SciELO - Scientific Electronic Library Online

 
vol.8 número1Whole genome sequencing for drug resistance determination in Mycobacterium tuberculosis índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Articulo

Indicadores

Links relacionados

  • En proceso de indezaciónCitado por Google
  • En proceso de indezaciónSimilares en Google

Compartir


African Journal of Laboratory Medicine

versión On-line ISSN 2225-2010
versión impresa ISSN 2225-2002

Afr. J. Lab. Med. vol.8 no.1 Addis Ababa  2019

http://dx.doi.org/10.4102/ajlm.v8i1.760 

REVIEW ARTICLE

 

Review of molecular subtyping methodologies used to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-Saharan Africa

 

 

Anthony M. SmithI, II

ICentre for Enteric Diseases, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa
IIFaculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Correspondence

 

 


ABSTRACT

BACKGROUND: In sub-Saharan Africa, molecular epidemiological investigation of outbreaks caused by antimicrobial-resistant enteric bacterial pathogens have mostly been described for Salmonella species, Vibrio cholerae, Shigella species and Escherichia coli. For these organisms, I reviewed all publications describing the use of molecular subtyping methodologies to investigate outbreaks caused by multidrug-resistant (MDR) enteric bacterial infections.
OBJECTIVES: To describe the use of molecular subtyping methodologies to investigate outbreaks caused by MDR enteric bacterial pathogens in sub-Saharan Africa and to describe the current status of molecular subtyping capabilities in the region.
METHODS: A PubMed database literature search (English language only) was performed using the search strings: 'Africa outbreak MDR', 'Africa outbreak multi', 'Africa outbreak multidrug', 'Africa outbreak multi drug', 'Africa outbreak resistance', 'Africa outbreak resistant', 'Africa outbreak drug', 'Africa outbreak antibiotic', 'Africa outbreak antimicrobial'. These search strings were used in combination with genus and species names of the organisms listed above. All results were included in the review.
RESULTS: The year 1991 saw one of the first reports describing the use of molecular subtyping methodologies in sub-Saharan Africa; this included the use of plasmid profiling to characterise Salmonella Enteritidis. To date, several methodologies have been used; pulsed-field gel electrophoresis analysis and multilocus sequence typing have been the most commonly used methodologies. Investigations have particularly highlighted the emergence and spread of MDR clones; these include Salmonella Typhi H58 and Salmonella Typhimurium ST313 clones. In recent times, whole-genome sequencing (WGS) analysis approaches have increasingly been used.
CONCLUSION: Traditional molecular subtyping methodologies are still commonly used and still have their place in investigations; however, WGS approaches have increasingly been used and are slowly gaining a stronghold. African laboratories need to start adapting their molecular surveillance methodologies to include WGS, as it is foreseen that WGS analysis will eventually replace all traditional methodologies.


 

 

Introduction

For molecular epidemiological investigation of outbreaks in sub-Saharan Africa caused by antimicrobial-resistant (including multidrug-resistant [MDR]) enteric bacterial pathogens, most published data describe pathogens belonging to the genus or species of Salmonella, Vibrio cholerae, Shigella and Escherichia coli. By 'molecular epidemiological investigation', I refer to the use of molecular subtyping techniques that analyse bacterial strains at the level of their nucleic acid and so gives an indication of genetic similarity of strains.1 Molecular subtyping is vital for accurate epidemiological investigations of bacterial infections.2 Molecular subtyping allows one to segregate unlike strains of the same species or serotype of bacteria and identify clones or clusters of bacteria (genetically related strains). Molecular subtyping allows one to track the spread of strains or clones and determine how local strains compare to those circulating worldwide.3 Molecular subtyping data is critical for successful epidemiological investigation of outbreaks of disease; in particular, outbreak-related cases of disease can be differentiated from sporadic cases of disease.4 Knowledge of the molecular epidemiology of bacterial infections provides information of major circulating (infecting) clones, so that in times of antimicrobial treatment and vaccine interventions, well-informed and educated decisions can be made to combat the disease.5

The mid-1990s saw the start of noteworthy publications describing the use of molecular subtyping techniques to investigate the molecular epidemiology of antimicrobial-resistant enteric bacterial pathogens in sub-Saharan Africa.6 These molecular subtyping techniques included plasmid profiling, ribotyping (southern blotting of restricted genomic DNA and probing with ribosomal genes), random amplified polymorphic DNA analysis (also called arbitrarily primed polymerase chain reaction [PCR]), enterobacterial repetitive intergenic consensus elements PCR, pulsed-field gel electrophoresis (PFGE) analysis, multiple-locus variable-number tandem-repeats analysis (MLVA), multilocus sequence typing (MLST), multilocus sequence analysis and whole-genome sequencing (WGS) analysis. Molecular subtyping data from sub-Saharan Africa is summarised in Table 1 and the countries involved are highlighted in Figure 1. To date, the most popular and well documented molecular subtyping techniques have included PFGE, MLVA, MLST and WGS analysis. Traditional molecular subtyping methodologies, such as PFGE, MLVA and MLST, have their advantages and disadvantages; these have been well reviewed elsewhere on numerous occasions.1,2,7,8,9,10,11 Some molecular subtyping techniques (such as ribotyping, random amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus elements PCR) may only allow a local comparison of bacterial strains within a single laboratory, because the workings of the methodology are difficult to standardise between laboratories, and so the data that is produced cannot be truly and accurately compared between laboratories. In contrast, a technique such as PFGE analysis, has been standardised by PulseNet International (http://www.pulsenetinternational.org/) and is successfully used for inter-laboratory comparison of subtyping data and global comparison of bacterial strains.4

Newer approaches to molecular subtyping involve WGS analysis.12,13,14 Analysis of WGS data for molecular epidemiological purposes can include multiple approaches; however, the more popular methods are whole-genome MLST and single nucleotide polymorphisms (SNP) analysis.15 WGS data is electronically portable and can easily be shared between laboratories, allowing for a global inter-laboratory comparison of bacterial strains. Current challenges surrounding WGS include how to standardise the quality of WGS data generated and how to standardise the analysis of WGS data, in order to allow for a successful inter-laboratory comparison of analysed WGS data. PulseNet International has taken on this challenge and published their vision for implementation of WGS for global foodborne disease surveillance and global analysis of enteric pathogens.15

The aim of the present manuscript is to review all publications that have described the use of molecular subtyping methodologies to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-Saharan Africa. The review focuses on enteric pathogens belonging to the genus and species of Salmonella, Vibrio cholerae, Shigella and Escherichia coli, because most molecular epidemiological investigations have been described for these microorganisms. The aim of this review is to inform the readers about the current status of molecular subtyping capabilities and activities in sub-Saharan Africa and finally suggest that the way forward for molecular subtyping in African laboratories is the implementation of WGS analysis as soon as possible.

A PubMed database literature search (English language only) was performed using the search strings: 'Africa outbreak MDR', 'Africa outbreak multi', 'Africa outbreak multidrug', 'Africa outbreak multi drug', 'Africa outbreak resistance', 'Africa outbreak resistant', 'Africa outbreak drug', 'Africa outbreak antibiotic', 'Africa outbreak antimicrobial'. These search strings were used in combination with genus and species names of the organisms listed above. All results were included in the review.

 

Nontyphoidal Salmonella

For nontyphoidal Salmonella (NTS), molecular epidemiological analysis of Salmonella Typhimurium and Salmonella Enteritidis are most commonly described in sub-Saharan Africa. One of the first reports using molecular subtyping techniques to investigate enteric bacterial pathogens isolated in sub-Saharan Africa was a 1991 study that used plasmid profiling (following restriction endonuclease digestion) to distinguish African isolates of Salmonella Enteritidis from United States (US) isolates.16 Molecular subtyping of NTS then progressed to PFGE analysis of MDR Salmonella Typhimurium isolated in Kenya, where 64 isolates were grouped into eight PFGE clusters and were described as multiclonal.17Salmonella Typhimurium accounts for a significant proportion of reported cases of NTS-associated invasive disease in sub-Saharan Africa. NTS-associated invasive disease has developed to become a leading public health challenge in sub-Saharan Africa.18,19 A predominant type of invasive Salmonella Typhimurium in sub-Saharan Africa is a MDR strain that has been designated ST313 (based on its MLST profile). Clonal spread of this MDR ST313 strain was first reported from Kenya and Malawi in 2009, with the strain showing resistance to ampicillin, chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin and kanamycin.20 In Kenya, a more recent analysis of Salmonella Typhimurium outbreak isolates, using WGS-SNP analysis, identified a single clade of MDR ST313 strains showing resistance to ampicillin, ceftriaxone, chloramphenicol, sulphamethoxazole and trimethoprim; extended-spectrum beta lactamase (ESBL) genes blaCTX-M-15, blaTEM-1 and blaOXA-1 were also harboured by the isolates.21 The MDR ST313 has further been reported from Malawi, Nigeria, Democratic Republic of the Congo and South Africa.22,23,24 Leekitcharoenphon and coworkers22 used MLST, PFGE and WGS-SNP analysis to describe a close relationship between MDR ST313 strains from Nigeria and Democratic Republic of the Congo; strains were isolated from invasive (blood) and non-invasive (stool) specimens; all isolates harboured blaTEM1b, catA1, strA/B, sul1, and dfrA1 genes coding for resistance to various classes of antimicrobial agents. Keddy and coworkers23 used MLST to investigate Salmonella Typhimurium isolates from South Africa and reported that NTS meningitis in South Africa was highly associated with the MDR ST313 strain.

Besides Salmonella Typhimurium, Salmonella Enteritidis is also a major player with regard to NTS-associated invasive disease in sub-Saharan Africa. Feasey and coworkers25 used WGS-SNP analysis to investigate 675 Salmonella Enteritidis isolates from 45 countries (including 28 African countries), to describe the existence of one global epidemic clade and two 'African' clades, of which the African clades are geographically confined to specific regions of sub-Saharan Africa. Both African clades show MDR, with an enlarged MDR virulence plasmid. Both African clades also show patterns of genomic degradation with a similarity to those shown by other host-restricted invasive Salmonella serotypes described in Africa: patterns of genomic degradation similar to the African Salmonella Typhimurium ST313 clade.

Pulsed-field gel electrophoresis analysis is commonly used to investigate outbreaks of Salmonella Enteritidis.26,27,28 Niehaus and coworkers26 reported on a Salmonella Enteritidis foodborne outbreak in South Africa, where human isolates and a food isolate were shown to have an indistinguishable PFGE pattern. Salmonella Enteritidis investigations have also included nosocomial outbreaks; Vaagland and coworkers28 investigated a nosocomial outbreak of neonatal Salmonella Enteritidis in Tanzania, with PFGE analysis of MDR (ampicillin, chloramphenicol and cefuroxime) isolates suggesting a clonal outbreak. More recently, MLVA has proven a suitable method to investigate the molecular epidemiology of Salmonella Enteritidis in South Africa; MLVA was used to investigate multiple foodborne outbreaks over the period 2013-2015; MLVA was able to cluster outbreak isolates according to distinct MLVA profiles.29 Nosocomial outbreaks have also been investigated in South Africa, where Salmonella Isangi and Salmonella Typhimurium have been involved.30,31 Wadula and coworkers30 used PFGE analysis to show clonality among ESBL-producing Salmonella Isangi in paediatric hospital wards. Smith and coworkers31 used PFGE analysis, MLVA and MLST to investigate an ESBL-producing Salmonella Typhimurium outbreak in a paediatric hospital ward; outbreak isolates showed indistinguishable molecular subtyping profiles including an MLST subtype of ST34, and showed resistance to ampicillin, ceftriaxone, trimethoprim, sulphamethoxazole, chloramphenicol and tetracycline.

 

Typhoidal Salmonella

In 2004, Kariuki and coworkers32 documented one of the first reports using molecular subtyping techniques to characterise MDR Salmonella Typhi isolated in Africa. They used PFGE analysis to investigate 102 Kenyan outbreak isolates of Salmonella Typhi and identified two distinct subtypes among the infecting isolates; 82% of the isolates were MDR with resistance to ampicillin, chloramphenicol, tetracycline, streptomycin and cotrimoxazole. When PFGE analysis is standardised, as is the case with PulseNet methodology employed by participating PulseNet International laboratories, then inter-laboratory comparison of PFGE patterns can occur, to allow for a global investigation of outbreak isolates and tracking of emerging strains. Such was the case reported by Smith and coworkers,4 who used PFGE analysis to investigate an outbreak of Salmonella Typhi associated with a restaurant in South Africa; the source of the infecting strain was a restaurant worker who was tracked to Australia where a Salmonella Typhi isolate was recovered from the individual; the isolate shared an indistinguishable PFGE pattern as compared to the pattern of the outbreak strain. Using the same scenario as described above (comparison of PFGE patterns within the PulseNet International laboratory network), Keddy and coworkers33 investigated a MDR (including resistance to fluoroquinolones) Salmonella Typhi isolate recovered from a South African patient who had interacted with a person who had recently travelled to Bangladesh; the PFGE pattern of the isolate had never previously been seen within the South African PFGE database, but the PFGE pattern was typical of that seen of isolates from the Indian subcontinent, evidence to support the view that the isolate originated in Bangladesh. Global tracking of Salmonella Typhi through inter-laboratory comparison of molecular subtyping data is particularly important in establishing the source (and spread) of MDR (and virulent) strains of Salmonella Typhi. An example of such a strain is Salmonella Typhi H58, a highly clonal MDR haplotype of Salmonella introduced from Asia into Africa and now currently spreading through many sub-Saharan African countries.5

Further investigation of Salmonella Typhi outbreaks using PFGE analysis have been reported from the Malawi-Mozambique border,34 in Uganda35,36 and in South Africa.37 In Uganda, Walters and coworkers36 investigated a prolonged waterborne outbreak in two neighbouring districts; they used PFGE analysis to document the clonal spread of MDR Salmonella Typhi from the Kasese district to the Bundibugyo district. Analysis of selected isolates showed MDR to ampicillin, chloramphenicol, cotrimoxazole, streptomycin and tetracycline. In South Africa, Keddy and coworkers37 investigated a waterborne outbreak in the Delmas area of South Africa in 2005; they used PFGE analysis and MLVA to show high relatedness among outbreak isolates. These outbreak isolates from 2005 were also shown to be highly related to isolates associated with a similar waterborne outbreak in the same area in 1993. Besides PFGE analysis, MLVA is often described as a useful molecular subtyping method for discrimination of strains belonging to the Salmonella species. Tau and coworkers38 described the development and evaluation of a MLVA assay for molecular subtyping of Salmonella Typhi in sub-Saharan Africa. They evaluated the MLVA assay on a panel of African isolates and showed that it had higher discriminatory power as compared to PFGE; the MLVA assay was able to differentiate outbreak isolates from sporadic isolates.

Whole-genome sequencing - single nucleotide polymor-phisms analysis has been increasingly used to investigate the molecular epidemiology of Salmonella Typhi in sub-Saharan Africa; a common theme has been the description of the MDR Salmonella Typhi H58 strain.5,39 Wong and coworkers5 used WGS-SNP analysis to investigate 1832 Salmonella Typhi isolates from 63 global countries, to identify one major MDR lineage (named H58) that has arisen and spread across Asia and Africa over the past 30 years. Multiple H58 transfers have occurred, moving from Asia into Africa. An MDR H58 epidemic has been described in sub-Saharan Africa where H58 lineages are displacing antimicrobial-susceptible strains. MDR H58 lineages are associated with resistance to ampicillin, trimethoprim, sulphonamides, chloramphenicol, streptomycin and tetracycline, including reduced susceptibility to fluoroquinolones.5,39 This MDR H58 lineage has now been described in the eastern and southern regions of sub-Saharan Africa including Kenya, Tanzania, Malawi and South Africa.5,38,39,40 Hendriksen and coworkers41 also used WGS-SNP analysis to investigate an outbreak of Salmonella Typhi in Zambia; most isolates showed resistance to ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and trimethoprim, while some isolates also had reduced susceptibility to fluoroquinolones. Isolates belonged to a new variant of the H58 haplotype, namely haplotype H58B. Interestingly, investigation of MDR Salmonella Typhi from Nigeria -western Africa, using WGS-SNP analysis, has not detected the H58 lineage; instead the majority of isolates belonged to a different lineage -H56 lineage, which carries genes coding for resistance to ampicillin, tetracycline, chloramphenicol and sulphamethoxazole (blaTEM1, catA1, tetB, dfrA1, sul1). This H56 lineage is relatively common across Africa, predominantly in western and central regions of sub-Saharan Africa.42

 

Vibrio cholerae

Ribotyping and enterobacterial repetitive intergenic consensus elements PCR were among the first molecular subtyping techniques used to characterise V. cholerae O1 in Africa.43,44 In 1996, Dalsgaard and coworkers43 documented the first molecular epidemiological investigations of MDR V. cholerae O1 in Africa. They used ribotyping to investigate outbreak isolates in Guinea-Bissau; strains isolated in 1994-1995 showed a ribotype pattern which was different fro strains isolated in 1987, suggesting that the 1994-1995 outbreak was as a result of the introduction of a novel strain into the country. Later, ribotyping analysis of V. cholerae O1 outbreak isolates from Senegal showed the same ribotype pattern as the 1994-1995 Guinea-Bissau outbreak strain, suggesting that Senegal acquired their outbreak strain from Guinea-Bissau.45 In 2001, Dalsgaard and coworkers46 reported on the clonal relationship (using ribotyping) of cholera isolates from Mozambican migrant workers associated with a 1998 outbreak that occurred in South African provinces bordering Mozambique. MDR V. cholerae O1 isolates were associated with the outbreak among the migrant workers and the isolates showed resistance to furazolidone, streptomycin, sulphamethoxazole, trimethoprim and tetracycline; isolates also showed the presence of class 1 integrons and the SXT element. Another analysis of Mozambican MDR isolates associated with the 1998 Mozambique/South Africa cholera outbreak confirmed the presence of the SXT element among isolates, with enterobacterial repetitive intergenic consensus elements PCR dividing isolates into two different molecular subtypes.47 Mobile genetic elements are mostly responsible for the molecular basis of MDR V. cholerae O1 in sub-Saharan Africa. Mobile genetic elements include transposable elements (SXT elements), integrons and conjugative plasmids. The SXT element is a self-transmissible element that integrates into the chromosome and carries genes encoding resistance to several antimicrobial agents, including chloramphenicol, sulfamethoxazole, streptomycin, trimethoprim and furazolidone. The SXT element has been reported from numerous countries in sub-Saharan Africa.46,48,49,50,51 In 2006, Scrascia and coworkers52 investigated V. cholerae O1 isolates from multiple outbreaks that occurred in Kenya during 1998 and 1999. Most isolates showed an identical ribotype profile and similar random amplified polymorphic DNA analysis profile suggesting a clonal origin for the outbreaks; isolates were resistant to chloramphenicol, spectinomycin, streptomycin, sulphamethoxazole and trimethoprim. Over the period 2008-2009, published data from Ghana, Cameroon, Ethiopia and Somalia reported similar trends (as described above) for MDR V. cholerae O1 isolates following analysis using ribotyping and random amplified polymorphic DNA analysis - that of each country reporting a clonal origin for outbreak isolates within their respective country.50,53,54,55

From 2007 onwards, trends in molecular subtyping of V. cholerae, started shifting to an increased use of PFGE analysis. In 2007, Keddy and coworkers56 used PFGE analysis to compare the relatedness of South African V. cholerae O1 isolates from a 2001-2002 epidemic to that of a 1980-1987 epidemic; PFGE analysis showed that isolates from the 1980-1987 epidemic were distinctly different to isolates from the 2001-2002 outbreak. In 2008, Smith and coworkers57 reported on the first cholera outbreak in Namibia over the