Resistance Profile From Staphylococcus Aureus And Pseudomonas Aeruginosa Obtained From Tracheostomized Children

The tracheostomized patients exhibit high risks of bacterial infections, because the tracheal tube acts as a gateway to these microorganisms. The objective was to characterize microbiologically the tracheal secretion of tracheostomized children, to evaluate the biofilm formation, and to study the phenotypic and molecular profile of antimicrobial resistance of Staphylococcus aureus and Pseudomonas aeruginosa isolated. The study collected 88 tracheal secretion samples. The material processed by phenotypic tests were performed for bacterial identification. For identification of the biofilm, the Congo red agar test and the plaque microtiter test were used, and the qPCR method was used to resistance verification. Were obtained 27 samples of S. aureus and 71 of P. aeruginosa . All S. aureus samples were positive for biofilm formation on Congo red agar test. In antibiogram test, S. aureus showed resistance to seven drugs. Regarding the identification of resistance genes, were amplificated bla Z in 42.8% from S. aureus and mec A in 28.6% of them. Pseudomonas aeruginosa presented resistance to eight drugs. The most frequent chromosomal genes were bla OXA with 66.7% and bla KPC with 58.3%. To plasmidial DNA, was highlighted bla NDM with 58.3% positive. on cefepime IMP; ceftazidime with bla bla SHV and bla IMP; imipenem bla SHV, bla CMY and bla TEM p-value The results obtained from the phenotypic antibiogram for the


Introduction
Tracheostomy consists of a surgical opening in the anterior tracheal wall, into which a tracheal cannula is inserted. It's indicated in respiratory obstruction cases, subglottic stenosis, tracheomalacia and craniofacial syndromes, as well as providing access to the lower respiratory tract in cases as neurological and neuromuscular diseases 1 .
Due the lack of protection as filtration, humidification and air heating, performed by the upper respiratory tract, tracheostomized patients exhibit high risks of virus and bacterial infections, because the tracheal tube acts as a gateway to these microorganisms 2,3,4 .
The contamination and the consequent lower respiratory tract colonization are facilitated, and the main bacteria found were Pseudomonas aeruginosa and Staphylococcus aureus 5,6 . Both microorganisms exhibit high resistance rates to β-lactams, the most commonly group used in medical routine 7 .
In addition, bacteria that are frequently found in tracheostomy tubes are biofilm-forming, such as S. aureus often associated with lower respiratory tract infections 1,3,8,9,10 .
Besides, others resistance factors dissemination can be attributed to the genetic mutational processes and genetic material exchange, because of selective environmental pressure and multiresistant clone's proliferation 10 . Through is possible the plasmids transport resistant genes, that codes antibacterial information's, becoming the bacteria multiresistant. The mechanisms that become bacteria resistant are decreased permeability of both wall and cytoplasmic membrane; target site antibiotics alteration; antibiotic expulsion by efflux pumps and β-lactamases enzymes production that inactivate the antibiotic. These mechanisms come from mutational processes and/or genetic material exchange 11,12 .
The indiscriminate antimicrobials use can lead to individual's normal microbiota alteration, which increases the risks of acquisition and colonization of resistant bacteria, as may select resistant microorganisms that exist in the airways. This may origins an increase in costs to the patient because of increased length of stay in hospitals, besides the prevention and treatment difficult 13 .
To prevent possible respiratory infections, tracheostomized patients should exchange tracheostomy tube once a month, but the non-availability of the cannula by the Brazilian Eighty-eight tracheal secretion samples were collected from 11 tracheostomized children, with age between zero months to eighteen years old, that using the tracheostomy cannula to ventilatory support and were not infected or with symptoms like fever, increased discharge, productive cough or any other unusual symptoms. Samples were collected prior to cannula removal and were taken twice each season, during the time of cannula replacement, which was performed between 30 and 45 days, according to the protocol.
The samples were collected from May 2017 to October 2018, the first being performed in autumn and the others in the following order: winter, spring and summer.
To collect of the secretion, the contents were aspirated with a syringe containing physiological solution, coupled to a probe, which was introduced into the tracheostoma, and the physiological solution was injected into it. The aspirated contents were deposited in a sterile vial and immediately sent for processing in the laboratory.

Microbiological analysis
Samples were sown in salty mannitol, MacConkey, chocolate and blood culture media.
Cultures were incubated for 24 hours at 37 °C under aerobic conditions. After this period, the morphocolonial and morphotintorial characterization was performed, as well as biochemical tests to identify isolated colonies, according to Procop and collaborators 14 and the ANVISA manual 15 .
Biofilm production was analyzed according to FREEMAN et al 16  For S. aureus, clindamycin resistance induction test, called D test, was performed to detected resistance to macrolides, lincosamines and streptogramins. Was also evaluated the oxacillin resistance mecA gene mediated. To evaluate the activity of β-lactamase resistance mediated by blaZ gene, was considered the penicillin halo measurement.

Resistance genes amplification
The samples with positive resistance, phenotypic tests were used to plasmid and chromosomal DNA extraction, according Pharmacia ® Flexiprep extraction kit manual.
For S. aureus, specific primers blaZ and mecA genes were designed for one of the authors.
Was also used primer for femA gene to confirm the S. aureus phenotypic identification, according to store sequences in GenBank 20,21 .

Statistical analysis
For percentage analysis and graphs, Microsoft Excel (Microsoft Corp., Redmond, WA, USA) was used. To associate the data obtained in the antibiogram test for phenotypic resistance and susceptibility, and the presence or absence of the resistance genes observed along with the seasons of the year, the MATLAB script (version 8.1, Natwick, USA) was applied using the test Chi-square.

Results
Among the 11 participants, a total of 88 tracheal secretion samples were obtained and  (Table 1). Enterobacter cloacae Providencia spp. Analyzing the number and diversity of bacterial species with respect to seasonality, it can be observed that in summer 25 different species of bacteria were isolated, followed by autumn and spring with 24 species and 18 species in winter. As for the number of isolated bacteria, autumn and spring were the seasons with the highest number of isolates, followed by winter and summer ( Figure 1). Observing the diversity individually, it is noteworthy that in three patients 27, 28 the isolated microbiota varied in all seasons in an equalized manner, which shows that the seasons may not have interference in the microbiota diversity (Table 2).  For the biofilm production tests, we chose the species of clinical importance that were most prevalent in the patients and were present in all seasons of the year. Thus, biofilm production was evaluated in 12 isolates of S. aureus.
Despite Corynebacterium spp. was the most prevalent specie, the phenotypic test was not performed to verify biofilm production, as this bacterium is not commonly associated with respiratory infections. Regarding the CNS, although they were also prevalent, when evaluated separately, they were not isolated in all seasons of the year.
To the experiments using Congo Red Agar, only isolates of S. aureus were tested, since the test does not show effectiveness for P. aeruginosa samples, including the positive control strain, known as biofilm-forming. All isolates of S. aureus (12) were positive in Congo red agar, and therefore all biofilm-forming.
Regarding the resistant genes amplification in the S. aureus tested samples, 42.8% (3/7) isolates containing blaZ in plasmid and chromosomal DNA was identified. The mecA gene was amplified in 28.6% (2/7) isolates. These genes were identified in 85.7% (6/7) different isolates. The femA gene was amplified in 100% of the samples (Table 3). Table 3  In the data obtained for P. aeruginosa, we can highlight resistance of 23.9% (17/71) for aztreonam and 12.7% (9/71) for imipenem ( Figure 4). In the phenotypic test to evaluate the β-lactamase activity, all were negative to ESβL, metallo-β-lactamase and carbapenemase.
Based on the resistance found in P. aeruginosa antibiogram test, the literature search showed 12 genes responsible for β-lactams resistance (dates not shown).

Discussion
The surgical opening in tracheostomized patients for the device installation causes a breakdown of the skin barrier and causes these microorganisms to become pathogenic 1, 5 .
To confirm the S. aureus specie identified, femA gene was used and was found in 100% of the samples. The phenotypic resistance to penicillin may be confirmed with the blaZ gene amplification, even on chromosomal DNA, that plasmidial DNA. The resistance acts in other types of penicillins as amoxicillin, ampicillin and piperacillin. However, stable penicillins such as oxacillin and methicillin, cephalosporins, β-lactamase inhibitors and carbapenems are not included 30,31 .
The methicillin resistance mediated by mecA gene can be phenotypically identified by cefoxitin, as identified in this study. This gene is responsible for the synthesis of a modified penicillin binding protein (PBP2a), which interferes with the formation of the bacterial cell wall, preventing its complete structuring 32 .
In SCCmec it's also possible to find resistance genes for macrolides, quinolones and lincosamines, which have antimicrobial resistance to CIP, ERI, CLI, GEN, among others, increasing the resistance to the group of macrolides, lincosamines and streptogramins, one of the most used in the treatment of staphylococcal infections. However, resistance to these antimicrobials has already been pointed out due to the constant vancomycin use and the easy acquisition of plasmids containing encoded genes to citoplasmatic membrane proteins that acts as efflux pump related to tetracycline 12,33,34 .
Inducible resistance to clindamycin was found in D test in 51.8% (14/27) of isolates, which limits its effectiveness as a treatment option in MRSA infections. Therefore, the application of D test in the laboratorial routine is useful to investigated possible clindamycin resistance that helps the clinician in the effective use of clindamycin when this was a therapeutic option 35 .
For P. aeruginosa, in the present study, phenotypic resistance were identified to the antibiotics ATM, IPM, CAZ, PPT, CPM, CIP and GEN, similar to Pires et al. 36  found is due the β-lactamases enzymes production and due the modification of the cell wall outer membrane permeability through the loss or reduction of porins, or by overexpression of efflux pumps present in the plasmatic membrane 37 .
In the P. aeruginosa samples, the most prevalent resistance genes encoding β-lactamase enzymes were blaOXA, blaKPC, blaVIM, blaCMY, blaTEM e blaSHV. The first two encode carbapenemases enzymes, blaVIM encodes metallo-β-lactamases, blaCMY ampicillins and the last two ESβL enzymes 37,38,39 . The "D" and "H" samples stood out due to the great genetic variation and according to previous bibliographic research, we can observe the variety of antimicrobials that are inactivated when there is the expression of the mentioned enzymes (Table 5).  Analyzing the amplitude of each enzyme resistance, the samples can be considered multiresistant 40 , because the genes simultaneous expression is capable of inactivate the action of AMC and PPT antimicrobials belonging to the class of β-lactams, from first generation of cephalosporins to fourth generation 32 .
The antibiogram tests results didn't have phenotypical resistance to the tested antibiotics.
This divergence occurs, because only the presence of the gene responsible for antimicrobial resistance isn't automatically linked to its expression 41 .
The antimicrobials β-lactams are the most used in bacterial respiratory tract infections, because they are highly effective and low toxicity 42 . Bellés et al. 43 identified that of the 160 patients studied (adults and children), 80 did antibiotic therapy with β-lactams, which in 16 of these was identified metallo-β-lactamase enzyme activity. In addition, national epidemiological studies evaluated 3728 gram positive and gram negative isolates, and P.
aeruginosa was responsible for 496 (13.3%) cases, being the third pathogen most frequent that presented 30.2% of IPM resistance 44 .
Carbapenems are the most important antibiotics in the treatment of multiresistant P.
aeruginosa infections. The resistance granted to this class makes treatment difficult 11,45 .
Regarding seasonality, there was no significant variation in the diversity of the microbiota throughout the year. This finding was similar to that described by Perez-Losada et al. 46 , Who also did not observe a difference in the tracheal secretion microbiota during the seasons.
It was expected to find changes in the microbiota mainly in winter, but the absence of this variation can be explained by the location of the tube, found in the trachea whose colonization differs when compared to the respiratory tract infections, where there is in fact a microbiological diversity that can be influenced by seasonality, due to greater contact with the external environment, as well as the presence of anatomical structures that contribute to microbiological control 47,48 .
The data from this study confirm that there is a relationship of infections occurring in lower respiratory tract of tracheostomized patients, with resistant bacteria. The cannula microbiota is influenced by the care of device hygiene and its permanence, when used for a long period, can lead to tracheal mucosa inflammation, increasing the risks of infections 1, 47, 49 .
Taechowisan et al. 50 verified the relationship between the phenotypic and genotypic resistance and found that those based on PCR did not completely correlate with the phenotypic resistance, which was also found in this study of bacterial isolates from tracheostomized children. Mohaman and Menon 51 also find coexistence of bla genes for metallo-β-lactamases in P. aeruginosa.
Researchers suggest the importance of changing the device regularly, since the patients in the present study performed this change once a month, and even so, a diversity of microorganisms was found, as well as biofilm-forming bacteria. Thus, if we consider patients who do not change the cannula frequently, they will probably present an intense and prolonged colonization, making the device a reservoir of bacteria, with the formation of persistent biofilm 52 .
In Brazil, tracheostomy tubes are not available free of charge by the Public Health System, making patients pay with the purchase of the device and often do not have the financial resources to do so, which makes monthly changes difficult 52 . In addition, the chances of biofilm adherence increase, resulting in the formation of granulation tissue, recurrent infections and failure in laryngotracheal reconstruction 53 . Due to the increased survival, tracheostomy has become a constant practice for those children who need mechanical ventilation, but the initial management is with endotracheal intubation. However, this intubation for long periods brings several problems to the patient, such as mucosal ulcers and ischemia of the larynx or trachea, which justifies the commonly indicated tracheostomy 58 .
The knowledge of the predominant microbiota in lower respiratory tract infections as a result of device use in tracheostomized patients, as well as the resistance profile of the most antimicrobial used, will help health professionals regarding the most appropriate therapy to be given children with traqueostomy, especially in the postoperative period, increasing the chances of airway restoration and decannulation, since local hygiene is not always performed as recommended 27 .

Conclusion
The S. aureus phenotypic tests identified resistance to β-lactams groups, macrolides, lincosamines, aminoglicosides and tetracyclines. In P. aeruginosa, the phenotypic tests identified resistence to all antibiotics tested.

Declarations
Ethics approval and consent to participate    Resistance genes amplification in Pseudomonas aeruginosa samples isolated from tracheostomized pediatric patients.