Phenotypic and Molecular Characterization of Non-typhoidal Salmonella Species in Humans and Animals in Central Ethiopia and Inhibition of Biofilm Formation Using Small Molecule Adenosine Mimetics
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Date
2016-06
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Addis Ababa University
Abstract
This dissertation reports prevalence, serotype distribution and phenotypic and genotypic
characteristics of non-typhoidal Salmonella (NTS) in humans and animals in central
Ethiopia and effect of small molecule adenosine mimetic compounds on Salmonella biofilm
formation. Farm level and animal level Salmonella prevalence was (7.6%, 2.3%) in dairy;
(14.6%, 4.7%) in poultry; and (42.6%, 4.4%) in swine farms. The prevalence was 7.2% in
diarrheic patients from primary health centers and 2.1% from hospitals. S.Typhimurium
(27.6%) was the most frequently isolated serotype, followed by S. Saintpaul (21.7%), S.
Virchow (18.4%) and S. Kentucky (6.6%). Salmonella isolation was significantly associated
with detection of diarrhea in dairy cattle (p=0.012), and consumption of raw vegetables in
humans (OR=1.91, 95% CI=1.29-2.83, χ2=4.74, p=0.025). Drug resistance was more
common in dairy farms in Addis Ababa than outside (p=0.009) and overall antimicrobial
resistance was more common in animals than in humans. Clonally related genotypes of
S.Virchow, S.Typhimurium, S.Kentucky, S.Braendurp and S. Miami were circulating among
humans and animals as determined by pulsed-field gel electrophoresis (PFGE). MLST
analysis showed 3 novel allele types and 5 novel sequence types among 21 strains examined.
The dominant beta-lactamase enzyme was blaTEM type. BlaOXA10 and blaCTX-15 were
detected only in a single MDR S. Concord strain. Double mutation in gyrA (Ser83-Phe and
Asp87-Gly) as well as parC (Thr57-Ser + Ser80-Ile) subunits of quinolone resistance
determining region (QRDR) was the primary mechanism for resistance to quinolones and
was detected in all S. Kentucky isolates resistant to both nalidixic acid and ciprofloxacin
from animals (n=8) and humans (n=2). Although decreased susceptibility to ciprofloxacin
and/or nalidixic acid was observed in some isolates, no mutation in QRDR nor plasmid
mediated quinolone resistance (PMQR) genes were detected. Majority of Salmonella
ii
isolates exhibited robust biofilm formation (89%) and displayed red dry and rough (RDAR)
morphotype. Detection of class 1 integron was correlated with expression of multicellular
behavior and the extent of MDR. Screening of an ATP-mimetic library, gave a single
compound (7955004) capable of significant inhibition of Salmonella enterica and
Acinetobacter baumannii biofilm formation. The compound was not bactericidal or
bacteriostatic toward S. Typhimurium nor cytotoxic to mammalian cells. GroEL and DeoD
were found to be the potential protein-binding targets of the compound as identified by
ATP-sepharose affinity matrix. Circulation of clonally related NTS serotypes in food
animals and humans, abundance of MDR in isolates from food animals, co-dominance of
MDR and multicellular behavior in Salmonella isolates in the study area, increased the risk
of spreading resistant Salmonella strains and resistance genes to human population.
Integrated surveillance of NTS in humans and animals and implementation of appropriate
pathogen control strategy along critical points in food animal production from farm to bench
is recommended. The identification of a lead compound with biofilm inhibitory capabilities
toward Salmonella provides a potential new avenue of therapeutic intervention against
Salmonella and other bacterial pathogens. Further activity guided evaluation of compound
7955004 and its derivatives with the goal of increasing its potency and broadening its
spectrum of activity against additional biofilm forming pathogens should be conducted.
Key words: Antimicrobial resistance, Biofilm, Non-typhoidal Salmonella, Prevalence,
Serotype
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Keywords
Antimicrobial resistance, Biofilm, Non-typhoidal Salmonella, Prevalence