Serovar distribution, antimicrobial resistance profiles, and PFGE typing of Salmonella enterica strains isolated from 2007–2012 in Guangdong, China

The genus Salmonella currently includes two species, Salmonella enterica and Salmonella bongori. Salmonella enterica is further divided into subspecies I (enterica), II (salamae), IIIa (arizonae), IIIb (diarizonae), IV (houtenae), and VI (indica). Serotyping is the traditional method for the subtyping and the differentiation of Salmonella isolates based on the Kauffmann–White scheme. Over 2500 Salmonella serotypes are currently known [1]. Salmonella enterica, the etiological agent of salmonellosis, is recognized as an endemic food-borne pathogen that causes a heavy global socioeconomic burden [2–4]. Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by Salmonella Typhi and Salmonella Paratyphi, is a bacteremic illness which occurs in strong, healthy hosts in addition to immunocompromised hosts and infants and is essentially dependent on the bacterial inoculum/infectious dose [5]. Non-typhoidal Salmonella (NTS) serovars (e.g., Salmonella Enteritidis, Salmonella Typhimurium and Salmonella 4,5,12:i:-) normally cause self-limiting diarrhea or gastroenteritis, which results in a significant disease burden, with an estimated 2.8 million cases of diarrhea and 93.8 million cases of gastroenteritis, worldwide, and 155,000 deaths each year [6]. The host range of NTS serovars is broad, including poultry and cattle. Infection caused by NTS is commonly due to contamination in the food chain and food poisoning in developing countries. Occasionally, NTS serovars cause secondary bacteremia, while in immunocompromised patients in sub-Saharan Africa, they cause high rates of bacteremia in children below 5 years old as well as those with HIV infection [5, 6].

Typhoid Salmonella caused 216,000 deaths globally in 2010 [2], whereas most cases of invasive, non-typhoid salmonellosis occur occasionally and are due to either Salmonella Typhimurium or Salmonella Enteritidis [7]. The resistance of NTS to ampicillin, chloramphenicol and sulphamethoxazole is usually high in Africa and the USA [8, 9]. The treatment of NTS, therefore, increasingly relies on fluoroquinolones or third-generation cephalosporins; however, these treatment options are threatened by reduced bacterial susceptibility to fluoroquinolones and extended-spectrum beta-lactam antibiotics, respectively [9, 10]. In Hong Kong, China, the rate of infections by NTS has been increasing since 1989, and the most common Salmonella serovar is Enteritidis [11]. The antimicrobial susceptibilities of 275 Salmonella Enteritidis strains isolated from 1986 to 1996 show that over 99% of the isolates are susceptible to 17 of the 19 antimicrobial agents tested [12]. From 2005 to 2010, Salmonella Enteritidis was the predominant serovar in Hong Kong; in that time, the strains that were not susceptible to ciprofloxacin increased significantly, from 39.3% to 63%, and the percentage of multidrug resistant (MDR) strains increased from 17.8% to 36.2% [13]. In an inland province, Henan, the two most common serovars among the isolates collected during 2006 and 2007 were Salmonella Typhimurium (27%) and Salmonella Enteritidis (17%) [14]. Our previous study on Salmonella infection in diarrhea patients in Guangdong province showed that Salmonella Typhimurium and Enteritidis were the most common serovars. Eighty percent of the 229 isolates were susceptible to cephalosporins and quinolones, while 59.3% of them were multidrug resistant strains [15–18]. However, these studies did not provide sufficient information concerning prevalence, serovar distribution, and antimicrobial susceptibility in Guangdong province. Reduced quinolone- and third-generation cephalosporin- susceptibility in NTS is also poorly documented [10, 19].

The current study describes serovar profiles and potential profile shifts, characterizes MDR isolates with a special focus on susceptibility to quinolones and third-generation cephalosporins and reveals associations between MDR profiles and PFGE fingerprint pattern.

The current study indicated the serovar profiles of Salmonella enterica in diarrheal outpatients, and confirmed widespread MDR and reduced ciprofloxacin and third-generation cephalosporin susceptibility among NTS isolates from Guangdong province. Although typhoid Salmonella occurred in a few cases, most of them showed no resistance to ciprofloxacin or third-generation cephalosporins. These results were in agreement with the general knowledge of the epidemiology of salmonellosis [6].

The serovar profile data showed that Salmonella Typhimurium became the dominant serovar in 2008. Salmonella 4,5,12:i:- is a monophasic variant of Salmonella Typhimurium, which is very similar at the molecular level [24, 25]. Therefore, the complex group of Salmonella Typhimurium & 4,5,12:i:- ranked first in endemic serovars of Salmonella in Guangdong. These data differ from those in Europe and Latin America where Salmonella Enteritidis is the most common serovar, followed by Salmonella Typhimurium [26, 27]. In contrast, the Guangdong data agree with those in Africa and North America where Salmonella Typhimurium seems to be more common than Salmonella Enteritidis [28]. However, we still do not know the exact reasons for this shifting from Salmonella Enteritidis to Salmonella Typhimurium and Salmonella 4,5,12:i:-. As we know, Salmonella Typhimurium can be found in a wide range of animal species and is most commonly observed in pigs, while the most common reservoirs of Salmonella Enteritidis are eggs and chickens [29]. Therefore, it seems likely that the wider reservoir pattern for Salmonella Typhimurium and Salmonella 4,5,12:i:- provides them with more opportunities to come in contact with people and other reservoirs, resulting in the fast expansion of these two serovars. Another possible explanation for the increased detection of Salmonella Typhimurium and Salmonella 4,5,12:i:- in Guangdong is the updated training program for sampling and culture in 2009. After training, the total number of sampling and isolates increased significantly in 2009 (Table 1). Meanwhile, we found that both Salmonella Typhimurium and Salmonella 4,5,12:i:- affected infants, whereas Salmonella Enteritidis affected adults. The different eating habits of infants and adults or special host adaptations may explain the observed distribution pattern. Salmonella Stanley and Derby were also commonly found in Guangdong as observed in other regions of the world [26, 28]. Several other serovars that are common in many countries were also found in Guangdong, such as Salmonella Rissen, Salmonella Weltevreden, Salmonella Infantis, Salmonella Agona, Salmonella Albany and Salmonella Newport [26, 28]. Likely, the frequent global travel, communication and trading that occur in Guangdong favor the transmission of these serovars, as similar trends have been verified in Asia [30], Africa and Europe [31].

The antimicrobial susceptibility analyses cover antimicrobial resistance patterns in 15 of 21 cities in Guangdong where the Salmonella situation had been poorly characterized. Indeed, previous data about Salmonellosis in Guangdong comprise few studies [15–18]. Comparing these studies illustrates the emergence of antimicrobial resistance except for the dramatic increase of resistance to ciprofloxacin and third-generation cephalosporins, which we have summarized in Guangdong province for the first time here. A similar increase in resistance to antibiotics was observed in fish and chicken in Guangdong [32]. The observed antibiotic resistance trend in Guangdong is very similar to those described in the Guangxi [33] and Henan provinces [14] in China. The major difference is the higher ciprofloxacin resistance observed in Henan than Guangdong (54% vs 8.05%). Meanwhile we found resistance to both ciprofloxacin and cephalosporins in Salmonella Typhimurium, Salmonella 4,5,12:i:- and Salmonella Enteritidis. Ciprofloxacin is currently the recommended the first-line antibiotic by the World Health Organization for the treatment of salmonellosis [34]. Third-generation cephalosporins are considered alternative drugs for salmonellosis treatment [34]. Hence, the resistance to fluoroquinolones and third-generation cephalosporins will pose a great challenge for the effective treatment of salmonellosis. Particularly, the dramatic increase of resistance to ciprofloxacin and third-generation cephalosporins in infants is a warning signal for a prudent application of these antibiotics in clinical settings. This situation in Guangdong seems completely different from particular areas in Africa where Salmonella are highly susceptible to antibiotics [35]. Therefore, close surveillance of Salmonella and their microbial resistance patterns is essential to prevent the expansion of MDR clonal populations [36]. Furthermore, the reduced ciprofloxacin susceptibility was, in all cases, associated with nalidixic acid resistance. Therefore, the detection of nalidixic acid resistance remains a valuable test to screen for reduced ciprofloxacin susceptibility in Guangdong.

PFGE typing implicated a relatively high diversity in all serotypes, indicating that the human cases were most likely sporadic; this interpretation agrees with unrelated epidemiological background of all of the cases [37]. We found few dominant PFGE patterns in past and recent isolates of Salmonella Typhimurium, Salmonella 4,5,12:i:-, Salmonella Enteritidis and Salmonella Stanley. Thus, these common patterns seem to have been constant over time. Meanwhile, resistant patterns of each predominant PFGE pattern also differed from each other. Therefore, the different PFGE patterns perhaps came from different clonal lineage. Even so, the obtained PFGE profiles and representative strains are available for comparison with profiles obtained of Salmonella from other countries to track the asymptomatic carriers and identify the circulation of major clones worldwide. However, as we did not determine the genotype (such as MLST or MLVA) of the dominant serovars, such as in the Salmonella Typhimurium and Salmonella 4,5,12:i:- isolates, we do not know if these serovars belong to the same lineages that are found in other continents; therefore, it would be of crucial importance to determine the genotype to link the possible transmission pathway of each predominant serovar in future studies [38].

In conclusion, Salmonella Typhimurium and Salmonella 4,5,12:i:- were the most common serovars isolated during the observation period from 2007 to 2012 in Guangdong, mainly causing salmonellosis in infants, whereas Salmonella Enteritidis mainly caused salmonellosis in adults. In 2008, a serovar shift from Salmonella Enteritidis to Salmonella Typhimurium became apparent. A high burden of multidrug resistant strains and an increasing incidence of quinolone and third-generation cephalosporin resistance were observed. Close surveillance of Salmonella and their microbial resistance patterns, using combined molecular tools, such as PFGE, multilocus sequence typing (MLST) and multiple locus VNTR analysis (MLVA), is therefore dedicate to prevent outbreaks of disease, track the potential transmission pathways and rationalize the antimicrobial therapy of salmonellosis.