Abstract:

Objective:

To describe the clinical and molecular characteristics of methicillin-resistantStaphylococcus aureus(MRSA) strains that were collected in theHospital Regional de Alta Especialidad de Veracruz(HRV).

Materials and methods:

A total of 107 MRSA strains from individual patients were examined. The strains were collected between September 2009 and September 2010. The clinical and demographic characteristics of patients were analyzed; molecular typing by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosomal cassette mec (SCCmec) typing and multilocus sequence typing were used to characterize the isolates.

Results:

Two PFGE patterns (NY/J and IB) were identified with 4 and 3 subtypes respectively. The isolates analyzed showed two SCCmec types (I and II) and two sequence types (ST): ST247 and ST5 related with the Iberian and New York/Japan clones respectively.

Conclusion:

This study establishes the presence of two very important clonal lineages of MRSA: New York/Japan and Iberian clone in the hospital environment.

Keywords:
MRSA; clones; Mexico

Resumen:

Objetivo:

Describir las características clínicas y moleculares de cepas deStaphylococcus aureusresistentes a meticilina (SARM) que fueron recolectadas en el Hospital Regional de Alta Especialidad de Veracruz (HRV).

Material y métodos:

Un total de 107 cepas de SARM fueron analizadas en el presente estudio. Las cepas estudiadas fueron recolectadas de septiembre de 2009 a septiembre de 2010. Las características clínicas y demográficas de los pacientes fueron analizadas; la tipificación molecular de las cepas se hizo por electroforesis en campos pulsados, casete cromosomal estafilococócico (SCCmec, en inglés) y secuenciación de múltiples locus.

Resultados:

Dos patrones electroforéticos (NY/J y IB) fueron identificados con 4 y 3 subtipos respectivamente. Los aislamientos analizados mostraron dos tipos de SCCmec (I y II) y dos secuencias tipo (ST): ST247 y ST5 relacionados con las clonas Ibérica y Nueva York/Japón respectivamente.

Conclusión:

Este estudio estableció la presencia en el medio hospitalario de dos linajes clonales de SARM importantes: Nueva York/Japón e Ibérico.

Palabras clave:
MRSA; clonas; México

Introduction

Methicillin-resistantStaphylococcus aureus(MRSA) has been a common nosocomial pathogen and has become a major problem in hospitals worldwide. It produces a wide variety of virulence factors that contribute to its ability to colonize and cause a wide range of diseases such as folliculitis, conjunctivitis, cellulitis, deep abscesses, osteomyelitis, meningitis, sepsis, endocarditis and pneumonia.¹1. Tacconelli E. Methicillin-resistant Staphylococcus aureus: source control and surveillance organization. Clin Microb Infect. 2009;15(suppl 7):31-8. https://doi.org/10.1111/j.1469-0691.2009.03096.x
https://doi.org/10.1111/j.1469-0691.2009... ^,22. Verkaik NJ, Dauwalder O, Antri K, Boubekri I, de Vogel CP, Badiou C,et al. Immunogenicity of toxins during Staphylococcus aureus infection. Clin Infect Dis. 2010;50(1):61-8. https://doi.org/10.1086/648673
https://doi.org/10.1086/648673... ^,33. Yamamoto T, Hung W, Takano T, Nishiyama A. Genetic nature and virulence of community associated methicillin-resistant Staphylococcus aureus. BioMed. 2013;3(1):2-18. https://doi.org/10.1016/j.biomed.2012.12.001
https://doi.org/10.1016/j.biomed.2012.12... Surveillance of MRSA infections has shown that there are two types of strains: healthcare-associated (HA-MRSA) or community-acquired (CA-MRSA) MRSA.^4,4. Peng H, Liu D, Ma Y, Gao W. Comparison of community- and healthcare-associated methicillin-resistant Staphylococcus aureus isolates at a Chinese tertiary hospital, 2012-2017. Sci Rep. 2018;8: 17916. https://doi.org/10.1038/s41598-018-36206-5
https://doi.org/10.1038/s41598-018-36206... ⁵5. Guimarães MA, Ramundo MS, Américo MA, de Mattos MC, Souza RR, Ramos-Júnior ES,et al. A comparison of virulence patterns and in vivo fitness between hospital- and community-acquired methicillin-resistant Staphylococcus aureusrelated to the USA400 clone. .Eur J Clin Microbiol Infect Dis. 2015;34:497-501. https://doi.org/10.1007/s10096-014-2253-1
https://doi.org/10.1007/s10096-014-2253-... The prevalence of MRSA in Mexico is high, with wide variations (24.1-80%).⁶6. Vega S, Dowzicky J. Antimicrobial susceptibility among Gram- positive and Gram-negative organisms collected from the Latin American region between 2004 and 2015 as part of the Tigecycline Evaluation and Surveillance Trial. Ann Clin Microbiol Antimicrob. 2017;16:2-16. https://doi.org/101186/s12941-017-0222-0
https://doi.org/101186/s12941-017-0222-0... A study carried in five hospitals of Monterrey, Nuevo León, Mexico, in 2013, detected 190 MRSA strains causing healthcare associated infection (HCAI).⁷7. Velazquez MME, Hernández SM, Contreras JF, Pérez CP, Villarreal TL. Surveillance of methicillin-resistant Staphylococcus aureus causing nosocomial infections in five medical centers of Monterrey, Nuevo Leon, Mexico from 2005-2009. Arch Med Res. 2013;44(7):570-4. https://doi.org/10.1016/j.arcmed.2013.09.001
https://doi.org/10.1016/j.arcmed.2013.09... Another study describes the first MRSA outbreak in a tertiary-care oncology hospital in Mexico City. The results showed that the source of this MRSA outbreak was a transferred female patient with a complicated bone-joint-prosthesis infection.⁸8. Cornejo JP, Volkow FP, Sifuentes OJ, Echániz AG, Díaz GA, Velázquez AC,et al. Tracing the source of an outbreak of methicillin-resistant Staphylococcus aureus in a tertiary-care oncology hospital by epidemiology and molecular methods. Microb Drug Resist. 2010;16(3):203-8. https://doi.org/10.1089/mdr.2010.0048
https://doi.org/10.1089/mdr.2010.0048...

MRSA strains are characterized by the presence of a mobile genetic element called staphylococcal chromosomal cassette (SCCmec); this element carries themecAgene that determines resistance to methicillin.⁹9. Baig S, Johannesen TB, Overballe-Petersen S, Larsen J, Larsen AR, Stegger M. Novel SCCmec type XIII (9A) identified in an ST152 methicillin-resistant Staphylococcus aureus. Infect Gen Evol. 2018;61:74-6. https://doi.org/10.1016/j.meegid.2018.03.013
https://doi.org/10.1016/j.meegid.2018.03... A small number of MRSA lineages have emerged from the transfer of SCCmecinto successful methicillin-susceptibleS. aureus(MSSA) clones. Using multilocus sequence typing (MLST), Enright and colleagues demonstrated that MRSA clones evolved from five different groups of related genotypes or clonal complexes, each arising from a distinct ancestral genotype.¹⁰10. Enright MC, Robinson A, Randle G, Feil EJ, Grundmann H, Spratt BG. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci. 2002;99(11):7687-92. https://doi.org/10.1073/pnas.122108599
https://doi.org/10.1073/pnas.122108599... The clonal framework of the MRSA population in different Mexican hospitals has been the subject of several studies. The ST5-MRSA-II-New York/Japan clone has been shown to be particularly successful in establishing itself in Mexico.⁷7. Velazquez MME, Hernández SM, Contreras JF, Pérez CP, Villarreal TL. Surveillance of methicillin-resistant Staphylococcus aureus causing nosocomial infections in five medical centers of Monterrey, Nuevo Leon, Mexico from 2005-2009. Arch Med Res. 2013;44(7):570-4. https://doi.org/10.1016/j.arcmed.2013.09.001
https://doi.org/10.1016/j.arcmed.2013.09... ^,88. Cornejo JP, Volkow FP, Sifuentes OJ, Echániz AG, Díaz GA, Velázquez AC,et al. Tracing the source of an outbreak of methicillin-resistant Staphylococcus aureus in a tertiary-care oncology hospital by epidemiology and molecular methods. Microb Drug Resist. 2010;16(3):203-8. https://doi.org/10.1089/mdr.2010.0048
https://doi.org/10.1089/mdr.2010.0048... ^,1111. Velazquez MME, Aires de Sousa M, Echániz AG, Solórzano SF, Miranda NG, Silva SJ,et al. Surveillance of methicillin-resistant Staphylococcus aureus in a pediatric hospital in Mexico City during a 7-year period (1997 to 2003): clonal evolution and impact of infection control. J Clin Microbiol. 2004;42(8):3877-80. https://doi.org/10.1128/JCM.42.8.3877-3880.2004
https://doi.org/10.1128/JCM.42.8.3877-38... ^,1212. Echániz AG, Velazquez MME, Aires de Sousa M, Morfín OR, Rodríguez NE, Carnalla BN,et al. Molecular characterization of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999-2003). Clin Microbiol Infect Dis. 2006;12(1):12-28. https://doi.org/10.1111/j.1469-0691.2005.01283.x
https://doi.org/10.1111/j.1469-0691.2005... ^,1313. Challagundla L, Reyes J, Rafiqullah I, Sordelli D, Echaniz AG, Velazquez MME,et al. Phylogenomic classification and the evolution of clonal complex 5 methicillin-resistant Staphylococcus aureus in the Western Hemisphere. Front Microbiol. 2018;9:1-14. https://doi.org/10.3389/fmicb.2018.01901
https://doi.org/10.3389/fmicb.2018.0190...

The aim of this study was to identify international clones of MRSA circulating in theHospital Regional de Alta Especialidad de Veracruz, Mexico (HRV), and to track changes in their prevalence during the study period (September 2009 to September 2010).

Materials and methods

Hospital setting

The HRV in Veracruz, Mexico, is a 360-bed tertiary-care teaching hospital with 36 wards. The hospital serves the population of the Veracruz metropolitan area, which has 811 671 inhabitants. The hospital also serves the rest of the state of Veracruz (population 7 643 194) as well as three neighboring states: Oaxaca, Chiapas and Tabasco. During the study period, the hospital had 100 972 first inquiries, 19 768 emergency consultations, 85 066 specialty consultations and 15 414 hospitalizations.

Bacterial strains

A total of 107 MRSA isolates from individual patients were analyzed in the present study. The strains were collected between September 2009 and September 2010. Identification was done at Sensititre ARIS 2X automated system (Thermo Fisher Scientific Inc., Waltham, MA USA.). All MRSA isolated from invasive and non-invasive diseases during the study period were sent to the Department of Vaccine Evaluation for molecular typing.

Antimicrobial susceptibility tests

We used the Sensititre ARIS 2X automated system to perform antimicrobial susceptibility tests for amoxicillin, ampicillin, cefepime, cefotaxime, cefuroxime, cephalothin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, imipenem, meropenem, oxacillin, penicillin, rifampin, teicoplanin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin, following the Clinical Laboratory Standards Institute guidelines.¹⁴14. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing: Twentieth Informational Supplement M100-S20. Wayne: CLSI, 2010.

Molecular typing

The genomic DNA was prepared as described previously; after digestion with SmaI endonuclease, the DNA was separated using a CHEF-DR II system (Bio-Rad, Birmingham, UK).¹⁵15. Chung M, Matthews LH, Tomasz A, Adamsson I, Aires de Sousa M, Camou T,et al. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb Drug Resist. 2000;6(3):189-98. https://doi.org/10.1089/mdr.2000.6.189
https://doi.org/10.1089/mdr.2000.6.189... The strains BK2464, HPV107, HDE288, and EMRSA-16, which represent the New York/Japan, Iberian, Pediatric and EMRSA-16-UK clones were included in each PFGE gel as controls. The control strains were kindly provided by Herminia de Lencastre from the Laboratory of Molecular Genetics of theInstituto de Tecnología Química e Biologica da Universidad Nova de Lisboa. The different clones were compared according to the criteria of Tenover.¹⁶16. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persin DH,et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol.1995;33(9):2233-9.Staphylococcal cassette chromosome mec element (SCC^mec) typing and multilocus sequence typing (MLST) based on sequencing internal fragments of sevenS. aureushousekeeping genes used to define the allelic profile and a sequence type (ST), according to the MLST database (http://www.mlst.net) were performed as described previously.¹⁷17. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol. 2000;38:1008-15.^,1818. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2002;46(7):2155-61. https://doi.org/10.1128/aac.46.7.2155-2161.2002
https://doi.org/10.1128/aac.46.7.2155-21...

Computer fingerprint analysis

A computer analysis of the banding patterns obtained by PFGE was done using the Gel Compar II software, v. 6.6.11 (Applied Maths Inc.) after visual inspection. The reference strainS. aureusNCTC 8325 was included in each gel to normalize the PFGE profiles. The Dice coefficients were calculated to compute the similarity matrix and were then transformed into an agglomerative cluster by the unweighted pair group method with arithmetic average (UPGMA).

Ethics statement

This research was performed according to the principles expressed in the Declaration of Helsinki with approval from the Ethics Committee of the National Institute of Public Health (Instituto Nacional de Salud Pública, INSP).

Results

MRSA isolates

Between September 2009 and September 2010, a total of 107 MRSA clinical isolates from theHospital Regional de Alta Especialidad de Veracruz, Mexico, were collected and analyzed. They were taken from 58 (54%) female patients and 49 (46%) male patients. The median age of the patients was 42 years (ranging from 1 to 80 years). The isolates were obtained from various body sites associated with S. aureus infections: blood (35; 33%), wound infections (33; 31%), bronchial aspirate (15; 14%), peritoneal fluid (7; 6%), cerebrospinal fluid (4; 4%), dialysis fluid (3; 3%) and other sources (10; 9%). The MRSA isolates were found in different wards: internal medicine (49; 46%), surgery (18; 17%), intensive care unit (19; 18%), traumatology (11; 10%), neurosurgery (4; 4%), emergency (2; 2%) and outpatient (4; 3%).

Antimicrobial susceptibility

All strains showed resistance (100%) to amoxicillin, ampicillin, cefepime, cefotaxime, cephalothin, clindamycin, erythromycin, imipenem, meropenem, cefuroxime, oxacillin and penicillin; some strains showed resistance to chloramphenicol (76%), gentamicin (16%), tetracycline (3%), trimethoprim/sulfamethoxazole (2%) and rifampicin (3%). No resistance was detected against vancomycin and teicoplanin.

Molecular typing

The PFGE pattern separated the MRSA strains into two clones: the New York/Japan clone (NY/J) and subtypes [NY/J28, NY/J35, NY/J31 and NY/J21] (97%) and the Iberian clone (IB), with three subtypes (IB1, IB2 and IB3 [3%]). The subtype NY28 was most abundant (75%). The results of the computer analysis of the banding patterns show a clear division between the two clone groups (NY/J and IB). The patterns of the NY/J clone and its subtypes had 79.7-88% similarity with the control strain BK2464 (representing the NY/J clone), while the profiles of the IB and subtypes showed 67.4-96.8% similarity with the control strain HPV107 (representing the IB clone) (figure 1). The NY/J clone was found in all wards of the hospital and was present throughout the study period. The IB clone was found only in the emergency (IB1) and intensive care units (IB2 and IB3) in December 2009, and April and May 2010, respectively. The SCCmec and MLST analysis showed the presence of two STs: ST247-I and ST5-II, related to the Iberian and New York/Japan clones, respectively.

Discussion

The wide availability of antibiotics and their indiscriminate use has exerted selective pressure on MRSA clones throughout the world.¹⁹19. Aung MS, Zi H, New KM, Maw WW, Aung MT, Min WW,et al. Drug resistance and genetic characteristics of clinical isolates of staphylococci in Myanmar: high prevalence of PVL among methicillin-susceptible S. aureus belonging to various sequence types. New Microbes New Infect. 2016;10:58-65. https://doi.org/10.1016/j.nmni.2015.12.007
https://doi.org/10.1016/j.nmni.2015.12.0... ^,2020. Szymanek MK, Mlynarczyk A, Dobrzaniecka K, Majchrzak K, Mierzwinska NE. Epidemiological and drug-resistance types of methicillin-resistant Staphylococcus aureus strains isolated from surgical and transplantation ward patients during 2010 to 2011. Transplant Proc. 2016;48(5):1414-17. https://doi.org/10.1016/j.transproceed.2016.03.015
https://doi.org/10.1016/j.transproceed.2... The present study shows evidence of the presence of two pandemic clones of MRSA circulating in the HRV: the New York/Japan clone (ST5-SCCmec-II) and the Iberian clone (ST247-SCCmec-I). The New York/Japan clone, described by some studies as the most common clone in the United States of America (USA) and Japan,²¹21. Chung M, Dickinson G, de Lencastre H, Tomasz A. International clones of methicillin-resistant Staphylococcus aureus in two hospitals in Miami, Florida. J Clin Microbiol. 2004;42(2):542-7. https://doi.org/10.1128/JCM.42.2.542-547.2004
https://doi.org/10.1128/JCM.42.2.542-547... has been described previously in other hospitals in Mexico.⁷7. Velazquez MME, Hernández SM, Contreras JF, Pérez CP, Villarreal TL. Surveillance of methicillin-resistant Staphylococcus aureus causing nosocomial infections in five medical centers of Monterrey, Nuevo Leon, Mexico from 2005-2009. Arch Med Res. 2013;44(7):570-4. https://doi.org/10.1016/j.arcmed.2013.09.001
https://doi.org/10.1016/j.arcmed.2013.09... ^,88. Cornejo JP, Volkow FP, Sifuentes OJ, Echániz AG, Díaz GA, Velázquez AC,et al. Tracing the source of an outbreak of methicillin-resistant Staphylococcus aureus in a tertiary-care oncology hospital by epidemiology and molecular methods. Microb Drug Resist. 2010;16(3):203-8. https://doi.org/10.1089/mdr.2010.0048
https://doi.org/10.1089/mdr.2010.0048... ^,1111. Velazquez MME, Aires de Sousa M, Echániz AG, Solórzano SF, Miranda NG, Silva SJ,et al. Surveillance of methicillin-resistant Staphylococcus aureus in a pediatric hospital in Mexico City during a 7-year period (1997 to 2003): clonal evolution and impact of infection control. J Clin Microbiol. 2004;42(8):3877-80. https://doi.org/10.1128/JCM.42.8.3877-3880.2004
https://doi.org/10.1128/JCM.42.8.3877-38... ^,1212. Echániz AG, Velazquez MME, Aires de Sousa M, Morfín OR, Rodríguez NE, Carnalla BN,et al. Molecular characterization of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999-2003). Clin Microbiol Infect Dis. 2006;12(1):12-28. https://doi.org/10.1111/j.1469-0691.2005.01283.x
https://doi.org/10.1111/j.1469-0691.2005... ^,1313. Challagundla L, Reyes J, Rafiqullah I, Sordelli D, Echaniz AG, Velazquez MME,et al. Phylogenomic classification and the evolution of clonal complex 5 methicillin-resistant Staphylococcus aureus in the Western Hemisphere. Front Microbiol. 2018;9:1-14. https://doi.org/10.3389/fmicb.2018.01901
https://doi.org/10.3389/fmicb.2018.0190... However, this is the first report of this clone in the South of Mexico, which demonstrates its ability for geographical spreading, probably favored by its resistance and virulence factors. The importance of this finding lies in the fact that the first resistant strain of MRSA with vancomycin minimum inhibitory concentration (MIC) of 1024ug/ml belongs to the New York/Japan clone.²²22. Weigel L, Clewell D, Gill S, Clark NC, McDougal LK, Flannagan SE,et al. Genetic analysis of a high-level vancomycin resistant isolate of Staphylococcus aureus. Science. 2003;302(5650):1569-71. https://doi.org/10.1126/science.1090956
https://doi.org/10.1126/science.1090956... Furthermore, Khokhlova and colleagues reported the first case of fatal community-acquired necrotizing pneumonia caused by MRSA strains associated with the New York/Japan MRSA clone.²³23. Khokhlova O, Tomita Y, Hung WC, Takano T, Iwao Y, Higuchi W,et al. Elderly infection in the community due to ST5/SCCmecII methicillin resistant Staphylococcus aureus (the New York/Japan clone) in Japan: panton-Valentine leukocidin-negative necrotizing pneumonia. J Microbiol Immunol Infect. 2015;48(3):335-9. https://doi.org/10.1016/j.jmii.2012.09.004
https://doi.org/10.1016/j.jmii.2012.09.0...

The pandemic Iberian clone (ST247-I), which accounted for 3% of the isolates examined in the present study, has been reported as a common clone in Europe although there has been a decrease in the incidence of this clone in several European countries.¹⁰10. Enright MC, Robinson A, Randle G, Feil EJ, Grundmann H, Spratt BG. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci. 2002;99(11):7687-92. https://doi.org/10.1073/pnas.122108599
https://doi.org/10.1073/pnas.122108599... ^,2424. Campanile F, Bongiorno D, Borbone S, Stefani S. Hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) in Italy. Ann Clin Microbiol Antimicrob. 2009;8. https://doi.org/10.1186/1476-0711-8-22
https://doi.org/10.1186/1476-0711-8-22... This is the first time that this clone has been detected in Mexico (three different subtypes; IB1 to IB3). Subtype IB1 was first isolated in the emergency ward from a patient with ear infection; the medical record showed that the infection had appeared after the patient returned from a travel to the USA. The MRSA strains of subtypes IB2 and IB3 were isolated later in the intensive care unit of the hospital from two more patients, showing that the Iberian clone had traveled from the external wards to the critical areas of the hospital. Previous studies have shown that the Iberian clone has spread widely in Europe and the USA, causing severe hospital outbreaks in these regions.²⁵25. Mato R, Santos SI, Venditti M, Platt DJ, Brown A. Chung M,et al. Spread of the multiresistant Iberian clone of methicillin-resistant Staphylococcus aureus (MRSA) to Italy and Scotland. Microb Drug Resist. 1998;4(2):107-12. https://doi.org/10.1089/mdr.1998.4.107
https://doi.org/10.1089/mdr.1998.4.107... ^,2626. Roberts RB, Tennenberg AM, Eisner W, Hargrave J, Drusin LM, Yurt R. Outbreak in a New York City teaching hospital burn center caused by the Iberian epidemic clone of MRSA. Microb Drug Resist. 1998;4(3):175-83. https://doi.org/10.1089/mdr.1998.4.175
https://doi.org/10.1089/mdr.1998.4.175... ^,2727. Stefani S, Varaldo PE. Epidemiology of methicillin-resistant staphylococci in Europe. Clin Microbiol Infect. 2003;9(12):1179-86. https://doi.org/10.1111/j.1469-0691.2003.00698.x
https://doi.org/10.1111/j.1469-0691.2003... MRSA strains of the Iberian clone were the first to develop intermediate resistance to vancomycin (VISA) and reduced sensitivity to teicoplanin.²⁸28. Heym B, Le Moal M, Armand LL, Nicolas MH. Multilocus sequence typing (MLST) shows that the ‘Iberian’ clone of methicillin-resistant Staphylococcus aureus has spread to France and acquired reduced susceptibility to teicoplanin. J Antimicrob Chemother. 2002;50(3):323-9. https://doi.org/10.1093/jac/dkf132
https://doi.org/10.1093/jac/dkf132... ^,2929. Krzysztoń RJ, Gniadkowski M, Połowniak PH, Hagmajer E, Hryniewicz W. The first Staphylococcus aureus isolates with reduced susceptibility to vancomycin in Poland. J Antimicrob Chemother. 2002;50:1065-1069. https://doi.org/10.1093/jac/dkf252
https://doi.org/10.1093/jac/dkf252... ^,3030. Cassone M, Campanile F, Pantosti A, Venditti M , Stefani S. Identification of a variant “Rome clone” of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit. Microb Drug Resist. 2004;10(1):43-9. https://doi.org/10.1089/107662904323047790
https://doi.org/10.1089/1076629043230477... ^,3131. Zribi M, Etienne J, El Euch D, Zribi H, Bes M, Meugnier H,et al. Detection of the first strain of glycopeptides intermediary Staphylococcus aureus in Tunis Rabta hospital. Pathol Biol. 2009;59(6):334-5. https://doi.org/10.1016/j.patbio.2009.10.002
https://doi.org/10.1016/j.patbio.2009.10... Glycopeptides are considered last-choice antibiotics against infections caused by MRSA and other Gram-positive microorganisms.

The antimicrobial resistance pattern to different antibiotics remained stable throughout the study period, similar to what has been observed in other studies.³²32. Khanal LK, Adhikari RP, Guragain A. Prevalence of methicillin resistant Staphylococcus aureus and antibiotic susceptibility pattern in a tertiary hospital in Nepal. J Nepal Health Res Counc. 2018; 16(2):172-4. https://doi.org/10.3126/jnhrcv16i2.20305
https://doi.org/10.3126/jnhrcv16i2.20305... ^,3333. Dilnessa T, Bitew A. Prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus isolated from clinical samples at Yekatit 12 Hospital Medical College, Addis Ababa, Ethiopia. BMC Infect Dis. 2016;16:1-9. https://doi.org/10.1186/s12879-016-1742-5
https://doi.org/10.1186/s12879-016-1742-... All strains were resistant to 12 antibiotics and only some strains showed different resistance levels to five antibiotics, as shown in the results. The multi-resistance of the New York/Japan clone and the Iberian clone has been reported in other studies.³⁴34. Conceição T, Aires de Sousa M, Füzi M, Tóth A, Pászti J, Ungvári E,et al. Replacement of methicillin-resistant Staphylococcus aureus clones in Hungary over time: a 10-year surveillance study. Clin Microbiol Infect. 2007;13(10):971-9. https://doi.org/10.1111/j.1469-0691.2007.01794.x
https://doi.org/10.1111/j.1469-0691.2007... ^,3535. Zaraket H, Otsuka T, Saito K, Dohmae S, Takano T, Higuchi W,et al. molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission. Microbiol Immunol. 2007;51(2):171-6. https://doi.org/10.1111/j.1348-0421.2007.tb03898.x
https://doi.org/10.1111/j.1348-0421.2007... However, our results showed that the Iberian clone strains (n=3) are susceptible to gentamicin and tetracycline, and resistant to rifampicin. The phenotype of the Iberian clone that showed resistance to rifampicin was found in clinical isolates of MRSA in 2004 in Spain, where the prevalence of this phenotype increased from 25% in 2004 to 45% in 2006. That study analyzed mutations of therpoBgene involved in the resistance to rifampicin; in this work an internal sequence of generpoB(432 bp) was amplified by PCR, this region includes the rifampicin resistance-determining; the PCR product was analyzed by DNA sequencing. The results showed mutational changes in 481His/Asn, 468Gln/Lys, 477Ala/Thr and 527Ile/Leu, the last three mutations associated with a high-level rifampicin resistance.³⁶36. Mick V, Domínguez MA, Tubau F, Liñares J, Pujol M, Martín R. Molecular characterization of resistance to rifampicin in an emerging hospital-associated methicillin-resistant Staphylococcus aureus clone ST228, Spain. BMN Microbiol. 2010;10:1-8. https://doi.org/10.1186/1471-2180-10-68
https://doi.org/10.1186/1471-2180-10-68...

Our study has several limitations. First, our collection is based on a small number of isolates collected in a short period of time. However, we do not believe there is a bias in the collection of isolates, because this was constant throughout the year of study. Second, prospective studies are needed to determine the incidence of infections due to MRSA in the HRV in order to implement measures aimed at preventing the dissemination of these clonal lineages. Despite this limitation, these data remain useful because they could be used primarily for monitoring trends of dissemination of these clones in Mexico, and could provide a basis for detection in other hospitals.

Several studies have shown that one or a few clones are predominant in each hospital, similar to what we found. In conclusion, this study establishes the presence of two very important clonal lineages of MRSA: New York/Japan and Iberian clone in hospital environment.

Acknowledgements

This work was supported by the INSP, Cuernavaca, Morelos, Mexico. Miguel Angel Ortiz Gil was a M. Sc. student from the Program of Infectious Diseases of the INSP, supported by the Consejo Nacional de Ciencia y Tecnología (Conacyt), number 22834.

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