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A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

Salmonella enterica , like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of Salmonella , namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the yfgL , engA , and yfgJ open reading frames in S. enterica serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in S. enterica serovar Enteritidis of yfgL , encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most Salmonella pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the Δ yfgL mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the Δ yfgL mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all Salmonella TTSS and, thus, in the virulence of Salmonella . Therefore, this outer membrane protein seems to be a privileged target for fighting Salmonella .

The susceptibility to beta -lactams and the beta -lactamase content of 110 Salmonella spp. blood isolates collected during 1997-98 in 19 European centers participating in the SENTRY Surveillance Program were studied. Thirty-one isolates (28%) were resistant to penicillins, due to production of TEM-1 (27 isolates), OXA-1 (three isolates) or TEM-1 + OXA-1 (one isolate). All OXA-1 producers and 10 TEM-1-producing isolates were also resistant to penicillin-clavulanic acid combinations. In the latter isolates, this phenotype was associated with increased production of TEM-1. Sixteen TEM-1-producing Salmonella Enteritidis isolates and one OXA-1-producing S. Typhimurium isolate were able to transfer beta -lactam resistance by conjugation.

In a matched cohort study, we determined the death rates associated with drug resistance in Salmonella Typhimurium. We linked data from the Danish Surveillance Registry for Enteric Pathogens with the Civil Registration System and the Danish National Discharge Registry. By survival analysis, the 2-year death rates were compared with a matched sample of the general Danish population, after the data were adjusted for differences in comorbidity. In 2,047 patients with S. Typhimurium, 59 deaths were identified. Patients with pansusceptible strains of S. Typhimurium were 2.3 times more likely to die 2 years after infection than persons in the general Danish population. Patients infected with strains resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline were 4.8 times (95% CI 2.2 to 10.2) more likely to die, whereas quinolone resistance was associated with a mortality rate 10.3 times higher than the general population.

To the Editor: Salmonella enterica serotype Typhimurium is one of the most common causes of bloodstream infection in sub-Saharan Africa (1). Among adults, the principal risk factor for invasive nontyphoidal Salmonella (iNTS) disease is advanced HIV infection; up to 44% of HIV-infected patients experience bacteremic recurrence through recrudescence of the original infection (2,3). Epidemics of iNTS disease in sub-Saharan Africa have been associated with a novel genotype of S. enterica ser. Typhimurium of multilocus sequence type (ST) 313 that is rarely seen outside the region and is associated with multidrug resistance (MDR) to chloramphenicol, cotrimoxazole, and ampicillin (4,5). As a consequence, ceftriaxone has become a key agent in the empirical management of nonfocal sepsis in Malawi (6). In March 2009, a 40-year-old HIV-infected and antiretroviral therapy–naive woman sought care in Blantyre, Malawi, with an MDR S. enterica ser. Typhimurium bloodstream infection. She was treated with ceftriaxone (2 g intravenously once daily) and discharged with oral ciprofloxacin (500 mg twice daily) for 10 days. She was readmitted 1 month later with recurrent fever. At this time, she had an MDR S. enterica ser. Typhimurium bloodstream infection with additional resistance to ceftriaxone and ciprofloxacin. In the absence of a locally available effective antimicrobial drug, she was treated with ceftriaxone, gentamicin, and high-dose ciprofloxacin but died shortly thereafter. To help clarify how this extended MDR S. enterica ser. Typhimurium emerged, we determined the molecular mechanisms underpinning this disturbing pattern of antimicrobial resistance (Technical Appendix). We conducted phenotypic drug susceptibility testing by disk diffusion on S. enterica ser. Typhimurium strains A54285 (initial presentation) and {"type":"entrez-protein","attrs":{"text":"A54560","term_id":"627945","term_text":"pir||A54560"}}A54560 (recurrence); both isolates were resistant to ampicillin, chloramphenicol, and cotrimoxazole, but {"type":"entrez-protein","attrs":{"text":"A54560","term_id":"627945","term_text":"pir||A54560"}}A54560 exhibited additional resistance to ceftriaxone, ciprofloxacin, and tetracycline. Paired-end sequencing of isolates A54285 (European Nucleotide Archive [ENA] accession number ERS035867) and {"type":"entrez-protein","attrs":{"text":"A54560","term_id":"627945","term_text":"pir||A54560"}}A54560 (ENA accession no. ERS035866) that were cultured 1 month apart showed no differences between the conserved regions of these genomes (Figure). The similarity of these S. enterica ser. Typhimurium genomes strongly suggests that this recrudescence occurred after incomplete clearance of the first infection; although re-infection from the same source is unlikely, it cannot be excluded. Comparison of the accessory genomes, however, showed an additional 300 kb DNA in {"type":"entrez-protein","attrs":{"text":"A54560","term_id":"627945","term_text":"pir||A54560"}}A54560. Figure Midpoint-rooted phylogenetic tree of published whole-genome sequence data from {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580-like Salmonella enterica serotype Typhimurium sequence type 313s from Malawi ... Plasmid extraction and gel electrophoresis of genomic DNA identified a plasmid migrating in the gel to a position approximately equivalent to 120 kb, the size of ST313 virulence plasmid pSLT-BT in both strains, but no 300-kb plasmid was visualized in the ceftriaxone- and ciprofloxacin-resistant strain ({"type":"entrez-protein","attrs":{"text":"A54560","term_id":"627945","term_text":"pir||A54560"}}A54560, data not shown), possibly because of the difficulty large plasmids have entering standard 1% agarose gels. However, ceftriaxone resistance was mobilized to Escherichia coli by conjugation at a frequency 6.5 × 10−2 transconjugants per donor at 26°C. This frequency dropped dramatically to ≈1 × 10−7 transconjugants per donor when conjugation was performed at 37°C. The presence of an IncHI2 plasmid in the transconjugants was confirmed by PCR for the IncHI2 region (7), and drug susceptibility testing confirmed that transconjugant clones acquired resistance to ceftriaxone, ciprofloxacin, and tetracycline. These data confirm the presence of an extended-spectrum β-lactamase (ESBL)–producing IncHI2 plasmid in strain {"type":"entrez-protein","attrs":{"text":"A54560","term_id":"627945","term_text":"pir||A54560"}}A54560 that is capable of conjugative transfer and suggest that the plasmid might have been acquired by residual index strain within the patient by transfer from an unknown donor bacterium. Partial decolonization of the patient’s gastrointestinal tract by ceftriaxone and fluoroquinolone antimicrobial therapy might have rendered it receptive to colonization by ESBL-producing bacteria, which we hypothesize donated the plasmid to the residual index strain. The transconjugant plasmid DNA was sequenced by using the PacBio RSII platform (Pacific Biosciences, Menlo Park, CA, USA; http://www.pacificbiosciences.com), which assembled as a single contiguous sequence of 309,406 bp, designated pSTm-BTCR (Technical Appendix Figure, ENA accession no. {"type":"entrez-nucleotide","attrs":{"text":"LK056646","term_id":"671685546","term_text":"LK056646"}}LK056646). We identified 331 predicted coding sequences, including 109 genes required for replication and transfer and 61 genes predicted to be associated with metabolism, membranes, virulence, antimicrobial resistance, and a toxin/antitoxin addiction system. We found an additional 160 predicted, hypothetical genes. Fifteen putative antimicrobial resistance genes were identified, predicted to encode resistance to; tetracycline (tetA(C), tetR(C)), β-lactams (blaCTX-M15, blaTEM-1b, blaOXA-30), chloramphenicol (catB3, catA1), aminoglycosides (strA, strB, aadA1, aacA4, aacC3), ciprofloxacin (qnrB1), ulfonamiides (sul2), and trimethoprim (dfrA14). In our experience, ESBL and fluoroquinolone-resistant iNTS remain extremely uncommon in Blantyre, Malawi. This is surprising because diverse ESBL genotypes were observed in other members of Enterobacteriaceae in Blantyre within a year after ceftriaxone came into common use locally (8). That IncHI2 plasmids transfer most efficiently at temperatures

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods.

A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

Whereas most nontyphoidal Salmonella (NTS) are associated with gastroenteritis, there has been a dramatic increase in reports of NTS-associated invasive disease in sub-Saharan Africa. Salmonella enterica serovar Typhimurium isolates are responsible for a significant proportion of the reported invasive NTS in this region. Multilocus sequence analysis of invasive S. Typhimurium from Malawi and Kenya identified a dominant type, designated ST313, which currently is rarely reported outside of Africa. Whole-genome sequencing of a multiple drug resistant (MDR) ST313 NTS isolate, D23580, identified a distinct prophage repertoire and a composite genetic element encoding MDR genes located on a virulence-associated plasmid. Further, there was evidence of genome degradation, including pseudogene formation and chromosomal deletions, when compared with other S. Typhimurium genome sequences. Some of this genome degradation involved genes previously implicated in virulence of S. Typhimurium or genes for which the orthologs in S. Typhi are either pseudogenes or are absent. Genome analysis of other epidemic ST313 isolates from Malawi and Kenya provided evidence for microevolution and clonal replacement in the field.

Background: Multidrug-resistant Salmonella serovar Concord infections have been reported from children adopted from Ethiopia. We interviewed patients, characterized the isolates, and gathered information about adoptions from Ethiopia to assess public health implications. Methods: Information about Salmonella Concord cases and adoptions were provided from Austria, Denmark, England (and Wales), Ireland, the Netherlands and the United States. Patients from Denmark and the United States were interviewed to determine the orphanages of origin; orphanages in Ethiopia were visited. Isolates were subtyped by pulsed-field gel electrophoresis and antimicrobial susceptibility; specific antimicrobial resistance genes were characterized. Results: Salmonella Concord was isolated from 78 persons from 2003 to 2007. Adoption status was known for 44 patients 2 indistinguishable isolates; one isolate from an Ethiopia adoptee. Antimicrobial susceptibility was per-formed on 43 isolates; 81% were multidrug-resistant (>= 3 agents). Multidrug-resistant isolates were from Ethiopian adoptees and were resistant to third and fourth generation cephalosporins and 14% had decreased susceptibility to ciprofloxacin. Conclusions: Improved hygiene and sanitation and more appropriate use of antimicrobial agents are needed in orphanages in Ethiopia. Culturing of stool specimens of children adopted from Ethiopia and appropriate hygiene may prevent further disease transmission.

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.

The susceptibility to beta -lactams and the beta -lactamase content of 110 Salmonella spp. blood isolates collected during 1997-98 in 19 European centers participating in the SENTRY Surveillance Program were studied. Thirty-one isolates (28%) were resistant to penicillins, due to production of TEM-1 (27 isolates), OXA-1 (three isolates) or TEM-1 + OXA-1 (one isolate). All OXA-1 producers and 10 TEM-1-producing isolates were also resistant to penicillin-clavulanic acid combinations. In the latter isolates, this phenotype was associated with increased production of TEM-1. Sixteen TEM-1-producing Salmonella Enteritidis isolates and one OXA-1-producing S. Typhimurium isolate were able to transfer beta -lactam resistance by conjugation.