Skip to main content
NCBI home page
Pathogens and Global Health logoLink to Pathogens and Global Health
. 2014 Jan;108(1):37–48. doi: 10.1179/2047773213Y.0000000118

Isolation and characterization of Salmonella enterica in day-old ducklings in Egypt

Kamelia M Osman 1, Sherif H Marouf 1, Tara R Zolnikov 2, Nayerah AlAtfeehy 3
PMCID: PMC4083166  PMID: 24548159

Abstract

Importing day-old ducklings (DOD) unknowingly infected with non-typhoid Salmonella (NTS) may be associated with disease risk. Domestic and international trade may enhance this risk. Salmonella enterica serovars, their virulence genes combinations and antibiotic resistance, garner attention for their potentiality to contribute to the adverse health effects on populations throughout the world. The aim of this study was to estimate the risk of imported versus domestic DOD as potential carriers of NTS. The results confirm the prevalence of salmonellosis in imported ducklings was 18.5% (25/135), whereas only 12% (9/75) of cases were determined in the domestic ducklings. Fourteen serovars (Salmonella enteritidis, Salmonella kisii, Salmonella typhimurium, Salmonella gaillac, Salmonella uno, Salmonella eingedi, Salmonella shubra, Salmonella bardo, Salmonella inganda, Salmonella kentucky, Salmonella stanley, Salmonella virchow, Salmonella haifa, and Salmonella anatum) were isolated from the imported ducklings, whereas only S. enteritidis, S. typhimurium, S. virchow, and S. shubra were isolated from the domestic ducklings. The isolated Salmonella serovars were 100% susceptible to only colistin sulphate and 100% resistant to lincomycin. The 14 Salmonella serovars were screened for 11 virulence genes (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by PCR. The invA, sopB, and bcfC genes were detected in 100% of the Salmonella serovars; alternatively, the gipA gene was absent in all of the isolated Salmonella serovars. The 11 virulent genes were not detected in either of S. stanley or S. haifa serovars. The results confirm an association between antibiotic resistance and virulence of Salmonella in the DOD. This study confirms the need for a country adherence to strict public health and food safety regimes.

Keywords: Antibiotic resistance, Day-old ducklings, Salmonella serovars, Virulence genes

Introduction

Ducks are frequently used by human populations throughout the world for a variety of reasons; duck meat and duck eggs are consumed for protein-specific dietary purposes, raised as pets for children, used for natural décor on small ponds or lakes, hunted in game preserves or conservation areas, and finally, used for entertainment at country fairs.1,2 In the United States of America (USA), 6.4 million households own and raise ducks in their backyards for meat, egg production, or as pets.3,4 Unfortunately, disease risks are associated with contact with ducks and may contribute to adverse health effects in people. Aside from food-borne infections, a cluster of non-typhoid Salmonella (NTS) human infections has also been associated with day-old ducklings (DOD).1–3,5–7 Salmonella outbreaks have intermittently occurred every few years since the 1950s when ducklings are purchased as pets during the Easter holiday season.8–10 One case in the USA confirmed a single mail-order poultry hatchery was responsible for an 8-year Salmonella outbreak and thousands of infections in 43 states.11 Salmonella typhimurium has been isolated from 40% of hatchlings and 1% of older ducklings in Taiwan, even though clear host species specific differences have also been detected.12

Salmonella has been previously isolated from imported DOD in Brazil and the USA.11,13,14 Additionally, a significant amount of the infected duck products contained multi-drug-resistant (MDR) foodborne pathogens.15 Because the prevalence of Salmonella in duck products poses a risk to human populations, an urgent need exists to investigate the prevalence, disease risk to human populations, and the global epidemiology of Salmonella serovars and specific clones. This information may be used to address Salmonella risk and promote evidence-based interventions in global public health.

A diversity of virulence factors have been previously described for Salmonella; these virulence factors are located on the bacterial chromosome, frequently as part of pathogenicity islands, on plasmids and prophages.16 Although all serovars of Salmonella enterica are considered pathogenic to man,17 the distribution of virulence genes in serovars is not well understood.18 An encompassing scientific literature review also confirms the negligible presence of research focussing on understanding virulence determinants associated with the isolated Salmonella serovars with special reference to DOD.

An accelerated dissemination of resistant pathogenic organisms from one geographic location to another has occurred as a result of globalization and international trade over the past decade.19 Yet, there is a limited amount of data concerning the prevalence of foodborne pathogens and associated antimicrobial susceptibility phenotypes among imported poultry.19 A very important issue that has been debated is whether the MDR strains are associated with virulence determinants20 and thus have the propensity to be more virulent than their susceptible counterparts; therefore, this study attempted to address this outstanding issue on whether genetic determinants for both antibiotic resistance and virulence genes could be harboured by the same transferable element and further confirm the association between antibiotic resistance and virulence in DOD.

Consequently, the objectives of the current study were to determine and establish baseline data on the prevalence of S. enterica serovars, virulence genes, gene association and combinations, virulence genes encoding serotypes, and antibiotic-resistant Salmonella phenotypes in imported and domestic DOD in Egypt.

Materials and Methods

Sampling and isolation

All imported bird samples were delivered to the Central Lab for Veterinary Quality Control on Poultry Production, Agriculture Research Center, Ministry of Agriculture. A total of 150 imported DOD were randomly collected from 50 boxes (25 birds per box; 3 birds/25 birds). Faecal samples were collected from the 150 imported DOD for culture and isolation of salmonellae; additionally, 150 faecal samples were taken from domestic DOD, which originated from both small-scale and commercial farms, whose production is primarily used for buying and selling DOD. The 300 faecal samples were analysed for Salmonella according to the ISO-6579-1993 standards.21 Twenty five grams of faeces were mixed with 225 ml of buffered peptone water (Oxoid Ltd, Hampshire, England) for pre-enrichment. After incubation at 37°C for 24 hours, 0.1 ml were transferred to 10 ml of selective Rappaport-Vassiliadis broth (Oxoid Ltd) and were incubated for 24 hours at 42°C. A loopful of broth culture was streaked on Rambach agar (Merck, Darmstadt, Germany), xylose lysine desoxycholate agar (XLD, Oxoid Ltd), and Hektoen enteric agar (Oxoid Ltd); agar plates were then incubated at 37°C for 24–48 hours Presumptive Salmonella colonies were identified on the basis of a Gram stain, catalase reaction, oxidase reaction, and oxidation/fermentation of glucose. Gram negative bacilli, catalase positive, oxidase negative, and capability of oxidation and fermentation of glucose were inoculated onto microtubes of API 20E strips (bioMérieux, Marcy L'Étoile, France) in accordance with the manufacturers' instructions. The bacteria were identified using the database API LAB Plus version 3.2.2 (bioMérieux). On each positive sample, an individual Salmonella isolate was typed and subjected to further serotyping analysis. Serotyping was performed according to the Kauffmann–White typing scheme22 using slide agglutination with standard antisera (Difco Laboratories, Detroit, MI, USA).

Phenotypic Virulence Assays

Invasiveness profiles

Congo red binding

The method of Qadir et al.23 was used to test invasiveness profiling. Congo red (0.003%, w/v) was incorporated into nutrient agar before autoclaving. Plates streaked with test strains were incubated at 37°C for 18 hours Colonies were examined for the presence (red, crb +ve) or absence (white, crb −ve) of Congo red binding.

Serum survival

Bacterial survival in normal, uninfected duck and chicken sera was assayed through Congo red binding.24 Counts were determined at times T  =  0 hour and T  =  2 hours following addition of the inoculum to 10% normal duck and chicken sera in single strength Hank's balanced salt solution (HBSS: 137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3, 5.6 mM d-glucose, 0.02% phenol red, distilled water to 1000 ml; membrane filtre sterilized (pore size: 0.45 μm). Cultures of bacteria (Salmonella enteritidis, S. typhimurium, Salmonella kisii, Salmonella gaillac, Salmonella uno, Salmonella eingedi, Salmonella shubra, Salmonella bardo, Salmonella inganda, Salmonella kentucky, Salmonella stanley, Salmonella haifa, Salmonella virchow, and Salmonella anatum in addition to the two untypable isolates) in the log growth phase were prepared in 1×phosphate buffered saline (PBS); 10 μl of each individual sample was added to 90 μl of freshly thawed, undiluted serum for a final bacterial concentration of 106 CFU/ml. The mixtures were incubated at 37°C with gentle rocking (20 rpm); sequentially, 10 μl of each sample was withdrawn at 45, 90, and 180 minutes. Serial dilutions of the samples were plated onto LB agar plates to determine the viable bacterial counts. Survival, expressed as serum resistance factor, was determined at either CFU at T  =  2 hours or CFU at T  =  0 hour. Mean results were based on three independent determinations.

MDCK and Vero cell ingestion assay

The ingestion assay on Madin Darby Canine kidney (MDCK) and Vero cells was conducted according to Raja et al.25 and were obtained from American Type Culture Collection, Rockville, Madison, USA. Cell free culture supernatants (CFCS) of S. enteritidis, S. typhimurium, S. kisii, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. stanley, S. haifa, S. virchow, and S. anatum, in addition to the two untypable isolates, were prepared. Each bacterial strain was grown in brain heart infusion (BHI) broth for 24 hours at 37°C. After incubation, an inoculum containing 5×107 CFU/ml was taken from which 100 μl was added to the wells containing Vero and MDCK cells. Bacterial cells were allowed to infect the epithelial cells for 30 minutes–12 hours at 37°C. A control assay with uninfected cell lines was also maintained. Live cells were removed, and cytological changes were observed using an inverted microscope.

Chicken embryo lethality assay

Embryo lethality was performed according to Wooley et al.26. Overnight broth cultures of each isolate (S. enteritidis, S. typhimurium, S. kisii, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. stanley, S. haifa, S. virchow, S. anatum, and two untypable isolates) were washed twice in PBS, resuspended, diluted in PBS, and inoculated (total volume of 0.1 ml) into the allantoic cavity of six 12-day-old embryonated chicken eggs. Six PBS inoculated and uninoculated control embryonated eggs were also included in embryo lethality tests. Eggs were candled daily; deaths were recorded as they occurred until the embryos were 18 days of age. Allantoic fluid, liver, spleen, heart, and the brain of each embryo were collected immediately upon death and used for reisolation of the infected Salmonella serotypes. Isolates causing embryo death of > 29, 10–29, and < 10% were considered virulent, moderately virulent, and avirulent isolates, respectively.

Mannose sensitive and mannose resistance haemagglutination assay

The tests were performed in the presence or absence of d-mannose at 37 and 18°C for 24 hours.27 Salmonella enteritidis, S. typhimurium, S. kisii, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. stanley, S. haifa, S. virchow, S. anatum, and the two untypable isolates were briefly grown on buffered glucose nutrient agar supplemented with 5% sheep blood. The bacteria were then suspended in PBS for a final concentration of approximately 1010 bacteria/ml; two-fold serial dilutions were created in 96-well round-bottom microtitre plates. An equal volume of 3% red blood cell (RBC) suspension (duck, chicken, guinea pig, and human type O) was added to each well of the bacterial suspensions. For mannose-resistant haemagglutination (MRHA) test, 20 μl of bacterial suspension was mixed with an equal volume of RBC suspension on a cavity glass slide and then, 20 μl of PBS was added to the mixture. Reactions were recorded as (+) for haemagglutination or (−) for no haemagglutination. A control, consisting of RBCs suspended in PBS, was included in the test.

To demonstrate mannose sensitive haemagglutination (MSHA) of the isolates, a 1% (w/v) d (+) mannose solution was used to examine the inhibition of haemagglutination. The effect of carbohydrates was assessed by adding 20 μl of a sugar solution to the bacteria-RBC suspension. A positive control was also included in each test; this control comprised equal volumes of bacterial suspension. If positive haemagglutination turned negative, inhibition was recorded. The highest dilution reciprocal of bacterial suspension with complete haemagglutination of RBCs was considered the endpoint. Reactions with titres above 1:4 were considered positive, indicating the presence of specific hemagglutins. Each test was duplicated throughout the haemagglutination assay.

Antimicrobial susceptibility testing by disc diffusion

To determine antibiotic resistance of the isolated Salmonella serotypes, each isolate was inoculated onto Muller–Hinton agar (Oxoid) and incubated at 37°C for 24 hours These techniques were conducted according to Clinical and Laboratory Standards Institute (CLSI)28 guidelines for disc diffusion techniques using commercial discs (Becton, Dickinson and Company, Sparks, Maryland, USA). After this procedure, the zones of inhibition were measured in order to assess resistance or susceptibility. The panel of antibiotic discs used in panel screens belonged to eight drug classifications. Selected antimicrobials were chosen based on their common use in treating or preventing Salmonella infection in humans. The antimicrobial agents and corresponding concentrations used in the study included ampicillin (10 mg), amoxicillin (20 mg), gentamicin (10 mg), neomycin (30 μg), streptomycin (10 mg), ciprofloxacin (5 μg), norfloxacin (5 μg), nalidixic acid (30 mg), lincomycin (30 μg), chloramphenicol (30 mg), colistin (10 mg), tetracycline (30 mg), trimethoprim (5 μg), and sulphamethoxazole+trimethoprim (23.75+1.75 mg).

Detection of virulence determinants

Detection of 11 virulence genes (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) in the 14 typable serotypes and 2 untypable isolates was conducted through PCR amplification. Table 1 outlines the primers, sequences, conditions, and predicted sizes of the amplified products. These virulence determinants represent either highly conserved (Salmonella pathogenicity islands, SPIs) or variable (prophages, plasmid) regions within the Salmonella genome. Bacterial strains of S. enteritidis (ATCC 13076) and Escherichia coli (ATCC 50034) were used as reference strains. Except sopE1, a positive control DNA from S. enterica serovar S. typhimurium strain LT2 was used for all targets.32 For sopE1, DNA from S. enterica serovar Hadar strain 99-0601 was used as the positive control.

Table 1. Virulence factor targets and primers, including nucleotide sequences, PCR conditions, and references.
Gene designation Location on SP1/gene function Oligonucleotide sequences (5′–3′) PCR conditionsa Product size (bp) References
Denaturing Annealing Extension
invA Type III secretion system apparatus SPI-1/invasion of macrophages gtg aaa tta tcg cca cgt tcg ggc aa tca tcg cac cgt caa agg aac g 94°C for 60 seconds 64°C for 30 seconds 72°C for 30 secondsb 284 29
avrA SPI-1/controls Salmonella-induced inflammation cct gta ttg ttg agc gtc tgg aga aga gct tcg ttg aat gtc c 95°C for 30 seconds 58°C for 30 seconds 72°C for 30 secondsb 422 30
ssaQ SPI-2/secretion system apparatus protein, component of second T3SS gaa tag cga atg aag agc gtc gtc c cat cgt gtt atc ctc tgt cag c 455
mgtC SPI-4/Mg2+ uptake tga cta tca atg ctc cag tga at att tac tgg ccg cta tgc tgt tg 677
siiD (Spi4D) Type I secretion/SPI-4 gaa tag aag aca aag cga tca tc gct ttg ttc acg cct ttc atc 655 31
sopB SPI-5/inositol polyphosphate, phosphatase that promotes macropinocytosis, regulates SCV localization, and promotes fluid secretion tca gaa gRc gtc taa cca ctc tac cgt cct cat gca cac tc 517 30
gipA Gifsy-1 bacteriophage/Peyer's patch-specific virulence factor acg act gag cag cgt gag ttg gaa atg gtg acg gta gac 518
sodC1 Gifsy-2 bacteriophage/periplasmic Cu, Zn-superoxide dismutases cgg gca gtg ttg aca aat aaag tgt tgg aat tgt gga gtc 424
sopE1 Cryptic bacteriophage/promotes membrane ruffling and disrupts tight junctions act cct tgc aca acc aaa tgc gga tgt ctt ctg cat ttc gcc acc 422
spvC pSLT/A phosphothreonine lyase required for complete virulence in murine models acc aga gac att gcc ttc c ttc tga tcg ccg cta ttc g 467
bcfC Chromosome/bovine colonization factor, fimbrial usher acc aga gac att gcc ttc c ttc tgc tcg ccg cta ttc g 95°C for 30 seconds 53°C for 30 seconds 72°C for 30 secondsb 467
Open in a new tab

a PCR was done for 35 cycles.

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

c SCV, Salmonella-containing vacuole.

Statistical analysis

The distribution of single or multi-drug antibiotic resistance phenotypes among serotypes was determined by a contingency table analysis; this table analysis included labelled rows  =  serotypes and columns resistance versus susceptible. The statistical significance of homogeneity in antimicrobial resistance patterns among the groups was assessed using Pearson's chi2 exact test using SAS version 9.2 (SAS, Cary, NJ, USA). Findings were considered statistically significant when P < 0.05. A contingency table allows researchers to test the proportion of resistance bacteria across different serotypes while using Pearson's chi2 test to assess the statistical significance between proportions. A high chi2 value confirms resistant phenotypes are not proportionately distributed among serotypes; although, some serotypes may have a higher frequency of resistant phenotypes than others.

Results

Prevalence and serotyping of Salmonella

The 14 isolated serotypes were identified as S. enteritidis, S. kisii, S. typhimurium, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. stanley, S. virchow, S. haifa, and S. anatum from the imported ducklings and S. enteritidis, S. typhimurium, S. virchow, and S. shubra from the domestic ducklings. The prevalence of salmonellosis in the imported ducklings was < 18.5% (25/135); serotyping of the Salmonella-isolated strains S. enteritidis and S. kisii confirmed a prevalence of 2.2% (3/135) in each strain. The prevalence decreased to 1.5% (2/135) in serotypes S. typhimurium, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. stanley, S. virchow, S. haifa, and S. anatum, while the one untypable isolate was < 1% (1/135). The prevalence of salmonellosis in tested local ducklings was 12% (9/75) and serotyping of the Salmonella-isolated strains, S. enteritidis, S. typhimurium, S. virchow, and S. shubra, recorded a prevalence of 2.7% (2/75) and 1.3% (1/75) in the untypable isolate.

Phenotypic Virulence Assays

Congo red binding, serum survival, MDCK, chicken embryo lethality assay, Vero cell ingestion assay, and haemagglutination tests

Interestingly, S. enteritidis, S. typhimurium, S. kisii, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. virchow, S. anatum, and the two untypable isolates displayed strong positive Congo red binding, were resistant to serum bactericidal activity, and survived for 3 hours in chicken and duck sera. Alternatively, S. stanley and S. haifa were weakly positive to Congo red binding (displayed by the orange colour) and were unable to survive over 1 hour in the chicken and duck sera; at the same time, the results confirmed 100% of susceptibility (after 12 hours of exposure to the Salmonella cytotoxin in cell free sonic lysate) and 100% death of the embryos during the periods of day-13 and day-17 in the two cell lines, Vero and MDCK.

The results of the tested serovars S. kisii, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. anatum, S. stanley, S. haifa, and the two untypable isolates were 100% MRHA to duck erythrocytes, whereas S. eingedi, S. stanley, S. bardo, S. kentucky, and S. haifa were 100% MRHA to chicken erythrocytes. In all, 100% MRHA to guinea pig erythrocytes was recorded for the serotypes S. kisii, S. uno, S. eingedi, S. stanley, S. inganda, S. kentucky, and S. haifa. Serotypes S. kisii, S. uno, S. eingedi, S. kentucky, and S. haifa were 100% MRHA to human group O erythrocytes.

Distribution of resistance to individual antimicrobial agents

All Salmonella serovars were resistant to more than one tested antimicrobial; the serovars were susceptible to only 1 (colistin sulphate) of the 14 antimicrobials (Table 2), but were all 100% resistant to lincomycin. Resistance frequencies varied depending on the antibiotic selection; all 14 Salmonella serovars and untypables exhibited significant diversity in their resistance patterns. From the eight different classes, S. typhimurium, S. shubra, S. eingedi, S. bardo, S. haifa, S. kentucky, and S. stanley were resistant to seven of them with the exception of polymyxin (colistin sulphate). Salmonella enteritidis, S. gaillac, and S. inganda were resistant to six different antimicrobial resistance profiles (aminoglycosides, fluoroquinolones, lincosamides, phenicols, tetracyclines, and sulphonamides). A wide variety of resistant antimicrobials occurred in the untypable Salmonella isolates; the six antimicrobial-resistant profiles included penicillins, aminoglycosides, fluoroquinolones, lincosamides, phenicols, and sulphonamides. Salmonella kisii and S. virchow isolates were resistant to only five antimicrobial resistance profiles (aminoglycosides, fluoroquinolones, lincosamides, phenicols, and sulphonamides). The least four antimicrobial-resistant profiles for S. uno included aminoglycosides, fluoroquinolones, lincosamides, and phenicols, whereas for S. anatum the least four resistant profiles included aminoglycosides, lincosamides, phenicols, and sulphonamides.

Table 2. Distribution of resistance to individual antimicrobial agents among sources and Salmonella enterica serovars in day-old ducklings (DOD).

Distribution of Salmonella resistance to antimicrobials
S. enteritidis 5/34 S. kisii 3/34 S. typhimurium 4/34 S. virchow 3/34 S. shubra 4/34 S. eingedi 2/34 S. bardo 3/34 S. uno 2/34 S. haifa 1/34 S. kentucky 1/34 S. stanley 1/34 S. gaillac 2/34 S. anatum 1/34 S. inganda 1/34 Untypable 2/34
Antimicrobials No. of resistant isolates
Penicillins
Ampicillin 0/5 0/3 2/4 0/3 3/3 2/2 3/3 0/2 1/1 1/1 1/1 0/2 0/1 0/1 1/2
Amoxicillin 0/5 0/3 3/4 0/3 3/3 2/2 3/3 0/2 1/1 1/1 1/1 0/2 0/1 0/1 0/2
Aminoglycosides
Gentamicin 0/5 0/3 0/4 0/3 2/3 1/2 1/3 0/2 0/1 1/1 1/1 2/2 0/1 0/1 0/2
Neomycin 1/5 1/3 0/4 0/3 1/3 1/2 1/3 0/2 1/1 1/1 1/1 2/2 1/1 1/1 1/2
Streptomycin 2/5 2/3 3/4 2/3 3/3 2/2 3/3 2/2 1/1 1/1 1/1 2/2 1/1 1/1 2/2
Fluoroquinolones
Ciprofloxacin 0/5 0/3 0/4 0/3 3/3 0/2 0/3 0/2 0/1 1/1 0/1 0/2 0/1 0/1 0/2
Nalidixic acid 2/5 2/3 3/4 1/3 2/3 2/2 1/3 1/2 1/1 1/1 1/1 2/2 0/1 1/1 1/2
Norfloxacin 0/5 0/3 0/4 0/3 1/3 1/2 0/3 0/2 0/1 1/1 1/1 2/2 0/1 0/1 1/2
Lincosamides
Lincomycin 5/5 3/3 4/4 3/3 3/3 2/2 3/3 2/2 1/1 1/1 1/1 2/2 1/1 1/1 2/2
Phenicols
Chloramphenicol 3/5 2/3 3/4 3/3 3/3 2/2 2/3 1/2 1/1 1/1 1/1 1/2 1/1 1/1 1/2
Polymyxin
Colistin sulphate 0/5 0/3 0/4 0/3 0/3 0/2 0/3 0/2 0/1 0/1 0/1 0/2 0/1 0/1 0/2
Tetracyclines
Tetracycline 1/5 0/3 1/4 0/3 3/3 2/2 3/3 0/2 1/1 1/1 1/1 2/2 0/1 1/1 0/2
Sulphonamides
Trimethoprim 1/5 1/3 2/4 0/3 3/3 2/2 2/3 0/2 1/1 1/1 1/1 1/2 1/1 0/1 1/2
Trimethoprim–sulfamethoxazole 2/5 2/3 3/4 1/3 3/3 1/2 2/3 0/2 1/1 1/1 1/1 0/2 0/1 1/1 1/2
Open in a new tab

Statistically significant correlations for resistance between the antimicrobials at the isolate level are presented in Tables 2 and 4. The distribution of resistance to the different antibiotics was largely dependent on the serovar identity. Significant resistance (P < 0.001) to amoxicillin (chi2  =  13.0) was associated with serovars S. eingedi, S. shubra, S. bardo, S. kentucky, S. stanley, and S. haifa. Chloramphenicol (chi2  =  13.0) confirmed significant resistance to serovars S. virchow, S. shubra, S. eingedi, S. haifa, S. kentucky, S. stanley, S. anatum, and S. inganda. Gentamicin (chi2  =  15.1) was associated with serovars S. kentucky, S. stanley, and S. gaillac. Neomycin (chi2  =  14.2) was associated with serovars S. kentucky, S. stanley, S. haifa, S. gaillac, S. anatum, and S. inganda. Ciprofloxacin (chi2  =  34.0) was associated with serovars S. shubra and S. kentucky. Tetracycline (chi2  =  26.8) was associated with serovars S. shubra, S. eingedi, S. bardo, S. haifa, S. kentucky, S. stanley, and S. gaillac. Finally, trimethoprim (chi2  =  12.2) was found to be associated with S. shubra, S. eingedi, S. haifa, S. kentucky, S. stanley, and S. anatum. Significant resistance (with a value of P < 0.003) was also recorded for ampicillin (chi2  =  8.5) that was associated with serovars S. shubra, S. eingedi, S. bardo, S. haifa, S. kentucky, and S. stanley. Streptomycin (chi2  =  8.8) was also observed to be associated with serovars S. shubra, S. eingedi, S. bardo, S. uno, S. haifa, S. kentucky, S. stanley, S. gaillac, S. anatum, S. inganda, and the untypable Salmonella isolate. Low significant resistance of sulphamethoxazole+trimethoprim (chi2  =  7.0; P < 0.008) was associated with serovars S. shubra, S. haifa, S. kentucky, S. stanley, and S. bardo; low resistance also occurred in nalidixic acid (chi2  =  6.5; P < 0.01), which was associated with serovars S. eingedi, S. haifa, S. kentucky, S. stanley, S. gaillac, and S. inganda.

Table 4. Distribution of virulence genes combinations in the different Salmonella serovars and antibiotic resistance phenotypes isolated from day-old ducklings (DOD).

Salmonella serotype Virulence genes combinationsa Antibiotic resistanceb
Isolated from imported ducklings
 S. enteritidis invA, avrA, mgtC, sodC1, sopB, spvC, bcfC Chl, Lin, Neo, Str, Tet, Sxt
 S. enteritidis invA, avrA, mgtC, sodC1, sopB, spvC, bcfC Chl, Lin, Neo
 S. enteritidis invA, sodC1, sopB, spvC, bcfC Lin
 S. typhimurium invA, sopB, sodC1, spvC, bcfC Lin, Str
 S. typhimurium invA, mgtC, ssaQ, sodC1, sopB, spvC, bcfC Amo, Amp, Chl, Lin, Na, Str, Tri, Sxt
 S. kissii invA, ssaQ, sopB, bcfC Chl, Lin, Na, Str, Tri, Sxt
 S. kissii invA, avrA, ssaQ, mgtC, sopB, bcfC Lin, Na, Neo, Sxt
 S. kissii invA, avrA, ssaQ, mgtC, sopB, bcfC Chl, Lin, Str
 S. virchow invA, avrA, ssaQ, mgtC, sopB, bcfC Chl, Lin, Nor
 S. uno invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Chl, Lin, Neo, Str
 S. uno invA, sopB, bcfC Lin, Str
 S. eingedi invA, avrA, ssaQ, mgtC, siiD, sopB, spvC, bcfC Amo, Amp, Chl, Lin, Na, Neo, Nor, Str, Tet, Tri, Sxt
 S. eingedi invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Amo, Amp, Chl, Gen, Lin, Str, Tet, Tri
 S. shubra invA, ssaQ, siiD, sopB, bcfC Amo, Amp, Chl, Gen, Lin, Na, Str, Tet, Tri, Sxt
 S. shubra invA, ssaQ, mgtC, siiD, sodC1, sopB, bcfC Amo, Amp, Chl, Gen, Lin, Na, Neo, Nor, Str, Tet, Tri, Sxt
 S. bardo invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Amo, Amp, Lin, Str, Tet, Tri, Sxt
 S. bardo invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Amo, Amp, Chl, Gen, Lin, Str, Tet
 S. inganda invA, avrA, ssaQ, mgtC, siiD, sopE1, sopB, bcfC Chl, Lin, Na, Neo, Str, Tet, Sxt
 S. kentucky invA, avrA, ssaQ, mgtC, siiD, sodC1, sopB, bcfC Amo, Amp, Chl, Cip, Gen, Lin, Na, Neo, Nor, Str, Tet, Tri, Sxt
 S. stanley The virulence genes invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC were not detected Amo, Amp, Chl, Gen, Lin, Na, Neo, Nor, Str, Tet, Tri, Sxt
 S. haifa Amo, Amp, Chl, Lin, Na, Neo, Str, Tet, Tri, Sxt
 S. gaillac invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Chl, Gen, Lin, Na, Neo, Nor, Str, Tet
 S. gaillac invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Gen, Lin, Na, Neo, Nor, Str, Tet, Tri
 S. anatum invA, ssaQ, mgtC, siiD, sopB, bcfC Chl, Lin, Neo, Str, Tri
    Untypable invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Amo, Lin, Neo, Str
Isolated from domestic ducklings
 S. enteritidis invA, avrA, mgtC, ssaQ, sodC1, sopB, spvC, bcfC Chl, Lin, Neo, Str, Tri, Sxt
 S. enteritidis invA, avrA, mgtC, ssaQ, sodC1, sopB, spvC, bcfC Lin
 S. typhimurium invA, sodC1, sopB, spvC, bcfC Amo, Amp, Chl, Lin, Na, Str, Tri, Sxt
 S. typhimurium invA, sopB, spvC, bcfC Amo, Chl, Lin, Na, Tet, Sxt
 S. virchow invA, avrA, ssaQ, mgtC, sopB, bcfC Chl, Lin, Na, Str
 S. virchow invA, ssaQ, sopB, bcfC Chl, Lin, Sxt
 S. shubra invA, ssaQ, mgtC, siiD, sopB, bcfC Amo, Amp, Chl, Gen, Lin, Str, Tet, Tri, Sxt
 S. shubra invA, ssaQ, mgtC, siiD, sopB, bcfC Amo, Amp, Neo, Chl, Na, Lin, Str, Tet, Tri, Sxt
    Untypable invA, avrA, ssaQ, mgtC, siiD, sopB, bcfC Chl, Lin, Na, Nor, Str, Tri, Sxt
Open in a new tab

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

b Amo: amoxicillin; Amp: ampicillin; Col: colistin sulphate; Tet: tetracycline; Cip: ciprofloxacin; Str: streptomycin; Nor: norfloxacin; Gen: gentamicin; Chl: chloramphenicol; Neo: neomycin; Lin: lincomycin; Na: nalidixic acid; Tri: trimethoprim; Sxt: trimethoprim+sulfamethoxazole.

Virulence genes

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) (Table 3). The PCR screening analysis detected the invA, sopB, and bcfC genes that were confirmed in S. enteritidis, S. typhimurium, S. kissii, S. virchow, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. gaillac, and S. anatum isolates and the two untypable isolates; on the contrary, the gipA gene was absent from all of the isolated Salmonella serovars (S. enteritidis, S. typhimurium, S. kissii, S. virchow, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. gaillac, and S. anatum), whereas the sopE1 gene (encoding a translocated effector protein) was detected in the S. inganda isolate, but remained undetected in the remainder of the Salmonella serovars. None of the 11 virulent genes were detected encoding either of the S. stanley and S. haifa isolates. The spvC gene, carried by the Salmonella virulence plasmid, was expressed in 10 isolates of the isolated salmonellae, 9 of which were on the S. enteritidis and S. typhimurium isolated from the imported and local ducklings. The sodC1 gene, located on a bacteriophage, in nine isolates of the tested salmonellae, was isolated from the imported and local ducklings; eight of these isolates were S. enteritidis and S. typhimurium.

Table 3. Distribution of the virulence genes among the Salmonella serovars isolated from day-old ducklings (DOD).

Salmonella serotypes Virulence genes
invA avrA ssaQ mgtC siiD sopB gipA sodC1 sopE1 spvC bcfC
From imported ducklings
 S. enteritidis + + ND + ND + ND + ND + +
 S. enteritidis + + ND + ND + ND + ND + +
 S. enteritidis + ND ND ND ND + ND + ND + +
 S. typhimurium + ND ND ND ND + ND + ND + +
 S. typhimurium + ND + + ND + ND + ND + +
 S. kisii + ND + ND ND + ND ND ND ND +
 S. kisii + + + + ND + ND ND ND ND +
 S. kisii + + + + ND + ND ND ND ND +
 S. virchow + + + + ND + ND ND ND ND +
 S. uno + + + + ND + ND ND ND ND +
 S. uno + ND ND ND ND + ND ND ND ND +
 S. eingedi + + + + + + ND + ND + +
 S. eingedi + + + + + + ND ND ND ND +
 S. shubra + ND + ND + + ND ND ND ND +
 S. shubra + ND + + + + ND ND ND ND +
 S. bardo + + + + + + ND ND ND ND +
 S. bardo + + + + + + ND ND ND ND +
 S. inganda + + + + + + ND ND + ND +
 S. kentucky + + + + + + ND ND ND ND +
 S. stanley ND ND ND ND ND ND ND ND ND ND ND
 S. haifa ND ND ND ND ND ND ND ND ND ND ND
 S. gaillac + + + + + + ND ND ND ND +
 S. gaillac + + + + + + ND ND ND ND +
 S. anatum + ND + + + + ND ND ND ND +
    Untypable + ND + + + + ND ND ND ND +
From domestic ducklings
    S. enteritidis + + + + ND + ND + ND + +
    S. enteritidis + + + + ND + ND + ND + +
    S. typhimurium + ND ND ND ND + ND + ND + +
    S. typhimurium + ND ND ND ND + ND ND ND + +
    S. virchow + + + + ND + ND ND ND ND +
    S. virchow + ND + ND ND + ND ND ND ND +
    S. shubra + ND + ND + + ND ND ND ND +
    S. shubra + ND + + + + ND ND ND ND +
    Untypable + + + + + + ND ND ND ND +
Open in a new tab

ND: not detected.

Virulence genes combinations

The 14 serovar collections provided significant diversity in the virulence genes combinations. The 11 genes were absent from the isolated S. stanley and S. haifa, which were isolated from the imported ducklings providing the first virulent gene combination. A second combination occurred in the S. eingedi (2/2), S. bardo (2/2), and S. kentucky (1/1). A third combination occurred in S. kissii (2/3), S. virchow (1/1), and S. uno serotypes (1/2). A fourth combination was recorded with S. enteritidis (2/3). A fifth combination occurred in S. enteritidis (1/3) and S. typhimurium (1/2). The two S. gaillac isolates had a common virulence gene combination, but varied from the other 34 isolates. Lastly, virulence gene repertoires occurred in S. anatum and the untypable isolate.

Association of antimicrobial resistance phenotype with virulence-associated genes

The presence of invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC genes in S. enteritidis, S. kissii, S. typhimurium, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. stanley, S. virchow, S. haifa, S. anatum, and the one untypable isolate in the imported ducklings, alongside the Salmonella isolated strains (S. enteritidis, S. typhimurium, S. virchow and S. shubra, and the untypable isolate) from the domestic ducklings, displayed various antimicrobial resistance patterns (Table 4). A detailed analysis displayed associations of resistance and susceptibility phenotypes with potential virulence genes. The study confirmed 14 tested antimicrobials were not associated with any of the 11 virulent genes, which were not detected in either of the S. stanley and S. haifa isolated from the imported ducklings; moreover, with the exception of S. stanley and S. haifa, the virulence-associated genes invA, sopB, and bcfC were found to be 100% associated with one antimicrobial resistance phenotype (lincosamide), which was not recorded previously. Association was also found between chloramphenicol and the virulence genes invA (94.1%; 32/34), sopB (71.8%; 23/32), and </emph>ssa</emph>Q (62.5%; 20/32).

Discussion

In spite of the long history of Salmonella and ducklings, literature on DOD is scarce.14,21,33–35 The high frequency of Salmonella recovery from imported DOD causes great public health concern due to the zoonotic potential of this pathogen and its economical importance to commercial poultry breeding.14 Our study provided evidence of Salmonella infection rates in imported and domestic DOD lower than those reported in previous studies14,34–36 and higher than those in a previous study in Egypt by Osman et al.21 and in Vietnam by Lam et al.34 In 2005, Tsai and Pi-Hung37 demonstrated that ducklings younger than 2 weeks of age had a significantly higher Salmonella prevalence rate than other age groups. Transovarian transmission is thought to be an important route of transmission to young birds; rodents and other vectors are also thought to play an important epidemiologic role in the transmission of Salmonella to the birds.38,39–41 Horizontal transmission at hatcheries during feeding, handling, and transportation is also known to contribute to the spread of Salmonella;10,38,42,43 in fact, Salmonella prevalence in hatcheries has been estimated between 20 and 60% for ducks.10,44 A study by Henry38 successfully confirmed the importance of hatchery contamination and Salmonella dissemination.

The 14 Salmonella serovars isolated from the imported DOD were substantially different from previous scientific literature.1–3,5–7,11,13,14,34,45 The isolated serovars of DOD in the current investigation have not been previously reported in Egypt;21 moreover, because contracting Salmonella causes adverse health effects and even mortality in humans,46 this study may provide additional evidence that may be used to address this impending global public health issue in the future.47,48 The high prevalence (5/34) of detected S. enteritidis is evident by the fact that serovar S. enteritidis may have contributed to filling the gap of an ecologic niche, apparent by the considerable increase in S. enteritidis prevalence after the eradication of biovar Gallinarum in the 1960s.49

The invA target gene is unique and present in all invasive strains of Salmonella,50 although generally absent from related genera such as Escherichia.51 Although the invA gene was absent in our isolated strains of S. stanley and S. haifa, it was available in the rest of the isolated serovars.52–54 Interestingly, the isolated S. stanley and S. haifa in our study lacked the 11 virulence genes. Therefore, in the absence of these 11 virulence genes (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC), we were interested in proving the virulence of S. stanley and S. haifa serovars and asked whether these serovars will present a comparable virulence phenotype as S. enteritidis, S. kissii, S. typhimurium, S. gaillac, S. uno, S. eingedi, S. shubra, S. bardo, S. inganda, S. kentucky, S. virchow, and S. anatum to Congo red binding, serum survival, MDCK and Vero cell ingestion, chicken embryo lethality, and MSHA and MRHA assays. The Vero and MDCK cell culture model was carried out in order to compare the abilities of clinical isolates of S. stanley and S. haifa that produce a disease status comparable to in vivo virulence and the results of the in vitro assay, which are often used as correlates of bacterial virulence phenotypes.25 The results of the pathogenicity assays and the association between phenotypic virulence assays and the invasiveness level of S. stanley and S. haifa serovars concluded their virulence that encouraged us to hypothetically speculate that these serovars may be using alternative invasive mechanisms.52,55,56

In the decades following the licencing of fluoroquinolones, an increased prevalence of quinolone-resistant salmonellae has been observed in clinical57,58 and poultry isolates worldwide;59 however, these same classes of antimicrobial agents are also administered in animals consumed by human populations, which leads to the inevitable development of resistant bacteria.60–62 The emergence of resistance to fluoroquinolones among NTS is of particular concern, because this class of antimicrobial agents constitutes the ‘drug of choice’ for treating potentially life-threatening Salmonella infections caused by multiple antibiotic-resistant strains.63,64 Salmonella typhimurium is among the serovars with the lowest average antimicrobial resistance in the present study, which is a positive indicator considering the nature of S. typhimurium, an exposure to cause severe human health effects; however, it is known that S. enteritidis is less prone to developing resistance than other serovars.65–67 The absence of resistance to ciprofloxacin and norfloxacin among serovars S. enteritidis, S. kissii, S. typhimurium, S. uno, S. eingedi, S. bardo, S. inganda, S. virchow, S. haifa, and S. anatum is of significant value because these two antimicrobials are the primary agents used against cases of invasive salmonellosis in humans.20 It was previously indicated that salmonellae exhibiting nalidixic acid-resistance is usually displayed with decreased susceptibility to ciprofloxacin.68–70 This was not evident in our investigation. Six and eight serovars were resistant to ampicillin and chloramphenicol, respectively; however, it should be noted that these antimicrobials are frequently used on Egyptian and European poultry farms71,72 and may be the cause of the resistance observed to the tested drugs in this study. The increase in resistance to streptomycin observed in the present study is not unexpected as this drug has been among the most frequently used antibiotics on poultry farms.73 None of the serovars identified were resistant to colistin sulphate, whereas ciprofloxacin was only resistant to serovars S. shubra and S. kentucky. This suggests a potentially effective treatment option for Salmonella infections. The direct association of virulence and resistance is determined in this study. In studies of the relationship between antibiotic resistance and pathogenicity, the direct comparison of sensitive and resistant Salmonella strains in virulence assays has been a favoured approach. Virulence and resistance are not independent properties but rather are closely related. In fact, this relationship can be seen in two ways: as genetic elements carrying genes associated with both virulence/resistance, and when the acquisition of resistance affects the virulence of the bacteria.74 Virulence and resistance might be contained in the same mobile genetic element.74 The fact that genetic determinants for both antibiotic resistance and virulence genes could be harboured by the same transferable element implies that there is a significant association between the presence of some virulence genes and antibiotic resistance.75,76 The majority of the molecular pathogenicity determinants are located on the chromosome or large virulence-associated plasmids,77,78 whereas antibiotic resistance genes are also often located on extrachromosomal genetic elements or in segments inserted within the chromosome that originate from other genomes.75 It has been reported that resistance plasmids carry genes encoding virulence factors.79 Also, antibiotic resistance and virulence genes can be linked in the same replicon, or eventually a single determinant can be involved in both virulence and resistance.79

Conclusion

It is difficult to compare prevalence estimates obtained from different studies regarding Salmonella across geographic regions; several factors must be considered when making such comparisons. While they may not reveal true differences in the distribution of Salmonella, one suggestion could include obtaining and perusing information obtained from management systems,10,80 raising practices, seasonal patterns, processing procedures, and varying techniques currently used to determine Salmonella prevalence.36

The emergence and development of antibiotic-resistant Salmonella in the DOD may be due to a variety of reasons. One such reason may be the practice of dipping hatching eggs in solutions containing antimicrobial agents or/and routine inoculation with antibiotics.39 Also, the occurrence and proliferation of antibiotic-resistant Salmonella in the ducklings have been linked to the common casual usage of antimicrobials in veterinary practice.73,81 In the present study, all the Salmonella serovars exhibited full resistance to lincomycin and total susceptibility to colistin sulphate; these antimicrobials are widely used in animal production environments for the treatment and prevention of disease as well as growth promotion and have been listed under the OIE list of antimicrobials of veterinary importance (Resolution No. XXVIII)82 as critically important antimicrobials used in human medicine.60,82

This study confirms the need for improved import control and testing83,84 in order to decrease the human health hazard risk associated with DOD.2–7,39

References

  • 1.Centers for Disease Control and Prevention (CDC) Multistate outbreaks of Salmonella infections associated with live poultry–United States, 2007. MMWR Morb Mortal Wkly Rep. 2009;58:25–9. [PubMed] [Google Scholar]
  • 2. CDC. Centers for disease control and prevention national center for emerging and zoonotic infectious diseases, division of foodborne, waterborne, and environmental diseases. [Page last updated: March 12, 2012a. Investigation update: multistate outbreak of human Salmonella Altona and Salmonella Johannesburg infections linked to chicks and ducklings]. AAP NEWS. 2012; 6(1):14. [Google Scholar]
  • 3.Centers for Disease Control and Prevention (CDC) Salmonellosis associated with chicks and ducklings – Michigan and Missouri, Spring 1999. MMWR Morb Mortal Wkly Rep. 2000;49:297–9. [PubMed] [Google Scholar]
  • 4.AVMA. American veterinary medical association: U.S. Pet Ownership & Demographics Sourcebook; 2012. ( https://www.avma.org/KB/Resources/Statistics/Pages/Market-research-statistics-US-Pet-Ownership-Demographics-Sourcebook.aspx) [Google Scholar]
  • 5.Centers for Disease Control (CDC) Salmonella hadar associated with pet ducklings – Connecticut, Maryland, and Pennsylvania, 1991. MMWR Morb Mortal Wkly Rep. 1992;41:185–7. [PubMed] [Google Scholar]
  • 6.Centers for Disease Control and Prevention (CDC) National Center for Emerging and Zoonotic Infectious Diseases (NCEZID). Page last updated: 18 April 2012. [Google Scholar]
  • 7.Centers for Disease Control and Prevention (CDC) Multistate outbreak of human Salmonella infections linked to live poultry in backyard flocks (Final Update). Atlanta, GA, USA. Page last updated: 26 October 2012. [Google Scholar]
  • 8.Anderson A, Bauer H, Nelson CB. Salmonellosis due to Salmonella Typhimurium with Easter chicks as likely source. J Am Med Assoc. 1955;158:1153–5. doi: 10.1001/jama.1955.02960130007003. [DOI] [PubMed] [Google Scholar]
  • 9.McCroan JE, McKinley TW, Brim A, Ramsey CH. Five salmonellosis outbreaks related to poultry products. Public Health Rep. 1963;78:1073–80. [PubMed] [Google Scholar]
  • 10.Hoelzer K, Switt AI, Wiedmann M. Animal contact as a source of human non-typhoidal salmonellosis. Vet Res. 2011;42:34. doi: 10.1186/1297-9716-42-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gaffga NH, Behravesh CB, Ettestad PJ, Smelser CB, Rhorer AR, Cronquist AB, et al. Outbreak of Salmonellosis linked to live poultry from a mail-order hatchery. N Engl J Med. 2012;366:2065–73. doi: 10.1056/NEJMoa1111818. [DOI] [PubMed] [Google Scholar]
  • 12.Yu CY, Chu C, Chou SJ, Chao MR, Yeh CM, Lo DY, et al. Comparison of the association of age with the infection of Salmonella and Salmonella enterica serovar Typhimurium in Pekin ducks and Roman geese. Poult Sci. 2008;87:1544–9. doi: 10.3382/ps.2008-00018. [DOI] [PubMed] [Google Scholar]
  • 13.Galletti MCM, Ribeiro SAM, Reis EMF, Doretto JRL, Orsi MA. Isolamento de Salmonella enterica subsp enterica serovar Kottbus em aves importadas. Conferência APINCO de Ciência e Tecnologia Avícolas. Anais Campinas:FACTA. 1999;36 [Google Scholar]
  • 14.Ribeiro SAM, Galleti MCM, Orsi MA, Ferrati AR, Mendonça AO, Doretto Júnior L, et al. Incidence of Salmonella in imported day-old ducklings. Brazil, 1998–2003. Rev Bras Ciên Avíc. 2006;8:39–43. [Google Scholar]
  • 15.Aarestrup FM, Rene S, Hendriksen JL, Katie G, Kathryn T, Patrick F, et al. International spread of multidrug-resistant Salmonella Schwarzengrund in food products. Emerg Infect Dis. 2007;13:726–31. doi: 10.3201/eid1305.061489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, et al. The complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature. 2001;413:852–6. doi: 10.1038/35101614. [DOI] [PubMed] [Google Scholar]
  • 17.Forshell LP, Wierup M. Salmonella contamination: a significant challenge to the global marketing of animal food products. Rev Sci Tech. 2006;25:541–54. [PubMed] [Google Scholar]
  • 18.Bhowmick PP, Devegowda D, Ruwandeepika HA, Arunasagar I, Karunasagar I. Presence of Salmonella pathogenicity island 2 genes in seafood-associated Salmonella serovars and the role of the sseC gene in survival of Salmonella enterica serovar Weltevreden in epithelial cells. Microbiology. 2011;157:160–8. doi: 10.1099/mic.0.043596-0. [DOI] [PubMed] [Google Scholar]
  • 19.Williams RJ. Globalization of antimicrobial resistance: epidemiological challenges. Clin Infect Dis. 2001;33(Suppl 3):S116–S7. doi: 10.1086/321835. [DOI] [PubMed] [Google Scholar]
  • 20.Gebreyes WA, Thakur S, Dorr P, Tadesse DA, Post K, Wolf L. Occurrence of spvA virulence gene and clinical significance for multidrug-resistant Salmonella strains. J Clin Microbiol. 2009;47:777–80. doi: 10.1128/JCM.01660-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Osman KM, Yousef AM, Aly MM, Radwan MI. Salmonella spp. infection in imported 1-day-old chicks, ducklings, and turkey poults: a public health risk. Foodborne Pathog Dis. 2010;7:383–90. doi: 10.1089/fpd.2009.0358. [DOI] [PubMed] [Google Scholar]
  • 22. Popoff MY. Antigenic formulas of the Salmonella serovars, 8th edn. W. C. C. f. R. a. R. o. Salmonella, Paris: Institut Pasteur; 2001. [Google Scholar]
  • 23.Qadir F, Hossain SA, Cizna'r I, Haider K, Ljungh A, Wadstrom T, et al. Congo red binding and salt aggregation as indicators of virulence in Shigella species. J Clin Microbiol. 1988;26:1343–8. doi: 10.1128/jcm.26.7.1343-1348.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Raghunathan D, Wells TJ, Morris FC, Shaw RK, Bobat S, Peters SE, et al. SadA, a trimeric autotransporter from Salmonella enterica serovar Typhimurium, can promote biofilm formation and provides limited protection against infection. Infect Immun. 2011;79:4342–52. doi: 10.1128/IAI.05592-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Raja RK, Ramesh N, Maripandi A. Invasion and interaction studies of Salmonella Typhimurium sub sp Enteritis in Vero and MDCK Cell Lines. Adv Biol Res. 2010;4:86–91. [Google Scholar]
  • 26.Wooley RE, Gibbs PS, Brown TP, Maurer JJ. Chicken embryo lethality assay for determining the virulence of avian Escherichia coli isolates. Avian Dis. 2000;44:318–24. [PubMed] [Google Scholar]
  • 27.Evans DJ, Jr, Evans DG, DuPont HL. Hemagglutination patterns of enterotoxigenic and enteropathogenic Escherichia coli determined with human, bovine, chicken, and guinea pig erythrocytes in the presence and absence of mannose. Infect Immun. 1979;23:336–46. doi: 10.1128/iai.23.2.336-346.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.CLSI. Performance standards for antimicrobial susceptibility testing. CLSI approved standard 2010; M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. [Google Scholar]
  • 29.Salehi TZ, Mahzounieh M, Saeedzadeh A. Detection of invA gene in isolated Salmonella from broilers by PCR method. Int J Poult Sci. 2005;4:557–9. [Google Scholar]
  • 30.Huehn S, La Ragione RM, Anjum M, Saunders M, Woodward MJ, Bunge C, et al. Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars relevant to human health in Europe. Foodborne Pathog Dis. 2010;7:523–35. doi: 10.1089/fpd.2009.0447. [DOI] [PubMed] [Google Scholar]
  • 31.Hauser E, Hebner F, Tietze E, Helmuth R, Junker E, Prager R, et al. Diversity of Salmonella enterica serovar Derby isolated from pig, pork and humans in Germany. Int J Food Microbiol. 2011;151:141–9. doi: 10.1016/j.ijfoodmicro.2011.08.020. [DOI] [PubMed] [Google Scholar]
  • 32.Marcus SL, Brumell JH, Pfeifer CG, Finlay BB. Salmonella pathogenicity islands: big virulence in small packages. Microbes Infect. 2000;2:145–56. doi: 10.1016/s1286-4579(00)00273-2. [DOI] [PubMed] [Google Scholar]
  • 33.Binh NX, Cuong NV, Khanh LTM, Hanh TX, To Phan TT, Ha PDH. Investigation of Salmonella and Escherichia coli infections of ducks in long an province, Vietnam. Khoa Hoc Ky Thuat Thu Y (Vet Sci Tech). 2000;7:29–34. [Google Scholar]
  • 34.Lam DN, Carles M, Tripodi A, Brugere-Picoux J, Bodin G. Étude bactériologique des infections par le genre Salmonella chez le canard dans la province de Can Tho (Viet Nam). Rev Méd Vét. 2000;151:955–64. [Google Scholar]
  • 35.Adzitey F, Huda N, Ali GR. Prevalence and antibiotic resistance of Campylobacter, Salmonella, and L. monocytogenes in ducks: a review. Foodborne Pathog Dis. 2012;9:498–505. doi: 10.1089/fpd.2011.1109. [DOI] [PubMed] [Google Scholar]
  • 36.Myint MS. Epidemiology of Salmonella contamination of poultry meat products: knowledge gaps in the farm to store products. Department of Veterinary Medical Sciences. Dissertation Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of doctor of Philosophy; 2004. Washington DC-Baltimore, USA. [Google Scholar]
  • 37.Tsai HJ, Pi-Hung H. The prevalence and antimicrobial susceptibilities of Salmonella and Campylobacter in ducks in Taiwan. J Vet Med Sci. 2005;67:7–12. doi: 10.1292/jvms.67.7. [DOI] [PubMed] [Google Scholar]
  • 38.Henry RR. Salmonella infection in ducks. In: Wray C, Wray A, editors. Salmonella in domestic animals, 1st edn. 2000. p. 157–68. CAB International North America. [Google Scholar]
  • 39.Lutful Kabir SM. Avian colibacillosis and salmonellosis: a closer look at epidemiology, pathogenesis, diagnosis, control and public health concerns. Int J Environ Res Public Health. 2010;7:89–114. doi: 10.3390/ijerph7010089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Backhans A, Fellström C. Rodents on pig and chicken farms – a potential threat to human and animal health. Infect Ecol Epidemiol. 2012;2 doi: 10.3402/iee.v2i0.17093. 17093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Umali DV, Lapuz RR, Suzuki T, Shirota K, Katoh H. Transmission and shedding patterns of Salmonella in naturally infected captive wild roof rats (Rattus rattus) from a Salmonella-contaminated layer farm. Avian Dis. 2012;56:288–94. doi: 10.1637/9911-090411-Reg.1. [DOI] [PubMed] [Google Scholar]
  • 42.Barrow PA, Lovell MA, Murphy CK, Page K. Salmonella infection in a commercial line of ducks; experimental studies on virulence, intestinal colonization and immune protection. Epidemiol Infect. 1999;123:121–32. doi: 10.1017/s0950268899002605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Foley SL, Nayak R, Hanning IB, Johnson TJ, Han J, Ricke SC. Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production. Appl Environ Microbiol. 2011;77:4273–9. doi: 10.1128/AEM.00598-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Chao MR, Hsien CH, Yeh CM, Chou SJ, Chu C, Su YC, et al. Assessing the prevalence of Salmonella enterica in poultry hatcheries by using hatched eggshell membranes. Poult Sci. 2007;86:1651–5. doi: 10.1093/ps/86.8.1651. [DOI] [PubMed] [Google Scholar]
  • 45.Bangtrakulnonth A, Pornreongwong S, Pulsrikarn C, Sawanpanyalert P, Hendriksen RS, Lo Fo Wong DM, et al. Salmonella serovars from humans and other sources in Thailand, 1993–2002. Emerg Infect Dis. 2004;10:131–6. doi: 10.3201/eid1001.02-0781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.WHO. Drug-resistant Salmonella Fact sheet N°139. Geneva: Food Safety Department WHO; 2005. [Google Scholar]
  • 47.Hendriksen R. Global epidemiology of non-typhoidal Salmonella infections in humans. PhD Thesis, National Food Institute Technical University of Denmark Mørkhøj Bygade 19 DK-2860 Søborg 2010. The work was supported by a grant 274-05-0117 from the Danish Research Agency and the World Health Organization Global Salm-Surv ( www.who.int/salmsurv) [Google Scholar]
  • 48. Global Foodborne Infections Network (GFN), 2012. Centers for Disease Control and Prevention National Center for Emerging and Zoonotic Infectious Diseases (NCEZID). Division of Foodborne, Waterborne, and Environmental Diseases. last updated: April 24, 2013. [1600 Clifton Rd. Atlanta, GA 30333, USA] [Google Scholar]
  • 49.Foley SL, Lynne AM, Nayak R.Salmonella challenges: prevalence in swine and poultry and potential pathogenicity of such isolates. J Anim Sci. 20088614 SupplE149–62. [DOI] [PubMed] [Google Scholar]
  • 50.Galán JE. Molecular genetic bases of Salmonella entry into host cells. Mol Microbiol. 1996;20:263–71. doi: 10.1111/j.1365-2958.1996.tb02615.x. [DOI] [PubMed] [Google Scholar]
  • 51.Bäumler AJ, Tsolis RM, Ficht TA, Adams LG. Evolution of host adaptation in Salmonella enterica. Infect Immun. 1998;66:4579–87. doi: 10.1128/iai.66.10.4579-4587.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Malorny B, Hoorfar J, Bunge C, Helmuth R. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl Environ Microbiol. 2003;69:290–6. doi: 10.1128/AEM.69.1.290-296.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Greig JD, Ravel A. Analysis of foodborne outbreak data reported internationally for source attribution. Int J Food Microbiol. 2009;130:77–87. doi: 10.1016/j.ijfoodmicro.2008.12.031. [DOI] [PubMed] [Google Scholar]
  • 54.Hadjinicolaou AV, Demetriou VL, Emmanuel MA, Kakoyiannis CK, Kostrikis LG. Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples. BMC Microbiol. 2009;9:97. doi: 10.1186/1471-2180-9-97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Galán JE, Curtiss R III. Cloning and molecular characterization of genes whose products allow Salmonella Typhimurium to penetrate tissue culture cells. Proc Nat Acad Sci USA. 1989;86:6383–7. doi: 10.1073/pnas.86.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Galán JE, Curtiss R III. Distribution of the invA, -BB, -CC, and -DD genes of Salmonella Typhimurium among other Salmonella serovars: invA mutants of Salmonella Typhi are deficient for entry into mammalian cells. Infect Immun. 1991;59:2901–8. doi: 10.1128/iai.59.9.2901-2908.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Schroeter A, Hoog B, Helmuth R. Resistance of Salmonella isolates in Germany. J Vet Med B. 2004;51:389–92. doi: 10.1111/j.1439-0450.2004.00795.x. [DOI] [PubMed] [Google Scholar]
  • 58.Wilson SJ, Knipe CJ, Zieger MJ, Gabehart KM, Goodman JE, Volk HM, et al. Direct costs of multidrug-resistant Acinetobacter baumannii in the burn unit of a public teaching hospital. Am J Infect Control. 2004;32:342–4. doi: 10.1016/j.ajic.2004.02.008. [DOI] [PubMed] [Google Scholar]
  • 59.Ellerbroek L, Narapati D, Phu Tai N, Poosaran N, Pinthong R, Sirimalaisuwan A, et al. Antibiotic resistance in Salmonella isolates from imported chicken carcasses in Bhutan and from pig carcasses in Vietnam. J Food Prot. 2010;73:376–9. doi: 10.4315/0362-028x-73.2.376. [DOI] [PubMed] [Google Scholar]
  • 60.Collignon P, Powers JH, Chiller TM, Kane AA, Aarestrup FM. World Health Organization ranking of antimicrobials according to their importance in human medicine: a critical step for developing risk management strategies for the use of antimicrobials in food production animals. Clin Infect Dis. 2009;49:132–41. doi: 10.1086/599374. [DOI] [PubMed] [Google Scholar]
  • 61.Aarestrup FM, Wegener HC, Collignon P. Resistance in bacteria of the food chain: epidemiology and control strategies. Expert Rev Anti Infect Ther. 2008;6:733–50. doi: 10.1586/14787210.6.5.733. [DOI] [PubMed] [Google Scholar]
  • 62. DANMAP 2011. The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme. DANMAP 2011 – Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. (Accessed 08/06/2013). [Google Scholar]
  • 63.Angulo FJ, Nargund VN, Chiller TC. Evidence of an association between use of anti-microbial agents in food animals and anti-microbial resistance among bacteria isolated from humans and the human health consequences of such resistance. J Vet Med B. 2004;51:374–9. doi: 10.1111/j.1439-0450.2004.00789.x. [DOI] [PubMed] [Google Scholar]
  • 64.Angulo FJ, Johnson KR, Tauxe RV, Cohen ML. Origins and consequences of antimicrobial-resistant nontyphoidal Salmonella: implications for the use of fluoroquinolones in food animals. Microb Drug Resist. 2000;6:77–83. doi: 10.1089/mdr.2000.6.77. [DOI] [PubMed] [Google Scholar]
  • 65.Jones YE, Chappell S, McLaren I, Davies RH, Wray C. Antimicrobial resistance in Salmonella isolated from animals and their environment in England and Wales from 1988–1999. Vet Rec. 2002;150:649–54. doi: 10.1136/vr.150.21.649. [DOI] [PubMed] [Google Scholar]
  • 66.Yang SJ, Park KY, Kim SH, No KM, Besser TE, Yoo HS, et al. Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization. Vet Microbiol. 2002;86:295–301. doi: 10.1016/s0378-1135(02)00009-3. [DOI] [PubMed] [Google Scholar]
  • 67.Yang B, Qu D, Zhang X, Shen J, Cui S, Shi Y, et al. Prevalence and characterization of Salmonella serovars in retail meats of marketplace in Shaanxi, China. Int J Food Microbiol. 2010;141:63–72. doi: 10.1016/j.ijfoodmicro.2010.04.015. [DOI] [PubMed] [Google Scholar]
  • 68.van Looveren M, Chasseur-Libotte ML, Godard C, Lammens C, Wijdooghe M, Peeters L, et al. Antimicrobial susceptibility of nontyphoidal Salmonella isolated from humans in Belgium. Acta Clin Belg. 2001;56:180–6. doi: 10.1179/acb.2001.028. [DOI] [PubMed] [Google Scholar]
  • 69.Wybot I, Wildemause C, Godard C, Bertrand S, Collard JM. Antimicrobial drug resistance in nontyphoid human Salmonella in Belgium: trends for the period 2000-2002. Acta Clin Belg. 2004;59:152–60. doi: 10.1179/acb.2004.022. [DOI] [PubMed] [Google Scholar]
  • 70.Hsueh PR, Teng LJ, Tseng SP, Chang CF, Wan JH, Yan JJ, et al. Ciprofloxacin-resistant Salmonella enterica Typhimurium and Choleraesuis from pigs to humans, Taiwan. Emerg Infect Dis. 2004;10:60–8. doi: 10.3201/eid1001.030171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Van Duijkeren E, Wannet WJ, Houwers DJ, Pelt WV. Antimicrobial susceptibilities of Salmonella strains isolated from humans, cattle, pigs and chickens in The Netherlands from 1984 to 2001. J Clin Microbiol. 2003;41:3574–8. doi: 10.1128/JCM.41.8.3574-3578.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Capita R, Alonso-Calleja C. Antibiotic-resistant bacteria: a challenge for the food industry. Crit Rev Food Sci Nutr. 2013;53:11–48. doi: 10.1080/10408398.2010.519837. [DOI] [PubMed] [Google Scholar]
  • 73. EAEMP. The European Agency for the evaluation of medicinal products. Antibiotic resistance in the European Union associated with the therapeutic use of veterinary medicines. EMEA/CVMP/342/99/-cor-Final; 1999. [Google Scholar]
  • 74.Soto SM. Relationship between virulence and antimicrobial resistance in bacteria. Rev Med Microbiol. 2009;20:84–90. [Google Scholar]
  • 75.Carattoli A. Plasmid-mediated antimicrobial resistance in Salmonella enterica. Curr Issues Mol Biol. 2003;5:113–2. [PubMed] [Google Scholar]
  • 76.Dione MM, Ikumapayi U, Saha D, Mohammed NI, Adegbola RA, Geerts S, et al. Antimicrobial resistance and virulence genes of non-typhoidal Salmonella isolates in The Gambia and Senegal. J Infect Dev Ctries. 2011;5:765–75. doi: 10.3855/jidc.1512. [DOI] [PubMed] [Google Scholar]
  • 77.Groisman EA, Ochman H. Pathogenicity islands: bacterial evolution in quantum leaps. . Cell. 1996;87:791–4. doi: 10.1016/s0092-8674(00)81985-6. [DOI] [PubMed] [Google Scholar]
  • 78.Hacker J, Blum-Oehler G, Muhldorfer I, Tschape H. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol. 1997;23:1089–97. doi: 10.1046/j.1365-2958.1997.3101672.x. [DOI] [PubMed] [Google Scholar]
  • 79.Martinez ZL, Baquero F. Interaction among strategies associated with bacterial infection: pathogenecity, epidemicity, and antibiotic resistance. Clin Microb Rev. 2002;15:647–79. doi: 10.1128/CMR.15.4.647-679.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80.Tizard I. Salmonellosis in wild birds. Semin Avian Exot Pet Med. 2004;13:50–66. [Google Scholar]
  • 81. FAO/WHO/OIE. Joint FAO/WHO/OIE expert meeting on critically important antimicrobials. Report of a meeting held in FAO, Rome, Italy, 26–30 November 2007. FAO, Rome, Italy, and WHO, Geneva, Switzerland; 2008. [Google Scholar]
  • 82. WHO. World Health Organization. Critically important antimicrobials for human medicine, 2nd Revision. WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR). Department of Food Safety and Zoonoses; 2009. Geneva, Switzerland. [Google Scholar]
  • 83. FAO/WHO. APPENDIX IX - Working papers of the Second Global Forum of Food Safety Regulators Second FAO/WHO Global Forum of Food Safety Regulators-Proceedings of the Forum. Food export control and certification Agenda Item 4.5 GF 02/8a Food and Agriculture Organization of the United Nations World Health Organization Rome, 2005. Second FAO/WHO Global Forum of Food Safety Regulators 12–14 October 2004, Bangkok, Thailand. Building effective food safety systems Proceedings of the Forum) WHO 2005. Drug-resistant Salmonella Fact sheet N°139 Revised April 2005. Food Safety Department WHO, Geneva. [Google Scholar]
  • 84. Anonymous. Institute of medicine (US) forum on microbial threats. Infectious disease movement in a borderless world: workshop summary. Atlanta, GA, USA: National Academies Press; 2010. p. 374–500. [PubMed] [Google Scholar]

Articles from Pathogens and Global Health are provided here courtesy of Taylor & Francis

RESOURCES