Characterization of extended-spectrum-β lactamases (esbls) and other resistant genes encoding bacteria from a rural community settlement

Antimicrobial resistance (AMR) is regarded as a global health threat, characterized by

rising resistant bacteria and rapid development of resistance genes to existing

antibiotics used in clinical and veterinary settings. AMR has negative impacts and

affects human, animal, and the environment. In Botswana, Africa, AMR is not well

understood, particularly in rural settings with poor sanitation. The misuse and overuse

of commonly used antibiotics like ß-lactam antibiotics, combined with the complex

environment in poor rural communities, could contribute to the high occurrence and

diversity of extended ß-lactamase (ESBL) encoding bacteria that are difficult to treat.

To understand the spectrum of AMR in Botswana, it is critical to use the one health

approach to characterize the occurrence and diversity of antibiotic resistance.

The study was conducted in the town of Palapye, in a rural Boseja ward, and focused

on ESBLs encoding bacteria and resistance genes isolated from various water, soil,

and healthy animal feces within a single household. In this study, the characterization

of ESBL encoding bacteria from a rural community settlement was explored using

two main approaches: the culture dependent (isolation) and culture independent

(genomics) methods. In the culture dependent method, viable bacteria from animal

feces and environmental samples (such as soil and pond water) were isolated using

selective agar, and then randomly selected isolates were tested for phenotypic

antibiotic resistance profiles on nutrient agar supplemented with six ß-lactam

antibiotics (penicillin (16 g/ml), ampicillin (32 g/ml), cephalosporin (32 g/ml),

meropenem (4 g/ml), cefotaxime (64 g/ml), and cefoxitin (32 g/ml)). Following that,

DNA from bacterial isolates and uncultured samples were explored using a culture

independent approach involving next generation sequencing (NGS) methods such as

whole genome, shotgun metagenomics sequencing method and bioinformatics tools.

Based on the output from NGS (whole genome sequence and shotgun metagenomics)

online analysis tools were used to assemble the raw reads into consensus contigs using

PATRIC (Unicycler). The contigs were further analyzed for antibiotic resistance genes

(ARGs), virulence and plasmids using ResFinder, CARD/RGI, VirulenceFinder and

PlasmidFinder bioinformatics programs, respectively. Additionally, the species

present in the whole genome bacterial isolate was determined by KmerFinder whereas

in the shotgun sequence data, taxonomic classification was achieved by Kraken2

which revealed bacterial diversity on animal feces and environmental sources samples.

A total of 21 samples from pond water (n=3), different animal feces (such as chicken,

dogs, ducks) (n=9) and different soil samples (n=9) which were collected in triplicates

from a single household were cultured to isolate viable microorganisms. Overall, both

selective media plates had growth for all the samples which were equally picked for

further characterization. A total of 336 isolates were randomly picked from the

triplicate plates from each sample source and were analyzed for antibiotic

susceptibility test against six ß-lactam antibiotics, where 42.9 % (144/3360) were from

different animal feces, 42.9 % (144/336), were from surrounding soil samples and 14.2

% (48/336) were from pond water. There was an overwhelming resistance to all the

six ß-lactam antibiotics across all 336 isolates from animal feces, surrounding soil

samples and pond exhibited 100 % resistance to penicillins (penicillin and ampicillin),

100 % resistance to cephalosporins (Cephalosporin, 2

nd generation cephalosporins:

Cefoxitin, 3rd generation cephalosporin: cefotaxime) and 100 % resistance to

carbapenem (meropenem).

The results of NGS of cultured and uncultured samples revealed a diversity of genes

encoding resistance to various antibiotics, including ß-lactam antibiotics (blaSHV,

blaOXA, blaTEM, blaOKP-B, blaCMY), tetracycline (tetB(P), tet(J), tet (W), tet(Q)), phenicol

(cat, catA3), aminoglycosides (aph(6)-Id, aac(6')-iid), macrolides (mef(A)),

trimethoprim (dfrA14, dfrA15), fluoroquinolone (OqxA, OqxB) and sulfonamide (sul

1, sul 2). Furthermore, plasmid groups revealed from the samples were ColpVC,

ColRNAI, Col (MG828), Col3M, Col (BS512), IncR, and IncFIB(K), and the

virulence related genes namely colicin gene (cia), tellurium ion resistance gene (ter

C), ferric aerobactin receptor gene (iutA), ABC transporter protein gene (mchF), Outer

membrane protein gene (traT), and glutamate decarboxylase gene (gad) were detected

from the various samples. Chromosomal mutations were also detected (gyrA, gyrB,

parA, OmpK35, OmpK36, OmpK37).

The bacterial genome sequencing revealed the cultured dog feces sample to be a mixed

culture containing five bacterial organisms namely, Proteus mirabilis strain

(CRPM10), Citrobacter sp. (RHB21-C05), Paeniclostridium sordellii strain

(AM370), Proteus mirabilis strain (AR_0059) and Proteus mirabilis strain

(PmSC1111) respectively. Taxonomic profiling from shotgun metagenomic analysis

revealed the presence of different microbial communities in animal feces (dog) and

environmental sources. The most prevalent species in animal feces, pond water and

soil sample were shown to be Klebsiella with 44 %, 49 % and 44 % respectively. It

was followed by Proteus species with relative abundance from animal feces (42 %),

pond water and soil with 39 % and 40% accordingly. Escherichia has shown to be the

least discovered across all samples with animal feces (4 %), pond water (4 %) and soil

sample (8 %).

This study remains critical in Africa, and highlights the importance of AMR

surveillance, and efforts towards the implementation of NGS to provide

comprehensive information on the occurrence and diversity of AMR and mobile

genetic elements from clinical and environmental sources. The research will also aid

in the recommendations for community education on antibiotic use, prevention, and

control measures in order to limit the spread of antibiotic resistance in community

settings.