Exploring the staphylococcus aureus in patients infected of the tertiary- care university hospital: results of the retrospective cohort study

Objective: To establish a baseline of knowledge regarding about inappropriate therapy, virulence and resistance in a cohort of patients infected with S. aureus. Methods: Retrospective cohort study in tertiary-care university hospital was employed to evaluate the risk factors and the impact of inappropriate therapy among patients with Staphylococcus aureus infections, resistance and virulence. To assess the presence of the genes was performed PCR. Results: Patients with MRSA were older and hospitalized 17 days longer than those with MSSA infection, which were in ICU with a bloodstream infection. 50.0% received inadequate antibiotic therapy and we found virulence factors associated with MRSA (mecA, LukS, fnbB and clfA genes). Conclusion: These data show that surveillance studies related to Staphylococcus aureus infections remain essential to identify resistance and inform policy on resistance.


Introduction
The epidemiology of Staphylococcus aureus is dynamic and has been presenting significant changes in recent years (OLIVEIRA;FARIA;LEVYAND, et al. 2001;AÑÓN;RODRÍGUEZ;TINAJAS, et al. 2004;PARKER, 2017;KOECH;COMO-SABETTI;BOXRUDE, 2019). Although Gram-negative bacteria are the most common source of nosocomial infections, S. aureus infections are still a substantial source of morbidity and mortality because they have the ability to induce inflammation and are responsible for superficial and invasive infections PELEG; HOOPER, 2010; SCHWEIZER.; EBER; LAXMINARAYAN, et al. 2011).
The treatment of infections caused by this microorganism is complicated by the high prevalence of multidrug-resistant strains, selected by the indiscriminate and inappropriate use of antibiotic, mainly associated with horizontal transfer of genes of resistance (HEGGERNDORNN; GOMES;SILVA, et al. 2016).
S. aureus still presents an arsenal of virulence factors that contribute to its survival and development, and it is responsible for the onset of clinical manifestations and severity of infections RICARDO-CALDERA; BUELVAS-DORIA; ESCOBAR-PÉREZ, et al. 2015). Although resistance plays an important role in the worsening of these infections, the pathogenic capacity of this microorganism is the result of the combined effect of the production of virulence factors, such as enzymes and toxins, and the ability to form biofilm, multiplication and dissemination in tissues and organs, besides its invasive capacity (CHAMBERS; DELEO, 2009;STEFANI;GOGLIO, 2010;BIMANAND, L.;TAHERIKALANI;JALILIAN, et al. 2018).
Reports of methicillin-resistant Staphylococcus aureus (MRSA) emerged in the 1960s, and currently, MRSA isolates are estimated with a prevalence of 25 of 50% or more in some hospitalization areas (INDRAWATTANA; SUNGKHACHAT;SOOKRUNG, et al. 2013;SANTAJIT;INDRAWATTANA, 2016). In recent years, due to the increased rate of detection of MRSA, empirical antibiotic treatment has become even more difficult, additionally, when the prescription occurs inappropriately, there is an increase in hospital stay, treatment costs and risk of death (PAUL; SHANI; MUCHTAR, et al. 2010;WEISS;FITZGERALD;BALAMUTH, et al. 2014;MICEK;KOLLEF, 2015;LI;LI;ZHANG, et al. 2019).
We aim to establish a baseline of knowledge regarding about inappropriate therapy, virulence and resistance in a cohort of patients infected with S. aureus.

Materials and methods Patients, settings and study design
The data base at our clinical microbiology laboratory was reviewed to identify patients with Staphylococcus aureus infections from November/2015 to April/2016 at Uberlandia University Hospital (Brazil), a 533-bed tertiary-care university hospital. For comparison between patients with Methicillinresistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA), only the first episode was analyzed.
A retrospective cohort study was employed to evaluate the risk factors and the impact of inappropriate therapy among patients with Staphylococcus aureus infections. During the surveillance period, 80 episodes of S. aureus infection were recovered from 55 patients. The main outcome was in hospital mortality and the procedure also assessed secondary outcomes, including the duration of hospital stays, admission to the Intensive Care Unit (ICU) and the use of central venous catheter and mechanical ventilation.
The data and the samples analyzed in the present study were obtained in accordance with the norms and approved by the Federal University of Uberlandia Ethics Committee (UFU), through license number 36601814.7.0000.5152 and 60374516.6.0000.5152.

Definitions
Hospital infection is acquired after the admission of the patient and it is manifested during hospitalization or after discharge, when it may be related to the hospitalization or hospital procedures (BRASIL, 1998). Inappropriate empirical therapy is the antimicrobial treatment with agents that did not have "in vitro" activity and/or treatment performed over 48 hours (DAIKOS; TSAOUSI; TZOUVELEKIS, et al. 2010).

Identification of species, antimicrobial susceptibility and molecular testing
Microbial identification and antimicrobial susceptibility testing were performed on a VITEK®II automated system (bioMérieux). To assess the presence of the mecA gene, LukS gene, fnbB and clfA genes in Staphylococcus aureus strains, a polymerase chain reaction (PCR) was performed, as described previously by Kondo et al. (2007), Said Salim et al. (2005) and Tristan et al. (2003), respectively. All PCRs were carried in a mastercycler personal (Eppendorf). To perform the PCR, only 38.8% of the isolates were chosen, due to the costs of molecular techniques. The criteria for selecting these were based on the antibiogram as follows: samples with the highest resistance profiles (in the cases of groups R1 and R2) and samples only with resistance to methicillin (in the case of group R3)

Statistical analysis
The evaluate of quantitative variables were performed by Student's T-Test. Categorical variables were expressed as mean and standard deviation. The statistical significance was defined by a value of P ≥ 0,05, using the statistical program GraphPad Prism 5.01.

Results and Discussion
Fifty-five patients (147 episodes with infection) were selected hospital-wide during surveillance period, S. aureus was the most common species (54.4%), of which 29.9% were oxacillin resistant followed by Gram-negative bacilli (38.8%). Most of this isolated (32.6%) were detected from patients on the ICU. A predominance of bloodstream infection (40.9%) among MRSA, and pneumonia (33.3% and 31.5%, respectively) among MSSA and Gram-negative bacilli was observed (Table 1). Based on the antibiotic susceptibility test, 11 antibiotypes (R1-R11) were identified among isolates of Staphylococcus aureus. The most isolated were assigned antibiotype R2 (30.0%) and R1 (18.7%). In R2 strains presented predominantly the genotype mecA+/ PVL-/ clfA+/ fnbB-(29.1%), with more isolates in lung, while the R1, 46.6% strains presented the genotype mecA+/ PVL-/ clfA-/ fnbB-with more isolates in wound. (Table 2). The table 3 shows the risk factors for Staphylococcus aureus infections. Although only the result of avera ge age presents significance, on what patients with MRSA had a mean age of 61.1 years and with MSSA, 37.7 years, other results showed important percentage differences. The majority of patients with resistant infection had ICU hospitalization (53.8%), made use of the inappropriate antibiotic therapy (50.0%) and used central venous catheter (61.5%), while the use of mechanical ventilation was higher in patients with sensitive infection (75.8%). The average hospitalization time and the average hospitalization time until diagnostic was also higher in patients with MRSA infection (44.5 days and 25.8 days, respectively). Analyzing and determining the importance of MRSA in research-limited countries is a great challenge, since today the study has demonstrated a more significant prevalence of Gram-negative bacilli as etiology of infections. However, our results agree with previous studies reporting a high burden of this phenotype among Staphylococcus aureus infection (ENRIGHT; ROBINSON;RANDLE, et al. 2002;WORLD HEALTH ORGANIZATION, 2015;FRIEDRICH, 2019).
Interestingly, in our assessment, MSSA was responsible for 33.3% of pneumonia and MRSA was predominant of bloodstream infection. Special attention was given to the Gram-negative bacilli, the main cause of pneumonia and urinary tract infection. This is particularly worrisome, since the literature shows that therapeutic options are scarce or ineffective to the multidrug-resistant organisms (ZARAGOZA; ARTERO;CAMARENA, et al. 2003;BASETTI;POULAKOU;RUPPE, et al. 2017).
MRSA is a well-recognized public health problem through the world (CIMOLAI, 2007;STRUELENS, 2009;CHESSA;HANAU;MAZZARELLO, 2015). From a hospital perspective, patients with MRSA tendent to be older, to have a longer hospital stay, to have a recent background of hospitalization, among others. The present study has detected and considered such factors, in which the patients with MRSA infection were significantly older and were hospitalized 17 days longer than those with MSSA infection, as well as in previous hospitalizations when the same comparison was made.
Empirical antibiotic therapy may appear to be a good treatment strategy before the microbiological result is available, however, as an inappropriate therapy can lead to increased antibiotic resistance ( therapy. The literature shows that the delay in initiating appropriate therapy may lead to a higher mortality rate (WI; RHEE;KANG, et al. 2018). Due to the small number of patients, the data collected in this study did not allow the specific examination of the effect of appropriate antibiotic therapy. However, knowing the prescribing patterns of antibiotics for S. aureus infections in our institution, may justify the results of similar mortality rates between MRSA and MSSA in our cohort, which leads us to assume that empirical antibiotic therapy may have been more effective against MRSA during the study period.
S. aureus is also a versatile pathogen in the production of several types of virulence factors, with the objective of destroying the host cells and causing infections in different ways (FOSTER; GEOGHEGAN;GANESH, et al. 2013;OTTO, 2014;WI;RHEE;KANG, et al. 2018;JIANG;YOU, 2018). Although our sample size was small, we found important virulence factors associated with MRSA. The fnbB genes responsible for binder production of fribronectin and clfA (POWERS; WARDENBURG, 2014), responsible for cloning factor encoding were positive in 8.8% and 44.1%, respectively, in MRSA strains. The lukS, responsible for the production of the PVL toxin was not present in any of the strains evaluated (MORAN; KRISHNADASAN;GORWITZ, et al. 2006), this should be found to be more prevalent in community strains.
In conclusion, although Gram-negative bacilli are predominant in nosocomial infections in Brazil, Staphylococcus aureus, particularly those resistant and more virulent, is still present in the hospital environment in important frequencies especially in ICU. Our further results confirm previous report showing a high frequency of inappropriate antibiotic therapy among MRSA associated with a very long hospitalization time.