Detection of β-lactamases among Multi-drug Resistant Enterobacteriaceae Colonising Gut of Normal Adults and Patient Population

Introduction

Members of the family Enterobacteriaceae are significant pathogens causing serious nosocomial and community-onset bacterial infections in humans. It is an alarming signal that increasing number of commensal strains are exhibiting multi-drug resistant genes. Enterobacteriaceae like Klebsiella pneumoniae and Escherichia coli are known for their propensity to carry resistance genes and hence were chosen as ideal to show commensal resistance in this study. Multi-drug resistant (MDRs) colonisers act as a reservoir for transmission of antibiotic resistance and are a major source of infection.¹ β-lactamase enzymes produced by these bacteria provide resistance towards β-lactam antibiotics, consisting of penicillins, carbapenems, cephalosporins and monobactams. All β-lactam antibiotics have a common four atom β-lactam ring that is hydrolysed by the lactamase enzymes inactivating antibacterial action of β-lactam antibiotics. The three main varieties of β-lactamases identified as the most widespread are ESBL (Extended Spectrum β Lactamase), AmpC β Lactamase and MBL (Metallo β Lactamase).²

Persistent exposure of bacterial strains to a multitude of β-lactams has induced dynamic and continuous production and mutation of β-lactamases in these bacteria, rendering previously powerful antibiotics powerless. It is imperative to take into concern, regular localised reports on the MDR status of each region before prescription of antibiotics, for a deeper understanding of the transmission dynamics of each antibiotic in a community.³ MDR status in regions in and around Karnataka has not received enough research attention; thus it is necessary to undertake this research to draw more attention to this burning issue. Since the majority of the existing research attention is towards the nosocomial spread of resistance, this study aimed to take a comparative approach and evaluate the hospitalized and the healthy group to determine the occurrence of MDR genes in the normal population.

Materials and Methods

The present prospective study was conducted in the Microbiology laboratory of a tertiary care center in Mangaluru, Coastal Karnataka from August 2019 to October 2019 and was approved by the Institutional Ethics Committee of our Institute and funded by RGUHS Undergraduate grant 2019 (UGMED267). The study included a total of 120 fecal strains of E. coli and Klebsiella.

a) Isolation of bacterial strains

The first set of 60 samples represented the pathogenic strains and were collected from hospitalized patients. Hospital sample codes were used to identify the patient samples; hence the identity of patients was maintained undisclosed throughout. The second set of 60 samples was collected from students and staff of our institute representing the normal (non-pathogenic) strains. Any subjects presenting with gastrointestinal symptoms and diarrhoea were excluded.

The common enteric bacteria, Klebsiella pneumoniae, and Escherichia coli were isolated and subjected to Antibiotic Susceptibility Testing by the Kirby Bauer disk diffusion method. Following six antibiotics disks were used to screen for resistance - Ampicillin (10 µg), Gentamicin (10 µg), Ciprofloxacin (5 µg), Cefotaxime (30 µg), Cefoparazone (30 µg) and Imipenem (10 µg). Isolates showing resistance to more than three classes out of the six tested were considered as multi-drug resistant.⁴

b) Detection of β-Lactamases

The strains were then specifically tested for ESBL, AmpC, and MBL production.

1) Extended Spectrum β-Lactamase: ESBL was detected by disc diffusion test using ceftazidime and ceftazidime/clavulanic acid discs. An increase in the inhibition zone of more than 5 mm was taken to be indicative of ESBL production.⁵ (Figure 1) 

2) AmpC beta Lactamase detection using AmpC disk test: Indicator strain, ATCC E. coli, adjusted to 0.5 McFarland was applied on Muller Hinton agar and a 30 µg Cefoxitin disc was placed on lawn culture. Sterile 6 mm blank filter paper discs moistened with sterile saline, inoculated with several colonies of test organism or control strains were placed beside the Cefoxitin disc, almost touching it, with the inoculated side in contact with the agar surface and incubated overnight at 37^oC. Indentation or flattening of the zone of inhibition was taken as a positive test, which indicated AmpC production (Figure 2).

3) Metallo β-Lactamase detection using Modified Hodge test (MHT): Indicator strain, ATCC E. coli, adjusted to 0.5 McFarland was applied on Muller Hinton agar. After drying, a 10 µg Imipenem disc was placed in the center of the plate on the lawn culture. Colonies of overnight cultured test strain was heavily streaked from the edge of the Imipenem disc to the periphery of the plate in four different directions. After incubation, MHT positive test showed the presence of a distorted zone (clover leaf-like indentation or zone of inhibition). MHT negative test showed no growth of the E. coli 25922 along the test organism growth streak within the disk diffusion, without an indentation of the zone (Figure 3).

This data on phenotypic characterization of antibiotic resistance was then tabulated to analyse β-lactamase production. The different β-lactamases and their respective percentages of detection (both from normal individuals and patients) are expressed as figures and graphs using Microsoft Excel.

Results

Out of the 120 strains included in the study, 26 Klebsiella pneumoniae strains and 94 Escherichia coli strains were isolated. Among sixty strains isolated from pathogenic samples, 45 were E. coli isolates and 15 were K. pneumoniae and among 60 strains from normal samples, 49 were Escherichia coli and 11 were Klebsiella, respectively.

On antibiotic susceptibility testing, Ampicillin resistance was the highest in both categories showing rates of 34 (56%) and 50 (83.3%) in normal and pathogenic strains, respectively. The details of the resistance pattern are represented in Figure 4.

Multi drug resistance (MDR) was shown by a total of 55 (45.8%) strains, out of which pathogenic strains showed higher rate of 31 (51.6%) as compared to normal strain rates of 24 (40%).

All 120 strains, subjected to β lactamase isolation showed rates of 77 (64.1%), from which 47 (78.3%) were pathogenic and 31 (51.6%) were from normal strains.

Details of various β lactamase producers are represented in Figure 5 and combination of β lactamase production in normal and pathogenic strains are shown in Table 1.

Discussion

Antibiotic resistance is a dangerous microbial threat and MDR bacteria are the most harmful propagators. Awareness about the alarming transmission rates of resistance is a must for doctors and general population alike. The occurrence of multi-drug resistant Entero-bacteriaceae was found to be 51.6% in hospitalized patients and 40% in healthy individuals who showed no pathogenic symptoms. Thus, a mean of 45.8% of isolates showed resistance to more than three antibiotics - Penicillins, Cephalosporins, and Aminoglycosides. This is a convincing fact that hospital drug-induced resistance affects the normal population dramatically due to the fast transmission dynamics. The rural study from Puducherry in 2015,¹ showed MDR rates of 30.1% which was found to be extremely high in a setting believed to have negligible access to antibiotics. The present study showed an even higher rate, most probably owing to the fact that it was conducted in an urban setting with consequently increased antibiotic exposure.

In a previous study on hospitalized patients showed MDR rates of 31.7% in a hospital in France in 2010.⁶ Another study from Chandigarh conducted in a semiurban setting with healthy candidates showed antibiotic resistance rates of 70.5% and MDR rates of just 2.4%.⁷ The human-to-human transfer is the common mechanism seen for resistance transmission in the nosocomial setting, while the spread in the community involves a mix of transmission types such as human to animal and vice versa. It is important to examine this ever-growing gene pool of resistance in the community as documenting and control of these strains will prove to be far more difficult.

Among the antibiotics included in AST (Antibiotic Susceptibility Test) in the present study, strains isolated from clinical infections exhibited a higher resistance rate of 83.3% to Ampicillin, while strains from normal subjects showed 56% resistance. Cefoparazone showed surprising results; the normal strains showed more resistance than the pathogenic strains- 35%and 23.3%, respectively. The normal strains also showed higher resistance than the pathogenic strains with Imipenem and Gentamicin, which too was an unexpected finding.

The human and animal alimentary tracts are vital reservoirs for ESBL-carbapenemases, and AmpC enzyme-producing Enterobacteriaceae as shown in various studies.³ About 78.3% of the pathogenic strains and 51.6% of the normal strains were positive for β lactamases in the current study which is a significant finding, with ESBL production being the most common type (61.6% in pathogenic strains and 41.6% in normal strains). In comparison, hospitalized patients in Morocco showed rates of 42.8% ESBL production.⁸ 35.6% of MDR isolates showed phenotypic ESBL in the Puducherry study conducted in a rural setting.¹ Around 26.1% of inpatients and 17.1% of asymptomatic individuals showed ESBL production in a study conducted in Saudi Arabia.⁹ An Argentinian community study had ESBL isolation rates of 18.9%.¹⁰ The rise in resistance through the years has occurred in conjunction with a lessening disparity in rates between hospitalized and healthy volunteers.^11-13

AmpC isolation rates from the current study were 31.6% and 20% in pathogenic and normal strains, respectively.

The Spanish study from 2014 revealed rates of 31% phenotypic AmpC production from healthy individuals.¹⁴ Another study involving healthy individuals from Turkey showed rates of just 5.3%.¹⁵ Hospitalized patients continued to show higher rates than healthy individuals.¹⁶

In the present study, MBL production rates in pathogenic and normal samples were found to be 25% and 16.6%, respectively. However, recent studies from Chandigarh and Delhi reported 18.1% and 9.9% MBL producers in hospitalized patients.^7,17 Other MBL studies, from Cairo and China showed genotypic isolation rates much lower than our study, with just 0.33% and 3.6%, respectively in the healthy group.^18,19 This implies the resistance conferred via carbapenemase production is much higher than anticipated and is in accordance with the high resistance shown to Imipenem during AST. As ESBL, Amp-C and MBL producers are now found even in normal commensals like Enterobacteriaceae,²⁰ the necessity of implementing judicious use of antibiotics and infection control practices to curb the dissemination of these isolates becomes crucial.

Conclusion

This study revealed high fecal MDR carriage rates even in normal individuals in and around Mangaluru. The β- lactamase production rates exceeded the reference literature values by a huge margin indicating an alarming microbial status in the area. Commensal bacteria like Enterobacteriaceae showing such drug resistance is worrisome. Moreover, the gap between rates of resistance between hospitalized patients and general population was extremely low, which is a proof of the rapid horizontal transmission dynamics of resistance and the increased presence of resistance gene carriers in the community. Environmental studies must be combined with routine sample screening to produce data so that dissemination of these isolates may be prevented. Financial support and sponsorship Funded by Rajiv Gandhi University of Health Sciences, Bangaluru (UGMED267) for undergraduate medical students.

Conflict of interest

Nil