Antimicrobial Resistance and Virulence of Staphylococcus spp. in patients from oncologic and non oncologic hospitals of Recife City/PE

Staphylococcus spp. is one of the major infection-associated bacteria within health care, especially in intensive care units, and one of principal cause of complication in cancer patients. This study compared the antimicrobial susceptibility profile and frequency of resistance (mecA, blaZ, ermA and ermC) and virulence (icaA, icaD and hlg) genes in Staphylococcus spp. from patients of Oncology Hospital (OH) and University Hospital (UH). The type of the ccr complex was assessed by PCR among the mecA positive isolates from the UH. Higher percentage of susceptible isolates, except for oxacillin and cefoxitin was found among the UH isolates and 27,3% vancomycin-resistant isolates were identified through the screening spot; 41 isolates displayed the MLSBc phenotype and five the MSLBi phenotype, and one isolate from the OH displayed the constitutive phenotype ermC gene. The ccr types I and II were identified with a higher frequency of ccr type I. No statistically significant difference was found in the frequency of the genes between the two groups of patients or in the two hospitals. Regarding the virulence genes, there was statistically significant difference when comparing the two hospitals.


Introduction
Bacterial infections are a major cause of complications in patients with hematological neoplasia and chemotherapyinduced neutropenia and bacteremia is the leading cause of death, either directly or by interfering with the chemotherapy chronogram (Montassier, et al., 2013;Quiles et., al, 2015;Jiang et al., 2020). Risk factors for infection in this group of patients are more expressive, because the immunocompromised related by disease itself, they are subjected to surgical treatment, chemotherapy and/or radiotherapy, where neutropenia is a major risk for infection in this population (Jiang et al., 2020).
In recent decades, the success of the treatment of malignancies in children and teenagers has increased, with up to 70 -80% of cases. Furthermore, the immunosuppression from the treatment makes these patients more susceptible to infections, which are the main cause of death among them. In these patients, bacteremia often results from infection by one or more microorganisms, mainly members of the Enterobacteriaceae family, Pseudomonas aeruginosa, Streptococcus and Staphylococcus (Quiles et., al, 2015). Over the last three decades, the proportion of Gram-positive microorganisms has increased because of the widespread use of permanent catheters (Montassier, et al., 2013).
It is noteworthy that the intensive care centers concentrate the highest incidence rates of hospital infections, and are emergency epicenters of microbial resistance, due to the hospitalization of seriously ill patients. These patients, in most cases, use a wide spectrum antimicrobials and are subjected to successive invasive procedures (Jiang et al., 2020). Occur more and more evidences supports the idea that neutropenic patients at low risk do not necessarily need hospitalization and antibiotic wide spectrum prolonged to bacterial invasive infections. The strategies for this group include antimicrobial short-term treatment with a consequent reduction in hospitalization and outpatient management with the use of oral antibiotics (Cagol et al., 2009), reinforcing the importance of proper characterization of the resistance profiles of bacterial strains.
S. aureus can evade the host immune system due to the existence of several virulence factors and some studies have shown differences in the production of virulence factors such as gamma-hemolysin (hlg) and biofilm (ica) between the strains isolated from different clinical specimens (Kord et al., 2018;Elboshra et al., 2020). However, these comparisons have not been related described in patients of oncology centers. The biofilm production may allow these isolates colonize the catheter and other devices, to evading the immune system and microorganisms might lead to an increased frequency of bacteremia and false-positive or false-negative blood cultures (Figueiredo et al., 2017;Kord et al., 2018).
In a retrospective study in an U.S., oncology hospital it was determined the existence of clones among isolates resistant groups related with the resistance data associated with clinical information suggestive of infection (Muldrew et al., 2008).
Among the several resistance mechanisms of Staphylococcus, the most common are the resistance to macrolides, aminoglycosides, beta-lactams and glycopeptides (Sangappa & Thiagarajan, 2012;Jenkins et al., 2015). The importance of these microorganisms depends on the toxins mediated virulence, its invasive nature and its antibiotics resistance profile (Jenkins et al., 2015;Elboshra et al., 2020). These characteristics are related to the diversity of clinical manifestations (Lim et al., 2012;Jenkins et al., 2015), however concerning the oncology patients, this is not yet clear.
The increasing of resistant strains with different virulence factors over the years indicates the potential failure of the use of antimicrobials in the treatment of infections due to the association of the resistance mechanisms of the microorganism and the resistance of the host immune response (Lim et al., 2012). The number of virulence associated genes harbored by a bacterial isolate results from the interaction between gene acquisition rates, the biological maintenance cost and the failure rate of the disease (Leclercq, 2002;Jenkins et al., 2015). Thus, the more severe S. aureus infections cannot be explained by the action of a certain virulence fator only, but rather by the action of several virulence factors during the infectious process. The survival and proliferation of a certain pathogen in the host is favored by mechanisms that enable evasion of the host immune mechanisms. Thus, strains that escape more efficiently the host defense mechanisms are the most prevalent. There is evidence that some clonal types are more virulent than others and they occur more frequently among patients than in healthy individuals (Leclercq, 2002;Spaan et al., 2014).
The proposal of this study was to characterize and comparing Staphylococcus spp. isolates from oncologic and nononcologic patients of two hospitals in the city of Recife-PE, regarding antimicrobials susceptibility profile, and the resistance and virulence genes content. This microbiological analysis will allow mapping the bacterial strains through resistance and virulence factors, which will facilitate tracing rational use of antimicrobials and treatment of the infections in the hospitals.

Methodology
The observacional study (Fontelles et al., 2009) compared Staphylococcus spp. isolates from oncological and nononcological patients from an oncology (OH) and a university hospital (UH) respectively in Recife, PE, Brazil during one year (2013) period. In the study, patients of male and female gender were included. The samples were collected during routine by the medical and nursing staff of the hospitals. The isolates were identified by macroscopic characteristics on 5% blood agar plates, Gram staining and the catalase, coagulase, DNAse and Mannitol salt agar tests (CLSI, 2014).

Screening of Oxacillin
Subcultures were made in nutrient agar plates of the isolates that were resistant to oxacillin and/or cefoxitin in disk diffusion technique. It was made direct suspension of colonies to obtain a turbidity to a standard solution of 0.5 McFarland scale. Was dipped into a 1μL inoculation loop in this suspension and proceeding with the inoculum in area with a diameter of 10 to 15 mm on plates containing Mueller Hinton agar medium with NaCl (4% v/v, 0.68 mol/L) and 6 μg/mL of oxacillin.
These plates were incubated at 35 °C for 24 hours being considered, after reading the results as: >1 colony = resistant. Were used as quality control standard strains for MRSA and MSSA: Staphylococcus aureus ATCC 29213 -Sensitive and Staphylococcus aureus ATCC 33591 -Resistant (CLSI, 2014).

Vancomycin Screening
All isolates were subjected to screening of vancomycin. These samples were inoculated into BHI broth and incubated at 35ºC to reach a 2 McFarland turbidity scale. One 1μL loop of this grow was spread in area of 10 to 15mm diameter on plates containing BHI agar supplemented with 6 μg/mL of vancomycin (OXOID) and incubated at 35 °C for 24 and 48 hours considering resistance as > 1 colony (Burnham, Weber & Dunne Jr, 2010;CLSI, 2014).

Total DNA Extraction
Total DNA was extracted from the isolates following Oliveira et al (2015) and the DNA obtained was quantified using nanodrop 2000 (Thermo Scientific).

Identification of the mecA gene by PCR
The presence of the mecA gene was assessed by PCR among the oxacillin and/or cefoxitin phenotypically resistant isolates using the primers described by Oliveira et al (2015). The PCR reactions were individually prepared in a final volume of 25µL containing: 20ng of genomic DNA, 20pmol of each primer, MgCl2 1,5mM, dNTP 200µM, 1U of Go Taq DNA polimerase (Promega, Brasil) and 5µL of Green Go Taq DNA polimerase buffer (Promega, Brasil). The reactions were performed in a thermocycler (Biometra), set for 35 cycles of one minute at 94°C, one minute at 55°C and two minutes at 72°C followed by a final step of 15 minutes at 72°C. As negative control, a tube containing all the components of the mixture without DNA and for positive control DNA from the strain Staphylococcus aureus ATCC 33591 -Resistant were used.

Identification of the betalactamase gene (blaZ) by PCR
The presence of the blaZ gene was assessed by PCR among the oxacilin and/or cefoxitin phenotypically resistant isolates using the primers described by Milheiriço et al (2011). The PCR reactions were individually prepared in a final volume of 25µL containing: 20ng of genomic DNA, 20pmol of each primer, 2,5µL of MgCl2 (25mM), 2,5µL dNTP (200µM), 1U of Go Taq DNA polimerase (Promega, Brasil) and 5µL of Green Go Taq DNA polimerase buffer (Promega, Brasil). The reactions were performed in a thermocycler (Biometra), set for 30 cycles composed of one minute at 92°C, one minute at 55°C and two minutes at 72°C followed by a final step of seven minutes ate 72°C. A tube containing all the components of the mixture without DNA was used as negative control and DNA from the isolate code 1171 from the Culture Collection from the Microbiology Departament from the FIOCRUZ-PE, positive for the gene blaZ confirmed by sequencing, was used as positive control.

Determining the type of SCCmec
The type of the ccr complex was assessed by multiplex PCR among the mecA positive isolates from the UH following a protocol based on Kondo et al (2007) as follows: PCR reactions were individually prepared in a final volume of 50µL containing: 20ng of genomic DNA, 10µM of each primer, 6,4µL of MgCl2 (25mM), 4,0µL dNTP (2,5mM), 1U of Go Taq DNA polimerase (Promega, Brasil) and 5µL of Green Go Taq DNA polimerase buffer (Promega, Brasil). The primers used are described in Table 1 and the PCR products are described in Chart 1. The reactions were performed in a termocycler (Biometra), programmed for 30 cycles composed of one minute at 95°C, one minute at 57°C and two minutes at 72°C followed by a step of seven minutes at 72°C.

Primer
Sequences Source: Authors.

Detection of ermA and ermC genes by PCR technique
The isolates erythromycin and clindamycin resistant (phenotype MLSBc) and erytromycin positive in the D test (phenotype MLSBi) were analysed for the presence of the ermA and ermC genes using the primers described by Lina et al (1999a): ermA F5'GTTCAAGAACAATCAATACAGAG3' and R5'GGATCAGGAAAAGGACATTTTAC3'; ermC:   (Kondo et al., 2007).
seconds at 49ºC, and 30 seconds at 72ºC. As negative control a tube containing all the components of the mixture without DNA and as positive control DNA from one isolate positive for the genes (ermA and ermC) confirmed by amplification and sequencing were used.

Detection of the icaA and icaD genes
The amplification of the icaA and icaD genes from the operon icaADBC, was performed using the primers described

Detection of the hlg gene
For amplification of the hlg gene the protocol described by Lina et al (1999b) was employed using the primers The products of all amplifications were electrophoresed in 1% agarose gels, stained with Blue-Green (LGC Biotechnology), visualized in UV transilluminator and scanned by Kodak 1D software version 3.5.2 (Scientific Imaging Systems, USA). For the products of the ccr complex amplification 2% agarose gels were used. The molecular weight marker 100bp DNA ladder (Invitrogen) was used.

Data Analysis
Clinical and microbiological data were introduced in SPSS Statistics 20 software for statistical analysis through frequency distribution, x² test and Fisher´s Test. The more frequent source of the samples was blood culture, followed by catheter tip in both hospitals (Table 2).
Related with the antimicrobial profile there was a higher percentage of susceptible isolates, except for oxacillin and cefoxitin among the 122 isolates from the UH (Table 3)    Source: Authors.
The occurrence of the blaZ and mecA genes was assessed by PCR among the methicillin and cefoxitin and/or oxacillin phenotypically resistant isolates. Among these from the Oncology Hospital 50 % and 77.7%, respectively were positive for the blaZ and mecA genes ( Table 5). The isolates analyzed from the University Hospital 45.7% were positive for the blaZ gene and 17% for the mecA gene (   Source: Authors. The isolates displaying the phenotypes MLSBc and MLSBi were analyzed by PCR for the detection of the ermA and ermC genes. Only one isolate was found harboring the constitutive phenotype ermC gene. The ermA gene wasn´t observed in Oncology hospital (Table 6). It was observed positive isolates for all genes in the studied phenotypes, except for the ermA gene in induced phenotype in the University Hospital (Table 6).
The occurrence of the genes icaAD and hlg was observed respectively in 25% and 42.8% in the isolates from the University Hospital and in 32.1% and 70% respectively in the isolates from the Oncology Hospital. Table 5 shows the distribution according to the source of origin.
In this study, there was no statistically significant difference in the occurrence of the resistance genes blaZ, mecA, ermA and ermC when compairing the two hospitals. Regarding the virulence genes, there was statistically significant difference when comparing the two hospitals. Chart 2 and 3 shows the results of the p value found in the static analysis.
Out of the five mecA positive isolates from the University Hospital analyzed by PCR for determination of the ccr complex type, four showed Type I ccr (ccrA1, 695bp) and one was Type II (ccrA2, 937 bp).

Discussion
Most Staphylococcus spp. carriers are asymptomatic and related the process of infection is usually associated with some factor which decreases the immune response of the individual, as invasive medical procedures, being this microorganism referred to as a main agents of bacteremia, often acquired in hospital environment (Muldrew et al., 2008). Among the users of central vascular catheter, S. aureus and CoNS are the more frequent microorganisms, accounting for 30% of the isolates (Muldrew et al., 2008Montassier, et al., 2013 in our study, the most frequent samples were blood culture and catheter tip with percentages of respectively 33,6% and 17,2% for the UH and 25,5% and 13,7% for OH, similar a study in public hospital in Brazil (Bride et al., 2019).

The frequency of isolation of S. aureus and its relationship with hospital infections reach high values in many
Brazilian hospitals, related of prevalence of MRSA strains varies from 40 to 80% (Lina et al., 1999b;Jones et al., 2013;Bride et al., 2019) and data from the Antimicrobial Surveillance Program show that MRSA reached 31% among the nosocomial and communitarian infections agents and are considered the more common among the most prevalent pathogens (Jones et al., 2013;Silveira et al., 2015). A survey in Brazilian hospitals showed 80% (Silveira et al., 2015) resistance of CoNS in blood cultures.
In our study, this was the most frequent type of sample; however, S. aureus was more prevalent in both hospitals.
Concerning the detection of the genes blaZ and mecA by PCR 50% and 77.7% of positive samples were found respectively in the OH while in the UH there was 45.7% of occurrence for the blaZ gene and 7% for the mecA gene. Some studies show that the rate of beta-lactamase production ranges from 55.7% to 92.6% for Staphylococcus Rabelo et al., 2013) and our results fit within these limits. Oliveira et al (2011) found high prevalence of beta-lactamase in their  Kang et al (2012), reported a prevalence of 48.4% of MRSA associated with bacteremia in cancer patients, a percentage lower than that from our work (77.7%) for oncology patients.
Another study conducted earlier in the same University Hospital, reported percentage slightly lower than ours, about 10% of the isolates were considered MRSA (Rabelo et al., 2014), suggesting that the control measures of this type of microorganism have not been fully effective. In this study, was noted the presence of strains resistant to methicillin only by phenotype methods for both hospitals, suggests the presence of other mechanisms of resistance or nonexpression of genes researched (Sangappa &Thiagarajan, 2012;Milheiriço et al., 2011;Andrade-Figueiredo & Leal-Balbino, 2016).
The profile of resistance to vancomycin in the present study among the isolates from the University and Oncology Hospitals described the percentage rate of 25% and 29.5% respectively. Staphylococci resistant to glycopeptides were described in Brazil, and isolation of vancomycin resistant S. aureus registered in São Paulo (Rossi et al, 2014;Panesso et al, 2015). Other study previously conducted in the University Hospital (Rabelo et al., 2014), reported 11% of resistant isolates using the screening spot method, but only among MRSA samples and the same hospital vanA gene was detected of isolates of S. epidermidis from nasopharyngeal secretion of health care workers (Bezerra Neto et al., 2018).
In the present study, the MLSBc phenotype found in 41 isolates (33.6%) from the University Hospital and eight isolates (15.7%) from the Oncology Hospital was predominant over the MLSBi phenotype found in five isolates (4.1%) from the University and six isolates (11.8%) from the Oncology Hospital. These findings are different from those from another study in Brazil that identified 71 (46.7%) MLSBc and five MLSBi (3.3%) isolates (Coutinho et al., 2010).
Antimicrobial susceptibility data are essential for appropriate therapy making imperative to perform the D test (Coutinho et al., 2010;Juyal et al., 2013). Categorizing a Staphylococcus spp. isolate as clindamycin sensitive without verifying if there is inducible resistance may result inadequate therapy. On the other hand, a negative result for inducible clindamycin resistance confirms the antimicrobial sensitivity and allow a correct therapeutic (Kumar et al., 2012).
Two studies conducted in Iran related with erythromycin-resistant genes described the frequency of ermC 35.2% and ermA 20.4% (Sedaghat et al., 2017) and Saderi, Emadi and Owlia (2011), found the opposite, the ermA gene was more frequent than the ermC gene, 76 (60.3%) and 69 (54.8%) respectively among the S. aureus isolates. Different results were obtained in our study, the presence of the ermC gene was higher than the ermA gene in the isolates of both Hospitals. In our study, no single positive isolate was found for the ermA gene in Oncology Hospital for the studied phenotypes. No single positive isolate was found for the induced phenotype at the University Hospital. The isolates that do not harbor the ermA or ermC genes, may possess the ermB gene. Due to a possible association of these genes the isolates harboring the ermA or ermC genes may also contain ermB. The presence of the ermB gene was not researched in our work because it occurs mainly among streptococci and enterococci (Leclercq, 2002). Furthermore, Coutinho et al (2010), reported a low frequency of the ermB gene.
In the present work, it was observed 42.8% and 70% of positive isolates for the hlg gene, respectively in the UH and OH, a higher frequency of 98.2% was found in hospital of India (Aggarwal et al., 2019), the data of UH was similar of Oliveira et al (2014) found 41.86% of methicillin-resistant CoNS isolates from blood cultures at another University Hospital of the same region and a study in Malaysia reported a percentage of 45% of methicillin-resistant isolates from patients and students from an university hospital (Lim et al., 2012). The gene hlg codes for the gamma toxin that has pro-inflammatory activity and is capable of lysing erythrocytes and leukocytes, which increases the toxigenic potential of the bacteria and the severity of the S. aureus and CoNS are able to produce biofilms and this process starts with the adhesion is mediated by the inter cell adhesin N-acetyl-glicosamine polimeric (PNAG) which is synthesized by enzymes encoded by genes present in the ica locus (Barbieri et al., 2015;Figueiredo et al., 2017). In our work, there was 25% and 32.1% of positive isolates for icaAD, respectively at the UH and OH. Higher positivity (83.5%) were detected in Palestinian hospital in isolates of S. aureus (Azmi, Qrei & Abdeen, 2019), positivity (79%) was found among inpatients and students in a hospital in Malaysia (Lim et al., 2012).
Another study reported 75% of positive isolates in samples from oncology patients with breast implants (Barbieri et al., 2015).
In this study, there was no statistically significant difference in the presence of the resistance genes (blaZ, mecA, ermA and ermC) when comparing the two hospitals whilst statistically significant difference was found in the presence of the virulence genes (icaA, icaD and hlg). This findind suggests that the oncology plays no role on the presence of resistance genes between the groups. On the other hand, the characteristics of the patient groups seems to be associated with the toxigenic and pathogenicity potential of the isolates studied.
The type of the ccr complex was assessed by PCR among the mecA positive isolates from the University Hospital and the ccr type I (ccrA1, 695bp) and type II (ccrA2, 937 bp) were found with higher frequency of ccr type I. Previous studies reported the occurrence of SCCmec types I, II, III, IV and V in Brazilian hospitals Oliveira et al., 2015; Andrade -Figueiredo & Leal-Balbino, 2016). Due to the small mumber of isolates, it was not be possible to make inferences about these findings. It is known that SCCmec types I and II are associated with strains of hospital origin, characterized by multiple antibiotic resistance besides to the betalactams, such as macrolides, aminoglycosides, tetracyclines, quinolones and rifampicin (Andrade- Figueiredo & Leal-Balbino, 2016).

Conclusion
The study thus suggests that Staphylococcus strains are drug resistant, virulent, and diverse irrespective of sources and place of isolation. These findings necessitate the continuous surveillance of multidrug-resistant and virulent S. aureus and monitoring of the transmission of infection. The antimicrobial monitoring is essential for the treatment of infections in each hospital.