Prevalence of Antibiotics Resistance in the Isolated Bacteria from Bronchial Washing Fluids in Ramadi Teaching Hospital, Iraq

The study proved defining the resistance of bacterial pathogens taken from the inferior respirational region from patients who were admitted to Ramadi Teaching Hospital. 76 samples of tracheal discharges were extracted. The highest percentage of bacterial isolate of males was Streptococcus pneumonia which about 26.2% for males. Generally, the lowest percentage is 4.8% for Staphylococcus spp. and Bordetella spp. The lowest percentage of females was 2% in the previous two genera. The outcome of the culture was determined by p value, the outcomes revealed that the growth of a pathogen with patients was equal to 0.782 for the Klebsiella pneumonia and the low p value appeared in the results of the Acinetobacter sp. strain level to 0. The confrontation of the antimicrobial is determined by the rate of MICs. The resistance appeared MIC ≥ 64 for Pseudomonas aeruginosa against antimicrobial Ciftazidim and resistance of Bordetella spp. showed MIC ≤ 0.5 against antimicrobial Colistin.


INTRODUCTION
Many pulmonologists analyze bronchial washing fluids to evaluate the etiology of hemoptysis by microbiological and cytological examinations (1). The bronchial washing samples are obtained for isolation bacteria by clinicians (2). Some studies exhibited rate of high drug resistant because of inadequate or noncompletion antibiotic treatment and infection control practices. These negative statuses may make recurrence the bacterial pulmonary infections which lead to developing the drug resistance (3).
The counts of the collected bacteria from lower respiratory tract infections in most of patients reached ≥ 10 3 cfu/ml in Alabama, USA in the period 1978-1981 (4). Patients aged 51-60 years old had 19.7% of Streptococcus pneumoniae isolates compared as other age groups that related with Lower respiratory tract infections. This bacteria was resistant 1100% to Erythromycin (5).
However, bronchial washing is a useful method to identify multi-resistant microorganisms when patients with bronchiectasis (2). Vitek 2 is a widely used commercial antimicrobial susceptibility testing system (6). The identification of the microbes using VITEK 2 present in the transtracheal washes is the best way to make the diagnosis (7). The VITEK 2 is an accuracy automated system to identify microbes and to test the susceptibility of antibiotics (7,8). Antimicrobial resistance may be caused by some random mutations in chromosomal DNA, or by transferring genes from resistant bacteria (9) or caused by transferring ribosomal DNA (10). Some recent studies have investigated emergence of antimicrobial resistance (AMR) in Iraq (11)(12)(13).
Therefore, a comprehensive investigation is required to uncover the scope of multi-drug resistance (MDR) problem among local population in Iraq. The current study aims to evaluate types of bacterial pathogens associated with Bronchitis patients, and to determine their antimicrobial susceptibility patterns using VITEK 2.

Collection and Isolation of Bronchial washing samples bacteria
This Laboratory-based reviewing investigation of Bronchial washing samples cultures and susceptibility tests were achieved at the Bacteriology Laboratory, in Department of Microbiology, Ramadi Teaching Hospital (TRH) within a period of one year between July 2017 and June 2018. One isolate per patient was randomly collected from different Bronchial clinical specimens of patients. About 76 isolates of human pathogenic microbes has isolated from 42 males and 34 females. The samples have been collected as a part of the routine clinical management of patients entered in various divisions of TRH. The collected samples were achieved in sterile containers by physicians and transferred to the laboratory within 30 min after the collection.
The bacterial samples were processed following the standard microbiological procedures by centrifugation, inoculating the precipitate on MacConkey Agar, Chocolate Agar, and Blood Agar and incubated at 37 °C aerobically. For creating micro-aerophilic conditions for the fastidious bacterium, the agar plates were put in in the candle jar, which provided 5%-10% CO2 concentration. The plates were examined for the finding bacterial colonies after 18-20 hr of incubation. Plates which did not exhibit any growth were further incubated for an additional 24 hr.

Identification of bacteria and Sensitivity test using VITEK 2 System
Organisms were identified by standard microbiological methods including morphology of colonies, staining, serological and biochemical tests. They were declared using Vitek 2. Antibiotic sensitivity test was conducted on pure culture isolates employing the VITEK system-2 Susceptibility tests with the Vitek 2 (bioMérieux, Inc., Durham, NC) system were performed using software version 5.01 and cards of AST (antimicrobial susceptibility testing) according to the manufacturer's instructions. Each isolate was tested concurrently with both methods using isolated colonies from a single 18 to 24 hr blood agar plate. Purity plates were prepared following inoculation of each test by sub-culturing an aliquot of inoculum suspension onto blood agar plates and incubating for 18-24 hr (6).

Statistical analysis
The statistical analysis was performed by using the Pearson Chi-square test and the analysis of variance (ANOVA). Data were analyzed using SPSS 17.0 (USA). Furthermore, the differences were significant at p< 0.01. The study was approved by Local Ethics committee in University of Anbar. Informed consent/assent was obtained from the patients (42 males and 34 females) in Teaching Ramadi Hospital in Ramadi. Table 1, about 76 isolates of human pathogenic microbes has isolated from 42 males and 34 females in Teaching Ramadi Hospital in Ramadi, Iraq from July 2017 and June 2018. About 8 genera have identified using the basic general methods. They were declared using Vitek 2. They are Streptococcus pneumonia, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus spp., Bordetella spp., Acinetobacter sp. and Oligella ureolytica. As in Table 1, the higher percent was 26.2% Streptococcus pneumonia in males, followed by 21.4%, 14.3% and 11.9% for Pseudomonas aeruginosa, Klebsiella pneumonia and Proteus mirabilis respectively. Staphylococcus spp. and Bordetella spp. had the lower percent 4.8%. In females, Streptococcus pneumonia, Klebsiella pneumonia and Bordetella spp. recorded the higher percent of pathogenic bacteria were 20.6% for each. However, the lower percent was recorded reached 2.9% for Oligella ureolytica. Acinetobacter sp. did not record any existence in the bronchial samples of female patients.

As in
Streptococcus pneumonia had higher percent reached 26.2% in males compared with 20.6% in females significantly (p<0.01). Oligella ureolytica record the lower percent 2.9% in females compared as 9.5% in males. About 7.1% of bacteria were Acinetobacter sp. appeared only in males. In females, Klebsiella pneumonia, Bordetella and Staphylococcus spp. recorded higher percentages 20.6%, 20.6% and 11.8% compared with 14.3%, 4.8% and 4.8% in males respectively. However, Proteus mirabilis and Pseudomonas aeruginosa recorded higher percentages 11.9% and 21.4% in males in comparison with 5.9% and 17.6% in females respectively. Generally, Streptococcus pneumonia has the highest percent 23.7% than other bacteria in males and females. However, Acinetobacter sp. has the lowest percent 3.9%.

DISCUSSION
Results of this study agrees with (2) who indicated to finding Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae in bronchial washing specimens of patients Bronchiectasis with in Seoul. Streptococcus pneumonia is the main responsible about Pneumonia and Bronchitis among Bacterial Pulmonary Infections (4), that agree with this study (Table 1). However, 20-40 years recorded the lower percentage reached 15.6%. results of this study for age group 41-60 near to results of (5).
The results of Table 3 agree with results of (11) who referred to resistance of Pseudomonas aeruginosa to Imipenem, Meropenem and Ciftazidim and resistance of Klebsiella pneumonia to Gentamicine and Cefepim. Also, the results of Streptococcus pneumonia are comptible with (5). Acinetobacter sp. exhibited resistance to Augmentin and Cefotaxim that may be due to the antibiotic resistant genes (12). Also, resistace of Staphylococcus spp. in this current study may be return to some antibiotic resistant genes (9). About 7% of Acinetobacter sp. isolated from sputum of patiants in some hospitals in Turkey and it was found resistant to many antibiotics (14) Pseudomonas aeruginosa isolated from bronchial washing showed resistance to Amikacin, Amox-Clav, Ampicillin, Cefazoline, Cefepime, Cefotaxime, Cefoxitin, Cefuroxime, Ciprofloxacin, Ertapenem, Fosfomycin, Gentamicin, Imipenem, Levofloxacin, Mezlocillin, Pip-Tazo, Tetracycline, Tobramycin, and Trimeth/Sulfa (3). Thus, using of antibiotic to treatment of some patients is changing due to the results of microorganisms in bronchial washing fluid (2).

CONCLUSION
The conclusion from this study is that bacterial infections are the record widespread diseases of the inferior respirational system among patients in hospitals in addition to bacteriological antibiotics cannot be the model resolution for the dealing of bacterial septicity, since it is a recurrent routine indications to the creation of a novel strain resistant to antimicrobial Similarly, the antibiotics are not expected a new effect in the case of various infections.