Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. RESULTS: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1ß and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. CONCLUSIONS: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

Antibacterial activity and action mode of chlorogenic acid against Salmonella Enteritidis, a foodborne pathogen in chilled fresh chicken.

The study evaluated the antibacterial activity of chlorogenic acid (CA) against Salmonella Enteritidis S1, a foodborne pathogen in chilled fresh chicken. Its minimum inhibitory concentration for S. Enteritidis S1 was 2 mM. 1 MIC CA treatment reduced the viable count of S. Enteritidis S1 by 3 log cfu/g in chilled fresh chicken. Scanning electron microscopy examination indicated that CA induced the cell envelope damage of S. Enteritidis S1. Following this, 1-N-Phenylnaphthylamine assay and LPS content analysis indicated that CA induced the permeability of outer membrane (OM). Confocal laser scanning microscopy examination further demonstrated that CA acted on the inner membrane (IM). To support this, the release of intracellular protein and ATP after CA treatment was also observed. CA also suppressed the activities of malate dehydrogenase and succinate dehydrogenase, two main metabolic enzymes in TCA cycle and electron transport chain. Thus, damage of intracelluar and outer membranes as well as disruption of cell metabolism resulted in cell death eventually. The finding suggested that CA has the potential to be developed as a preservative to control S. Enteritidis associated foodborne diseases.

Dimethyl adenosine transferase (KsgA) contributes to cell-envelope fitness in Salmonella Enteritidis.

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.

Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection.

Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin-antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S. Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect.

Novel insights from molecular docking of SdiA from Salmonella Enteritidis and Escherichia coli with quorum sensing and quorum quenching molecules.

Quorum sensing is a cell-to-cell communication mechanism leading to differential gene expression in response to high population density. The autoinducer-1 (AI-1) type quorum sensing system is incomplete in Escherichia coli and Salmonella due to the lack of the AI-1 synthase (LuxI homolog) responsible for acyl homoserine lactone (AHL) synthesis. However, these bacteria encode the AHL receptor SdiA (a LuxR homolog) leading to gene regulation in response to AI-1 produced by other bacteria. This study aimed to model the SdiA protein of Salmonella enterica serovar Enteritidis PT4 578 based on three crystallized SdiA structures from Enterohemorrhagic E. coli (EHEC) with different ligands. Molecular docking of these predicted structures with AHLs, furanones and 1-octanoyl-rac-glycerol were also performed. The available EHEC SdiA structures provided good prototypes for modeling SdiA from Salmonella. The molecular docking of these proteins showed that residues Y63, W67, Y71, D80 and S134 are common binding sites for different quorum modulating signals, besides being conserved among other LuxR type proteins. We also show that AHLs with twelve carbons presented better binding affinity to SdiA than AHLs with smaller side chains in our docking analysis, regardless of the protein structures used. Interestingly, the conformational changes provided by AHL binding resulted in structural models with increased affinities to brominated furanones. These results suggest that the use of brominated furanones to inhibit phenotypes controlled by quorum sensing in Salmonella and EHEC may present a good strategy since these inhibitors seem to specifically compete with AHLs for binding to SdiA in both pathogens.

Functional characterization of glucosamine-6-phosphate synthase (GlmS) in Salmonella enterica serovar Enteritidis.

Salmonella is a threat to public health due to consumption of contaminated food. Screening of a transposon library identified a unique mutant that was growth and host cell binding deficient. The objective of this study was to determine the functional role of glucosamine-6-phosphate synthase (GlmS) in the biology and pathogenesis of Salmonella. To examine this, we created a glmS mutant (ΔglmS) of Salmonella and examined the effect on cell envelope integrity, growth, metabolism, and pathogenesis. Our data indicated ΔglmS was defective in growth unless media were supplemented with D-glucosamine (D-GlcN). Examination of the bacterial cell envelope revealed that ΔglmS was highly sensitive to detergents, hydrophobic antibiotics, and bile salts compared to the wild type (WT). A release assay indicated that ΔglmS secreted higher amounts of ß-lactamase than the WT in culture supernatant fractions. Furthermore, ΔglmS was attenuated in cell culture models of Salmonella infection. Taken together, this study determined an important role for GlmS in the pathogenesis and biology of Salmonella.

[Deletion-mutant construction, prokaryotic expression and characterization of peptidal-prolyl cis-trans isomerase SlyD from Salmonella enteritidis].

Objective: Salmonella enterica serovar enteritidis is an important food-borne pathogen of human and animal. To further study the function of SlyD associated with virulence and regulation in stress responses of Salmonella Enteritidis, we constructed slyD gene-deletion mutant,, expressed it in E. coli, and characterized the PPIase enzyme obtained. Methods: The slyD gene-deletion mutant of Salmonella enteritidis C50041 was constructed by suicide plasmid mediated homologous recombination. Salmonella enteritidis slyD prokaryotic expression vector was carried out in E. coli, and PPIase activity of recombination SlyD was measured in protease-coupling assay with chymotrypsin. For amino acids conservation studies, functional domain searches and secondary structure predictions, the BLAST, SMART, TMHMM, SignalP, PHD and SWISS MODEL were used. Results: Salmonella enteritidis C50041 ΔslyD mutant strain was successfully constructed. The growth rate of slyD-deleted strain was identified consistent with its parent strain C50041. A soluble recombinant SlyD protein was expressed in Escherichia coli BL21(DE3) cells and confirmed by SDS-PAGE. Catalytic activity confirmed that the SlyD protein was biologically active. Bioinformatic analysis showed that Salmonella Enteritidis SlyD as a multifaceted protein including three separated domains, the FKBP type peptidal-prolyl cis-trans isomerase domain, the IF chaperone domain and the metal-binding domain. Conclusion: Salmonella enteritidis C50041 ΔslyD mutant strain and soluble SlyD protein was obtained, and the present study may provide a basis for further study of the role of SlyD in Salmonella enteritidis.

Community-onset pyomyositis caused by a Salmonella enterica serovar enteritidis sequence type 11 strain producing CTX-M-15 extended-spectrum ß-lactamase.

We report a case of community-onset pyomyositis due to Salmonella enterica serovar Enteritidis in South Korea. The isolated strain was resistant to extended-spectrum cephalosporins and harbored sequence type 11 coproducing CTX-M-15 extended-spectrum ß-lactamase (ESBL). Physicians should be alert for early diagnosis and appropriate treatment since ESBL-producing nontyphoidal Salmonella infections are difficult to treat without initiation of appropriate empirical antibiotics.

The endoplasmic reticulum-based acetyltransferases, ATase1 and ATase2, associate with the oligosaccharyltransferase to acetylate correctly folded polypeptides.

The endoplasmic reticulum (ER) has two membrane-bound acetyltransferases responsible for the endoluminal N(ϵ)-lysine acetylation of ER-transiting and -resident proteins. Mutations that impair the ER-based acetylation machinery are associated with developmental defects and a familial form of spastic paraplegia. Deficient ER acetylation in the mouse leads to defects of the immune and nervous system. Here, we report that both ATase1 and ATase2 form homo- and heterodimers and associate with members of the oligosaccharyltransferase (OST) complex. In contrast to the OST, the ATases only modify correctly folded polypetides. Collectively, our studies suggest that one of the functions of the ATases is to work in concert with the OST and "select" correctly folded from unfolded/misfolded transiting polypeptides.

Investigating the physiological roles of low-efficiency D-mannonate and D-gluconate dehydratases in the enolase superfamily: pathways for the catabolism of L-gulonate and L-idonate.

The sequence/function space in the D-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10(3)-10(4) M(-1) s(-1)) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10-10(2) M(-1) s(-1)) for dehydration of D-mannonate and/or D-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087-4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

Liver abscess caused by CTX-M-55-type extended-spectrum ß-lactamase (ESBL)-producing Salmonella enteritidis.

Liver abscesses secondary to Salmonella species are rarely described in the general population. We herein describe a case of a liver abscess caused by CTX-M-55-type extended-spectrum ß-lactamase (ESBL)-producing Salmonella enteritidis, which has not been reported in the literature. A 54-year-old male was admitted due to a high fever and was clinically diagnosed with a liver abscess. Culture of the fluid from the liver abscess revealed CTX-M-55-type ESBL-producing S. enteritidis. Although the patient underwent percutaneous transhepatic abscess drainage and antibiotic therapy, he died one month later. It should be noted that liver abscesses are potentially fatal depending on the causative pathogen.

Dimethyl adenosine transferase (KsgA) deficiency in Salmonella enterica Serovar Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens.

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.

Analysis of pulsed field gel electrophoresis profiles using multiple enzymes for predicting potential source reservoirs for strains of Salmonella Enteritidis and Salmonella Typhimurium isolated from humans.

We reported previously on a highly discriminatory pulsed field gel electrophoresis-based (PFGE) subtyping scheme for Salmonella enterica serovar Enteritidis (SE) and Salmonella Typhimurium (ST) that relies on combined cluster analysis of up to six restriction enzymes. This approach allowed for the high-resolution separation of numerous poultry-derived SE and ST isolates into several distinct clusters that sorted along several geographical and host-linked boundaries. In this study, 101 SE and 151 ST strains isolated from poultry, swine, beef, mouse, and produce origins were combined with 62 human SE and ST isolates of unknown sources. PFGE profiles were generated across six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for human SE and ST isolates. The combined six-enzyme UPGMA trees of SE and ST revealed six separate origins of North American human SE isolates including one association with a "cosmopolitan" cluster of SEs from poultry originating in Scotland, Mexico, and China. In the case of ST, human isolates assorted readily along host lines rather than geographical partitions with the majority of human STs clustering in a larger group of STs of potential porcine origin. Such observations may underscore the ecological importance of poultry and pork reservoirs for SE and ST transmission to humans, respectively. In an examination of the relationship between enzyme diversity and congruence among enzymes, pairwise genetic diversity ranged from 6.5% to 9.7% for SE isolates and, more widely, from 17.5% to 27.4% for ST isolates. Phylogenetic congruence measures singled out XbaI, BlnI, and SfiI as most concordant for SE while XbaI and SfiI were most concordant among ST strains. Thus, these data provide the first proof of principal for concatenated PFGE, when coupled with sufficient enzyme numbers and combinations, as one effective means for predicting geographical and food source reservoirs for human isolates of these two highly prevalent Salmonella serovars.

Prevalence of quinolone resistance and mutations in the topoisomerase genes in Salmonella enterica serotype Enteritidis isolates from Serbia.

The prevalence of quinolone resistance was studied in Salmonella enterica serotype Enteritidis isolates collected during 2005-2010 in Southern Backa County, Serbia. A total of 878 clinical isolates were examined, among which 19 (2.2%) nalidixic acid (NAL)-resistant S. Enteritidis were detected by selection on agar plates containing 64 mg/L NAL. Antimicrobial susceptibility of the isolates was tested by the agar dilution method. According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, ciprofloxacin (CIP) resistance was not present in the strains. Multiple drug resistance was rare, and resistance to NAL was most often present as a single resistance property. All but one NAL-resistant S. Enteritidis showed reduced susceptibility to CIP [i.e. minimum inhibitory concentration (MIC)≥0.125 mg/L]. This isolate of human origin had a CIP MIC of 0.064 mg/L and DNA sequencing revealed that it contained an Asp87Gly gyrA mutation. Most of the remaining isolates had MICs for NAL and CIP of 256 mg/L and 0.256 mg/L, respectively. Mutations in the Asp87 codon resulted in substitutions to Asn in most of the isolates, but Asp87Gly and Ser83Phe exchanges were also detected. No mutations were present in the gyrB, parC or parE genes. Although CIP resistance was absent, reduced susceptibility characterised by mutations in gyrA was apparent among S. Enteritidis isolates from Serbia.

Deletion of the aceE gene (encoding a component of pyruvate dehydrogenase) attenuates Salmonella enterica serovar Enteritidis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen. From a transposon insertion mutant library created previously using S. Enteritidis 10/02, one of the mutants was identified to have a 50% lethal dose (LD(50) ) at least 100 times that of the parental strain in young chicks, with an attenuation in a poorly studied gene encoding a component of pyruvate dehydrogenase, namely the aceE gene. Evaluation of the in vitro virulence characteristics of the ΔaceE∷kan mutant revealed that it was less able to invade epithelial cells, less resistant to reactive oxygen intermediate, less able to survive within a chicken macrophage cell line and had a retarded growth rate compared with the parental strain. Young chicks vaccinated with 2 × 10(9) CFU of the ΔaceE∷kan mutant were protected from the subsequent challenge of the parental strain, with the mutant colonized in the liver and spleen in a shorter time than the group infected with the parental strain. In addition, compared with the parental strain, the ΔaceE∷kan mutant did not cause persistent eggshell contamination of vaccinated hens.

Association of attenuated mutants of Salmonella enterica serovar Enteritidis with porcine peripheral blood leukocytes.

In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.

Characterization of the first extended-spectrum beta-lactamase-producing nontyphoidal Salmonella strains isolated in Tehran, Iran.

The infections caused by Salmonella remain a significant public health problem throughout the world. beta-Lactams and fluoroquinolones are generally used to treat invasive Salmonella infections, but emergence and spread of antibiotic-resistant strains are being increasingly notified in many countries. In particular, detection of extended-spectrum beta-lactamases (ESBLs) in Salmonella spp. is a newly emerging threat worldwide. This study was carried out to characterize beta-lactamase-producing Salmonella strains identified in Tehran, Iran. Over the 2-year period from 2007 to 2008, 6 of 136 Salmonella isolates recovered from pediatrics patients, including three Salmonella enterica serotypes Enteritidis (S. Enteritidis) and three S. Infantis, showed an ESBL-positive phenotype. Polymerase chain reaction and sequencing were used to identify the genetic determinants responsible for ESBL phenotypes. The Salmonella isolates were also compared by pulsed-field gel electrophoresis. All ESBL-producing strains, but one, carried the bla(CTX-M-15) gene. Moreover, three of four strains that proved to be positive for a bla(TEM) gene were producing a TEM-1 beta-lactamase. Two strains of S. Infantis tested positive for a previously unidentified CTX-M and TEM ESBL, respectively. All ESBL-producing strains carried the insertion sequence ISEcp1 gene. Except for one strain of serotype Infantis, all strains were able to transfer the ESBL determinants by conjugation. Distinct, but closely related, pulsed-field gel electrophoresis patterns were observed among the strains belonging to both serotypes. This study reports for the first time the emergence and characterization of ESBL-producing S. Enteritidis and Infantis strains in Iran.

RfaB, a galactosyltransferase, contributes to the resistance to detergent and the virulence of Salmonella enterica serovar Enteritidis.

In this study, a deletion mutant of rfaB (DeltarfaB) was observed to be susceptible to sodium dodecyl sulfate and less tolerant to bile salts. In addition, pre-incubation in 10% bile salts increased bacterial tolerance to 30% bile salts. We also showed that the DeltarfaB mutant invaded HeLa cells less than the wild type and resulted in a lower ratio of intracellular bacteria. Competitive infection of mice showed that the DeltarfaB mutant was defective in the colonization of host organs and was cleared more quickly in fecal shedding. Transforming of a plasmid containing a wild-type allele of rfaB (pRB3-rfaB) partially rescued the defect of the DeltarfaB mutant. The results suggest that RfaB, which is responsible to add the glycosyl residue to the core lipopolysaccharide, contributes to the tolerance to detergent and the virulence of Salmonella enterica serovar Enteritidis.

Characterization of TEM-, SHV- and AmpC-type beta-lactamases from cephalosporin-resistant Enterobacteriaceae isolated from swine.

Extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacteriaceae are an increasing problem in human medicine and an emerging problem in the veterinary field. Our study, therefore, focused on assessing the prevalence of beta-lactamases isolated from swine. Sixty-six Salmonella enterica serovar Typhimurium (S. Typhimurium), 33 Salmonella enterica serovar Enteritidis (S. Enteritidis), 26 Klebsiella pneumonia (K. pneumoniae) and 130 Escherichia coli (E. coli) pig isolates collected from 1999-2006 were screened for beta-lactam resistance by the disk diffusion test (DDT) and micro-broth dilution. Among the isolates, five E. coli and five K. pneumoniae exhibited reduced susceptibility to the cephalosporins tested. PCR, plasmid profiling and Southern blot hybridization showed the presence of multiple beta-lactamases in these isolates of animal origin. Hybridization patterns of the DHA-1 specific probe indicated that dissemination of DHA-1 related beta-lactamases could be attributed to plasmids of one common size among the enteric microbes of animal origin. To the best of our knowledge, this study reports the first identification of SHV-28 and DHA-1 from microbes of animal origin.

aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity.

In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.