An eight-year study of Shigella species in Beijing , China : serodiversity , virulence genes , and antimicrobial resistance

Introduction: This study was conducted to determine the prevalence of serotypes, virulence factors, and antimicrobial resistance patterns of Shigella spp. in Beijing, China, from 2004 to 2011. Methodology: Real-time PCR assays were used to detect virulent genes, and the Kirby-Bauer disk diffusion method was used to evaluate antimicrobial resistance. Results: Among the total of 1,652 Shigella isolates, S. sonnei (57.1%) was the predominant species, followed by S. flexneri (42.3%), S. dysenteriae (0.4%), and S. boydii (0.2%). Nineteen serotypes were discovered among S. flexneri strains. The virulence gene ipaH was the most frequent, followed by sen and set. The presence of set showed significant difference in two dominant serogroups, S. flexneri and S. sonnei. Over 90% of Shigella isolates showed resistance to at least three drugs with widened spectrum. High-level antimicrobial resistance to single and multiple antibiotics was more common among S. sonnei than S. flexneri. Conclusion: There was an obvious serotype change and a dramatic increase of antibiotic resistance in Shigella prevalence in Beijing.


Introduction
Shigellosis, as a major source of diarrhea, is a global human health problem.Any of four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) can cause shigellosis.S. sonnei and S. boydii, associated with mild illness of short duration, most often occur in developed countries; S. flexneri is the most prevalent species in developing countries, and S. dysenteriae is known to cause sporadic outbreaks and epidemics worldwide, with severe complications and very high mortality [1][2][3].In China, Shigella spp. is the most frequently isolated diarrheal pathogen, accounting for up to 1.7 million episodes of bacillary dysentery annually, with an estimated 200,000 patients admitted to hospitals [4].
Virulence genes responsible for the pathogenesis of shigellosis are often multifactorial and coordinately regulated.IpaH is present in multiple copies on both the invasion plasmid and on the chromosome, and is responsible for the modification of host response to infection.Another two genes encoding enterotoxins, set and sen, have roles in altering electrolyte and water transport in the small intestine during the initial watery phase of shigellosis [5,6].Knowledge about the distribution of the three genes is limited, especially for the last two, which has been reported only in S. flexneri serotypes in some detail [7,8].The purpose of this study is to describe the prevalence of various Shigella species, the distribution of three virulence genes, and the local antibiotic resistance patterns in Beijing, the capital of China, between 2004 and 2011.

Shigella isolates and bacteriological examination
A hospital-based active surveillance was conducted in 16 districts of Beijing from 2004 to 2011.The sentinel hospitals collected stool specimens of outpatients, with a monthly enrollment number of 20-30 patients per district.For isolation of Shigella, the fecal swabs were plated directly onto Salmonella-Shigella agar and incubated at 37°C for 16 to 24 hours.After culture and screening, the biochemical identification was confirmed with the VITEK 2 Compact instrument (bioMérieux, Marcy l'Etoile, France).Finally, serologic identification was performed by the slide agglutination test.

Statistical analysis
Statistical analysis was performed with SPSS version 11.5 software.Comparison of proportions and statistical significance were calculated using the twotailed Chi-square test.
The trends in 19 serotypes of S. flexneri are described in Figure 2. The seven most frequently isolated serotypes (in order of prevalence: 4c, 2a, 1a, 2b, 5b, var x, 4) were responsible for 94.1% of all S. flexneri episodes, whereas any other serotype was  present in less than 1.0%.For three major serotypes -4c, 1a, and 2a -the former two showed a pronounced decrease with occasional inter-annual variations, while the third had an obvious fluctuation over time, with peaks in 2009 and 2010.The other infrequently observed serotypes, such as 2b, 4, 5b, var x and so on, varied in different years.Statistically significant shifts in the relative proportions of S. flexneri serotypes were observed during the period.
The detection of the virulence genes from 274 Shigella strains revealed that ipaH (96.3%) was the most frequent, followed by sen (85.8%) and set (46.0%) (Supplementary Table 1).Of note, all the strains positive for set were observed with the presentence of both ipaH and sen.Of the 274 strains, 43.1% were found to be positive for ipaH+set+sen+, 85.8% for ipaH+sen+, 46.0% for ipaH+set+, and 43.1% for set+sen+.The prevalence of the three virulence genes among diverse Shigella spp. was analyzed.The ipaH gene was detected in 97.0% of 135 S. flexneri isolates, 99.2% of 133 S. sonnei isolates, one of four S. boydii isolates, and none of two S. dysenteriae isolates.The set gene was present in 88.1% of S. flexneri isolates and in 5.3% of S. sonnei isolates, whereas it was absent in S. boydii and S. dysenteriae isolates.The sen gene was present in 88.9% of S. flexneri isolates, 85.7% of S. sonnei isolates, one S. boydii isolate, and none of the S. dysenteriae isolates.The set gene showed the difference of distribution in two dominant serogroups, S. flexneri (88.1%) and S. sonne (5.3%) (p < 0.001).As for ipaH and sen, there were no statistically significant differences in different Shigella species (p > 0.05).
Antimicrobial drug testing was carried out on 872 Shigella isolates, including 495 S. sonnei, 369 S. flexneri, as well as three S. dysenteriae and three S. boydii isolates (Supplementary Table 2).Resistance to one or more drugs was observed in 98.9% of the 872 isolates.The highest resistance was detected for NAL (90.7%), followed by TET (89.9%),AMP (86.9 %), and SXT (81.5%).Moderate resistance of Shigella isolates to GEN (49.2%) also was detected; however, resistance to NOR or OFX was relatively low (9.9% and 9.7%, respectively).

Discussion
Shigellosis is the most important cause of diarrhea worldwide, especially in underdeveloped and developing nations with substandard hygiene and poor quality water supplies.In this study, we found an unexpectedly complex landscape of circulating Shigella strains in Beijing.S. flexneri was the most common of the four species from 2004 to 2006, whereas S. sonnei has replaced S. flexneri as the most prevalent serogroup since 2009.These findings differ from those of studies conducted in developing countries [9] and other provinces in China [10], where S. flexneri was still the most frequently isolated species; however, our results match the findings in developed countries [2,11] and Thailand [12], which is rapidly becoming industrialized.The reason for this change is not known, but improvement of environmental conditions, hygiene habits, and water supplies should be considered.The significant increase in percentage of S. sonnei accompanied by the obvious reduction in S. flexneri may reflect that Beijing has undergone considerable socioeconomic changes during the period, with the infection pattern having changed from that of a developing city to that of a developed city.
Amongst the S. flexneri isolates, there were a surprisingly wide range of 19 serotypes, with 4c and 2a being the most prevalent serotypes.S. flexneri 2a is still the most common in many developing countries, including China [1,4,12].It should be noted that S. flexneri serotype 4c has remained in continuous existence over time in Beijing, although it has rarely been reported in other studies.We found statistically significant shifts in S. flexneri serotypes between observation years, consistent with previous works in India and Chile [13,14].The variety in Shigella species and serotypes shows high heterogeneous characteristics in temporal distribution, which further emphasizes the importance of identifying isolates to serotype level for the implementation of effective control strategies.
In the present study, the prevalence of ipaH was independent of the four different species of Shigella tested, confirming previous works that the gene is highly conserved various serotypes [9].The set and sen genes were found to be widely distributed in various S. flexneri serotypes, unlike in previous studies where they were almost exclusively found in S. flexneri 2a isolates and rarely in other species or serotypes [5][6][7][8]15].Overall, the present study detected ipaH, set, and sen in all the prevalent serotypes of S. flexneri; however, the presentence of set in S. sonnei was much lower than in sen and ipaH.Working on temporal variations of diverse species, our results enhance knowledge on the prevalence and distribution of these genes, which might assist in developing markers for Shigella of different types.
This study demonstrated the drastically increasing spectrum of antimicrobial resistance in Shigella isolates.All Shigella species also exhibited a very high rate of resistance (80%-90%) to older generation antimicrobials such as NAL, TET, AMP, and SXT.Although a similar pattern of resistance has been reported in our region and in other Asian countries [4,16,17], the frequency of resistance reported here is higher than frequencies observed earlier, and the trend to MDR is more pronounced [3,4].Cephalosporins and fluoroquinolones were two popular empirical options to treat severe gastrointestinal infections caused by pathogenic bacteria.Our finding of a 17.8% resistance rate to the third-generation cephalosporins (CTX) was in agreement with observations from India and Cameroon, where resistance rates of 12% and 20%, respectively, were reported [16,18].Additionally, our study found a 10% resistance rate to other fluoroquinolones such as CIP, NOR, and OFX.It has been noted that in other countries with strict antimicrobial controls (e.g., the United States), less than 1% of Shigella isolates were resistant to cephalosporins and fluoroquinolones [19].In China, the use of antimicrobial drugs has been poorly regulated and the indiscriminate overuse of antibiotics remains a serious issue that might be responsible for the increase of multiple antimicrobial resistance at an alarming speed.
In the present study, we found that S. sonnei was more frequently resistant to NAL, TET, and SXT, alone or in combination, than was S. flexneri.Other studies showed that S. sonnei had lower rates of resistance, compared with S. flexneri, to most of the antibiotics [19].A possible explanation is that infections due to S. sonnei are becoming more and more common than infections due to the other Shigella species in Beijing, making exposure to selective pressure from antibiotics more likely.
In summary, this study provides initial data on the prevalence and distribution of ipaH, set, and sen genes in a wide variety of Shigella isolates over an eightyear period.S. sonnei has replaced S. flexneri as a dominant species with high rates of multidrug resistances in Beijing.Continuous local monitoring of resistance patterns is necessary for effective therapy and control measures against shigellosis.

Figure 1 .
Figure 1.Distribution of Shigella species from 2004 to 2011 in Beijing

Figure 2 .
Figure 2. Distribution of Shigella flexneri serotype from 2004 to 2011 in Beijing

Table 2 .
Chi-square test was calculated by two predominant serotypes, S. flexneri and S. sonnei; S. dysenteriae and S. boydii were not included because of too few cases.Antimicrobial resistance of Shigella strains by species **Chi-square test was calculated by two predominant serotypes, S. flexneri and S. sonnei; S. dysenteriae and S. boydii were not included because of too few cases.Supplementary

Table 3 .
Predominant multidrug resistance patterns of Shigella strains by species

Predominant resistance pattern No. of isolates (%) resistant to multidrug
Chi-square test was calculated by two predominant serotypes, S. flexneri and S. sonnei; S. dysenteriae and S. boydii were not included because of too few cases.