Molecular characterization of Salmonella and Salmonella specific bacteriophages from cattle and beef in the North West Province South Africa
Abstract
Salmonella species are currently one of the most widespread foodborne pathogens that
claim millions of lives yearly nationwide and therefore they pose severe challenges in
humans in developing as well as developed countries. In addition, Salmonella infections
are difficult to treat due to the ·emergence and wide spread distribution of antibiotic
resistant strains. Treatment failures amplify the need to explore the virulence
capabilities of other antibacterial agents such as bacteriophages to serve as alternative
antimicrobial agents, especially in resolving the war against resistant bacteria strains.
The aim of this study was to isolate, characterise and determine the virulence
capabilities of Salmonella species and Salmonella specific bacteriophages from cattle
faeces and raw beef obtained from some supermarkets and butcheries in the North
West Province, South Africa against environmental antibiotic resistant isolates.
A total of 300 presumptive Salmonella isolates were isolated from 160 samples (cattle
faeces and raw beef) using Salmonella Shigella agar. Typical pale yellow colonies with
black spots on their centres were subjected to both preliminary and confirmatory tests
specific for Salmonella species. The antibiotic resistance profiles of the Salmonella
strains were determined against a panel of 12 antimicrobial agents. Multiple antibiotic
resistance phenotypes and cluster analysis of antibiotic resistance inhibition zone
diameter data was used to determine the phenotypic relationship of isolates from
different sources. PCR assays were used to determine the pathogenicity of the isolates
in order to assess their impact on consumers. Genetic similarities among isolates from
different sampling stations and/or sources were assessed by determining the restriction
fragment length polymorphic (RFLP) patterns of the 16S rRNA gene fragments
amplified from isolates in the study. Furthermore, morphological characterisation of
Salmonella specific bacteriophages was achieved using the double agar layer technique
and electron microscopy. The findings of the study suggest a high presence 140 (46%)
of Salmonella species in both cattle faeces and raw beef obtained from the different
locations.
All the isolates were Gram negative rods while a significantly large proportion 291 (97%)
were oxidase negative and only 12 (4%) of the isolates produced H2S. Eight (34.8%)
representative isolates were identified as Salmonella species. Of the 300 presumptive
isolates a large proportion 159 (53%) were positive for Salmonella based on
agglutination with the polyvalent O antiserum and belonged to groups A-G, while 185
(61.7%) were positive for the polyvalent H antiserum. Salmonella species specific 16S
rRNA PCR analysis indicated that 128 (42 .7%) were positively identified. Despite the
fact that only a small proportion 60 (20%) of the isolates were confirmed as Salmonella
species based on the presence of the f/iC gene sequence while 80 (26.7%) had the f/iB
gene fragment.
Large proportions (62.4% to 94.3%) of the isolates from both cattle faeces and beef
samples were most often resistant to Erythromycin, Rifampicin, Penicillin, Ampicillin and
Cephalexin. On the contrary, these isolates showed very little resistance (4.3% to
37.9%) against Cefixime, Nalidixic Acid, Gentamicin, Ciprofloxacin, Tetracycline,
Norfloxacin and Chloramphenicol. MAR phenotypes E-RP-PG-CFXM, AP-E-RP-PGV
CFXM, RP-PG-CFXM, AP-CFM-E-RP-PG-CFXM, T-E-RP-PG-CFXM and AP-CFM-T-ERP-
PG-CFXM were dominant among these isolates. On the other hand, phenotypes
AP-CFM-NA-CIP-NOR-E-RP-PG, E-RP-PG-CFXM and T-E-RP-PG-CFXM were
predominant among isolates obtained from Rustenburg. Despite the fact that phenotype
AP-CFM-T-E-RP-PG-CFXM was observed only in 1 isolate obtained from Mafikeng, a
cause for concern is that this isolate was resistant to 7 of the 12 antibiotics tested. In
addition the AP-CFM-NA-CIP-NOR-E-RP-PG was not only dominant among isolates
from Rustenburg but the isolates were resistant to eight different antimicrobial agents.
Two major clusters (cluster 1 and cluster 2) that contained four sub-clusters (1A, 1 B, 2A
and 2B) were obtained. Generally three sub-clusters (1A, 1 B and 2A) were mixed since
they had isolates from almost all the different sites sampled.
Large proportions 38 (27.1%) and 46 (30.7%) were positive for the spvC and invA
virulence genes respectively. RFLP patterns of 16S rRNA gene fragments indicated that
the band sizes ranged from 50 bp and 572 bp and from 50 bp to 300 bp for EcoRI and
Haelll respectively. The great similarities in the antibiotic resistance profiles of
Salmonella isolates from the different locations indicated that isolates share similarly
antibiotic exposure histories. In addition, the similarities in the RFLP patterns of 16S
rRNA gene fragments also revealed that the data may be of great epidemiological
importance and therefore be very useful in identifying the source of contamination.