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FREQUENCY OF COMMUNITY-ACQUIRED METHICILIN-RESISTANT Staphylococcus aureus IN A TERTIARY CARE HOSPITAL IN PERU

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 crosssectional 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.


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 (1)(2)(3) . 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 (3)(4)(5) . 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 (4) . The most prevalent CA-MRSA clone is USA300, which characteristically carries SCCmec type IV and is usually resistant only to β-lactams (5)(6) .
In Peru, some imported cases of CA-MRSA were described between 2010-2011 (7) . 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 (8) .
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.

Motivation for the study: Community methicillin-resistant
Staphylococcus aureus causes infections with poor response to antibiotics, mainly skin and soft tissue infections. Information in Peru on its presence in hospitals is insufficient, so we sought to determine its frequency in order to establish the necessary preventive biosecurity measures and its resistance profile to identify antibiotics for empirical use.

Main findings:
The study confirms the presence of said bacterium in our setting.

Implications:
It is necessary to maintain epidemiological surveillance measures to prevent its spread.

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 (9) . S. aureus strain ATCC 29213 was used as quality control.
For molecular analysis, DNA was extracted according to the methodology described by Bouillaut et al (10) . 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 al (11) . 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 al (12) .
Those isolates that had discordance between the cefoxitin resistance pattern and the presence of mecA, or whose were considered HA-MRSA (13) .

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.
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 (14,15) . Brazil and Venezuela report the highest frequencies in the region, with 62% and 57%, respectively (15) . In the case of Peru, a frequency of 50%-54% has been reported (14,15) . 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 (16) . Subsequently, a study that included S. aureus isolates from all sources in three referral hospitals in Lima (17) 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 (15) .
From the molecular point of view, the SCCmec type of MRSA has a varied distribution in Latin America (18,20) . 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 (15) .
In Peru, a multicenter study conducted in Lima (17) 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 (17,18) . 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 (8,14) . 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 (15,20) . 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 (19) . Subsequently, it was described  that its production could be considered a marker for identification of resistant strains, especially CA-MRSA (19) .
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 (17) 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.