Molecular characterization and antibiotic resistance of Salmonella in children with acute gastroenteritis in Abuja , Nigeria

Introduction: In Nigeria, acute gastroenteritis in children under five years of age is a major cause of mortality and morbidity; identification and characterization of microbial agents of acute gastroenteritis, including Salmonella, remains a powerful tool for effective management, surveillance, and control. Methodology: Diarrheal stool samples were directly plated onto differential and selective media to isolate Salmonella. Extended-spectrum beta-lactamases were screened using the double disk diffusion technique and by PCR targeting the blaTEM and blaCTX-M genes. Pulsed-field gel electrophoresis (PFGE) was performed usingthe PulseNet Canada Laboratory protocol for molecular subtyping using the restriction enzymes XbaI and BlnI. Results: The serotypes identified were Salmonella enterica serovar Zanzibar (n = 5), Salmonella Brancaster (n = 3), and one isolate of Salmonella Enteritidis (phage type 1). The following levels of resistance were found among the Salmonella strains: amoxicillin, five strains (55.6%); amoxicillin-clavulanic acid, two strains (22.2%); cephalexin, five strains (55.6%); and cefuroxime, five strains (55.6%). Intermediate resistance was found in five strains (55.6%) only to amoxicillin-clavulanic acid. All isolates were susceptible to nalidixic acid, ciprofloxacin, and ceftriaxone, and no ESBL-producing Salmonella were detected. Conclusions: Our findings demonstrated the involvement of three Salmonella serovars in acute gastroenteritis; resistance to penicillins and cephalosporins was common.


Introduction
Salmonella infections are associated with acute gastroenteritis, one of the most common alimentary diseases; they are caused by the consumption of contaminated water and food, especially meat [1].The prevalence rate of acute diarrhea in Nigeria is 18.8%, one of the worst in sub-Saharan Africa.It accounts for over 16% of child deaths in Nigeria and an estimated 150,000 deaths -chiefly amongst children under five years of age -occur annually due to this disease, mainly caused by poor sanitation and hygiene practices.These diseases are especially common in developing countries with poor hygiene and sanitation and with limited access to safe drinking water [2].The identification of the pathogenic organisms responsible for these diseases is highly crucial for surveillance, prevention, and control activities.TEM β-lactamases are encoded by plasmid-mediated bla TEM gene clusters, which have various coding region frameworks and types of promoters [3].The bla TEM gene cluster encodes by plasmid mediation resistance mechanisms such as penicillinase hyper production, extendedspectrum beta-lactamases (ESBL), or inhibitorresistant TEM beta-lactamase [4].Variations in sugar content of the O-polysaccharide contribute to the wide variety of antigenic types between species and even strains of Gram-negative bacteria [5].The variability defined by immunologic characterization of two surface structures, O-polysaccharide (O antigen) and flagellin protein (H antigen), provides the basis for serotyping Salmonella spp.into serogroups [6].Polymorphism in the bla TEM genes has already been noted for clinical isolates with > 150 variants [7].The highly polymorphic bla TEM gene has been targetedas a 713 molecular marker to detect antimicrobial resistance associated with Salmonella serovars in acute gastroenteritis in Abuja.
Antimicrobial resistance in enteric pathogens is of great importance in developing countries, where the rate of diarrheal disease is high.The progressive increase in antimicrobial resistance among enteric pathogens in developing countries is becoming a critical area of concern.Among the bacterial causes of diarrheal illnesses, Salmonella spp.continues to be a major public health problem [8].Although most Salmonella infections are self-limiting, serious sequelae, including systemic infection and death, can occur [9].In potentially life-threatening cases, the antibiotics of choice are fluoroquinolones and extended-spectrum cephalosporins [10].These cephalosporins are commonly used in the treatment of Salmonella infections in children because of their pharmacodynamic properties and the very low prevalence of resistance to these agents.Very few studies have reported on the problem of ESBL in Africa in general and in Nigeria in particular.There have been reports of SHV-12 in Salmonella enterica serotype in Mali [10] and TEM-3 in S. Tyhimurium in Morocco [11].
The aim of this study was to determine serotype, antibiotic resistance, and extended-spectrum betalactamase (ESBL) gene production of Salmonella species associated with childhood acute gastroenteritis in Federal Capital Territory Abuja, Nigeria.In addition, the study evaluated the molecular characteristics and clonal relatedness among the strains.

Microbiological diagnosis
Stool samples were collected in sterile plastic cups, with prior instruction given for correct sampling.The samples were obtained by spontaneous emission, and given to the investigator.They were inoculated within two hours of collection onto selective and differential media: MacConkey (MAC) agar, Salmonella Shigella (SS) agar, and xylose lysine deoxycholate (XLD) agar, using a calibrated inoculating loop in the spread plate method.The media were then incubated aerobically at 35°C for 18 to 24 hours.Samples were also inoculated into selenite cystine enrichment broth -which favors the development of potentially pathogenic microorganisms and contains substances that inhibit the native flora [12] -and incubated at 35°C for 8 to 12 hours before re-inoculation onto MCK and SS agars.
Colonies with a presumptive Salmonella morphology as per the phenotypic characteristics in the different culture media were identified biochemically according to the following procedure [13]: five probable Salmonella colonies were selected from the SS or XLD agars, inoculated into 3.5 mL brain-heart infusion broth (BHI), incubated at 35°C for 10 minutes, and then inoculated onto the different media.Biochemical tests were then carried out for: the determination of glucose and lactose; gas formation and the production of ferrous sulfite from sodium thiosulfate in Kligler agar; the presence of cytochrome oxidase; citrate utilization; urea hydrolysis in Christensen's agar; the presence of phenylalanine deaminase; the liberation of indole; acid production from sugar fermentation; the decarboxylation of lysine, ornithine, and arginine; and the utilization of malonate as the sole carbon source.In addition, a nutrient agar plate was inoculated from each BHI broth and incubated at 35°C for 24 hours; the colonies were used for the oxidase test.

Serotyping and phage typing
Serotyping and phage typing of isolates were performed at the National Microbiology Laboratory (NML), Public Health Agency (PHC) of Canada according to the Kauffmann-White scheme and the recommendations of Edwards and Ewing [14].

Molecular characterization
The serovars that were resistant to either amoxicillin-clavulanic acid, cephalexin, or cefuroxime alone or in combinations were screened by PCR using two primer sets sequence specific for bla TEM and bla CTX-M genes to amplifythe DNA templatefor genes coding for ESBL production [3].
DNA was extracted from Salmonella isolates using Wizard Genomic DNA purification Kit (Promega, Fitchburg, USA) according to manufacturer's specifications.Oligonucleotide pairs that amplified 708 bp and 593 bp products of bla TEM and bla CTX-M genes, respectively, were used (Table 1).PCR amplification employed a final volume of 25 μL containing 1.5 mM MgCl 2, 100μM deoxyribonucleoside triphosphate, 0.2μM of each oligonucleotide, and 1U of Taq DNA polymerase.The PCR amplification reaction for the detection of bla TEM consisted of 36 cycles of 30 seconds of denaturation at 94°C, 30 seconds of hybridization at 42°C, and 60 seconds of extension at 72°C, with a final extension 714 step at 72°C for 10 minutes [3].The bla CTX-M gene was identified with PCR conditions similar to those described for bla TEM , except for the alteration of the annealing temperature to 58°C [3].The amplified products were visualized electrophoretically on 2% agarose gel [15].For quality control, S. branderup H9812 ATCC BAA-664 was used.

Pulsed-field gel electrophoresis analysis
Pulsed-field gel electrophoresis (PFGE) was performed usingthe standardized protocol of PulseNet Canada Laboratory for molecular subtyping by using the restriction enzymes XbaI and BlnI according to the standardized protocol for E. coli (O157:H7).Salmonella switch times of 2.2-63.8seconds was used for the molecular typing of Salmonella isolates [15].S. braenderup strain H9812 (ATCC BAA 664) was used as a reference strain.DNA macrorestriction profiles were analyzed with BioNumerics software version 2.5 (Applied Maths, Saint Martens-Latem, Belgium) using a band tolerance of 1.5% and optimized at 1.0%.Dendrograms were constructed on the basis of the unweighted pair group method of averages, position tolerance of 1%.Clusters were defined as DNA patterns sharing ≥ 85% similarity [16].PFGE pattern designations were assigned according the PulseNet Canada database.

Ethical considerations
Ethical approvals were obtained from the University of Abuja Teaching Hospital Gwagwalada, Abuja, National Hospital Abuja, and Health and Human Services Secretariat of the Federal Capital Administration.Informed consent was obtained from the parents of the patients who were to participate in the study.A questionnaire was used to collect epidemiological data following the principles proposed by the model ethical protocol in accordance with the Helsinki declaration [18].

Results
The nine strains of Salmonella isolates were typed into three serovars with specific antigenic formulae.The serotypes identified were Salmonella Zanzibar (n = 5), Salmonella Brancaster (n = 3), and one strain of Salmonella Enteritidis.The S. Enteritidis strain was phage type 1; the phage types of S. Zanzibar and S. Brancaster were not determined (Table 2).
There was no genetic diversity in the PFGE patterns of XbaI digested chromosomal DNA of the five isolates of S. Zanzibar, as all had the same restriction pattern.The PFGE patterns of the five strains of S. Zanzibar isolated appeared identical and hence genetically related, as did the three strains of S. Brancaster isolates (Table 3).
The following levels of resistance were found among the Salmonella strains: amoxicillin, five strains (55.6%); augmentin, two strains (22.2%); cephalexin, five strains (55.6%); and cefuroxime, five strains (55.6%).Intermediate resistance was found in five strains (55.6%)only to amoxicillin-clavulanic acid (Augmentin).All the serotypes isolated were susceptible to nalidixic acid, ciprofloxacin, and ceftriaxone (Table 4a).In all, seven antimicrobial resistant phenotypes were observed among the three Salmonella serovars.All the Salmonella serovars with the exception of one S. Zanzibar strain were resistant to two or more antibiotics.Pan susceptibility was detected in only one strain each of S. Zanzibar and S. Brancaster.Similarly, a multidrug-resistant pattern to four antibiotics (AMX, AUG, CEPH, and CEFU) was detected in only one strain each of these serovars (S.Zanzibar and S. Brancaster).Among the three Salmonella serovars, common resistant profile patterns to three antibiotics (AMX, CEPH, and CEFU) were observed (Table 4b).Although among the Salmonella serovars, multidrug resistance was observed in five (55.6%) of the Salmonella serovars (two S. Zanzibar, two S. Brancaster) and in the only S. Enteritidis serovar, none exhibited resistance MICs to tested antimicrobial agents according to the current CLSI breakpoints.In addition, all the serotypes exhibited susceptible zone inhibition diameters ranging from 32-34 mm (minimum and maximum) to ceftazidime, cefotaxime, and cefpodoxime alone or in combination with clavulanic acid.No ESBL-producing Salmonella isolates identified as either TEM or CTX-M-type ESBLs genes were detected in any of the isolates.Screening of isolates for bla SHV and AmpC betalactamase production was not performed in this study.Enteritidis).The different patterns, sources, year of isolation, and number of strains are indicated.The Zanzibar and Brancaster isolates were the first from north-central Nigeria to be ever typed using PFGE.The five S. Zanzibar isolates all hade the same restriction patterns using XbaI (no genetic diversity was identified), but one isolate had a different BlnI restriction pattern than the other four.Four Zanzibar isolates were from mucoid-type diarrhea.All three S. Brancaster isolates assigned to the same PFGE pattern had the same XbaI and BlnI patterns.Of the three Brancaster isolates, two isolates were from the municipal area and one was from the peri-urban area.No comparative assessment of S. Enteritidis was possible, but the lone isolate was recovered from Abuja Municipality and was phenotypically phage type 1.

Discussion
This study characterized Salmonella species recovered from children with acute gastroenteritis, and determined antibiotic susceptibility patterns and extended-spectrum beta-lactamase production using polymerase chain reaction, pulse field gel electrophoresis, micro dilution, and double disk diffusion techniques.Notably, a rare serotype incriminated in human Salmonella infections, S. Zanzibar, had the highest prevalence (55.6%) in this study.The first reported human infection with the serotype S. Zanzibar was in 1976 from the stool culture of an eight-year-old boy with gastroenteritis in Ankara [19].Thereafter, it has been documented to be associated with adult infections in North Scotland [20], and more recently in Tunis [21].Notably, multidrug resistant S. Brancaster isolated from poultry has been reported in Dakar, Senegal [22].The reported epidemiology of S. Brancaster in the literature is rather limited [23].However, its occurrence in our study is in consonance with reported isolation from human cases in the Toscana region of Italy [24], in the United States [25], and in Senegal, on the West African coast [24].Recently, resistance to spectinomycin and streptomycin by S. Zanzibar isolated from chicken muscle was observed in Dakar, Senegal [26].
The choice of antimicrobial agents evaluated in this study was based on reported resistance of Salmonella isolates to commonly used antimicrobials [27].The results of this study showed that 77.8% of the Salmonella isolates tested for antimicrobial susceptibility were resistant to two or more antibiotics.Although a previous study in Nigeria had reported a resistance rate of 61.0% [28], other authors from Ethiopia [29] recently reported a resistance rate of 83.3%, which is, comparatively, a higher proportion of multidrug resistance.The finding of good antimicrobial activity for ciprofloxacin is in line with earlier reports from Lagos, Nigeria [28].
The identification of no ESBL-producing Salmonella serovars is consistent with the findings of some previous studies in developed and developing countries.No ESBLs were detected among human Salmonella isolates in multicenter surveys conducted by a France hospital-based network in 1994 (n= 2,622) and 1997 (n=2,464) [30].Similarly, in China, no ESBL-producing isolates were identified among human diarrheal Salmonella isolates from 28 hospitals in Henan Province in 2006 [31].More recently, non-ESBL-producing Salmonella isolates were identified in another multicenter study conducted in India that monitored in vitro susceptibility of community-or hospital-acquired Gram-negative bacilli in intraabdominal infections to identify early changes in susceptibility patterns, with a focus on ESBL producers.[32].Therefore, the antibiotic resistance exhibited by the Salmonella serotypes isolated as observed from the disk diffusion susceptibility tests may have been conferred by a mechanism for β-lactam resistance other than those analyzed in this study.Conversely, the identification of non-ESBL-producing Salmonella isolates may be inadvertent, but not altogether unusual.

Conclusions
Molecular characterization helps scientists to understand the nature of the epidemiology of infections caused the different Salmonella serotypes.Our findings have demonstrated the involvement of three Salmonella strains in acute gastroenteritis; ciprofloxacin, ceftriaxone, and nalidixic acid remain the antibiotics of choice in the management of acute gastroenteritis caused by the three strains of Salmonella isolates.The study further demonstrated the absence of extended-spectrum beta-lactamase producing Salmonellae.718 contribution in collecting the samples for study, and the staff of the Department of Biological Science, University of Abuja, for the logistic support.We also thank Laboratoire de Contrôle des Eaux et Denrées Alimentaires de l'Institut Pasteur de Tunis in Tunis, Tunisia for their valuable input into this study.

Figure 1
illustrates phylogenic dendrogram of PFGE patterns obtained using the restriction enzymes XbaI and BlnI showing percent similarity calculated by the Dice similarity index of PFGE restriction endonuclease digestion profiles among the nine Salmonella isolates (S.Zanzibar, S. Brancaster, S.

Figure 1 .
Figure 1.Dendogram showing the cluster analysis on the basis of XbaI-PFGE and BInI-PFGE of the three Salmonella serovars.ND: Not determined;NHA: National Hospital Abuja; MDH: Maitama Distict Hospital; NGH: Nyanya General Hospital

Table 1 .
Primers used in identifying ESBL gene production in Salmonella serogroups

Table 2 .
Demographic and phenotypic characteristics of Salmonella serotypes isolated from diarrheic children younger than five years of age in FCT, Abuja, Nigeria

Table 3 .
The PFGE patterns of XbaI and BlnI digested chromosomal DNA of Salmonella serovar isolates from diarrheagenic children younger than five years of age in FCT Abuja, Nigeria

Table 4b .
Antibiotic resistance pattern of nine strains of Salmonella serovars isolated from children younger than five years of age in FCT, Abuja Nigeria.
AMX: amoxicillin; CEPH: cephalexin; CEFU: cefuroxime; CEFTR: ceftriaxone; AUG: Augmentin (amoxicillin-clavulanic acid) 716 Comparatively, one isolate out the five S. Zanzibar isolates had a different BlnI restriction pattern than the other four (relating to a single band shift).The three isolates of S. Brancaster had the same restriction patterns for XbaI and BlnI digested chromosomal DNA, respectively.No comparison could be made for serovar Enteritidis clonality based on PFGE, as only a single patient isolate was recovered.