Evaluation of the Activities of Antimicrobial Agents on Multi-drug Resistant Gram Positive Bacteria Isolated from Intensive Care Units

highest rates of nosocomial infections are observed in intensive care units (ICUs), which are also the units in which the most critically ill patients are treated and the highest mortality rates are detected. The hospital-acquired infections that are seen in ICUs comprise almost 20-25% of total nosocomial infections. Antimicrobial treatment and invasive procedures, applied in ICUs, and also the immunocompromised status make patients sensitive against infections [2].

highest rates of nosocomial infections are observed in intensive care units (ICUs), which are also the units in which the most critically ill patients are treated and the highest mortality rates are detected.The hospital-acquired infections that are seen in ICUs comprise almost 20-25% of total nosocomial infections.Antimicrobial treatment and invasive procedures, applied in ICUs, and also the immunocompromised status make patients sensitive against infections [2].
Antimicrobial agents have had crucial effects on the nosocomial infections.Approximately 25 to 35% of hospitalized patients take systemic antibiotics [3].However, it has become absolutely clear that the major hospital-acquired pathogens either are inherently resistant to clinically useful antimicrobial agents or possess the ability to acquire resistance.Every major class of bacterial pathogens has demonstrated an ability to develop resistance to one or more commonly used antimicrobial agents [4].The usage of antimicrobial agents tends to create selective pressure that promotes the emergence of resistant organisms and predisposes patients to colonization with such organisms.
Gram-positive bacteria are the second most common cause of nosocomial infections with Staphylococcus aureus being the predominant pathogen.There has been an increase in the rate of antibiotic resistant bacteria associated with nosocomial infections in the ICU [5].Bacteria develop resistance when they acquire new genetic material [6,7].The genetic material that encodes resistance is transferred to other strains.Methicillinresistant S. aureus (MRSA) causes up to 60% of nosocomial infection in the ICU [8].A broad-spectrum antibiotic such as Vancomycin is usually prescribed for treatment of MRSA.However, Vancomycin-resistant Enterococci and isolated cases of vancomycin-resistant S. aureus have been reported [9].Therefore our objective is to evaluate MICs of the new and more efficent antibiotics which can be used for treatment of Gram positive multi-drug resistant microorganisms.

Abstract
Multi-drug resistant Gram-positive bacteria are responsible for nosocomial infections.It is important to show the minimal inhibitory concentrations and new antibiotic resistance profiles of these bacteria to antimicrobials which are commonly used in treatment.The aim was to evaluate the new and more efficient antibiotics minimal inhibitory concentrations which can be used for treatment of Gram positive multiple drug resistant microorganisms.
In this study, 337 Gram positive bacterial strains were isolated from clinical samples of 192 patients who were hospitalized at intensive care units.Minimum Inhibitory Concentration (MIC) values of antibiotics were investigated in 72 Gram positive bacterial strain.Broth macrodilution and disc diffusion methods were performed to evaluate the activities of antibiotics against Gram positive bacteria.
All the 72 Gram positive bacterial strains including 38 coagulase negative Staphylococci, 18 Staphylococcus aureus and 16 Enterococci strains were sensitive to tigecycline, linezolid and daptomycin.All coagulase negative staphylococci and Staphylococcus aureus strains were sensitive to vancomycin and teicoplanin.Additionally, 2 Enterococci strains were resistant to vancomycin, while 1 strain was resistant and 1 strain was intermediately sensitive to teicoplanin.
Vancomycin, teicoplanin, tigecycline, linezolid and daptomycin might be useful and good alternative for the treatment of multi-drug resistant bacterial infections.Different antibiotic resistance profiles and the MIC's of antibiotics should be searched for the treatment of intensive care unit patients.

Introduction
Evaluation of the Activities of Some Antimicrobial Agents on Multi-drug Resistant Gram Positive Bacteria Isolated from Intensive Care Units.
One of the most important issues encountered in the infectious disease practice are hospital-acquired infections also referred to as 'nosocomial infections' [1].These infections can be described as those occurring within 48 hours of hospital admission, 3 days of following discharge or 30 days after an operation.The

Microbial identification
The samples obtained from the clinics were cultured onto 5% sheep blood agar, Eosin Methylene Blue (EMB) agar and chocolate agar in incubator at 37 °C in aerobic conditions.After 24 hours incubation, Gram staining method was performed on bacterial colonies and preparations were examined under the microscope.Catalase, coagulase and L-pyrrolidonyl arylamidase (PYR) tests were performed to identify Gram positive bacteria.After these procedures, the bacterial strains were identified by using an automated microorganism identification system (Vitek 2 compact biomerieux) to confirm the manual procedures.At the end of these tests; Staphylococcus aureus, Enterococcus spp.and coagulase-negative Staphylococci (CNS) strains were detected.

Antibacterial activity assays
The antibacterial activity was tested by using agar disc diffusion technique to determine the diameter of growth inhibition zones while broth macrodilution method was used to determine the Minimum Inhibitory Concentration (MIC).
The disc diffusion method was employed for the determination of antimicrobial activities [11].Briefly, bacterial strains were grown overnight in Mueller-Hilton broth at an incubation temperature of 36.5°C.The bacterial concentration was standardized to 3×10 8 CFU ml -1 using the Mc Farland scale.Antimicrobial activity was evaluated by measuring the diameters of inhibition zones.The bacterial strains and the antibiotics (Bioanalyse, Ankara, Turkey) used for disc diffusion method is shown in Table 1.
For the broth macrodilution assay [12], culture broth was placed into five 5ml sterile tubes using the following concentrations; 1.tube: 1:1, 2.tube: 1:2, 3.tube: 1:4, 4.tube: 1:6 and final tube: 1:8 cfu/ml and then 1x10 6 CFU/ml of the bacterial colonies isolated from clinical samples were equally added to all tube series.The bacterial standardized inoculum (30 μl) was poured into the tubes, homogenized and incubated at 36.5°C for 24 hours.Samples taken from the tubes were inoculated into petri dishes containing Brain-Heart Infusion Broth to verify bacterial growth.Inhibition of bacterial growth was visible as a clear broth and the presence of growth was detected by the presence of turbidity.The lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism was evaluated as the MIC value.MIC 50 is the minimal inhibitory concentration that inhibited 50% of bacterial strains, while MIC 90 is the minimal inhibitor concentration that inhibited 90% of them.The bacterial strains tested, antibiotics used and MIC testing ranges are shown in Table 2.

Results
Out of 72 Gram positive bacteria, 38 (53%) CNS, 18 (25%) S. aureus and 16 (22%) Enterococcus spp were identified as hospital-acquired agents.About 70% of these pathogens were isolated from intensive care units, while the remaining isolates were obtained from the burn intensive care unit (26%) and organ transplantation unit (4%).Sample distribution of Gram positive bacteria isolated from ICU are shown Table 3.
According to the antibiotic susceptibility test results, 86.8% of CNS (Staphylococcus saprophyticus and Stapylococcus epidermidis) strains were resistant to methicillin (MRCNS).The most sensitive antibiotics were rifampin, chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole for MRCNS and MSCNS strains.On the other hand, 77.7% of S. aureus strains were resistant to methicillin (MRSA).The most sensitive antibiotics were rifampin and trimethoprim-sulfamethoxazole for MRSA and MSSA strains.All MRSA and MSSA strains were evaluated as sensitive for vancomycin, teicoplanin, tigecycline, linezolid and daptomycin by macrodilution broth method.MIC 50 and MIC 90 values can be found in Table 4 for MRCNS, MSCNS, MSSA, MRSA and Enterococcus spp.
All bacterial strains (n:72) were sensitive to Tigecycline, Linezolid and Daptomycin antibiotics.However, 2 Enterococcus strains were resistant to vancomycin.The most effective antibiotic was daptomicin according to the MIC 90 value.The antibiotic sensitivity rates of all bacterial strains are shown in Table 5.

Discussion
ICUs are the most important units when compared with the

Streptococcus spp
Testing ranges (μg/ml) 0.12-4 1-32 0.12-4 0.5-16 0.12-4 Sensitivity (μg/ml) other units of the hospital regarding hospital-acquired infections and resistant bacterial strains.The hospital-acquired infections detected in these units spread quickly [13].The description of nosocomial infections in the ICU is necessary to determine the epidemiologic properties and treatment approaches.Additionally, to determine the bacterial agents and antibiotic susceptibilities is really important by the way of reducing the rates of morbidity and mortality [14].62% of the positive isolates were Gram negative bacteria, 47% were Gram positive bacteria and 19% were fungus respectively at one of the big clinical study named as "European Prevalence of Infection in Intensive Care (EPIC II)" that was performed in 75 countries and 1265 ICUs.These data pointed out that Gram negative bacteria generally were isolated from ICUs [15].
S. aureus and Enterococcus strains are the most isolated microorganism respectively when looking at the distribution of Gram positive bacteria isolated from ICUs.The data obtained from our study were in the same line with the current literature in the terms of isolation rates of bacterial strains.S. aureus, CNS and Enterococcus spp.were isolated from the clinical samples and most of these bacteria were isolated from blood samples of the patients who were hospitalized in ICUs [16,17].
Most of the bacteria isolated from ICUs such as Acinetobacter spp., Klebsiella spp.(for Gram negative) and MRSA, MRCNS, Enterococcus spp.(for fram positive) were resistant to most antimicrobial agents especially colistin and tigecycline all over the world.The extensive use of broad-spectrum antibiotics can cause the selection of multidrug-resistant bacteria in the infections seen in ICU patients.Additionally, the general situaiton of ICU patients, the presence of underlying disease, the length of hospital stay, endogenous and resistant bacteria carriage of inpatients and application of more surgical procedures are some reasons of increasing number of resistant bacteria [18].
In the light of literature reviews, methicillin resistance is seen up to 80% in especially S. aureus and CNS in Turkey.It was reported that these 2 bacterial groups were resistant to methicillin at the rate of 60-100% [19].Some Staphylococcus strains that showed intrinsic heterogenous resistance were often resistant to non-beta-lactam antibiotics (erythromycin, clindamycin, tetracycline, sulfonamides, fluoroquinolones and aminoglycosides).This situation can cause serious problems especially in staphylococcus-dependent nosocomial infections.As shown in some studies that methicillin resistant staphylococcus strains have increased resistance to most antibiotic groups such as aminoglycosides, tetracyclines, lincosamides, glycopeptides and quinolones.This situation raises the difficulty in treating multi-drug-resistant Staphylococci [20,21].
On the other hand, Enterococcus strains are reported the second most common agent after S. aureus for nosocomial infections.The observation of the intrinsic and acquired resistance to most of the antibiotics in Enterococci may lead to critical problems in the terms of Enterococci isolation frequency which is increasing day to day [22].
The increased number of multidrug-resistant bacterial isolation frequency in the units that specific patient groups such as ICU showed that antimicrobial agents which are durable for resistance mechanisms should be used in clinics.It has been seen that there were no resistance to any Stapylococci strains in terms of glycopeptides antibiotics, when the literature review was performed in the similar studies [23][24][25].
Consequently, there is an increased number of antibiotic resistance and Gram positive bacteria isolation from ICUs as nosocomial infection agents in our country and all around the world.It would be beneficial for all hospitals to search for the properties of agents and their known susceptibility patterns and wait for the results of microbiological culture and antibiotic susceptibility test before starting the treatment.These multidrug resistant Gram positive bacteria particularly restrict the treatment options.Methicillin resistance strains for example MRSA and MRCNS were also showed high-resistance to other antibiotic such as teicoplanin, tigecyclin and linezolid as well as methicillin.For this reason, we must use glycopeptides on these infections however, it has to be known that this glycopeptides usage may increase vancomycin and teicoplanin resistances which have non-dangerous levels for today on Enterococci and Staphylococci.Vancomycin and teicoplanin resistance for these two bacteria should be continuously monitored.As well as glycopeptides, it is seen as a good treatment alternative to use tigecycline, daptomycin and linezolid which are sensitive against all Gram positive cocci because of their efficiency on low minimal inhibitor concentration values.

Table 1 :
The bacterial strains and the antibiotics used for disc diffusion method.

Table 2 :
The bacterial strains and the antibiotics used for broth macrodilution method.

Table 3 :
Clinical sample numbers of gram positive bacteria obtained from patients in the ICU.

Table 4 :
MIC50and MIC 90 values of gram positive bacteria isolated from ICUs.

Table 5 :
The antibiotic sensitivity rates of all bacterial strains.