Bacterial Isolates from Endotracheal Aspirates and their Antimicrobial Resistance Pattern in Patients from Intensive Care Unit

ANDREEA LOREDANA GOLLI1, FLOAREA MIMI NITU2*, MARIA BALASOIU3, ROXANA MARIA NEMES4, SORIN IOAN TUDORACHE5, BEATRICE MAHLER BOCA6, MIHAI OLTEANU2 1University of Medicine and Pharmacy Craiova, Department of Public Health, 2 Petru Rares Str, 200349, Craiova, Romania 2 University of Medicine and Pharmacy Craiova, Department of Pneumology, 2 Petru Rares Str, 200349, Craiova, Romania 3 University of Medicine and Pharmacy Craiova, Laboratory Department, 2 Petru Rares Str, 200349, Craiova, Romania 4 Titu Maiorescu University, Faculty of Medicine, Department of Physiopathology, 22 Dambovnicului Str., 031593, Bucharest, Romania 5 Titu Maiorescu University, Faculty of Medicine, Department of Anatomy, 22 Dambovnicului Str., 031593, Bucharest, Romania 6 Carol Davila University, Faculty of Medicine, Department of Pneumology, 8 Eroii Sanitari Blvd., 020021, Bucharest, Romania

Antimicrobial resistance is a major world-wide problem in the intensive care unit (ICU), including in Romania, the emergence and spread of antimicrobial resistance (AMR) being now considered a global public health threat [1].
Hospital acquired infections (HAIs) have been shown to occur about 5 to 10 times more in the patients admitted in ICUs, which are critically ill patients [2].
In the European Union, about 25 000 patients die each year from infections caused by selected multidrug-resistant bacteria and the associated costs are estimated at about 1.5 billion euros per year [3].
According to the European Antimicrobial Resistance Surveillance Network (EARS-Net), Improving Patient Safety in Europe (IPSE) and ECDC data, Romania is one of the South-Eastern European countries with one of the highest prevalence rates of MDR pathogens. [4,5] A worrying phenomenon in Romania is also the existence of the MDR-TB and XDR-TB cases in socio-economic conditions (malnutrition, agglomeration, stress), with the doubling of number of cases of XDR-TB in the last years [6,7,8], including the cases of extrapulmonary tuberculosis which originates from the hematogenous metastatic affects developed during the prime TB infection period [9]. Therefore, the present study was designed to identify the bacterial profile and determine the antimicrobial resistance pattern of tracheal isolates among patients admitted to the ICU.

Materials and methods
The research is a retrospective study, which includes * email: dr_nitumimi@yahoo.com, 0722491034 All authors have equally contributed to the paper and they should be regarded as main authors.
the determination of pathogens involved in respiratory infections of patients admitted to the intensive care unit (ICU) of Craiova Emergency Clinical County Hospital, Romania. Data were collected from January 2017 to December 2017 from the clinical pathology databases of the hospital, including culture sensitivity reports of the patients admitted to the ICU in the studied period. Samples included endotracheal tube aspirates from 734 patients of ICU. There were included in the study only those samples which were positive by culture. The identification of the isolated strains on the clinical specimens received from ICU patients was carried out in the Hospital's Laboratory of Microbiology. The analyse of the resistance patterns for the action of the appropriate antibiotics was performed using Vitek 2 Compact system and diffusion method.
Information about gender and age of the patients, site of infection and antimicrobial resistance pattern were collected from Hospital's Information System and from the available hospital records.
Data were entered and analysed using Microsoft Excel. Continuous variables like age are expressed as mean±STDEV. The pattern of micro-organisms and gender were analysed and expressed as percentages. The χ2 test was used for count data, and p<0.05 meant the difference was statistically significant.

Results and discussions
From January to December 2017, there were analysed specimens of endotracheal aspirates collected from 734 patients hospitalized in ICU. The mean age of the patients was 59.49±22.39 years, 301 women (41.01%) and 433 men (58.99%). Distribution of subjects by age group reflects the largest proportion of patients over 65 years (52.45%) (table 1).
It is a retrospective study and the patients signed the informed consent for analysis and treatment.
A total of 985 bacterial isolates were obtained, excluding cases where it was more than one isolate of the same pathogen from the same patient. Among these, 712 isolates (72.28%) were Gram negative and 273 isolates (27.71%) were Gram positive bacteria.
Referring to the total number of samples collected by gender, isolation rates indicates a higher value for male patients, especially for NFB, E.coli, Klebsiella, Acinetobacter spp. Pseudomonas aeruginosa, CoNS -Coagulase-negative staphylococci (table 2).  In terms of germ distribution by age group, there was a larger number in the case of elderly patients (over 65 years), the difference being statistically significant (p<0.001) (table 3). The highest percentage was registered for CoNS (70.40%), followed by Pseudomonas aeruginosa (63.15%) and NFB (59.47%).
42.10% of the Klebsiella spp. strains were isolated in patients over 65 years, with 13 cases being recorded in children ≤18 years. Only 2 Acinetobacter strains were isolated in children (0-18 years), 6 of Pseudomonas aeruginosa and 10 of MRSA.
In our study we have analysed the percentage of multidrug-resistant (MDR) strains among the endotracheal aspirates from ICU patients, by taking into consideration resistance to at least three different antibiotic groups: aminoglycosides, cephalosporins, carbapenems, tetracyclines and fluoroquinolones. 85.88% from the Acinetobacter spp. strains were MDR. High rates of MDR were found for Klebsiella spp. (70.04%) and Pseudomonas aeruginosa (66.25%) while almost all of the isolated NFB strains were MDR (98.82%).
The antibiotic resistance rates of the isolates are summarized in tables 4-6. The combined resistance to multiple antimicrobial groups observed for Klebsiella spp. is consistent with European Centre for Disease Prevention and control (ECDC). The majority of infections caused by K. pneumoniae are healthcare-associated and the most common resistance phenotype was combined resistance to three key antimicrobial groups: fluoroquinolones, thirdgeneration cephalosporins and aminoglycosides [11].
Around 85% from the Klebsiella spp. strains isolated in our study were resistant to first generation cephalosporins, around 60% to second-generation, over 70% to thirdgeneration and over 65% to fourth-generation cephalosporins (table 4). Over 50% of the Klebsiella spp. strains were resistant to meropenem and ertapenem, and almost a third to imipenem, consistent to CDC analysis which places Romania between the three countries with the highest carbapenems resistance [11]. Also 45% of the strains were resistant to ciprofloxacin and third to amoxicillin/clavulanic acid. Around 30% of E. coli isolates were resistant to amoxicillin/clavulanic acid and meropenem and over 60% to first-generation cephalosporins.
In our research were identified 35.07% MDR MRSA strains in the Gram-positive group, in the conditions in which this pathogen has been the most important cause of antimicrobial-resistant healthcare-associated infections worldwide, with higher percentages in the southern and south-eastern parts of Europe [11].
Over 80% from the isolated strains of coagulase-negative staphylococci (CoNS) were resistant to tetracycline, clindamycin and oxacillin, and over 95% to penicillin (table 5) Linezolid was the most effective drug among Gram positive isolates.
A high resistance to the cephalosporins (between 48-78%), has been highlighted in the case of Pseudomonas aeruginosa strains. Half of the strains were resistant also to ciprofloxacin and over 60% to carbapenems (meropenem and ertapenem) (table 6).
A very high level of resistance was found for the tested strains of other nonfermenting Gram negative bacilli (other  According to our study, for the Acinetobacter strains it was found a high resistance to the carbapenems (77-80%) and to third and fourth-generation cephalosporins (cefotaxime -92.94%, ceftazidime -76.47%, cefepime -78.82%), and also to ciprofloxacin (81.17%).
Over 80% of the Proteus strains were resistant to ceftazidime, ceftriaxone and cefuroxime, 68.42% to cefepime and almost 60% to ciprofloxacin.
Our study included patients with respiratory infections hospitalized in ICU, including all age groups, with the aim of highlighting the MDR strains.
The research revealed that the most common isolated pathogen was Klebsiella spp. (23.04%), followed by NFB-Glucose-nonfermenting Gram-negative bacilli (17.25%) and MRSA (13.60%). Also in other researchers' studies Klebsiella ranks first among isolated germs [12], while a similar percentage was also highlighted for this pathogen, but occupying the second place in the hierarchy of the most frequent pathogens involved in respiratory infections of patients hospitalized in ICU, after Acinetobacter spp. [13]. After other researchers, Staphylococcus aureus was the commonest isolate [14].
Predominantly Gram-negative germs, found in our study, is consistent with the results of other researchers [13,15].
Although antibiotics are considered to be the most effective method of fighting against infections, their empirical, indiscriminate, prolonged, or incorrect usage contributes significantly to the selection of MDR strains [16,17]. Antimicrobial resistance (AMR) is a serious threat to public health and patient safety in Europe, leading to mounting healthcare costs, patient treatment failure, and deaths [11,18,19].
Our study revealed that almost two-thirds of the strains of Klebsiella spp. are MDR, in the conditions in which, according to the European Antimicrobial Resistance Surveillance Network (EARS-Net), more than one third (34.5%) of the Klebsiella pneumoniae isolates reported in 2016 were resistant to at least one of the antimicrobial groups under regular surveillance (fluoroquinolones, third generation cephalosporins, aminoglycosides and carbapenems). In a research conducted by Radji et al [20], K. pneumoniae was also found to be multidrug resistant to the third generation cephalosporins and quinolone antibiotics. An increasing carbapenem resistance rate for Klebsiella, Acinetobacter and Pseudomonas was reported in their study by Akter et al [21].
A small percentage of E. coli strains have been carbapenem resistant, consistent with EARS-Net. The results are consistent with analyses from the European Centre for Disease Prevention and Control [11].
One-third of MRSA strains have been MDR, while both Coagulase-negative Staphylococci (CoNS) and MRSA showed resistance to penicillin in almost all patients, consistent also with other findings [22]. MRSA remains a major cause of healthcare-associated infections worldwide, with higher percentages in the southern and south-eastern parts of Europe [23].
Our study revealed more than half of the Pseudomonas strains resistant to carbapenems and fluoroquinolones, outcomes consistent with other findings [2,24,25], and also to third and fourth-generation cephalosporins, despite the fact that there were significant decreases of the mean percentages for fluoroquinolone resistance, aminoglycoside resistance and carbapenem resistance during the period 2013 to 2016 [23], In our study it was found also a very high level of resistance of Acinetobacter strains to the carbapenems and cephalosporins, which is consistent with the results from other studies [2,26,27]. 85.88% from the Acinetobacter spp. strains were MDR, which draws again attention to the fact that antimicrobial resistant Acinetobacter spp is a public health concern due to the severe limitation of treatment and infection control options [23]. Our study has identified a very high percentage of NFB strains resistant to all generations of cephalosporins, piperacillin/tazobactam, carbapenems, almost all strains being MDR. A worrying phenomenon in Romania is also the existence of the MDR-TB and XDR-TB cases in socioeconomic conditions (malnutrition, agglomeration, stress), with the doubling of number of cases of XDR-TB in the last years, including the cases of extrapulmonary tuberculosis which originates from the hematogenous metastatic affects developed during the prime TB infection period [28][29][30] All these observations are in line with the results of other studies we have conducted to identify germs involved in the production of infections in patients hospitalized in ICU. [31,32].

Conclusions
The study revealed that lower respiratory tract infections in ICU hospitalized patients are increasing and the majority of isolates belong to MDR category. Regardless of the age category, males were more affected by Klebsiella, NFB, Acinetobacter, Pseudomonas, MRSA in terms of bacterial isolates, compared with females.
To prevent the proliferation of MDR strains, a strict hospital infection control policy and the surveillance of antibiotic prescription are necessary.