Community Acquired Multi-drug Resistant Clinical Strains from Tracheal Aspirates of Patients in Hospital Settings in Dhaka, Bangladesh

Background: Antimicrobial resistance is a multi-sectoral problem which poses a major threat in the treatment of infectious diseases especially in developing countries like Bangladesh. Multidrug-resistant (MDR) bacteria along with extremely drug resistant (XDR) bacteria have emerged as major clinical and therapeutic dilemma in the treatment of tracheal infections in hospitals here. Thus the aim of this study was to document the incidence of MDR and XDR producing β-lactamases in clinical isolates from tracheal aspirates of patients in Dhaka, Bangladesh. Methods: Two hundred clinical isolates from tracheal aspirates were identified and their antibiotic susceptibility profiles were evaluated by using the VITEK 2 system following the Clinical and Laboratory Standards Institute guidelines. Patient information on diagnosis, sex, age was obtained from hospital data. Results: Of 200 clinical, non-duplicate bacterial isolates obtained, Pseudomonas aeruginosa was the most frequent pathogens (N=61/200, 30.5%) followed by Acinetobacter baumannii (N=58/200, 29%), Klebsiella pneumoniae (N=45/200, 22.5%), Streptococcus pneumoniae (N = 15/200, 7.5%), Escherichia coli (N=10/200, 5%), Staphylococcus aureus (N=4/200, 2%), Proteus spp (N=3/200, 1.5%), Enterobacter spp (N=2/100, 1%), Citrobacter spp (1/200, 0.5%), Providencia spp (N=1/200, 0.5%). Of 20 different antibiotics tested, highest number of isolates (N=172/200, 86%) showed resistance to third generation cephalosporin cefixime, however least number of isolates showed resistance to polymixin antibiotics-colistin (N=25/200, 12.5%) and polymixinB (N=12/200, 6%) . The patients’ ages ranged between 1 month to 95 years with the gender distribution of 133 (66.5%) males and 67 (33.5%) females. patients of age-group (old adults) ≥60 years (N=123/200, 61.5%). Of 200 clinical isolates, 43 (21.5%) were XDR and 125 (62.5%) were MDR bacteria. Of 200 clinical isolates, the synthesis of extended spectrum β-lactamases (ESBL) and carbepenemase were detected in 59 (29.5%) and 98 (49%) strains respectively. Conclusion: Tracheal infections caused by MDR and XDR pathogens among patients are high at hospital settings in Bangladesh. Therefore, there is an urgent need for constant surveillance and interventions in Bangladesh in order to prevent further spreading of those resistant organisms.


Introduction
Respiratory infections are the leading cause of global morbidity and mortality from infectious diseases worldwide [1]. Community acquired pneumonia (CAP), nosocomial pneumonia and acute and chronic bronchial infections in patients with chronic obstructive pulmonary disease (COPD) and bronchiectasis are known as the most common respiratory diseases those are responsible for elevated morbidity and mortality rate [2]. Lower respiratory tract infections like tracheal infections are caused by both of Gram-positive and Gram-negative bacteria. The emergence of multidrug-resistant (MDR) bacteria poses a major threat in hospital settings [3]. The most frequent multidrug-resistant bacteria associated with tracheal infections are Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and other Enterobacteriaceae [4].
Antibiotic resistance is an increasingly serious threat to global public health that threatens our ability to treat common infectious diseases, resulting in prolonged illness, disability and death (5). In recent years, several studies have reported an increased number of bacteria causing both hospital-acquired and community-acquired infections [3,6]. Enterobacteriaceae including K. pneumoniae, E. coli as well as Enterobacter spp. along with other bacteria such as P. aeruginosa and A.
However, Gram-positive organisms such as Staphlylococcus aureus which is a common causative agent of severe infections in health facilities and in the community become resistant to first-line drugs [5]. Patients infected with methicillin-resistant Staphylococcus aureus (MRSA) are estimated to be 64% more likely to die than people with a non-resistant form of the infection whereas MRSA are also reported to cause tracheal infections [5,10]. For the treatment of lifethreatening infections caused by Enterobacteriaceae which are resistant to carbapenems, colistin is used as the last resort of treatment [11]. However, resistance to colistin has been detected recently in several countries, making infections untreatable those are caused by such bacteria [5,11].
Resistance to broad spectrum β-lactams mediated by extended spectrum β- This study aimed to assess multidrug-resistance among Gram-negative and Grampositive bacteria those are responsible to cause lower respiratory tract infections in Dhaka, Bangladesh to guide treatment protocols along with to determine the existence of ESBL, carbapenemase production in multi-drug and extensively-drug resistant bacterial strains isolated from tracheal aspirates. The data further provides a baseline for future comparative studies.

Study:
The study was conducted between January 2018 and June 2019, in Dhaka Central International Medical College and Hospital in Dhaka, Bangladesh from where tracheal aspirates specimens (N=200) were aseptically collected from patients (N=200; Male=133, Female=67) then subsequently transported to microbiology laboratory of Primasia University for bacterial isolation and identification, phenotypic determination of antibiotic susceptibility, identification of multidrug resistant (MDR), extremely drug resistant (XDR), pan-drug resistant (PDR) organisms along with detection of ESBL and carbapenemase production. Information on diagnosis, sex, age was obtained from patients' records.

Demographic characteristics of patients with bacterial infections
The patients' ages ranged between 1 month to 95 years with the gender distribution  (Table 4).

Discussion
Antimicrobial resistance (AMR) is a major problem to global public health that requires action across all government sectors and society [5]. Infections caused by resistant bacteria responsible for longer duration of illness, additional tests and use of more expensive drugs rather than those infections which are caused by nonresistant bacterial species [5]. Our study observed the prevalence of tracheal infections was highest among the patients of old adults whose age was ≥60 years; however least susceptibility to these infections was noticed in young adults of age group 13-19 years. The annual incidence of pneumonia in the elderly people is four-times higher than that of younger populations reported elsewhere [23]. Tracheal infection was found to be more prevalent in males rather than in females. A. baumannii is an important nosocomial pathogen in healthcare facilities and has become one of the most significant microorganisms causing infections in hospitalized patients in last few decades [28]. A study conducted at the Dhaka Medical College Hospital (DMCH) showed 96% strains of A. baumannii isolated from endotracheal aspirates collected from patients, were multidrug resistant [29].
Another study carried in Square Hospitals Ltd. showed 90 % of the A. baumannii strains isolated from the patients with lower respiratory tract infections, were multidrug resistant [30]. In our study, 57% A. baumannii were MDR and 29% were XDR indicating an alarming situation. Moreover, 83% A. baumannii were found to be resistant to amoxicillin-clavulanic acid and cefixime whereas only 12% showed resistance to polymixin B.
The highest incidence rate of respiratory tract infection was caused by A. baumannii (25%) followed by Pseudomonas spp. (15%) and Klebsiella spp. (10%) [31]. Our findings correlate with these reports though P. aeruginosa was to be found as a predominate organisms causing respiratory tract infections. In our study, it was observed that among 45 strains of K. pneumoniae, 71% were MDR and 20% were XDR. K. pneumoniae was the most common causative agent of nosocomial pneumonia where the presence of MDR K. pneumoniae strains was prevalent [32].
However, other studies showed the most prevalent organism causing tracheal infections was Enterobacter spp. followed by P. aeruginosa [33][34]. Though only 1% strains causing tracheal infection were Enterobacter spp in our study, those were multidrug resistant. Moreover, it was observed methicillin resistant S. aureus (MRSA)was found to beresponsible for tracheal infections. A report stated elsewhere that MRSA is a cause of lung infection including airway infection, communityacquired pneumonia and hospital-acquired pneumonia [35]. Among the Gramnegative bacteria causing chronic respiratory disease, E. coli is considered one of the major respiratory threats [36]. Our study showed 20% E. coli strains were XDR and 50% were MDR. S. pneumoniae is an important causative agent of chronic respiratory disease including tracheal infections that result in higher rate of morbidity and mortality due to MDR S. pneumoniae [37]. It was observed 93% strains of S. pneumoniae causing tracheal infection were resistant to cefixime whereas 80% strains were MDR.
The present study observed highest number of strains of both Gram-positive and Gram-negative bacteria showed resistance to third generation cephalosporins, however the most effective antibiotics were polymixin antibiotics especially colistin and polymixin B along with tigecyclin. These findings correlate with other reports where colistin was reported as an effective drug in the treatment of infections caused by MDR bacteria [38][39].
In the recent years, antimicrobial resistance mediated by ESBL-and carbapenemase has been found to be ubiquitous [40] and the current dissemination of these enzymes makes it mandatory to understand this phenomenon especially because of the higher mortality, morbidity, and increased health treatment costs associated with resistance to β-lactams [41]. The increasing rate of dissemination of carbapenemase in Bangladesh has been documented with the isolation of clinical A. baumannii, P. aeruginosa and K. pneumonia [42]. The present study showed the synthesis of ESBL and carbepenemase were detected in 29.5% and 49% strains respectively where it was noticed most of the antibiotics tested were non-effective against ESBL and carbapenemase producer, however, polymixin B, colistin, tigecyclin were found as effective antibiotics against ESBL and carbepemase producers.

Conclusion
The study demonstrated high prevalence of β-lactamase producing multidrug resistant bacteria implicated in the tracheal infections diagnosed among patients.
Infections were common among the elderly people and predominantly caused by P. aeruginosa followed by A. baumannii, K. pneumonia, Streptococcus spp during the period of our study. Appropriate and justified use of antimicrobial agents should be ensured in controlling the growing danger of antimicrobial drug resistance. Therefore,there is an urgent need for constant surveillance and interventions in Bangladesh in order to prevent further spreading of those resistant organisms.
Further studies at molecular level will be required to determine the mechanism(s) of resistance by genotypic methods.

Declarations
Medical College and Hospital from whom the clinical strains were isolated. We would like to thank Bangladesh Council of Scientific and Industrial Research (BCSIR) for the generous support.

Competing interests:
The authors declare that they have no competing interests.

Availability of data and materials:
Additional information of the study can be made available from the corresponding author on request where necessary.

Authors' contributions:
The study was co-conceptualized and jointly designed by FTJ, JA, TF and MMHS. JA collected the data and undertook laboratory analysis with the help from SS, TF and MJF. FTJ and MMHS analyzed and interpreted the data with assistance from AKD, SA, MZR, ANC and MM. All the authors contributed in preparation and submission of manuscript. All authors read and approved the final manuscript.

Funding:
This study did not receive any specific grant from any funding agencies in the public, commercial or not-for-profit sectors.

Ethical approval and consent to participate:
Ethical clearance was approved by Ethical Committee, Bangladesh Council of Scientific and Industrial Research (BCSIR), Rajshahi. Verbal consent was taken from all participants in this study and from parents or guardians for minors' patients after explanation of the procedure and the purpose of the study.       Percentages of total patients in each age group. The prevalence of tracheal infections was hi Prevalence of MDR and XDR isolates causing tracheal infections was showed in the pie chart. Distribution of frequently isolated MDR (grey bars) and XDR (black bars) bacterial strains cau Figure 6 Distribution of β-lactamase among major bacterial strains causing tracheal infections. The pr