Antibiotic resistance pattern of bacterial isolates retrieved from febrile neutropenic patients with hematological disorders

Antibiotic resistance is nowadays becoming a threat in the treatment of immunosuppressed patients. The aim of this study was to nd out the antibiotic resistance pattern of bacteria isolated from febrile neutropenic patients with hematological disorders so that it would help to select the empirical antibiotic for prompt effective treatment of the febrile neutropenic patients. A cross-sectional descriptive study was conducted at a tertiary care hospital of Nepal from October 2018 to November 2019. Blood was drawn aseptically in blood culture bottles. The bacteria were identied by standard microbiological methods with observation of colony morphology, gram staining and biochemical tests of bacteria. The antibiotic susceptibility tests were done by Kirby Bauer disc diffusion method. Extended Spectrum Beta Lactamase (ESBL) and Metallo Beta Lactamase (MBL) producers, and Methicillin Resistant Staphylococcus aureus (MRSA) were detected by phenotypic methods.


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Background Febrile neutropenia is a common observation among patients undergoing treatment of hematological malignancies. It leads to prolonged hospital stays, increase in medical costs and increase in mortality in spite of therapeutic advances available including broad spectrum antibiotics, antifungals, antivirals and granulocyte colony stimulating factors (1).
Neutropenia is the predisposing factor for bacteremia as neutrophil is the rst line of defense mechanism against bacterial infection (2). Neutropenia occurs in the hematological patients after cytotoxic chemotherapy that suppresses the hematopoietic system. Often fever is the only sign of infection in neutropenic patients as neutropenia reduces the signs and symptoms of infection (2).
Bacteremia is the important cause of fever in neutropenic patients (3). Emergence of antimicrobial resistance has become a global problem (4). Treatment of bacteremia due to so called "Superbugs", MDR pathogens, is becoming a clinical challenge especially in neutropenic patients. Microbiological pro le of bacteremia in febrile neutropenic patients is unknown on the onset of fever. The rst choice empirical antibiotic for the treatment must be selected on the basis of local epidemiological bacterial isolates and resistance patterns (5).
Emergence and spread of antimicrobial resistance is growing rapidly in South Asian countries (6). Nepal is one of those countries with high burden of antibiotic resistance (7). There is lack of data regarding antimicrobial surveillance from Nepal. To our best knowledge, this is probably the rst report on microbial surveillance in febrile neutropenia patients with hematological malignancies from Nepal.

Methods
This cross-sectional descriptive study was conducted in patients admitted in the hematology ward at a tertiary care hospital in Kathmandu, Nepal from October 2018 to November 2019. Ethical approval was obtained from the Institutional Review Committee of the hospital. Oral informed consent was obtained from all the enrolled patients.

Patient's enrollments
The patients admitted in hematology ward with hematological disorders were selected for the study. Only the patients with febrile neutropenic conditions were enrolled in the study. In this study, febrile condition was de ned as rise in temperature of the body more than 38 0 C (100.4 0 F) which lasted for more than one hour (5). Neutropenia was de ned as absolute neutrophil count < 0.5 × 10 9 /L (500 cells/mm 3 ) (5).

Blood cultures and bacteremia
Blood was drawn aseptically in blood culture bottles from BD BACTEC™ (Becton, Dickinson and company). The bottles were incubated in BD BACTEC™ Fx model instrument. A patient was considered as blood culture positive if > 1 bottle of the same patient grew same organism. Since only aerobic vials were used, isolation of anaerobic bacteria was excluded from this study.

Microbiological procedures
The positive blood culture vials were then sub cultured onto 5% sheep blood agar and MacConkey agar media plates. The media plates were then incubated at 37 0 C for 24 hours. The organism was identi ed by gram staining, by its morphological characteristics and using biochemical tests. The antibiotic susceptibility tests were done by following the disk diffusion method (modi ed Kirby-Bauer method) on Mueller Hinton agar (Hi-Media, India) following standard procedures recommended by the Clinical and Laboratory Standards Institute (CLSI), Wayne, USA (8).
The antibiotics used for Gram negative bacteria were amikacin, amoxicillin, ce xime, ceftriaxone, cefepime, cipro oxacin, cotrimoxazole, piperacillin-tazobactam, imipenem, meropenem, tigecycline and polymixin B. In case of Gram-positive organisms, the antibiotics used were amikacin, amoxicillin, cloxacillin, cephalexin, ceftriaxone, cipro oxacin, erythromycin, clindamycin, cotrimoxazole and vancomycin. The drugs were chosen so as to classify the organisms as MDR, XDR (extensively drugresistant) and PDR (pandrug -resistant) as per the international expert proposal for interim standard de nitions for acquired resistance by the European Centre for Disease Prevention and Control & Centre for Disease Control and Prevention (4).
MDR is de ned as non-susceptibility to at least one agent in three or more antimicrobial categories. XDR is de ned as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories). PDR is de ned as non-susceptibility to all agents in all antimicrobial categories (i.e. no agents tested as susceptible for that organism) (4).
ESBL and MBL enzymes were detected by double disc combined synergy method. For ESBL detection, cefotaxime and cefotaxime + clavulanic discs were used (8). For MBL detection, imipenem and imipenem + EDTA discs were used (9). Cefoxitin discs were used for the detection of MRSA (8).

Statistics
The demographic data of patients (age, sex and disease type) and the results of microbiological investigations were entered in a computer program. SPSS 20.0 (SPSS Inc., IBM Co., Chicago, IL, USA) software was used to enter and analyze the data. Descriptive analysis was done by calculating frequency and percentages.

Patient demographics
From October 2018 to November 2019, 214 febrile neutropenic episodes from 91 patients were observed and enrolled in this study. A bacterium isolated from a single patient with same antibiotic sensitivity pattern was considered as single pathogen. The age of patient ranged from 4 to 77 years. Of the total patients, 74.7% of patients were male and 25.3% were females (Table 1). Bacterial isolates Of the total 214 blood samples, 33.9% (71) yielded the bacterial growth. Gram negative bacteria were isolated from 23.8% of total samples and Gram-positive bacteria were isolated from 9.3% of the total samples. Among Gram negative isolates, E. coli was the predominant pathogen and S. aureus was the predominant pathogen among Gram positive isolates (Table 2). All the Gram-negative isolates were susceptible to polymixin B and majority of the isolates were susceptible to tigecycline. Besides tigecycline and polymixin B, amikacin was the drug towards which Gram-negative bacteria showed the lowest resistance (35.2%) ( Table 3). Imipenem and meropenem are considered as a drug of choice for MDR bacteria but a marked resistance was observed towards them in this study (41.1% and 37.2% respectively). All the Gram-negative bacteria were sensitive towards polymixin B (Table 3).  About 19.6% of total Gram-negative bacteria isolated were found to be ESBL producers and 19.6% of them were found to be MBL producers (Table 4). One isolate of K. pneumoniae was found to be both ESBL and MBL producer (Table 4). A total of 34 isolates of Gram-negative bacteria were found to be MDR which was 66.7% of total Gram-negative bacteria isolated (Table 5). About 33.3% of the Gram-negative bacteria were found to be XDR and none of the isolates was found to be PDR. Most of K. pneumoniae (90%) were MDR and about 88.2% of E. coli were MDR (Table 5). Gram-positive bacteria in the study were found to be less resistant to antibiotics as compared to Gramnegative bacteria. All Enterococcus spp. bacteria were found to be sensitive to all antibiotics those were used. None of the Gram-positive bacteria were resistant to vancomycin. As the Gram-negative isolates, majority of the Gram-positive bacteria were sensitive to amikacin (Table 6). But marked resistance was found towards other antibiotics used (Table 6).  About 41.6% of S. aureus were found to be MRSA ( Table 7). None of Enterococcus spp. was found to be MDR but majority of S. aureus and CONS were found to be MDR with few XDR as well. None of the Grampositive bacteria were found to be PDR (Table 8).

Discussion
Increasing antimicrobial resistance is the emerging global issue. This study has pointed towards increasing antibiotic resistance among the bacteria isolated from febrile neutropenic patients. Most of the bacteria isolated in the current study were found to be MDR. About 66.7% of total gram-negative bacteria and 50% of the total gram-positive bacteria were found to be MDR. The incidence in this study is higher than the study done in India by Babu K G et al in 2014 (1) (11). In our study, the rate of ESBL producers and MBL producers was found to be lesser than that of MDR. It points towards additional mechanisms of antibiotic resistance acquired by MDR bacteria in addition to ESBL and MBL production such as resistance due to decreased antibiotic penetration and e ux, changes in target sites or resistance due to global cell adaptations (12).
Gram-negative bacteria were more predominant than gram positive bacteria in our study. Similar ndings were reported by Babu K G et al from India (1). However, in studies conducted in developed countries such as Japan and America, they have found Gram-positive bacteria to be more predominant than Gramnegative bacteria (3). E. coli was the predominant Gram-negative bacteria followed by K. pneumoniae and S. aureus was the predominant Gram-positive bacteria isolated in the current study. These ndings are concordant with other reports conducted elsewhere in febrile neutropenic patients (3,(13)(14)(15)(16)(17)(18).
Though marked resistance towards the antibiotics was found in our study, the resistance percent towards the antibiotics is lesser than in the study done by Parajuli et al in Nepal in critical unit patients where amikacin resistance ranged from 45-100%, piperacillin-tazobactam resistance ranged 48-92%, imipenem resistance 19.3-86% and meropenem resistance ranged 19-84% (10).
Piperacillin-tazobactam is the preferred initial antibiotic for febrile neutropenic patients in our institution. However, this study has shown marked resistance against piperacillin-tazobactam. Hence there is a prompt necessity to switch to another antibiotic with high sensitivity for effective treatment of febrile neutropenic patients.

Conclusion
Rapidly growing antibiotic resistance is a great concern in the modern medicine. This global problem is threatening the e cacy of the treatment of febrile neutropenic patients. Every institution must have periodic surveillance of the microbial data of bacteremia in febrile neutropenic patients in order to select the most sensitive antibiotic at that particular time for appropriate treatment of febrile neutropenic conditions. Availability of data and materials

Abbreviations
The datasets (tables) supporting the conclusion of this article are included within the article. The raw data of the study are available from the corresponding author on reasonable request.

Ethical approval
Ethical approval was obtained from the Institutional Review Committee of Civil Service Hospital, Nepal.
Blood culture and antibiotic sensitivity is considered as routine test for febrile neutropenic patients. Hence written consent was not needed from the patients. Oral informed consent was obtained from all enrolled patients.