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Antibiogram and Detection of ESBL Production in Klebsiella species Isolated from Various Clinical Samples

Klebsiella species belonging to the family Enterobacteriaceae, are known to cause a variety of human infections such as pneumonia, urinary tract infections, septicaemia, soft tissue infections and are an important cause of healthcare associated infections. The emergence of multidrug resistant Klebsiella is a growing problem worldwide. Klebsiella spp. are one of the most common organisms to carry plasmids encoding ESBL enzyme. They are associated with higher rates of treatment failure and death. In this study, we intend to detect the different species of Klebsiella causing infections and analyze their antibiogram including extended spectrum beta lactamase production. A retrospective study was conducted in the department of Microbiology in a tertiary care hospital. Patient samples in which Klebsiella species was isolated were included in the study. Sample processing and isolate identification was done using standard laboratory techniques. Antimicrobial susceptibility testing and ESBL screening was performed by Kirby Bauer‟s disc diffusion method as per CLSI guidelines. A total of 210 Klebsiella spp. were isolated. Sputum (35.7%) was the most frequently received specimen and 94.8% isolates were K. pneumonia. Highest sensitivity was seen towards imipenem. Among urinary isolates, norfloxacin was the least effective drug. 68.6% isolates were ESBL producers. There are limited therapeutic options for treatment with multidrug resistant organisms. Hence, it is becoming very important to prevent colonization and infection by implementing and periodically reinforcing appropriate infection control measures. K e y w o r d s

K. pneumoniae is a primary pathogen capable of causing pneumonia, urinary tract infections and liver abscess in otherwise healthy people. However, most infections especially multidrug resistant infection caused by K.pneumoniae are healthcare associated infections such as wound infections, biliary tract infections, peritonitis, meningitis and device related infections. K. oxytoca, like K.pneumoniae can cause a variety of hospital acquired infections (Mandell et al., 2010).
Over the last 15 years, numerous outbreaks of infection with organisms producing extended spectrum ß Lactamase (ESBL) enzyme have been observed worldwide (Slden et al., 1971). Klebsiella spp. are one of the most common organisms to carry plasmids encoding ESBL enzyme and are associated with higher rates of treatment failure and death (Mandell et al., 2010). Studies in Karnataka have showed detection rates of ESBL producing K. pneumoniae to vary from 9.6% to 81.1% (Rao et al., 2014).
In this study, we intend to detect the different species of Klebsiella associated with infectious diseases in our hospital and analyze their antimicrobial resistance patterns including extended spectrum beta lactamase production. This will help in the selection of effective antibiotics for empirical treatment of Klebsiella infections as well as generating data on the prevalence of antibiotic resistance in our hospital.

Materials and Methods
A retrospective study was conducted in the department of Microbiology at a tertiary care centre in northern Karnataka from January 2016 to December 2017. Data of specimens such as sputum, urine, pus, blood, body fluids like peritoneal fluid, pleural fluid, synovial fluid, etc. received from patient"s in the microbiology laboratory for culture during the study period were scrutinized. Patient samples in which Klebsiella species was isolated were included in the study. Sample received from the same patient twice and yielding the same growth was considered as a single isolate.
All samples were inoculated onto blood agar and MacConkey"s agar and incubated overnight at 37 0 C. Urine samples were inoculated by "standard loop technique" using a calibrated loop. Preliminary identification of the organism was based on presence of large, mucoid, nonhemolytic colonies on blood agar and large, mucoid, pink lactose fermenting colonies on MacConkey"s agar and Gram"s stain showing short and stout Gram negative bacilli. Antimicrobial susceptibility testing was performed by Kirby Bauer"s disc diffusion method on Mueller Hinton agar. Selection of antimicrobials to be tested and interpretation was done according to Clinical Laboratory Standards Institute (CLSI) guidelines. The antimicrobials tested were cefotaxime, ceftazidime, cefepime, piperacillin, piperacillin-tazobactam, imipenem, amikacin, gentamicin, ciprofloxacin, levofloxacin andtrimethoprim-sulfamethoxazole.
Only urinary isolates were also tested against nitrofurantoin and norfloxacin. Quality control was done using Escherichia coli ATCC 25922 (Clinical Laboratory Standards Institute, 2014).
Screening for Extended spectrum beta lactamase (ESBL) producing Klebsiella spp. was done as per CLSI guidelines. The isolate was tested with ceftazidime (30µg), ceftriaxone (30 µg) and cefotaxime (30 µg) by disk diffusion. If the zone size for ceftazidime was < 22 mm and cefotaxime was < 27 mm, the isolate was an ESBL producer (Clinical Laboratory Standards Institute, 2014).
Descriptive statistics like percentage and proportions were used to describe data and difference was tested using chi square test.

Results and Discussion
During the study period, a total of 210 Klebsiella spp. were isolated in the microbiology laboratory. The age and sex distribution of the patients has been depicted in Table 1. Men were more frequently infected. Male to female ratio was 1.3: 1. The age group most commonly infected with Klebsiella spp. was from 61 to 70 years (19.5%), followed by 21 to 30 years (16.2%).
Out of the 210 Klebsiella spp., 199 (94.8%) isolates were K. pneumoniae and 11 (5.2%) were K. oxytoca. These isolates were obtained from various clinical samples received in the microbiology laboratory. Figure 1 shows the various samples that yielded both the species of Klebsiella. Overall, sputum (35.7%) was the most frequently received specimen, followed by urine (26.2%), pus (22.4%), blood (10%), endotracheal aspirate (3.8%) and body fluids (1.9%). It was seen that K. oxytoca was most frequently isolated from urine while K. pneumoniae was most commonly isolated from sputum. Among the isolates from blood, 16 out of 21 were from neonates in Neonatal Intensive Care Unit.
Antibiotic sensitivity testing was interpreted as per CLSI guidelines. The sensitivity pattern of the isolates is depicted in Figure 2. Highest sensitivity was seen towards imipenem, whereas least effective drug was ciprofloxacin and among urinary isolates, norfloxacin was the least effective drug. Phenotypic screening of extended spectrum beta lactamase (ESBL) producers as per CLSI guidelines is done routinely in the microbiology laboratory. It was seen that 68.6 % of isolates were ESBL producers by screening test. In the present study, a small proportion of resistance to imipenem (3.3%) was also noted.
The age and gender distribution of patients infected with Klebsiella species was studied (Table 1). In our study, maximum patients infected were in the age group of 61-70 years. Similar results have been reported by several other investigators (Das and Debnath, 2015; Thomas and Ramyashree, 2016;Chakraborty et al., 2016). There was no statistically significant difference between the number of male and females infected and no difference was observed in the age distribution.
In this study, majority of the infections were due to K. pneumoniae (94.8%), followed by K. oxytoca (5.2%). These findings are in concurrence with the fact that K pneumoniae is the most common pathogen amongst In the present study, pneumonia was the most common infection followed by UTI. We noticed most of the isolates from blood were from neonates admitted in the Neonatal Intensive Care Unit [NICU] and all were due to K. pneumoniae. K. pneumoniae causing neonatal septicaemia has been well documented (Zaidi et al., 2005). A previous study in the same hospital also found Klebsiella pneumoniae as the most common cause of neonatal septicaemia (Bhurle and Solabannavar, 2014)

Fig.2 Antimicrobial Susceptibility Pattern of Klebsiella isolates
The various species of Klebsiella exhibit different levels of susceptibility to antibiotics employed in treatment. In the present study, the antibiogram of Klebsiella isolates showed higher resistance against cephalosporins and fluoroquinolones. Similar results were reported by other researchers (Panta et al., 2013). Screening for extended-spectrum ß lactamase (ESBL) showed that 68.6% of the isolates were ESBL producers. Higher resistance to both cephalosporins as well as quinolones could be because the plasmids that contain the ESBL gene also contain resistance genes for quinolones (Paterson et al., 2000).
Sridhar Rao et al., (2014) reported a wide variation in detection rates of ESBL across regions. The authors stated that it could be due to a varied degree of exposure of these organisms to beta lactam antimicrobials or because of varied transferability of plasmids in the nature or due to the differences in the methodology employed (Rao et al., 2014). In the present study, the high proportion of ESBL producers could be because only screening test was performed.
96.7% of the isolates were sensitive to imipenem. As it was a retrospective study, carbapenem resistance was not being confirmed. 100% sensitivity to imipenem was observed by the author in her previous study in the same setup (Bhurle and Solabannavar, 2014). 3.3% resistance is observed to imipenem in the present study, this gradual increase in resistance is an impending threat and cause for concern. This could be attributed to the increased use of carbapenems in the recent years.
Over the last 2 decades, the incidence of infections caused by multidrug resistant Klebsiella strains has increased significantly and this emergence of antimicrobial resistance has become a global health problem (Gupta et al., 2003). Multidrug resistance strains causes challenges in healthcare and increases the morbidity and mortality. It increases the duration of hospital stay and cost incurred to the patient (Thomas and Ramyashree, 2016). The commonest reason for development of resistance is the high frequency use of antibiotics for empirical treatment thus strict adherence to antibiotic policy for empiric therapy is the need of the hour (Gupta et al., 2003). Continuous monitoring of multidrug resistant organisms and their resistance pattern will help in managing the infections efficiently and also help in formulation of antibiotic policies.