mup clo rif
1308a 06/15/2009 10 HA II A1 USA100 5/5 cip cli ery mup clo rif
1260a 09/17/2008 10 HA II A2 USA100 5/5 cip cli ery
1263a 09/22/2008 11 HA II A2 USA100 5/5 cip cli ery
1275a 12/02/2008 ICU HA II A2 USA100 5/5 cip cli ery
1238a 05/26/2008 8 HA II A3 USA100 5/5 cip cli ery mup
1240a 05/27/2008 Em HA II A3 USA100 5/5 cip cli ery mup
1284a 02/02/2009 Em HA II A4 USA100 5/5 cip cli ery
1285a 02/16/2009 11 HA II A4 USA100 5/5 cip cli ery
1301a 05/27/2009 IU HA II A5 USA100 5/5 cip cli clo ery
B (9) 1229a 02/09/2008 11 HA IV B1 USA400 1/1 cip cli ery
1237a 05/20/2008 11 HA IV B1 USA400 1/1 cip cli ery
1307a 06/12/2009 ICU HA IV B1 USA400 1/1 cip clo
1231a 02/13/2008 11 HA IV B2 USA400 1/1 cip
1282a 01/22/2009 ICU HA IV B2 USA400 1/1 cip clo
1268a 09/26/2008 10 HA IV B3 USA400 1/1 cip cli ery gen
1283a 01/22/2009 ICU HA IV B3 USA400 1/1 cip clo
1295a 05/25/2009 9 HA IV B4 USA400 1/1 -
1302a 05/21/2009 ICU HA IV B5 USA400 1/1 cip clo
F (1) 1306a 06/04/2009 10 HA II F ND 105/5 cip cli clo ery
Hospital 2 (13)
A (1) 1087a 01/20/2008 11 HA II A1 USA100 5/5 cip cli ery mup
B (3) 1094a 01/16/2008 9 CA IV B1 USA400 1/1 cip cli ery
1187a 06/12/2008 8 HA IV B1 USA400 1/1 cip cli clo ery
1100a 01/27/2008 Em CA IV B2 USA400 1/1 cip cli ery
C (5) 1214a 06/26/2009 7 HA IV C1 USA800 5/5 -
1318a 08/16/2008 Em HA IV C2 USA800 5/5 cip
1324a 08/08/2008 Em HA IV C3 USA800 5/5 cip
1328a 12/23/2008 Em HA IV C4 USA800 5/5 cip cli ery
1326a 12/25/2008 9 HA IV C4 USA800 5/5 -
D (1) 1314a 11/08/2008 7 HA IV D ND 484/30 cip cli ery
E (1) 1092a 02/22/2008 9 HA III E ND 889/5 cip clo ery gen sut tec
G (1) 1212a 06/02/2009 ICU HA II G ND 3050/45 cip cli ery
H (1) 1219a 06/06/2009 8 HA II H ND 221/221 cip cli ery

ICU, intensive care unit; Em, emergency; ND, not determined; HA, hospital acquired; CA, community acquired; SCCmec, Staphylococcal cassette chromosome mec; PFGE, pulsed field gel electrophoresis; ST, sequence type; CC, clonal complex; cip, ciprofloxacin; cli, clindamycin; ery, erythromycin; mup, mupirocin; tec, teicoplanin; clo, chloramphenicol; rif, rifampin; gen, gentamicin; sut, sulfamethoxazole/trimethoprim.

In our study, 139 S. aureus isolates from BSI obtained at two different public hospitals in Rio de Janeiro city were characterized regarding their antimicrobial resistance and clonal profile. We verified that almost 70% of the BSI isolates from the military public hospital (H1) carried the SCCmecII and were related to the USA100 lineage. All but one isolate were assigned as USA400 lineage. This lineage appears to have survival and growth advantage since it has remained for years as a major hospital-associated lineage in USA and Japan1,12 showing the good adaptability of such clone, even in different geographic areas.

At H2, a reference teaching public hospital, a polyclonal profile was observed for the MRSA isolates. Interestingly, we previously found a similar higher clonal diversity among MRSA isolates at a private hospital.3 These findings may be a reflection of the occurrence of fewer outbreaks due to adequate infection control measures at this particular institution, as found in this teaching public hospital. Padoveze et al.6 conducted a cross-sectional study evaluating a collection of 153 hospitals from five different Brazilian regions. The authors showed that a minimal structure is necessary for an effective prevention of hospital infections, specially the presence of an active HAIPCC, as well as sterilization services, hand hygiene resources, and a microbiology laboratory.

Caboclo et al.3 compared S. aureus isolates from two health institutions in Rio de Janeiro between 2004 and 2007. One of these institutions was the same military hospital (H1) of the current study. The authors showed a dissemination of the USA100, USA400, USA800 and BEC lineages at this military institution. Moreover, around 60% of the isolates were from the BEC lineage, showing that at the time this lineage was still highly present. Simultaneously, similar results were found in a study conducted at a private tertiary care hospital in São Paulo where 40% (13/33) of MRSA isolates belonged to BEC and 21% were related to the USA100 lineage.13 At H2, Cavalcante et al.14 showed that the BEC lineage was responsible for around 30% of all MRSA isolates, between 2005 and 2006. In the present study, carried out between 2008 and 2009, complete absence of the BEC lineage was verified in both public institutions evaluated, showing that certain global MRSA lineages are replacing BEC. Caiaffa-Filho et al.5 recently evaluated a collection of 50 consecutive MRSA BSI isolates in a São Paulo tertiary care teaching hospital, between October and December 2010, and found that a single PFGE clone related to the USA100 lineage was disseminated, almost replacing the BEC in that institution during the study period. This finding highlights a possible change in the epidemiological profile of MRSA in Brazilian hospitals.

MRSA isolates were more frequently found at H1 than in the teaching hospital (H2) being associated to a MDR profile and mupirocin resistance. In Brazil, despite the legislation mandating the implementation of HAIPCC in the health system, the lack of qualified professionals, the growing health costs, and limited availability of financial resources are of great impact in the infection control.6 Therefore, the differences regarding resistance rates observed between MRSA isolates from the H1 and H2, as well as the prevalence of specific SCCmec types, may be a reflection of the policy for control and prevention of infections and use of antimicrobials in the hospitals evaluated. Interestingly, the prevalent USA100 clone presenting MDR profile isolated from H1 had already been described as a MRSA-daptomycin-resistant isolate with vancomycin MIC of 4 µg/mL, also causing BSI,15 confirming the ability of this lineage to acquire resistance determinants. Moreover, this lineage was also associated with mupirocin resistance, a drug used for decolonizing MRSA nasal carriage.2 Although only two isolates have showed high levels of mupirocin resistance (MIC > 1024 µg/mL), both high- and low-level resistance have been associated with S. aureus decolonization failure.2 As showed in a study conducted in China,2 USA100/ST5 isolates associated with mupirocin resistance lead to possible outbreaks. The dissemination of mupirocin-resistant MRSA isolates among H1 patients in our study also highlights the importance of the judicious use of mupirocin among the hospitalized patients.

MRSA, as well as MSSA isolates, have been reported as important causes of nosocomial infections, such as BSI.1 The present study showed that S. aureus isolates from the CCs 1 and 5, regardless their methicillin-resistance status, had similar clonality at both hospitals. According to Diep and Otto,16 the emergence of the MRSA lineages around the world may be related to the successful conversion of certain MSSA isolates into MRSA isolates by the acquisition of SCCmec. The presence of MSSA and MRSA isolates presenting the same CCs and/or STs have already been observed in Rio de Janeiro hospitals,4 indicating the ability of certain lineages to acquire the mec cassette thus providing an advantage to their spreading in health institutions.

In conclusion, although both institutions evaluated in the present study are public hospitals, the military hospital showed dissemination of the USA100/ST5/CC5/SCCmecII lineage associated to multidrug resistance, including to mupirocin. On the other hand, the teaching hospital presented polyclonal and non-multidrug resistant MRSA isolates. These differences may reflect the policy of control and prevention of infections and/or use of antimicrobials employed in each hospital evaluated. Moreover, complete substitution of the BEC/ST239/SCCmecIII by other lineages was found in both hospitals, highlighting a change in the epidemiological profile of MRSA in Brazilian hospitals.

Ethics statement

The present study was approved by the Research Ethics Committee under No. 159/07.

1The authors contributed equally to this work.

Acknowledgments

This study was supported by grants from: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Fundação Universitária José Bonifácio (FUJB) and Programa de Núcleos de Excelência (PRONEX).

We acknowledge the contribution of PhD Rosana Barreto Rocha Ferreira (Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro) for providing language help and of the student Juliana Curityba de Mello Campos for the help in some experiments.

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Received: July 21, 2016; Accepted: September 28, 2016

*Corresponding author. E-mail address: santoskrn@micro.ufrj.br (K.R. dos Santos).

Conflicts of interest

The authors declare no conflicts of interest.

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