Phenotypic and Molecular Characterizations of Salmonella Species in Ethiopia
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Date
2008-12
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Addis Ababa University
Abstract
Background: Salmonella infections are very common and an important public health problem in many parts of the world. In sub-Saharan Africa there is very little direct data on strain type or antibiotic resistance. Research to date, as well as unpublished reports from different health institutions in Ethiopia, has indicated that salmonellosis is a common problem and the extensive use of the first line drugs has led to the development of multiple drug resistance at a level which could pose a serious problem in the near future.
OBJECTIVES: The study was aimed at defining the serovars responsible for Salmonella infection, estimating the level of antibiotic resistance, investigate molecular basis of resistance and observing molecular polymorphism among Salmonella species isolated from children in Ethiopia.
MATERIALS AND METHODS: Pediatric patients (n=1225) presenting with diarrhoea or fever from pediatric out patient department of Tikur Anbessa University Hospital, Addis Ababa (n= 825) and Jimma University Hospital, south west Ethiopia (n= 400) were investigated for enteric pathogens during January 2006 to June 2008. Forty eight Salmonella isolates collected in Tikur Anbessa hospital between January, 2004 and December, 2005 from similar age group were analyzed together. Stool specimens were collected for
microscopic examination and culture. In addition blood specimens were obtained for culture. Brain Heart Infusion broth was used for blood culture and Selenite F broth enrichment followed by plating on Deoxycholate agar (DCA) and Xylose lysin deoxycholate agar (XLD) were used for stool culture. Identification of Salmonella species was carried out using API-20E, serology with antisera and strain were characterized using multilocus sequence typing (MLST). Based on resistance pattern, site of collection and type of specimen in which the isolate was detected, S. Concord isolates were selected and investigated for plasmid profile, incompatibility grouping, pulsed field gel electrophoresis (PFGE), MLST profile and fliC gene sequencing using standard procedures. In addition, 48 previously collected Salmonella isolates before the commencement of the present study were sero-grouped and serotyped.
RESULTS: A total of 463 entropathogens were isolated from 1225 pediatric patients. The isolates were: 65 Salmonella species, 61 Shigella species and 337 parasites. Among the 113 xi Salmonella isolates (65 + 48 previously collected), serogroup C, B, D and E were isolated at a frequency of 78.8%, 11.5%, 8% and 1.8% respectively. Most of the Salmonella isolates
were from stool (68%) and the rest were from blood (32%). No isolate was detected from both blood and stool of the same patient. A total of 12 serotypes were identified namely; S. Concord (82), S. Colindale (1), S. Gatow (3), S. Laronchelle (1), S. Garoli (1), S. Colorado (1), S. Typhimurium (7) S. Paratyphi B(2), S. Haifa (1) S. Typhi (2) S. Enteritidis (4), and S. Butantan (2). Eighty nine percent of the group C isolates were S. Concord. S. Concord isolates were highly resistant to ampicillin, trimethoprimsulfamethoxazole,
ceftriaxone, amoxicillin, chloramphenicol, gentamicin, and tetracycline.
Low resistance rate was observed for nalidixic acid and ofloxacin and there was no resistance to ciprofloxacin by disk diffusion test. However, E-test result indicated the presence of one resistant, one intermediate and twenty two (26.8%) Concord isolates which showed reduced susceptibility to ciprofloxacin. The extended spectrum beta lactamase (ESBL) screening test result showed that 98.8% of S. Concord were positive for ESBL production. Plasmid analysis showed that all characterized S. Concord isolates harbored
multiple copies of small and large plasmids. The molecular weight of plasmids varied from less than five to 170 kb with 120, 118 and 95 kb being the most prevalent. Plasmid replicons A/C, I1 and incFI were found in the majority of the isolates. Different plasmid studies indicated that A/C and incFI replicons are associated with multi drug resistance (MDR) Salmonella isolates. A total of 16 pulse field gel electrophoresis (PFGE) profiles were seen among the 23 Concord group and 5 non Concord isolates. Every sequence type (ST) had a unique PFGE profile which indicates that S. Concord in Ethiopia is in an endemic situation, rather than a spread of a clonal type (has many different point sources/reservoirs). The same ST and PFGE types were found in Addis Ababa and in Jimma suggesting movement of infected people/reservoirs between the two cities. Multilocus sequence typing (MLST)
analysis showed the presence of a total of seven STs among 58 isolates. ST533, ST534 and ST599 are single locus variants. Because they differ in only one of the seven loci, they are closely related genetically and make a single Concord group. Distantly related serotypes like Gatow and Colindale had sequence types that differ by more than one allele from Concord
group. Molecular serotyping using sequencing of the fliC gene was able to differentiate xii between S. Haifa and the S. Concord group of isolates indicating the possibility of its role in molecular serology. For our strain collection, it seems therefore that fliC sequencing can complement MLST for classification and strain differentiation.
CONCLUSION AND RECOMMENDATIONS: Salmonellosis in children in these two regions of Ethiopia is mainly due to non-typhoidal salmonellae particularly with S. Concord. This is different from other countries where S. Enteritidis and S. Typhimurium accounted for the majority of salmonellosis. Both phenotypic and genotypic characterization indicated that S. Concord is highly resistant and present in endemic situation in Ethiopia. The presence of
many multi-drug resistant strains containing genes for ESBL production and the emergence of reduced susceptibility to ciprofloxacin in this study posed a major concern in the search for efficient antimicrobial therapy of Salmonella infections in the near future. Therefore more comprehensive studies should be designed to trace its source and distribution within the country and to monitor antibiotic resistance pattern over time.
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Keywords
Phenotypic and Molecular, Salmonella Species