Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4–5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic‐membrane‐located inhibitor of proton‐driven F₁F₀ ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin‐resistant (Nov^R) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with Nov^R gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug‐treated bacteria. The Salmonella cytosol reaches pH 5–6 in response to an external pH of 4–5: the ATP‐dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP‐dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid‐mediated impairment of the negative supercoiling activity of gyrase.     

First outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta in Italy

Aims:  To provide an epidemiologic interpretation of a suspected outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta strains isolated from humans and from the leftovers of the implicated foods (cream, dairy‐based desserts and eggs). Methods and Results:  We have correlated the similarity between the strains through genotyping with Pulsed Field Gel Electrophoresis (PFGE), studying antimicrobial sensitivity patterns and epidemiological investigation. The clonal origin of the outbreak was confirmed by all laboratory tests. PFGE analysis of the restriction profiles obtained with XbaI and SpeI revealed a certainly correlation from the strains isolated from the various sources, while the antimicrobial sensitivity pattern was the same in all cases, with all strains sensitive to all antibiotics tested. Conclusions:  Poor hygiene conditions in the facility concerned, lack of hygiene in food handling, high summer temperatures and positive cultures from asymptomatic staff could all be implicated in the infection, with food being the means through which it spread. Significance and Impact of the Study:  This study describes the first outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta (Salmonella Berta) reported in Italy. It confirms the importance of correlating epidemiological investigations with genotyping and phenotyping to understand the dynamics of infection.     

Inhibition of the type III secretion system by syringaldehyde protects mice from Salmonella enterica serovar Typhimurium

The invasiveness of Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely associated with the Salmonella pathogenicity island (SPI)‐encoded type Ⅲ secretion system (T3SS), which can directly inject a series of effector proteins into eukaryotic cells to enable bacterial infection. In this study, syringaldehyde was identified as an effective inhibitor of the S. Typhimurium T3SS using an effector protein‐lactamase fusion reporter system. Syringaldehyde treatment could inhibit the expression of important effector proteins (SipA, SipB and SipC) at a concentration of 0.18 mM without affecting bacterial growth. Additionally, significant inhibition of bacterial invasion and cellular injury was observed following the syringaldehyde treatment in the co‐infection system of HeLa cells and S. Typhimurium. Furthermore, treatment with syringaldehyde provided systemic protection to mice infected with S. Typhimurium, reducing mortality (40.00%) and bacterial loads and relieving caecal damage and systemic inflammation. The results presented in this study indicate that syringaldehyde significantly affects T3SS activity and is a potential leading compound for treating S. Typhimurium infections.     

Fluoroquinolone resistance of Escherichia coli and Salmonella from healthy livestock and poultry in the EU

The potential for transmission of antibiotic-resistant enteric zoonotic bacteria from animals to humans has been a public health concern for several decades. Bacteria carrying antibiotic resistance genes found in the intestinal tract of food animals can contaminate carcasses and may lead to food-borne disease in humans that may not respond to antibiotic treatment. It is consequently important to monitor changes in antimicrobial susceptibility of zoonotic and commensal organism; in this context, there are a number of veterinary monitoring programmes that collect bacteria in food-producing animals at slaughter and determine their susceptibility against antibiotics relevant for human medicine. The data generated are part of the risk analysis for potential food-borne transmission of resistance. There has been much debate about the use of fluoroquinolones in veterinary medicine, and so, this review will consider the fluoroquinolone data from two surveys and compare them to national surveillance programmes. At the outset, it must be pointed out that there is, however, a lack of agreement between several programmes on what is meant by the term 'fluoroquinolone resistance' through use of different definitions of resistance and different resistance breakpoints. An additional aim of this paper is to clarify some of those definitions. Despite the debate about the contribution of antibiotic use in veterinary medicine to the overall resistance development in human pathogens, the data suggest that clinical resistance to fluoroquinolones in Escherichia coli and nontyphoidal Salmonella is generally uncommon, except for a few countries. Ongoing surveillance will continue to monitor the situation and identify whether this situation changes within the respective animal populations. For the benefit of both the epidemiologist and the clinician, it would be strongly advantageous that national monitoring surveys report both percentages of clinical resistance and decreased susceptibility.     

Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash waters and colonization in mice

Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host–microorganism interactions. Here, we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and biofilm formation in leafy-green vegetable wash water. The opgGH mutant strain, which was defective in OPG biosynthesis, initiated the growth at a slower rate in wash waters obtained from spinach, lettuce and green collard and severely impaired biofilm formation. The lack of OPG synthesis did not influence biofilm formation by the opgGH mutant in low-nutrient low-osmolarity laboratory media. In coculture experiments initiated with equal proportions of cells, the opgGH mutant was outnumbered by the wild-type strain under the planktonic as well as the biofilm growth conditions. The opgGH mutant strain poorly colonized mouse organs when introduced orally along with the wild-type strain. This is the first report demonstrating the role of OPGs of Salmonella in competitive colonization of biofilms, planktonic cultures and mouse organs.     

Inactivation of Salmonella enterica serovar enteritidis in shell eggs by sequential application of heat and ozone

Aims:  To assess the contribution of ozone to lethality of Salmonella enterica serovar Enteritidis in experimentally inoculated whole shell eggs that are sequentially treated with heat and gaseous ozone in pilot-scale equipment.
Methods and Results:  Whole shell eggs were inoculated with small populations of Salmonella Enteritidis (8·5 × 10⁴–2·4 × 10⁵ CFU per egg) near the egg vitelline membrane. Eggs were subjected to immersion heating (57°C for 21 min), ozone treatment (vacuum at 67·5 kPa, followed by ozonation at a maximum concentration of approx. 140 g ozone m⁻³ and 184–198 kPa for 40 min) or a combination of both treatments. Survivors were detected after an enrichment process or enumerated using modified most probable number technique. Ozone, heat and combination treatments inactivated 0·11, 3·1 and 4·2 log Salmonella Enteritidis per egg, respectively.
Conclusions:  Sequential application of heat and gaseous ozone was significantly more effective than either heat or ozone alone. The demonstrated synergy between these treatment steps should produce safer shell eggs than the heat treatment alone.
Significance and Impact of the Study:  Shell eggs are the most common vehicle for human infection by Salmonella Enteritidis. Many cases of egg-related salmonellosis are reported annually despite efforts to reduce contamination, including thermal pasteurization of shell eggs and egg products. Treatment with ozone-based combination should produce shell eggs safer than those treated with heat alone.     

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

AIMS: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracycline-resistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. METHODS AND RESULTS: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l-1 and a 10% increase in E. coli with an MIC > 50 mg l-1 during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 microg g-1 faeces during treatment. CONCLUSION: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.     

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA,the soluble component of the ZnuABC transporter

BackgroundUnder conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake.MethodsAntibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15 Å resolution.ResultsTwo di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound.ConclusionsTwo molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella.General SignificanceThe data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.     

Insights into PG‐binding,conformational change,and dimerization of the OmpA C‐terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi

Salmonella enterica serovar Typhimurium can induce both humoral and cell‐mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N‐terminal eight‐stranded β‐barrel transmembrane domain and a C‐terminal domain (OmpA^CTD). The OmpA^CTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpA^CTD from two pathogens: S. typhimurium (STOmpA^CTD) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpA^CTD), in closed form. In the open form of STOmpA^CTD, an aspartate residue from a long β2‐α3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpA^CTD and in the structure of BbOmpA^CTD, a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpA^CTD, suggest a large conformational change that includes an extension of α3 helix by ordering a part of β2‐α3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpA^CTD suggesting PG‐anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpA^CTD, or possibly that of full length STOmpA.     

Detection of Salmonella enterica serovar Typhimurium by selective amplification of fliC, fljB, iroB, invA, rfbJ, STM2755, STM4497 genes by polymerase chain reaction in a monoplex and multiplex format

The aim of the work was to specifically differentiate S. typhimurium from other closely related Salmonella serovars by monoplex or multiplex PCR and to detect it from water and food samples. Genes targeted were invA, iroB, STM4497, STM2755, fliC, fljB and rfbJ and evaluated on 58 Salmonella standard serovars/strains including 9 S. typhimurium strains, 7 suspected Salmonella isolates and 8 other organisms as negative controls. Both invA and iroB showed a uniform amplification with all serovars of S. enterica group. STM2755 and STM4497 gene based PCR’s specifically exhibited amplification in all the nine confirmed S. typhimurium strains. The rfbJ PCR produced amplification with confirmed S. typhimurium strains, in addition showed reaction with S. abony. Both STM4497, STM2755 PCR’s and rfbJ could identify two of the seven biochemically suspected Salmonella isolates that were later confirmed to be S. typhimurium on the basis of sequence data. PCR for fliC genes had amplification exhibited by a large number of serovars of the S. enterica group, including S. typhimurium strains but not to S. brunei, S. newporti, S. abony and S. weltevreden. fljB was detected in all strains of S. enterica and E. coli with the exception of S. typhi. fljB and fliC were amplified in 6/7 and 5/7 presumptive Salmonella isolates. The same PCR’s were converted into two multiplex formats for simultaneous identification of the Salmonella genus, S. enterica group and S. typhimurium as a species. The first multiplex set comprised on invA, iroB, STM4497, STM2755 and the IAC. The second multiplex set comprised of invA, iroB, fljB, fliC, rfbJ along with IAC. The detection limit for S. typhimurium in the two multiplex PCR sets was in the range of 350–400 cfu/PCR reaction and that of DNA around 2 pg. The multiplex PCR (format 1) was first evaluated on spiked water, chicken and mutton samples and the detection limit for S. typhimurium was in the range of 100 cfu/100 ml, <60 and <50 cfu/gm, respectively. Further evaluation of multiplex PCR (format 1) was undertaken on 50 natural samples of chicken, eggs, litter, soil etc. and the comparison done with conventional culture isolation and identification procedure. The multiplex PCR could identify the presence of Salmonellla in three samples and the same three samples also yielded Salmonella by the conventional method. Therefore, the presently described multiplex PCR can serve as an alternative to the tedious time-consuming procedure of Salmonella culture and identification in food safety laboratories.