Trends in Antimicrobial Resistance in a Tertiary Care Hospital of Assam, India

Antimicrobial resistance (AMR) in bacterial pathogens has emerged as a challenge in health care settings resulting in high rates of morbidity and mortality. The aim of the present study was to describe the trends and burden of AMR in a tertiary care hospital. A retrospective observational study was undertaken from October 2018 to March 2021 in a clinical microbiology laboratory where local priority pathogens and their antimicrobial resistance patterns were analyzed. Organism identification and antimicrobial susceptibility testing were performed as per Clinical and Laboratory Standards Institute guidelines. Out of 9948 isolates, Enterobacteriaceae (58%) were mostly isolated followed by Staphylococci (18.6%), Non-fermenting gram negative bacilli (NFGNB) (13.7%), and Enterococci (8.4%) respectively. Highest isolation was from inpatient department (61.3%); 31.5% from outpatient, and 7.2 % from intensive care units. Klebsiella pneumoniae (26.9%) was most isolated organism, mostly from respiratory samples; Escherichia coli was isolated mostly from urine (40.7%). Almost half of the Enterobacteriaceae isolates were extended spectrum beta-lactamase producers while >50% of Enterobacteriaceae and NFGNB isolates were resistant to one or more Carbapenems. Frequency of Methicillin resistant Staphylococcus aureus was 44.7% , Vancomycin resistant Enterococci was 1.2%. A rising trend of resistance to cephalosporins and carbapenems along with fluoroquinolones was observed. Our study has witnessed a high prevalence of Gram negative pathogens with increasing resistance to commonly applied antimicrobials during the surveillance period which can act as a guiding tool in devising local antimicrobial priorities, antibiotic policy, and proper antimicrobial prescribing practices.


INTRODUCTION
Antimicrobial resistance (AMR) in bacterial pathogens has emerged as a challenge in health care settings resulting in high rates of morbidity and mortality worldwide. 1 Globally about 700,000 people die every year due to AMR related illness; death toll will reach an estimated 10 million by 2050. 2 The emergence and rapid spread of drug-resistant pathogens especially of multidrug resistant (MDR) and pandrug-resistant bacteria (also known as "superbugs") has caused infections which are not responding to treatment with existing antimicrobial agents. 3 Due to increasing use of broad-spectrum antibiotics like Carbapenems which are usually reserved for treatment of MDR infections, resistance among Gram-negative bacteria has become a global problem. 4 Instances of Colistin resistance have also been reported in many countries, for which no effective antibiotic treatment is available at present. 3 In 2014, the World Health Organization (WHO) declared AMR as a global health security threat and emphasized the urgent need of reliable surveillance data for formulating strategies and actions to contain AMR. 5 Due to huge burden of infectious diseases in India, our country is one of the largest consumers of antibiotics in the world. 6 The southern and northern regions of India has witnessed high prevalence of carbapenemresistant bacterial infections. 7 On the other hand, the states of Mizoram, Tripura, Nagaland, and Meghalaya of northeast India reported below 5% prevalence of carbapenem resistance by virtue of less developed healthcare and lower amounts of carbapenem use. 8 Due to lack of nationwide data on AMR, there was a limitation for intensive response against AMR in India. 9 To overcome this deficiency and to collect nationwide representative data, Antimicrobial Resistance Surveillance & Research Network (AMRSN) was initiated by the Indian Council of Medical Research (ICMR), New Delhi in 2013. 9 Under AMRSN,a hospital based or laboratory based surveillance system was established at major tertiary care centers of India to monitor the antimicrobial susceptibility trends of clinically important pathogen groups similar to the WHO priority pathogen list released in 2017. 10 Being a part of AMRSN network since 2018, our laboratory has been monitoring, generating and disseminating AMR data besides developing the local antibiotic policy and infection prevention and control protocol. Since no published literature is available describing the AMR trends in Assam till date, this study was performed to describe the trends and burden of AMR and to provide useful information to clinicians and policy makers.

Surveillance of antimicrobial resistance in clinical isolates
All significant clinical isolates i.e. pure culture isolates or isolates recovered from successive culture belonging to the following bacterial groups are included in the surveillancemembers of the family Enterobacteriaceae, Non-fermenting Gram negative bacilli (NFGNB)-Pseudomonas aeruginosa and Acinetobacter baumannii, Staphylococcus aureus, Enterococcus species, enteric pathogens including Shigella species, Salmonella species, and Vibrio species.

Inclusion criteria
• All significant bacterial isolates from clinically defined infections. • For multiple isolates of a single species from the same patient, only the first isolate has been included. • For outbreak, only one strain (outbreak strain) has been included in surveillance. Outbreak strain is defined as a group of isolates that were both epidemiologically related and thus presumed to be clonally related. 11

Exclusion criteria
• Bacterial isolates from colonization without infection. Colonization is defined as presence of microorganism on/in a host, with growth and multiplication of the organism, but without causing any disease. 12 • For outbreaks, all except the index strain(i.e. bacterial strain isolated from the first documented patient of an outbreak)were excluded from surveillance listing. • For multiple isolates of a single species from the same patient, all isolates except the first isolate were excluded. The isolates have been classified as outpatient-isolated from patients attending Outpatient department (OPD)s, inpatient-isolated from patients admitted in wards and high dependency areas like Intensive Care Unit (ICUs) and high dependency unit HDUs. For every isolate, the following information was noted: study number, age, gender, clinical diagnosis, OPD/ward/ ICU, date of collection of specimen, and nature of specimen (blood, pus, urine, etc). For the ICU cases, the data of admission and the antibiotic therapy was also noted. All the collected data were entered into the i-AMRSS tool of ICMR, Govt. of India and analysis could be done as permissible to regional centre of the AMRSN.

Microbiological identification
Routine microbiological investigations were performed and species identification was performed using standard biochemical reactions or Vitek2 automated system (Biomerieux) method using standard operating procedures Bacteriology of ICMR-AMRSN.

RESULTS
A total of 9948 culture isolates were recovered during the period of analysis. Location wise distribution showed most isolates (61.3%; n=6098) were from wards; 31.5% (n=3133)

A n t i m i c ro b i a l re s i sta n c e p a tte r n s o f Enterobacteriaceae
A substantially higher resistance rate was observed for Cephalosporin among Enterobacteriaceae isolates followed by resistance against Fluoroquinolones. Carbapenems also found resistant in an important fraction of Enterobacteriaceae isolates. (Table 2) Overall, 50% of Enterobacteriaceae isolates were resistant to 3rd generation cephalosporins due to ESBL production (1328/2237 of Escherichia coli and 1067/2673 of Klebsiella spp.).

Antimicrobial resistance patterns of NFGNB
Acinetobacter baumannii (n=550) isolates showed a higher resistant rate mostly to Cephalosporin, followed by Piperacillin-Tazobactam and carbapenems. (Table 2) Pseudomonas aeruginosa (n=590) isolates showed comparatively better susceptibility rate than other isolates.

Carbapenem resistance in Gram negative isolates
Overall carbapenem resistance among Enterobacteriaceae isolates of this study was 58.8% while frequency among NFGNBs was 56.0%. The carbapenem resistant Acinetobacter baumannii, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa isolates also showed higher resistance frequency to other antimicrobials viz. aminoglycosides, fluoroquinolones, cephalosporins, trimethoprim-sulfamethoxazole and Piperacillin-Tazobactam in comparison to carbapenem susceptible isolates. Colistin was found susceptible to all 261 nos. of gram negative isolates tested for MIC.

Resistance patterns in Gram positive pathogens
The frequency of MRSA observed during the period of study was 44.7% (799 out of 1786 isolates). Vancomycin resistant Staphylococcus aureus was not found during this period of study. Higher frequency of resistance to all the antimicrobials was observed among the MRSA isolates in comparison to Methicillin Susceptible Staphylococcus aureus (MSSA) isolates. (Table  3) Among the Enterococci isolates, resistance frequency to Ampicillin, Ciprofloxacin, High level aminoglycoside (Gentamicin 120 µg) and Nitrofurantoin were higher in Enterococcus faecium isolates. Vancomycin resistant Enterococci were observed in 1.2 per cent (10 of 817) of isolates. (Table 3)

Susceptibility trend (2018-2020) in different groups of organism
Enterobacteriaceae isolates showed an increasing trend of susceptibility to Amikacin and decreasing trend to Piperacillin Tazobactam. (Figure 1) The NFGNB isolates showed a decreasing trend of susceptibility to carbapenems & cephalosporins. (Figure 2) The trend is almost static in Staphylococcus aureus isolates except for a slight decrease in susceptibility to Trimethoprim-sulfamethoxazole. (Figure 3) Enterococci isolates, on the other hand witnessed an increasing trend of susceptibility to Ampicillin and Ciprofloxacin over the years. (Figure 4)

Distribution of Carbapenemase genes
During the study period one or more carbapenemase gene targets were detected in 79 (28.2 %) of the 280 study isolates using PCR. (Table 4)

DISCUSSION
Various AMR surveillance studies in India have been providing key information about antimicrobial resistance in various parts of the country. 9,[18][19][20] The present study is first of its kind in the Eastern Assam region of North Eastern India; has been performing AMR surveillance since October, 2018. Our study has witnessed an increasing prevalence of Gram Negative pathogens with increasing resistance to Cephalosporin, Fluoroquinolones, and Carbapenem during the surveillance period. The finding of gram negative dominance has also played a vital role in formulating the institutional antibiotic policy. WHO 19 A recent study from Mizoram, North East Indian state reported as low as 11.3% carbapenem resistance. 21 Another recent study from southern part of Assam reported 26.2% of ESBL positive Escherichia coli urine isolate and 12.6% carbapenemase producers which is considerably lower than our finding. 22 Though isolates are less in number; a very high frequency of Fluoroquinolones resistance (>95 per cent) was observed among the Shigella species in this study. Mogasale et al. from South India also reported a high frequency of Fluoroquinolones resistance in Shigella species (75 per cent). 18 Among the Gram positive pathogens, higher frequency (44.7%) of Methicillin resistant Staphylococcus aureus(MRSA) is observed in comparison to an earlier study in 2016-17 (35.3%) reported by Walia K et al. 9 The present study is concordant with their study in non-susceptibility of Staphylococcus aureus isolates to Penicillin (91% vs 93% respectively). But Ciprofloxacin non-susceptibility is in decreasing trend (63% vs 79%). Mogasale et al. from South India reported 53% Methicillin resistant Staphylococcus aureus in their study which is higher than the present study; also reported 1.72 per cent Vancomycin resistant Staphylococcus aureus (VSA). 18 VSA was not detected in the present study.
Vancomycin resistant Enterococci (VRE) isolates were relatively less in our study compared to those reported by Walia K et al. 9 & Mogasale et al. 18 (1.2 per cent, 11.9 per cent & 13.3 per cent respectively). VRE infection in hospital settings is a threat-the source due to cross contamination among patients through health care workers and ICU patient with VRE colonized in gut. 23

Molecular characterization of all drug resistant
genes could not be reported in detail as only few isolates were tested. 2. About AMR pattern of bacterial isolates from community acquired and hospital acquired infections could not be commented as the archived data are impermissible due to the ongoing surveillance program of AMRSN. 3. AMR pattern of fungal isolates could not be analyzed as number of isolates of each organism was less (n= <30); which can be addressed in a phased manner of coming period of AMR surveillance. I n c o n c l u s i o n , t h e l e a d i n g pathogens Escherichia coli, Klebsiella species, Acinetobacter baumannii, and Methicillinresistant Staphylococcus aureus have high AMR in this region with an increasing trend of high level of resistance towards third generation cephalosporins, and carbapenems. The regional AMR data provided herein are of great importance to policy makers in making informed decisions for the treatment of bacterial infections and developing appropriate antibiotics prescription guidelines to curb the unnecessary use of broad-spectrum antibiotics, thereby slowing down the rapidly increasing trend of AMR and even multi-drug resistance.