Antimicrobial Resistance and Virulence-Associated Genes of Salmonella enterica Subsp. enterica Serotypes Muenster,Florian,Omuna,and Noya Strains Isolated from Clinically Diarrheic Humans in Egypt

Abstract

Four serotypes recovered from clinically diarrheic human faecal samples (Salmonella Muenster, Salmonella Florian, Salmonella Omuna and Salmonella Noya) were investigated for the presence of 11 virulence genes (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) and their association with antibiotic resistance. The 4 Salmonella serotypes lacked virulence genes gipA and spvC. Resistance to 7 of the 14 antimicrobials was detected. The frequency of resistance, to lincomycin and streptomycin (100% of the Salmonella Muenster [2/5], Salmonella Florian [1/5], Salmonella Omuna [1/5], and Salmonella Noya [1/5] isolates), chloramphenicol (100% of the Salmonella Muenster [2/5] and Salmonella Florian [1/5] isolates) and trimethoprim–sulfamethoxazole (100% of the Salmonella Florian [1/5] and Salmonella Omuna [1/5] isolates) was an outstanding feature. With the rest of the antibiotics, the four Salmonella serotypes exhibited a great diversity in their resistance patterns. Overall, the four Salmonella serotypes were resistant to more than one antimicrobial. The antimicrobials to which the Salmonella Muenster, Salmonella Florian, and Salmonella Omuna isolates were resistant, contributed to five different antimicrobial resistance profiles. The virulence associated genes invA, ssaQ, siiD, sopB, and bcfC genes were 100% associated with certain antimicrobial resistance phenotypes (streptomycin and lincosamide) not recorded previously, and secondly, the presence of invA, avrA, ssaQ, mgtC, siiD, sopB, and bcfC was associated with resistance to chloramphenicol. The results of this study will help in understanding the spread of virulence genotypes and antibiotic resistance in Salmonella in the region of study.

Introduction

S almonella enterica has been recognized as a cause of intestinal disease for many years.⁴² S. enterica remains the primary cause of reported food poisoning worldwide and recent years have seen massive outbreaks.^28,46 A recent study estimated that ∼93.8 million human cases of nontyphoidal gastroenteritis and 155,000 deaths occur due to S. enterica infection around the world each year.²⁹ Data on salmonellosis are scarce in many countries of Asia, Africa, and South and Central America where only 1% to 10% of cases are reported.³⁶ Rates have been reported to vary considerably by geographic region as in the USA and Europe.^24,25 Large differences in the most commonly isolated serotypes between regions, with lesser differences between countries within the same region were recorded.^11,13,15,20 Most cases of invasive nontyphoidal Salmonella (NTS) disease across Africa are due to either Salmonella Typhimurium or Enteritidis although investigators at some sites report contributions from other serotypes, such as Salmonella Isangi in South Africa, Salmonella Concord in Ethiopia, and Salmonella Stanleyville and Salmonella Dublin in Mali.^11,15,27 The analyses conducted by Hendriksen²⁰ from 37 countries, with the exception of the Oceania and North American regions, ranked Salmonella serotypes Enteritidis and Typhimurium as the most common and second most common serotype, respectively. Although the relative importance of Salmonella Enteritidis and Typhimurium is decreasing globally,²⁰ other serotypes, such as Newport, Infantis, Virchow, Hadar, and Agona are increasing in addition to Enteritidis and Typhimurium.^3,20,21,33

Salmonella is a leading cause of foodborne illness worldwide through the consumption of food products of animal origin. Several lines of evidence indicate that antibiotic-resistance among human Salmonella infections results from the use of antimicrobial agents in food animals.^2,9,10,43 The first suggestion that antibiotic use in livestock led to antibiotic-resistant bacteria was in 1951 when a streptomycin resistant generic intestinal bacteria was isolated from turkeys that had been fed with streptomycin as a growth-promoting supplement.³⁹ Antibiotic resistance, especially to the most commonly used antimicrobials in humans is of critical concern in African countries where multidrug-resistance NTS strains are among the most frequent causes of bacteraemia in children.⁸ Due to the increasing resistance of this bacterium to conventional antimicrobial agents (ampicillin and trimethoprim/sulfamethoxazole) used in the treatment of salmonellosis, amoxicillin/clavulanate, third generation cephalosporins, and fluoroquinolones have become further treatment options.^7,8 A very important issue that has been debated is whether the multidrug resistant (MDR) strains are associated with virulence determinants, and thus, have the propensity to be more virulent than their susceptible equivalents.¹⁴ The fact that genetic determinants for both antibiotic resistance and virulence genes could be harbored by the same transferable element⁴ implies that there is an association between antibiotic resistance and virulence.^8,14,27 The association between the invasion-associated loci with antibiotic resistance genes suggests that, the overall observations indicate that the virulence plasmid may act as a general platform for multiple and varied capture of antibiotic, heavy metal, and antiseptic resistance.^23,27,31 Such association could have an impact on the spread of resistance clones of Salmonella.^8,14 Consequently, assessing the presence of virulence genes as well as the antibiotic resistance status in Salmonella serotypes would be useful to better understand its pathogenicity.^8,14

Although some Salmonella serotype virulotyping studies have been undertaken across Europe,²⁶ USA and Mexico,^40,44 Africa,^8,27,34 and Asia,³⁵ little is known of the distribution of virulence and antibiotic resistance phenotypes in strains across Egypt as one of the Middle East countries. The aim of this study was to determine the distribution of virulence genes and antimicrobial resistance within Salmonella field strains isolated from diarrheic humans in Egypt. The isolated field strains were screened for 11 potential virulence factors (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction (PCR).

Materials and Methods

Study and case participants

Specimens' collection from cases was from both male and female diarrheic patients from all age categories attending the Outpatient Department of Internal Medicine, Kasr AlAini hospital, Faculty of Medicine, Cairo University, Egypt during 2011. Written informed consent was obtained from all patients by trained health workers. Information provided included the frequency of episodes of diarrhea and whether or not antibiotic or other forms of medication has been used. The study protocol and data handling were approved by the Faculty of Medicine, Kasr AlAini Hospital, Cairo University, Egypt Ethics committee.

Isolation and identification of Salmonella

Fresh rectal swabs were collected from 1,500 patients presented with acute diarrhea in the tertiary referral facility outpatient clinic in Cary-Blair transport medium per patient. These samples were transported on ice pack to the laboratory where bacteriological analysis was done within 24 hr. The rectal swabs were first pre-enriched in buffered peptone water (Oxoid) overnight at 37°C. About 1 ml of this suspension was then added to 9 ml of tetrathionate brilliant green broth (Oxoid) (enrichment). After overnight incubation at 37°C, a drop of this suspension was spread on brilliant green agar (Oxoid). Colonies typical of Salmonella were sub-cultured onto tryptic soya agar (BBL, Becton Dickinson) slants and subjected to a series of biochemical tests for identification of Salmonella spp. The isolates were archived at −80°C in Luria-Bertani broth (Sigma-Aldrich) containing 15% glycerol. Serotyping of the isolates was done at the National Salmonella and Escherichia Centre, Ministry of Health, Cairo, Egypt. For PCR, a loop of bacterial culture was resuspended in 50 μl of water, and DNA was released from bacterial cells by boiling the suspension for 20 min. After the mixture was spun for 1 min in a microfuge at 14,000 g, 2 μl of the supernatant was taken as a template DNA for PCR. PCR was carried out in 20 μl volumes by using PCR Master Mix from Qiagen according to the manufacturer's instructions.

PCR virulotyping

All isolates were screened by PCR analysis for the presence or absence of 11 selected virulence genes. The primers designed in this study for PCR detection of various virulence genes are listed in Table 1. PCR amplifications of invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC genes were performed as described previously.^19,26,38 The PCR cycling conditions consisted 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C and 1 min extension at 72°C. The specificity of the oligonucleotide primers were detected by using bacterial strain of Salmonella Enteritidis (ATCC 13076) and Escherichia coli (ATCC 50034) as reference strains.

Table 1.

Virulence Factor Targets and Primers, Including Nucleotide Sequences, PCR Conditions, and References

		PCR conditions
References	Product size (bp)	Extension	Annealing	Denaturing	Oligonucleotide sequences (5'-3')	Location on SPI1/Gene function	Gene designation
38	284	72°C for 30 sec^a	64°C for 30 sec	94°C for 60 sec	gtg aaa tta tcg cca cgt tcg ggc aa tca tcg cac cgt caa agg aac g	Type III secretion system apparatus SPI-1/Invasion of macrophages	invA
19,26	422	72°C for 30 sec^b	58°C for 30 sec	95°C for 30 sec	cct gta ttg ttg agc gtc tgg aga aga gct tcg ttg aat gtc c	SPI-1/Controls Salmonella-induced inflammation	avrA
	455				gaa tag cga atg aag agc gtc gtc ccat cgt gtt atc ctc tgt cag c	SPI-2/Secretion system apparatus protein, component of second T3SS	ssaQ
	677				tga cta tca atg ctc cag tga atatt tac tgg ccg cta tgc tgt tg	SPI-4/ Mg²⁺ uptake	mgtC
	655				gaa tag aag aca aag cga tca tcgct ttg ttc acg cct ttc atc	Type I secretion/SPI-4	siiD (Spi4D)
26	517				tca gaa gRc gtc taa cca ctctac cgt cct cat gca cac tc	SPI-5/Inositol polyphosphate phosphatase that promotes macropinocytosis, regulates SCV localization, and promotes fluid secretion	sopB
	518				acg act gag cag cgt gagttg gaa atg gtg acg gta gac	Gifsy-1 bacteriophage/Peyer's patch-specificVirulence factor	gipA
	424				cgg gca gtg ttg aca aat aaagtgt tgg aat tgt gga gtc	Gifsy-2 bacteriophage/Periplasmic Cu, Zn-superoxide dismutases	sodC1
	422				act cct tgc aca acc aaa tgc ggatgt ctt ctg cat ttc gcc acc	Cryptic bacteriophage/Promotes membrane ruffling and disrupts tight junctions	sopE1
	467				acc aga gac att gcc ttc cttc tga tcg ccg cta ttc g	pSLT /A phosphothreonine lyase required for complete virulence in murine models	spvC
	467	72°C for 30 sec^b	53°C for 30 sec	95°C for 30 sec	acc aga gac att gcc ttc cttc tgc tcg ccg cta ttc g	Chromosome/Bovine colonization factor, fimbrial usher	bcfC

PCR was done for 35 cycles.

After 30 cycles, final extension step of 4 min at 72°C was performed.

PCR, polymerase chain reaction.

Antimicrobial susceptibility tests

Inhibitors of the cell wall synthesis (aminopenicillins, colistin), protein synthesis (gentamicin, streptomycin, tetracyclines, chloramphenicol, lincomycin and neomycin), and nucleic acid synthesis (norfloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, trimethoprim, and nalidixic acid) were employed for inhibition tests. The antimicrobial susceptibility of Salmonella isolates of this study was determined by standard agar disc diffusion technique in accordance with the Clinical and Laboratory Standards Institute (CLSI).⁵ The panel of antibiotic disks (Becton, Dickinson and Company) used in the panel screens belonged to eight drug classes. Ranked by both OIE^9,10 and WHO^9,10,43 lists, the following antimicrobials were chosen given their common use in treating and preventing Salmonella infection in poultry and human and because they represented a variety of antimicrobial classes. The antimicrobial agents tested and their corresponding concentrations were as follows: ampicillin (10 mg), amoxicillin (20 mg), gentamycin (10 mg), neomycin (30 μg), streptomycin (10 mg), lincomycin (30 μg), chloramphenicol (30 mg), colistin (10 mg), tetracycline (30 mg), trimethoprim (5 μg), sulphamethoxazole+trimethoprim (23.75+1.75 mg). In addition, the resistance to three fluoroquinolones was tested: ciprofloxacin (5 μg), norfloxacin (5 μg) and nalidixic acid (30 mg). Antibiotic discs were placed onto Mueller-Hinton Agar (Difco Laboratories) (six discs in each plate) and the plates were incubated for 24 to 48 hr at 37°C under microaerophilic conditions. After the incubation, the diameter of the halos was measured. Multidrug resistant isolates are defined as those resistant to more than three classes of antibiotics.¹⁹ Susceptibility and resistance were determined according to the interpretation criteria to E. coli standard (ATCC No. 25922) established by CLSI.

Results

Incidence

In this study, four S. enterica subsp. enterica serovars were recovered in <2% of all Enterobactereaceae isolated in the hospital. The four isolated serotypes (Table 2) recovered from fecal samples of different patients during 2011 were: Salmonella Muenster (two isolates), Salmonella Florian (one isolate), Salmonella Omuna (one isolate) and Salmonella Noya (one isolate).

Table 2.

Distribution of the Virulence Genes Among the Salmonella Serotypes

		Virulence genes
Salmonella serotypes	Number of isolates	invA	avrA	ssaQ	mgtC	siiD	sopB	gipA	sodC1	sopE1	spvC	bcfC
Muenster	2	2	2	2	2	2	2	NE	2	2	NE	1
Florian	1	1	1	1	1	1	1	NE	NE	1	NE	1
Omuna	1	1	NE	1	NE	1	1	NE	NE	NE	NE	1
Noya	1	1	1	1	1	1	1	NE	NE	NE	NE	1
Total	5	5 (100%)	4 (80%)	5 (100%)	4 (80%)	5 (100%)	5 (100%)	0 (0%)	2 (40%)	3 (60%)	0 (0%)	4 (80%)

NE, not expressed.

Virulotyping

All isolates were screened by PCR analysis for the presence or absence of 11 selected virulence genes (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC). The results recorded in Table 2 indicate that, five of the virulence genes, invA, ssaQ, siiD, sopB (carried by Salmonella pathogenicity islands SPIs), and bcfC (fimbria-related) were detected in all the Salmonella isolated serotypes of this study (100%). In contrast, all of the isolated Salmonella serotypes lacked the spvC gene (carried by Salmonella virulence plasmid) and the gipA gene (encoding a Peyer's patch-specific virulence factor, GipA) was absent in all of the Salmonella isolated serotypes. Except for the Salmonella Omuna serotype, all isolates also carried the avrA gene located in SPI1. Likewise, the mgtC gene (carried by Salmonella pathogenicity islands SPIs) was found in all isolates belonging to Salmonella Muenster, Salmonella Florian and Salmonella Noya, whereas for sodC1 gene (located on a functional lambda-like bacteriophage known as Gifsy-2) only the Salmonella Muenster isolates tested were positive. The sopE1 gene (encoding a translocated effector protein) was detected in only 1/2 of the Salmonella Muenster isolates (Table 2). Altogether, in these serotype collections, five different combinations of virulence genes were detected (Tables 2 and 4). All four Salmonella serotypes tested were not represented by identical virulence gene repertoires.

Distribution of resistance to individual antimicrobial agents

Considering the Table 3, it seems that there is an observed resistance for at least 9/14 antibiotics. All Salmonella serotypes Muenster (2/2), Florian (1/1), Omuna (1/1), and Noya (1/1) were 100% resistant to lincomycin and streptomycin. The two Salmonella serotypes Muenster (2/2) and Florian (1/1) isolates were 100% resistant to chloramphenicol, while Florian (1/1) and Omuna (1/1) isolates were 100% resistant to trimethoprim–sulfamethoxazole. With the rest of the antibiotics, the four Salmonella serotypes (4/4) exhibited a great diversity in their resistance patterns.

Table 3.

Distribution of Resistance to Individual Antimicrobial Agents Among Salmonella enterica Serotypes in Human (n=5)

	Distribution of resistance to antimicrobials
Antimicrobials	Salmonella Muenster (n=2/5)	Salmonella Florian (n=1/5)	Salmonella Omuna (n=1/5)	Salmonella Noya (n=1/5)
Penicillins
Ampicillin	0/2	0/1	1/1	0/1
Amoxicillin	0/2	0/1	0/1	0/1
Aminoglycosides
Gentamicin	1/2	0/1	0/1	0/1
Neomycin	2/2	0/1	0/1	0/1
Streptomycin	2/2	1/1	1/1	1/1
Fluoroquinolones
Ciprofloxacin	0/2	0/1	0/1	0/1
Nalidixic acid	0/2	0/1	1/1	0/1
Norfloxacin	0/2	0/1	0/1	0/1
Lincosamides
Lincomycin	2/2	1/1	1/1	1/1
Phenicols
Chloramphenicol	2/2	1/1	0/1	0/1
Polymyxin
Colistin sulphate	0/2	1/1	0/1	0/1
Tetracyclines
Tetracycline	1/2	0/1	0/1	0/1
Sulphonamides
Trimethoprim	0/2	1/1	1/1	0/1
Trimethoprim–sulfamethoxazole	1/2	1/1	1/1	0/1

n, resistant isolates.

Overall, the four Salmonella serotypes were resistant to more than one antimicrobial indicating multiple resistances (≥2 antimicrobial types). The antimicrobials to which Salmonella Muenster (2/2), Florian (1/1) and Omuna (1/1) isolates were resistant, contributed to five different antimicrobial resistance profiles of which aminoglycosides, lincosamides and sulphonamides were common to the three serotypes. Salmonella Muenster isolates exhibited an additional phenicols and tetracyclines profile, while Salmonella Florian displayed a phenicols and polymyxin profile and Salmonella Omuna revealed a penicillin and fluoroquinolones profile. The Salmonella Noya isolate (1/1) was the least in its resistance patterns as seen in the two different antimicrobial resistance profiles (aminoglycosides and lincosamides).

Association of antimicrobial resistance phenotype with virulence-associated genes

Analysis of the presence of the invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC genes in the five isolates with various antimicrobial resistance patterns was conducted as shown in Table 4. A detailed analysis displayed associations of resistance/susceptibility phenotypes with potential virulence genes. Firstly, the virulence associated genes invA, ssaQ, siiD, sopB, and bcfC genes were 100% associated with streptomycin and lincosamide antimicrobial resistance phenotypes. This result has never been recorded. Secondly, the presence of invA, avrA, ssaQ, mgtC, siiD, sopB, and bcfC was associated with resistance to chloramphenicol.

Table 4.

Distribution of Virulence Genes Combinations in the Different Salmonella Serotypes and Antibiotic Resistance Phenotypes

Salmonella serotypes	Virulence genes combination	Resistance phenotype	Number of isolates (%)
Muenster	inv A, ssa Q, sii D, sop B , bcf C, avrA, mgtC, sodC1, sopE1	Chl, Gen, Lin, Neo, Str	1/2 (50)
Muenster	inv A, ssa Q, sii D, sop B , bcf C, avrA, mgtC, sodC1	Chl, Lin, Neo, Nor, Str	1/2 (50)
Florian	inv A, ssa Q, sii D, sop B , bcf C, avrA, mgtC, sopE1	Chl, Col, Lin, Str, Tri, Sxt	1/1 (100)
Omuna	inv A, ssa Q, sii D, sop B, bcf C	Amp, Lin, Na, Str, Tri, Sxt	1/1 (100)
Noya	inv A, ssa Q, sii D, sop B , bcf C, mgtC, avrA,	Lin, Str	1/1 (100)

Bold indicates where the frequency of the virulence genes is consistent.

Amp, ampicillin; Col, colistin sulphate; Tet, tetracyclin; Cip, ciprofloxacin; Str, streptomycin; Nor, norfloxacin; Gen, gentamycin; Chl, chloramphenicol; Neo, neomycin; Lin, lincomycin; Na, nalidixic acid; Tri, trimethoprim; Sxt, trimethoprim-sulfamethoxazole.

Discussion

Although 2,600 serotypes of Salmonella enterica have been identified,^1,12,22,42 most human infections are caused by a limited number of serotypes. During the last decade, there have been changes in the relative contributions of different serotypes to the overall Salmonella burden on human health.²⁰ Salmonella Typhimurium and Enteritidis are the most common causes of human salmonellosis worldwide, although other serotypes have been reported to be more prevalent in some regions.^22,45 However, the epidemiology of human NTS or enterocolitis is dominated by at least 150 Salmonella serotypes.³⁶ Shifts in prevalence of specific strain types and serotypes can reflect the influence of international travel and trade of animals and food products, and can therefore, serve as useful epidemiologic markers.^1,45 Although the needs assessment found that institutions in Africa, Middle East, Asia and Southeast Asia were more likely to report difficulty in obtaining antisera, especially for serotyping unusual Salmonella strains, than were institutions in other regions, yet we were able to detect four Salmonella serotypes that were not previously reported in Egypt, Salmonella Muenster (2/4), Florian (1/4), Omuna (1/4), and Noya (1/4). Salmonella enterica serotype Muenster is rarely identified from humans.⁴¹ The possible role of domestic animals in infecting humans and on the prevalence of Salmonella Muenster in the USA, Canada, and France was studied by several authors.⁴¹ The isolation of Salmonella Muenster in the present investigation suggests that the human cases were commonly associated with consumption of raw milk and cow manure⁴¹ or food raw materials contaminated with Salmonella Florian.²⁹ Egypt raises native crops and animals that are then consumed locally. This locally produced food may harbor a greater diversity of less commonly reported serovars.²⁰ There are many potential reasons why the isolates serotyped may differ between countries. An apparent reason may be due to geographical, cultural, or socioeconomic factors in a country with a truly low proportion of serotyped Salmonella due to a low number of human cases.²⁵ Although it is essential that data from different countries are comparable, yet few countries report data on the societal and economic impact of salmonellosis. Comparisons with other studies should be made with caution due to differences in monitoring schemes and/or study design, laboratory methodologies, quality of the antisera available, an under-detection bias or reflects true differences in NTS illnesses.^{20–22,24,25} Outpatient visitors to the Faculty of Medicine, Abou ElReesh Hospital, Kasr Al Ainy, where the strains were obtained and subjected to analyses in the present investigation were Arab patients and Egyptians who engage in various occupational activities with a result in that, the potential sources of contamination vary. Kasr Al Ainy location (30°2′14′′N 31°12′28′′E; 19.6 km²) has a special nature in its containment of a diversity in its demography and commercial activities and is a very busy commercial area where a wide diversity of activities and which are abundant in the area, in the form of grocery stores, butcheries, shops selling live poultry, dairies, cafeterias, fresh fruit, produce, and vegetable retail shops constituting .a source of high risk occupational hazard.

The distribution of 11 virulence genes that play an important role for invasion, enterotoxin production and pathogenesis in the host were investigated. The invA gene, detected in the five isolated strains in the present investigation, is an invasion gene conserved within the S. enterica serotypes and is an useful marker for molecular detection of Salmonella by PCR.⁸ The findings on SSPs and SpvC genes carried by Salmonella Enteritidis strains prevalent in Europe, USA, and some African and Asian countries are readily available.^{8,26,34,35,40,44} However, such data regarding strains prevalent in Egypt has never been published. This study documents the first results on the presence of nine potential virulence effectors, invA, avrA, ssaQ, mgtC, siiD, sopB, sodC1, sopE1, and bcfC genes and the absence of spvC and gipA genes in Salmonella Muenster, Floria, Omuna and Noya. In a previous study,²⁶ the same 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) were screened in five predominant Salmonella serotypes isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar) where most of these virulotypes were restricted to only one or two serotypes and that the virulotype did not vary significantly with host source or geographical location. We also observed that, some virulence patterns were serotype specific, for example, Salmonella Muenster (1/2), Salmonella Florian (1/1), Salmonella Omuna (1/1), and Salmonella Noya (1/1) strains shared a common virulotype, which was characterized by the presence of five genes (invA, ssaQ, siiD, sopB, and bcfC). The bcfC fimbrial gene was present in 100% of our human isolates a two-fold higher percentage than that recorded by Graziani et al.¹⁶ The prevalence of Salmonella sopB and sopE genes among different serotypes of S. enterica isolated from cases of enteritis in man was investigated by several authors.^8,32,37 Recently, Dione et al.⁸ found that the sopE gene was common in serotypes isolated from diarrheic children, chicken, sheep, and goats in The Gambia. Thus, the expression of the sopE gene in our investigation might implicate the sopE gene in diarrheic children in Egypt. It should be noted that, while the Salmonella plasmid virulence (spv) operon,^17,46 which consists of five genes (spvRABCD),^14,17,46 potentiates the systemic spread of the NTS strains and aids in its replication in extra-intestinal sites^14,17 yet, spvC was not expressed in our investigation.

Antibiotic resistance, especially to the most commonly used antimicrobials in humans is of critical concern in African countries where multidrug-resistance NTS strains are among the most frequent causes of bacteraemia in children.^{8,11,15,27,34} The emergence of resistance to fluoroquinolones among nontyphoid Salmonella is of particular concern, since this class of antimicrobial agents constitutes the drug of choice for treating potentially life-threatening Salmonella infections caused by multiple-antibiotic resistant strains in adults.² Cephalosporins are the drugs of choice for children because they cannot be treated with fluoroquinolones. Salmonella exhibiting resistance to cephalosporins is an emergent problem worldwide.^42,43 We found that the most common multidrug resistance phenotype reported was to streptomycin, lincomycin, chloramphenicol, and trimethoprim+sulfamethoxazole. Reports from other countries were found to exhibit an increasing frequency of ciprofloxacin-resistant isolates⁴⁵ in contrast to the situation in Egypt. Also, interestingly of the 14 commercial antimicrobial agents tested, only two (lincomycin and streptomycin) proved to be 100% effective against all isolates and which was not previously pointed out.

In our investigation, we found that, there was significant association between invA, ssaQ, siiD, sopB, and bcfC virulence genes and resistance to the commonly used antibiotics in Egypt (Table 4) as previously observed in Africa^8,27 and other parts of the world¹⁴ but with different associations between virulence genes and antibiotics. The significant association between sopB, virulence gene and resistance to commonly used antibiotics in Egypt, namely amoxicillin, trimethoprim+sulfamethoxazole, tetracycline, trimethoprim, and streptomycin was also recorded in Senegal and The Gambia.⁸ Although the invasiveness of strains with an association pattern between spv and ACSSuT has been documented previously,^14,27 yet, we found that one of the most important genes in the spv operon (spvC) associated with MDR is absent from all diarrheic patients. Therefore, since this virulence gene was not detected, we could hypothetically imply that our isolates could also exhibit such epidemic potential and that carriage of invA, ssaQ, siiD, sopB, and bcfC together with the MDR phenotype enables these strains to be of primary clinical importance compared to those MDR strains carrying the spvC gene or part of the operon. These associations could be explained based on the mechanisms involved in the pathogenicity and the acquisition of resistance genes by Salmonella. The majority of the molecular pathogenicity determinants are located on the chromosome or large virulence-associated plasmids,¹⁸ whereas antibiotic resistance genes are also often located on extrachromosomal genetic elements or in segments inserted within the chromosome that originate from other genomes.⁴ It has been reported that resistance plasmids carry genes encoding virulence factors.³⁰ As antibiotic resistance and virulence genes can be linked in the same replicon, eventually a single determinant can be involved in both virulence and resistance.³⁰

In conclusion, the isolates tested positive for five to nine virulence genes, implying that these strains have the necessary virulence gene capable of playing an important role in causing severe infection. Because the emerging clone (sopB and sopE) was dominant in all levels of the food chain, including fish and broiler chickens, it is possible that the emerging clone was originally introduced from a marine or poultry source.^8,35 It is likely that environmental selective pressure caused by use of antimicrobial drugs to treat infections and promote growth of livestock has led to the distribution of appropriate resistant strains.⁴² There is a need of continuous surveillance and sharing of antimicrobial susceptibility data for Salmonella among countries worldwide⁶ to ensure the effectiveness of control programmes. To assist national and international efforts aimed at control of Salmonella, cooperation between the countries of the Middle East region should focus on the analyses of the distribution of the most prevalent Salmonella serotypes, virulence determinants and MDR. The carriage of virulence genes among MDR strains may increase the propensity of such strains to be of major clinical relevance. Our results and those previously recorded^{8,14,23,27,31} have shown the possible importance with regard to the association of resistance and virulence genes in Salmonella leading to a better understanding of the full mechanism of the pathogenicity of Salmonella. The present study has a few limitations. First, the conclusions made from this study are from a limited number of isolates. It would be desirable to investigate a greater population of Salmonella isolates for this purpose. Second, travel histories were not available; therefore, for isolates from patients without information about travel history, we could not be sure whether the isolates tested originated in Egypt or whether they were imported from another country. Third, no data are available on high risk occupations of the patients. Fourth, despite the limitations, these data provide baseline information, and context for future research and surveillance efforts. Antimicrobial sensitivity testing of this limited number of Salmonella isolates has provided some baseline information on the resistance of Salmonella to individual and multiple antimicrobial.

Footnotes

Disclosure Statement

No competing financial interests exist.

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