Investigation of a foodborne outbreak of Shigella sonnei in Ireland and Northern Ireland, December 2016: the benefits of cross-border collaboration and commercial sales data.

OBJECTIVES: To describe a cross-border foodborne outbreak of Shigella sonnei that occurred in Ireland and Northern Ireland (NI) in December 2016 whilst also highlighting the valuable roles of sales data and international collaboration in the investigation and control of this outbreak. STUDY DESIGN: A cross-border outbreak control team was established to investigate the outbreak. METHODS: Epidemiological, microbiological, and environmental investigations were undertaken. Traditional analytical epidemiological studies were not feasible in this investigation. The restaurant chain provided sales data, which allowed assessment of a possible increased risk of illness associated with exposure to a particular type of heated food product (product A). RESULTS: Confirmed cases demonstrated sole trimethoprim resistance: an atypical antibiogram for Shigella isolates in Ireland. Early communication and the sharing of information within the outbreak control team facilitated the early detection of the international dimension of this outbreak. A joint international alert using the European Centre for Disease Control's confidential Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD) did not reveal further cases outside of the island of Ireland. The outbreak investigation identified that nine of thirteen primary case individuals had consumed product A from one of multiple branches of a restaurant chain located throughout the island of Ireland. Product A was made specifically for this chain in a food production facility in NI. S. sonnei was not detected in food samples from the food production facility. Strong statistical associations were observed between visiting a branch of this restaurant chain between 5 and 9 December 2016 and eating product A and developing shigellosis. CONCLUSIONS: This outbreak investigation highlights the importance of international collaboration in the efficient identification of cross-border foodborne outbreaks and the value of using sales data as the analytical component of such studies.

Characterization of bacteriophages used in the Salmonella enterica serovar Enteritidis phage-typing scheme.

The 16 Salmonella enterica serovar Enteritidis (S. Enteritidis) typing phages (SETPs) used in the Laboratory of Enteric Pathogens (Health Protection Agency, London, UK) phage-typing scheme have not previously been characterized in detail. We have examined the adsorption properties of the phages with respect to a number of S. enterica serovars and defined phage morphology with electron microscopy. PFGE was used to estimate overall genome size and banding patterns generated by electrophoresis following restriction endonuclease digestion of the genome with HindIII were compared. PCR amplification and sequencing of selected genes was performed. The 16 phages comprise three morphotypes, Podoviridae (SETP1, 8, 10, 14, 15 and 16), Siphoviridae (SETP3, 5, 7, 11, 12 and 13) and Myoviridae (SETP2, 4, 6 and 9). All Podoviridae and Siphoviridae, but not Myoviridae, adsorbed to the O12 lipopolysaccharide antigen of Salmonella serogroups B (4,12) and D(1) (9,12). The genome sizes for the Podoviridae and Siphoviridae (PFGE-A) were approximately 42 kb. The genome size for Myoviridae SETP2, 4 and 9 was 36.5 kb, and for myovirus SETP6 was 27 kb. HindIII digestion of phage DNA produced 9 distinct patterns of 8 to 11 bands. Relationships between phages based on digest patterns were consistent with those defined by morphology. The Podoviridae had homologues of several P22 genes while the Siphoviridae had homologues of several genes present in the sequenced siphovirus SETP3 (EF177456). This study represents an initial step in characterizing the molecular basis that underlies the widely used S. Enteritidis typing scheme.

Inhibition of DNA binding of the NF-Y transcription factor by the pyrrolobenzodiazepine-polyamide conjugate GWL-78.

Many genes involved in cell cycle control have promoters that bind the heterotrimeric transcription factor NF-Y. Several minor-groove binding drugs have been shown to block interactions of transcription factors with cognate DNA-binding sequences. We showed previously that noncovalent minor-groove binding agents block interactions of NF-Y with the promoter of topoisomerase IIalpha (topo IIalpha). In this study, we investigated the ability of GWL-78, a pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugate, to inhibit the binding of NF-Y to DNA. Electrophoretic mobility shift assays showed that GWL-78 could displace NF-Y bound to several CCAAT motifs within promoters of genes involved in cell cycle progression. DNase I footprinting of the topo IIalpha promoter confirmed binding of GWL-78 to AT-rich sequences corresponding to the preferred binding site of NF-Y. Incubation with GWL-78 resulted in displacement of NF-Y binding to DNA. Chromatin immunoprecipitation assays on the topo IIalpha promoter showed that GWL-78 was able to enter the nucleus and interact with specific DNA sequences. Treatment of NIH3T3 cells with GWL-78 resulted in a block of cell cycle progression, which did not involve activation of p53. Thus, agents such as GWL-78 may be useful in modulating transcription and blocking cellular proliferation.

Sequence recognition in the minor groove of DNA by covalently linked formamido imidazole-pyrrole-imidazole polyamides: effect of H-pin linkage and linker length on selectivity and affinity.

The polyamide N-formamido imidazole-pyrrole-imidazole (f-ImPyIm) binds with an exceptionally high affinity for its cognate site 5'-ACGCGT-3' as a stacked, staggered, and noncovalent cooperative dimer. Investigations are presented into its sequence specificity and binding affinity when linked covalently as an H-pin "dimer". Five f-ImPyIm cross-linked analogues with six to nine methylene linkers and an eight-linked ethylene glycol linker were examined to investigate the effect of linkage and linker length on DNA binding. Thermal denaturation studies on short DNA hairpins showed preferential binding by both f-ImPyIm (DeltaTm = 7.8 degrees C) and its cross-linked derivatives (DeltaTm > 30 degrees C) at 5'-ACGCGT-3', indicating sequence specificity was retained on linkage. DNase I footprinting confirmed strict cognate site selectivity and demonstrated that affinity increased with linker length (f-ImPyIm-9 = f-ImPyIm-8 = f-ImPyIm-EG-8 > f-ImPyIm-7 > f-ImPyIm-6). The eight- and nine-linked derivatives bound at 100-fold lower concentrations at the cognate site relative to f-ImPyIm-6, and with 10-fold higher affinity than unlinked f-ImPyIm. Use of an ethylene glycol linkage in f-ImPyIm-EG-8 to improve solubility slightly increased the cognate site affinity relative to those of f-ImPyIm-8 and f-ImPyIm-9, although some selectivity was lost at high ligand concentration. CD demonstrated that cognate site binding by eight and nine-linked compounds occurred in the minor groove. SPR analysis gave a binding affinity (K) for f-ImPyIm-EG-8 at the cognate site of 2 x 10(10) M-1, representing a 100-fold increase relative to that of f-ImPyIm. This study demonstrates that the high-affinity cooperative binding of f-ImPyIm can be enhanced significantly by suitable covalent linkage, while maintaining its strict cognate site selectivity.

Clonal expansion may account for high levels of quinolone resistance in Salmonella enterica serovar enteritidis.

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.

Synthesis of novel DNA cross-linking antitumour agents based on polyazamacrocycles.

We are seeking to develop more effective alkylating agents as antitumour agents. In previous work conformationally restricted nitrogen mustards were synthesised containing piperidine or pyrrolidine rings. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation and the effects of varying the distances between the two alkylating sites were studied. Some efficient cross-linkers of naked DNA were prepared but few of these compounds exhibited significant cytotoxicity in human tumour cells in vitro. We have extended this work by making tri- and tetra-azamacrocyclic compounds containing two to four potential alkylating sites. Most of these compounds were powerful DNA alkylating agents and showed cytotoxicity (IC(50) values 6-100microM) comparable with chlorambucil (45microM) and melphalan (8.5microM). In particular the cyclen derivative 2a was more than 10(4) times more effective at cross-linking DNA (2a XL(50)<<10nM) than chlorambucil (XL(50) 100microM), and showed significant cytotoxicity in human tumour cells in vitro.

A novel design strategy for stable metal complexes of nitrogen mustards as bioreductive prodrugs.

Tumor hypoxia provides a key difference between healthy and cancerous cells. It can be exploited to produce drug selectivity, offering a reductase-rich environment for prodrug activation. Nitrogen mustard drugs are cytotoxic, but usually unselective. Polyamine mustards are candidates for conversion into hypoxia-selective prodrugs via complexation with metals. Reduction to a less stable complex can free the active drug. The novel Cu(II) complexes of N-mustard derivatives of 1,4,7-triazacyclononane (tacn), 1,4,7,10-tetraazacyclododecane (cyclen), and 1,4,8,11-tetraazacyclotetradecane (cyclam) were assessed in vitro as hypoxia-selective cytotoxins. The cyclen mustard complex showed 24-fold selectivity as a hypoxia-selective bioreductive prodrug, with an IC50 value of 2 microM against the lung tumor cell line A549. Reversible redox behavior and stability of the cyclen-Cu(II) complex in aqueous solution correlated with good hypoxia selectivity. The two other related complexes showed irreversible redox behavior and low aqueous stability and were not hypoxia-selective. The use of macrocyclic nitrogen mustard complexes represents a promising new strategy in the design of hypoxia-selective cytotoxins.

Antimicrobial resistance and phage types of human and non-human Salmonella enterica isolates in Ireland, 1998-2003.

Between 1998 and 2003, 5,161 isolates (3,182 human) of Salmonella enterica were received by the National Salmonella Reference Laboratory of Ireland. Serotyping, antimicrobial susceptibility testing and phage typing were performed by standard methods. The number of isolates of S. enterica serovar Typhimurium decreased from 579 (80%) in 1998 to 208 (19%) in 2003, while S. enterica serovar Enteritidis increased from 59 (8%) in 1998 to 219 (20%) in 2003. Definitive (DT) phage types 104 and DT104b accounted for a declining proportion of all Salmonella Typhimurium isolates (from n = 523 [90%] in 1998 to 126 [60%] in 2003). Numbers of Salmonella Enteritidis phage type 4 declined from 50 (85%) in 1998 to 59 (27%) in 2003. Twenty-eight isolates of typhoidal Salmonella were received with a history of recent travel in 17 cases. Resistance to multiple (four or more) antimicrobial agents was related to serotype and, where applicable, phage type, and was common in Salmonella Typhimurium. Salmonella Typhimurium predominated among isolates from cattle and pigs (n = 213 [58%]), while Salmonella Livingstone (n = 327) and S. Kentucky (n = 227) were predominant in isolates from poultry (total n = 554 [43%]). This paper discusses trends, and their implications, in Irish salmonella isolates since the establishment of the Reference Laboratory.

DNA sequence recognition in the minor groove by crosslinked polyamides: The effect of N-terminal head group and linker length on binding affinity and specificity.

Development of sequence-reading polyamides or "lexitropsins" with comparable DNA-binding affinities to cellular proteins raises the possibility of artificially regulated gene expression. Covalent linkage of polyamide ligands, with either a hairpin motif or crosslinking methylene bridge, has greatly improved binding affinity by ensuring their side-by-side register. Whereas hairpin polyamides have been investigated extensively, the optimized structure of crosslinked polyamides remains to be determined. This study examines a series of thiazole-imidazole-pyrrole (TIP) monomers and crosslinked dimers to evaluate the effects on selectivity and binding affinity of different N-terminal head groups attached to the leading thiazole ring and differing methylene linker lengths. Quantitative footprinting of a DNA sequence, containing potential match and mismatch sites for both maximum overlap and one-residue stagger binding modes, allowed measurement of binding constants at each putative site. Within an N-terminal amino TIP series, C7 and C8-linked compounds bound most strongly to these sites, whereas maximum binding affinity was observed for a C6 linker with a formyl head group. A C5 linker gave weak binding with either head group. A hydrogen or acetyl head group abrogated binding. Binding was confirmed by gel shift analyses. The highest specificity for the maximum overlap site over the one-residue stagger was observed with TIP-C7-amino. Selectivity of the leading thiazole was modulated by the head group, with N-terminal formyl TIP exhibiting up to 3-fold specificity for AGT over TGT, suggesting that N-formyl-thiazole may provide sequence discrimination of adenine over thymine. Moreover, the leading head group and methylene linker length significantly influences the binding characteristics of crosslinked polyamides.

Synthesis of new homochiral bispyrrolidines as potential DNA cross-linking antitumour agents.

We are seeking to develop more effective bifunctional alkylating agents as antitumour agents. We previously synthesized conformationally restricted nitrogen mustards containing one piperidine ring, then bispiperidine derivatives were designed and prepared with varying lengths of carbon chain between the two rings and structure-activity relationships in these systems were studied. A bispiperidine with the shortest bridge of two carbon atoms was the most reactive bifunctional alkylating agent. In order to extend this work and investigate the effects of a change in the size of the heterocyclic systems, new bispyrrolidine salts 17-23 with chloromethyl groups at the 2-positions and a bridge between the two nitrogen atoms of 2-8 carbon atoms were synthesized from L-proline so that only the LL-enantiomers were produced. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation with different distances between the two alkylating sites. All of the bispyrrolidines were efficient cross-linkers of naked DNA apart from those with three-carbon (18) and four-carbon (19) bridges, in contrast to the results with the bispiperidines. A piperazine derivative 24 with two potential alkylating sites was also shown to be an efficient cross-linker, as was an alicyclic compound 25 with six carbon atoms between the two alkylating sites. Compounds 26 and 30 with an extra carbon atom between the nitrogen and the leaving group were not cross-linkers, as expected if aziridinium ion formation is crucial for cross-linking ability. The preformed aziridine 27 with a further alkylating site was an efficient cross-linker. Compounds 28-29 with only one potential alkylating centre were not cross-linkers of DNA. None of the compounds, however, produced significant cytotoxicity in human tumour cells in vitro.

Molecular characterization of Irish Salmonella enterica serotype typhimurium: detection of class I integrons and assessment of genetic relationships by DNA amplification fingerprinting.

Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica. In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated (n = 48). Phage types DT104b (n = 3), -193 (n = 9), -195 (n = 6), -208 (n = 3), -204a (n = 2), PT U302 (n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.

An in vitro evaluation of flow from multihole epidural catheters during continuous infusion with four different infusion pumps.

We have observed in vitro the distribution of flow from 10 identical multihole epidural catheters during continuous infusion with four different infusion pumps. The pumps chosen were the B Braun Perfusor Secura FT syringe driver and three volumetric infusion pumps utilising different pumping mechanisms (Dekra 3000 BL, Graseby 500 and CADD-Prizm). These pumps infused 0.9% saline through each catheter at 5 ml.h-1, 15 ml.h-1, 50 ml.h-1 and 99 ml.h-1 for 3 min. The number of holes through which flow occurred and the catheter hole where flow predominated during each test were recorded. The pressure waveform generated during each infusion was displayed and the peak pressure recorded. In 38 of the 160 tests (24%) the largest proportion of flow was seen at the hole closest to the catheter tip. The CADD pump generated multihole flow during significantly more tests (p < 0.0001) than the other pumps and produced significantly higher driving pressures (p < 0.001) at all infusion rates compared with the Graseby and Perfusor pumps. The CADD was the only pump to produce flow from all three holes of the catheter at 5 ml.h-1.

Cross-linking and sequence-specific alkylation of DNA by aziridinylquinones. 3. Effects of alkyl substituents.

The cytotoxicities and DNA cross-linking abilities of several alkyl-substituted diaziridinylquinones have been investigated. The cytotoxicities were determined in DT-diaphorase-rich (H460 and HT29) and -deficient (H596 and BE) cell lines. It was shown that the cytotoxicities in these cell lines correlated with the relative rates of reduction by the purified human enzyme and with the cross-linking efficiencies. The rates of reduction by DT-diaphorase were more dependent on the structures of the compounds than the reduction potentials, as determined by cyclic voltammetry. A computer model was also used to explain high efficiency of cross-linking and the GNC sequence selectivity of the reduced methyl-substituted diaziridinylquinones.

DNA alkylation and interstrand cross-linking by treosulfan.

The anti-tumour drug treosulfan (L-threitol 1,4-bismethanesulphonate, Ovastat) is a prodrug for epoxy compounds by converting non-enzymatically to L-diepoxybutane via the corresponding monoepoxide under physiological conditions. The present study supports the hypothesis that this conversion of treosulfan is required for cytotoxicity in vitro. DNA alkylation and interstrand cross-linking of plasmid DNA is observed after treosulfan treatment, but this is again produced via the epoxide species. Alkylation occurs at guanine bases with a sequence selectivity similar to other alkylating agents such as the nitrogen mustards. In treosulfan-treated K562 cells, cross-links form slowly, reaching a peak at approximately 24 h. Incubation of K562 cells with preformed epoxides shows faster and more efficient DNA cross-linking.

Synthesis of new bifunctional compounds which selectively alkylate guanines in DNA.

The aim of this work was to develop new bifunctional alkylating agents which damage DNA in a selective manner. In order to extend our previously published work on conformationally restricted nitrogen mustards containing one piperidine ring, new bispiperidine derivatives were designed with varying lengths of carbon chain between the two rings and structure-activity relationships in these systems were studied. Thus samples of new bispiperidine salts 22-26 with chloromethyl groups at the 2-positions and a bridge between the two nitrogen atoms of 2-6 carbon atoms were synthesized. The analogous new bis(p-nitrophenylcarbamates) 17-21 were also prepared. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation with different distances between the two alkylating sites. The bispiperidines 22-24 were shown to alkylate guanines at the 7-position in the major groove of DNA more selectively than melphalan. The bispiperidine 22 with the shortest two carbon bridge was the most reactive but it was less cytotoxic than melphalan in a human colon carcinoma cell line (IC50 value approximately 30 microM) and in a human chronic myeloid leukaemia cell line (IC50 value approximately 12 microM). The most cytotoxic compound in the latter cell line was the carbamate 17, with an IC50 value of approximately 0.3 microM, and carbamates 17, 19 and 20 were most potent in a panel of human ovarian carcinoma cell lines. These compounds also showed circumvention of acquired cisplatin resistance in three paired cell lines. The carbamates 17-21, however, were less efficient at alkylating and cross-linking naked DNA than the bispiperidines 22-26.

A monofunctional derivative of melphalan: preparation, DNA alkylation products, and determination of the specificity of monoclonal antibodies that recognize melphalan-DNA adducts.

Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was

Cross-linking and sequence specific alkylation of DNA by aziridinyl quinones. 2. Structure requirements for sequence selectivity.

The cytotoxicities and DNA sequence selectivity for guanine-N7 alkylation of 22 mono- and disubstituted 2,5-diaziridinyl-1,4-benzoquinones have been investigated. Several quinones produced patterns of alkylation following reduction with a selectivity for 5'-TGC-3' sequences. This sequence selectivity appeared to be dependent only on the presence of a hydrogen in position-6 of the quinone. A computer model, based on published crystallographic data, was used to explain this selectivity. The sequence selective quinones were generally more cytotoxic that the quinones which reacted randomly.