Abstract: The multidrug resistant Klebsiella is becoming an increasingly common clinical entity and has presented a global clinical problem. The present research aimed to observe the prevalence of infection and multidrug resistance pattern of Klebsiella in Chittagong region, Bangladesh. A total of 99 Klebsiella isolates associated with Klebsiella infection were isolated and identified according to the standard microbiological method from 502 individual patients. The most prevalent infection caused by Klebsiella isolates was urinary tract infection and male were more vulnerable to Klebsiella infection than female. Female of age 31-41 years group were found to be very much prone to Klebsiella associated infection whereas male has highest percentage on age group greater than 50 which was 58%. The sensitivity tests to Klebsiella were carried out with 5 antibiotics such as, Azythromycin, Amoxicillin, Cefexime, Nalidixic acid and Ciprofloxacin on Mueller-hinton agar by modified Kirby-bauer disc diffusion technique. Among the Klebsiella isolates, 92, 87, 60 and 42% were susceptible to Ciprofloxacin, Azythromycin, Cefixime and Nalidixic acid, respectively. Nearly 81% Klebsiella isolates exhibited resistance to amoxicillin and 58% isolate to Nalidixic acid. Azithromycin and Ciprofloxacin showed tremendous level of sensitivity to Klebsiella spp. The present study revealed the incidence of resistance to available antibiotics is currently relatively high, which implies a total lack of antibiotics for treatment of life-threatening infections caused by this MDR Gram-negative ‘superbug’.
INTRODUCTION
Klebsiella species are considered to be major opportunistic pathogens which can cause nosocomial infections. K. pneumoniae is an important cause of human infections among all Klebsiella species, followed by K. oxytoca, K. ozaenae and K. rhinoscleromatis (Podschun and Ullmann, 1998). In recent years, Klebsiella species have become important and frequent pathogens in hospital-associated infections. The range of clinical diseases includes nosocomial pneumonia, urinary tract infections and bacteremia in immunocompromised humans (Chou et al., 2004). The emergence of multidrug resistant Klebsiella pneumoniae is a growing up problem around the world (Deris et al., 2012). Emergence and spread of antimicrobial resistance is an inevitable consequence of widespread use of antibiotics in medical practice (Finch et al., 2012). Carriage of Klebsiella and the development of multidrug-resistant bacteria have been increased due to frequent use of broad-spectrum antibiotics in hospitalized patients (Paterson and Bonomo, 2005). Antibiotic resistance is one of the major public health concerns in this century declared by World Health Organization (WHO) (Lachmayr et al., 2009). It is one of the major causes of failure in the treatment of infectious diseases that results in increased morbidity, mortality and costs of health care (Bouza and Cercenado, 2002). Furthermore, the high prevalence of bacterial infections and extensive use of antibiotics incline to higher the percentage of antimicrobial drug resistance in developing countries like Bangladesh (Okeke et al., 1999; Col and O’Connor, 1987; O’Brien, 1992). In the South- East Asia (SEA) region, antimicrobial resistance (AMR) is a burgeoning and greatly ignored problem (WHO, 2011).
Surveillance of antibiotics is important to the control of resistance and Klebsiella pneumoniae infection. Therefore, this study was conducted to isolate, identify and determine the sensitivity pattern of Klebsiella from clinical specimens in Chittagong which is the second largest city of Bangladesh.
MATERIALS AND METHODS
Sample collection: A total number of 434 urine, 25 swab, 37 pus and 6 sputum, samples from August 2011-July 2012 were included in the study. All clinical samples had taken from prescribed patients who submitted their samples to Chevron Clinical Laboratory and Surgiscope Hospital. All clinical specimens were collected and processed according to standard operating procedures.
Isolation and identification of Klebsiella: Clinical isolates of Klebsiella were identified by their morphological characteristics on MacConkey agar media. The large mucoid colonies obtained from MacConkey’s agar plate were processed for Gram staining. In Gram staining, all the isolates were G- and rod shaped.
Biochemical characterization: The biochemical tests employed were IMViC (Indole, Methyl red, Voges Proskeaur, Citrate), H2S production on TSI agar, Motility test, Oxidase test, Urease test, fermentation of Lactose and Mannitol. For biochemical tests standard procedures were used (Cruickshank, 1980).
Antibiotic susceptibility test: Identified Klebsiella isolates were then subjected to sensitivity analysis against five antibiotics on Mueller-hinton agar by modified Kirby-bauer disc diffusion technique. Zone of inhibition are measured according to the criteria suggested by NCCLS (2000). The antibiotics that were used included Ciprofloxacin (05 μg), Nalidixic acid (30 μg), Amoxicillin (30 μg), Azythromycin (15 μg) and Cefixime(10 μg).
Statistical analysis: Analysis was performed by using Statistical Package for Social Science (SPSS Version 16) software and Excel Office program for the statistical analysis of this study. The t-test was done as a test of significance.
RESULTS AND DISCUSSION
The comprehensive data analysis of Klebsiella infected patients in different age group suggested the prevalence of multidrug resistant Klebsiella is increasing sharply. The prevalence of Klebsiella infections varies in different geographical regions which create an alarming situation nowadays (Riaz et al., 2012). From the screened cases, Klebsiella was found in 19.72%. Similar results were found earlier by Riaz et al. (2012), Lina et al. (2007) and Sarathbabu et al. (2012) where Klebsiella was mostly the etiologic agent.
| Fig. 1: | Prevalence of Klebsiella according to gender classification |
| Fig. 2: | Frequency of specimen sources in different genders caused by Klebsiella spp. |
In this study, samples in the form of urine, swab, pus and sputum were collected from 502 individuals including male and female. From these 502 patients sample, 99 gave positive results for Klebsiella associated infection. In the case of Klebsiella, males (57%) had a higher incidence as compared to females (42%) considering gender classification. It depicted that male were more vulnerable to Klebsiella infection than female (Fig. 1). Janda and Abbott (2006) reported that males are more susceptible to infection with Klebsiella spp. than females.
In this study, data of Klebsiella had arranged according to clinical samples. The most prevalent infection caused by Klebsiella isolates was urinary tract infection followed by swab or pus. Riaz et al. (2012) reported that urine is a most common source of UTI infection. However, in the case of urine, pus and sputum, males were more affected with 37, 7 and 5% of Klebsiella, respectively. In swab samples, 9% Klebsiella affects females (Fig. 2).
To determine the correlation of Klebsiella infection with sex and age, the patient histories were analyzed. It was observed that maximum age group the percentage of female patients was higher than male.
| Fig. 3: | Percentage of male and female patients by Klebsiella in different age groups in this study |
Male has highest percentage on age group greater than 50 which was 58%. In the age group 31-40 years, female patients had the top most percentage (approx. 29%) which was the highest figure compare to other age group (Fig. 3). In males, the age groups between 50+ and 70+ had larger frequency of beta-lactamase producing Klebsiella infections (Riaz et al., 2012). On the other hand, high frequency in females was in age groups of 70+ and 20-30+. Lina et al. (2007) reported men and women of elderly group were found to be very much prone to UTI. In Pakistan, both males and females in the age group of 50+ were more commonly infected by multidrug resistant bacteria (Bashir et al., 2008). Lindback et al. (2010) revealed that there are interrelations between old age and antibiotic resistance.
Month wise observation of 99 Klebsiella infection cases showed that, during the year, the percentage of female and male patients were fluctuating. Moreover, June-July got the peak number of patients. During June, female and male patients were 19.05 and 12.28%, respectively and in July female and male patients were 19.05 and 15.79%. Between November and December, the amounts of female patients decrease sharply. In the month of November and December male patients were 8.77 and 7.02%. This data suggested that, the onset of the diseases was high in June and July.The number of female patients were significantly high in both June and July and low in January and December (Fig. 4). Atmospheric condition of Bangladesh may play the vital factor for Klebsiella infection.
The problem of bacterial antibiotic resistance emerged as soon as the first antibiotics become available for clinical use (Raco and Barez, 1998). All the isolates were tested for antibacterial susceptibility. Among positive isolates, 92 and 87% were susceptible to Ciprofloxacin and Azythromycin respectively whereas only 60% was susceptible to Cefixime and 42% to Nalidixic acid. Nearly 81% Klebsiella isolates exhibited resistance to Amoxicillin and 58% isolate to Nalidixic acid.
| Fig. 4: | Month wise observation of patients by Klebsiella infection in this study |
| Fig. 5: | Antibiotic resistance/sensitivity pattern of the Klebsiella spp. isolates recovered from patients |
This data suggested that, Amoxicillin, Cefexime, Nalidixic acid were significantly resistant to Klebsiella. But Azithromycin, Ciprofloxacin showed tremendous level of sensitivity to Klebsiella (Fig. 5). Lina et al. (2007) reported, all of the K. pneumoniae isolates were noticed to be resistant to Ampicillin and Amoxicillin and 86% isolates were resistant to Azithromycin. Sikarwar and Batra (2011) found that K. pneumoniae were resistant to Ciprofloxacin and Nalidixic acid which observed approximately 45-54% isolates. The major reason of this spread of resistance is likely due to inappropriate prescribing of antibiotics, incorrect dose or duration of use and antibiotic use without prescription.
CONCLUSION
The emergence of Multidrug-resistant (MDR) Klebsiella is inevitable and is a major public health threat worldwide. In Chittagong, numerous hospitals have experienced outbreaks of infections caused by MDR Klebsiella. This study suggested that, 92, 87, 60 and 42% Klebsiella isolates were susceptible to Ciprofloxacin, Azythromycin, Cefixime and Nalidixic acid respectively. Nearly 81% Klebsiella isolates exhibited resistance to Amoxicillin and 58% isolate to Nalidixic acid. Azithromycin and Ciprofloxacin showed tremendous level of sensitivity to Klebsiella spp. Therefore, the present study provides an idea about the prevalence of Klebsiella as superbug in Chittagong city, which needs special attention for improving the health care systems of Bangladesh.
ACKNOWLEDGMENTS
We would like to express utmost gratitude to Chevron Diagnostics and Surgiscope Hospital, Chittagong for their support during the study. Special thanks to Dr. Tipu Sultan, Microbiology Department, Chittagong Medical College and S.M. Murshid Ul Alam, Department of Genetic Engineering and Biotechnology, University of Chittagong for their co-operation during the study.