Two novel antibiotic resistance genes, tet(44) and ant(6)-Ib, are located within a transferable pathogenicity island in Campylobacter fetus subsp. fetus

New tetracycline and streptomycin resistance genes, tet(44) and ant(6)-Ib, were identified in Campylobacter fetus subsp. fetus within a transferable pathogenicity island that is typically unique to Campylobacter fetus subsp. venerealis. The 640-amino-acid tetracycline resistance determinant, Tet 44, belongs to a class of proteins that confers resistance to tetracycline and minocycline by ribosomal protection. The 286-amino-acid streptomycin resistance determinant, ANT(6)-Ib, belongs to a family of aminoglycoside nucleotidyltransferases. The resistance phenotypes were demonstrated by gene inactivation and expression.

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms.

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.

Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.

Comparison of Salmonella enterica serovar Typhimurium colitis in germfree mice and mice pretreated with streptomycin.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of bacterial enterocolitis. Mice are generally protected from Salmonella serovar Typhimurium colonization and enterocolitis by their resident intestinal microflora. This phenomenon is called "colonization resistance" (CR). Two murine Salmonella serovar Typhimurium infection models are based on the neutralization of CR: (i) in specific-pathogen-free mice pretreated with streptomycin (StrSPF mice) antibiotics disrupt the intestinal microflora; and (ii) germfree (GF) mice are raised without any intestinal microflora, but their intestines show distinct physiologic and immunologic characteristics. It has been unclear whether the same pathogenetic mechanisms trigger Salmonella serovar Typhimurium colitis in GF and StrSPF mice. In this study, we compared the two colitis models. In both of the models Salmonella serovar Typhimurium efficiently colonized the large intestine and triggered cecum and colon inflammation starting 8 h postinfection. The type III secretion system encoded in Salmonella pathogenicity island 1 was essential in both disease models. Thus, Salmonella serovar Typhimurium colitis is triggered by similar pathogenetic mechanisms in StrSPF and GF mice. This is remarkable considering the distinct physiological properties of the GF mouse gut. One obvious difference was more pronounced damage and reduced regenerative response of the cecal epithelium in GF mice. Overall, StrSPF mice and GF mice provide similar but not identical models for Salmonella serovar Typhimurium colitis.

Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of gastrointestinal infections. The host's innate immune system and a complex set of Salmonella virulence factors are thought to contribute to enteric disease. The serovar Typhimurium virulence factors have been studied extensively by using tissue culture assays, and bovine infection models have been used to verify the role of these factors in enterocolitis. Streptomycin-pretreated mice provide an alternative animal model to study enteric salmonellosis. In this model, the Salmonella pathogenicity island 1 type III secretion system has a key virulence function. Nothing is known about the role of other virulence factors. We investigated the role of flagella in murine serovar Typhimurium colitis. A nonflagellated serovar Typhimurium mutant (fliGHI) efficiently colonized the intestine but caused little colitis during the early phase of infection (10 and 24 h postinfection). In competition assays with differentially labeled strains, the fliGHI mutant had a reduced capacity to get near the intestinal epithelium, as determined by fluorescence microscopy. A flagellated but nonchemotactic cheY mutant had the same virulence defects as the fliGHI mutant for causing colitis. In competitive infections, both mutants colonized the intestine of streptomycin-pretreated mice by day 1 postinfection but were outcompeted by the wild-type strain by day 3 postinfection. Together, these data demonstrate that flagella are required for efficient colonization and induction of colitis in streptomycin-pretreated mice. This effect is mostly attributable to chemotaxis. Recognition of flagellar subunits (i.e., flagellin) by innate immune receptors (i.e., Toll-like receptor 5) may be less important.

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ? 4/5 methylated genes), MSI (MSI-H: ? 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

Island Invasion by a Threatened Tree Species: Evidence for Natural Enemy Release of Mahogany (Swietenia macrophylla) on Dominica, Lesser Antilles

Despite its appeal to explain plant invasions, the enemy release hypothesis (ERH) remains largely unexplored for tropical forest trees. Even scarcer are ERH studies conducted on the same host species at both the community and biogeographical scale, irrespective of the system or plant life form. In Cabrits National Park, Dominica, we observed patterns consistent with enemy release of two introduced, congeneric mahogany species, Swietenia macrophylla and S. mahagoni, planted almost 50 years ago. Swietenia populations at Cabrits have reproduced, with S. macrophylla juveniles established in and out of plantation areas at densities much higher than observed in its native range. Swietenia macrophylla juveniles also experienced significantly lower leaf-level herbivory (~3.0%) than nine co-occurring species native to Dominica (8.4–21.8%), and far lower than conspecific herbivory observed in its native range (11%–43%, on average). These complimentary findings at multiple scales support ERH, and confirm that Swietenia has naturalized at Cabrits. However, Swietenia abundance was positively correlated with native plant diversity at the seedling stage, and only marginally negatively correlated with native plant abundance for stems ≥1-cm dbh. Taken together, these descriptive patterns point to relaxed enemy pressure from specialized enemies, specifically the defoliator Steniscadia poliophaea and the shoot-borer Hypsipyla grandella, as a leading explanation for the enhanced recruitment of Swietenia trees documented at Cabrits.

The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer

In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high-level microsatellite instability (MSI-H). Cases with MSI-H or high-level CpG island methylator phenotype (CIMP-H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference.