Prevalence and characterization of extended-spectrum beta-lactamases-producing Salmonella enterica isolates in Saragossa, Spain (2001-2008)

We analyzed the prevalence of resistance to extended-spectrum cephalosporins (ESCs) among clinical strains of Salmonella enterica collected by the Laboratory of Clinical Microbiology in the University Clinical Hospital Lozano Blesa in the region of Aragón (Spain), for which very few epidemiological information exists. A total of 2,092 strains of S. enterica were identified in stool samples from patients with gastroenteritis. Five isolates showed an extended-spectrum beta-lactamase (ESBL) phenotype: four isolates of S. enterica serotype Virchow harbored the ESBL-encoding bla(CTX-M-9) gene and an isolate of serotype Enteritidis carried a bla(CTX-M-1) gene, which, to the best of our knowledge, is described here for the first time in this serotype of S. enterica. The five ESC-resistant isolates were also resistant to spectinomycin, streptomycin, kanamycin, sulfonamides, tetracycline, and trimethoprim as well as to nalidixic acid. The ESBL isolate of serotype Enteritidis, however, remained susceptible to kanamycin and nalidixic acid. A class 1 integron of 1.5?kb was detected for the four serotype Virchow isolates with the gene cassette dfrA16-aadA2. The bla(CTX-M-9) gene was carried by an ～300-kb IncHI2 conjugative plasmid in the case of the S. enterica serotype Virchow isolates. The bla(CTX-M-1) gene was carried by an ～100-kb IncI1-N conjugative plasmid for the serotype Enteritidis ESC-resistant isolate. All the four ESC-resistant strains of S. enterica serotype Virchow clustered together in a XbaI pulsed-field gel electrophoresis, which also revealed a strong similarity between them and some pulsotypes of S. enterica serotype Virchow from France.     

Stabilization of a plasmid coding for a heterologous antigen in Salmonella enterica serotype typhi vaccine strain CVD908-htrA by using site-specific recombination

A gene cassette incorporating the crs-rsd site-specific recombination system from the Salmonella enterica subsp. enterica serovar Dublin virulence plasmid improved the inheritance in S. enterica serotype Typhi strain CVD908-htrA of a multicopy plasmid expression vector. Use of this recombination cassette may improve expression of heterologous antigens from multicopy plasmid expression vectors in attenuated bacterial vaccine strains.     

Molecular methods for the epidemiological typing of Salmonella enterica serotype Typhi from Hong Kong and Vietnam

A total of 217 and 73 strains of Salmonella enterica serotype Typhi isolated from 1985 to 1997 in Hong Kong and in 2 months of 1989 and 1990 in Vietnam, respectively, were studied. These isolates were typed by plasmid profile analysis, plasmid fingerprinting, ribotyping with PstI, and total DNA fingerprinting with NarI. There appeared to be no major outbreak of typhoid fever in Hong Kong during the study period since there was considerable heterogeneity among the isolates. Isolates from Hong Kong were different from those from Vietnam. Thirty-seven percent of Vietnamese isolates belonged to two predominant clones, with the rest being heterogeneous in nature. Total DNA fingerprinting supplemented with ribotyping could be a reliable and rapid method for epidemiological typing of S. enterica serotype Typhi.     

Characterization of the first extended-spectrum beta-lactamase-producing Salmonella isolate identified in Canada

A single Salmonella enterica serovar Typhimurium isolate with an UT2 phage type producing an extended-spectrum beta-lactamase (ESBL) was identified in Canada in 2000. The isolate harbored two plasmids, one containing a bla(TEM-1) gene and the other containing a bla(SHV-2a) gene. The ESBL gene was located on a 70-kb transferable plasmid which also carried tetracycline and trimethoprim resistance elements.     

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.     

Occurrence of extended-spectrum β-lactamase-producing Salmonella enterica in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans

Among the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum β-lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a bla _SHV-2 gene. The bla _CTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the bla _CTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other bla _CTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals.     

一株不典型山夫登堡沙门氏菌的鉴定及其16SrRNA序列分析

目的对一株从广州市食品从业人员肛拭子中分离的蔗糖发酵型沙门氏菌进行鉴定。方法应用培养特性试验、生化分析、血清型鉴定,及16S rRNA序列分析进行鉴定。结果该菌培养特性及生化特性符合沙门氏菌定义,但与普通沙门氏菌在蔗糖利用、硫化氢产生上存在差异。血清型鉴定为山夫登堡沙门氏菌（Salmonella enterica Subsp·enterica serovar Senftenberg）,16S rRNA序列分析为肠道沙门氏菌亚种（Salmonella enterica subsp）。结论该菌为一株蔗糖发酵型、硫化氢阴性的不典型山夫登堡沙门氏菌。     

Profile of Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:- strains causing food-borne infections in New York City

Strains of newly emerging Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:- causing food-borne infections, including a large food poisoning outbreak (n = 86) characterized by persistent diarrhea (14% bloody), abdominal pain, fever, and headache, were examined. The organisms were found in the stool samples from the patients. The biochemical profile of the organisms is consistent with that of S. enterica subsp. I serotypes, except for decreased dulcitol (13%) and increased inositol (96%) utilization. Twenty-eight percent of the strains showed resistance to streptomycin, sulfonamides, or tetracycline only; all three antimicrobial agents; or these agents either alone or in combination with ampicillin, trimethoprim, and trimethoprim-sulfamethoxazole. None of the serotype 4,5,12:i:- strains showed resistance or decreased susceptibility to chloramphenicol or ciprofloxacin. On pulsed-field gel electrophoresis (PFGE), the strains showed 11 or 12 resolvable genomic fragments with 18 banding patterns and three PFGE profile (PFP) clusters (i.e., PFP/A, PFP/B, and PFP/C). Seventy-five percent of the isolates fingerprinted were closely related (zero to three band differences; similarity [Dice] coefficient, 86 to 100%); 63% of these were indistinguishable from each other (PFP/A(1)). PFP/A(1) was common to all strains from the outbreak and 11 hospital sources. Strains from six other hospitals shared clusters PFP/B and PFP/C. PFP/C(4), of the environmental isolate, was unrelated to PFP/A and PFP/B. Nine band differences (similarity coefficient, 61%) were noted between PFP/A(1) and PFP/E of the multidrug-resistant S. enterica subsp. enterica serotype Typhimurium definitive type 104 strains. Whether these emerging Salmonella strains represent a monophasic, Dul(-) variant of serotype Typhimurium or S. enterica subsp. enterica serotype Lagos or a distinct serotype of S. enterica subsp. I is not yet known. Some of the phenotypic and genotypic properties of the serotype 4,5,12:i:- strains are described here.     

Use of molecular subtyping in surveillance for Salmonella enterica serotype typhimurium

BACKGROUND: Because Salmonella enterica serotype typhimurium is the most common serotype isolated from persons with salmonellosis in the United States, it is difficult to detect unusual clusters or outbreaks. To determine whether molecular subtyping could be useful in public health surveillance for S. enterica serotype typhimurium, the Minnesota Department of Health initiated the routine use of pulsed-field gel electrophoresis (PFGE) of isolates. METHODS: Beginning in 1994, all S. enterica serotype typhimurium isolates submitted by clinical laboratories to the Department of Health were subtyped by PFGE. A standard questionnaire was used to interview patients about possible sources of infection. RESULTS: From 1994 through 1998, 998 cases of infection with S. enterica serotype typhimurium were reported to the Minnesota Department of Health (4.4 cases per 100,000 person-years). PFGE was performed on 958 of the isolates (96 percent), and 174 different patterns were identified. Sixteen outbreaks with a common source were identified, accounting for 154 cases. PFGE subtyping made it possible to confirm 10 outbreaks that involved small numbers of cases in institutional settings. Of six larger, community-based outbreaks, four would probably not have been recognized without PFGE subtyping. These four outbreaks accounted for 96 of the 154 culture-confirmed outbreak cases (62 percent). Fifty-six of 209 isolates tested for antimicrobial susceptibility (27 percent) were resistant to at least five antimicrobial agents. The multidrug-resistant isolates identified had unique PFGE patterns. CONCLUSIONS: Routine molecular subtyping of S. enterica serotype typhimurium by PFGE can improve the detection of outbreaks and aid in the identification of multidrug-resistant strains. Combining routine molecular subtyping with a method of rapid communication among public health authorities can improve surveillance for S. enterica serotype typhimurium infections.     

Characterization of Salmonella enterica serotype Typhimurium isolates from human, food, and animal sources in the Republic of Ireland

A potential epidemic clone of Salmonella enterica serotype Typhimurium DT104, and the possible emergence of S. enterica serotype Typhimurium DT104b, has been identified from the characterization of 67 S. enterica serotype Typhimurium strains from three sources, human gastroenteritis isolates, isolates from food samples, and veterinary isolates, by antimicrobial resistance profiling, phage typing, and pulsed-field gel electrophoresis. Resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline was found in 77.6% of these strains.     

Genetic evolution of the Spanish multidrug-resistant Salmonella enterica 4,5,12:i:- monophasic variant

We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpson's index of diversity [SID]=0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID=0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS26. We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS26 at plasmid pU302L, could be the ancestor of the variant in Spain.     

Typhoid fever associated with acute appendicitis caused by an H1-j strain of Salmonella enterica serotype Typhi

While most strains of Salmonella enterica serotype Typhi, the etiologic agent of typhoid fever, have only a phase 1 flagellar antigen, H1-d, variations of the flagellar antigen have been observed. Although H1-j strains (one of the flagellar antigen variants) account for 10 to 50% of S. enterica serotype Typhi strains found in Indonesia, there have been no published data to suggest its existence in other parts of the world. We describe a case of typhoid fever associated with acute appendicitis caused by an S. enterica serotype Typhi H1-j strain in a Chinese woman in Hong Kong. A gram-negative, motile rod was recovered from her blood and stool cultures. Conventional biochemical tests and the Vitek system (GNI+) showed that the bacterium was S. enterica serotype Typhi. The isolate agglutinated with poly(O), 9O, Vi and H1-j Salmonella antisera but not with poly(H) antisera. The patient developed antibodies against only S. enterica serotype Typhi O antigens but not against H1-d antigen by the Widal test. Flagellin C gene (fliC) sequencing showed a 261-bp deletion in the fliC gene of the isolate, confirming that the isolate possessed the H1-j antigen. The patient had no past history of travel to Indonesia or personal contact with any Indonesian. She recovered with appendectomy and antibiotic treatment. Further studies should be performed to determine the prevalence of this unusual S. enterica serotype Typhi strain in our locality.     

Analysis of molecular epidemiology of Chilean Salmonella enterica serotype enteritidis isolates by pulsed-field gel electrophoresis and bacteriophage typing

Human Salmonella enterica serotype Enteritidis infections emerged in Chile in 1994. S. enterica serotype Enteritidis phage type 1 isolates predominated in the north, and phage type 4 isolates predominated in the central and southern regions. A study was planned to characterize this epidemic using the best discriminatory typing technique. Research involved 441 S. enterica serotype Enteritidis isolates, including clinical preepidemic samples (n = 74; 1975 to 1993) and epidemic (n = 199), food (n = 72), poultry (n = 57), and some Latin American (n = 39) isolates. The best method was selected based on a sample of preepidemic isolates, analyzing the discriminatory power (DP) obtained by phage typing and randomly amplified polymorphic DNA and pulsed-field gel electophoresis (PFGE) analysis. The highest DP was associated with BlnI PFGE-bacteriophage typing analysis (0.993). A total of 38 BlnI patterns (B patterns) were identified before the epidemic period, 19 since 1994, and only 4 in both periods. Two major clusters were identified by phylogenetic analysis, and the predominant B patterns clustered in the same branch. Combined analysis revealed that specific B pattern-phage type combinations (subtypes) disappeared before 1994, that different genotypes associated with S. enterica serotype Enteritidis phage type 4 had been observed since 1988, and that strain diversity increased before the expansion of S. enterica serotype Enteritidis in 1994. Predominant subtype B3-phage type 4 was associated with the central and southern regions, and subtype B38-phage type 1 was associated with the north (P < 0.0001). Food and poultry isolates matched the predominant S. enterica serotype Enteritidis subtypes, but isolates identified in neighboring countries (Peru and Bolivia) did not match S. enterica serotype Enteritidis subtypes identified in the north of Chile. The results of this work demonstrate that genetic diversity, replacement, and expansion of specific S. enterica serotype Enteritidis subtypes were associated with epidemic changes.     

Nosocomial outbreak caused by Salmonella enterica serotype Livingstone producing CTX-M-27 extended-spectrum beta-lactamase in a neonatal unit in Sousse, Tunisia

In this study, we report an outbreak of Salmonella enterica serotype Livingstone resistant to extended-spectrum cephalosporins that occurred in a neonatal ward of the maternity department of Farhat Hached Hospital, Sousse, Tunisia, in 2002. A total of 16 isolates were recovered from 16 babies hospitalized in the ward during the period 1 to 16 July. All these babies developed diarrhea, and three of them developed septicemia. All the isolates demonstrated resistance to ceftriaxone and ceftazidime due to the production of an extended-spectrum beta-lactamase (ESBL). The isolates were also resistant to aminoglycosides (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin) and sulfamethoxazole-trimethoprim. DNA profiles were determined by pulsed-field gel electrophoresis using the XbaI and SpeI endonucleases and by ribotyping with PstI digestion. They yielded the same patterns, showing that the outbreak was caused by a single clone. The ESBL was identified as CTX-M-27 by sequencing of PCR products and by isoelectric focusing. The ESBL resistance was transferred by a 40-kb conjugative plasmid. The mobile insertion sequence ISEcp1 was found to be located upstream of bla(CTX-M-27) in the same position as that known for a bla(CTX-M-14) sequence. A new gene named dfrA21, encoding resistance to trimethoprim and carried by a 90-kb plasmid, was characterized. The dfrA21 gene was inserted as a single resistance cassette in a class I integron. The babies were treated with colistin, and all except two recovered. The outbreak came to an end when appropriate actions were taken: patient isolation, hand washing, and disinfection of the ward.     

Hatchery-borne Salmonella enterica serovar Tennessee infections in broilers.

A substantial increase in the prevalence of 5. enterica serovar Tennessee was observed in broiler flocks in Denmark at the turn of the year 1994 and in the following months. Epidemiological data indicated that a single hatchery was involved in spreading of the infection. Molecular characterization of S.enterica serovar Tennessee isolates from Danish broilers (1992 to 1995), the suspected hatchery and strains from various other sources included for comparison was initiated in order to trace the source of infection of the broilers. In general, strains of S.enterica ser. Tennessee showed only minor genotypic variation. Three different ribotypes were demonstrated when EcoRI was used for digestion of DNA. Two types were obtained by the use of HindIII. Nine different plasmids and seven different plasmid profiles were demonstrated. A 180 kb plasmid was, however, only demonstrated in isolates from broilers and the hatchery. Sixty-nine per cent of the broiler isolates obtained during the period 1992 to 1995 harboured this plasmid and 88% of the hatchery isolates contained a plasmid of the same size. An increased number of the broiler isolates (79%) contained this plasmid at the turn of 1994. Restriction enzyme analysis of the plasmid ensured that the plasmids from broilers and the hatchery were identical. By analysis of cleaning and disinfection procedures and by sampling of different control points in the hatchery it was shown that S.enterica ser. Tennessee had colonized areas of the hatchers which were protected from routine cleaning and disinfection. Subsequent inclusion of these areas into the sanitation programme resulted in the elimination of S. enterica ser. Tennessee from the hatchers, and a decreasing prevalence of S.enterica ser. Tennessee was observed in broiler flocks during the following months.     

Epidemic typhoid in vietnam: molecular typing of multiple-antibiotic-resistant Salmonella enterica serotype typhi from four outbreaks

Multidrug-resistant Salmonella enterica serotype Typhi isolates from four outbreaks of typhoid fever in southern Vietnam between 1993 and 1997 were compared. Pulsed-field gel electrophoresis, bacteriophage and plasmid typing, and antibiotic susceptibilities showed that independent outbreaks of multidrug-resistant typhoid fever in southern Vietnam are caused by single bacterial strains. However, different outbreaks do not derive from the clonal expansion of a single multidrug-resistant serotype Typhi strain.     

Analysis of Salmonella enterica serotype-host specificity in calves: avirulence of S. enterica serotype gallinarum correlates with bacterial dissemination from mesenteric lymph nodes and persistence in vivo

Host and bacterial factors that determine whether Salmonella serotypes remain restricted to the gastrointestinal tract or penetrate beyond the mucosa and cause systemic disease remain largely undefined. Here, factors influencing Salmonella host specificity in calves were assessed by characterizing the pathogenesis of different serotypes. Salmonella enterica serotype Dublin was highly virulent intravenously, whereas S. enterica serotype Choleraesuis was moderately virulent. Both serotypes were virulent in calves infected orally. In contrast, S. enterica serotypes Gallinarum and Abortusovis were avirulent by either route. Serotypes Dublin, Gallinarum, and Abortusovis colonized the intestinal tract 24 h after oral inoculation, yet only serotype Dublin was consistently recovered from systemic tissues. Serotypes Dublin and Gallinarum invaded bovine intestines in greater numbers and induced greater enteropathogenic responses than serotypes Choleraesuis and Abortusovis. However, only serotype Dublin was able to persist within the intestinal mucosa, and use of a novel cannulation model demonstrated that serotype Dublin was able to pass through the mesenteric lymph nodes in greater numbers than serotype Gallinarum. Together, these results suggest that initial interactions with the intestinal mucosa do not correlate with host specificity, although persistence within tissues and translocation via efferent lymphatics appear to be crucial for the induction of bovine salmonellosis.     

Evaluation of a modified single-enzyme amplified fragment length polymorphism (SE-AFLP) technique for subtyping Salmonella enterica serotype Enteritidis

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). Pulsed field gel electrophoresis (PFGE) is generally acknowledged to be the most discriminating typing method for Salmonella, but only a restricted variety of PFGE types has been described for S. enterica serotype Enteritidis. In the present study, a modification of the SE-AFLP typing method was used to investigate both outbreak and apparently sporadic isolates of S. enterica serotype Enteritidis belonging to different PTs and/or PFGE types. The method proved to be as discriminatory as PFGE when combined with phage typing, and provided subtyping data consistent with epidemiological information. Although the modified SE-AFLP typing method did not prove to achieve a superior discriminatory ability in resolving clusters, it has a high enough throughput for use in outbreak investigations. This method can be used in combination with other typing methods to obtain epidemiologically relevant subtyping data on S. enterica serotype Enteritidis.     

Use of biochemical kinetic data to determine strain relatedness among Salmonella enterica subsp. enterica isolates

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:-:- strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of > or =95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.