Automatic Deep Learning-Based Pipeline for Automatic Delineation and Measurement of Fetal Brain Structures in Routine Mid-Trimester Ultrasound Images.

INTRODUCTION: The aim of this study was to develop a pipeline using state-of-the-art deep learning methods to automatically delineate and measure several of the most important brain structures in fetal brain ultrasound (US) images. METHODS: The dataset was composed of 5,331 images of the fetal brain acquired during the routine mid-trimester US scan. Our proposed pipeline automatically performs the following three steps: brain plane classification (transventricular, transthalamic, or transcerebellar plane); brain structures delineation (9 different structures); and automatic measurement (from the structure delineations). The methods were trained on a subset of 4,331 images and each step was evaluated on the remaining 1,000 images. RESULTS: Plane classification reached 98.6% average class accuracy. Brain structure delineation obtained an average pixel accuracy higher than 96% and a Jaccard index higher than 70%. Automatic measurements get an absolute error below 3.5% for the four standard head biometries (head circumference, biparietal diameter, occipitofrontal diameter, and cephalic index), 9% for transcerebellar diameter, 12% for cavum septi pellucidi ratio, and 26% for Sylvian fissure operculization degree. CONCLUSIONS: The proposed pipeline shows the potential of deep learning methods to delineate fetal head and brain structures and obtain automatic measures of each anatomical standard plane acquired during routine fetal US examination.

Natural History of Anal HPV Infection in Women Treated for Cervical Intraepithelial Neoplasia.

Women with high-grade squamous intraepithelial lesions/cervical intraepithelial neoplasia (HSIL/CIN) are at high risk of anal human papillomavirus HPV infection, and it has also been suggested that self-inoculation of the virus from the anal canal to the cervix could explain HPV recurrence in the cervix after treatment of HSIL/CIN. We aimed to evaluate the bidirectional interactions of HPV infection between these two anatomical sites. We evaluated 68 immunocompetent women undergoing excisional treatment for HSIL/CIN. Immediately before treatment, samples from the anus and the cervix were obtained (baseline anal and cervical HPV status). Cervical HPV clearance after treatment was defined as treatment success. The first follow-up control was scheduled 4-6 months after treatment for cervical and anal samples. High resolution anoscopy (HRA) was performed on patients with persistent anal HPV infections or abnormal anal cytology in the first control. Baseline anal HPV was positive in 42/68 (61.8%) of the women. Anal HPV infection persisted after treatment in 29/68 (42.6%) of the women. One-third of these women (10/29; 34.5%) had HSIL/anal intraepithelial neoplasia (AIN). Among women achieving treatment success, cervical HPV in the first control was positive in 34.6% and 17.6% of the patients with positive and negative baseline anal HPV infection, respectively (p = 0.306). In conclusion, patients with persisting anal HPV after HSIL/CIN treatment are at high risk of HSIL/AIN, suggesting that these women would benefit from anal exploration. The study also suggests that women with anal HPV infection treated for HSIL/CIN might be at higher risk of recurrent cervical HPV even after successful treatment.

Effect of Fractional Carbon Dioxide vs Sham Laser on Sexual Function in Survivors of Breast Cancer Receiving Aromatase Inhibitors for Genitourinary Syndrome of Menopause: The LIGHT Randomized Clinical Trial.

Importance: Survivors of breast cancer present more severe symptoms of genitourinary syndrome of menopause (GSM) than patients without history of breast cancer. Recently, new treatments, such as vaginal laser therapy, have appeared, but evidence of their efficacy remains scarce. Objective: To assess the safety and efficacy of carbon dioxide (CO2) vs sham vaginal laser therapy after 6 months of follow-up in survivors of breast cancer with GSM receiving aromatase inhibitors. Design, Setting, and Participants: This prospective double-blind sham-controlled randomized clinical trial with two parallel study groups was performed during October 2020 to March 2022 in a tertiary referral hospital. Survivors of breast cancer using aromatase inhibitors were assessed for eligibility, and eligible patients were randomized into the 2 treatment groups. Follow-up was conducted at 6 months. Data were analyzed in July 2022. Interventions: All patients from both groups were instructed to use the first-line treatment (FLT) based on nonhormonal moisturizers and vaginal vibrator stimulation. Patients for each group were allocated to 5 monthly sessions of fractional CO2 laser therapy (CLT) or sham laser therapy (SLT). Main Outcomes and Measures: The primary outcome was sexual function, evaluated through Female Sexual Function Index (FSFI) score. Other subjective measures of efficacy included a visual analog scale of dyspareunia, vaginal pH, a Vaginal Health Index, quality of life (assessed via Short-Form 12), and body image (assessed with the Spanish Body Image Scale). Objective measures of efficacy included vaginal maturation index, vaginal epithelial elasticity (measured in Pascals) and vaginal epithelial thickness (measured in millimeters). Measures were assessed before and after the intervention. Tolerance (measured on a Likert scale), adverse effects, and estradiol levels were recorded. Results: Among 211 survivors of breast cancer assessed, 84 women were deemed eligible and 72 women (mean [SD] age, 52.6 [8.3] years) were randomized to CLT (35 participants) or SLT (37 participants) and analyzed. There were no statistically significant differences between groups at baseline. At 6 months, both groups showed improvement in FSFI (mean [SD] score at baseline vs 6 months: CLT, 14.8 [8.8] points vs 20.0 [9.5] points; SLT, 15.6 [7.0] points vs 23.5 [6.5] points), but there was no significant difference between CLT and SLT groups in the improvement of sexual function evaluated through the FSFI test overall (mean [SD] difference, 5.2 [1.5] points vs 7.9 [1.2] points; P = .15) or after excluding women who were not sexually active (mean [SD] difference, 2.9 [1.4] points vs 5.5 [1.1] points; P = .15). There were also no differences between improvement of the 2 groups at 6 months of follow-up in the other assessed subjective outcomes, including dyspareunia (mean [SD] difference, -4.3 [3.4] vs -4.5 [2.3]; P = .73), Vaginal Health Index (mean [SD] difference, 3.3 [4.1] vs 5.0 [4.5]; P = .17), body image (mean [SD] difference, -3.7 [4.5] vs -2.7 [4.8]; P = .35), and quality of life (mean [SD] difference, -0.3 [3.6] vs -0.7 [3.2]; P = .39). Similarly, there were no differences in improvements in objective outcomes, including vaginal pH (mean [SD] difference, -0.6 [0.9] vs -0.8 [1.2]; P = .29), vaginal maturation index (mean [SD] difference, 10.2 [17.4] vs 14.4 [17.1]; P = .15), vaginal epithelial thickness (mean [SD] difference, 0.021 [0.014] mm vs 0.013 [0.012] mm; P = .30), vaginal epithelial elasticity (mean [SD] difference, -1373 [3197] Pascals vs -2103 [3771] Pascals; P = .64). There were significant improvements in the overall analysis regardless of group in many outcomes. The 2 interventions were well tolerated, but tolerance was significantly lower in the CLT group than the SLT group (mean [SD] Likert scale score, 3.3 [1.3] vs 4.1 [1.0]; P = .007). No differences were observed in complications or serum estradiol levels. Conclusions and Relevance: In this randomized clinical trial, vaginal laser treatment was found to be safe after 6 months of follow-up, but no statistically significant differences in efficacy were observed between CLT and SLT. Trial Registration: ClinicalTrials.gov identifier: NCT04619485.

De Novo Assembly of the Dirofilaria immitis Genome by Long-Read Nanopore-Based Sequencing Technology on an Adult Worm from a Canine Cardiopulmonary Dirofilariosis Case.

Dirofilaria immitis is a zoonotic parasitic nematode that infects domestic and wild canids, among its vertebrate hosts. The genetic analysis of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such as the study of resistance to macrocyclic lactone. We expanded the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform, followed by the study of macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110 contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci previously characterized as being associated to macrocyclic lactone resistance selection pressure showed that four have a genotype associated with either some loss of efficacy or the resistance phenotype. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health.

Adenomyosis is an independent risk factor for complications in deep endometriosis laparoscopic surgery.

Deep endometriosis (DE) occurs in 15-30% of patients with endometriosis and is associated with concomitant adenomyosis in around 25-49% of cases. There are no data about the effect of the presence of adenomyosis in terms of surgical outcomes and complications. Thus, the aim of the present study was to evaluate the impact of adenomyosis on surgical complications in women with deep endometriosis undergoing laparoscopic surgery. A retrospective cohort study including women referred to the endometriosis unit of a referral teaching hospital. Two expert sonographers preoperatively diagnosed DE and adenomyosis. DE was defined according to the criteria of the International Deep Endometriosis Analysis group. Adenomyosis was considered when 3 or more ultrasound criteria of the Morphological Uterus Sonographic Assessment group were present. Demographical variables, current medical treatment, symptoms, DE location, surgical time, hospital stay and difference in pre and post hemoglobin levels were collected. The Clavien-Dindo classification was used to assess surgical complications, and multivariate analysis was performed to compare patients with and without adenomyosis. 157 DE patients were included into the study; 77 (49.05%) had adenomyosis according to transvaginal ultrasound (TVS) and were classified in the A group, and 80 (50.95%) had no adenomyosis and were classified in the noA group. Adenomyosis was associated with a higher rate of surgical complications: 33.76% (A group) vs. 12.50% (noA group) (p < 0.001). Multivariate analysis showed a 4.56-fold increased risk of presenting complications in women with adenomyosis (CI 1.90-11.30; p = 0.001) independently of undergoing hysterectomy. There was a statistically significant association between the number of criteria of adenomyosis present in each patient and the proportion of patients presenting surgical complications (p < 0.001). Adenomyosis is an independent preoperative risk factor for surgical complications in DE surgery after adjustment for known demographic, clinical and surgical risk factors.

Occurrence and molecular characterization of human pegivirus-1 (HPgV-1) viremia in healthy volunteer blood donors from Northern Portugal.

Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family and the Pegivirus genus. Despite having been discovered 25 years ago, there is still much to know regarding HPgV-1 clinical impact, as this virus is currently not associated with any pathology. Yet, HPgV-1 prevalence and molecular characterization are still unknown in many countries, including Portugal. To fill in this knowledge gap, this study aimed to determine the occurrence and molecular characterization of HPgV-1 in a group of healthy blood donors from the north of Portugal. Blood samples from 465 Portuguese blood donors were collected from a major Hospital Center in the north of Portugal. RNA was extracted and an initial nested RT-PCR was performed targeting the conserved 5'-untranslated region  region of the HPgV-1 genome. A second nested RT-PCR targeting the E2 region was performed for genotyping. Only one sample tested positive for HPgV-1 RNA, resulting in a prevalence of approximately 0.22%. Phylogenetic analyses confirmed the characterization as genotype 2, the most prevalent in Europe.

Vaginal laser therapy for genitourinary syndrome of menopause - systematic review.

BACKGROUND: Genitourinary syndrome of menopause (GSM) can have a great impact on the quality of life (QOL), and affects between 53.8% and 90% of postmenopausal women. The literature suggests that vaginal laser therapy could be an effective treatment for GSM symptoms, but its efficacy and safety have not been established and international societies do not endorse its use. Despite that, there has been an increase in the use of vaginal laser therapy globally over the last decade. OBJECTIVE: The objective of this review is to evaluate the literature which assesses the efficacy and safety of the vaginal laser therapy in the treatment of GSM. METHODS: A comprehensive literature search was conducted electronically using Embase and PubMed to retrieve studies assessing evidence for the efficacy and safety of vaginal laser therapy for GSM or vulvovaginal atrophy up to June 2021. RESULTS: A total of 64 studies were finally included in the review. There were 10 controlled intervention studies, 7 observational cohort and cross-sectional studies and 47 before-after studies without a control group. CONCLUSION: Vaginal laser seems to improve scores on the Visual Analogue Scale (VAS), Female Sexual Function Index (FSFI) and Vaginal Health Index (VHI) in GSM over the short term. Safety outcomes are underreported and short-term. Further well-designed clinical trials with sham-laser control groups and evaluating objective variables are needed to provide the best evidence on efficacy.

Genitourinary Syndrome of Menopause Assessment Tools.

New therapeutic options are being considered to treat genitourinary syndrome of menopause (GSM), such as vaginal laser, ospemifene, or prasterone, but there is no explicit agreement in the scientific community for its use. Some concerns have arisen on how to evaluate the improvement of GSM symptoms. In 2003, the FDA suggested possible end points for this purpose: change in severity of symptoms, change in vaginal pH, and change in vaginal maturation index (VMI). Contrarily, the most common assessment tools used to quantify severity and improvement of GSM nowadays are the visual analog scale of GSM symptoms, the vaginal health index, and the female sexual function index. In our opinion, subjective and objective variables to evaluate GSM can be differentiated, and not many of the considered objective outcomes are used in the recent literature assessing GSM. There is the possibility that some therapies present only subjective improvement, giving place to a possible placebo effect that is not being evaluated. To conclude, there is a demand to evaluate whether vaginal pH and VMI are enough to assess objectively GSM changes or new objective approaches should be audited.

Placental growth factor testing in the management of late preterm preeclampsia without severe features: a multicenter, randomized, controlled trial.

BACKGROUND: In women with late preterm preeclampsia, the optimal time for delivery remains a controversial topic, because of the fine balance between the maternal benefits from early delivery and the risks for prematurity. It remains challenging to define prognostic markers to identify women at highest risk for complications, in which case a selective, planned delivery may reduce the adverse maternal and perinatal outcomes. OBJECTIVE: This trial aimed to determine whether using an algorithm based on the maternal levels of placental growth factor in women with late preterm preeclampsia to evaluate the best time for delivery reduced the progression to preeclampsia with severe features without increasing the adverse perinatal outcomes. STUDY DESIGN: This parallel-group, open-label, multicenter, randomized controlled trial was conducted at 7 maternity units across Spain. We compared selective planned deliveries based on maternal levels of placental growth factor at admission (revealed group) and expectant management under usual care (concealed group) with individual randomization in singleton pregnancies with late preterm preeclampsia from 34 to 36+6 weeks' gestation. The coprimary maternal outcome was the progression to preeclampsia with severe features. The coprimary neonatal outcome was morbidity at infant hospital discharge with a noninferiority hypothesis (noninferiority margin of 10% difference in incidence). Analyses were conducted according to intention-to-treat. RESULTS: Between January 1, 2016, and December 31, 2019, 178 women were recruited. Of those women, 88 were assigned to the revealed group and 90 were assigned to the concealed group. The data analysis was performed before the completion of the required sample size. The proportion of women with progression to preeclampsia with severe features was significantly lower in the revealed group than in the concealed group (adjusted relative risk, 0.5; 95% confidence interval, 0.33-0.76; P=.001). The proportion of infants with neonatal morbidity was not significantly different between groups (adjusted relative risk, 0.77; 95% confidence interval, 0.39-1.53; P=.45). CONCLUSION: There is evidence to suggest that the use of an algorithm based on placental growth factor levels in women with late preterm preeclampsia leads to a lower rate of progression to preeclampsia with severe features and reduces maternal complications without worsening the neonatal outcomes. This trade-off should be discussed with women with late preterm preeclampsia to allow shared decision making about the timing of delivery.

Cell-Free DNA Testing: What Is the Reason Why High-Risk Women Choose It?

OBJECTIVE: The aim of the study wasto describe the past medical history, sociodemographic, and pregnancy characteristics of women at high risk for aneuploidy and to determine which factors are related to her choice of cell-free DNA (cfDNA) testing instead of invasive diagnostic testing. METHODS: We conducted a prospective descriptive study including pregnant women from the Western Barcelona public health area at high risk for fetal aneuploidy, defined as a trisomy 21 or 18 risk between 1/10 and 1/250 at the combined first-trimester or at the second-trimester biochemical screening. During 1 year (December 2018 to November 2019), these women were asked to fill in a confidential questionnaire about her past medical history, demographic and pregnancy characteristics, and her opinion about termination of the pregnancy after a counseling consultation with a maternal-fetal medicine specialist in which advantages and disadvantages of both testing methods, cfDNA or diagnostic testing, were discussed. Logistic regression analysis was used to determine which factors were related with cfDNA uptake. RESULTS: During the study period, 82 pregnant women filled the questionnaire. The median maternal age was 39.6 years (interquartile range [IQR] 37.3-40.9 years), and 73 (89%) of them were 35 years or older. Forty-three (52%) women opted for cfDNA testing, while 39 (48%) chose invasive diagnosis. In a logistic regression analysis, the use of assisted reproductive techniques (OR 13.03; 95% CI: 1.47-115.56; p = 0.021) and Latin American origin (OR 6.66; 95% CI 1.73-25.66; p = 0.006) were shown to be related to a higher cfDNA uptake. In contrast, nonreligious women (OR 0.21; 95% CI: 0.06-0.72; p = 0.013) and a favorable opinion about termination of pregnancy (OR 0.23; 95% CI: 0.06-0.92; p = 0.037) were related with a lower uptake. CONCLUSION: Half of the pregnant women at high risk for fetal aneuploidy opted for cfDNA testing. The main reason to choose cfDNA was avoiding the risk of pregnancy loss. Women using assisted reproductive techniques and those of Latin American origin preferred cfDNA testing, while nonreligious women and those with a favorable opinion on termination pregnancy preferred invasive testing.

High-Quality Draft Genome Sequences of Marine Fish Gut Bacillus sp. Strains ABP1 and ABP2 with Nonstarch Polysaccharide Hydrolytic Potential.

Here, we present the genome sequences of two environmental Bacillus strains with broad hydrolytic capacity toward different nonstarch polysaccharides (NSPs) that were isolated from the gut of marine fish fed NSP-rich diets. Several genes that may contribute to the NSP-degrading behavior were identified through in silico analysis.

Quantification of dose-mortality responses in adult Diptera: Validation using Ceratitis capitata and Drosophila suzukii responses to spinosad.

Quantitative laboratory bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest control and to determine the presence of resistance traits. We used a radioactive tracer based on 32P-ATP to estimate the volume of a droplet ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 µl. Females ingested a ~20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 µl and females ingested ~30% greater volume than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females, respectively, when sex-specific variation in body weight was considered. Using the same process for D. suzukii, the LD50 value was estimated at 2.927 ng a.i./insect, or 1.994 ng a.i./mg based on a mean body weight of 1.67 mg for both sexes together. We conclude that this technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of pestiferous Diptera.

The use of stable and unstable green fluorescent proteins for studies in two bacterial models: Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris.

Fluorescent proteins have been used to track plant pathogens to understand their host interactions. To be useful, the transgenic pathogens must present similar behaviour than the wild-type isolates. Herein, a GFP marker was used to transform two plant pathogenic bacteria, Agrobacterium and Xanthomonas, to localize and track the bacteria during infection. The transgenic bacteria were evaluated to determine whether they showed the same fitness than the wild-type strains or whether the expression of the GFP protein interfered in the bacterial activity. In Agrobacterium, the plasmid used for transformation was stable in the bacteria and the strain kept the virulence, while Xanthomonas was not able to conserve the plasmid and transformed strains showed virulence variations compared to wild-type strains. Although marking bacteria with GFP to track infection in plants is a common issue, works to validate the transgenic strains and corroborate their fitness are not usual. Results, presented here, confirm the importance of proper fitness tests on the marked strains before performing localization assays, to avoid underestimation of the microbe population or possible artificial effects in its interaction with the plant.

Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants.

BACKGROUND: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear. RESULTS: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants. CONCLUSIONS: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

Translocation and functional analysis of Pseudomonas savastanoi pv. savastanoi NCPPB 3335 type III secretion system effectors reveals two novel effector families of the Pseudomonas syringae complex.

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

Identification of novel virulence genes and metabolic pathways required for full fitness of Pseudomonas savastanoi pv. savastanoi in olive (Olea europaea) knots.

Comparative genomics and functional analysis of Pseudomonas syringae and related pathogens have mainly focused on diseases of herbaceous plants; however, there is a general lack of knowledge about the virulence and pathogenicity determinants required for infection of woody plants. Here, we applied signature-tagged mutagenesis (STM) to Pseudomonas savastanoi pv. savastanoi during colonization of olive (Olea europaea) knots, with the goal of identifying the range of genes linked to growth and symptom production in its plant host. A total of 58 different genes were identified, and most mutations resulted in hypovirulence in woody olive plants. Sequence analysis of STM mutations allowed us to identify metabolic pathways required for full fitness of P. savastanoi in olive and revealed novel mechanisms involved in the virulence of this pathogen, some of which are essential for full colonization of olive knots by the pathogen and for the lysis of host cells. This first application of STM to a P. syringae-like pathogen provides confirmation of functional capabilities long believed to play a role in the survival and virulence of this group of pathogens but not adequately tested before, and unravels novel factors not correlated previously with the virulence of other plant or animal bacterial pathogens.