Antimicrobial Resistance Dynamics in Chilean Shigella sonnei Strains Within Two Decades: Role of Shigella Resistance Locus Pathogenicity Island and Class 1 and Class 2 Integrons.

Shigellosis is an enteric infectious disease in which antibiotic treatment is effective, shortening the duration of symptoms and reducing the excretion of the pathogen into the environment. Shigella spp., the etiologic agent, are considered emerging pathogens with a high public health impact due to the increase and global spread of multidrug-resistant (MDR) strains. Since Shigella resistance phenotype varies worldwide, we present an overview of the resistance phenotypes and associated genetic determinants present in 349 Chilean S. sonnei strains isolated during the periods 1995-1997, 2002-2004, 2008-2009, and 2010-2013. We detected a great variability in antibiotic susceptibility patterns, finding 300 (86%) MDR strains. Mobile genetic elements (MGE), such as plasmids, integrons, and genomic islands, have been associated with the MDR phenotypes. The Shigella resistance locus pathogenicity island (SRL PAI), which encodes for ampicillin, streptomycin, chloramphenicol, and tetracycline resistance genes, was detected by PCR in 100% of the strains isolated in 2008-2009 but was less frequent in isolates from other periods. The presence or absence of SRL PAI was also differentiated by pulsed-field gel electrophoresis. An atypical class 1 integron which harbors the bla OXA-1 -aadA1-IS1 organization was detected as part of SRL PAI. The dfrA14 gene conferring trimethoprim resistance was present in 98.8% of the 2008-2009 isolates, distinguishing them from the SRL-positive strains isolated before that. Thus, it seems an SRL-dfrA14 S. sonnei clone spread during the 2008-2009 period and declined thereafter. Besides these, SRL-negative strains harboring class 2 integrons with or without resistance to nalidixic acid were detected from 2011 onward, suggesting the circulation of another clone. Whole-genome sequencing of selected strains confirmed the results obtained by PCR and phenotypic analysis. It is highlighted that 70.8% of the MDR strains harbored one or more of the MGE evaluated, while 15.2% lacked both SRL PAI and integrons. These results underscore the temporal dynamics of antimicrobial resistance in S. sonnei strains circulating in Chile, mainly determined by the spread of MGE conferring MDR phenotypes. Since shigellosis is endemic in Chile, constant surveillance of antimicrobial resistance phenotypes and their genetic basis is a priority to contribute to public health policies.

SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Emergence of Plasmid-Borne dfrA14 Trimethoprim Resistance Gene in Shigella sonnei.

The most common mechanism of trimethoprim (TMP)-resistance is the acquisition of dihydrofolate reductase enzyme resistant to this drug. Previous molecular characterization of TMP-genes resistance in Chilean isolates of Shigella sonnei searching for dfrA1 and dfrA8, showed solely the presence of dfrA8 (formerly dhfrIIIc). However, these genetic markers were absent in S. sonnei strains further isolated during an outbreak in 2009. To identify the TMP-resistance gene in these strains, a genomic DNA library from a TMP-resistant (TMP(R)) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP(R) clone was sequenced by primer walking, identifying the presence of the dfrA14 gene in the sul2-strA'-dfrA14-'strA-strB gene arrangement, harbored in a native 6779-bp plasmid. The same plasmid was isolated by transforming with a ~4.2 MDa plasmid extracted from several TMP(R) S. sonnei strains into Escherichia coli. This plasmid, named pABC-3, was present only in dfrA14-positive strains and was homologous to a previously described pCERC-1, but different due to the absence of an 11-bp repetitive unit. The distribution of dfrA1, dfrA8, and dfrA14 TMP-resistance genes was determined in 126 TMP(R) S. sonnei isolates. Most of the strains (96%) carried only one of the three TMP-resistance genes assessed. Thus, all strains obtained during the 2009-outbreak harbored only dfrA14, whereas, dfrA8 was the most abundant gene marker before outbreak and, after the outbreak dfrA1 seems have appeared in circulating strains. According to PFGE, dfrA14-positive strains were clustered in a genetically related group including some dfrA1- and dfrA8-positive strains; meanwhile other genetic group included most of the dfrA8-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean S. sonnei strains. To our knowledge, dfrA14 gene associated to a small non-conjugative plasmid was detected for the first time in Shigella. Apparently, the strain causing the outbreak must have been introduced, changing drastically the genetic distribution of trimethoprim resistance in Chilean S. sonnei strains.

Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp.

Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events.

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

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.

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.

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 DeltaSPI-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.

Spontaneous excision of the Salmonella enterica serovar Enteritidis-specific defective prophage-like element phiSE14.

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an approximately 12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5' end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of phiSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of phiSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of phiSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that phiSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of phiSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.

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.

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.