Antimicrobial resistance (AMR) is a worldwide public health concern. While it is
evident that the use of antibiotics creates selection pressure for the evolution of
antibiotic resistance genes, there are still considerable knowledge gaps relating to the
status quo of antibiotic use, emergence of resistant pathogens in different livestock
production systems and spread within human and animal communities.
This thesis includes a survey of antibiotic use in the dairy sector within a specific area
of Zambia and analysis of AMR and virulence factors in E. coli isolated from dairy
cattle and diarrhoea human patients with the following objectives.
1. To investigate the usage of antibiotics in the dairy sector and the drivers for
use.
2. To determine the prevalence and patterns of antimicrobial resistance in E. coli
isolated from faecal samples of dairy cattle.
3. To use whole genome sequencing (WGS) to investigate the molecular
epidemiology of resistance determinants in E. coli strains isolated from both
dairy cattle and humans.
4. To assess the zoonotic potential of isolated E. coli focusing on Shiga toxin-producing
E. coli (STEC) and relationship to STEC associated with clinical
disease in the UK.
In view of these objectives, the first part of the work was carried out in Zambia and
involved a questionnaire, a field survey, isolation of E. coli from dairy cattle faecal
samples and phenotypic testing for AMR. In addition, E. coli isolates were obtained
from another study that was focused on human patients presenting with diarrhoea at
the University Teaching Hospital in Lusaka. The second part involved whole genome
sequencing and molecular analyses of E. coli for resistance and virulence genotypes at
the Roslin Institute (UK). For the field study, a stratified random sample of 104 farms
was studied, representing approximately 20% of all dairy farms in the region. On each
farm, faecal samples were collected from a random sample of animals and a
standardised questionnaire on the usage of antibiotics was completed. An E. coli
isolate was obtained from 98.67% (371/376) of the sampled animals and tested for
resistance against the six types of antibiotics (tetracycline, ampicillin,
sulfamethoxazole/trimethoprim, cefpodoxime, gentamicin and ciprofloxacin). These
E. coli were then analysed together with those from humans for genotypes in the
laboratory and from Illumina short read whole genome sequences using bioinformatics
tools.
Tetracylines and penicillin were the commonly used antibiotics in dairy herds. This
finding was in line with the resistance phenotypes detected in E. coli isolated from the
dairy cattle. The most prevalent AMR was to tetracycline (10.61; 95%CI: 7.40-13.82),
followed by ampicillin (6.02; 95%CI: 3.31-8.73), sulfamethoxazole/ trimethoprim
(4.49; 95%CI: 2.42-6.56), cefpodoxime (1.91; 95%CI: 0.46-3.36), gentamicin (0.89;
95%CI: 0.06-1.84) and ciprofloxacin (0%). The risk analysis indicated that AMR was
associated with livestock diseases (lumpy skin disease and foot rot), exotic breeds
(Jersey and Friesian), location, farm size and certain management practices.
Analysis of whole genome sequences showed that isolates from humans had both
higher levels and a greater diversity of resistance alleles than the cattle isolates.
Common genotypes in both populations were: tetA (16%), tetB (10%), tetC (2%) for
cattle isolates with tetA (32%), tetB (22%) and tetD (1%) in human isolates. Other
common genotypes were blaTEM (56%), sul1 (29%), sul2 (66%), strA4 (57%) and
strB1 (64%) in isolates of human origin while blaTEM (15%), sul1 (3%), sul2 (17%),
strA4 (13%) and strB1 (19%) were in the cattle isolates.
Whilst the E. coli isolates from cattle encoded resistance to common antibiotics of
limited significance to human clinical medicine, isolates from humans had additional
extended spectrum beta-lactamases (blaOXA, blaCMY, blaNDM, and blaDHA, blaOKP and
blaCTX-M) that encode for resistance to essential antibiotics such as third generation
cephalosporins and carbapenems. This was an evidence that AMR is an ongoing public
health subject in Zambia but the exclusivity of certain resistances in the human
population points to limited or no exchange of genotypes between E. coli of human
origin and those from cattle. AMR in humans was probably independently selected by
the use of antibiotics of clinical importance such as cephalosporin and
fluoroquinolones.
The virulence analysis focused on STEC, 11% (41/371) of E. coli isolates from cattle
contained Shiga toxin genes (stx) while none (0/73) of the human isolates were
positive. Phylogenetic analysis showed a random distribution of bovine STEC, with
no indication of clonal spread. Although 89% (16/18) of the STEC tested had a
cytotoxic effect on Vero cells, indicative of Shiga toxin production, only three (O45,
O111, O157) belonged to one of the seven serogroups (O26, O157, O111, O103,
O121, O145 and O45) associated with life-threatening enterohaemorrhagic E. coli
(EHEC) infections in humans. In line with this, only the O157 serotype encoded a type
3 secretion system. This shows that, while Stx-encoding strains are common in these
dairy herds of Zambia, they are not strain backgrounds known to pose an immediate
threat to human health as they lack colonisation factors that are found in typical human
EHEC. However, we must remain vigilant as emergence of EHEC strains in these
animals remains an ever-present threat