Molecular Characterization of Extended-Spectrum Cephalosporinase-Producing Salmonella enterica Serovar Choleraesuis Isolates from Patients in Thailand and Denmark

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla_CMY-2 containing incA/C or bla_CTX-M-14 containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla_CTX-M-14 gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A “travel-associated” spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.Salmonella enterica is a common cause of human gastroenteritis and bacteremia worldwide (18, 31), and a wide variety of animals, particularly food animals, have been identified as reservoirs for non-Typhi Salmonella (11, 22, 23). Although approximately 2,600 serovars of Salmonella enterica have been identified, most human infections are caused by a limited number of serovars, and in general these infections are self-limiting. Some Salmonella serovars, including Salmonella Choleraesuis (swine) and Salmonella Dublin (cattle), which are adapted to a specific animal host, have a propensity to cause extraintestinal infections in humans. Compared to those with other serovars of non-Typhi Salmonella, infections with these serovars are associated with higher rates of bacteremia, meningitis, and mortality (4, 5, 24). For patients with severe salmonellosis, antimicrobial chemotherapy may be life-saving. Due to the increasing prevalence of fluoroquinolone resistance, extended-spectrum cephalosporins are increasingly used for the treatment of Salmonella infections in humans (17, 21, 25) and especially for children, for whom treatment of highly resistant Salmonella infections with fluoroquinolones is not advised, since such treatment has been associated with treatment failures (12, 13, 21). Therefore, these compounds have been designated critically important for human health by the World Health Organization (10).We recently reported that the prevalence of human infections with Salmonella serovar Choleraesuis in Thailand increased from 1.5% (n = 87) in 1994 to 9.2% (n = 190) in 2006 (19). The group of people at highest risk for these infections was those between 6 and 40 years of age in the central region of Thailand (19). A 2007 study of Salmonella serovar Choleraesuis isolates from Thailand observed an increasing resistance to both extended-spectrum cephalosporins (ceftriaxone) and fluoroquinolones. Fifty-four isolates obtained between 2003 and 2005 were tested, of which 30% were found to be resistant to an extended-spectrum cephalosporin (ceftriaxone) (25).To date, only two reports, both from Taiwan, have described mechanisms for extended-spectrum cephalosporin resistance in Salmonella serovar Choleraesuis. The first report was published in 2004 with the discovery of the bla_CMY-2 AmpC β-lactamase gene located on a 140-kb F-like plasmid (6). The following year, the same authors detected bla_CTX-M-3 in a Salmonella serovar Choleraesuis isolate from a patient admitted to a university hospital (30). In 2007, a massive increase of fluoroquinolone- and ceftriaxone-resistant Salmonella serovar Choleraesuis isolates was described in Thailand (25).In Taiwan, the usage of antimicrobials in veterinary medicine and as growth promoters in animal feed may have promoted the emergence of resistance (5). Likewise, in Thailand, the extended-spectrum cephalosporin ceftiofur is used as a growth promoter in swine production (25). However, data on antimicrobial usage in disease prevention and as growth promoters are not accessible in both countries.The objective of the present study was to characterize the mechanisms responsible for the emergence of resistance to extended-spectrum cephalosporins in isolates of Salmonella serovar Choleraesuis recovered from patients in Thailand and Denmark.Additional objectives were to determine the clonality of the isolates resistant and susceptible to cephalosporins (ceftriaxone and cefoxitin) using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing and to find biological evidence of transmission through international travel.     

Subtyping of Salmonella enterica serovar typhimurium outbreak strains isolated from humans and animals in Iceland

A total of 75 Salmonella enterica serovar Typhimurium strains of various (mainly human and animal) origins were typed by pulsed-field gel electrophoresis (PFGE) and phage typing. These strains were collected during an outbreak in Iceland in 1999 and 2000. The typing revealed that 84% of the strains belonged to the same PFGE and phage type (PT), namely, PFGE type 1Aa and PT 1.     

Interaction between Host Cells and Septicemic Salmonella enterica Serovar Typhimurium Isolates from Pigs

Salmonella enterica serovar Typhimurium is an important pathogen in swine and is also a frequently reported zoonotic agent. The objective of this study was to characterize isolates of S. enterica serovar Typhimurium associated with septicemia in swine and to compare them to isolates recovered from clinically healthy pigs. We were particularly interested in comparing the two groups of isolates for their ability to adhere to and invade host cells, to be phagocytized and survive in monocyte cells, to induce apoptosis, and to adhere to intestinal mucus. Their surface properties were also evaluated by interactions with solvents. The isolates recovered from diseased animals were shown to invade intestinal epithelial cell lines at a higher rate (P = 0.003) than isolates from healthy pigs. Septicemic isolates were phagocytized by human monocytes at a higher rate than isolates from healthy pigs (P = 0.009). The mean percentages of phagocytosis were significantly lower for human monocytes than for porcine monocytes (P = 0.02 and P = 0.008, respectively) for isolates from both diseased and healthy animals. Healthy animal isolates were phagocytized more by porcine monocytes at 15 min (P = 0.02) than septicemic isolates. No difference between isolates from septicemic pigs and isolates from healthy pigs was detected for other tested parameters. These results suggest that septicemic isolates have a particular pattern of invasion.In pigs, Salmonella strains are related to significant animal infections associated with clinical signs and economic losses but are mainly associated with a carrier state, becoming a reservoir for human infections (3). Infection and/or silent carriage of Salmonella in pigs is an important public health concern. Multiresistance to antibiotics is often associated with Salmonella enterica serovar Typhimurium (14). In order to develop control measures, it is important to characterize these isolates and better understand the pathogenesis of infection.The pattern of infection by Salmonella is oral ingestion of the bacteria followed by passage through the mucus which covers the epithelial cells to invade mucous membranes and cause disease (35). The intestinal mucus can then serve as the initial binding site for bacteria. Initial adhesion is mainly a physicochemical process founded on nonspecific interactions (van der Waals and coulombic interactions) (17, 33). This type of adhesion can be reversible or not, and surface properties of some bacteria have been show to influence nonspecific interactions with host cells. The first steps of infection are adhesion on the surface, firm attachment, and penetration into intestinal epithelial cells. The invasion of intestinal epithelial cells is believed to be a very important step related to the virulence of Salmonella strains associated with infections (5).After invasion of the epithelial cells, the bacteria reach the subepithelial lymph tissue and the lamina propria, where Salmonella cells meet host immune cells (28). The overall phagocytic process can be divided into at least two main parts. First, bacteria must adhere to the phagocyte surface in a process called adherence. The second step of phagocytosis involves internalization or ingestion of the adherent particle. Following initial adhesion to phagocytes, special bacterial cell surface structures recognize receptors on the target cell surface (17, 33). The virulence genes of S. enterica serovar Typhimurium located on SPI-1, which encode a type III protein export machinery, are necessary for invasion of either nonphagocytic (5) or phagocytic (28) cells. Salmonella is able to induce cell death in macrophages in two different ways (32). Rapid activation of programmed macrophage cell death depends on SipB and SPI-1, whereas delayed induction of apoptosis in infected macrophages is SPI-1 independent (32). The results reported by van der Velden et al. indicate that ompR and a functional SPI-2-encoded type III protein secretion apparatus are required for delayed induction of apoptosis (32). The survival in phagocytes is an important step to induce septicemia in pigs, causing clinical signs similar to those in humans (16, 31); therefore, this animal model may be used to study human salmonellosis. Salmonella is able to survive and replicate in phagocytic cells, and this is an essential component of the virulence of these bacteria (1). S. enterica serovar Typhimurium cells that have invaded the macrophage by phagocytosis are able to replicate intracellularly (28) and induce apoptosis (32). In the past 10 to 15 years, an increased number of cases of clinical salmonellosis associated with Salmonella serovar Typhimurium were observed in pigs (11).The aim of this study was to characterize isolates of S. enterica serovar Typhimurium associated with septicemia in swine and to compare them to isolates recovered from clinically healthy pigs. We were particularly interested in comparing the two groups of isolates in regard to their abilities to adhere to host cells, invade host cells, be phagocytized, survive in cells, generate apoptosis, adhere to intestinal mucus, and adhere to solvents.     

Variability in antimicrobial resistance among Salmonella enterica strains from fattening pigs and sows

The aim of this study was to determine the prevalence of antibiotic resistance and different resistance patterns for Salmonella isolates collected from Belgian sows and fattening pigs at different ages and at slaughter. The most frequently isolated serotypes were S. Typhimurium (42.3%), S. Derby (25.1%), S. Goldcoast (7.3%), and S. Infantis (4.8%). All 901 isolates were submitted to antimicrobial susceptibility testing for 14 compounds using the disc agar diffusion test. The highest percentage of resistance was found to oxytetracycline (34.2%), streptomycin (32.5%), sulfamethizole (27.6%), and ampicillin (24.9%). Three of the isolates showed resistance to cephalosporins and none to second-generation fluoroquinolones. Multiresistance (resistance to > or =2 antimicrobials) was observed in 33.2% of the strains. Differences in resistance patterns were observed between and within serotypes as well as genotypes. A significant lower proportion (p < 0.01) of resistant strains was recovered in fecal samples from sows (23/56) than from fattening pigs during the weaning (30/30), growing (79/85), and finishing periods (45/52). The proportion of resistant strains recovered from fecal samples taken at the herd (154/167) was higher than in the samples collected at the slaughterhouse (75/140). When designing antimicrobial resistance surveillance programs for Salmonella in pigs, it is important to take multiple samples within each herd from both sows and fattening pigs at different time points.     

Characterization of Salmonella enterica serotype newport isolated from humans and food animals

Salmonella enterica serotype Newport isolates resistant to at least nine antimicrobials (including extended-spectrum cephalosporins), known as serotype Newport MDR-AmpC isolates, have been rapidly emerging as pathogens in both animals and humans throughout the United States. Resistance to extended-spectrum cephalosporins is associated with clinical failures, including death, in patients with systemic infections. In this study, 87 Salmonella serotype Newport strains were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing and examined for the presence of class 1 integrons and bla(CMY) genes. Thirty-five PFGE patterns were observed with XbaI, and three of these patterns were indistinguishable among isolates from humans and animals. Fifty-three (60%) Salmonella serotype Newport isolates were identified as serotype Newport MDR-AmpC, including 16 (53%) of 30 human isolates, 27 (93%) of 29 cattle isolates, 7 (70%) of 10 swine isolates, and 3 (30%) of 10 chicken isolates. However, 28 (32%) Salmonella serotype Newport isolates were susceptible to all 16 antimicrobials tested. The bla(CMY) gene was present in all serotype Newport MDR-AmpC isolates. Furthermore, the plasmid-mediated bla(CMY) gene was transferable via conjugation to an Escherichia coli strain. The transconjugant showed the MDR-AmpC resistance profile. Thirty-five (40%) of the isolates possessed class 1 integrons. Sequence analyses of the integrons showed that they contained aadA, which confers resistance to streptomycin, or aadA and dhfr, which confer resistance to trimethoprim-sulfamethoxazole. One integron from a swine isolate contained the sat-1 gene, which encodes resistance to streptothricin, an antimicrobial agent that has never been approved for use in the United States. In conclusion, Salmonella serotype Newport MDR-AmpC was commonly identified among Salmonella serotype Newport isolates recovered from humans and food animals. These findings support the possibility of transmission of this organism to humans through the food chain.     

Multidrug resistance in Salmonella enterica serotype Typhimurium from humans in France (1993 to 2003)

The aim of this study was to determine the distribution of the antimicrobial resistance phenotypes (R types), the phage types and XbaI-pulsed-field gel electrophoresis (PFGE) types, the genes coding for resistance to beta-lactams and to quinolones, and the class 1 integrons among a representative sample of Salmonella enterica serotype Typhimurium isolates collected from humans in 2002 through the French National Reference Center for Salmonella (NRC-Salm) network. The trends in the evolution of antimicrobial resistance of serotype Typhimurium were reviewed by using NRC-Salm data from 1993, 1997, 2000, and 2003. In 2002, 3,998 isolates of serotype Typhimurium were registered at the NRC-Salm among 11,775 serotyped S. enterica isolates (34%). The most common multiple antibiotic resistance pattern was resistance to amoxicillin, chloramphenicol, streptomycin and spectinomycin, sulfonamides, and tetracycline (ACSSpSuTe R type), with 156 isolates (48.8%). One isolate resistant to extended-spectrum cephalosporins due to the production of TEM-52 extended-spectrum beta-lactamase was detected (0.3%), and one multidrug-resistant isolate was highly resistant to ciprofloxacin (MIC > 32 mg/liter). We found that 57.2% of the isolates tested belonged to the DT104 clone. The main resistance pattern of DT104 isolates was R type ACSSpSuTe (83.2%). However, evolutionary changes have occurred within DT104, involving both loss (variants of Salmonella genomic island 1) and acquisition of genes for drug resistance to trimethoprim or to quinolones. PFGE profile X1 was the most prevalent (74.5%) among DT104 isolates, indicating the need to use a more discriminatory subtyping method for such isolates. Global data from the NRC-Salm suggested that DT104 was the main cause of multidrug resistance in serotype Typhimurium from humans from at least 1997 to 2003, with a roughly stable prevalence during this period.     

Prevalence of Salmonella enterica in Poultry and Eggs in Uruguay during an Epidemic Due to Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is frequently associated with food-borne disease worldwide. Poultry-derived products are a major source. An epidemic of human infection with S. Enteritidis occurred in Uruguay, and to evaluate the extent of poultry contamination, we conducted a nationwide survey over 2 years that included the analysis of sera from 5,751 birds and 12,400 eggs. Serological evidence of infection with Salmonella group O:9 was found in 24.4% of the birds. All positive sera were retested with a gm flagellum-based enzyme-linked immunosorbent assay, and based on these results, the national prevalence of S. Enteritidis infection was estimated to be 6.3%. Salmonellae were recovered from 58 of 620 pools made up of 20 eggs each, demonstrating a prevalence of at least 1 in every 214 eggs. Surprisingly, the majority of the isolates were not S. Enteritidis. Thirty-nine isolates were typed as S. Derby, 9 as S. Gallinarum, 8 as S. Enteritidis, and 2 as S. Panama. Despite the highest prevalence in eggs, S. Derby was not isolated from humans in the period of analysis, suggesting a low capacity to infect humans. Microarray-based comparative genomic hybridization analysis of S. Derby and S. Enteritidis revealed more than 350 genetic differences. S. Derby lacked pathogenicity islands 13 and 14, the fimbrial lpf operon, and other regions encoding metabolic functions. Several of these regions are present not only in serovar Enteritidis but also in all sequenced strains of S. Typhimurium, suggesting that these regions might be related to the capacity of Salmonella to cause food-borne disease.Salmonella enterica is a major cause of food-borne disease worldwide (14, 18, 46). Poultry-derived products, particularly chicken eggs, are considered a major source of human infection with Salmonella (2, 20, 38). Chickens can be infected with many different serovars of Salmonella. Of these, S. enterica serovars Pullorum and Gallinarum (S. Pullorum and S. Gallinarum, respectively) are host specific and represent a major concern for the poultry industry but have no impact on public health. Other S. enterica serovars frequently isolated from chickens, such as Typhimurium, Enteritidis, and Heidelberg, can infect a wider range of hosts and frequently reach the human food chain, causing food-borne disease.A peculiar epidemiological feature of human salmonellosis is that epidemics are commonly associated with a particular prevalent serovar of S. enterica that shows temporal and geographical variation. Until the 1980s, S. enterica serovar Typhimurium (S. Typhimurium) was the serovar most commonly isolated from humans worldwide, but by the late 1980s, S. enterica serovar Enteritidis (S. Enteritidis) emerged as the most common cause of salmonellosis in Europe, and during the 1990s, it became the most prevalent serovar in many countries worldwide (9, 22, 33, 40, 43). The reasons for this worldwide serovar shift are still not understood, and several hypotheses have been proposed, including the existence of a rodent reservoir for S. Enteritidis or the epidemiological change induced by vaccination of poultry against the closely related bacterium S. Gallinarum (47).In Uruguay, S. Typhimurium was the most frequently isolated serovar until 1994, and S. Enteritidis was only sporadically isolated (3, 24, 37). In 1995, a first outbreak of S. Enteritidis occurred, starting an epidemic that lasted almost 10 years. This outbreak was traced back to sandwiches prepared with contaminated mayonnaise that were distributed nationwide by a local catering service. According to data provided by the national public health authorities, the outbreak affected an estimated 600 individuals countrywide. From then on, several other outbreaks of various sizes occurred and S. Enteritidis was identified as the cause in 89% of Salmonella food poisoning episodes. In most of these cases (80%, according to official records), eggs or chicken meat was identified as the source of infection. From 1997 to 2004, S. Enteritidis was the most frequently identified serovar in Uruguay, accounting for more than 50% of the strains received each year at the National Salmonella Center and for more than 85% of the strains isolated from humans (3). After 2005, there was a dramatic reduction in the number of S. Enteritidis outbreaks, and this year was considered the end of the epidemic. Over the last 3 years, S. Typhimurium has become the serovar most frequently associated with isolated cases of food poisoning, and S. Derby and S. Panama have been sporadically isolated. Nevertheless, S. Enteritidis is still the serovar most frequently associated with outbreaks in the country.S. Enteritidis frequently colonizes the alimentary tracts of chickens without causing disease. However, it can produce a systemic infection in young chicks that may further lead to the infection of egg contents (13, 51). With the aim of knowing the prevalence of S. Enteritidis infection in poultry, we designed and conducted a countrywide serological and microbiological survey of chicken flocks and commercially available eggs from 2000 to 2002, and the results are presented here. An unexpected result of the survey was a higher prevalence of S. Derby than S. Enteritidis in eggs, particularly because while the latter was identified as the etiological agent of the epidemic there were no reports of human infections with S. Derby in the same period of time. This suggested a low capacity of S. Derby isolates to infect humans; thus, we performed a genomic comparison of the two serovars to search for genetic differences that could be the basis of such marked differences in epidemiological behavior. We found that S. Derby lacks several genomic regions related to virulence, suggesting that these regions could be involved in the capacity of Salmonella to cause food-borne disease.     

Extended-spectrum-beta-lactamase (TEM-52)-producing strains of Salmonella enterica of various serotypes isolated in France

From 2002 to 2003, four isolates of Salmonella enterica serotypes Typhimurium, Enteritidis, Blockley, and Panama, isolated in France from patients with gastroenteritis, were found to produce extended-spectrum beta-lactamase TEM-52. The study showed the bla(TEM-52) gene to be located in a Tn3-like structure and carried by 100- or 32-kb conjugative plasmids.     

Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France

During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum beta-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The bla(CTX-M-9) gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum beta-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.     

Interplay between the QseC and QseE Bacterial Adrenergic Sensor Kinases in Salmonella enterica Serovar Typhimurium Pathogenesis

The bacterial adrenergic sensor kinases QseC and QseE respond to epinephrine and/or norepinephrine to initiate a complex phosphorelay regulatory cascade that modulates virulence gene expression in several pathogens. We have previously shown that QseC activates virulence gene expression in Salmonella enterica serovar Typhimurium. Here we report the role of QseE in S. Typhimurium pathogenesis as well as the interplay between these two histidine sensor kinases in gene regulation. An S. Typhimurium qseE mutant is hampered in the invasion of epithelial cells and intramacrophage replication. The ΔqseC strain is highly attenuated for intramacrophage survival but has only a minor defect in invasion. However, the ΔqseEC strain has only a slight attenuation in invasion, mirroring the ΔqseC strain, and has an intermediary intramacrophage replication defect in comparison to the ΔqseE and ΔqseC strains. The expressions of the sipA and sopB genes, involved in the invasion of epithelial cells, are activated by epinephrine via QseE. The expression levels of these genes are still decreased in the ΔqseEC double mutant, albeit to a lesser extent, congruent with the invasion phenotype of this mutant. The expression level of the sifA gene, important for intramacrophage replication, is decreased in the qseE mutant and the ΔqseEC double mutant grown in vitro. However, as previously reported by us, the epinephrine-dependent activation of this gene occurs via QseC. In the systemic model of S. Typhimurium infection of BALB/c mice, the qseC and qseE mutants are highly attenuated, while the double mutant has an intermediary phenotype. Altogether, these data suggest that both adrenergic sensors play an important role in modulating several aspects of S. Typhimurium pathogenesis.     

Random amplified polymorphic DNA and phenotyping analysis of Salmonella enterica serovar enteritidis isolates collected from humans and poultry in Uruguay from 1995 to 2002

Molecular and phenotyping techniques were applied to study Salmonella enterica serovar Enteritidis strains both from human cases of infection and of avian origin isolated in Uruguay from 1995 to 2002. A group of 62 isolates was subjected to random amplified polymorphic DNA (RAPD) assay and analysis of antibiotic resistance patterns. Twenty-one of these strains were further characterized by phage typing and analysis of their protein expression profiles. RAPD fingerprinting with five different primers discriminated 10 different genetic profiles. Of the 62 strains tested, 48 had a single major genetic profile, whereas the other nine profiles were evenly distributed among the other strains. The genetic diversity was greater among strains of animal origin than among isolates of human origin. Comparative examination of the results obtained by RAPD analysis and phenotypic analysis and by strain source provided evidence of the reliable discriminatory power of RAPD analysis in our study. Six avian isolates with antibiotic resistance were detected: two were nalidixic acid resistant and four had a particular beta-lactam resistance pattern. The last four isolates all had the same unusual phage type (phage type 4b); however, RAPD analysis differentiated them into two groups. Two isolates with unique RAPD profiles were recovered from distinct human cases, suggesting that the technique differentiates unrelated strains. Overall, the results show the existence of a predominant genetic type that is present in poultry and that is transmitted to humans. There are also several other genotypes, but only a few of them could be recovered from human sources, suggesting the existence of different pathogenic traits among strains circulating in the country.     

Genomic Signature of Multidrug-Resistant Salmonella enterica Serovar Typhi Isolates Related to a Massive Outbreak in Zambia between 2010 and 2012

Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole-genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the multilocus sequence type (MLST), haplotype, plasmid replicon, antimicrobial resistance genes, and genetic relatedness by single nucleotide polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients, with a fatality rate of 0.5%. Most (83.0%) isolates were multidrug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype, H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes, catA1, bla_TEM-1, dfrA7, sul1, sul2, strA, and strB, and fragments of the incompatibility group Q1 (IncQ1) plasmid replicon, the class 1 integron, and the mer operon. The genomic analysis revealed 415 SNP differences overall and 35 deletions among 33 of the isolates subjected to whole-genome sequencing. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India by 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 of the 33 isolates sequenced belonged to a tight clonal group distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with the short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak, during which occasional other S. Typhi lineages, including sensitive ones, continued to cocirculate. The common view is that the emerging global S. Typhi haplotype, H58B, containing the MDR IncHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harboring a chromosomally translocated region containing the MDR islands of IncHI1 plasmid has emerged in Zambia. This could change the perception of the term “classical MDR typhoid” currently being solely associated with the IncHI1 plasmid. It might be more common than presently thought that S. Typhi haplotype H58B harbors the IncHI1 plasmid or a chromosomally translocated MDR region or both.     

Genetic diversity of clinical and environmental strains of Salmonella enterica serotype Weltevreden isolated in Malaysia

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.     

Genotyping of Salmonella enterica serovar Typhi strains isolated from 1959 to 2006 in China and analysis of genetic diversity by genomic microarray

Aim

To determine the genotype of Salmonella enterica serovar Typhi (S. Typhi) strains in China and analyze their genetic diversity.

Methods

We collected S. Typhi strains from 1959 to 2006 in five highly endemic Chinese provinces and chose 40 representative strains. Multilocus sequence typing was used to determine the genotypes or sequence types (ST) and microarray-based comparative genomic hybridization (M-CGH) to investigate the differences in gene content among these strains.

Results

Forty representative S. Typhi strains belonged to 4 sequence types (ST1, ST2, ST890, and ST892). The predominant S. Typhi genotype (31/40) was ST2 and it had a diverse geographic distribution. We discovered two novel STs – ST890 and ST892. M-CGH showed that 69 genes in these two novel STs were divergent from S. Typhi Ty2, which belongs to ST1. In addition, 5 representative Typhi strains of ST2 isolated from Guizhou province showed differences in divergent genes.

Conclusion

We determined two novel sequence types, ST890 and ST892, and found that ST2 was the most prevalent genotype of S. Typhi in China. Genetic diversity was present even within a highly clonal bacterial population.Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative, human-restricted enteroinvasive pathogen that causes typhoid fever (1,2). The harm caused by S. Typhi has been greatly reduced by the application of antibiotics, but typhoid fever is still a common disease in tropical and subtropical regions, and many drug-resistant strains of S. Typhi have been discovered. In the recent years, there have been more than 16 million cases reported annually worldwide. Even in the United States and other developed countries, there are still outbreaks of typhoid fever caused by S. Typhi (3). This is in part due to the ability of S. Typhi to rapidly evolve through either horizontal gene transfer mechanisms or produce a cloud of related strains by using highly mutable genes (4). Thus, there is an urgent need for improved molecular diagnostics to discriminate among the large numbers of related strains. There are many methods for genotyping of Salmonella, and polymerase chain reaction (PCR)-based typing methods are very prevalent. A multiplex PCR-based reverse line blot hybridization system can enhance outbreak investigations and surveillance of Salmonella infections (5). Recently, real-time PCR-based single nucleotide polymorphism typing method has been used for global epidemiological analysis of S. Typhi (6).Multilocus sequence typing (MLST), which is based on the analysis of DNA sequence polymorphisms in a group of housekeeping genes, is the most widely used method for bacterial strain genotyping (7). Each unique sequence of a housekeeping gene is assigned an allele number, and an allele profile of a strain is defined as the set of allele numbers for that strain. Each unique allele profile is assigned a sequence type (ST) number. Strains that have the same ST number are identical at all of the sequenced loci and are considered to be members of the same clone. MLST, unlike earlier molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), has high discriminative power, allows easy standardization of data acquisition and analysis across laboratories, and has a high degree of portability of the resulting sequence data (8). MLST was used to investigate the genotype of S. Typhi as early as in 2002 (9). Up to now, 51 S. Typhi strains have been recorded in the Salmonella enterica MLST Database (10) and classified into 8 STs (ST1, ST2, ST3, ST8, ST890, ST892, ST911, and ST980), but most of them belong to ST1 (15/51) and ST2 (29/51).One well accepted definition of procaryotic species, based on whole-genome DNA-DNA hybridization, is that it is an entity comprised of strains sharing a reassociation value of approximately 70% or greater (11). Genomic diversity and relatedness of closely related organisms has since recently been determined with microarrays, which have higher resolution than traditional DNA-DNA hybridization methods (12). Microarray-based comparative genomic hybridization (M-CGH) is in widespread use in relatedness determination of procaryotic species (13-15). CGH usually uses the whole genome open reading frame (ORF) array-based hybridization approach (16).Typhoid fever is endemic in developing countries, such as China. However, there are few reports of genotyping of S. Typhi in China (17). These reports mostly used PFGE, which is currently the method of choice for genotyping of sporadic or epidemic Salmonella isolates. S. typhi strains isolated from Shenzhen in China showed 22 distinct PFGE patterns with variable genetic diversity (17). We speculated that genetic diversity of S. Typhi strains may be largely present throughout the last several decades in China. Therefore, we collected S. Typhi strains from 1959 to 2006 and chose 40 strains representative in terms of genetic diversity isolated from 5 highly endemic Chinese provinces. The aim of this study was to identify the genotype of 40 representative S. Typhi strains by MLST and evaluate their genetic diversity by M-CGH.     

Enterotoxic, cytotoxic, necrotic and lethal activities in cell-free extracts of Salmonella strains isolated from humans

Unconcentrated cell-free sonic extracts from thirty Salmonella strains isolated from the faeces and blood of humans were investigated for the production of enterotoxins in various tests (Vero cell, infant mouse, rabbit skin permeability and rabbit ileal loop), as well as for lethal activity in adult mice. Sonic extracts from 23 (76.7%) strains were lethal for mice, 21 (70%) increased skin permeability and 3 (10%) showed necrotizing activity for the rabbit skin. No Salmonella strain producing typical Escherichia coli toxins, such as thermolabile (LT) or thermostable (STa) enterotoxins, Verotoxin (VT) or cytotoxic necrotizing factor (CNF) cytotoxins, were detected. Non-repetitive fluid accumulation in rabbit loops was obtained when unconcentrated sonic extracts from 10 selected strains were assayed in seven rabbits. Growth of Salmonella in casamino acid yeast extract medium, followed by treatment of bacterial cells with polymyxin B, was demonstrated to be a rapid and sensitive method for releasing the delayed permeability factor.     

Antimicrobial susceptibilities of salmonella strains isolated from humans,cattle, pigs,and chickens in the Netherlands from 1984 to 2001

We monitored antimicrobial susceptibility data for Salmonella strains isolated from humans, cattle, pigs, and chickens in The Netherlands from 1984 to 2001 in order to provide insight into the dynamics of resistance over time. The strains were tested for their susceptibilities to seven antimicrobial agents by the agar diffusion method. Resistance was most common in Salmonella enterica subsp. enterica serovar Typhimurium. Among the strains from humans, pigs, and chickens, it was found that the level of resistance of serovar Typhimurium strains to tetracycline, ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole increased from 1984 to 2001. This increase could be attributed to the emergence of multidrug-resistant serovar Typhimurium DT 104. Among the strains from cattle, it was found that the level of resistance of serovar Typhimurium strains, which was already very high in the 1980s, declined during the study period to the same levels as those for the strains from the other species from 1996 to 2001. Serovar Enteritidis isolates remained susceptible during the entire survey period. Among serovar Paratyphi B variation Java strains isolated from chickens, resistance to furazolidone, flumequine, trimethoprim-sulfamethoxazole, and ampicillin emerged, although furazolidone was not used after 1990. Together, the data indicate that the levels and patterns of resistance differed considerably between Salmonella serovars isolated from one host species.