ABSTRACT

In order to determine the frequency of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates and to describe the antimicrobial resistance pattern and genotype, a cross-sectional study was conducted in 2017 at the Hospital Nacional Cayetano Heredia in Lima, Peru. We found a MRSA prevalence of 46.1% in the 115 analyzed S. aureus isolates; most were reported from different secretions (26.4%) and blood (18.9%). We found high co-resistance (>75%) to clindamycin, erythromycin, gentamicin and ciprofloxacin. Regarding SSCmec typification, most of the isolates were identified as hospital-acquired MRSA (HA-MRSA) and a minority of them as CA-MRSA (2.6%). Despite its low prevalence when compared to other Latin American countries (27%), epidemiological surveillance is recommended to control local CA-MRSA dissemination.

Keywords:
Staphylococcus aureus; Methicillin-resistant Staphylococcus aureus; prevalence; epidemiological surveillance; Peru

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) was initially described as a bacterium associated with nosocomial infections in patients with prolonged hospital stay, recent surgery, dialysis requirement or presence of invasive medical devices ¹1. DeLeo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated meticilin-resistant Staphylococcus aureus. Lancet. 2010; 375(9725):1557-1568. doi: 10.1016/S0140-6736(09)61999-1.
https://doi.org/10.1016/S0140-6736(09)61...

2. Shorr AF. Epidemiology of Staphylococcal resistance. Clin Infect Dis. 2007,45(3):1-6. doi: 10.1086/519473.
https://doi.org/10.1086/519473.... ^-33. Naimini TS, LeDell KH, Como-Sabetti K, Borchardt SM, Boxrud DJ, Etienne J, et al. Comparison of community - and health care - associated methicillin - resistance Staphylococcus aureus infection. JAMA. 2003; 290(22):2976-84. doi: 10.1001/jama.290.22.2976.
https://doi.org/10.1001/jama.290.22.2976... . Hospital-acquired MRSA (HA-MRSA) is characterized by being resistant to several families of antimicrobials and carrying the mecA gene, contained in the staphylococcal chromosomal cassette mec (SCCmec) type I, II and III ³3. Naimini TS, LeDell KH, Como-Sabetti K, Borchardt SM, Boxrud DJ, Etienne J, et al. Comparison of community - and health care - associated methicillin - resistance Staphylococcus aureus infection. JAMA. 2003; 290(22):2976-84. doi: 10.1001/jama.290.22.2976.
https://doi.org/10.1001/jama.290.22.2976...

4. Mediavilla JR, Chen L, Mathema B, Kreiswirth BN. Global epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). Curr Opin Microbiol. 2012;15(5):588-595. doi: 10.1016/j.mib.2012.08.003.
https://doi.org/10.1016/j.mib.2012.08.00... ^-55. Inglis B, Matthews PR, Stewart PR. The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance. J Gen Microbiol. 1988;134(6):1465-9. doi: 10.1099/00221287-134-6-1465.
https://doi.org/10.1099/00221287-134-6-1... . However, in the 1990s, the first cases of community-acquired MRSA infections (CA-MRSA) began to be described in the USA in people without nosocomial risk factors; subsequently infections spread to all continents ⁴4. Mediavilla JR, Chen L, Mathema B, Kreiswirth BN. Global epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). Curr Opin Microbiol. 2012;15(5):588-595. doi: 10.1016/j.mib.2012.08.003.
https://doi.org/10.1016/j.mib.2012.08.00... . The most prevalent CA-MRSA clone is USA300, which characteristically carries SCCmec type IV and is usually resistant only to β-lactams ⁵5. Inglis B, Matthews PR, Stewart PR. The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance. J Gen Microbiol. 1988;134(6):1465-9. doi: 10.1099/00221287-134-6-1465.
https://doi.org/10.1099/00221287-134-6-1... ^-66. Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, et al. Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus Infections in Adults and Children. Clin Infect Dis. 2011;53(3):e18-55. doi: 10.1093/cid/ciq146.
https://doi.org/10.1093/cid/ciq146... .

In Peru, some imported cases of CA-MRSA were described between 2010-2011 ⁷7. Garcia C, Deplano A, Denis O, León M, Siu H, Chincha O, et al. Spread of community-associated methicillin-resistant Staphylococcus aureus to Peru. J Infect. 2011; 63(6): 482-3. doi: 10.1016/j.jinf.2011.09.001.
https://doi.org/10.1016/j.jinf.2011.09.0... . A study conducted during 2011-2014, using blood cultures, showed that the most prevalent clone in northern South America was the Latin American variant of USA300 (USA300-LV), described in 79%, 72% and 50% of MRSA infections in Colombia, Ecuador and Venezuela, respectively; although no cases were found in Peru ⁸8. Seas C, Garcia C, Salles MJ, Labarca J, Luna C, Alvarez-Moreno C, et al. Staphylococcus aureus bloodstream infections in Latin America: results of a multinational prospective cohort study. J Antimicrob Chemother. 2018; 73(1):212-22. doi: 10.1093/jac/dkx350.
https://doi.org/10.1093/jac/dkx350... .

The aim of the study was to determine the frequency of CA-MRSA in S. aureus isolates from patients in a hospital in Lima, Peru, and to describe their molecular and antimicrobial resistance characteristics.

THE STUDY

Design and population

A cross-sectional and descriptive study was conducted during 2017 in patients from the Hospital Cayetano Heredia (HCH) in Lima, Peru. This is a complexity level III-1 public hospital, which has outpatient consultation by specialties and 367 hospital beds. During this period, all S. aureus isolates reported in blood, fluid or body secretion by the hospital’s microbiology laboratory from pediatric or adult inpatients and outpatients were collected.

Microbiological and molecular analysis

The isolates were transferred to the Tropical Medicine Institute “Alexander von Humboldt” for identification, according to conventional diagnostic procedures. Antimicrobial susceptibility was performed through the Kirby Bauer method and the following antimicrobials were used: cefoxitin, ceftaroline, gentamicin, erythromycin, tetracycline, ciprofloxacin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, rifampicin and linezolid, considering the standard cut-off points ⁹9. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests. 13th ed. CLSI standard M02. Wayne, PA: CLSI; 2018.. S. aureus strain ATCC 29213 was used as quality control.

For molecular analysis, DNA was extracted according to the methodology described by Bouillaut et al¹⁰10. Bouillaut L, McBride SM, Sorg JA. Genetic manipulation of Clostridium difficile. Curr Protoc Microbiol. 2011;9;1-20. doi: 10.1002/9780471729259.mc09a02s20.
https://doi.org/10.1002/9780471729259.mc... . The detection of methicillin resistance by identification of the mecA gene and subsequent typification of SCCmec (types I, II, III, IVa, IVb, IVc and V) were carried out by multiplex PCR, following the methodology described by Zhang et al¹¹11. Zhang K, McClure J-A, Elsayed S, Louie T, Conly JM. Novel Multiplex PCR assay for characterization and concomitant subtyping of Staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005;43(10):5026-33. doi: 10.1128/JCM.43.10.5026-5033.2005.
https://doi.org/10.1128/JCM.43.10.5026-5... . Likewise, the lukF.PV and lukS-PV genes, which encode Panton-Valentine leukocidin (PVL), were detected by the PCR method, following the procedures described by Lina et al¹²12. Lina G, Piémont Y, Godail-Gamot F, Bles M, Peter MO, Gauduchon V, et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999;29(5):1128-32. doi: 10.1086/313461.
https://doi.org/10.1086/313461... .

Those isolates that had discordance between the cefoxitin resistance pattern and the presence of mecA, or whose SCCmec could not be identified were sent to the Henry Ford Hospital Infectious Disease Research Laboratory (Detroit, Michigan) for typification.

MRSA was defined according to the detection of the mecA gene. Because a clinical definition was not used, we defined whether the MRSA isolate was community-acquired based on the type of SCCmec and the presence of genes encoding PVL: if isolates carried SCCmec type IV or V and had the lukF.PV and lukS-PV genes, they were considered CA-MRSA. Those carrying SCCmec type I, II and III, independent of the presence of lukF.PV and lukS-PV genes, were considered HA-MRSA ¹³13. Lakhundi S, Zhang K. Methicillin-Resistan Staphylococcus aureus: molecular characterization, evolution, and epidemiology. Clin Microbiol Rev. 2018;31(4):e00020-18. doi: 10.1128/CMR.00020-18.
https://doi.org/10.1128/CMR.00020-18... .

Statistical analysis

We used Windows XP Excel 2007 to collect information on the origin of the reported S. aureus strains. STATA SE 16 was used for statistical analysis of the data. A descriptive analysis with frequencies and percentages was conducted.

Ethical Considerations

Samples and patient data were processed and stored under strict confidentiality. Each S. aureus isolate was assigned a code, and the database was stored with a password, to which only the principal researchers had access. The HCH Institutional Ethics Committee approved the study in 2016, with code 021-017.

FINDINGS

During 2017, 152 S. aureus isolates were reported (only one per patient), of which 120 were analyzed in this study. Of these, five isolates were excluded: four had discordant findings between cefoxitin susceptibility and the presence of the mecA gene, and one was S. haemolyticus.

Of the 115 isolates positive for S. aureus, most were obtained from unspecified secretions (21.7%), followed by blood (20.0%), tracheobronchial secretions (14.8%) and skin (14.8%) (Table 1). Of the isolates, 46.1% were identified as MRSA. Among the MRSA isolates (n = 53), the distribution of SCCmec types was as follows: I (79.2%), III (1.9%), and IV (7.5%); no isolates with SCCmec type II and V were found. In addition, in six isolates (11.3%) the SCCmec type could not be determined (Table 2).

Regarding the antibiotic susceptibility profile, among methicillin-sensitive S. aureus isolates (MSSA, n = 62), the highest frequency of resistance was found for erythromycin (22.2%), gentamicin (17.2%) and clindamycin (11.1%). On the other hand, the majority of isolates (>75%) of MRSA showed resistance to clindamycin, erythromycin, gentamicin and, in addition, ciprofloxacin; this co-resistance was more common in isolates carrying SCCmec type I and III (n = 43) (Table 2).

Genes encoding PVL were identified in ten isolates (8.7%): six MSSA isolates (9.7%) and four MRSA isolates (7.5%). Of the latter, three isolates were categorized as CA-MRSA, because they carried SCCmec type IV; while only 1 corresponded to HA-MRSA, because it carried SCCmec type I (Table 3).

DISCUSSION

We analyzed 115 strains (75.7%) of the 152 isolates reported during 2017. Within these, a high frequency of MRSA isolates was found (46.1%) and the frequency of CA-MRSA was 2.6%.

Previous multicenter studies that have evaluated S. aureus strains according to their antimicrobial susceptibility and genotype have shown a high prevalence (>40%) of MRSA in Latin America, with a heterogeneous distribution among countries ⁽¹⁴14. Garcia C, Rijnders MI, Bruggeman C, Samalvides F, Stobberingh EE, Jacobs J. Antimicrobial resistance and molecular typing of Staphylococcus aureus bloodstream isolates form hospitals in Peru. J Infect. 2012;65(5):406-11. doi: 10.1016/j.jinf.2012.06.009.
https://doi.org/10.1016/j.jinf.2012.06.0... ^,1515. Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A Prospective cohort multicentre study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American Countries. Antimicrob Agents Chemother. 2017;61(10):E00816-17. doi: 10.1128/AAC.00816-17.
https://doi.org/10.1128/AAC.00816-17... . Brazil and Venezuela report the highest frequencies in the region, with 62% and 57%, respectively ¹⁵15. Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A Prospective cohort multicentre study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American Countries. Antimicrob Agents Chemother. 2017;61(10):E00816-17. doi: 10.1128/AAC.00816-17.
https://doi.org/10.1128/AAC.00816-17... . In the case of Peru, a frequency of 50%-54% has been reported ¹⁴14. Garcia C, Rijnders MI, Bruggeman C, Samalvides F, Stobberingh EE, Jacobs J. Antimicrobial resistance and molecular typing of Staphylococcus aureus bloodstream isolates form hospitals in Peru. J Infect. 2012;65(5):406-11. doi: 10.1016/j.jinf.2012.06.009.
https://doi.org/10.1016/j.jinf.2012.06.0... ^,1515. Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A Prospective cohort multicentre study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American Countries. Antimicrob Agents Chemother. 2017;61(10):E00816-17. doi: 10.1128/AAC.00816-17.
https://doi.org/10.1128/AAC.00816-17... . The frequencies described are similar to those found in our study; however, the comparison with these studies ^{(14, 15)} is limited, since only cases of bacteremia were considered. To date, no studies have been conducted in the region that consider all types of isolates.

There are few local studies that evaluate the distribution of MRSA strains and their molecular typification. One research conducted at the same hospital as our study, that considered all S. aureus isolates, described a 68% frequency of MRSA in 2002 ¹⁶16. Seas C, Hernandez K, Ramos R, Bazan E, Rodriguez I, Torres A, et al. Oxacillin-resistant and multidrug-resistant Stapylococcus aureus in Lima, Peru. Infect Control Hosp Epidemiol, 2006; 27:198-200. doi: 10.1086/500650.
https://doi.org/10.1086/500650... . Subsequently, a study that included S. aureus isolates from all sources in three referral hospitals in Lima ¹⁷17. Tamariz J, Agapito J, Horna J, Tapia E, Vicente W, Silva M, et al. Staphylococcus aureus resistente a meticilina adquirido en la comunidad aislado en tres hospitales de Lima-Perú. Rev Med Hered. 2010;21(1):4-10. doi: 10.20453/rmh.v21i1.1139.
https://doi.org/10.20453/rmh.v21i1.1139... showed an overall frequency of 58%, obtaining the molecular characteristics of HA-MRSA in almost all isolates. This shows that the presence of MRSA continues to be prevalent in Peruvian hospitals, and measures should be implemented to contain its dissemination ¹⁵15. Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A Prospective cohort multicentre study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American Countries. Antimicrob Agents Chemother. 2017;61(10):E00816-17. doi: 10.1128/AAC.00816-17.
https://doi.org/10.1128/AAC.00816-17... .

From the molecular point of view, the SCCmec type of MRSA has a varied distribution in Latin America ¹⁸18. Martínez JRW, Diaz L, Rojas M, Rios R, Hanson B, Rivas LM. 556. Phylogenomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) Chilean-Cordobes clone in Latin America. Open Forum Infect Dis. 2019; 6(Supp 2):S263-4. doi: 10.1093/ofid/ofz360.625.
https://doi.org/10.1093/ofid/ofz360.625.... ^,2020. Reyes J, Rincón S, Diaz L, Panesso D, Contreras DA, Zurita J, et al. Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America. Clin Infect Dis. 2009; 49(12):1861-7. doi: 10.1086/648426.
https://doi.org/10.1086/648426... . In the northern countries of South America, such as Colombia and Ecuador, the USA300 clone carrying SCCmec type IV is the most frequent, followed by the Chilean-Cordovan clone, carrying SCCmec type I; while in countries such as Peru and Chile it has been reported that the majority (>90%) of S. aureus isolates correspond to the Chilean-Cordovan clone ⁽¹⁵15. Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A Prospective cohort multicentre study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American Countries. Antimicrob Agents Chemother. 2017;61(10):E00816-17. doi: 10.1128/AAC.00816-17.
https://doi.org/10.1128/AAC.00816-17... .

In Peru, a multicenter study conducted in Lima ¹⁷17. Tamariz J, Agapito J, Horna J, Tapia E, Vicente W, Silva M, et al. Staphylococcus aureus resistente a meticilina adquirido en la comunidad aislado en tres hospitales de Lima-Perú. Rev Med Hered. 2010;21(1):4-10. doi: 10.20453/rmh.v21i1.1139.
https://doi.org/10.20453/rmh.v21i1.1139... revealed that MRSA carrying SCCmec type I were found with a frequency of 75.2%; furthermore, these isolates showed resistance to ciprofloxacin, clindamycin, erythromycin and gentamicin, similar to what was found in our study. Our findings are similar to previous studies in hospitals in Lima, which confirms that MRSA carrying SCCmec type I non-PVL-producing is the most common nosocomial clone in our setting ¹⁷17. Tamariz J, Agapito J, Horna J, Tapia E, Vicente W, Silva M, et al. Staphylococcus aureus resistente a meticilina adquirido en la comunidad aislado en tres hospitales de Lima-Perú. Rev Med Hered. 2010;21(1):4-10. doi: 10.20453/rmh.v21i1.1139.
https://doi.org/10.20453/rmh.v21i1.1139... ^,1818. Martínez JRW, Diaz L, Rojas M, Rios R, Hanson B, Rivas LM. 556. Phylogenomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) Chilean-Cordobes clone in Latin America. Open Forum Infect Dis. 2019; 6(Supp 2):S263-4. doi: 10.1093/ofid/ofz360.625.
https://doi.org/10.1093/ofid/ofz360.625.... . Regarding the emergence of CA-MRSA in the last decade in Peru, several multicenter studies have shown that this is a very infrequent event in Peruvian hospitals ⁸8. Seas C, Garcia C, Salles MJ, Labarca J, Luna C, Alvarez-Moreno C, et al. Staphylococcus aureus bloodstream infections in Latin America: results of a multinational prospective cohort study. J Antimicrob Chemother. 2018; 73(1):212-22. doi: 10.1093/jac/dkx350.
https://doi.org/10.1093/jac/dkx350... ^,1414. Garcia C, Rijnders MI, Bruggeman C, Samalvides F, Stobberingh EE, Jacobs J. Antimicrobial resistance and molecular typing of Staphylococcus aureus bloodstream isolates form hospitals in Peru. J Infect. 2012;65(5):406-11. doi: 10.1016/j.jinf.2012.06.009.
https://doi.org/10.1016/j.jinf.2012.06.0... . In this study, 2.6% of the isolates were found to have molecular characteristics of CA-MRSA, in contrast to neighboring countries such as Brazil, Ecuador, Venezuela and Colombia, where a prevalence of around 27% has been reported ¹⁵15. Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A Prospective cohort multicentre study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American Countries. Antimicrob Agents Chemother. 2017;61(10):E00816-17. doi: 10.1128/AAC.00816-17.
https://doi.org/10.1128/AAC.00816-17... ^,2020. Reyes J, Rincón S, Diaz L, Panesso D, Contreras DA, Zurita J, et al. Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America. Clin Infect Dis. 2009; 49(12):1861-7. doi: 10.1086/648426.
https://doi.org/10.1086/648426... . This situation requires continuous surveillance because if this proportion increases significantly, there would be an impact on the empirical antibiotic treatment for skin and soft tissue infections, its most frequent presentation.

Likewise, this study found a frequency of genes encoding PVL of 8.7%, with greater distribution in the MSSA group. This exotoxin was first described in 1932 in sensitive strains, as a factor associated with severe skin infections and necrotizing pneumonia ¹⁹19. Panton PN, Valentine FCO. Staphylococcal toxin. Lancet. 1932; 219:506-508. doi: 10.1016/S0140-6736(01)24468-7.
https://doi.org/10.1016/S0140-6736(01)24... . Subsequently, it was described that its production could be considered a marker for identification of resistant strains, especially CA-MRSA ¹⁹19. Panton PN, Valentine FCO. Staphylococcal toxin. Lancet. 1932; 219:506-508. doi: 10.1016/S0140-6736(01)24468-7.
https://doi.org/10.1016/S0140-6736(01)24... . However, this theory is currently controversial, since the genes encoding PVL are reported in HA-MRSA and MSSA strains. A study conducted in three hospitals in Lima ¹⁷17. Tamariz J, Agapito J, Horna J, Tapia E, Vicente W, Silva M, et al. Staphylococcus aureus resistente a meticilina adquirido en la comunidad aislado en tres hospitales de Lima-Perú. Rev Med Hered. 2010;21(1):4-10. doi: 10.20453/rmh.v21i1.1139.
https://doi.org/10.20453/rmh.v21i1.1139... described a low production of PVL (9.1% of the isolates analyzed), with a higher distribution in the MSSA group than in the MRSA group, which is similar to what was found in our study. These findings favor the current theory that the presence of PVL is not a reliable marker for the identification of CA-MRSA strains.

The first limitation of this study was that the number of isolates analyzed was small, which could have altered the true prevalence of CA-MRSA isolates. This was due to the limited availability of reported S. aureus isolates during the study period and, probably, to the fact that this institution does not perform systematic culture sampling in cases with suspected skin and soft tissue infection, the most frequent presentation of this infection, or that it is performed after the initiation of antibiotics. The second limitation of the study was that the complete review of medical records was not included, which prevented us from completing the clinical definition of HA-MRSA and CA-MRSA. In addition, this study did not allow us to evaluate whether the samples obtained corresponded to cases of infection or colonization, which would provide more information on the impact of the presence of this bacterium in our setting. In addition, there were 11.3% of MRSA isolates in which the SSCmec type could not be identified. The third limitation is related to the external validity of the study. The study was conducted in a public referral hospital located in the northern area of the city of Lima, so the information described only corresponds to that population. This, together with the small number of samples analyzed, limits the extrapolation of the results. However, the intention of our study is to draw attention to the importance of epidemiological surveillance of multidrug-resistant bacteria.

Our study shows a low frequency of CA-MRSA in Lima. However, we consider that close epidemiological surveillance should be continued and studies should be expanded, even more so in the context of increasing migration from countries with higher prevalence.

Acknowledgements:

Dr. Marcus Zervos (Division Head - Division of Infectious Diseases, Henry Ford Hospital, Detroit, Michigan, USA) and MT (ASCP) Mary Beth Perri (Infectious Disease Research Laboratory, Henry Ford Health System, Detroit, Michigan, USA) for their support in the molecular analysis of the study.

References

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