Salmonella Enteritidis foodborne infection induces altered placental morphometrics in the murine model.

INTRODUCTION: Salmonella foodborne disease during pregnancy causes a significant fetal loss in domestic livestock and preterm birth, chorioamnionitis and miscarriage in humans. These complications could be associated with alterations in placental structure. This study was aimed to determine how a low dose of Salmonella Enteritidis during late gestation affects placental histomorphometric in mice. METHODS: We used a self-limiting enterocolitis murine model. BALB/c pregnant animals received a low dose of Salmonella Enteritidis (3-4 x 102 CFU/mouse) on gestational day (GD) 15. At day 3 post infection bacterial loads, serum cytokines expression and placental histomorphometrics parameters were analyzed. RESULTS: We found that a sub-lethal infection with Salmonella induced a significant drop in fetal weight -to-placental weight-ratio and an increase in the placental coefficient. After bacterial inoculation maternal organs were colonized, inducing placental morphometric alterations, including increased placental thickness, reduced surface area, and diminished major and minor diameters. Also, foci of necrosis accompanied by acute leukocyte infiltration in decidual zone, reduction of vascular spaces and vascular congestion in labyrinth zone, were also evident in placentas from infected females on GD 18. Our data shows that placentas from infected mothers are phenotypically different from control ones. Furthermore, expression of IFN-gamma and IL-6 was up regulated in response to Salmonella in maternal serum. DISCUSSION: Our findings demonstrate that a low dose of Salmonella during late gestation alters the placental morphometry leading to negative consequences on pregnancy outcome such as significant reduction in fetal body weight.

Colonization by multidrug-resistant organisms in long-term care facilities in Italy: a point-prevalence study.

OBJECTIVES: To determine prevalence and risk factors for colonization by multidrug-resistant organisms (MDROs) in long-term care facility (LTCF) residents in Italy. Genotypes of MDRO isolates were investigated. METHODS: A point-prevalence study was conducted at 12 LTCFs located in four Italian cities (2 February to 14 March 2015). Rectal swabs, faeces and nasal/auxiliary swabs were cultured for extended-spectrum ß-lactamase (ESBL)- and/or carbapenemase-producing Enterobacteriaceae, Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA) respectively. Antimicrobial susceptibility testing, detection of ESBL and/or carbapenemase genes and molecular typing of MDROs were performed. Risk factors for colonization were determined by univariate and multivariate analysis. RESULTS: A total of 489 LTCF residents aged ≥65 years were enrolled. The prevalence of colonization by ESBL-producing Enterobacteriaceae, MRSA and C. difficile was 57.3% (279/487), 17.2% (84/487) and 5.1% (21/409) respectively. Carriage rate of carbapenemase-producing Enterobacteriaceae was 1% (5/487). Being bedridden was a common independent risk factor for colonization by all MDROs, although risk factors specific for each MDRO were identified. ESBL-producing Escherichia coli carriage was associated with the sequence type (ST) 131-H30 subclone, but other minor STs predominated in individual LTCF or in LTCFs located in the same city, suggesting a role for intrafacility or local transmission. Similarly, MRSA from LTCF residents belonged to the same spa types/ST clones (t008/ST8 and t032/ST22) commonly found in Italian acute-care hospitals, but infrequent spa types were recovered in individual LTCFs. The prevalent C. difficile PCR ribotypes were 356/607 and 018, both common in Italian acute-care hospitals. CONCLUSIONS: MDRO colonization is common among residents in Italian LTCFs.

IncI1 plasmids associated with the spread of CMY-2, CTX-M-1 and SHV-12 in Escherichia coli of animal and human origin.

Fourteen plasmids carrying blaCTX -M-1, blaSHV -12 or blaCMY -2 genes from Escherichia coli of both avian and human origin were analysed. IncI1 plasmids were largely predominant. Plasmid mutilocus sequence typing and comparative analysis revealed that the blaCMY -2 -ST12-IncI1 plasmids from avian E. coli were identical to those previously found in Salmonella from humans, but different to those associated with human E. coli. The IncI1-ST3 plasmids carrying blaCTX -M-1 or blaSHV -12 were related to those previously identified in avian E. coli, but different to those identified in human E. coli. Overall, no plasmids shared by E. coli of both origin (human/avian) were identified; however, further investigations are needed.

Ciprofloxacin-resistant, CTX-M-15-producing Escherichia coli ST131 clone in extraintestinal infections in Italy.

Quinolone and ß-lactam resistance mechanisms and clonal relationships were characterized among Escherichia coli isolates resistant to ciprofloxacin and extended-spectrum cephalosporins associated with human extra-intestinal infections in Rome. The E. coli. ST131 clone was found to be prevalent. This clone invariably carried a specific pattern of substitutions in the topoisomerase genes and all isolates but one produced CTX-M-15. One ST131 isolate produced SHV-12. The new ST131 variant described here is of particular concern because it combines fluoroquinolone resistance and chromosomally encoded CTX-M-15.

Dam and its role in pathogenicity of Salmonella enterica.

Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella enterica. Lack of Dam methylation causes severe attenuation in animal models. It has been proposed that dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. In this review, we focus our attention on the role of methylation by Dam protein in regulating bacterial gene expression and virulence in Salmonella enterica.

Sublethal infection with Salmonella Enteritidis by the natural route induces intestinal and joint inflammation in mice.

Reactive arthritis (ReA) is a sterile inflammation triggered by a distal mucosal infection, which suggests a contribution from bacterial products. Investigation on the pathogenesis of ReA is difficult because of the limited studies that can be performed in humans; therefore the availability of animal models is crucial. We hereby describe a murine model for studying the early stages of Salmonella-induced ReA. BALB/c mice infected by the natural route with a sublethal dose of S. Enteritidis showed long lasting gut inflammation, synovitis in the knee joint and a significant increase of CD4+ lymphocytes in the draining popliteal lymph nodes. S. Enteritidis infection induced histological changes in intact knees and exacerbated inflammation in previously damaged joints. Experiments performed with S. Enteritidis DeltainvG mutant suggest that the proinflammatory signalling mediated by Salmonella TTSS-1 in the gut is required for the induction of joint sequelae. Since this model is highly reproducible and easy to perform, it provides great potential for investigating both host and bacterial contributions to the early stages of ReA.

Salmonella enterica serovar Enteritidis dam mutant induces low NOS-2 and COX-2 expression in macrophages via attenuation of MAPK and NF-kappaB pathways.

Although dam mutants of Salmonella have been proposed as live vaccines, their capacity to trigger cell inflammatory cascades has not been fully elucidated. We investigated in detail the ability of Salmonella enterica dam mutant to activate the signalling pathways of the inflammatory response in RAW 264.7 cells. Apoptosis in macrophages treated with Salmonella dam mutant was low. Similarly, the expression of both NOS-2 and COX-2 and subsequently the production of NO and PGE(2) was significantly reduced. Also, Salmonella dam mutant induced an attenuated activation of the inflammatory signalling pathway as indicated by the reduced degradation of IkappaBalpha and IkappaBbeta and the low IkappaBalpha phosphorylation found. In addition, translocation of p65 to the nucleus was notably impaired and the amount of phosphorylated p44, p42 and p38 MAPKs was clearly reduced in extracts from dam-infected macrophages. These results indicate that the lack of ERK and p38 phosphorylation at the proper time in dam-infected cells notably reduces the engagement of subsequent signalling pathways involved in the full activation of NF-kappaB in response to infection. Taken together, these results suggest that Salmonella activation of both signalling cascades in the inflammatory response is a mechanism requiring Dam protein participation.

SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium are synthesized at late stages of infection in mice.

Salmonella pathogenicity island (SPI)-1 is essential for invasion of non-phagocytic cells, whereas SPI-2 is required for intracellular survival and proliferation in phagocytes. Some SPI-1 effectors, however, are induced upon invasion of both phagocytic and non-phagocytic cells, suggesting that they may also be required post-invasion. In the present work, the presence was analysed of SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium in vitro and in vivo during murine salmonellosis. Tagged (3xFLAG) strains of S. enterica serovar Typhimurium were inoculated intraperitoneally or intragastrically to BALB/c mice and recovered from the spleen and mesenteric lymph nodes of moribund mice. Tagged proteins were detected by SDS-PAGE and immunoblotting with anti-FLAG antibodies. In vitro experiments showed that SPI-1 effector proteins SipA, SopA, SopB, SopD and SopE2 were secreted under SPI-1 conditions. Interestingly, it was found that S. enterica serovar Typhimurium continued to synthesize SipA, SopB, SopD and SopE2 in colonized organs for several days, regardless of the route of inoculation. Together, these results indicate that SPI-1 effector proteins may participate in the late stages of Salmonella infection in mice.

Host response to a dam mutant of Salmonella enterica serovar enteritidis with a temperature-sensitive phenotype.

The temperature-sensitive dam mutant strain of Salmonella enterica serovar Enteritidis SD1 is highly attenuated and induces innate and protective immunity in mice. SD1 activates NF-kappaB and induces gamma interferon secretion. Early interaction of the SD1 mutant with intestinal epithelial cells was associated with ruffling of enterocytes. Invading bacteria were found inside Peyer's patches after inoculation.

Involvement of intestinal inducible nitric oxide synthase (iNOS) in the early stages of murine salmonellosis.

Local induction of inducible nitric oxide synthase (iNOS) and apoptosis was examined in the intestine of mice infected with virulent Salmonella enterica serovar Enteritidis 5694 (S. enteritidis) and its attenuated derivative mutant E/1/3. Both, intestinal iNOS mRNA expression and iNOS activity showed a peak at 4 h only in animals receiving the virulent S. enteritidis. Aminoguanidine treatment abrogated intestinal epithelial damage produced by virulent S. enteritidis and diminished apoptosis at the tips of the villi. Unlike the virulent strain, mutant E/1/3 induced massive iNOS expression in Peyer's patches, these findings may be related to its protective capacity. Our results suggest that intestinal iNOS participates in the early response to intestinal infection and that the final effect depends on the nature of the insult.

VMP1 expression correlates with acinar cell cytoplasmic vacuolization in arginine-induced acute pancreatitis.

BACKGROUND: Recently, we described the cloning of VMP1 (vacuole membrane protein 1). In vitro expression of VMP1 promotes formation of cytoplasmic vacuoles followed by cell death. In order to test if VMP1 expression is related to the cytoplasmic vacuolization of the acinar cells during acute pancreatitis, we studied the in vivo expression of the new gene during arginine-induced acute pancreatitis. METHODS: Male Wistar rats injected with 500 mg/100 g of L-arginine were time-course sacrificed and pancreas tissue removed. RESULTS: Northern blot analysis showed maximal induction of VMP1 after 24 h remaining high after 48 h of arginine administration. Significant increase in the number of TUNEL-stained cells were found at those periods. After 24 and 48 h of arginine administration, light micrographs from thin plastic toluidine blue sections revealed numerous vacuoles in the cytoplasm of acinar cells. In situ hybridization studies showed high expression of VMP1 in acinar cells with cytoplasmic vacuolization. VMP1 mRNA highly and significantly correlated with vacuole formation. CONCLUSION: These results suggest that VMP1 expression may be involved in the cytoplasmic vacuolization of acinar cells during the early stage of acute pancreatitis.

Nitric oxide and apoptosis induced in Peyer's patches by attenuated strains of Salmonella enterica serovar Enteritidis.

Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection by Salmonella spp. In this work we investigated the ability of attenuated strains of Salmonella enterica serovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyer's patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within the first few hours after immunization seemed to correlate with the protective capacity of mutant E/1/3 of S. enterica serovar Enteritidis. It was found that nonprotective mutant C/2/2, which was unable to induce intestinal NO production, also failed to induce apoptosis in PP. Moreover, aminoguanidine treatment at the time of immunization resulted in inhibition of the NO production and apoptosis induced by protective mutant E/1/3 and completely abolished protection against challenge. These results suggest that the induction of iNOS in the intestinal mucosa by attenuated mutant E/1/3 of S. enterica serovar Enteritidis at the time of immunization is necessary to generate a protective immune response.

Molecular and genomic analysis of genes encoding surface-anchored proteins from Clostridium difficile.

The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79--685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79--685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.

Characterization of non-type B Haemophilus influenzae strains isolated from patients with invasive disease. The HI Study Group.

Forty-one non-type b Haemophilus influenzae isolates from cases of invasive disease were characterized. By PCR capsular genotyping, 33 nonencapsulated strains, 4 type f isolates, and 4 b(-) strains were identified. By pulsed-field gel electrophoresis, the nonencapsulated isolates exhibited great genetic heterogenicity, whereas the type f and the b(-) strains seemed to have a clonal spread. Occurrence of the hifA gene was found by PCR in 18% of the nonencapsulated, 50% of the b(-), and all of the type f strains. Hemagglutinating fimbriae were generally expressed by nonencapsulated isolates when fimbrial gene hifA was present. Two nonencapsulated isolates not susceptible to ampicillin were detected; no strains were positive for beta-lactamase production.

Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes.

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.

Orally administered attenuated Salmonella enteritidis reduces chicken cecal carriage of virulent Salmonella challenge organisms.

Chickens were immunized orally with 10(9)cfu of the temperature-sensitive (T(s)) mutant E/1/3 of Salmonella enteritidis at 1, 2, 3 and 7 days of age. The animals were challenged with wild-type strains of Salmonella of different serotypes 7 or 14 days following immunization. Chickens receiving multiple oral doses of the vaccine strain showed no signs of disease. Immunized animals shed the vaccine strain for at least 2 weeks after the last inoculation; on the other hand, colonization by the attenuated mutant of internal organs such as spleen and liver was limited. Early exposure of the immunized animals to the virulent bacteria resulted in a reduced cecal colonization by the pathogen. Visceral invasion by the wild-type strain of S. enteritidis or S. gallinarum was drastically diminished in birds challenged 14 days after immunization. Significant differences in the number of these Salmonella were found in the cecal contents, spleen and liver of immunized birds compared with the control animals. In addition, cecal colonization by the virulent strain was reduced in birds challenged with S. typhimurium. These results demonstrate that immunization of newly hatched chickens with live attenuated T(s) mutant E/1/3 of S. enteritidis is safe and reduces Salmonella shedding.

Characterization of surface layer proteins from different Clostridium difficile clinical isolates.

In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall. In the present paper six C. difficile strains isolated from cases and healthy carriers were studied. By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C. difficile strains. In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry. The S-layer proteins from the different strains were isolated and characterized. Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36-56 kDa. Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses. N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins. These data indicate that the presence of S-layer on C. difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity.

Vaccination of chickens with a temperature-sensitive mutant of Salmonella enteritidis.

One-day old chickens were inoculated with temperature-sensitive mutant E/1/3 of S. enteritidis. Two routes of inoculation were used: oral and intraperitoneal (ip). One group of chickens were given two oral inoculations (oral-oral). A second group received two ip inoculations (ip-ip). A third group received the first dose orally and the second ip (oral-ip) and the fourth group was given the first dose ip and the second dose orally (ip-oral). The vaccine strain was safe even when inoculated at high doses, and induced strong protection against virulent S. enteritidis strain after oral challenge. Results show that vaccination with mutant E/1/3 reduced the number of animals shedding the pathogen after challenge. Furthermore, animals immunized oral-oral and oral-ip showed a significant reduction in cecal and spleen colonization by virulent Salmonella.

Immunogenicity issues in the quality control of the new acellular pertussis vaccines.

Over the last few years our laboratory has been assessing the consistency of production of different batches of acellular pertussis vaccines to be marketed in Italy. Central to this is immunogenicity assay of the lots under control compared with those of a reference vaccine with documented clinical efficacy.However, the current assays based on the assessment of antibody (Ab) response in the mouse are unrelated to mechanisms of protection in children. The absence of a clear correlation between Ab responses and protection has also been documented in recent clinical trials. On this basis, we are currently considering the possibility of adding to the established criteria of immunogenicity in mice based on Ab responses, information from studies on cell-mediated immune responses to the vaccine constituents.