Antimicrobial resistance , virulence genes and molecular subtypes of S . Enteritidis isolated from children in Shanghai

Introduction: Antimicrobial resistance of Salmonella poses a key threat to public health worldwide. Salmonella Enteritidis (S. Enteritidis) (119 isolates) from children under 10 years old with diarrhea in Shanghai from 2010-2012 were characterized for antimicrobial susceptibility, virulence gene profiles and pulsed field gel electrophoresis (PFGE) patterns. Methodology: The minimum inhibitory concentration for the 119 S. Enteritidis isolates was determined using an agar dilution method. The presence of virulence genes was detected by polymerase chain reaction (PCR). All the isolates with antimicrobial resistance were subjected to PFGE analysis. Results: Among these isolates, 71.4% (85) were resistant to sulfafurazole, 59.7% (71) were resistant to ampicillin, 47.1% (56) were resistant to streptomycin, 7.6% (9) were resistant to ceftiofur and 3.4% (4) were resistant to ceftriaxone. Multidrug resistance (MDR) was found in 63.9% (76) of the isolates; 23 resistance profiles were identified. All isolates harbored the ssaQ and sopE virulence genes in the 16 virulence profiles (VPs); VP1 accounted for 70.59% of the 119 isolates. There were 57 PFGE patterns among the 92 isolates tested, mainly grouped into five clusters (A to E). All of the 76 MDR isolates carried multiple virulence genes. Conclusions: Our study provides useful microbiological data for the successful treatment of S. Enteritidis infections in Shanghai. Although broad spectrum antimicrobials may be useful in the treatment of invasive S. Enteritidis infections, clinicians need to be aware of common microbiological traits, because of the high prevalence of MDR.


Introduction
Salmonella enterica subspecies enterica is one of the most common foodborne pathogens [1], causing more than 93 million illnesses and 155,000 deaths worldwide, 85% of which were related to contaminated food [2,3].Salmonella enterica serovars are recognized as a common cause of childhood infections all over the world, particularly gastroenteritis, bacteremia, and typhoid (enteric) fever [4].Notably, the incidence rate among children under the age of five (69.5 infections per 100,000 children) was higher than the average incidence rate of Salmonella enterica infection, which was 17.6 illnesses per 100,000 people [5].In 2001-2003, an outbreak of Salmonella (Newport, Poona and Sandiego) in children was reported in the United States, in which 70% of the infected patients were under 10 years old and 31% were infants under 1 year old [6].A recent review indicates that Salmonella Enteritidis (S.Enteritidis) and Salmonella Typhimurium (S.Typhimurium) cause approximately 80% of Salmonellosis in children under 5 [7].S. Enteritidis is a predominant serovar in both developing and developed countries [8] and the most common serovar in clinical cases among children [9].Because children have relatively low immunity, they are susceptible to S. Enteritidis infection, especially children aged under 10 years.Therefore, children were chosen as subjects for this study of antibiotic resistance in S. Enteritidis in Shanghai.
Clinical treatment of severe salmonellosis is based on the prescription of antibiotics, including ampicillin, third and fourth generation cephalosporins and fluoroquinolones [10].However, Salmonella isolates with multidrug resistance (MDR, defined as resistance to three or more antimicrobials) have been found [11] and had increased to 70% by early in this century [12].The spread of resistant Salmonellae is a particular concern for pediatricians because of the limited therapy options available for infants and children.The study of antimicrobial resistance in Salmonellae, especially in S. Enteritidis, has played an important role in public health research in the last two decades [13].It is essential to continue to update information on antimicrobial treatments for children with invasive bacterial infections.
Shanghai is the economic and financial center of China and one of its largest cities, with more than 24 million inhabitants.The Shanghai Center for Disease Control and Prevention (SCDC) is one of eight sites in China supported by the WHO Global Foodborne Infectious Diseases Network (WHO GFN).However, the level of antimicrobial resistance in the most common serovar S. Enteritidis for children in a large metropolitan population like Shanghai was unknown.The incidence of these infections has been underreported; updated data is needed to develop rational strategies for the clinical treatment of S. Enteritidis infection.
The objective of this study was to determine the antimicrobial susceptibility, virulence profile and genetic relatedness of S. Enteritidis isolates from children in Shanghai collected from 2010 to 2012.

S. Enteritidis isolates
The S. Enteritidis isolates (n = 119) used in this study were collected from 11 hospitals located in eight major districts of Shanghai, (Jinshan, Baoshan, Changning, Xuhui, Jingan, Songjiang, Hongkou, Putuo), China.All isolates from local hospitals were obtained from children under the age of 10 showing diarrhea and infection, during the years from 2010-2012.API 20Etest strips (bioMerieux Vitek, Marcyl'Étoile, France) were used for strain identification.All isolates were serotyped with commercial antiserum (S&A Reagents Laboratory, Bangkok, Thailand) and the serotypes were assigned according to the Kauffmann-White scheme [14].The clinical isolates were stored in 25% glycerol at -80°C until use.

PCR detection of virulence genes
DNA was extracted from the S. Enteritidis isolates using a bacteria genomic DNA extraction kit (Tiangen, Beijing, China) according to the manufacturer's instructions.DNA concentration and purification was measured using Nanodrop 2000c.The DNA was tested for ten virulence genes by PCR (Table 1) [18][19][20][21][22][23].PCR reactions were carried out in a volume of 25 μL containing 12.5 μL 2×Taq Plus Master Mix (Vazyme Biotech, Nanjing, China), 9 μL ddH2O, 1 μL of forward and reverse primers and 1.5 μL DNA template.Negative controls using dH2O instead of template DNA were included in all PCR reaction sets.PCR products were analyzed using electrophoresis in 1.5% agarose gels stained with ethidium bromide and visualized under UV light.

Pulsed-field gel electrophoresis (PFGE)
According to the protocol developed by the Centers for Disease Control and Prevention (CDC) for Molecular Subtyping of Salmonellae, all resistant isolates (92) were subjected to PFGE analysis (Pulse-Net, CDC, Atlanta, GA, USA) (CDC 2012).Briefly, agarose-embedded DNA was digested with 50 U of restriction enzyme Xba I (TaKaRa, Dalian, China) for 1.5-2 hours at 37°C.The restriction fragments were separated by electrophoresis in 0.5 × TBE buffer at 14°C for 18-19 hours using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA, USA) with the pulse times set to 2.16 -63.8 sec.Salmonella Braenderup H9812 was used as a molecular reference
A total of 57 PFGE patterns were observed in 92 isolates with a similarity of position of DNA fragments ranging between 47.0% and 100%.Seventy-six PFGE profiles were grouped into five clusters (A to E).The predominant cluster was cluster C, which contained 27 isolate, 19 of which had the same pattern.Isolates from children in different age groups were represented in the the number to be zero.Resistance % represents the proportion of resistant isolates (number).MIC50: The minimum inhibitory concentration that inhibits the growth of 50% of the isolates.MIC90: The minimum inhibitory concentration that inhibits the growth of 90% of the isolate.577 same patterns; there was no correlation between the S. Enteritidis PFGE profile and patient age.

Discussion
We found high levels of antimicrobial resistance to sulfisoxazole (71.4%), ampicillin (59.7%) and streptomycin (47.1%) in S. Enteritidis isolated.These results were similar to findings from Beijing, China on clinical samples of children from 2010 to 2014 showed resistance to sulfisoxazole in 54.3% of the isolates, resistance to ampicillin in 60.0% and resistance to streptomycin in 42.9% [24].Indeed, the antimicrobial resistance rates of sulfisoxazole, ampicillin and streptomycin were even higher in our study, which may indicate that the antimicrobial resistance rates had increased during 2010, 2011 and 2012.Approximately 63.9 % of the isolates were resistant to at least three antimicrobials.The predominant resistance pattern (AMP-STR-SUL) was similar to that in the previous study in Beijing [24].Despite the similarity in the pattern of resistance, the frequency was higher in our study and the trend of multidrug resistance was more obvious.It is worth mentioning that one of the 119 isolates in our study exhibited resistance to nine antimicrobial agents.The high level of MDR found in our research serves as a warning for other major cities in China [24,25] and should be considered when reviewing appropriate treatment options.
The prevalences of the virulence genes sopB and spvC were 97.48% and 88.24% in our study, almost identical to a previous report on poultry isolates [26].The plasmid-associated gene spvC is often detected in S. Enteritidis and a few other Salmonella serovars; it was found in more than 80% of the isolates we tested [27].The avrA, ssaQ, mgtC, siiD, and sopB genes were highly conserved and were genetic markers for the presence of the SPI 1-5 in Salmonella [28].The high percentage of these virulence genes in our study also indicated that they were widespread and highly conserved.Information on the distribution of virulence genes can also reflect and improve the understanding of potential risks for human infections.PFGE genotyping can be used to ascertain the homology of the same serotype isolates and determine whether they were derived from an outbreak or not.PFGE analysis in our study indicated that the S. Enteritidis isolates did not cluster by age, isolation date or gender, as isolates from different ages clustered with the same PFGE pattern.For example, in cluster C, eight isolates recovered from children who were 11 months to 9 years of age shared an identical pattern.These isolates were recovered between 2010 and2012, suggesting that the general characteristics of the Salmonella population did not change substantially during these years.PFGE typing of S. Enteritidis also showed a relatively high diversity indicating that the cases were sporadic and not part of any major outbreak.In our study, the same PFGE pattern was found in different Salmonella isolates from different months.This indicates this particular strain is successful in its current niche over a longer period of time, in comparison to the sporadic strains, and could pose a risk of an epidemic outbreak of Salmonella infection in the future.As mentioned, S. Enteritidis strains had a high prevalence in poultry products [29].It was very likely that children have been exposed to such animals or have consumed poultry-related foods in these urban areas.Hence, supervision of children's diet, such as ensuring that chicken is properly cooked, should be strengthened.Meanwhile, children should wash their hands frequently to reduce the possibility of infection.

Conclusions
Our study provided data on the distribution of antimicrobial resistance in S. Enteritidis isolates from children with clinical disease in Shanghai from 2010-2012.Most isolates (85) had some antimicrobial resistance and some (76) were MDR.Furthermore, isolates taken from children of different ages on different isolation dates had the same PFGE patterns, indicating these could be linked.These results highlight the need for continuous surveillance and monitoring of potential epidemic outbreaks of MDR S. Enteritidis in China.They also provide a useful view of antimicrobial resistance patterns needed by pediatricians to decide on treatment options.If antimicrobial treatment is necessary, the third-generation cephalosporins (ceftriaxone and ceftiofur) can still be considered for the treatment of S. Enteritidis infections in children.

Figure 1 .
Figure 1.XbaⅠPFGE patterns, antimicrobial resistance profiles and virulence gene profiles of isolates in this study.A black box indicates resistance to a particular antimicrobial or positive for a particular virulence gene.

Table 2 .
Resistance patterns of the 119 S. Enteritidis isolates.
The DNA bands were visualized under UV light.An unweighted pair group method with arithmetic averages (UPGMA) was employed, using BioNumerics 7.6 (Applied Maths, Saint-Martens-Latem, Belgium) to generate dendrograms for pattern analysis.The Dice coefficient and tolerance was set to 1.5%.

agent Distribution (no.) of MIC (μg/mL) among the 119 isolates
The number in the table represent the number of isolates at each dilution level.Blanks within the table indicate

Table 4 .
Virulence gene profiles among the 119 S. Enteritidis strains.