Cecilia S. Toro

Single Multiplex PCR Assay To Identify Simultaneously the Six Categories of Diarrheagenic Escherichia coli Associated with Enteric Infections

ABSTRACT We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx1, stx2, and eae), enteropathogenic (eae and bfp), enterotoxigenic (stII and lt), enteroinvasive (virF and ipaH), enteroaggregative (aafII), and diffuse adherent (daaE) Escherichia coli in stool samples.

Contribution of the Type VI Secretion System Encoded in SPI-19 to Chicken Colonization by Salmonella enterica Serotypes Gallinarum and Enteritidis

Salmonella Gallinarum is a pathogen with a host range specific to poultry, while Salmonella Enteritidis is a broad host range pathogen that colonizes poultry sub-clinically but is a leading cause of gastrointestinal salmonellosis in humans and many other species. Despite recent advances in our understanding of the complex interplay between Salmonella and their hosts, the molecular basis of host range restriction and unique pathobiology of Gallinarum remain largely unknown. Type VI Secretion System (T6SS) represents a new paradigm of protein secretion that is critical for the pathogenesis of many Gram-negative bacteria. We recently identified a putative T6SS in the Salmonella Pathogenicity Island 19 (SPI-19) of Gallinarum. In Enteritidis, SPI-19 is a degenerate element that has lost most of the T6SS functions encoded in the island. In this work, we studied the contribution of SPI-19 to the colonization of Salmonella Gallinarum strain 287/91 in chickens. Non-polar deletion mutants of SPI-19 and the clpV gene, an essential T6SS component, colonized the ileum, ceca, liver and spleen of White Leghorn chicks poorly compared to the wild-type strain after oral inoculation. Return of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. In contrast, transfer of SPI-19 from Gallinarum to Enteritidis resulted in transient increase in the colonization of the ileum, liver and spleen at day 1 post-infection, but at days 3 and 5 post-infection a strong colonization defect of the gut and internal organs of the experimentally infected chickens was observed. Our data indicate that SPI-19 and the T6SS encoded in this region contribute to the colonization of the gastrointestinal tract and internal organs of chickens by Salmonella Gallinarum and suggest that degradation of SPI-19 T6SS in Salmonella Enteritidis conferred an advantage in colonization of the avian host.

Genetic analysis of antibiotic-resistance determinants in multidrug-resistant Shigella strains isolated from Chilean children

A total of 162 clinical isolates of Shigella collected from children in a semi-rural community of Chile were examined for the presence of genetic determinants of resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim. Ampicillin resistance was most frequently associated with the presence of bla(OXA) in S. flexneri and with bla(TEM) in S. sonnei. The bla(OXA) gene but not bla(TEM) was located in class 1 integrons. The dhfrIa gene encoding for resistance to trimethoprim was associated to class 2 integrons and detected exclusively in S. flexneri, whereas dhfrIIIc was found in all S. sonnei strains and in 10% of the S. flexneri isolates. Cat, coding for choramphenicol resistance, and bla(OXA) genes were located in the chromosome in all cases, whereas tetA gene, coding for tetracycline resistance, and bla(TEM), dhfrIa and dhfrIIIc genes were found either in the chromosome or in conjugative plasmids. Our results show a heterogenous distribution of antibiotic-resistance determinants between S. flexneri and S. sonnei.

Salmonella typhi mutants defective in anaerobic respiration are impaired in their ability to replicate within epithelial cells

By using MudJ (Kan, lac)-directed operon fusion technology, mutants of Salmonella typhi whose gene expression is induced under anaerobic growth conditions were isolated. Characterization of their phenotypes and regulatory properties revealed that two of the mutants were unable to use nitrate as a terminal electron acceptor in the absence of oxygen, suggesting that they were defective in nitrate reductase activity. Anaerobic induction of these fusions did not further increase in response to nitrate. Strains carrying an additional mutation in oxrA were constructed. They showed a lower level of beta-galactosidase expression both aerobically and anaerobically; however, the ratios of anaerobic induction remained unaltered. These MudJ insertions mapped to the 17-19 min region of the chromosome. Based upon their phenotypes and mapping, one of the mutants probably possessed a modC (chlD)::MudJ insertion and the other a moaA (chlA)::MudJ insertion. A third mutant was unable to use either nitrate or fumarate as a terminal electron acceptor. All three mutants showed a reduced ability to enter into and proliferate within HEp-2 epithelial cells. The oxrA mutation enhanced entry and proliferation of both the wild-type cells and the three mutants. Taken together, these results suggest that anaerobic respiration plays a role in S. typhi invasiveness.

Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium

ABSTRACT Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models.

Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol.

We studied a clinical isolate of Salmonella typhi (strain 1895) characterized by resistance to 200 micrograms of chloramphenicol per ml despite the absence of chloramphenicol-inactivating activity. The outer membrane protein profile analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a deficiency of one of the major protein species which may serve as a porin for entry of chloramphenicol. When the strain was transformed with a plasmid encoding chloramphenicol acetyltransferase, chloramphenicol added to the culture was not inactivated, suggesting a drastic reduction of permeability towards the drug. Moreover, transformants bearing a plasmid coding for the Escherichia coli OmpF porin became considerably more susceptible to chloramphenicol (40 micrograms/ml). On the other hand, transformants carrying a plasmid encoding the Salmonella typhi ompC gene remained as resistant to the drug as the parental strain, even though they overexpressed OmpC. These findings indicate that the lack of OmpF plays a major role in the resistance to chloramphenicol in strain 1895. Images

Genetic Characterization of Antibiotic Resistance Genes Linked to Class 1 and Class 2 Integrons in Commensal Strains of Escherichia coli Isolated from Poultry and Swine

The aim of this research was to identify the presence of integrons among Escherichia coli strains isolated from poultry and swine and to characterize the topological association of these integrons with resistance genes and assess their potential ability to transfer these elements by conjugation. One hundred and seventy-two strains of E. coli were isolated. Their resistance to tetracycline, streptomycin, sulfamethoxazole-trimethoprim, ciprofloxacin, and enrofloxacin was studied by plate dilution. In resistant strains the presence of integrons and resistance genes was assessed by PCR. In the variable region, genes aadA1, dfrA1, and qnr were analyzed. Also, presence of tetA, tetB, and sul1 was assessed. Transference of these genes and integrons in vitro was evaluated by conjugation assays, using E. coli J53 Az(r) as recipient strain. Seventy-eight percent and 83% of the poultry and swine strains, respectively, were resistant to at least one of the studied antimicrobials. Of the isolated strains 91 presented integrons. Resistance genes detected within the integrons were aadA1, dfrA1, and sat1. Gene qnr was not detected. Genes tet and sul1 were identified in 105 and 53 strains, respectively. Seven strains transferred their resistance determinants by conjugation. The results verify the high percentage of antibiotic resistance in the E. coli strains isolated, and these represent a reservoir of resistance genes and integrons.

Global Regulation of the Salmonella enterica Serovar Typhimurium Major Porin, OmpD

The OmpD porin is the most abundant outer membrane protein in Salmonella enterica serovar Typhimurium and represents about 1% of total cell protein. Unlike the case with the less abundant OmpC and OmpF porins, the stoichiometry of OmpD in the outer membrane does not change in response to changes in osmolarity. The abundance of OmpD increases in response to anaerobiosis and decreases in response to low pH, conditions encountered by serovar Typhimurium during the infection of its murine host. By constructing an operon fusion of the lacZY genes with the ompD promoter, we show that the abundance of OmpD in the outer membrane is regulated primarily at the level of transcription and is subject to catabolite repression. In response to anaerobiosis, the abundance of OmpD in the outer membrane also appears to be controlled posttranscriptionally by a function dependent on Fnr.

A chromosomal region surrounding the ompD porin gene marks a genetic difference between Salmonella typhi and the majority of Salmonella serovars

In this work it is shown that the majority of Salmonella serovars most frequently associated with the systemic infection of vertebrate hosts produce a major outer-membrane porin, OmpD. However, OmpD is absent from the outer-membrane protein profiles of Salmonella typhi strain Ty2 and 26 clinical isolates of S. typhi examined by SDS-PAGE. To determine whether the ompD gene is present in S. typhi, primers internal to the ompD coding sequence were used to amplify the gene by PCR. With the exception of S. typhi strains, the ompD gene was amplified from the genomes of all Salmonella serovars tested. Consistently, a specific ompD probe did not hybridize with DNA isolated from the S. typhi strains. Taken together, these results demonstrate that S. typhi does not produce OmpD due to the absence of the ompD gene. Furthermore, it was investigated whether the deletion of ompD extended to smvA. This gene is adjacent to ompD in the Salmonella typhimurium chromosome and encodes a protein involved in the resistance to methyl viologen, a superoxide-generating agent. Although PCR failed to amplify the smvA gene from the S. typhi strain Ty2 genome, it was possible to amplify it from the chromosome of the clinical strains. On the other hand, hybridization analyses showed that the smvA gene is present in all the S. typhi strains tested. In contrast to the other Salmonella serovars, S. typhi strain Ty2 and the clinical isolates showed sensitivity to methyl viologen, suggesting that smvA gene is inactive in S. typhi. In conclusion, the ompD-smvA region is variable in structure among Salmonella serovars. It is hypothesized that the absence of ompD may suggest a role in host specificity.

Shigella enterotoxin‐2 is a type III effector that participates in Shigella‐induced interleukin 8 secretion by epithelial cells

We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.