Typhimurium Isolates Research Articles

Plasmid-borne colistin resistance gene mcr-1 in a multidrug resistant Salmonella enterica serovar Typhimurium isolate from an infant with acute diarrhea in China

BackgroundAntimicrobial resistance of Salmonella enterica is a major global concern. Recent findings suggest that colistin as a last resort treatment for multidrug-resistant gram-negative bacteria is seriously threatened by the report of plasmid-mediated colistin resistance gene mcr-1 in China. MethodsA total of 827 S. Typhimurium isolates were recovered from 4 cities of China, including Henan, Shanghai, Zhejiang, and Hubei provinces. Subsequently, mcr-1 presence was identified by PCR screening. Antimicrobial susceptibility testing was performed by broth microdilution using a 96-well microtiter plate. Plasmid conjugation transfer experiments were conducted using Escherichia coli J53 as the recipient. ResultsOnly one mcr-1 positive strain from the stool sample of an infant with acute diarrhea was isolated. Apart from colistin, the mcr-1-positive isolate showed co-resistance to the third-generation cephalosporins, ampicillin, nalidixic acid, tetracycline, chloramphenicol, sulfisoxazole, gentamicin, and cefotaxime revealing a multidrug-resistant phenotype. This strain harbored mcr-1 on a 227 kb IncHI2 plasmid, termed pJZ26, which could be transferred to E. coli J53. In addition to mcr-1, pJZ26 coharbored other resistance genes, including aph(4)-Ia, aac(3)-IVa, fosA, floR, sul2, and blaCTX-M-14. Compared with p2474-MCR1 and pHYEC7-IncHI2, pJZ26 contains an additional 4.6 kb fragment harboring the resistance gene tet(A) and its regulator tetR located on TnAs1 transposable element, which could mediate resistance to tetracycline. ConclusionsThese findings highlight that the fact the mcr-1–harboring plasmid pJZ26 has a high potential to disseminate the mcr-1 gene and further challenge the clinical treatment.

Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources.

Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species.

Antimicrobial resistance and related gene analysis of Salmonella from egg and chicken sources by whole-genome sequencing

Whole-genome sequencing (WGS) is a valuable tool in research on foodborne pathogens. In this study, a total of 143 isolates of Salmonella serotypes Enteritidis, Typhimurium, and Heidelberg sourced from eggs and chickens were analyzed for their antimicrobial resistance profiles using WGS data. The isolates carried high rate of genes resistant to aminoglycoside (70.63%), tetracycline (26.57%), fosfomycin (25.17%), sulfonamides (23.78%), and β-lactamases (15.38%); and aadA was the most frequently observed antimicrobial resistance gene (ARG). Antimicrobial resistance varies by Salmonella serotypes, with Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis) isolates being highly resistant to aminoglycoside (particularly streptomycin); Salmonella ser. Typhimurium more resistant to aminoglycoside, tetracycline, and sulfonamides; and Salmonella ser. Heidelberg more resistant to aminoglycoside and fosfomycin. Salmonella ser. Typhimurium isolates presented more varieties of ARG than Salmonella ser. Enteritidis and Salmonella ser. Heidelberg. Our data showed that 5 isolates of Salmonella ser. Typhimurium and Salmonella ser. Heidelberg contained ARG resistant to ≥ 5 antimicrobials. In addition, 23 Salmonella isolates carried ARG resistant to 4 antimicrobials.

MLST based serotype prediction for the accurate identification of non typhoidal Salmonella serovars.

Traditional serotyping based on the phenotypic variation of O- and H-antigen remains as the gold-standard for the identification and classification of Salmonella isolates for last 70years. Although this classification is a globally recognized nomenclature, huge diversity of Salmonella serotypes have made the serovar identification to be very complex. Seven gene multilocus sequence typing (MLST) on the other hand can provide serovar prediction as well as the evolutionary origin between the serovars. In this study non typhoidal Salmonella (NTS) strains (n = 45) isolated from clinical samples (blood, faeces and pus) were identified by traditional phenotypic serotyping and biochemical testing. All the tested Salmonella isolates were designated as serovar Typhimurium based on phenotyping. However, by MLST 60% (27/45) of the isolates were S. Typhimurium, 35.5% (16/45) were S. Agona (ST13), 2.2% (1/45) were S. Kentucky (ST198) and 2.2% (1/45) were S. Saintpaul (ST27). MLST analysis assigned S. Typhimurium isolates as ST36 (18/127), ST19 (7/27) and ST313 (2/27). Mismatches in serovar designation between MLST database and phenotypic serotyping can be due to the misinterpretation of phenotypic serotyping as the antigenic structures of S. Typhimurium, S. Agona differs by a surface antigen. MLST based phylogeny of study isolates showed clustering according to sequence types. Concordance between MLST based sequence type and phenotypic serotype is important to provide insights into genetic population structure of Salmonella.

Quinolone resistance among Salmonella Kentucky and Typhimurium isolates in Tunisia: first report of Salmonella Typhimurium ST34 in Africa and qnrB19 in Tunisia.

Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness. A total of 1404 isolates of S. Kentucky (n=1059)/S. Typhimurium (n=345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes. According to our selection criteria (NAL=resistance phenotype; CIP=resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type. Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia. This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia.

Anti-quorum sensing and antibacterial activities of Satureja sahendica hydroalcoholic extract against avian isolate of Salmonella Typhimurium

Quorum sensing (QS) is a cell density dependent mechanism used by many pathogenic bacteria for regulating virulence gene expression. Inhibition or interruption of QS by herbal remedies has been suggested as a new strategy for fighting against antibiotic resistant bacteria. The aim of this study was to evaluate the antibacterial activity of Satureja sahendica hydroalcoholic extract (SSHE) against Salmonella Typhimurium (S. Typhimurium) isolates, and to assess the impact of sub-inhibitory concentrations of this extract on the QS-associated gene (sdiA) expression. Using the Soxhlet method, hydroalcoholic extract of S. sahendica leaves was prepared and antimicrobial effects of the SSHE were determined by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methodS. The reverse transcription quantitative PCR (RT-qPCR) assay was used to analyze the expression of sdiA in 20 S. Typhimurium isolates from poultry flocks in response to the treatment of sub-inhibitory concentrations of SSHE at 60-min time point. The MIC values of SSHE against S. Typhimurium isolates were ranged from 0.29-4.68 mg/ml-1 and MBC values were ranging from 75-150 mg/ml-1. The results also indicated that the expression of sdiA gene was reduced in S. Typhimurium isolates triggered by the treatment of SSHE comparatively with those from the control cultureS. Findings suggest that SSHE possess the antibacterial and anti-QS activity and can be used to control the expression of virulence genes in pathogenic bacteria, such as S. Typhimurium.

Characterization of Salmonella spp. isolated from chickens in Central China

BackgroundSalmonella is an important zoonotic pathogen, and chickens are one of its main hosts. Every year, Salmonella infections pose a serious threat to the poultry industry in developing countries, especially China. In this study, a total of 84 Salmonella isolates recovered from sick and healthy-looking chickens in central China were characterized by serotyping, MLST-based strain typing, presence of potential virulence factors, and antimicrobial resistance profiles.ResultData showed that the main serotypes of Salmonella isolates in central China were Salmonella enterica serovar Gallinarum biovar Pullorum, Salmonella enterica serovar Gallinarum biovar Gallinarum, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, and among them, S. Pullorum was the dominant type in both sick and healthy-looking chickens, accounting for 43.9 and 46.5%, respectively, while S. Enteritidis was only found in healthy-looking chickens. All isolates exhibited higher resistance rates to ampicillin (97.6%), tetracycline (58.3%) and colistin (51.2%), and among these isolates, 49.5% were resistant to more than three drugs in different combinations. S. Enteritidis was the most severe multidrug-resistant serotype, which showed higher resistance rates to colistin, meropenem and ciprofloxacin. Multilocus sequence typing (MLST) revealed that S. Gallinarum and S. Enteritidis isolates were clustered in clade 1, which belonged to two and one STs, respectively. All S. Typhimurium isolates were clustered in clade 3, and belonged to three STs. However, S. Pullorum were distributed in three clades, which belonged to 7 STs. Twenty-seven virulence-associated genes were detected, and expected cdtB, which was absent in all the isolates, the other 26 genes were conserved in the closely related Salmonella serogroup D (S. Enteritidis, S. Pullorum, and S. Gallinarum).ConclusionSalmonella serogroup D was the major subgroup, and S. Pullorum was the most common type in sick and healthy-looking chickens in central China. Drug resistance assays showed serious multiple antimicrobial resistances, and S. Enteritidis was the most severe drug-resistant serotype. MLST showed that there was correlation between serotypes and genotypes in most Salmonella isolates, except S. Pullorum, which showed complicated genetic diversity firstly. These results provide important epidemiological information for us to control Salmonella in chickens.

High relatedness of invasive multi-drug resistant non-typhoidal Salmonella genotypes among patients and asymptomatic carriers in endemic informal settlements in Kenya.

Invasive Non-typhoidal Salmonella (iNTS) disease is a major public health challenge, especially in Sub-Saharan Africa (SSA). In Kenya, mortality rates are high (20–25%) unless prompt treatment is instituted. The most common serotypes are Salmonella enterica serotype Typhimurium (S. Typhimurium) and Salmonella enterica serotype Enteritidis (S. Enteritidis). In a 5 year case-control study in children residing in the Mukuru informal settlement in Nairobi, Kenya, a total of 4201 blood cultures from suspected iNTS cases and 6326 fecal samples from age-matched controls were studied. From the laboratory cultures we obtained a total of 133 S. Typhimurium isolates of which 83(62.4%) came from cases (53 blood and 30 fecal) and 50(37.6%) from controls (fecal). A total of 120 S. Enteritidis consisted of 70(58.3%) from cases (43 blood and 27 fecal) and 50(41.7%) from controls (fecal).The S. Typhimurium population fell into two distinct ST19 lineages constituting 36.1%, as well as ST313 lineage I (27.8%) and ST313 lineage II (36.1%) isolates. The S. Enteritidis isolates fell into the global epidemic lineage (46.6%), the Central/Eastern African lineage (30.5%), a novel Kenyan-specific lineage (12.2%) and a phylogenetically outlier lineage (10.7%). Detailed phylogenetic analysis revealed a high level of relatedness between NTS from blood and stool originating from cases and controls, indicating a common source pool. Multidrug resistance was common throughout, with 8.5% of such isolates resistant to extended spectrum beta lactams. The high rate of asymptomatic carriage in the population is a concern for transmission to vulnerable individuals and this group could be targeted for vaccination if an iNTS vaccine becomes available.

The phenotypic and molecular characteristics of antimicrobial resistance of Salmonella enterica subsp. enterica serovar Typhimurium in Henan Province, China

BackgroundSalmonella enterica subsp. enterica serovar Typhimurium infections continue to be a significant public health threat worldwide. The aim of this study was to investigate antibiotic resistance among 147 S. Typhimurium isolates collected from patients in Henan, China from 2006 to 2015.Methods147 S. Typhimurium isolates were collected from March 2006 to November 2015 in Henan Province, China. Antimicrobial susceptibility testing was performed, and the resistant genes of ciprofloxacin, cephalosporins (ceftriaxone and cefoxitin) and azithromycin were detected and sequenced. Clonal relationships were assessed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE).ResultsOf the 147 isolates, 91.1% were multidrug resistant (MDR), with 4.1% being resistant to all antibiotic classes tested. Of concern, 13 MDR isolates were co-resistant to the first-line treatments cephalosporins and ciprofloxacin, while three were also resistant to azithromycin. Seven PFGE patterns were identified among the 13 isolates. All of the isolates could be assigned to one of four main groups, with a similarity value of 89%. MLST assigned the 147 isolates into five STs, including two dominant STs (ST19 and ST34). Of the 43 ciprofloxacin-resistant isolates, 39 carried double gyrA mutations (Ser83Phe, Asp87Asn/Tyr/Gly) and a single parC (Ser80Arg) mutation, including 1 isolate with four mutations (gyrA: Ser83Phe, Asp87Gly; parC: Ser80Arg; parE: Ser458Pro). In addition, 12 isolates not only carried mutations in gyrA and parC but also had at least one plasmid-mediated quinolone resistance (PMQR) gene. Among the 32 cephalosporin-resistant isolates, the most common extended-spectrum β-lactamase (ESBL) gene was blaOXA-1, followed by blaCTX-M, blaTEM-1, and blaCMY-2. Moreover, the mphA gene was identified in 5 of the 15 azithromycin-resistant isolates. Four MDR isolates contained ESBL and PMQR genes, and one of them also carried mphA in addition.ConclusionThe high level of antibiotic resistance observed in S. Typhimurium poses a great danger to public health, so continuous surveillance of changes in antibiotic resistance is necessary.

Prevalence, serotypes, and antimicrobial resistance of Salmonella isolates from patients with diarrhea in Shenzhen, China

BackgroundSalmonella is one of the main causative agents of diarrhea which results in substantial disease burden. To determine the prevalence, serotype distribution, and antimicrobial resistance profiles of clinical Salmonella isolates in Shenzhen, a 6-year surveillance study was conducted.ResultsA total of 297 (5.7%) Salmonella strains were isolated from stool samples from 5239 patients. Among the 42 serotypes identified, serotype Typhimurium was the most common one which represented 39.7% of the isolates (118), followed by serotype Enteritidis (71, 23.9%), London (12, 4.0%), 4, 5, 12: i: - (11, 3.7%), and Senftenberg (8, 2.7%). A high frequency of resistance was found in ampicillin (70.6%), piperacillin (64.5%), tetracycline (63.5%), and streptomycin (54.3%). Resistance to ampicillin and tetracycline was observed in 95.3% of S. Typhimurium isolates; and nalidixic acid in 93.1% of S. Enteritidis isolates. Resistance to 5 or more antimicrobial agents was found in 78.8% of S. Typhimurium and 69.0% of S. Enteritidis isolates. A decreased susceptibility to ciprofloxacin and levofloxacin was associated with amino acid alteration in gyrA gene. Point mutations without amino acid changes were seen in gyrB, parC, and parE genes.ConclusionsA broad range of serotypes are responsible for Salmonellosis in Shenzhen, with Enteritidis and Typhimurium being the most common serotypes. The high level of antibiotic resistance is of public health significance and ongoing monitoring combined with rational use of antibiotics are recommended. Point mutations in gyrA gene might play an important role in the resistance to fluoroquinolones.

High genetic similarity between non-typhoidal Salmonella isolated from paired blood and stool samples of children in the Democratic Republic of the Congo.

BackgroundNon-typhoidal Salmonella (NTS) serotypes Typhimurium and Enteritidis are a major cause of bloodstream infections in children in sub-Saharan Africa but their reservoir is unknown. We compared pairs of NTS blood and stool isolates (with the same NTS serotype recovered in the same patient) for genetic similarity.MethodsBetween November 2013 and April 2017, hospital-admitted children (29 days to 14 years) with culture-confirmed NTS bloodstream infections were enrolled in a cross-sectional study at Kisantu Hospital, DR Congo. Stool cultures for Salmonella were performed on a subset of enrolled children, as well as on a control group of non-febrile hospital-admitted children. Pairs of blood and stool NTS isolates were assessed for genetic similarity by multiple-locus variable-number of tandem repeats (MLVA) and genomics analysis.ResultsA total of 299 children with NTS grown from blood cultures (Typhimurium 68.6%, Enteritidis 30.4%, other NTS 1.0%) had a stool sample processed; in 105 (35.1%) of them NTS was detected (Typhimurium 70.5%, Enteritidis 25.7%, other NTS 3.8%). A total of 87/105 (82.9%) pairs of blood and stool NTS isolates were observed (representing 29.1% of the 299 children). Among 1598 controls, the proportion of NTS stool excretion was 2.1% (p < 0.0001). MLVA types among paired isolates were identical in 82/87 (94.3%) pairs (27.4% of the 299 children; 61/66 (92.4%) in Typhimurium and 21/21 (100%) in Enteritidis pairs). Genomics analysis confirmed high genetic similarity within 41/43 (95.3%) pairs, showing a median SNP difference of 1 (range 0–77) and 1 (range 0–4) for Typhimurium and Enteritidis pairs respectively. Typhimurium and Enteritidis isolates belonged to sequence types ST313 lineage II and ST11 respectively.ConclusionNearly 30% of children with NTS bloodstream infection showed stool excretion of an NTS isolate with high genetic similarity, adding to the evidence of humans as a potential reservoir for NTS.

Molecular Relatedness of Salmonella enterica Typhimurium Isolates from Feces and an Infected Surgical Wound.

PurposeSalmonella enterica serovar Typhimurium infection is common in foodborne diseases, but its isolation from surgical incisions is rare. Our aim in this study was to trace the transmission source of a surgical incision infected with S. Typhimurium in a Yunnan Province hospital patient and elucidate the underlying molecular mechanisms of antibiotic resistance.MethodsPrimers were designed to amplify the drug-resistance genes using polymerase chain reaction (PCR). Susceptibility to antibiotics was determined using Etest strips. Macrorestriction profiles were analyzed using pulsed-field gel electrophoresis (PFGE) and XbaI. The two isolates were characterized using agglutination tests and multilocus sequence typing (MLST).ResultsMLST analysis revealed that S. Typhimurium isolates SM043 and SM080 belonged to the same genotype, ST34, and PFGE revealed that SM043 and SM080 had high similarity. The isolates were both resistant to third-generation cephalosporins. SM043 harbored the antibiotic resistance genes blaCTX-M-15, blaTEM-1, qnrS-1, qnrB, and acc-3, whereas blaCTX-M-15, blaTEM-1, blaCMY-2, qnrS-1, and acc-3 were detected in SM080.ConclusionThe surgical incision infection by S. Typhimurium may have been hospital-acquired. Thus, it is critical to strengthen hospital sanitation by addressing hand hygiene and sterilization of the operational environment to avoid outbreaks of nosocomial Salmonella infections.

Combined effects of Allium sativum and Cuminum cyminum essential oils on planktonic and biofilm forms of Salmonella typhimurium isolates.

Sa lmonella typhimurium (S. typhimurium) represents an important global public health problem and has the ability to survive under desiccation conditions in foods and food processing facilities for years. The aim of this study was to investigate the effects of Allium sativum (A. sativum) and Cuminum cyminum (C. cyminum) essential oils (EOs) against planktonic growth, biofilm formation and quorum sensing (QS) of S. Typhimurium isolates, the strong biofilm producers. The major components of EOs were determined by gas chromatography-mass spectrometry (GC-MS). Biofilm formation of S. Typhimurium isolates was measured by crystal violet staining. Then, the effects of the EOs on the planktonic cell growth (using determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)), measurement of the synergistic effects of EOs (using checkerboard method), biofilm formation (using microtiter-plate test and scanning electron microscope (SEM)), and expression of QS and cellulose synthesis genes (using quantitative real-time PCR) were assessed. Finally, tetrazolium-based colorimetric (MTT) assay was used to examine EOs cytotoxicity on the Vero cell line. GC-MS analysis showed that terpineol, carene and pinene in C. cyminum EO and sulfur compounds in A. sativum EO were the major components of the plant extract. The Geometric mean of MIC values of the A. sativum and C. cyminum were 0.66 and 2.62 μLmL-1, respectively. The geometric means of the fractional inhibitory concentration index (FICi) for both EOs were calculated as 1.05. The qPCR results showed that MIC/2 concentrations of both EOs significantly down-regulated of QS (sdiA and luxS) and cellulose synthesis (csgD and adrA) genes. Scanning electron microscopy showed the EOs reduced the amount of S. Typhimurium mature biofilm. In general, we showed that C. cyminum and A. sativum EOs can be considered as the potential agents against planktonic and biofilm form of S. Typhimurium without any concern of cytotoxic effect at 4 MIC concentrations on the eukaryotic Vero cells.

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000–2013

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants).Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59.SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.

A Plasmid-Encoded FetMP-Fls Iron Uptake System Confers Selective Advantages to Salmonella enterica Serovar Typhimurium in Growth under Iron-Restricted Conditions and for Infection of Mammalian Host Cells.

The resistance plasmid pUO-StVR2, derived from virulence plasmid pSLT, is widespread in clinical isolates of Salmonella enterica serovar Typhimurium recovered in Spain and other European countries. pUO-StVR2 carries several genes encoding a FetMP-Fls system, which could be involved in iron uptake. We therefore analyzed S. Typhimurium LSP 146/02, a clinical strain selected as representative of the isolates carrying the plasmid, and an otherwise isogenic mutant lacking four genes (fetMP-flsDA) of the fetMP-fls region. Growth curves and determination of the intracellular iron content under iron-restricted conditions demonstrated that deletion of these genes impairs iron acquisition. Thus, under these conditions, the mutant grew significantly worse than the wild-type strain, its iron content was significantly lower, and it was outcompeted by the wild-type strain in competition assays. Importantly, the strain lacking the fetMP-flsDA genes was less invasive in cultured epithelial HeLa cells and replicated poorly upon infection of RAW264.7 macrophages. The genes were introduced into S. Typhimurium ATCC 14028, which lacks the FetMP-Fls system, and this resulted in increased growth under iron limitation as well as an increased ability to multiply inside macrophages. These findings indicate that the FetMP-Fls iron acquisition system exceeds the benefits conferred by the other high-affinity iron uptake systems carried by ATCC 14028 and LSP 146/02. We proposed that effective iron acquisition by this system in conjunction with antimicrobial resistance encoded from the same plasmid have greatly contributed to the epidemic success of S. Typhimurium isolates harboring pUO-StVR2.

Occurrence of Salmonella spp. in eggs from backyard chicken flocks in Portugal and Romania - Results of a preliminary study

The aim of this study was to conduct a preliminary investigation on the occurrence of Salmonella spp. in eggs from chickens raised in backyards in Portugal and Romania. A lack of compliance with safety practices by chicken owners, was demonstrated, especially in Portugal, as 96% of the eggs were visibly dirty and 92.5% were stored at room temperature. In Romania the 202 analysed eggs were Salmonella free, whereas in Portugal six of the 200 eggs sampled were positive for Salmonella spp. (3%). A positive egg for Salmonella spp. was found in 10.7% of the 56 backyard flocks sampled in Portugal. One egg exhibited contamination both in the shell-membrane mixture and in its content, while in the remaining eggs, the pathogen was found either in the shell-membrane (n = 2) or in the yolk and white mixture (n = 3). The serotypes S. Typhimurium (with identical PFGE patterns) and S. Enteritidis were isolated from five eggs and one egg, respectively. Whilst S. Enteritidis was sensitive to the 14 antibiotics tested, S. Typhimurium isolates presented divergent antimicrobial resistant phenotypes and three were classified as multi-drug resistant.

Characterization of Salmonella enterica Isolates from Diseased Poultry in Northern China between 2014 and 2018.

Salmonella infection not only causes acute and chronic diseases in poultry flocks, but the infected poultry are among the most important reservoirs for a variety of Salmonella serovars frequently transmitted to humans. This study aimed to investigate the occurrence of Salmonella spp. in local poultry farms in China. Samples (n = 4255), including dead-in-shell embryos, culled day-old-hatchings and 1- to 4-week-old diseased birds, were collected for Salmonella culture from broiler chicken, meat-type duck and pigeon farms in northern China between 2014 and 2018. A total of 103 Salmonella were isolated. S. enterica serovar Enteritidis and S. Typhimurium were the most prevalent serovars, representing 53.4% and 34.9% of the isolates, respectively. Serovar diversity was the highest in ducks, with the S. Apeyeme being isolated for the first time from duck tissues. All isolates were characterized by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). MLST showed that all S. Enteritidis isolates shared the same sequence type (ST11), and Typhimurium showed several rare STs in addition to ST19. In comparison, PFGE showed better discrimination for S. Enteritidis and S. Typhimurium isolates, with nine distinct pulsotypes being observed. The isolates exhibited varying degrees of resistance to 15 tested antimicrobials and identified S. Enteritidis isolates (98.18%) with multiple antimicrobial resistance were a cause for concern. Our data on invasive Salmonella infection in meat-type poultry in local farms can be used to identify sources and factors associated with Salmonella spread in poultry and the associated food chain.

Whole-Genome Sequences of 66 Incompatibility Group FIB Plasmid-Carrying Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources.

Sixty-six Salmonella enterica serovar Typhimurium isolates carrying incompatibility group FIB (IncFIB) plasmids were sequenced to further characterize the IncFIB plasmid-encoded factors associated with virulence and antimicrobial resistance genes. In addition to the IncFIB plasmid, many of these isolates harbored additional plasmids encoding virulence and antimicrobial resistance genes.

Pathogenicity and Competitive Fitness of Salmonella enterica Serovar 4,[5],12:i:- Compared to Salmonella Typhimurium and Salmonella Derby in Swine.

Since 2014, Salmonella 4,[5],12:i:- has emerged as the most common serovar of Salmonella enterica identified from swine samples submitted to veterinary diagnostic laboratories in the United States. To compare the pathogenicity of S. 4,[5],12:i:- in swine to the known pathogenic Salmonella Typhimurium and lesser pathogenic Salmonella Derby, 72 pigs (20 per Salmonella serovar treatment and 12 controls) were inoculated with either S. Typhimurium, S. 4,[5],12:i:-, S. Derby, or sham-inoculated and followed for up to 28 days thereafter via rectal temperature, fecal scoring, and fecal culture. Animals were euthanized on days 2, 4, or 28 to determine the gross and histopathologic signs of disease and tissue colonization. The results clearly demonstrate that for the isolates selected, serovar 4,[5],12:i:- possesses similar ability as serovar Typhimurium to cause clinical disease, colonize the tonsils and ileocecal lymph nodes, and be shed in the feces of infected swine past resolution of clinical disease. To compare the competitive fitness of S. 4,[5],12:i:- to S. Typhimurium in swine when co-infected, 12 pigs were co-inoculated with equal concentrations of both S. Typhimurium and S. 4,[5],12:i and followed for up to 10 days thereafter. When co-inoculated, serovar 4,[5],12:i:- was consistently detected in the feces of a higher percentage of pigs and at higher concentrations than serovar Typhimurium, suggesting an increased competitive fitness of 4,[5],12:i:- relative to serovar Typhimurium when inoculated simultaneously into naïve pigs. Whole genome sequencing analysis of the isolates used in these studies revealed similar virulence factor presence in all S. 4,[5],12:i:- and S. Typhimurium isolates, but not S. Derby, providing additional evidence for similar pathogenicity potential between serovars 4,[5],12:i:- and Typhimurium. Altogether, this data strongly supports the hypothesis that S. 4,[5],12:i:- is a pathogen of swine and suggests a mechanism through increased competitive fitness for the increasing identification of Salmonella 4,[5],12:i:- in swine diagnostic samples over the past several years.

Occurrence and Characterization of Salmonella Isolated from Table Egg Layer Farming Environments in Western Australia and Insights into Biosecurity and Egg Handling Practices.

The aim of this study was to investigate the occurrence and distribution of Salmonella in commercial layer farming environments of 26 flocks belonging to seven egg businesses (free-range and barn-laid) in Western Australia (WA). Between November 2017 and June 2018, a total of 265 environmental samples of dust, feed, water, pooled feces, and boot swabs were tested for detection of Salmonella according to standard culture-based methods. Isolates were assayed for serovar and subtyped by multilocus sequence typing (MLST). Salmonella spp. were recovered from 35% (93/265) of all tested samples. Dust (53.8%, 28/52) and pooled fecal (54.5%, 18/33) samples provided the highest Salmonella recovery rates. Nine different Salmonella serovars were characterized across the positive (n = 93) environmental samples, of which S. Typhimurium (60/93, 64.5%) and S. Infantis (21/93, 22.5%) were the most prevalent. MLST revealed that all S. Typhimurium isolates were of sequence type ST-19. Microbiological screening of Salmonella was not routinely practiced in any of the surveyed egg businesses. Some of the egg businesses exhibited variable levels of compliance with basic biosecurity measures as well as high-risk egg handling practices. Egg businesses in WA should be encouraged to adopt a voluntary program of environmental sampling and verification testing for Salmonella. Such voluntary programs will aid in supporting solutions for the management of this pathogen in the human food chain.