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Emergence of Colistin Resistant Gram-Negative Bacteria in a Tertiary Care Rural Hospital in 2019

Introduction Antibiotic resistance, which started in the 1970s among Gramnegative bacteria, is a crucial global problem. The rapid increase in the prevalence of Gram-negative pathogens that are resistant to fluoroquinolones and aminoglycosides as well as all beta lactams, including carbapenems, monobactam, cephalosporins and broad-spectrum penicillin, has prompted to solve this challenge by one of the oldest antibiotics Colistin in the polymyxin groupis using as a last therapeutic optionfor critically ill patients suffering from infections caused by multidrug resistant gram negative bacteria. Colistin (polymyxin E) is a polypeptide bactericidal agent available in two forms for clinical use as (polymyxin B and polymyxin E). Colistin sulfate and colistimethate sodium. Colistimethate sodium is to hydrolyze to colistin sulfate on parenteral administrationand acts like bactericidal drug by disrupting lipopolysaccharide (LPS) molecules of gram negative bacterial outer membrane. It is also established that neither new development of antimicrobials nor an alternative drug working activity against multidrug-resistant (MDR) gram-negative bacteria, all are bound to widespread use of colistin. As a result, there are reports of emergence of older antibiotics like colistin resistance to gram negative bacteria of Enterobacteriaceae.


Introduction
Antibiotic resistance, which started in the 1970s among Gramnegative bacteria, is a crucial global problem. 1 The rapid increase in the prevalence of Gram-negative pathogens that are resistant to fluoroquinolones and aminoglycosides as well as all beta lactams, including carbapenems, monobactam, cephalosporins and broad-spectrum penicillin, has prompted to solve this challenge by one of the oldest antibiotics Colistin in the polymyxin groupis using as a last therapeutic optionfor critically ill patients suffering from infections caused by multidrug resistant gram negative bacteria. Colistin (polymyxin E) is a polypeptide bactericidal agent available in two forms for clinical use as (polymyxin B and polymyxin E). Colistin sulfate and colistimethate sodium. Colistimethate sodium is to hydrolyze to colistin sulfate on parenteral administrationand acts like bactericidal drug by disrupting lipopolysaccharide (LPS) molecules of gram negative bacterial outer membrane. 2 It is also established that neither new development of antimicrobials nor an alternative drug working activity against multidrug-resistant (MDR) gram-negative bacteria, all are bound to widespread use of colistin. As a result, there are reports of emergence of older antibiotics like colistin resistance to gram negative bacteria of Enterobacteriaceae. 3,4,5 The colistin resistant aerobic Gram-negative bacteria70.75% cases was found in an intensive care unit patient in Netherlands. 6 A study in Italy found 50% colistin resistant E.coli. 7 Considering the widespread occurrence of colistin resistance and the impending danger associated with it, we screened gram-negative bacteria belonging to the family Enterobacteriaceae isolated from the patients of Khwaja Yunus Ali medical College Hospital has prompted more accurate and careful monitoring of resistance to this polypeptide. 1

Materials and Methods
It was Laboratory based prospective study carried out at the Department of Microbiology of Laboratory Services, Khwaja Yunus Ali Medical College Hospital, Enayetpur, Sirajganj, Bangladesh from January 2019 to March 2019 (3 months). The clinical specimens from which Colistin resistant pathogenic isolates were included in the study. The colistin resistant pathogens isolated repeatedly from the same patient's repeat specimen were excluded from study to avoid duplication of isolate. In the Department of Microbiology 640 clinical specimens were received from different wards like ICU, CCU during this period. The specimens included were urine, stool, blood, pus, sputum, throat swab and aspirated fluid. Processing of the specimens was done on blood agar,chocolate agar, Mac Conkey's agar and eosin methylene blue agar (EMB Agar) media (Merck, Germany) for double checking and taking pure culture. 8 After incubation of plates at 37°C for 18-24 hours, the isolated colonies were identified by Gram staining and standard biochemical tests such as triplesugar iron (TSI) and Simmons citrate. 9 Antimicrobial susceptibility testing (AST) was performed using disk diffusion method (Kirby-Bauer) on Mueller-Hinton agar (Merck) plates according to the Clinical and Laboratory Standards Institute (CLSI) Guidelines. 10 The tested antibiotic panel were meropenem, ampicillin, amoxicillin, amoxiclav, amikacin, azithromycin, azitreonam, ceftriaxone, cefixim, cefotaxime, cefuroxin, cephradine, cephtazidime, ciprofloxacin, Clindamycin, gentamicin, trimethoprim/sulfamethoxazole, nitrofurantoin, levofloxacin, teicoplanin, vancomycin and colistin. The phenotype of Enterobacteriaceae was defined as MDR according to the International Expert proposal for Interim Standards Guidelines. 11 This study was approved by the Institutional Review Board (IRB) and Ethics Committee of Khwaja Yunus Ali Medical College Hospital. In the microbiology laboratory all the isolates were found to be multiple-drug-resistant by the disk diffusion method. Throughout this study, results were interpreted using the CLSI guidelines. Total 156 gram negative bacilli were isolated inthe study period. While 37 (23.7%) were sensitive to colistin while 119 (76.3%) were resistant. (Figure 2)

Discussion
The spread of antibiotics resistance to a wide variety of antibiotics such as beta-lactams, aminoglycosides and carbapenemsis a global challenge to the health systems. To cope up this vital problem colistin is increasingly used as one of the last available treatment options for patients with severe infections caused by MDR Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa. 12 However, its nephrotoxicity and neurotoxicity impacts have reduced its application as aroutine prescribed drug. 13 Colistin resistance follows the increasing trend in consumption of colistin in human medicine, especially in countries with high rates of carbapenem-resistant gram-negative bacilli, including Italy. 14 Now, acquired resistance to colistin is extremely worrying considering that colistin is used as a last resort antibiotic against carbapenem-resistant Gram-negative bacteria, especially Enterobacteriaceae. 15 During the 1 st quarter of 2019, in this study 20% and 76% Gram negative bacilli found resistant to meropenem and colistin, respectively. Whereas the two years earlier study carried out in Tamil Nadu of our neighboring country India a quite different scenario of resistance was found as 65% and 33% Gram-negative bacteria resistant to meropenem and colistin, respectively. 15 Indiscriminate antibiotic use is the cause of rapid spread of carbapenem and colistin resistance among Gram-negative bacteria has become a major threatfor the treatment of infectious diseases.
In a study from Singapore during 2006, 30% of P. aeruginosa isolates were found resistant to colistin. 16 But in our study far more colistin resistance 81% found in P. aeruginosa.This highly significant difference in colistin resistance from Singapore's one-year study on only 102 strains of Enterobacteriaceae may be explained as it was carried out 13 year ago. Now it must be increased as its resistance spread rapidly. Our study carried during three months on 156 isolated gram-negative bacilli in comparison to one year. This proves emergence of colistin resistance is far grievers than other country of the world.
Colistin resistance in gram negative bacilli develops by mutational mechanism was thought but by research the transmission through plasmid mediated colistin resistance (mcr-1 gene) was reported 1 st in China on 18 November 2015 in food animals, foods and humans. 17,18

Conclusion
This document is unique, as it is the first in vitro Colistinresistance report of short period study from a tertiary health care delivery center of Bangladesh. Colistin resistance is an alarming concern because it is used as last resort of treatment in healthcare facilities. From the results of this study, we recommend, strict infection control guidelines and antimicrobial steward ship should be implemented to overcome the new resistance spread and without susceptibility testing no antibiotics should be prescribed.