ABSTRACT
Infections caused by non-typhoidal Salmonella (NTS) are common around the world, with high morbidity and mortality rates recorded annually. Salmonella serovars harbor plasmids of various sizes which may play roles in antibiotic resistance and virulence. The aim of this study was to profile and determine the role of plasmids in ciprofloxacin resistance and virulence of Salmonella serovars. Using alkaline lysis method 25 NTS serovars from food animals and humans were assayed for plasmids. Isolates ability to resist healthy human serum, bind congo red, produce hemolysin and susceptibility to ciprofloxacin before and after plasmid curing were evaluated. Mobility of plasmids was determined by conjugation. Fifteen (60%) of the 25 Salmonella serovars harbored plasmids with sizes ranging from 0.4 to 38.4 kb. S. Budapest serovars harbored 5–9 plasmids, while S. Essen and S. Mura had six plasmids each. S. Chomedey and a S. Budapest serovar were sensitive to ciprofloxacin after plasmid curing while other serovars remained resistant to ciprofloxacin after plasmid curing. All Salmonella isolates had the ability to withstand human serum before and after plasmid curing, however, some serovars lost their ability to bind congo red after plasmid curing. All Salmonella isolates that initially displayed hemolysin activity retained their ability after curing. Thirteen (86.7%) of the 15 serovars that harbored plasmids conjugatively transferred their plasmids to E. coli K-12 (DH5α). Having Salmonella serovars that harbor transferrable plasmids in the food chain can drive antibiotic resistance and enhanced virulence of otherwise less virulence strains.
KEYWORDS: Plasmid, serovar, salmonella, virulence, ciprofloxacin, conjugation
Introduction
Foodborne illness elicited by non-typhoidal Salmonella (NTS) remains a great threat to the health of children, immunocompromised and the elderly mainly in resource-limited countries in sub-Saharan Africa [1,2]. NTS infection is predominantly zoonotic since outbreaks have been linked to consumption of contaminated foods majorly from animal origin [3]. Several studies have reported Salmonella species harboring plasmid coding for serovar specific virulence and antibiotic resistance [4,5]. Though some Salmonella serovars like S. serotype Hader, S. serotype Paratyphi, S. serotype Infantis and most rare serovars have been said to be usually free of any plasmids, however, most serovars associated with animal and human disease harbor plasmids of various sizes [5]. Some of these plasmids are known to be conjugative, hence transferable while others are nontransferable. In a recent study Klemm et al. [6], reported the emergence of an extensive drug-resistant S. Typhi that possessed a promiscuous plasmid encoding resistance to fluoroquinolones, third-generation cephalosporins and three first-line drugs including trimethoprim-sulfamethoxazole, chloramphenicol and ampicillin in Sindh, Pakistan. The plasmid belongs to the IncY class and harbored blaCTX-M-15 and qnrS genes with sequence identity similar to plasmids found in other enteric bacteria. To establish an infection, Salmonella must first evade their host’s defense mechanisms. This they accomplish by various virulence factors that are both chromosomal and plasmid mediated [4,5,7]. Some of these factors include pef (plasmid-encoded fimbriae) for host recognition and invasion, cytolysin A (clyA) for hemolytic activity, rck genes that encodes resistance to complement-mediated killing (serum resistance) [8–12]). In Nigeria, ciprofloxacin is commonly prescribed in typhodial and NTS infections however resistance to this antibiotic is emerging. Although plasmid-mediated antibiotic resistance has been extensively reported [14], their role in the mediation of other virulence properties has not been fully studied. This current study was carried out to profile and determine their role in ciprofloxacin resistance, human serum resistance, expression of curli fimbriae and hemolysin activity in Salmonella serovars of human and food animal origin.
Materials and methods
Isolates source
A total of 25 NTS serovars previously isolated from apparently healthy food handlers, cattle, sheep and chicken in Lagos Nigeria [15] were used for this study.
Plasmid extraction
Plasmids were extracted according to Kado and Liu [16], with modifications using the alkaline sodium dodecyl sulfate (SDS) method. Single colonies of pure isolates were inoculated into Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, UK) and incubated at 37°C overnight. One milliliter was dispensed into a 1.5 mL Eppendorf tube and centrifuged for 2 min at 13,000 rpm. Supernatant was discarded and the pellet was washed by re-suspending in sterile water and centrifuged at 13,000 rpm for 2 min, after which the supernatant was discarded. Three hundred microliters (300 µL) of TENS solution (Tris 25 mM, EDTA 10 mM, NaOH 0.1 N and SDS 0.5%) was added to the suspended pellet and the tubes were inverted until solution became slimy. Thereafter, 150 µL of sodium acetate solution was added and vortexed then centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to 1.5 mL Eppendorf tube to which 900 µL of ice-cold absolute ethanol was added. This was vortexed and centrifuged at 13,000 rpm for 10 min. The supernatant was discarded and white pellet was dissolved in 1,000 µL of 70% ethanol and centrifuged at 13,000 rpm for 5 min. This was repeated and the final pellet was dried and thereafter 40 µL of TE buffer + RNase was added and kept at −20°C. Twenty-five microliters (10 µL) of extracted plasmids were loaded into wells of 0.8% agarose gel and electrophoresed at 100 V for 2 h with a 1 kb Hind III ladder. Bands were visualized using a trans-illuminator (Cleaver Scientific Ltd). Molecular weight of plasmids was estimated insilico at www.insilico.ehu.es
Plasmid curing
Single colonies of isolates were inoculated into nutrient broth supplemented with 2% SDS, 0.1% acridine orange (Electran) and incubated at 37°C for 24 h after which they were plated out on nutrient agar, incubated at 37°C for another 24 h [17]. Colonies obtained were then harvested and subjected to plasmid extraction.
Antibiotic susceptibility testing of cured isolates
Antimicrobial susceptibility testing of plasmid cured isolates to ciprofloxacin (5 µg) was performed using the disk diffusion method according to the European Committee on Antimicrobial Susceptibility [18]. Salmonella isolates were inoculated into Brain heart infusion (BHI) broth (Oxoid, Basingstoke, UK) and incubated at 37°C for 18 h after which they were subcultured on Muller-Hinton agar (Hi-Media India) at 37°C for 24 h. Two distinct colonies were emulsified in 5 mL of sterile physiological saline and adjusted with more physiological saline to 0.5 McFarland standard. Cell suspension was applied unto the surface of Muller-Hinton agar using a sterile swab stick and ciprofloxacin (5 µg) was applied using a sterile forceps. Escherichia coli ATCC 25,922 was used as quality control organism and the zone of inhibition was measured interpreted as Resistant (R) and Sensitive (S).
Resistance to human serum assay
Bacterial isolates with plasmid and plasmid cured bacterial isolates were grown on BHI broth overnight and bacterial suspension was adjusted to 0.5 Marfaland standard to obtain 108 bacteria/mL using phosphate buffer saline (PBS). Two hundred microliter (200 µL) of PBS was added to a tube containing 200 µL of bacterial suspension and 100 µL of blood serum from a healthy human subject was added and incubated at 37°C for 0–3 h. Suspension was plated out on nutrient agar and incubated at 37°C overnight and colonies were counted [19].
Screening of NTS serovars for hemolysin activity
Bacterial isolates with plasmid and plasmid cured bacterial isolates were cultured on blood agar that contained 5% sheep blood which was overlaid with nutrient agar after 4-h incubation at 37°C to obtain a micro-aerobic condition. This was further incubated for 72 h at 37°C during which culture was examined for Alpha-hemolysis (partial hemolysis with green or brown coloration), Beta-hemolysis (complete hemolysis with a clear zone) or Gamma-hemolysis (no hemolysis) daily [20].
Congo red binding activity (expression of adhesive fimbriae)
Isolates from overnight culture in BHI broth were inoculated unto tryptone soya agar supplemented with 0.004% Congo red dye and incubated at 37°C for 24 h. Congo red positive Salmonella isolates were identified by the appearance of red colonies ([19]; [21]).
Conjugation experiment
Mobility of plasmid was determined through conjugation experiment adapting the procedure of Olusesen et al. [14]. Potential donor Salmonella serovars with plasmids were cultured in Luria Bertani (LB) broth (Carl Roth GmbH) for 16 h at 37°C and recipient organism (F−) Escherichia coli K-12 (DH5α) was cultured in SOC medium (LB + 2.5 mM KCl, 20mM MgSO4, 20mM glucose) for 16 h at 37°C. Ten microliters of both broth culture of recipient and donor was cultured onto 2 mL of LB and incubated for 24 h at 37°C. Transconjugants were selected on MacConkey agar (Oxoid Basingstoke, UK) supplemented with rifampicin overnight at 37°C. Colonies of recipient were picked and purified then plasmid extraction was carried out.
Data analysis
Graphics was performed using GraphPad Prism software 5.01 (La Jolla, CA)
Results
Plasmid profiling
Several plasmids with molecular weights ranging from 0.4 to 38.4 were detected in 15 (60%) of 25 Salmonella serovars evaluated for plasmid (Table 1 and Figure 1). Multiple plasmids ranging from 2 to 9 were observed in some serovars, while S. Typhimurium and S. Chomedey had a single low molecular weight and high molecular weight plasmid, respectively. Of the 15 Salmonella serovars that harbored plasmid, 12 displayed hemolysin activity, 5 were able to bind Congo red while are resistant to ciprofloxacin Table 2.
Table 1.
Plasmid sizes and number of plasmids detected in Salmonella serovars.
| S/N | Code | Serovar | Number of Plasmid | Estimated Molecular Sizes of Plasmid (Kb) |
|---|---|---|---|---|
| 1 | CO19 | S. Budapest | 0 | – |
| 2 | F5 | S. Budapest | 6 | 9.3, 3.7, 2.9, 2.2, 2.1 & 1.6 |
| 3 | Fe | S. Budapest | 9 | 8.5, 6.6, 5.1, 4.0, 3.1, 2.9, 2.2, 2.0 & 1.7 |
| 4 | A2 | S. Dabou | 2 | 14.1 & 0.4 |
| 5 | A115 | S. Carno | 0 | – |
| 6 | H363 | S. Typhimurium | 1 | 2.4 |
| 7 | H117 | S. Chagoua | 0 | – |
| 8 | CF3 | S. Budapest | 5 | 9.3, 6.6, 4.0, 2.6 & 2.0 |
| 9 | E14 | S. Budapest | 7 | 7.8, 4.0, 2.4, 3.1, 2.9, 2.0 &1.5 |
| 10 | H44 | S. Portland | 0 | – |
| 11 | A152 | S. Budapest | 5 | 14.6, 7.2, 5.6, 4.4 & 3.4 |
| 12 | S51 | S. Wien | 2 | 15.3 & 10.9 |
| 13 | S57 | S. Essen | 5 | 15.3, 10.9, 7.2, 6.1 & 4.7 |
| 14 | CF10 | S. Budapest | 0 | – |
| 15 | S56 | S. Essen | 6 | 35.3, 14.1,10.9, 7.2, 6.1 & 4.7 |
| 16 | H63 | S. Mowanjum | 0 | – |
| 17 | S61 | S. Mura | 6 | 14.1, 10.9, 6.6, 5.6, 4.4 & 3.4 |
| 18 | S81 | S. Essen | 0 | – |
| 19 | S38B | S. Essen | 5 | 19.7, 15.3, 11.9, 10.0 & 6.1 |
| 20 | S28 | S. Chomedey | 1 | 38.4 |
| 21 | A11 | S. Tennyson | 3 | 23.2, 11.9 & 3.7 |
| 22 | D25 | S. Wichita | 0 | – |
| 23 | S76 | S. Orion | 0 | – |
| 24 | D6 | S. Agodi | 0 | – |
| 25 | AF6 | S. Essen | 2 | 19.7 & 10.9 |
Figure 1.
Plasmids of molecular weight 0.4kb to 35.3kb detected in Salmonella serovars. Lane M: Lambda DNA Hind III digest, Lane 2, 3, 8, 9, 11, 14: S. Budapest, Lane 13, 15, 18: S. Essen, Lane 4: S. Dabou, Lane 12: S. Wien, Lane 17: S. Mura.
Table 2.
Ciprofloxacin susceptibility, hemolysin activity and Congo red binding ability before and after plasmid curing.
| Before Plasmid Curing |
After Plasmid Curing |
||||||
|---|---|---|---|---|---|---|---|
| Code | Serovar | CIP | HA | CRBA | CIP | HA | CRBA |
| A2 | S. Dabou | ND | + (α) | − | ND | + (α) | − |
| F5 | S. Budapest | R | + (α) | + | R | + (α) | − |
| CF3 | S. Budapest | R | + (α) | + | R | + (α) | − |
| Fe | S. Budapest | R | − | + | R | − | − |
| A152 | S. Budapest | R | + (α) | − | S | + (α) | − |
| A11 | S. Tennyson | R | − | − | R | − | − |
| AF6 | S. Essen | R | + (α) | − | R | + (α) | − |
| S56 | S. Essen | R | + (α) | − | R | + (α) | − |
| S28 | S. Chomedey | R | + (β) | − | S | + (β) | − |
| S38B | S. Essen | ND | + (α) | − | ND | + (α) | − |
| S61 | S. Mura | ND | + (α) | − | ND | + (α) | − |
| S57 | S. Essen | ND | + (α) | − | ND | + (α) | − |
| S51 | S. Wien | ND | − | − | ND | − | − |
| E14 | S. Budapest | ND | + (α) | + | ND | + (α) | − |
| H363 | S. Typhimurium | ND | + (β) | + | ND | + (β) | − |
CIP- Ciprofloxacin, HA-Hemolysin Activity, CRBA-Congo Red Binding Ability, ND-Not Determined, α – Alpha Hemolysis, β – Beta Hemolysis
Plasmid curing
All isolates were effectively cured of their plasmids when treated with acridine orange and SDS. Ciprofloxacin susceptibility tests revealed that S. Chomedey and S. Budapest (A152) were sensitive after plasmid curing while S. Budapest (F5), S. Budapest (CF3) and S. Budapest (Fe) were still resistant to ciprofloxacin after plasmid curing (Table 2). All Salmonella isolates that initially displayed hemolysin activity retained their ability even after plasmid curing. They all showed alpha (α) hemolysis with the exception of S. Chomedy and S. Typhimurium that showed beta (β) hemolysis. However, S. Budapest (F5), S. Budapest (CF3), S. Budapest (Fe), S. Budapest (E14) and S. Typhimurium (H363) lost their ability to bind Congo red as shown in Table 2
Human serum resistance assay
Figure 2 show the results of Salmonella isolates serum resistance assay before and after plasmid curing experiment. Serovars possessed the ability to resist healthy human serum at 0 h and 3 h before and after plasmid curing. Average growth rate of bacteria in serum during the logarithm phase was 0.73logCFU/mL/h and a growth rate constant (µ) of 1.61. This indicates that serum resistance was chromosomally mediated.
Figure 2.
Human serum resistance activity. B0 = Plate count at 0 h before plasmid curing, A0 = Plate count at 0 h after plasmid curing, B3 = Plate count at 3 h before plasmid curing, A3 = plate count at 3 h after plasmid curing. Value = Mean ± SEM (n = 2).
Conjugative transfer of plasmid
Of 15 Salmonella isolates harboring plasmids, 13 (86.7%) were able to transfer their plasmids to the recipient E. coli (DH5α). All plasmids of S. Budapest (Fe), S. Budapest (E14), S. Budapest (F5), S. Budapest (A152), S. Essen (S56), S. Wien (S51) and S. Dabou were transferred. S. Essen (S38B) and S. Essen (S57) transferred only two and one of their plasmid respectively, while S. Typhimurium (H363) and S. Chomedey did not transfer their plasmids (Table 3)
Table 3.
Number of plasmids transferred to transconjugants.
| Transconjugant |
||||
|---|---|---|---|---|
| S/N | Code | Serovar | Number of Plasmid | Number of Plasmids transferred |
| 1 | S28 | S. Chomedy | 1 | 0 |
| 2 | F5 | S. Budapest | 6 | 6 |
| 3 | Fe | S. Budapest | 9 | 9 |
| 4 | E14 | S. Budapest | 7 | 7 |
| 5 | S38B | S. Essen | 5 | 2 |
| 6 | S51 | S. Wien | 2 | 2 |
| 7 | A2 | S. Dabou | 2 | 2 |
| 8 | A11 | S. Tennyson | 3 | 3 |
| 9 | H363 | S. Typhimurium | 1 | 0 |
| 10 | S61 | S. Mura | 6 | 6 |
| 11 | S57 | S. Essen | 5 | 1 |
| 12 | S56 | S. Essen | 6 | 6 |
| 13 | CF3 | S. Budapest | 5 | 5 |
| 14 | AF6 | S. Essen | 2 | 2 |
| 15 | A152 | S. Budapest | 5 | 5 |
Discussion
In the present study, various plasmids of molecular weight ranging from 0.4 kb to 38.4 kb were isolated from NTS serovars from food animals and humans. Isolates harbored single to multiple (2–9) plasmids, some of which conferred ciprofloxacin resistance. Occurrence of plasmid was more in isolates from food animal origin compared to those from humans. Only one isolate S. Typhiumrium (H363) isolated from human harbored a 2.4 kb plasmid. This is in line with the findings of Akinyemi et al. [14]. In a previous study, Olusesan et al. [14] reported Salmonella spp. harboring plasmids of estimated molecular weight ranging from 0.145 kb to 23.13 kb in Zaria Nigeria. Though there has been draught of data on plasmid-mediated quinolone resistance among Salmonella serovars in Africa as revealed from a recent systematic review and meta-analysis by Tadesse et al. [22]. However, in this study two isolates S. Budapest (A152) and S. Chomedey were observed to have lost their ciprofloxacin resistance after plasmid curing indicating plasmid-mediated resistance. Similarly, Fashae et al. [23], reported observed reduced susceptibility to ciprofloxacin in Salmonella serovars isolated from chicken in Ibadan Nigeria which were plasmid mediated as informed from the detection of qnr genes. In this study, all isolates with the exception of three S. Budapest (Fe), S. Tennyson and S. Wien were positive for hemolysin activity. Their hemolysin activity was sustained after plasmid curing, suggesting that hemolysin activity could also be chromosomally mediated besides plasmid that has been reported in some serovars. Hemolysin activity is a vital virulence indicator which is widespread among Salmonella serovars including isolates from fish, meat, meat products and clinical samples [24]. Libby et al. [25], reported a hemolysin encoded by a plasmid in Salmonella required for survival within macrophages. Of the 15 Salmonella serovars that possessed plasmids, five had the ability to bind congo red. However, this ability was lost after plasmid curing indicating that this ability is plasmid mediated. Congo red imitates host tissue factor in vitro, which infers the ability of Salmonella isolates to bind and invade host intestinal epithelium, hence establishing infection [20]. Salmonella serovars were able to resist healthy human serum before and after plasmid curing. This implies that there are intrinsic factors other than plasmid that elicit resistance to compliment-mediated killing in Salmonella as reported by Heffernan et al. [8]. Conjugation was successful among Salmonella serovars studied. Some serovars transferred all their plasmids of various sizes to recipient E. coli K-12 (DHα) as was observed in S. Budapest (Fe), S. Budapest (E14), S. Budapest (F5), S. Budapest (A152), S. Essen (S56), S. Dabou and S. Chomedey. This is in line with the observation of Adeleye [26],who reported the conjugal transfer of single and multiple antibiotic resistance plasmids of Salmonella and Shigella spp. to recipient E. coli. In a similar study Akinyemi et al. [27], also reported the transfer of a 23kb plasmid of S. Typhi that conferred multiple antibiotic resistance on recipient E. coli j53-2. Transfer of mobile plasmids among bacteria species as observed in this case portends grave public health risk as both antimicrobial resistance and virulence genes can be acquired by other bacteria species that are otherwise not having these genes.
Conclusion
Salmonella remains a major foodborne pathogen and public health risk. Having Salmonella serovars that harbor transferrable plasmids in the food chain poses a greater public risk as these may drive antibiotic resistance resulting in treatment failures and enhanced virulence of otherwise less virulence strains. Hence, good personal hygiene and the use of antibiotics in animal feed should be discouraged.
Acknowledgments
We thank Dr. Ayorinde James of the Department of Biochemistry, College of Medicine University of Lagos for providing us with the recipient E. coli K-12 (DH5α) for this work.
Disclosure statement
No potential conflict of interest was reported by the authors.
Ethical approval
Ethical approval for this research was obtained from the Human Research and Ethical Committee (HREC) of the Lagos University Teaching Hospital with code number ADM/DCST/HREC/APP/1118 and Nigerian Institute of Medical Research – Institutional Review Board, with project number IRB/12/180.
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