Untitled Document

Multi-drug resistance among the Escherichia coli isolated from
suspected cases of septicemia

Shoorashetty Manohar Rudresh1*, Giriyapur Siddappa Ravi2
1*Associate Professor, 2 Professor, Department of Microbiology, ESIC MC PGIMSR & MH, Rajajinagar, Bengaluru, Karnataka

Year : 2021 | Volume : 4 | Issue : 1 | Page : 14 | DOI -10.46319/RJMAHS.2021.v04i01.003

Abstract
Aim: Bacterial blood stream infections (BSI) are major cause of mortality and morbidity. The Escherichia coli (E. coli) is the common gram-negative bacteria (GNB) causing BSIs. The occurrence of multi-drug resistance among E. coli causing blood stream infections has increased over a decade. Early detection and management of cases will decrease the mortality.  Methods: A total of 5000 blood cultures were received from September 2015 to August 2016 from suspected cases of septicemia. The blood culture was done using BactAlert 3D automated system. Any growth was identified by conventional biochemical tests. The antibiotic susceptibility pattern was determined according CLSI guidelines. The ESBL, AmpC and MBL were detected. Results: Among the 248 positive blood cultures, 195 were gram negative bacteria and 53 were gram positive bacteria. A total of 26 E. coli were isolated, accounting for 13.3% of GNB. The E. coli had highest resistance to β-lactam antibiotics (92%) followed by ciprofloxacin (85%) and cotrimoxazole (62%). They showed a good sensitivity to colistin (100%), carbapenems (92%) and amikacin (89%). The extended spectrum β-lactamase (ESBL) production was seen in 65%, AmpC production in 19% and MBL production was seen in 8% of isolates. Conclusion: Increased proportion of E. coli bacteremia was noted compared to previous studies. The occurrence of multi-drug resistance and resistance to 3rd generation cephalosporins limits the treatment options.
Key words: Septicemia, Escherichia coli, Bacteriaemia, multi-drug resistance, Extended spectrum β-lactamase.

Introduction
The bloodstream infections (BSIs) remain a common cause of hospitalization.[1,2]  Both Gram-positive and Gram-negative bacteria (GNBs) can cause BSIs. Among the GNBs, Escherichia coli (E. coli) is the leading cause of BSI. It ranks first as a cause of community-acquired BSI and second as a cause of hospital-acquired BSI.[2, 3, 4]
The E. coli is the part of the normal commensal gutmicrobiota of healthy human populations.  However, some strains have specific virulence factors which makes them to infect intestinal or extraintestinal sites. The isolates which have capacity to gain access in to the tissue and survive are known as extraintestinal pathogenic E. coli and they cause a variety of infection, including urinary tract infections (UTI), sepsis, and neonatal meningitis.[4] The E. coli blood stream infections most commonly arise as a complication of focal infections of the urinary or gastrointestinal tract.
The emergence of E. coli, that produces extended spectrum β-lactamases, AmpC β-lactamases and carbapenemases are of most concern.[3] Such strains of E. coli also carry drug resistance genes for other antibiotics, making them multi-drug resistance. The antibiotics recommended for empirical antimicrobial therapy will be ineffective against this infection. The infections due to MDR E. coli infections lead to a longer hospital stay, economic loss, and higher mortality.[5]
Hence the present study was conducted to know the proportion of blood culture yielding E. coli and to determine the antibiotic susceptibility pattern of the isolates.
Materials and Methods
This is a prospective study on blood cultures sent to Clinical Microbiology laboratory from September 2015 to August 2016.
Blood culture: The blood samples collected from suspected cases of bacteriaemia was inoculated into the BacT/ALERT Standard Aerobic blood culture bottles (bioMérieux Inc., Durham, NC, USA) and incubated in BacT/ALERT automated blood culture system. Any bottle which was flagged as positive was sub-cultured on 5% sheep blood agar and MacConkies agar and incubated at 37° C in ambient air for 18-24 hours. The growth was identified to species level using standard laboratory techniques.
Antibiotic susceptibility testing: The antibiotic susceptibility testing was done according to CLSI M100-S16 guidelines. The antibiotic discs used were gentamicin (GEN), amikacin (AK), cotrimoxazole (COT), aztreonam (AT), amoxacillin-clavulanic acid (AMC), cefoxitin (CX), ceftazidime (CAZ), cefotaxime (CTX), piperacillin (PI), piperacillin-tazobactam (PIT), cefaperazone (CPZ), cefaperazone sulbactam (CFS), imipenem (IMP) and meropenem (MRP). The discs were purchased from HiMedia laboratories Pvt Ltd, Mumbai. A lawn of 0.5 McFarland standard inoculums was made on Mueller Hinton agar and antibiotic discs were placed within 15 minutes. The plates were incubated at 37ºC in ambient air for 18-24 hours. The zone diameters were interpreted according to CLSI guidelines.[6]
Extended spectrum β-lactamase detection:[6] ESBL detection was done using CLSI described phenotypic confirmatory test along with the routine antibiotic susceptibility testing. An isolate was confirmed for ESBL producer, if ceftazidime zone was enhanced to ≥5mm on addition of clavulanic acid.
AmpC detection:[7] AmpC disc test was done according to Black JA et al. Briefly, a 0.5 McFarland lawn of E. coli ATCC 25922 was made on MHA. The cefoxitin disc was placed at center and a sterile filter paper disc impregnated with the test organism was placed adjacent to it facing the organism side down. Any indentation of the zone of cefoxitin was considered as AmpC β-lactamase production.
MBL detection:[8] MBL detection was done according to Yong D et al. Briefly, the imipenem and imipenem with 0.5 M EDTA discs were placed along with routine antibiotic susceptibility testing. A zone difference of ≥5mm between imipenem with EDTA with imipenem alone was interpreted as MBL production.
Results
A total of 5000 consecutive, non-duplicate blood cultures were received from suspected cases of septicemia over period of one year. A total of 248 (4.96%) blood cultures grew bacteria. Among the 248, 195 (3.9) were Gram negative bacteria and 53 (1.06%) were gram positive bacteria. The most common gram-negative bacteria isolated were S Typhi (n=60; 30.8%), non-fermenting gram negative bacteria (n=45; 23.1%) and E. coli (n=26; 13.3%).
A total of 26 (13.3%) strains of Escherichia coli were isolated with overall incidence of 0.52%. All the positive blood cultures were flagged within 48 hours of loading to automated blood culture system. The mean age group of patients affected was 56 years with range of 0 to 75 years. A total of 19 patients were between 29 to 65 years, 5 patients more than 65 years and two patients less than one year age. The female (15; 58%) were more commonly affected compared to males (11; 42%) and male to female ratio was found to be 1:1.36. The patients infected with E. coli septicemia were admitted in ICU (13; 50%), NICU (1; 4%) and various inpatient wards (12;46%).
The antibiotic resistance pattern of the isolates is shown in Table 1. The isolates were most commonly resistant to β-lactam antibiotics (92%) like ceftazidime, cefotaxime, piperacillin, cefaperazone and amoxacillin-clavulanic acid followed by ciprofloxacin (n=22; 85%) and cotrimoxazole (n=16; 62%). The isolates showed good sensitivity for colistin (n=26; 100%) imipenem (n=24; 92%), meropenem (n=24; 92%) and amikacin (n=23; 89%). The overall resistance to 3rd generation cephalosporins was found to be 88.5%.  Among the isolates tested, 17 (65%) were confirmed for extended spectrum β-lactamase (ESBL) production, 5 (19%) isolates were positive for AmpC β-lactamase production and 2 (8%) isolates were positive for metallo β-lactamase production (Table 2). The multi-drug resistance was found among 10 (38.5%) strains of E. coli. Among these MDR isolates 7 were ESBL produces, 2 were MBL producers and 1 was AmpC producer.
Table 1: Antibiotic resistance pattern of E. coli


Sl No.

Antibiotic

Resistance [n (%)]

1

Amoxacillin-clavulanic acid

24 (92)

2

Ceftazidime

24 (92)

3

Cefotaxime

24 (92)

4

Piperacillin

24 (92)

5

Cefaperazone

24 (92)

6

Ciprofloxacin

22 (85)

7

Cotrimoxazole

16 (62)

8

Gentamicin

10 (39)

9

Piperacillin-tazobactam

9 (35)

10

Cefaperazone-sulbactam

8 (31)

11

Cefoxitin

7 (27)

12

Amikacin

3 (11)

13

Imipenem

2 (8)

14

Meropenem

2 (8)

Table 2: The β-lactamase production among E. coli

 

Frequency

Percent

AmpC

5

19

ESBL

17

65

MBL

2

8

NONE

2

8

Total

26

100

Discussion
Escherichia coli is an important agent in causing blood stream infections. The global increase in the incidence of blood stream infections due to multi drug resistant E. coli has been noted in last decade. There is an urgent need for large scale studies defining the epidemiology of E. coli sepsis.
In the current study the proportion of blood cultures positive for gram negative bacteria was found to be 3.9%. The study showed E. coli being the third most common (13.3%) isolate among gram negative bacteria. Bajaj et al9 studied a total of 6265 blood cultures and found E. coli as second most common organism (6.64%) causing blood stream infections among gram negative bacteria.  The overall incidence of E. coli septicemia in our institution was found to be 0.52%. A recent study conducted by Ragupathi NDK et al[10] showed an overall occurrence of E. coli in blood culture to be 0.11%.
Majority of the infected patients with E. coli bacteriaemia required ICU admission. The mean age group of patients affected with E. coli bacteriaemia was found to be 56 years (range 0 to 75 years). The burden of the disease was more among the patients aged 29 years and above. The females (58%) were more commonly affected compared to males (42%). Many studies have shown the incidence of E. coli infections increasing age and more often associated with females.[11, 13]
The increasing antimicrobial resistance among E. coli especially for 3rd generation cephalosporins (3GC) and carbapenems has been noted since past decade. Most of the isolates showing resistance to 3GC were also resistance to other groups of antibiotics limiting the therapeutic options. In the current study 88.5% of isolates were resistant to 3GC, followed by ciprofloxacin (85%) and cotrimoxazole (62%). All the strains were sensitive to colistin and followed by carbapenem antibiotics (92.3%). Many studies have shown the 3GC resistance ranging from 16.7% to 80%.[4,9,13] Extended spectrum β-lactamase production was found in 17 (64%) E. coli followed by AmpC (19%) and MBL (8%).
The multi-drug resistance was found among 38.5% (n=10) isolates. The ESBL and MBL producing E. coli isolates often showed resistance to multiple classes of antibiotics.  Previous studies have shown poor outcomes and higher death rates among ESBL producing multi-drug resistant E. coli infection.[5, 13] This was due to the limited therapeutic options available for infected patients.
Conclusion
The overall incidence of the E. coli was increased when compared to the previous studies. The mean age affected was above 30 years with female preponderance. High incidence of resistance to β-lactam antibiotics and its association with multi-drug resistance limits the therapeutic options.
Financial support and sponsorship: This study was a part of research work funded by Rajiv Gandhi University of Health Sciences (RGUHS) Adv Research Grants No. M107/ 2015-16.
Conflict of interest: Nil
References
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Address for Correspondence: Dr Rudresh SM, Associate Professor, Dept of Microbiology, ESIC MC PGIMSR & MH, Rajajinagar, Bengaluru – 10. E-mail : rudreshsm@gmail.com


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