Excess length of hospital stay due to healthcare acquired infections: methodologies evaluation.

BACKGROUND: Healthcare acquired infections (HAI) cause an increase of burden and in particular excess length of hospital stay (LOS) accounts for approximately up to 90% of total costs. Therefore accurate estimation of extra hospital stay due to healthcare acquired infections is very important. METHODS: The authors carried out a review comparing the principal methods internationally used for estimating the excess LOS attributable to healthcare acquired infections. RESULTS: The methods described and analysed are: 1) Implicit physician assessment; 2) appropriateness evaluation protocol; 3) unmatched case-control; 4) matched case-control; 5) regression analysis; 6) multistate model. The various methodologies are described underlining advantages and limits which researchers need to know before starting any economic analysis. CONCLUSIONS: Overall, studies taking into account the time-dependent nature of HAI show to give more precise and reliable results.

Surveillance of healthcare acquired infections by "alert microorganisms": preliminary results.

BACKGROUND: We evaluated the trend of four years (2015 - 2018) of "alert organisms" surveillance carried out at the 450 bed teaching hospital S. Andrea in Rome. METHODS: All patients with an "Alert organism" isolation were screened. In accordance with definitions used by the Centers for Disease Control patients with an "alert organism" isolation were evaluated for infection or colonization, by an infection control team (ICT). RESULTS: Between April 2015 and December 2018 a total 4,762 specimens with "Alert organism" isolation were screened and 1,601 patients were surveyed and included in the study. Overall 780 (48.8%) patients developed an healthcare acquired infection (HAI) at our institution, whereas 311 (19.4%) entered with a community acquired infection, 254 (15.8%) with an infection acquired in another healthcare setting and 256 (16.0%) resulted simply colonized. The 780 patients who developed an HAI at our institution presented 878 infectious episodes and the isolation of 931 microorganisms. C. difficile infections were the most common (27.2%), followed by 21.3% respiratory tract infections, 16.9% urinary tract infections, 15.5% surgical site infections, 12.5% bloodstream infections, 3.6% ulcers and 3.0% others. Among HAI group Gram negatives (54.1%) were more frequent than Gram positives (45.9%), whereas in patients entering in the hospital already with a community infection Gram positives overpassed Gram negatives (58.7% vs. 41.3%; p<0.001). Most common pathogens responsible for HAI were C. difficile (25.6%), Klebsiella spp. (25.5%), MRSA (19.6%) and Acinetobacter spp. (15.3%). Notably 30.0% HAI at other institutions were represented by C. difficile. Impressively, >40% of community acquired infections were related to MRSA. CONCLUSIONS: The present study provided some useful insight into the major multi-resistant pathogens epidemiology at our institution. The Authors succeeded in organizing a multidisciplinary ICT that created a partnership feeling with the hospital personnel.

Contact precaution procedures in healthcare facilities.

BACKGROUND: Multidrug resistance is an established and growing worldwide public health problem, since few therapeutic options remain available. MRSA is the leading Gram-positive organism which has spread both in the community and healthcare environment. Gram-negative bacteria, either fermenter (enterobacteriaceae) or nonfermenter, pose a major challenge to the healthcare providers because they can express a wide multidrug resistance. METHODS: Specific keywords combinations were analitically searched in PubMed and Scopus databases. Publications concerning contact precaution procedures were reviewed. RESULTS: The review on infection control and isolation precautions was carried out focusing on bundles that could help healthcare personnel to improve their action. CONCLUSIONS: This paper clearly refers to measures in order to control the spread of infectious disease. We provided some synthetic tables that could improve healthcare workers knowledge and help them to apply all fundamental concepts in infection control.

Point prevalence surveys on healthcare acquired infections in medical and surgical wards of a teaching hospital in Rome.

BACKGROUND: Healthcare acquired infections (HAI) are an important cause of morbidity and mortality in hospitals worldwide. Aim of the study was to analyze nine years surveillance activity, carried out by point prevalence surveys from 2007 to 2015, in a 450-bed teaching hospital in Rome. METHODS: Point prevalence surveys were carried out every year in the medical and surgical wards following the same methodology. In accordance with definitions used by the Centers for Disease Control, all infections occurred more than 48 hours after hospital admission were considered HAI, and included in the study. Baseline characteristics, clinical features, isolated pathogens (only for the period 2011-2015) and antimicrobial resistance were recorded. RESULTS: During the nine years point prevalence surveys a total 2,840 patients were enrolled. Overall 136 (4.79%) patients developed 180 (6.34%) HAI. The most frequent HAI were respiratory tract infections (RTI), which accounted for 35.0% of all HAI, followed by surgical site infections (SSI) 22.2%, urinary tract infections (UTI) 19.4%, bloodstream infections (BSI) 17.2%, and others 6.1%. HAI related to major invasive risk procedures were also evaluated. SSI/patients undergoing surgery 3.99%, UTI/ patients with urinary catheter 4.17% and BSI/patients with CVC 9.42%. Over one-half of all patients surveyed (1,532, 53.9%) were receiving antibiotics at the time of our study. Among them 892 (58.2%) for treatment, 641 (41.8%) for prophylaxis. In the latter group, 109 (17.0%) underwent extra-short term, 89 (13.9%) short term and 443 (69.1%) a long term prophylaxis. During the period 2011-2015 out of 110 HAI episodes 71 (64.5%) were confirmed microbiologically. In total 106 pathogens were isolates, Gram-negative bacteria (63.2%) were isolated more frequently than Gram-positive bacteria (28.3%). CONCLUSIONS: The overall HAI prevalence in our hospital was consistent with those reported in other studies in Italy. The study underlined the role of Gram-negative bacteria in HAI and the need for antimicrobial stewardship. It also provided useful baseline data for rational priorities in allocation of resources, for further infection control activities.

Healthcare Associated Infections: educational intervention by "Adult Learning" in an Italian teaching hospital.

BACKGROUND: An educational intervention for HAI prevention based on a combination of training, motivation and subsequent application in the current clinical practice in an Italian teaching hospital. METHODS: In 2015-2016 a pilot mandatory training on HAI targeted to HCWs was organized in the 450 bed teaching hospital Sant'Andrea in Rome. By adopting the "Impact/control matrix" prioritization tool, the relative level of impact (risk in causing or favoring HAI) and control (possibility for HCWs to prevent HAI) attributed by the participants to the issues associated to HAI during their working groups was evaluated. RESULTS: Overall, 34 physicians, 43 nurses and 15 non clinical professionals participated actively in seven courses, identifying 58 different issues related to HAI, which were reported 128 times. Results showed frequently that, within the same type of issue, HCW referred various levels of impact (risk in causing or favoring HAI) and personal control (possibility for HCW to prevent HAI). Overall staff shortage was the most reported problem by HCW in our hospital. Also hand washing was regarded as a main problem, but HCW expressed the feeling that individuals could act more successfully on this issue (high or medium control). Results showed that staff frequently did not know how to handle correctly visitors, similarly many colleagues expressed some difficulty in communicating information to patients and relatives on HAI. Surprisingly, "antimicrobial therapy" and "excessive invasive procedures" were not particularly highlighted by the personnel. HCW expressed satisfaction for the course approac. CONCLUSIONS: The study showed an overall good level of knowledge regarding the importance and principles of infection control in our teaching hospital HCW. However personnel perceived a variability in the impact of many issues on HAI and even more on the personal possibility to control their effect. In order to improve HCW compliance with HAI prevention programs, the "Adult Learning" model seems to be very useful.

Impact of low-level gestational exposure to organophosphate pesticides on neurobehavior in early infancy: a prospective study.

BACKGROUND: National data suggest widespread gestational exposure to organophosphate pesticides (OPs) based on the detection of OP metabolites in the urine of pregnant women. Associations with early infant neurobehavior are largely understudied, with only two studies reporting abnormal reflexes in newborns in association with gestational exposure to OPs. Our objective was to utilize biological markers of OP metabolites in pregnant women and a comprehensive assessment of infant neurobehavior to determine the association of gestational exposure to OPs with neurobehavioral outcomes during early infancy. METHODS: Among a cohort of 350 mother/infant pairs, we measured six common dialkylphosphate metabolites of OP pesticides in maternal urine, at two times during pregnancy (16 w & 26 w gestation), then calculated aggregate concentrations of diethylphosphate, dimethylphosphate, and total dialkyphosphate metabolites. We measured infant neurobehavior at about five weeks of age using the NICU Network Neurobehavioral Scale (NNNS), a comprehensive assessment of neurobehavior in young infants. Analyses of associations between gestational exposure to OPs and neurobehavior at five weeks included multiple linear and logistic regression. RESULTS: After adjustment for covariates, higher creatinine-corrected urinary concentrations of diethylphosphate metabolites were associated with improved attention and reduced lethargy and hypotonia in young infants. Higher creatinine-corrected urinary concentrations of total dialkylphosphate metabolites were associated with fewer signs of autonomic stress. Women who were white, married, had advanced education, and reported more frequent consumption of fresh fruits and vegetables had higher concentrations of OP metabolites during pregnancy. CONCLUSIONS: In this sample of pregnant women whose urinary concentrations of dialkylphosphate metabolites are representative of national exposure levels, we found no detrimental effects of gestational exposure to OPs on neurobehavioral outcomes among young infants. These results are important as they suggest there may be minimal to no detectable adverse impact of low level prenatal OP exposure on the neurobehavior of young infants.

Factors predicting the outcome of allergen-specific nasal provocation test in children with grass pollen allergic rhinitis.

Background: Nasal provocation testing (NPT) is a reference methodology to identify the culprit allergen in patients with allergic rhinitis. Selecting the right allergen for NPT is particularly difficult in poly-sensitized patients with seasonal allergic rhinitis (SAR). Predictors of NPT outcomes may facilitate the proper use of this test or even substitute it. Objective: To identify predictors of grass pollen NPT outcome from an array of clinical data, e-diary outcomes, and allergy test results in poly-sensitized pediatric patients with SAR. Methods: Poly-sensitized, SAR patients with grass pollen allergy, participating in the @IT.2020 pilot project in Rome and Pordenone (Italy), participated in a baseline (T0) visit with questionnaires, skin prick testing (SPT), and blood sampling to measure total (ImmunoCAP, TFS, Sweden) and specific IgE antibodies to grass pollen extracts and their major allergenic molecules (ESEP, Euroimmun Labordiagnostika, Germany). During the pollen season, patients filled the AllergyMonitor® e-diary app measuring their symptoms, medication intake, and allergy-related well-being via the Visual Analogue Scale (VAS). After the pollen season (T1), patients answered clinical questionnaires and underwent a nasal provocation test (NPT) with grass pollen extract. Results: We recruited 72 patients (age 14.3 ± 2.8 years, 46 males) sensitized to grass and/or other pollens, including olive (63; 87.5%) and pellitory (49; 68.1%). Patients positive to grass pollen NPT (61; 84.7%), compared to the negative ones, had worse VAS values in the e-diary, larger SPT wheal reactions, and higher IgE levels, as well as specific activity to timothy and Bermuda grass extracts, rPhl p 5 and nCyn d 1. A positive NPT to grass pollen was predicted by an index combining the specific activity of IgE towards Phl p 5 and Cyn d 1 (AUC: 0.82; p < 0.01; best cut-off ≥7.25%, sensitivity 70.5%, specificity: 90.9%). VAS results also predicted NPT positivity, although with less precision (AUC: 0.77, p < 0.01; best cut-off ≥7, sensitivity: 60.7%, specificity: 81.8%). Conclusions: An index combining the specific activity of IgE to rPhl p 5 and nCyn d 1 predicted with moderate sensitivity and high specificity the outcome of a grass pollen NPT in complex, poly-sensitized pediatric patients with seasonal allergic rhinitis. Further studies are needed to improve the index sensitivity and to assess its usefulness for NPT allergen selection or as an alternative to this demanding test procedure.

FLOTAC can detect parasitic and pseudoparasitic elements in reptiles.

Reptiles have increased in popularity worldwide; snakes and lizards are frequently used as pets. As a consequence of their popularity, the interest of the scientific community in these animals has increased. In order to acquire epidemiological data on the parasitic infections affecting reptiles in Italy a survey was carried out in 125 snakes and 25 lizards bred in the Campania region of southern Italy. Individual fecal samples were collected and FLOTAC was used for copromicroscopic diagnosis. Eimeriidae, oxyurids, strongylids, other gastro-intestinal nematodes and pulmonary nematodes were the most representative parasites found. Eggs of pseudoparasites (mites, oxyurids and trichurids affecting rodents) were also found. The use of FLOTAC for diagnosis of parasitic infections in reptiles has demonstrated to be a rapid and sensitive test to improve diagnosis and acquire new information on the parasitological fauna of reptiles.

High-throughput sample preparation for the quantitation of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea, and propylenethiourea in human urine using 96-well-plate automated extraction and high-performance liquid chromatography-tandem mass spectrometry.

Acephate, methamidophos, o-methoate, and dimethoate are organophosphorus pesticides, and ethylenethiouria and propylenethiourea are two metabolites from the bisdithiocarbamate fungicide family. They are some of the most widely used pesticides and fungicides in agriculture both domestically and abroad. The existing high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the measurement of these compounds in human urine was improved by using a 96-well plate format sample preparation; the use of HPLC-MS/MS was comparable with a concentration range of 0.125 to 50 ng/ml. Deuterium-labeled acephate, ethylenethiouria, and methamidophos were used as internal standards. The sample preparation procedure, in the 96-well format with a 0.8-ml urine sample size, uses lyophilization of samples, followed by extraction with dichloromethane. The analytes were chromatographed on a Zorbax SB-C3 (4.6 × 150 mm, 5.0-µm) column with gradient elution by using 0.1% formic acid in aqueous solution (solvent A) and 0.1% formic acid in methanol (solvent B) mobile phase at a flow rate of 1 ml/min. Quantitative analysis was performed by atmospheric pressure chemical ionization source in positive ion mode using multiple-reaction monitoring of the precursor-to-product ion pairs for the analytes on a TSQ Quantum Ultra HPLC-MS/MS. Repeated analyses of urine samples spiked with high (15 ng/ml), medium (5 ng/ml), and low (1 ng/ml) concentrations of the analytes gave relative SDs of <13%. The limits of detection were in the range of 0.004-0.01 ng/ml. The method also has high accuracy, high precision, and excellent extraction recovery. Furthermore, the improved sample preparation method decreased the cost and labor required while effectively doubling the analytic throughput with minimal matrix effect.

Idiotypes expressed early in experimental Schistosoma mansoni infections predict clinical outcomes of chronic disease.

In murine Schistosoma mansoni infections, schistosome-specific cross-reactive idiotypes (CRI) are present in the sera of mice with moderate splenomegaly syndrome (MSS) at 20 wk after infection. In contrast, sera from animals that have the more severe hypersplenomegaly syndrome (HSS) at 20 wk of infection do not express these CRI in their sera. To examine when these regulatory CRI first appear in mice that eventually develop MSS, sera from infected animals were monitored for CRI from 1.5 to 20 wk of infection. In mice that eventually developed MSS, CRI were detected by 5 to 6 wk after infection, plateaued by 8 to 10 wk, and persisted through 20 wk of infection. Animals that developed HSS pathology or that died before 20 wk of infection never expressed CRI. Moreover, CRI levels present in the sera of mice at 6 wk of infection were inversely correlated with splenomegaly and hepatic fibrosis, but not with parasitologic measures, at 20 wk after infection. These results suggest that critical events occur very early in some schistosome infections that induce the production of regulatory idiotypes and that the presence or absence of these idiotypes predicts, and possibly determines, subsequent morbidity.

Neonatal idiotypic exposure alters subsequent cytokine, pathology, and survival patterns in experimental Schistosoma mansoni infections.

Exposure to maternal idiotypes (Ids) or antigens might predispose a child to develop an immunoregulated, asymptomatic clinical presentation of schistosomiasis. We have used an experimental murine system to address the role of Ids in this immunoregulation. Sera from mice with 8-wk Schistosoma mansoni infection, chronic (20-wk infection) moderate splenomegaly syndrome (MSS), or chronic hypersplenomegaly syndrome (HSS) were passed over an S. mansoni soluble egg antigen (SEA) immunoaffinity column to prepare Ids (8WkId, MSS Id, HSS Id). Newborn mice were injected with 8WkId, MSS Id, HSS Id, or normal mouse immunoglobulin (NoMoIgG) and infected with S. mansoni 8 wk later. Mice exposed to 8WkId or MSS Id as newborns had prolonged survival and decreased morbidity compared with mice that received HSS Id or NoMoIgG. When stimulated with SEA, 8WkId, or MSS Id, spleen cells from mice neonatally injected with 8WkId or MSS Id produced more interferon gamma than spleen cells from mice neonatally injected with HSS Id or NoMoIgG. Furthermore, neonatal exposure to 8WkId or MSS Id, but not NoMoIgG or HSS Id, led to significantly smaller granuloma size and lower hepatic fibrosis levels in infected mice. Together, these results indicate that perinatal exposure to appropriate anti-SEA Ids induces long-term effects on survival, pathology, and immune response patterns in mice subsequently infected with S. mansoni.

Urinary complications from breast cancer metastasis: case report and review of the literature.

Breast cancer is the most prevalent malignant disease among women, with the exception of non-melanoma skin cancers. Malignant breast tumours metastasise to lungs, bone, liver, lymph nodes and skin, but the literature also reports few cases of unusual metastases such as to the bladder. We present the case of a 57-year-old woman affected by lobular invasive breast cancer and complaining of high urinary frequency with nicturia. To date this is the seventh reported case of isolated metastatis of breast carcinoma to the bladder.

Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave-assisted solvent extraction (MASE) and high-performance liquid chromatography-mass spectrometry.

Animal models, consistent with the hypothesis of direct interaction of paraquat (PQ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with specific areas of the central nervous system have been developed to study Parkinson's disease (PD) in mice. These models have necessitated the creation of an analytical method for unambiguous identification and quantitation of PQ and structurally similar MPTP and 1-methyl-4-phenylpyridinium ion (MPP+) in brain tissue. A method for determination of these compounds was developed using microwave-assisted solvent extraction (MASE) and liquid chromatography-mass spectrometry. Extraction solvent and microwave conditions such as power and time were optimized to produce recoveries of 90% for PQ 78% for MPTP and 97% for its metabolite MPP+. The chromatographic separation was performed on a C8, column and detection was carried out using an ion trap as an analyzer with electrospray ionization. Mass spectrometer parameters such as heated capillary temperature, spray voltage, capillary voltage and others were also optimized for each analyte. Analysis was done in selective ion-monitoring (SIM) mode using m/z 186 for PQ, m/z 174 for MPTP, and m/z 170 for MPP+. The method detection limit for paraquat in matrix was 100 pg, 40 pg for MPTP, and 20 pg MPP+.

Mapping of the conserved antigenic domains shared between potato apyrase and parasite ATP diphosphohydrolases: potential application in human parasitic diseases.

Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.

Concentrations of urinary pesticide metabolites in small-scale farmers in Chiang Mai Province, Thailand.

Our research goal was to assess exposure to currently used pesticides among small-scale male farmers residing in two topographically different areas in Chiang Mai Province, Thailand. Farmers (N=136) were recruited from Pong Yaeng subdistrict (N=67) and Inthakhin subdistrict (N=69). Each farmer provided a morning urine void for the analysis of 30 urinary metabolites of insecticides, herbicides, and fungicides. Farmers in Pong Yaeng had significantly higher urinary concentrations of metabolites of organophosphorus insecticides and ethylene bisdithiocarbamates, while farmers from Inthakhin had significantly higher concentrations of malathion, 2,4-D, alachlor, and parathion or EPN metabolites. Based upon the metabolites measured in the urine of the farmers, chlorpyrifos and pyrethroid insecticides seemed to be commonly used across both communities; no significant differences in metabolite concentrations of these insecticides were observed between the two farmer groups. The presence of methamidaphos in the urine of farmers suggests that, despite a ban on its use, methamidaphos continues to be used in the communities. A similar finding with metabolites of methyl parathion must be further investigated. Overall, our results suggest that while each community may use different pesticides, Thai farmers are exposed to a wide variety of pesticides with a broad range in exposure magnitude. Furthermore, age, field size, crop production type, and the use of protective equipment were found to be potential factors influencing the degree of exposure.

Immune responses during human Schistosomiasis mansoni. Evidence for antiidiotypic T lymphocyte responsiveness.

We present a method for the examination of antiidiotypic cell-mediated reactivity during chronic human infections. Pooled and individual sera from patients with schistosomiasis mansoni were purified on immunoaffinity columns of schistosomal egg antigens (SEA). The eluates contained anti-SEA antibodies, but not SEA. These antibody preparations, and their F(ab)2 fragments, stimulated dose-dependent proliferation of peripheral blood mononuclear cells (PBMN) and T lymphocytes from some, but not all active or former schistosomiasis mansoni patients, and could do so autologously. Stimulation required presentation by plastic-adherent cells. The eluates did not stimulate PBMN from persons who had never had schistosomiasis. Affinity-purified anti-SEA antibodies from former patients (cured for greater than 10 yr) did not stimulate PBMN from patients with active infections. Reabsorption on SEA columns removed stimulatory activity from the eluates. We propose that multiclonal, SEA-related idiotypes expressed by some anti-SEA antibodies stimulate proliferation of T lymphocytes that express antiidiotypic specificities.

Distribution and determinants of urinary biomarkers of exposure to organophosphate insecticides in Puerto Rican pregnant women.

Globally, human exposures to organophosphate (OP) insecticides may pose a significant burden to the health of mothers and their developing fetuses. Unfortunately, relevant data is limited in certain areas of the world concerning sources of exposure to OP insecticides in pregnant populations. To begin to address this gap in information for Puerto Rico, we studied repeated measures of urinary concentrations of 10 OP insecticide metabolites among 54 pregnant women from the northern karst region of the island. We also collected demographic data and self-reported information on the consumption of fruits, vegetables, and legumes in the past 48 h before urine collection and home pest-related issues. We calculated the distributions of the urinary biomarkers and compared them to women of reproductive age from the general U.S. population. We also used statistical models accounting for correlated data to assess within-subject temporal variability of the urinary biomarkers and to identify predictors of exposure. We found that for all but two metabolites (para-nitrophenol [PNP], diethylthiophosphate [DETP]), 50th or 95th percentile urinary concentrations (the metric that was used for comparison was based on the biomarker's detection frequency) of the other eight metabolites (3,5,6-trichloro-2-pyridinol [TCPY], 2-isopropyl-4-methyl-6-hydroxy-pyrimidine, malathion dicarboxylic acid, diethylphosphate, diethyldithiophosphate, dimethylphosphate, dimethylthiophosphate [DMTP], dimethyldithiophosphate) were somewhat lower in our cohort compared with similarly aged women from the continental United States. TCPY, PNP, DETP, and DMTP, which were the only urinary metabolites detected in greater than 50% of the samples, had poor reproducibility (intraclass correlation coefficient range: 0.19-0.28) during pregnancy. Positive predictors of OP insecticide exposure included: age; marital or employment status; consumption of cherries, grape juice, peanuts, peanut butter, or raisins; and residential application of pesticides. Further research is needed to understand what aspects of the predictors identified influence OP insecticide exposure during pregnancy.

Global regulators ExpA (GacA) and KdgR modulate extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp. carotovora.

The production of the main virulence determinants, the extracellular plant cell wall-degrading enzymes, and hence virulence of Erwinia carotovora subsp. carotovora is controlled by a complex regulatory network. One of the global regulators, the response regulator ExpA, a GacA homolog, is required for transcriptional activation of the extracellular enzyme genes of this soft-rot pathogen. To elucidate the mechanism of ExpA control as well as interactions with other regulatory systems, we isolated second-site transposon mutants that would suppress the enzyme-negative phenotype of an expA (gacA) mutant. Inactivation of kdgR resulted in partial restoration of extracellular enzyme production and virulence to the expA mutant, suggesting an interaction between the two regulatory pathways. This interaction was mediated by the RsmA-rsmB system. Northern analysis was used to show that the regulatory rsmB RNA was under positive control of ExpA. Conversely, the expression of rsmA encoding a global repressor was under negative control of ExpA and positive control of KdgR. This study indicates a central role for the RsmA-rsmB regulatory system during pathogenesis, integrating signals from the ExpA (GacA) and KdgR global regulators of extracellular enzyme production in E. carotovora subsp. carotovora.

Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora.

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.