Identification of multidrug-resistant salmonella plasmid variants using polymerase chain reaction (PCR) based replicon typing technique in Ghana
Ellis Owusu-Dabo, Godfred Acheampong, Michael Owusul, Alex Owusu-Ofori, Isaac Osei, Nimako Sarpong, Se Eun Park, Florian Marks
Africa Health Agenda International Journal . 2019;1:6

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Identification of multidrug-resistant salmonella plasmid variants using polymerase chain reaction (PCR) based replicon typing technique in Ghana

Cite this: Africa Health Agenda International Journal. 2019;1:6.

Received: 20 Feb 2019 - Accepted: 06 Aug 2019 - Published: 12 Dec 2019

Key words: salmonella Typhi, non-typhoidal salmonella, multidrug resistance, polymerase chain reaction, replicon typing

© Ellis Owusu-Dabo et al. Africa Health Agenda International Journal - . This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Corresponding author: Ellis Owusu-Dabo, Department of Global and International Health, School of Public Health, Kwame Nkrumah University of Science and Technology, Ghana (

Identification of multidrug-resistant salmonella plasmid variants using polymerase chain reaction (PCR) based replicon typing technique in Ghana

Ellis Owusu-Dabo1,&, Godfred Acheampong2, Michael Owusul2,3, Alex Owusu-Ofori4,5, Isaac Osei2, Nimako Sarpong6, Se Eun Park7, Florian Marks7


1Department of Global and International Health, School of Public Health, Kwame Nkrumah University of Science and Technology, Ghana, 2Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana, 3Department of Medical Laboratory Technology, Kwame Nkrumah University of Science and Technology, Ghana, 4Department of Clinical Microbiology, Kwame Nkrumah University of Science and Technology, Ghana, 5Komfo Anokye Teaching Hospital, Kumasi, Ghana, 6Agogo Presbyterian Hospital, Agogo, Ghana, 7Department of Epidemiology, International Vaccine Institute, South-Korea



&Corresponding author
Ellis Owusu-Dabo, Department of Global Health, School of Public Health, Kwame Nkrumah University of Science and Technology, Ghana




Introduction: Salmonella infections cause significant morbidity and mortality, especially in resource-limited countries. The situation is worsened by widespread presence of multidrug resistant (MDR) strains, largely encoded on conjugative plasmids, but with little knowledge about how these plasmids are identified especially in low-middle income countries. We present findings of various plasmid variants possibly encoding MDR salmonella from Ghana.


Methods: this was a cross-sectional study involving individuals suspected of having salmonella infection presenting at two major hospitals in Ghana. Blood, stool and oropharyngeal specimens (OPS) were taken from consenting individuals between May, 2016 and January, 2018. Identification of salmonella was done using standard microbiological procedures. Total DNA was extracted from MDR salmonella isolates and PCR-based replicon typing (PBRT) performed using 30 replicons representative of the major incompatibility groups among Enterobacteriaceae.


Results: of 2,376 samples collected, 101 (4.3%) salmonella were isolated. Multidrug-resistant salmonella was detected in 34 (33.7%) strains; S. Typhi (67.6%), NTS (32.4%). Four different incompatibility (Inc) groups were identified by PBRT. Eleven S. Typhi bacteremic isolates (32.4%), harboured plasmids with Inc group HI1 of target size 534bp. The most predominant replicon (13/34; 38.2%) belonged to IncU plasmid. Non-typhoidal salmonella harboured 1 rare IncX2 plasmid and 8 IncFIIS plasmids known to encode resistance to carbapenems, colistins and several virulence genes.


Conclusion: this study shows presence of circulating plasmid variants likely to confer MDR in salmonella from clinical isolates. This is the first time Ghana has reported 3 (IncU, IncFIIS, IncX) of 4 variants and these are similar to those circulating in Africa but one (IncFIIS).



Introduction    Down

Salmonella infection has been a global problem for centuries. It is among the top four important bacterial agents of diarrhoeal illness reported globally [1], and caused by one of several serovars of gram negative facultative anaerobic Salmonella bacteriae. Cases of the infection are usually mild, especially in the case of NTS infection. However, it can be life-threatening, depending on host immune factors present and the type of serovar involved [2]. Generally, the disease is frequently characterized by abdominal pain, fever, diarrhoea, nausea and/or vomiting [3]. In Ghana, the most predominant Salmonella infections are caused by Salmonella enterica serovar Typhi (S. Typhi), and two other non-typhoidal Salmonella (S. Typhimurium and S. Enteritidis) [4, 5]. The emergence and spread of resistant strains of the organism have posed several health and economic threats globally. Of note is the occurrence of multidrug-resistant (MDR) Salmonella strains, defined as resistance to at least three classes of anti-Salmonella drugs [6, 7]. These MDR strains are known to be encoded on conjugative plasmids [8, 9], however, knowledge about the different variants of plasmids associated are poorly understood in Ghana. In the meantime, plasmids increase bacterial genetic diversity through the acquisition and loss of genes, especially those responsible for antibiotic resistance and/or virulence within the bacterial population [10]. A special property exhibited by plasmids which is more suitable for their classification is Incompatibility (Inc) grouping [11]. Incompatibility is a manifestation of relatedness, sharing common replication controls/equipartitioning elements. Thus, plasmids that are incompatible with each other are assigned to the same incompatibility group. The role of antimicrobial resistant plasmids is extremely vital as it allows for diffusion of these MDR Salmonella strains into different environments to be traced [11]. Knowledge of specific plasmids associated with antimicrobial resistance and virulence in bacterial host are able to be monitored when identified early [12]. While low-middle income countries of Africa (including Ghana) and Asia bear a greater brunt of the disease burden, very little is known about the various plasmid variants existing in those areas and their consequential diffusion within the environment. This study therefore sought to identify plasmids likely to be encoding MDR Salmonella into incompatibility groups (Inc groups) using a multiplex polymerase chain reaction (PCR) based replicon-typing approach for the first time in Ghana.



Methods Up    Down

Study design and area: this was a cross-sectional study conducted from May 2016 to January 2018 as part of a larger study that is examining the burden of severe typhoid in sub-Saharan Africa involving six countries (Ghana, Burkina Faso, Democratic Republic of Congo, Ethiopia, Nigeria and Madagascar). However, the present report is only from Ghana. In Ghana, there were two participating major hospitals, one rural (Agogo Presbyterian Hospital (APH)), and the other a major referral hospital referred to as Komfo Anokye Teaching Hospital (KATH) in Kumasi. Census data and hospital records were used to define the study catchment areas. The Agogo Presbyterian hospital typically serves patients living within and around the Asante-Akim North District (AND) of Ashanti region whereas KATH provides services for patients living within Kumasi metropolis (KM) and beyond.


Study population and sample size estimation: the study population comprised patients of all ages and gender attending the selected local health facilities presenting with symptoms of Salmonella infection such as fever (≥ 38°C tympanic and/or ≥ 37.5°C axillary) of at least three days’ duration, headache, stomach cramp and nausea. The minimum sample size expected was 150 based on 6.5%, 2.3% and 1.3% prevalence of Salmonella in blood, stool and oropharyngeal specimens (OPS), respectively using precision of 5% and standard normal deviation of 1.96 corresponding to 95% confidence interval [13-15]. However, we oversampled from the target population to take care of any population variation that may have inadvertently been ignored and to allow for adequate study power. In all a total of 2,376 samples was used for the study.


Ethical clearance: the consent process was at different levels. First we sought permission from all patients and their relatives before samples were taken from them. Children older than 7yrs but younger than 18yrs gave their assent together with parental/guardian consent before they were enrolled into the study. The study was explained to participants and their consent sought by means of signature and/or thumb printing. The main study protocol was reviewed and approved by the Committee for Human Research Publications and Ethics (CHRPE) at the School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST).


Sample collection and transportation: eight milliliters (adult) and 3ml (pediatric) of venous blood were collected using a 23G and 21G butterfly needles, respectively and inoculated directly into a blood culture bottle (Becton Dickinson (BD), Franklin Lakes, NJ USA). Stool samples were collected into 30ml sterile screw cap stool containers. About 1g (loopful) of it was inoculated into selenite broth on site before transportation. Again, OPS were collected from recruits using sterile floq swabs. The stool specimen and OPS were transported immediately from both sites to Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) laboratory at 4°C whiles the blood culture bottles were transported at room temperature.


Bacteria identification from clinical specimens


Bacteria cultures: a total of 2,376 samples (blood-1400, stool-418 and OPS-558) were collected from study participants from both hospitals. Variance in sample numbers in the three samples was as a results of unwillingness of study participants to provide stool and OPS specimens more readily compared to the blood samples. Blood samples collected into culture bottles were incubated in BACTECTM 9050 blood culture system at 35°C for five days or until positive signal was detected. Direct stool and OPS were partly plated on xylose lysine deoxycholate (XLD) agar and incubated overnight (18-24hours) at 35-37°C. Other portions of stool samples (already inoculated in selenite broth) and OPS enriched in selenite broth were also incubated overnight at 35-37°C for recovery of injured Salmonella. Inoculated stool specimen and OPS were streaked onto XLD agar after 24hours of incubation using sterile 10µl bacteriological loop.


Identification of Salmonella: positive blood culture samples were plated onto 3 standard growth media: blood agar (BA - Columbia agar base supplemented with 5% sheep blood), chocolate agar (CA) and macConkey (Mac) agar (BD, USA) under sterile working condition. MacConkey and blood agar were incubated overnight under aerobic conditions at 35-37°C whiles CA plates were incubated in candle jar for microaerophilic condition at the same temperature. Any bacterial growth on all 3 media observed as small creamy non-hemolytic colonies on BA and CA, and its corresponding Mac plate giving small transparent non-lactose fermenting colonies were noted for further testing. On XLD agar, any pink colonies with black pigments from stool and OPS were investigated for Salmonella identification. Pure colonies were sub-cultured from XLD onto blood agar for biochemical tests. Gram negative short rods gave a presumptive identification. Biochemical tests were performed as follows: triple sugar iron agar (TSI) – red slope on yellow butt (R/Y), with strong/weak hydrogen sulphide (H2S) production and gas/no gas seen; negative indole and urease tests; positive or negative citrate test and SIM test - sulphide (+/-), indole (-) and motility (+). Again, latex agglutination test (Oxoid, UK) was performed on the presumptive Salmonella isolates to check for agglutination after emulsifying pure colonies in commercially prepared latex antisera for 10 seconds. Confirmation of Salmonella was done by Analytical Profile Index (API) 20E (Biomerieux, France). Finally, serotyping was performed to identify which serovar of Salmonella was invloved.


Antimicrobial susceptibility testing: the antibiotics chosen for testing were based on current treatment regimens for Salmonella infections as well as clinical and laboratory standards institute [16] standards. Susceptibility to ampicillin (10µg), amoxiclav (amoxicillin & clavulanic acid; 20/10µg), ceftriaxone (30µg), trimethoprim/sulfamethoxazole (1.25/23.75µg), ciprofloxacin (5µg), gentamicin (10µg), tetracycline (30µg), chloramphenicol (30µg), ceftazidime (30µg), cefotaxime (30µg) and nalidixic acid (30µg) was tested on Mueller-Hinton agar (BD, USA) using the Kirby-Bauer disc diffusion method. Isolates resistant to 3 or more classes of antibiotics were regarded as MDR.


Quality control: escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC 14028 were set up together with the test organisms to control media, biochemical tests and potency of antibiotic discs.


DNA extraction: total DNA was extracted on all MDR Salmonella isolates from blood, stool and OPS using spherolyse DNA extraction kit according to manufacturer’s instructions (Hain-life Science, Germany).


Plasmid incompatibility profiling: PCR-based replicon typing (PBRT) method was used to detect plasmid variants encoding MDR in Salmonella isolates using PBRT 2.0 kit (Diatheva, Italy). This kit contains novel set of 8 panels (designated as M1, M2, M3, M4, M5, M6, M7 and M8) of specific PCR assays optimized to perform 8 multiplex PCRs for the amplification of 30 replicons (Table 1). These replicons/plasmid variants represent the major plasmid incompatibility groups and replicase genes identified on resistance plasmids circulating in Enterobacteriaceae [17,18]. Total DNA preparations were used as templates along with incompatibility groups within 8 panels in order to amplify specific target regions (Table 1).


PCR mastermix preparation: for 1 sample, 23.8µl PCR mix from tube M1 was pipetted into an empty sterile 1.5ml Eppendorf tube. 0.2µl DNA polymerase was added and mixed gently by vortexing. Two extra 1.5ml tubes each containing 23.8µl and 0.2µl PCR mix from tube M1 and DNA polymerase, respectively were added to serve as positive and negative control tubes. The same procedure was performed for the rest of the PCR mixes (M2 - M8). One microlitre of template DNA and controls were added to their respective PCR mix tubes containing DNA polymerase.


Polymerase Chain Reaction (PCR): amplification of targeted replicons within the extracted samples was performed by conventional multiplex PCR using a 96-well verity thermal cycler (Applied Biosystems, USA). The following amplification conditions were used to run all the 8 panels (M1 - M8): 1 cycle of initial denaturation at 95°C for 10 minutes, 30 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds, elongation at 72°C for 1 minute and final extension cycle at 72°C for 5 minutes. The final amplicons were kept on hold at 4ºC prior to collection for agarose gel electrophoresis.


Agarose gel electrophoresis: ten microlitres each of the PCR amplicon was mixed with 2µl loading buffer (bromophenol blue) and the final volume loaded onto a 2.5% agarose gel containing 2μl of ethidium bromide. Positive and negative controls were also loaded for each set (M1-M8), including 12µl of molecular ladder specific for low range (100-1000bp). Electrophoresis was initiated for 2 hours at 100V (Figure 1). Visualisation of the gel was done by ultraviolet (UV) transilluminator and captured onto a desktop computer connected to it using Infinity® software.


Statistical Analysis: a paper-based questionnaire was administered to record each participant’s biodata. Blood, stool and OPS were taken from each consented participant and those positive for MDR Salmonella were subjected to PBRT to determine the presence of plasmid variant(s) encoding MDR. Data were collected, entered into Microsoft excel and exported to STATA version 12 (Stata Corp, USA) for analysis. Descriptive statistics was used to summarize the distribution of various variables into tables and graphs. Differences between discrete variables were analysed using chi-square with 95% confidence interval and 5% margin of error.



Results Up    Down

General characteristics of the study population: during the study period of May, 2016 to January, 2018, a total number of 4,561 participants were approached and screened for eligibility. However, 1,400 were eligible and consented to take part in the study (Table 2). Overall, a total number of 2,376 samples were collected from the two study sites. One thousand four hundred blood, 418 stool and 558 OPS representing 58.9%, 17.6% and 23.5%, respectively were obtained from the study participants. At KATH alone, of 680 samples received, 364 (53.5%) were blood samples for culture, 159 (23.4%) were stool and 157 (23.1%) OPS specimens. Of 1,696 samples collected from APH, blood culture samples were 1,036 (61.1%), 259 stool (15.3%) and 401 OPS (23.6%).


Salmonella identification from clinical specimens: of 2,376 samples collected and analysed, 101 (4.3%) confirmed Salmonella isolates were obtained from blood (n=84), stool (n=13) and OPS (n=4). Salmonellae were classified either as serovar Typhi or Non-typhoidal Salmonella (NTS). Of 101 Salmonella isolates, 68 (67.3%) S. Typhi and 16 (15.8%) NTS were isolated from blood; 3 (2.9%) S. Typhi and 10 (9.9%) NTS from stool; and 1 (0.9%) S. Typhi and 3 (2.9%) NTS isolated from OPS. Children (between <1 to 19 years) were predominantly infected with both S. Typhi (68/72) and NTS (27/29) than adults.


Antimicrobial susceptibility profile of Salmonella isolates: all Salmonella isolates from blood, stool and OPS were tested to determine their antimicrobial susceptibility to selected antibiotics (Table 3). Trimethoprim/Sulfamethoxazole showed highest rate of resistance (27/72; 37.5%) in S. Typhi whiles no resistance was observed in gentamicin and 2 cephalosporins (ceftazidime and ceftriaxone). Conversely, among those NTS infected samples, ampicillin recorded the highest resistance (14/29; 48.3%) whiles ciprofloxacin, gentamicin and 3rd generation cephalosporins showed excellent activity (Table 3). Thirty-four (33.7%) of the 101 Salmonella isolates exhibited multidrug-resistance. Overall mean age (mean ± SE) of individuals infected with any MDR Salmonella was 6.5 ± 1.0 (p = 0.0001). Mean ages of individuals infected with MDR S. Typhi and NTS were 7.9 ± 1.2 and 2.5 ± 1.7 (p = 0.0001), respectively. S. Typhi MDR (23/34; 67.6%) was twice as many as non-typhoidal Salmonella MDR (11/34; 32.4%). The multidrug-resistance distribution of the Salmonella identified showed 16 (47.1%), 11 (32.3%) and 7 (20.6%) isolates were resistant to 3, 4 and 5 antibiotics, respectively (Table 4).


Incompatibility profiling: PCR-based replicon typing produced 4 different plasmid incompatibility (Inc) groups that could be encoding Salmonella MDR in the clinical samples tested. Two of these Inc groups (IncHI1 and IncU) were detected in MDR S. Typhi from the blood. Specifically, 11 (32.4%) isolates, harboured IncHI1 of target size 534bp (Figure 1 A). Majority (13/34; 38.2%) of the MDR S. Typhi isolates tested possessed replicon of size 843bp belonging to IncU (Figure 1 D). Overall mean ages of individuals with MDR S. Typhi possessing IncHI1 and Inc U were 8.8 ± 1.8 and 9.5 ± 1.6, respectively. Individuals between the ages of 6-10 were mostly infected with both IncHI1 and Inc U S. Typhi (Table 5). Male children (8/11; 72.7%) infected with S. Typhi were predominantly found to be encoding plasmids with Inc HI1 replicons than female children (3/11; 27.3%) (Table 5). Patients who visited APH were significantly infected with MDR Salmonella from the Inc HI1 plasmid group (p=0.01) (Table 5). Unlike S. Typhi, NTS bacteremia yielded 8 (23.5%) isolates belonging to Inc FIIS of target size 260bp (Figure 1 B) with female children of age group <1 – 5 years mostly affected (5/8; 62.5%) (Table 5). Again, children between the ages of <1 – 5 years were found to be significantly prone to harbour IncFIIS plasmid than the older groups (p=0.01) (Table 5). Incompatibility group Inc X2 was detected in only 1 female NTS isolate (Figure 1 C). Multireplicon isolates were detected in 4 of the samples tested: 1 isolate (NTS) harboured both IncX2 and IncFIIS plasmids whereas 3 (all S. Typhi) harboured plasmids in the IncU and IncHI1 groups (Table 6).



Discussion Up    Down

This study found appreciable numbers of MDR cases of both Salmonella Typhi and non-typhoidal Salmonella in the two study populations. Four plasmid variants were identified in MDR S. Typhi (IncHI1 and IncU) and NTS (IncFIIS and IncX2). Children less than 15 years were significantly associated with MDR Salmonella belonging to IncU and IncFIIS plasmids. Again, individuals seeking medical care in rural Ghana were found to be more likely to possess IncHI1 plasmid than those from the urban area. Globally, Salmonella infection poses a significant public health threat. Enteric fever disease occurs throughout the tropics and sub-tropics of Africa, Asia and South America, with a high disease burden in South-East Asia. However, NTS has a high burden in sub-Sahara Africa with moderate infection in South-East Asia [19-21]. In Ghana, annual cumulative S. Typhi incidence for children <5 years was reported in a study as 3.3 per 1000 and that of NTS as 25 per 1000 population, giving a clear indication of an alarmingly high rate of Salmonella infection in the country [22].Antibiotic resistances in all genera of disease-causing bacteria have increased tremendously in recent times [7, 23]. There is therefore the need by the scientific community to undertake routine surveillance of microbial populations to determine the extent of these resistances. Inexpensive first line drugs (ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole) used for Salmonella treatment in the past are no longer effective, paving way for third generation cephalosporins and fluoroquinolones [24, 25]. In this study, 101 clinical isolates of S. Typhi (71.3%) and NTS (28.7%) were investigated for their susceptibility to antibiotics used in the management or/and treatment of Salmonella infection in Ghana. Of the 101 Salmonella bacteria isolated, 34 (33.7%) were classified as MDR out of which 23 (67.6%) and 11 (32.4%) were MDR S. Typhi and NTS, respectively. This is consistent with high rates of MDR S. Typhi reported in India (97%), Cambodia (90%), Iraq (83%), Nigeria (34%) and Egypt (36%) and NTS reported in Cambodian (33%) and Ugandan (15.4%) populations [26-30]. The impact of MDR is very devastating; leading to increased morbidity and mortality, especially in children under age 5 years (as seen in this study) and those who are malnourished [31]. This study adds to the body of evidence that mass immunization (in the case of typhoid fever) in endemic areas, rational use of antibiotics, improvement in public sanitary conditions, promotion of safe drinking water and food handling practices are still lacking [32]. The rate of ciprofloxacin (a fluoroquinolone) and other cephalosporins resistance in both S. Typhi and NTS remained low, similar to previous studies conducted in Ghana [13, 22, 33, 34] but significantly higher in other countries [35, 36] including Democratic Republic of Congo [37] and Kenya [38]. In Ghana, prices of ciprofloxacin and ceftriaxone are expensive; limiting their widespread use and that might explain why resistance to these drugs is still low. To better understand how resistant bacterial strains emerge and spread, it is important to know the molecular basis of their resistance and how the resistant determinants (usually plasmids) are characterized. The action(s) of plasmids are regulated by their replication controls (replicons) and these have proven to be an effective way to characterize them into their incompatibility groups [11].


More often than not, resistance to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline (AMPCSXTTET) are heavily encoded on plasmids with incompatibility group Inc HI1 [7, 9, 39]. These agree partly with our study as 6 out of 11 MDR S. Typhi isolates that belonged to IncHI1 produced resistance pattern AMPCSXTTET. However, 5 other MDR isolates of IncHI1 yielded different antibiotic resistance patterns (Table 6). Again, we identified MDR Salmonella strains belonging to other incompatibility groups (IncFIIS and IncU) conferring resistance with pattern AMPCSXTTET that had never been reported in Ghana and the rest of Africa. This indicates that plasmids belonging to incompatibility group IncHI1 is not the sole source of resistance to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline as previously thought. Plasmids with incompatibility group IncF encountered in clinical bacteria strains are mostly associated with the spread of antimicrobial resistance and virulence genes [40], key among them include the blaCTX-M-15 and IncFIme plasmids, respectively [17]. Characterisation of some bacteria strains, especially Salmonella enterica, Shigella sp., enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC) and enteroinvasive E. coli (EIEC) by their virulence traits such as bacteriocins, siderophores, adhesion factors and cytotoxins are specifically encoded on IncF plasmids [40]. A subtype of IncF plasmid identified in this study, IncFIIS, is responsible for carrying transfer, MDR and virulence functions [40, 41]. The most predominant replicon detected in this study belonged to the incompatibility group IncU, similar to a study reported in Brazil [42]. Plasmids in this group are commonly isolated from many clinical and environmental strains of Escherichia coli and Aeromonas sp. with a wide global distribution [43, 44]. These plasmid replicons have a broad-host-range and are capable of carrying transfer and multidrug-resistance functions [40, 45]. Aside IncU plasmids encoding MDR in this study, other studies have reported that many IncU plasmids encode plasmid-mediated quinolone resistance determinants: qnrS2 and aac(6’)-Ib-cr [42, 46, 47]. A variant of narrow-host-range IncX plasmid we identified, IncX2, is a rare plasmid type commonly found in Enterobacteriaceae [48]. IncX2 plasmids are receiving much attention lately as they harbor and transfer carbapenem and colistin resistant genes such as blaNDM, blaKPC and mcr-1 [49]. Furthermore, a study in Nigeria shows that IncX plasmids serve as viable platform for dissemination of plasmid-mediated quinolone resistance (qnr) genes, especially qnrS1 [50]. It is worthy of note that IncHI1 and IncU plasmids encoding MDR serovar Typhi were more prevalent in male children within the 6-10 years age group, while IncFIIS and IncX2 found in MDR NTS were predominantly found in female children less than 1 to 5 years. However, it remains unclear why male children infected with MDR S. Typhi had high preponderance to IncHI1 and IncU whereas female children had high affinity to IncX2 and IncFIIS plasmid variants, although they were statistically not significant. This could suggest that genotypic distribution of both MDR Typhi and NTS might possibly be age and gender dependent, which could be beneficial for vaccination programmes that targets these age groups. Further, the likelihood that an MDR infection from rural Ghana could possess plasmid with incompatibility group HI1 is high compared to those living in urban.



Conclusion Up    Down

This study reports on plasmid variants in two study populations in Ghana that might possibly confer multiple drug-resistance in typhoidal and non-typhoidal Salmonella serovars from clinical isolates. Some variants are similar to those already known circulating in Africa (IncHI1, IncX2 and IncU) while one is not yet known (IncFIIS). Further studies should look into using a more rigorous technique such as multi-locus sequence typing (MLST) and/or whole genome sequencing to characterize plasmids associated with MDR to provide more comprehensive data on plasmid-mediated MDRs across the regions in Ghana and Africa.

What is known about this topic

  • Multidrug-resistant (MDR) Salmonella enterica infection in humans causes significant economic burden on patient as a result of treatment failure and subsequently death;
  • These MDR Salmonella strains are largely encoded on conjugative plasmids.

What this study adds

  • Cases of invasive MDR typhoidal and non-typhoidal Salmonella are still alarming;
  • The use of molecular typing technique to classify plasmids possibly encoding MDR Salmonella has revealed 4 plasmid variants circulating within the study sites in Ghana;
  • To strengthen health systems, antimicrobial resistance surveillance and distribution of MDR plasmids should be given attention, and this information could help direct interventional measures to where they are needed most and advocate for a strong surveillance system to be put in place to strengthen and monitor the progressive diffusion of plasmid-mediated MDR Salmonella strains in Ghana and Africa at large.



Competing interests Up    Down

The authors declare no competing interests.



Authors´ contributions Up    Down

Conception and designing the experiments: Ellis Owusu-Dabo, Michael Owusul, Godfred Acheampong, and Florian Marks. Performing the experiments: Michael Owusul & Godfred Acheampong. Data collection and analysis: Michael Owusul, Godfred Acheampong, and Isaac Osei. Contributing reagents/materials/analysis tools: Ellis Owusu-Dabo Michael Owusul and Se Eun Park. Writing the paper: Godfred Acheampong & Michael Owusul. Critical review of the manuscript: Alex Owusu-Ofori, Isaac Osei, Nimako Sarpong Ellis Owusu-Dabo Michael Owusul.



Acknowledgments Up    Down

This study was funded by Bill and Melinda Gates Foundation. We thank the study participants for their immense contribution and co-operation. We are also grateful for the continuous endeavors of the field workers and laboratory staff of the Kumasi Center for Collaborative Research (KCCR) and affiliated contributors, without whose efforts this research would not have been possible. Again, thank you to all staff of APH and KATH for their support.



Tables and figure Up    Down

Table 1: PCR mix and targeted amplicon sizes

Table 2: demographic attributes of study participants

Table 3: rate of Salmonella resistance against antibiotics tested

Table 4: multidrug-resistance pattern of Salmonella strains isolated

Table 5: effect of socio-demographics on detected plasmid variants

Table 6: MDR Salmonella strains by PCR-based inc/rep typing method

Figure 1: representative multiplex agarose gel (2.5%)



References Up    Down

  1. WHO. Salmonella (Non-typhoidal). 2017. Accessed on October 8, 2017.

  2. Eng SK, Pusparajah P, Ab Mutalib NS, Ser HL, Chan KG, Lee, LH. Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science. 2015; 8(3):284-293. Google Scholar

  3. Gal-Mor O, Boyle EC, Grassl GA. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Frontiers in microbiology. 2014;5:391. PubMed | Google Scholar

  4. Andoh LA, Ahmed S, Olsen JE, Obiri-Danso K, Newman MJ, Opintan JA et al. Prevalence and characterization of Salmonella among humans in Ghana. Tropical medicine and health. 2017;45:3. PubMed | Google Scholar

  5. Al-Emran HM, Krumkamp R, Dekker DM, Eibach D, Aaby P, Adu-Sarkodie Y et al. Validation and identification of invasive Salmonella serotypes in Sub-Saharan Africa by multiplex polymerase chain reaction. Clinical Infectious Diseases. 2016;62(suppl1):S80-S82. PubMed | Google Scholar

  6. Espié E, De Valk H, Vaillant V, Quelquejeu N, Le Querrec F, Weill F. An outbreak of multidrug-resistant Salmonella enterica serotype Newport infections linked to the consumption of imported horse meat in France. Epidemiology & Infection. 2005 April 8;133(2):373-376. PubMed | Google Scholar

  7. Mills-Robertson F, Addy ME, Mensah P, Crupper SS. Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana. FEMS microbiology letters. 2002;215(2):249-253. PubMed | Google Scholar

  8. Mills-Robertson F, Crupper S, Addy M, Mensah P. Antibiotic resistance and genotyping of clinical group B Salmonella isolated in Accra, Ghana. Journal of applied microbiology. 2003 Jan 13;94(2):289-294. PubMed | Google Scholar

  9. Shanahan PM, Jesudason MV, Thomson CJ, Amyes SG. Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India. Journal of Clinical microbiology. 1998 June; 36(6):1595-1600. PubMed | Google Scholar

  10. Francia MV, Varsaki A, Garcillán-Barcia MP, Latorre A, Drainas C, de la Cruz F. A classification scheme for mobilization regions of bacterial plasmids. FEMS microbiology reviews. 2004 Feb; 28(1):79-100. PubMed | Google Scholar

  11. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. Journal of microbiological methods. 2005 June 2;63(3):219-228. PubMed | Google Scholar

  12. Johnson TJ, Wannemuehler YM, Johnson SJ, Logue CM, White DG, Doetkott C et al. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates. Applied and environmental microbiology. 2007;73(6):1976-1983. PubMed | Google Scholar

  13. Labi A-K, Obeng-Nkrumah N, Addison NO, Donkor ES. Salmonella blood stream infections in a tertiary care setting in Ghana. BMC infectious diseases. 2014;14:3857. PubMed | Google Scholar

  14. Feglo P, Frimpong E, Essel-Ahun M. Salmonellae carrier status of food vendors in Kumasi, Ghana. East African medical journal. 2004 July;81(7):358-361. PubMed | Google Scholar

  15. Gravel J, Ceroni, D, Lacroix, L, Renaud, C, Grimard, G, Samara, E et al. Association between oropharyngeal carriage of Kingella kingae and osteoarticular infection in young children: a case–control study. Canadian Medical Association Journal. 2017;189(35):E1107-E1111. PubMed | Google Scholar

  16. CLSI. Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement. CLSI standards for antimicrobial susceptibility testing . 2015. Accessed on October 8, 2017

  17. Villa L, García-Fernández A, Fortini D, Carattoli A. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. Journal of antimicrobial chemotherapy. 2010;65(12):2518-2529. PubMed | Google Scholar

  18. García-Fernández A, Villa L, Carta C, Venditti C, Giordano A, Venditti M et al. Klebsiella pneumoniae ST258 producing KPC-3 identified in Italy carries novel plasmids and OmpK36/OmpK35 porin variants. Antimicrobial agents and chemotherapy. 2012 Apr;56(4):2143-5. PubMed | Google Scholar

  19. Buckle GC, Walker C, Black RE. Typhoid fever and paratyphoid fever: Systematic review to estimate global morbidity and mortality for 2010. J Glob Health. 2012;2(1):10401. PubMed | Google Scholar

  20. Gill CJ, Hamer DH. Foodborne illnesses. Current treatment options in gastroenterology. 2001;4(1):23-38. PubMed | Google Scholar

  21. Gilchrist JJ, MacLennan CA, Hill AV. Genetic susceptibility to invasive Salmonella disease. Nature reviews Immunology. 2015;15(7):452-63. PubMed | Google Scholar

  22. Nielsen MV, Sarpong N, Krumkamp R, Dekker D, Loag W, Amemasor S et al. Incidence and characteristics of bacteremia among children in rural Ghana. PloS one. 2012;7(9):e44063. PubMed | Google Scholar

  23. Laxminarayan R, Chaudhury RR. Antibiotic resistance in India: drivers and opportunities for action. PLoS medicine. 2016;13(3):e1001974. PubMed | Google Scholar

  24. Lee K, Yong D, Yum, JH, Lim YS, Kim HS, Lee BK et al. Emergence of multidrug-resistant Salmonella enterica serovar typhi in Korea. Antimicrobial agents and chemotherapy. 2004; 48(11):4130-4135. PubMed | Google Scholar

  25. Yoon HJ, Cho SH and Kim SH. A case of multidrug-resistant Salmonella enterica serovar typhi treated with a bench to bedside approach. Yonsei medical journal. 2009;50(1):147-151. PubMed | Google Scholar

  26. Balaji V, Sharma A, Ranjan P, Kapil A. Revised ciprofloxacin breakpoints for Salmonella Typhi: its implications in India. Indian journal of medical microbiology. 2014;32(2):161. PubMed | Google Scholar

  27. Rahman B, Wasfy M, Maksoud M, Hanna N, Dueger E, House B. Multi-drug resistance and reduced susceptibility to ciprofloxacin among Salmonella enterica serovar Typhi isolates from the Middle East and Central Asia. New microbes and new infections. 2014;2(4):88-92. PubMed | Google Scholar

  28. Vlieghe ER, Phe T, De Smet B, Veng CH, Kham C, Bertrand S, Vanhoof R et al. Azithromycin and ciprofloxacin resistance in Salmonella bloodstream infections in Cambodian adults. PLoS neglected tropical diseases. 2012;6(12):e1933. PubMed | Google Scholar

  29. Mathew B, Ugboko H, De N. Prevalence of Multidrug Resistant Salmonella enterica Serovar Typhi in Kaduna Metropolis, Kaduna, Nigeria. International Journal of Current Microbiology and Applied Sciences. 2015;4(9):323-335. Google Scholar

  30. Odoch T, Wasteson Y, L’Abée-Lund T, Muwonge A, Kankya C, Nyakarahuka L et al. Prevalence, antimicrobial susceptibility and risk factors associated with non-typhoidal Salmonella on Ugandan layer hen farms. BMC veterinary research. 2017;13(1):365. PubMed | Google Scholar

  31. Kumar R, Gupta N, Shalini. Multidrug-resistant typhoid fever. Indian J Pediatr. 2007;74(1):39-42. PubMed | Google Scholar

  32. Zaki SA, Karande S. Multidrug-resistant typhoid fever: a review. The Journal of Infection in Developing Countries. 2011;5(5):324-337. PubMed | Google Scholar

  33. Marks F, Adu-Sarkodie Y, Hünger F, Sarpong N, Ekuban S, Agyekum A et al. Typhoid fever among children, Ghana. Emerg Infect Dis. 2010;16(11):176-78. PubMed | Google Scholar

  34. Groß U, Amuzu SK, Ciman RD, Kassimova I, Groß L, Rabsch W et al. Bacteremia and antimicrobial drug resistance over time, Ghana. Emerg Infect Dis. 2011 Oct;17(10):1879–1882. PubMed | Google Scholar

  35. Klemm EJ, Shakoor S, Page AJ, Qamar FN, Judge K, Saeed DK et al. Emergence of an extensively drug-resistant Salmonella enterica serovar Typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins. MBio. 2018;9(1):e00105-18. PubMed | Google Scholar

  36. Yan JJ, Chiou CS, Lauderdale TL, Tsai SH, Wu JJ. Cephalosporin and ciprofloxacin resistance in Salmonella, Taiwan. Emerging infectious diseases. 2005;11(6):947. PubMed | Google Scholar

  37. Phoba MF, Barbé B, Lunguya O, Masendu L, Lulengwa D, Dougan G. Salmonella enterica serovar Typhi producing CTX-M-15 extended spectrum ß-lactamase in the Democratic Republic of the Congo. Clinical Infectious Diseases. 2017;65(7):1229-1231. PubMed | Google Scholar

  38. Kariuki S, Revathi G, KiiruJ, Mengo D.M, Mwituria J, Muyodi J et al. Typhoid in Kenya is associated with a dominant multidrug-resistant Salmonella enterica serovar Typhi haplotype that is also widespread in Southeast Asia. Journal of clinical microbiology. 2010;48(6):2171-2176. PubMed | Google Scholar

  39. Rowe B, Ward LR, Threlfall EJ. Multidrug-resistant Salmonella typhi: a worldwide epidemic. Clinical Infectious Diseases. 1997;24(Suppl_1):S106-S109. PubMed | Google Scholar

  40. Johnson TJ, Nolan LK. Pathogenomics of the virulence plasmids of Escherichia coli. Microbiology and Molecular Biology Reviews. 2009;73(4):750-774. PubMed | Google Scholar

  41. Bielak EM. Diversity and epidemiology of plasmids from Enterobacteriaceae from human and non-human reservoirs. Kgs Lyngby: Technical University of Denmark (DTU), 2012: 188. Google Scholar

  42. Seribelli AA, Ballaben AS, Ferreira JC, Medeiros MIC, Darini ALC, Falcao JP. IncU plasmid harbouring blaCTX-M-8 in multidrug-resistant Shigella sonnei in Brazil. Journal of global antimicrobial resistance. 2018;14:99-100. PubMed | Google Scholar

  43. Piotrowska M, Popowska M. Insight into the mobilome of Aeromonas strains. Frontiers in microbiology. 2015;6:494. PubMed | Google Scholar

  44. Marti E, Balcázar JL. Multidrug resistance-encoding plasmid from Aeromonas sp. strain P2G1. Clinical Microbiology and Infection. 2012;18(9):E366-E368. PubMed | Google Scholar

  45. Dang B, Xu Y, Mao D, Luo Y. Complete nucleotide sequence of plasmid pNA6 reveals the high plasticity of IncU family plasmids. Gene. 2016; 591(1):74-79. PubMed | Google Scholar

  46. Nordmann P, Poirel L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy. 2005; 56(3):463-469. Google Scholar

  47. Carattoli A. Plasmids and the spread of resistance. International Journal of Medical Microbiology. 2013; 303(6-7):298-304. PubMed | Google Scholar

  48. Fortini D, Fashae K, Villa L, Feudi C, García-Fernández A, Carattoli A. A novel plasmid carrying blaCTX-M-15 identified in commensal Escherichia coli from healthy pregnant women in Ibadan, Nigeria. Journal of global antimicrobial resistance. 2015;3(1):9-12. PubMed | Google Scholar

  49. Guo Q, Su J, McElheny CL, Stoesser N, Doi Y, Wang M. IncX2 and IncX1-X2 hybrid plasmids coexisting in FosA6-producing Escherichia coli. Antimicrobial agents and chemotherapy. 2017;61(7) pii: e00536-17. PubMed | Google Scholar

  50. Sumrall ET, Gallo EB, Aboderin AO, Lamikanra A, Okeke IN. Dissemination of the transmissible quinolone-resistance gene qnrS1 by IncX plasmids in Nigeria. PloS one. 2014;9(10):e110279. PubMed | Google Scholar






























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