Identification and characterization of methicillin-resistant Staphylococcus spp. isolated from surfaces near patients in an intensive care unit of a hospital in southeastern Brazil

Abstract INTRODUCTION: Contaminated hospital environments contribute to the transmission of microorganisms associated with healthcare. Contaminated surfaces handled by patients or healthcare professionals are a source of microorganism transmission by hand. Methicillin-resistant Staphylococcus bacteria are among the main agents responsible for increasing healthcare-associated infections in Brazil and worldwide. METHODS: The objective of this study was to screen and characterize methicillin-resistant Staphylococcus spp. on surfaces near patients in an intensive care unit. Microbiological samples, collected from ten beds in an intensive care unit with five sampling sites, were inoculated into a methicillin-resistant Staphylococcus aureus chromogenic medium. MALDI-TOF and PCR analyses were used to identify the bacteria. Antimicrobial susceptibility was determined using the disk diffusion test. The presence of the mecA gene was investigated using PCR. RESULTS: We observed that 44 out of the 50 sampling sites presented grown isolates in the methicillin-resistant Staphylococcus aureus medium. The incidence of isolated microorganisms on the right side rail, left side rail, tables, infusion pump keypad, and cardiac monitor were 18.8 %, 36.7 %, 10.9 %, 2.4 %, and 31 %, respectively. The 42 isolates included in this study were identified as coagulase-negative Staphylococcus. All of these microorganisms were multidrug-resistant and mecA gene-positive. CONCLUSIONS: This study identified the presence of methicillin-resistant coagulase-negative Staphylococcus on the beds of an intensive care unit, providing evidence for the necessity of assertive actions to decrease the risk of healthcare-associated infections at the site.


INTRODUCTION
Healthcare-associated infections (HAIs) are infections that patients acquire while receiving healthcare 1 . HAIs include crossinfection among patients infected or colonized with pathogenic bacteria, which can be transmitted from patient to patient directly or indirectly through vomit, devices, healthcare professionals, companions, and the environment around the patients 2 . HAIs have an impact on morbidity, mortality, and length of patient stay in nosocomial units, leading to increased mean costs of hospitalization 3 . The environment surrounding the patients is an important source of HAI microorganisms. Surfaces such as side rails, medical equipment, support tables, and curtains, among others, can become reservoirs of harmful microorganisms that can be transmitted to patients either directly, when the patient comes into contact with the medium, or indirectly, by contamination of the hands and gloves of health professionals 4 . Surface cleaning and disinfection are extremely important to reduce the occurrence of microbial contamination and consequently the risk of HAIs. This cleaning process is highly complex and multidimensional, involving the physical friction of the surfaces to remove organic and inorganic materials, and then using a disinfectant solution, as well as monitoring strategies to ensure proper hygiene procedures 5,6 .
Bacteria of the genus Staphylococcus are the main cause of HAIs; their high prevalence on the skin of patients facilitates infection after a medical procedure breaks the barrier, or may be associated with decreased immunity. Coagulase-negative Staphylococcus (CoNS) strains are the main etiological agents of a series of infectious processes acquired in a hospital environment. In 2018, CoNS were the main causative agents of catheter-associated bloodstream infection in ICUs in Brazil 7 . The occurrence of these CoNS-related HAIs has several predisposing factors, including the immune and health status of the patient and the environment close to the patient, which can be a source of both origin and transmission 8,9 . Another characteristic that makes CoNS-related HAIs worrisome is their increased resistance to beta-lactam antibiotics, especially methicillin 10 .
Resistance mechanisms against this class of antibiotics, developed by bacteria of the genus Staphylococcus spp., include two specificities that are important for their prevalence: the production of the beta-lactamase enzyme encoded by the blaZ gene, and the modified effector mechanism of beta-lactam antibiotics with the modification of the PBP binding protein into PBP2A protein, which has a low affinity for penicillin-binding, encoded by the mecA gene and its homologs, including mecALGA251 10 . As the main human skin colonizer with a high survival capacity in inert, inhospitable environments with low substrate levels, CoNS often colonize the environment near the patients. They can be differentiated from S. aureus at the species level through PCR identification of the femA gene (an essential factor for methicillin resistance). This gene encodes a 48-50 kDa protein recognized as a specific factor and is contained in the chromosomes of these pathogens 11 . Thus, the objective of this study was to screen and characterize methicillinresistant Staphylococcus spp. on surfaces near patients in the ICU of a hospital in southeastern Brazil.

Microorganism sampling and collection sites
The samples included in this study were composed of microorganisms isolated from hospital ICU bed surfaces, not involving patients (or their data) or any approach to health professionals. The sample collection was authorized by the hospital where the research was conducted.
The study was conducted in a general tertiary-level hospital located in the southeast region of Brazil. It has 397 beds and 3 ICUs, of which the general ICU, containing 10 beds, was chosen for sample collection. The rationale for choosing the general ICU was based on the fact that this environment receives patients from different hospital sectors and with different clinical conditions, increasing the variability of their microbiota, with a greater chance of analyzing the real epidemiological profile of the institution. Each of the 10 ICU beds included 5 sampling sites: right side rail (RS), left side rail (LS), bedside table (BT), infusion pump keypad (PK), and cardiac monitor (CM) (Figure 1). These points were chosen based on the frequency with which they are manipulated/touched, acting as potential contamination sources for healthcare professionals and patients 12,13 . Surface sampling was performed around ICU bed surfaces before any cleaning procedure was performed. The sampling procedure was performed with patients inside the room. The samples were collected on October 24, 2019, using swabs immersed in saline solution for sampling a 5 cm² area. Subsequently, the swabs were depleted on Petri dishes containing a methicillinresistant Staphylococcus aureus (MRSA) chromogenic growth medium (Probac do Brasil, Brazil). The plates were incubated at 37°C for 24 h. After the incubation period, colony-forming units (CFUs) were counted and registered on a form.

Microorganism identification
Sampling sites where no growth could be observed were excluded from the study. A total of 42 colonies from different sampling sites were analyzed using the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) proteomic identification approach in the AQUACEN laboratory of the Veterinary School of the Federal University of Minas Gerais (UFMG), according to the methodology previously described by Assis et al. 14 , using a Microflex TM MALDI-TOF MS device (Bruker Daltonics, USA). Furthermore, the genomic PCR method was used to identify S. aureus at the species level through the detection of the femA marker gene, as described by Xavier et al. 11

Genomic analyses
The bacteria were selected on plates containing MRSA medium, transferred to Brain Heart Infusion (BHI) broth (Laborclin, Brazil), inoculated onto mannitol salt agar (Laborclin, Brazil) plates, and incubated at 37°C for 24 h. The growing colonies were analyzed using the DNA extraction procedure described by Gu et al. 15 The integrity and quantification of the extracted DNA were verified by 1.0 % (w/v) agarose gel electrophoresis. This material was used for PCR . All primers used in this study were synthesized by GenOne Biotechnologies, Brazil.
PCR tests were used for femA gene detection, a species-specific marker for S. aureus, and mecA gene detection, a specific marker for methicillin resistance, as described by Xavier et al. 17 The reactions were performed using a mix containing 2× Go Taq Green Master Mix® (Promega Corporation, USA), 10 µM of each primer, and 50 ng of DNA, with a final reaction volume of 50 µL. PCR was performed in a Veriti Thermal Cycler (Applied Biosystems,USA). The PCR thermal conditions were described by Xavier et al. 17 The amplicons were visualized on 1.5 % (w/v) agarose gel stained with ethidium bromide and photo-documented. S. aureus ATCC 43300, S. aureus ATCC 29213, and S. aureus 25923 strains were used as controls for PCR.

Antimicrobial sensitivity profile
The susceptibility to beta-lactam antibiotics was determined by the disk diffusion test according to the Clinical & Laboratory Standards Institute (CLSI) guidelines 16

RESULTS
This study demonstrated environmental surface contamination through the collection of microbiological samples from 50 surfaces near patients in an ICU. We observed that 44 out of the 50 samples grew on MRSA medium-containing plates. Although they grew on MRSA medium, two samples did not grow in mannitol salt agar and were therefore excluded.
Proteomic genus identification confirmed that all isolates were Staphylococcus bacteria; however, species identification was only possible for 45.22 % (19/42) of the isolates. The species isolated from the surface samples of this study were all CoNS. The microorganisms identified were S. haemolyticus (11), S. epidermidis (4), S. capitis (2), and S. cohnii (1) ( Table 1).
Due to the low species identification index obtained by MALDI-TOF, PCR was used to trace the femA gene, a species-specific marker for S. aureus, in all isolates. Control S. aureus strains were used to standardize the PCR for positive femA gene identification ( Figure 3A). None of the isolates were femA gene-positive ( Table 1), confirming the species identified using the MALDI-TOF and excluding the possibility that the strains could be identified as S. aureus.
Although the isolated strains were not S. aureus, they all grew on MRSA medium-containing plates, and their resistance to methicillin was tested at the genomic level by PCR screening for the mecA gene (Figure 2). Control S. aureus strains were used to standardize the PCR to screen for mecA gene-positive samples ( Figure 3A). Among all the isolates, 90.47 % (38/42) were mecA gene-positive, confirming the phenotypic growth result in MRSA medium, even if they were not S. aureus strains. Figure 3B shows the mecA gene PCR results of representative isolates.
The antimicrobial susceptibility test showed the presence of extensively drug-resistant strains (   Table 2).

DISCUSSION
The presence of bacterial contamination on inanimate surfaces in a hospital environment has attracted the interest of healthcare professionals in order to relate these microorganisms to HAIs.
Gram-positive bacteria have been researched as they are more prevalent in these environments, especially in ICUs. Most of the studies on this subject show environmental contamination by Staphylococcus aureus 17 .
CoNS represent a group of microorganisms that are the main definitive or transitory colonizers of human skin and mucous membranes. However, as they live in balance with the human 5/7  This study analyzed 50 samples collected in the ICU of a general hospital. Each of the 10 beds had 5 collection sites chosen according to the frequency they are handled by the multidisciplinary team; therefore, they are considered potential sources of contamination.
Of the 50 samples collected, 42 (84 %) showed bacterial growth due to the presence of microorganisms that commonly colonize hands, which spread CoNS on the equipment and surfaces near the patient. In this work, the sampled surfaces were not subjected to any disinfection process prior to sampling collection, since the objective was to track the presence or absence of Staphylococcus bacteria. Of the equipment and surfaces analyzed, LS and CM were the most contaminated. A study by Moraes et al. also reported high contamination rates in infusion pumps 21 19 .
PCR analysis of the femA gene showed no presence of Staphylococcus aureus in any of the samples, confirming the above result, which did not identify the presence of S. aureus. The mecA gene was present in 95 % (40/42) of the samples. It was absent in only 3 S. haemolyticus samples and 1 S. epidermis sample. Oxacillin resistance without the mecA gene may occur for other reasons, such as beta-lactamase enzyme production 22,23 .
All samples showed resistance to 3 or more classes of antimicrobials, and 16 samples showed resistance to at least 10 of the 11 antimicrobials tested. This high resistance rate corroborates the literature regarding the high capacity of CoNS to develop antimicrobial resistance 17,18 .
These results show that the equipment and surfaces analyzed are CoNS reservoirs and thus should be considered effective transmission and dissemination means of HAIs in the analyzed ICU. Our work was limited to investigating only the role of some ICU surfaces considered potential sources for transmission and dissemination of HAIs. It could be considered a study limitation once CDC guidelines for environmental infection control 25 suggest other aspects that should be also investigated as key points related to HAI incidence. Knowledge of airborne sampling techniques to monitor the effectiveness of air, ventilation, and water systems performance, and other methods for the management of HAIs are also recommended by the CDC guidelines 25 .
We also considered the limitation of this study regarding surface microorganism sampling planning. However, the results reflected the presence of multidrug-resistant microorganisms on the analyzed hospital surfaces. A comprehensive study with a larger number of samples, including collections at other points (patients' and healthcare professionals' hands), also including air sampling and co-relating with patient data, could better elucidate the profile of the bioburden found in the hospital.
Despite these limitations, the results should be a warning for healthcare professionals, hygiene service companies, and healthcare institution managers regarding the importance of correct hand hygiene and adequate cleaning and decontamination of the environment and equipment, as the isolation of CoNS suggests failures in the process of surface and equipment cleaning and disinfection performed daily in ICUs. Furthermore, the failure in the cleaning and disinfection process indicates that the sampling sites could eventually be reservoirs for several microorganisms that could have an impact on public healthcare. This makes it even more important for hospitals to strive for excellence when it comes to infection control in order to protect workers and patients as well as their family members. Hand hygiene combined with the correct decontamination procedure of surfaces and equipment is fundamental to control the spread of microorganisms and HAIs.