AMR componentInvestigations in the component antimicrobial resistance revealed that there were substantial differences with respect to antimicrobial use between the peri-urban semiintensive production in Wakisu and the extensive production in Soroti. Nevertheless, antimicrobial resistance was frequent in bacteria from both regions including resistance to antimicrobials of special relevance to humans, such as 3rd and 4th generation cephalosporins and fluoroquinolones. 1. ILRI
AMRshort forAntimicrobial resistance graduate fellows Irene Mbatidde and Dickson Ndoboli have co-authored their first PhD paper on the risk factors of
AMRshort forAntimicrobial resistance occurrence in the two poultry production systems in Uganda. Besides supervising the PhD student Irene Mbattide
BfRshort forGerman Federal Institute for Risk Assessment carried out in depth analysis of
E. colishort forEscherichia coli isolates that were resistant to colistin or 3rd generation
Colistin-resistant E. coliPresumptive colistin-resistant
E. coli mixed cultures were inoculated in peptone water supplemented with 2
mgshort formilligram/L colistin and incubated overnight at 37°C while shaking. DNA was isolated from the enrichments and screened for mobile colistin resistant (
mcr) genes and positive isolates were identified by MALDI-TOF mass spectrometry. Next-generation sequencing was performed on
mcr-positive
E. colishort forEscherichia coli isolates on a NextSeq 500 Illumina platform. The antimicrobial resistance genes harboured by the bacteria were determined using the AMRfinder and Resfinder databases. The sequence types, virulence factors, mobile genetic elements, pathotypes, phylogroups, serotypes, and phylogenetic relatedness were determined. Further in-depth characterisation of selected isolates using long-read sequencing and plasmid conjugation experiments were performed. Lastly, the minimum inhibitory concentration (MIC) of colistin and 14 other key antibiotics was determined using broth microdilution. Four
mcr-1 positive isolates were obtained from the semi-intensive production system and one from the free-ranging, all showing MIC values of 8mg/L, well above the cut-off. Additional phenotypic resistance to ampicillin, ciprofloxacin, gentamicin, sulfamethoxazole, tetracycline, and trimethoprim, and nalidixic acid was observed. The
mcr genes were identified on IncI2 plasmids, and conjugation experiments proved that the colistin resistance was transmissible to other bacteria. Long-read sequencing is planned for the isolates. The low occurrence of multi-drug resistance with transmissible colistin resistance in poultry farms is encouraging but does highlight the need for continuous monitoring and urgent interventions to reduce the use of these critically important antibiotics in livestock.
Cefotaxime-resistant E. coliSelective isolation of ESBL-
E. coli from the mixed cultures received at the
BfRshort forGerman Federal Institute for Risk Assessment was performed on MacConkey agar supplemented with cefotaxime. Suspected
E. colishort forEscherichia coli isolates were confirmed using MALDI-TOF mass spectrometry, and genomic DNA was extracted from confirmed isolates. Short-read whole genome sequencing was performed on a NextSeq 500 Illumina platform. The antimicrobial resistance genes (ARGs) were determined using the AMRFinder and Resfinder databases. Further in-depth characterisation comprised the determination of sequence types, virulence factors, mobile genetic elements, pathotypes, phylogroups, serotypes, and phylogenetic relatedness using core genome multi-locus sequence typing. ESBL-
E. coli were obtained from 152/402 farms, with 72 being from Soroti and 80 from Wakiso. Genes that confirm resistance to folic acid antagonists, aminoglycosides and tetracyclines were commonly found in isolates from both semi-intensive and free-ranging poultry production systems. Analyses on the data are currently on-going and long-read sequencing on selected isolates is also planned. The widespread presence of ESBL-
E. coli and a diverse array of resistance genes observed in poultry farms highlights the need for strengthening
AMRshort forAntimicrobial resistance surveillance to monitor trends and further investigations to identify areas for targeted
AMRshort forAntimicrobial resistance mitigating interventions.
2.
VPH component Leptospires Single-locus sequence typing (SLST) was performed to determine
Leptospira genomospecies and those positive further investigated using MLST to determine sequence types (ST) and likely serogroups. The genomospecies
L. borgpetersenii (11 in cattle and one in goat),
L. kirschneri (five in cattle and four in sheep) and
L. interrogans (one in a pig) were determined using SLST. Thirteen novel sequence types of leptospires have been identified from this study and several new alleles of housekeeping genes in the
Leptospira MLST scheme 1 were also registered and publicly available on the PubMLST database (https://pubmlst.org/organisms/
leptospira-spp). These entries are the first ever from Uganda, and this knowledge will greatly help in characterizing future leptospires from the region which is important in studying leptospiral diversity and transmission dynamics. However, future studies that isolate leptospires and characterize using whole genome sequencing are imperative.
The ILRI graduate fellow Lordrick Alinaitwe has published one paper in
PLoS Neglected Tropical Diseases investigating the prevalence of leptospires and associated risk factors for in various hosts Uganda.
Heshort forhelium also submitted another paper which is currently under review in Scientific Reports exploring the genetic diversity of leptospires from the slaughterhouses in Uganda.
Heshort forhelium has successfully defended his PhD thesis acquiring his doctoral degree from the University of Bern.
Heshort forhelium has also presented in various international conferences.
Brucellae DNA isolation was performed using a protocol modified at the
BfRshort forGerman Federal Institute for Risk Assessment and tailored for long-read sequencing. Whole genome sequencing was performed using short-read sequencing and the resulting assemblies were generated using
BfRshort forGerman Federal Institute for Risk Assessment pipelines. Genomic characterization to identify species, sequence types,
AMRshort forAntimicrobial resistance genes, virulence factors, and phylogenetic relatedness was performed using
BfRshort forGerman Federal Institute for Risk Assessment pipelines.
B. melitensis was identified in ten samples and
B. abortus in four samples. Most of these isolates were obtained from cattle (four
B. abortus and two
B. melitensis), followed by goats (five
B. melitensis), humans (two
B. melitensis), and sheep (one
B. melitensis). The isolates showed a few variations in their genetic makeup depending on which host source.
B. melitensis samples from humans and cattle shared similar genomes as evidenced by very few shared single nucleotide polymorphisms (SNPs). The
B. melitensis obtained from sheep (from Mbale which is a town near neighbouring Kenya) showed to be very different from those from other sources.
Both
B. abortus and
B. melitensis share similar virulence genes, with the exception of the
bmaA gene that was only present in
B. abortus isolates and is associated with adherence to host cells. There were also no classically antimicrobial resistance genes (ARGs) and plasmids associated with the strains. However, five chromosomal efflux-related genes were detected in all isolates and are known to confer tolerance to some biocides and natural resistance to some antibiotics, a few of which are necessary for the treatment of uncomplicated human brucellosis. This knowledge can contribute to refining control strategies such as antibiotic therapy.
We lastly supplemented the short-read sequencing of one
B. abortus and
B. melitensis isolate with long-read sequencing, thereby representing the two
Brucella species found in the study. A hybrid-assembly from both sequencing approaches resulted in the first reliable reference sequences of
B. abortus and
B. melitensis from the region, an important resource for future studies on brucellae in Africa.
The ILRI graduate fellow James Bugeza has drafted his third doctoral paper based on these results and is currently being revised by co-authors in readiness for submission.
Heshort forhelium has also written his doctoral thesis and will soon defend his work at Makerere University.
Salmonellae The 380
Salmonella isolates were characterized by plating out and serotyping using the White-Kauffmann-Le Minor scheme. Non-typhoidal salmonellae were identified from the
BfRshort forGerman Federal Institute for Risk Assessment (n=24) and were shipped to the Institute for Animal Hygiene and Environmental Health, Freie Universität Berlin, for whole genome characterisation.