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Methicillin resistance and vancomycin susceptibility pattern among blood isolates of Staphylococcus aureus

How to cite this article: Rodríguez-Pineda J, Terrazas-Estrada JJ, Urdez-Hernández E, Hernández-Sánchez EA, Sánchez-Tejeda SL. Methicillin resistance and vancomycin susceptibility pattern among blood isolates of Staphylococcus aureus. Rev Med Inst Mex Seguro Soc. 2016;54(1):48-51.



Received: August 14th 2014

Accepted: September 1st 2015

Methicillin resistance and vancomycin susceptibility pattern among blood isolates of Staphylococcus aureus

Jonathan Rodríguez-Pineda,a,† José Juan Terrazas-Estrada,b Elena Urdez-Hernández,a Eva Aurora Hernández-Sánchez,c Sandra Leticia Sánchez-Tejedad

a,†Servicio de Infectología Adultos, Hospital de Infectología

bServicio de Infectología, Hospital de Especialidades

cSección de Bacteriología, Laboratorio Clínico, Hospital de Infectología

dSección de Bacteriología, Laboratorio Clínico, Hospital de Especialidades

Centro Médico Nacional La Raza, Instituto Mexicano del Seguro Social, Distrito Federal, México

Communication with: Elena Urdez-Hernández

Thelephone: (55) 5211 27 73


Background: Staphylococcus aureus is capable of acquiring resistance against all antimicrobial agents. Vancomycin has been the cornerstone therapy for serious methicillin-resistant S. aureus infections. However, vancomycin treatment failures have been reported.

Methods: From March to August 2010, S. aureus blood isolates were included to determine methicillin-resistance and the vancomycin susceptibility by using a standard microdilution method. To detect methicillin-resistance, a Mueller-Hinton agar plate added with oxacillin 4 µg/mL and 2 % NaCl, and an agglutination test were used. Growth of S. aureus on the agar plate and/or reactive agglutination defined a methicillin-resistant organism. Vancomycin susceptibility was assessed by determining the minimal inhibitory concentration (MIC) in Muelller-Hinton agar plates prepared with dilutions ranging from 16 to 0.5 µg/mL.

Results: A total of 25 blood-isolates of S. aureus were included. A 60 % was methicillin-resistant. All isolates were vancomycin-susceptible (MIC ≤ 2 µg/mL) showing the next MICs distribution: 48 % ≤ 0.5 µg/mL; 44 % 1 µg/mL, and 8 % 2 µg/mL.

Conclusion: The high proportion of methicillin-resistance among S. aureus and the presence of vancomycin susceptible phenotypes (MIC of 2 µg/mL) not only claim for an enforcement of standard precautions and antimicrobial control, but also for a regular surveillance of vancomycin susceptibility pattern using a reference method.

Keywords: Vancomycin resistance; Methicillin resistant Staphylococcus aureus.

Staphylococcus aureus (SA) has been shown to have both a great ability to develop resistance to all antimicrobials and to cause serious infections in people, particularly when the bacterium acquires methicillin resistance (MRSA).1 To treat these mainly nosocomial infections, vancomycin has been the glycopeptide of choice for nearly 50 years. However, since 1999, cases of therapeutic failure have been reported2-6 among MRSA-infected patients whose minimum inhibitory concentration (MIC) to vancomycin has been determined not only as resistant (MIC ≥16 µg/mL) and intermediate (MIC 4-8 µg/mL), but also within the range of susceptibility (MIC ≤ 2 µg/mL). Although the decrease in susceptibility to glycopeptide is a trait that usually occurs in MRSA, it can also be generated among S. aureus that are methicillin-sensitive.7 This study was conducted to determine the resistance to methicillin and the vancomycin susceptibility profile of S. aureus using a reference method.


Staphylococcus aureus isolated from blood cultures in the Bacteriology Laboratory of the Hospital de Infectología of Centro Médico Nacional La Raza, member of Instituto Mexicano del Seguro Social (IMSS), were included from March to August 2010. The microorganisms were identified by the standard method.8

Antimicrobial susceptibility was determined by agar dilution of the antimicrobial in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI).9

Methicillin resistance was investigated in two ways. The first, by a Mueller-Hinton agar plate with oxacillin salt for analysis (Sigma Chemical Company, St. Louis, Missouri, USA) at a concentration of 4 µg/mL plus NaCl at 2%. The inoculum was prepared based on a suspension equivalent to McFarland’s standard 0.5, from bacteria studied with 24 hours of development in blood agar; dilutions were made to obtain a final inoculum of 1x104 CFU, which was deposited on the plate. The control strains were S. aureus ATCC 29213, methicillin-sensitive, and S. aureus ATCC 43300, methicillin-resistant. After air drying, the plates were incubated at 35 °C for 24 hours. The development of ≥ 1 CFU defined the isolate as methicillin-resistant. The second resistance determination was made by detecting PBP2a encoded by the methicillin-resistance gene (mecA), by the Slidex MRSA test, in which latex particles sensitized with a monoclonal antibody against PBP2a were put in contact with the microorganisms, according to the manufacturer's instructions (bioMerieux, Durham, North Carolina, United States). Evidence of agglutination defined the bacteria as methicillin-resistant.    

Susceptibility to vancomycin was determined in Mueller-Hinton agar plates with vancomycin salt (Sigma Chemical Company, St. Louis, Missouri, USA) diluted to decreasing concentrations from 16 to 0.5 µg/mL. After the preparation of a MacFarland equivalent 0.5 suspension, the plates were inoculated with 104 CFU of clinical isolates, with subsequent incubation at 35 °C for 24 hours. Enterococcus faecalis ATCC 29212, vancomycin-sensitive, and E. faecalis ATCC 51299, also vancomycin-resistant, were included as controls. The growth of at least 1 CFU characterized the organism as resistant. The categories considered were: sensitive (MICX ≤ 2 µg/ mL), intermediate (MIC 4-8 µg/mL) and resistant (MIC ≥ 1 µg/mL) to vancomycin.


Over six months, 25 isolates of Staphylococcus aureus were detected in blood cultures. 60% of Staphylococcus expressed methicillin resistance, both from its development in oxacillin plates and its agglutination. Vancomycin MICs were distributed as follows: 48%, ≤ 0.5 µg /mL; 44%, 1 µg/mL; and 8%, 2 µg/mL. MIC50 and MIC90 were 1 µg/mL. Therefore, the 25 isolates were categorized as vancomycin-sensitive (MIC ≤ 2 µg /mL).


Among nosocomial bacteremia, Staphylococcus aureus occupies the first place as a causative agent, both in America and in Europe,10 with a higher morbidity and mortality when the organism is resistant to methicillin.11,12 Therefore, vancomycin has been considered choice for treating serious infections caused by MRSA for more than four decades. However, decreased glycopeptide susceptibility in such microorganisms13 and the association with treatment failures3,4 have worried doctors in recent years.

The present study found that although 60% of S. aureus isolated from blood was methicillin-resistant, 100% was sensitive to vancomycin.

Regarding methicillin-resistance, the ratio found was higher than that observed in other communities, such as the European Union, where the rate among the countries ranges from 1 to more than 40%,14 and Australia, where this proportion was 20%.15 In Mexico, the rate reported from 2000 to 2004, at the Instituto Nacional de Ciencias Médicas y de la Nutrición Salvador Zubirán, reached 45%.16 This difference could be determined by the flexibility with which nosocomial epidemiological control measures are applied, considering that the main reservoirs of MRSA are infected or colonized subjects, and that bacterial transmission happens predominantly from person to person.17 The standard method for the epidemiological control of endemic MRSA involves the following strategies: identifying carriers, eliminating reservoirs, stopping transmission, and reducing the use of antibiotics.17 Although these strategies are complementary, strict adherence to standard precautions (third strategy), particularly hand disinfection for health care workers after patient contact or handling contaminated materials, could prevent most MRSA cross-transmission without the primary need to identify carriers. However, average adherence of health personnel to hand hygiene recommendations is close to 40%.18 Therefore, in communities where MRSA control policies include universal screening, combined with physical isolation and decolonization of the carrier patient, MRSA bacteremia rates have declined,19 while in countries where only standard precautions apply, these rates remain high. Increasing adherence to hand hygiene is a prized goal, but difficult when considering that the determinants of this behavior, while they have been studied,20 have not yet been defined.   

With respect to vancomycin susceptibility, all staphylococci were sensitive. However, the rate of microorganisms with MIC ≥ 1 µg/mL was 52% (13 of 25). This ratio is below the 86-92% range, based on the SENTRY Programa de Vigilancia Antimicrobiana (universal program to monitor infectious diseases) during 2002-2006 respectively, for other Latin American medical centers.21 Such a difference could be determined by two intrinsic characteristics of the medical units: the selective pressure exerted by the use of vancomycin, or the method of determining said antimicrobial susceptibility. The selective pressure of vancomycin dates from 1958, when its clinical use was approved. However, it was not until 22 years later that the gradual increase in the use of the antimicrobial began, up 100 times from 1975 to 1995.22 Two clinical entities have predominately justified the widespread use of vancomycin, pseudomembranous colitis, and infections caused by MRSA and Streptococcus pneumoniae, which is penicillin-resistant.23 In the first, the parenteral glycopeptide began to be used orally before going on the market. In the second, infections caused by MRSA are particularly important, for which vancomycin has been considered the glycopeptide of choice in the parenteral treatment of serious infections since 1980.24 The procedure used to determine vancomycin susceptibility depends on the resources of the medical units. The reference method is dilution in broth or agar.9 However, the E test and automated methods are most commonly used in clinical laboratories. While the E test categorizes susceptible microorganisms as "intermediate", automated methods such as Vitek 2 classify bacteria with intermediate susceptibility as “vancomycin-sensitive".25,26

As such, since this work and SENTRY both determine vancomycin susceptibility with the reference method, the lower proportion of isolates found with MIC ≥ 1 µg/mL (52 versus 86-92%) could relate to lower selective pressure on the bacterial population studied.  

In conclusion, all isolates tested were sensitive to vancomycin; however, 8% (2 of 25) showed an MIC of 2 µg/mL. This fact is important because it shows the current existence of a phenotype associated with increased mortality among patients with bacteremia caused by MRSA, whose vancomycin MIC is ˃ 1 µg/mL when treated with that glycopeptide.6


Although few isolates were studied, the proportion of methicillin-resistance and the existence of vancomycin-sensitive phenotypes, while associated with poor treatment response among patients with Staphylococcus aureus, demand not only the continuous reinforcement of standard precautions and antimicrobial control, but also the systematic monitoring of the pattern of vancomycin susceptibility with a reference method.

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Conflict of interest statement: The authors have completed and submitted the form translated into Spanish for the declaration of potential conflicts of interest of the International Committee of Medical Journal Editors, and none were reported in relation to this article.

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