Microbes and Infectious Diseases Isolation and molecular identification of Salmonella with high multidrug resistance to first line typhoid antibiotics in Southwest Cameroon

Background: The prevalence of typhoid fever in Cameroon is high. However, studies on susceptibility of Salmonella to the recommended antibiotics are few and not extensive. This study investigated the susceptibility of Salmonella isolated from clinical specimens in the South West region of Cameroon and the genes responsible for their antibiotic resistance. Methods: Salmonella was isolated from clinical specimens collected from some medical laboratories in the study area by culture on Salmonella Shigella agar. Identification and resistance genes analysis were done using biochemical tests, PCR and gene sequencing. Disc diffusion test was done using 11 commercial antibiotics and minimum inhibitory concentrations determined by microdilution test to assess antibiotic susceptibility profile. The Multi-Antibiotic Resistance (MAR) index values were calculated. Results: Salmonella was isolated from 50 out of 115 specimens, of which 46 (92 %) were multidrug resistant (MDR) with MAR indices > 0.2. The MDR isolates showed high resistance to penicillins and low resistance to ciprofloxacin. PCR revealed that antibiotic resistance was largely due to the tem-1 , Sul 1 , dfrA1 , floR genes. Sequencing revealed double mutations at codons 83 (Ser83-Phe) and 87 (Asp87-Gly) and one mutation at codon 83 (Ser83-Trp) in the quinolone resistance determining regions of the gyr A gene in two isolates. Conclusion: This study has revealed presence of MDR Salmonella enterica isolates in Southwest Cameroon with several resistance genes . This finding will guide use of antibiotics in treatment of Salmonella infections in the study area and justifies the search for new efficacious antibacterials against the MDR strains.


Introduction
Salmonella enterica causes gastroenteritis, enteric (typhoid and paratyphoid) fevers in humans and animals [1]. Salmonella infections were initially treated with first line drugs ampicillin, sulphamethoxazole-trimethoprim, and chloramphenicol. Due to emergence of multidrug resistant (MDR) Salmonella strains, these drugs have become less effective [2]. This led to a shift to third generation cephalosporins (ceftriaxone) and fluoroquinolones (ciprofloxacin). However, resistance to ciprofloxacin has also emerged with reported decreased susceptibility to the drug [3]. The World Health Organisation recommends use of fluoroquinolones as optimal treatment choice in areas showing resistance to first line drugs and use of third generation cephalosporins in severe MDR cases [4,5]. The prevalence of MDR bacteria continues to rise worldwide leading to increased incidence of treatment failure, increased morbidity, mortality as well as increased healthcare cost [6].
In Cameroon, typhoid fever is highly prevalent due to poor sanitation and fecal contamination of water sources [7]. Also, increasing levels of antimicrobial resistance in Salmonella has been reported. A study in the Northwest region of Cameroon showed a significant decrease in sensitivity to ciprofloxacin and all first line antibiotics [8]. Also, a five years retrospective study of medical records reported a high degree of multidrug resistance in Salmonella in Buea health district in the Southwest region [9]. This has been attributed to indiscriminate use of antibiotics, inadequate dosing and poor compliance to treatment regimens, circulation of low quality drugs, no susceptibility testing in some health facilities and irrational use of the antibiotics. Studies on antibiotic susceptibly of Salmonella in Cameroon have been few, limited to culture and susceptibility testing without molecular characterization of resistant isolates and have not been extensive in geographical coverage. Determination of resistant genes would reveal the mechanisms of antimicrobial resistance and is vital in guiding treatment and monitoring evolution of resistance. Increasing resistance justifies the search for new efficacious antibacterials and alternative treatments. This study aimed to investigate the antibiotic susceptibility and the implicated genes of clinical Salmonella isolates collected in the South West region of Cameroon using susceptibility testing and molecular techniques.

Materials and Methods
Purification and identification of Salmonella isolates Culture media comprising Salmonella Shigella (SS) agar, Nutrient agar, Mueller Hinton agar and broth (Liofilchem, Italy) were prepared strictly according to manufacturer's instructions. A total of 115 clinical samples were obtained from medical laboratories in the District hospital and JET Medicare in Kumba; Regional Hospital Buea; the Regional Hospital, District Hospital and Grosmelf Laboratory in Limbe, and District Hospital Tiko) in the South West Region. The specimens were sub-cultured 2-3 times on SS agar to obtain uniform colonies prior to characterization. Colonies were subjected to a series of biochemical tests using the API (Analytical Profile Index) 20E test kit (Biomerieux, France).

Antibiotics susceptibility testing
Antibiotic susceptibility testing was done by the Kirby Bauer disc diffusion method [1] [4], using the broth microdilution method [11]. Antibiotics were purchased from a certified pharmacy in Buea, capital city of the South West region and all tablets ground to obtain fine powder. A stock solution of 1024 µg/mL was prepared (in broth) and diluted serially two-fold giving concentrations from 1 to 512µg/mL. The assay was set up in a 96 well microtiter plate in duplicate. In wells containing antibiotic (100 µL), 100 µL of inoculum (5 x 10 5 CFUs/well) was added giving final concentrations of 0.5 to 256 µg/mL. Negative (bacteria, no antibiotic) and positive (25 µg/mL gentamycin) control wells were included. The plates were incubated at 37 o C for 24 hours. Then 20 µL bromothymol blue was added and incubated for an additional 4 hours to indicate bacterial growth by a colour change from blue to yellowish. The minimum bactericidal concentration (MBC) was determined by sub-culturing all wells with inhibited growth on nutrient agar using a sterile wire loop. Plates were incubated at 37 o C for 24 hours and observed for any growth and MBC was considered as the lowest concentration that showed no bacterial colonies [1].

Extraction of genomic DNA
Bacterial genomic DNA was isolated from all 50 resistant Salmonella isolates by conventional boiling centrifugation as described [12]. Briefly, a single colony from a pure nutrient agar culture was grown overnight at 37 °C in 1mL Luria Bertani (LB) broth. Bacterial cells were pelleted by centrifugation at 14,000 rpm for 5mins in a microcentrifuge (Eppendorf centrifuge 5415D, Germany). The supernatant was discarded and the pellet re-suspended in 500µL deionized distilled water by vortexing (Vorte-2 Genie, Scientific Industries, Inc, USA) and centrifuged for 5mins at 14,000rpm. The supernatant was discarded, 200µL of sterile deionized distilled water was added and boiled for 10 mins in a heating block at 100 °C (Lab exchange, Germany), then immediately chilled on ice for 5 mins. The sample was then centrifuged for 5 mins at 14,000rpm at 4 °C (Eppendorf centrifuge, 5415D). The resulting supernatant was carefully transferred to a new Eppendorf tube. The DNA quality was determined by running a 1% gel and stored at -20°C for further use.

Molecular characterization of clinical isolates of Salmonella
Confirmation of Salmonella species was done using primers specific to invA gene by polymerase chain reaction (PCR) amplification as described [13]. Salmonella isolates (n = 50) were further subjected to molecular characterization for the identification of resistance-associated genes (n = 16) according to reported method [14]. The isolates were tested for the presence of genes encoding resistance to beta-lactams, chloramphenicol, sulphonamides, trimethoprim and mutation in gyrA and parC for fluoroquinolone resistance using specific primers ( Table 1). The PCR was performed in 25 µL volumes containing 12.5µL One Taq Quickload 2x Master mix (New England Biolabs, UK), 0.5µL each of forward and reverse primers, 9.5µL distilled water and 2µL template DNA. DNA amplifications were carried out using an Eppendorf 2720 PCR thermocycler (Applied BioSystems, USA) under the following reaction conditions: initial denaturation at 95 °C for 5 mins followed by 35 cycles of enaturation at 95 °C for 2mins, annealing at various primer annealing temperatures for 30s, extension at 68 °C for 1.5min and final extension at 68°C for 5 mins. The amplified products were resolved on 1.5 or 2% agarose gel stained with ethidium bromide (0.5µg/mL) and visualized using a photogel documentation system (BIORAD Molecular Imager R GelDoc™XR,) with Image Lab™ Software.

Sequencing of DNA
Polymerase chain reaction amplification was carried out to detect the presence of gyrase and topoisomerase IV genes in Salmonella using primers (Table 1). DNA sequencing was performed by automated sequencer services (Genewiz, Germany) to detect mutations in the quinolone resistance determining regions (QRDRs) of gyr A and parC. The results were analyzed using Bioedit software version 7.0 where the nucleotides of gyrA and parC from QRDR were compared to reference nucleotides of Salmonella isolates available in National Center for Biotechnology Information (NCBI) GenBank with accession nos. X78977 and AE008878, respectively.

Data analysis
Diameters of zones of inhibition and MIC values were interpreted based on reference values [11]. Proportions of resistant isolates were reported as percentage of total resistant isolates. Multi-antibiotic resistance (MAR) indices were determined using the where, A = number of antibiotics that each isolate was resistant to, B = total number of antibiotics tested [15]. Indices > 0.2 were considered as high risk (indicate MDR).

Antibiotic susceptibility of Salmonella isolates
All 50 isolates showed resistance to at least one antibiotic. Norfloxacin, imipenem and ceftriaxone displayed high activity while high resistance was detected to methicillin and ampicillin ( Table 2). Multi drug resistance was recorded in 36 (72%) isolates, (resistant to at least one antibiotic in three of the chemical classes), (Table 2), while 7 (14%) isolates showed extensive drug resistance (> 5 antibiotic classes), [16]. In terms of the MAR index, 45 (90%) isolates had values > 0.2 indicating multidrug resistance. The 6 isolates that displayed MDR in the diffusion test were used for the MIC assay for antibiotics of four classes ( Table 3). The clinical isolates were most sensitive to ciprofloxacin with an average MIC of 63.79 µg/mL, and resistant to all first line antibiotics with MIC values several folds above their MIC breakpoints for resistance which further confirmed the isolates were MDR.

PCR detection of Salmonella and resistanceassociated genes
All 50 isolates were confirmed as Salmonella by the amplification of invA gene by PCR, which generated an amplicon of 284 base pairs (Figure 1). From the PCR, the detection frequency of the resistance genes ranged from 2 to 96 % (Table 4) with the highest being 48 (96.0 %) of the 50 isolates positive for the tem-1 gene (Figure 2), followed by sul1 and dfrA1 genes in 40 strains each (80.0%) while the least was the cat2 gene in one isolate (2.0 %). Meanwhile oxa-1, sul2, sul3, dfra14, dfrB, cat1 and cat3 genes were not detected in any isolate.

Detection of gene mutations
The isolated Salmonella strains were analysed for mutations in gyrA and parC genes for fluoroquinolone resistance. Of the 50 isolates, only 12 revealed presence of gyrA and parC genes. Seven of them were Salmonella typhimurium (BU03, BU04, BU06, BU13, BU15, BU53 and BU54), four Salmonella typhi (BU05, BU07, BU11 and BU70) and one Salmonella paratyphi A (BU10). Following sequencing of ten isolates and alignment of sequences to the references in the NCBI GenBank, point mutations were found in two isolates. S. typhimurium (BU03) had double mutations at codon 83 (Ser83-Phe) and codon 87 (Asp87-Gly) while S. typhimurium (BU06) had one mutation at codon 83 (Ser83-Trp) with no mutation detected among the parC genes. BU03 and BU06 contained no mutation at codon 119 of gyrA whereas no non synonymous mutations and some synonymous mutations were detected in parC genes of all ten isolates.

Discussion
Recent increases in antibiotic-resistant bacteria have prompted the scientific community to perform routine surveillance of microbial populations to determine the extent of the resistance [1]. Previously, typhoid fever was treated successfully with inexpensive drugs (ampicillin, chloramphenicol, sulphamethoxazole-trimethoprim), but the emergence of antibiotic resistance to these first line drugs have made treatment ineffective and very costly [9]. Routine antibiotic susceptibility testing is necessary for effective treatment of typhoid fever to determine which antibiotics effectively kill the bacterium [2].
In this study, 50 clinical Salmonella isolates were examined for their susceptibility to various antibiotics and the presence of drug resistanceassociated genes. Eleven antibiotics belonging to 8 different antibiotic classes and 17 genes were tested in this study. For the antibiogram results, 48 (96%) Salmonella isolates were multidrug resistant. The penicillins displayed the highest level of resistance hence should be excluded in the treatment of typhoid fever. Other first line antibiotics recorded lower but considerable levels of resistance ( Table 2).
The two fluoroquinolones (ciprofloxacin and norfloxacin) showed percentage resistance of 36% and 0% respectively. Interestingly, Khadka et al. [1] recorded no resistance for co-trimoxazole (sulphamethoxazole-trimethoprim) compared to 64% in this study. This difference could be due to variations in the factors accounting for emergence of resistance such as misuse of antibiotics among others.
The lower resistance to fluoroquinolones, amoxicillin-clavulanic acid and imipenem supports their continued use in the treatment of typhoid fever. The MIC values confirmed the resistance recorded in the disc diffusion test with values several folds above the standard breakpoints except ciprofloxacin which showed sensitivity to one isolate and values close the its breakpoint [11]. These findings confirm earlier reports and constitute a strong basis to search for more efficacious antibacterials.
Analysis of resistance genes in the clinical isolates of Salmonella strains (n= 50) revealed the presence of the tem gene in almost all the isolates. This suggests that resistance to β-lactamase antibiotics is mediated by the tem gene only which encodes a βlactamase enzyme. The oxa-1 gene was not detected in any isolate whereas β-lactamase resistance in Salmonella isolates were attributed to blaTEM-1 and blaoxa-1 genes [17,18] and El-Tayeb et al. [14], revealed the presence of carb-like gene (carbenicillinase) in isolates that exhibited resistance to β-lactam antibiotics.
It was found that isolates that exhibited resistance to sulphamethoxazole-trimethoprim were associated with presence of dfrA1 and sul1 genes only, indicating both genes are responsible for the resistance. This report was supported by another study which revealed presence of dfrA1 only [14] but in contrast to the study by Zishiri et al. [19], which revealed the presence of sul1 and sul2 genes. The resistance to chloramphenicol is highly associated with the acquisition and expression of efflux pumps that reduce toxic levels of the drug in bacterial cells. The resistance to phenicols in this study was associated with the presence of cat2, cmlA, cmlB and floR genes. However, floR gene was most prevalent in isolates (n=40) suggesting that this gene is responsible for chloramphenicol resistance. This finding is similar to other studies that reported the presence of floR gene in various Salmonella Pathogenicity island-1 [14,20].
The main mechanism of resistance to fluoroquinolones in Gram-negative bacteria is point mutations in genes coding for DNA gyrase (gyrA and gyr B) and topoisomerase IV (parC and parE). These mutations alter the binding sites of gyrase or topoisomerase which are targets of antimicrobial agents [21,22]. In the present study, both gyrA and parC genes were detected in 12 Salmonella isolates, indicating emerging resistance to fluoroquinolones in the study area. This finding is in contrast to a study in Saudi Arabia by El-Tayeb et al. which detected few isolates with gyrA and parC genes [14].
Sequencing detected the QRDR of gyrA gene in two Salmonella isolates which has been reported in other studies [17,23,24]. Results of this study suggest that parC mutations may not be necessary to .generate high level resistance to ciprofloxacin. However, Eaves et al. [25], found that isolates with mutation in both gyrA and parC were more susceptible to ciprofloxacin than isolates with a mutation in gyrA alone. Findings of this study differ from those of El-Tayeb et al.  [26]. In this study, it was found that mutations in gyrA gene led to subsequent changes in amino acids at codon 83 from serine to phenylalanine or serine to tryptophan and codon 87 from aspartic acid to glycine. These findings could explain the mechanism of resistance to fluoroquinolones associated with changes in NDA gyrase. The rest of the nucleotide substitutions resulted in no changes in amino acid sequence thus being synonymous mutations since they do not cause a change in amino acid.

Conclusion
This study has revealed considerable presence of multidrug and extensively resistant Salmonella enterica isolates in Southwest Cameroon, with increasing resistance to fluoroquinolone and cephalosporin antibiotics. This resistance is largely due to the Tem-1, Sul 1, dfrA1, floR and to a lesser extent gyrA genes. This finding justifies the exploration of alternative approaches in the use of available antibiotics and the search for new efficacious antibacterials for treatment of Salmonella infections.