The Arabidopsis thaliana AtHMA1 protein is a member of the P IB -ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplas- Plant Ca 2ϩ and heavy metal-ATPases belong to the superfamily of P-ATPases (1, 2). Their common characteristic is the presence of a phosphorylated intermediary in the catalytic cycle. In Arabidopsis, the heavy metal-ATPases belong to the P IB subfamily and normally have eight predicted transmembrane domains, whereas the Ca 2ϩ -ATPases are part of the P IIA and P IIB subfamilies and have ten predicted transmembrane domains (2). A common feature among the P IB -ATPases is the presence of a CPX motif, which is though to play a role in metal translocation, as well as putative metal-binding domains located at the amino or carboxyl terminus (3). On the other hand, P IIB ATPases have a calmodulin-binding domain that regulate their activity; however, the P IIA do not have this domain (4). The most important difference between the calcium and heavy metal-ATPases is their substrate specificity (1). However, in vitro metal transport studies performed with membrane fractions isolated from seedlings suggest that one or more members of the superfamily of P-ATPases are capable of transporting calcium and heavy metals. The studies showed competitive inhibition between active transport of heavy metals (such as copper and cadmium) and calcium.mic2 Interestingly, both transport activities were inhibited by the sesquiterpene lactone thapsigargin, a potent and specific inhibitor of SERCA 3 -type pumps (5-7). To date no one has described plant ion pumps that transport calcium and heavy metals in biochemical terms, nor have scientists described genes encoding for ion pumps inhibited by thapsigargin or plant mutants with thapsigargin-sensitive/tolerant phenotypes. Based on the results of biochemical assays suggesting the existence of thapsigargin-sensitive Ca 2ϩ /heavy metal-ATPase, we searched for potential candidate proteins in the Arabidopsis genome. This was made possible by the highly conserved "stalk segment" or S3 sequence adjacent to the third transmembrane segment of the SERCA pumps (8, 9) composed of amino acids DEF-GEQLSK (5-7). This sequence was almost complete and was annotated as a stalk segment (using the topology prediction software ARAMEMNON) in the Arabidopsis heavy metal pump AtHMA1 (At4g37270). This pump belongs to the subclass of zinc/cobalt/cadmium/lead-ATPases and is the most divergent metal pump of the Arabidopsis P IB -ATPases (1, 2, 10, 11) (see Fig. 1). It lacks an amino-terminal heavy metal-binding domain, such as GMXCXXC or GICC(T/S)SE, which is often found in other members of the group. It has an intramembranous SPC instead of the CP(C/H/S) motif located at the putative metal transporting site of P IB -ATPases (11, 12). The pump possesses other structural characteristics related to heavy metal binding and transport, such as a poly-H motif commonly found in zinc-binding pro...

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Salmonella enterica subsp. enterica serotype Enteritidis is a major cause of human salmonellosis worldwide; however, little is known about the genetic relationships between S. Enteritidis clinical strains and S. Enteritidis strains from other sources in Chile. We compared the whole genomes of 30 S. Enteritidis strains isolated from gulls, domestic chicken eggs, and humans in Chile, to investigate their phylogenetic relationships and to establish their relatedness to international strains. Core genome multilocus sequence typing (cgMLST) analysis showed that only 246/4,065 shared loci differed among these Chilean strains, separating them into two clusters (I and II), with cluster II being further divided into five subclusters. One subcluster (subcluster 2) contained strains from all surveyed sources that differed at 1 to 18 loci (of 4,065 loci) with 1 to 18 single-nucleotide polymorphisms (SNPs), suggesting interspecies transmission of S. Enteritidis in Chile. Moreover, clusters were formed by strains that were distant geographically, which could imply that gulls might be spreading the pathogen throughout the country. Our cgMLST analysis, using other S. Enteritidis genomes available in the National Center for Biotechnology Information (NCBI) database, showed that S. Enteritidis strains from Chile and the United States belonged to different lineages, which suggests that S. Enteritidis regional markers might exist and could be used for trace-back investigations. IMPORTANCE This study highlights the importance of gulls in the spread of Salmonella Enteritidis in Chile. We revealed a close genetic relationship between some human and gull S. Enteritidis strains (with as few as 2 of 4,065 genes being different), and we also found that gull strains were present in clusters formed by strains isolated from other sources or distant locations. Together with previously published evidence, this suggests that gulls might be spreading this pathogen between different regions in Chile and that some of those strains have been transmitted to humans. Moreover, we discovered that Chilean S. Enteritidis strains clustered separately from most of S. Enteritidis strains isolated throughout the world (in the GenBank database) and thus it might be possible to distinguish the geographical origins of strains based on specific genomic features. This could be useful for trace-back investigations of foodborne illnesses throughout the world.

Escherichia coli O26 is the second most important enterohemorrhagic E. coli (EHEC) serogroup worldwide. Serogroup O26 strains are categorized mainly into two groups: enteropathogenic (EPEC) O26, carrying a locus of enterocyte effacement (LEE) and mostly causing mild diarrhea, and Shiga-toxigenic (STEC) O26, which carries the Shiga toxin (STX) gene (stx), responsible for more severe outcomes. stx-negative O26 strains can be further split into two groups. One O26 group differs significantly from O26 EHEC, while the other O26 EHEC-like group shows all the characteristics of EHEC O26 except production of STX. In order to determine the different populations of O26 E. coli present in U.S. cattle, we sequenced 42 O26:H11 strains isolated from feedlot cattle and compared them to 37 O26:H11 genomes available in GenBank. Phylogenetic analysis by whole-genome multilocus sequence typing (wgMLST) showed that O26:H11/H ؊ strains in U.S. cattle were highly diverse. Most strains were sequence type 29 (ST29). By wgMLST, two clear lineages could be distinguished among cattle strains. Lineage 1 consisted of O26:H11 EHEC-like strains (ST29) (4 strains) and O26:H11 EHEC strains (ST21) (2 strains), and lineage 2 (36 strains) consisted of O26: H11 EPEC strains (ST29). Overall, our analysis showed U.S. cattle carried pathogenic (ST21; stx 1 ؉ ehxA ؉ toxB ؉ ) and also potentially pathogenic (ST29; ehxA ؉ toxB ؉ ) O26:H11 E. coli strains. Furthermore, in silico analysis showed that 70% of the cattle strains carried at least one antimicrobial resistance gene. Our results showed that whole-genome sequence analysis is a robust and valid approach to identify and genetically characterize E. coli O26:H11, which is of importance for food safety and public health. IMPORTANCEEscherichia coli O26 is the second most important type of enterohemorrhagic E. coli (EHEC) worldwide. Serogroup O26 strains are categorized into two groups: enteropathogenic (EPEC) carrying LEE, causing mild diarrhea, and Shiga toxigenic (STEC) carrying the stx gene, responsible for more severe outcomes. However, there are currently problems in distinguishing one group from the other. Furthermore, several O26 stx-negative strains are consistently misidentified as either EHEC-like or EPEC. The use of whole-genome sequence (WGS) analysis of O26 strains from cattle in the United States (i) allowed identification of O26 strains present in U.S. cattle, (ii) determined O26 strain diversity, (iii) solved the misidentification problem, and (iv) screened for the presence of antimicrobial resistance and virulence genes in the strains. This study provided a framework showing how to easily and rapidly use WGS information to identify and genetically characterize E. coli O26:H11, which is important for food safety and public health.

BackgroundThermotolerant Campylobacter is among the more prevalent bacterial pathogens that cause foodborne diseases. This study aimed at evaluating the occurrence of thermotolerant Campylobacter contamination in chicken carcasses and processing plant stations (chilling water, scalding water, defeathering machinery, evisceration machine, and transport crates) in two of the Chilean main slaughterhouses. In addition, the isolation rates of thermotolerant Campylobacter during evisceration and following chiller processing were compared.ResultsThe overall slaughterhouse contamination with thermotolerant Campylobacter was 54%. Differences were evident when the results from each plant were compared (plant A and plant B was 72% and 36%, respectively). The sampling points with the greatest contamination rates in both plants were after evisceration (90% and 54%, for plants A and B respectively). The decrease of thermotolerant Campylobacter contamination after chilling was significant (2 and 1.6 logs for plant A and B respectively P < 0.05).ConclusionOur findings indicate that chilling process has a limited effect in the final products Campylobacter contamination because poultry enter the slaughter processing with high counts of contamination. This may represent a health risk to consumers, if proper cooking practices are not employed. The levels and frequencies of Campylobacter found during the processing of Chilean poultry appear to be similar to those reported elsewhere in the world.