P-DOR, an easy-to-use pipeline to reconstruct bacterial outbreaks using genomics.

SUMMARY: Bacterial Healthcare-Associated Infections (HAIs) are a major threat worldwide, which can be counteracted by establishing effective infection control measures, guided by constant surveillance and timely epidemiological investigations. Genomics is crucial in modern epidemiology but lacks standard methods and user-friendly software, accessible to users without a strong bioinformatics proficiency. To overcome these issues we developed P-DOR, a novel tool for rapid bacterial outbreak characterization. P-DOR accepts genome assemblies as input, it automatically selects a background of publicly available genomes using k-mer distances and adds it to the analysis dataset before inferring a Single-Nucleotide Polymorphism (SNP)-based phylogeny. Epidemiological clusters are identified considering the phylogenetic tree topology and SNP distances. By analyzing the SNP-distance distribution, the user can gauge the correct threshold. Patient metadata can be inputted as well, to provide a spatio-temporal representation of the outbreak. The entire pipeline is fast and scalable and can be also run on low-end computers. AVAILABILITY AND IMPLEMENTATION: P-DOR is implemented in Python3 and R and can be installed using conda environments. It is available from GitHub https://github.com/SteMIDIfactory/P-DOR under the GPL-3.0 license.

Clinical isolates of ST131 blaOXA-244-positive Escherichia coli, Italy, December 2022 to July 2023.

The dissemination of carbapenemase-producing Escherichia coli, although still at low level, should be continuously monitored. OXA-244 is emerging in Europe, mainly in E. coli. In Italy, this carbapenemase was reported from an environmental river sample in 2019. We report clinical isolates of OXA-244-producing ST131 E. coli in four patients admitted to an acute care hospital in Pavia, Italy. The association of this difficult-to-detect determinant with a globally circulating high-risk clone, ST131 E. coli, is of clinical relevance.

Rapid spread of a novel NDM-producing clone of Klebsiella pneumoniae CC147, Northern Italy, February to August 2023.

New Delhi metallo-beta-lactamase (NDM)-producing Klebsiella pneumoniae (Kp) ST147 caused a large multi-hospital outbreak in Italy from 2018 to 2021. We describe a new ST6668 NDM-producing Kp clone, belonging to CC147, which rapidly spread across hospitals in the Pavia province (Northern Italy) from February to August 2023. Genomic analyses revealed that ST6668 is different from ST147 and fast evolving. As shown here, genomic surveillance programmes are useful for tracking the spread of new clones with reduced susceptibility to most antibiotics.

Concomitant Resistance to Cefiderocol and Ceftazidime/Avibactam in Two Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Lung Transplant Patients.

In this study, we present two cases of Klebsiella pneumoniae, one KPC-33- and one NDM-1-producing, showing resistance to cefiderocol and ceftazidime/avibactam, collected in the intensive care unit of a hospital in Northern Italy from two patients who had recently undergone lung transplantation. Whole-genome sequencing was performed to investigate the molecular features of these strains.

Pan-pathogen deep sequencing of nosocomial bacterial pathogens in Italy in spring 2020: a prospective cohort study.

BACKGROUND: Nosocomial infections pose a considerable risk to patients who are susceptible, and this is particularly acute in intensive care units when hospital-associated bacteria are endemic. During the first wave of the COVID-19 pandemic, the surge of patients presented a significant obstacle to the effectiveness of infection control measures. We aimed to assess the risks and extent of nosocomial pathogen transmission under a high patient burden by designing a novel bacterial pan-pathogen deep-sequencing approach that could be integrated with standard clinical surveillance and diagnostics workflows. METHODS: We did a prospective cohort study in a region of northern Italy that was severely affected by the first wave of the COVID-19 pandemic. Inpatients on both ordinary and intensive care unit (ICU) wards at the San Matteo hospital, Pavia were sampled on multiple occasions to identify bacterial pathogens from respiratory, nasal, and rectal samples. Diagnostic samples collected between April 7 and May 10, 2020 were cultured on six different selective media designed to enrich for Acinetobacter baumannii, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, and DNA from each plate with positive growth was deep sequenced en masse. We used mSWEEP and mGEMS to bin sequencing reads by sequence cluster for each species, followed by mapping with snippy to generate high quality alignments. Antimicrobial resistance genes were detected by use of ARIBA and CARD. Estimates of hospital transmission were obtained from pairwise bacterial single nucleotide polymorphism distances, partitioned by within-patient and between-patient samples. Finally, we compared the accuracy of our binned Acinetobacter baumannii genomes with those obtained by single colony whole-genome sequencing of isolates from the same hospital. FINDINGS: We recruited patients from March 1 to May 7, 2020. The pathogen population among the patients was large and diverse, with 2148 species detections overall among the 2418 sequenced samples from the 256 patients. In total, 55 sequence clusters from key pathogen species were detected at least five times. The antimicrobial resistance gene prevalence was correspondingly high, with key carbapenemase and extended spectrum ß-lactamase genes detected in at least 50 (40%) of 125 patients in ICUs. Using high-resolution mapping to infer transmission, we established that hospital transmission was likely to be a significant mode of acquisition for each of the pathogen species. Finally, comparison with single colony Acinetobacter baumannii genomes showed that the resolution offered by deep sequencing was equivalent to single-colony sequencing, with the additional benefit of detection of co-colonisation of highly similar strains. INTERPRETATION: Our study shows that a culture-based deep-sequencing approach is a possible route towards improving future pathogen surveillance and infection control at hospitals. Future studies should be designed to directly compare the accuracy, cost, and feasibility of culture-based deep sequencing with single colony whole-genome sequencing on a range of bacterial species. FUNDING: Wellcome Trust, European Research Council, Academy of Finland Flagship program, Trond Mohn Foundation, and Research Council of Norway.

Dynamics of viral DNA shedding and culture viral DNA positivity in different clinical samples collected during the 2022 mpox outbreak in Lombardy, Italy.

BACKGROUND: Mpox virus (MPXV) has recently spread outside of sub-Saharan Africa. This large multicentre study was conducted in Lombardy, the most densely populated Italian region accounting for more than 40% of Italian cases. The present study aims to: i) evaluate the presence and the shedding duration of MPXV DNA in different body compartments correlating the MPXV viability with the time to onset of symptoms; ii) provide evidence of MPXV persistence in different body compartment as a source of infection and iii) characterize the MPXV evolution by whole genome sequencing (WGS) during the outbreak occurred in Italy. MATERIAL AND METHODS: The study included 353 patients with a laboratory-confirmed diagnosis of MPXV infection screened in several clinical specimens in the period May 24th - September 1st, 2022. Viral isolation was attempted from different biological matrices and complete genome sequencing was performed for 61 MPXV strains. RESULTS: MPXV DNA detection was more frequent in the skin (94.4%) with the longest median time of viral clearance (16 days). The actively-replicating virus in cell culture was obtained for 123/377 (32.6%) samples with a significant higher viral quantity on isolation positive samples (20 vs 31, p < 0.001). The phylogenetic analysis highlighted the high genetic identity of the MPXV strains collected, both globally and within the Lombardy region. CONCLUSION: Skin lesion is gold standard material and the high viral load and the actively-replicating virus observed in genital sites confirms that sexual contact plays a key role in the viral transmission.

The COVID-19 Pandemic Sparked Off a Large-Scale Outbreak of Carbapenem-Resistant Acinetobacter baumannii from the Endemic Strains at an Italian Hospital.

Acinetobacter baumannii is a nosocomial pathogen that poses a serious threat due to the rise of incidence of multidrug-resistant (MDR) strains. During the COVID-19 pandemic, MDR A. baumannii clones have caused several outbreaks worldwide. Here, we describe a detailed investigation of an MDR A. baumannii outbreak that occurred at Policlinico San Matteo (Pavia, Italy). A total of 96 A. baumannii strains, isolated between January and July 2020 from 41 inpatients (both SARS-CoV-2 positive and negative) in different wards, were characterized by phenotypic and genomic analyses combining Illumina and Nanopore sequencing. Antibiotic susceptibility testing revealed that all isolates were resistant to carbapenems, and the sequence analysis attributed this to the carbapenemase gene blaOXA-23. Virulence factor screening unveiled that all strains carried determinants for biofilm formation, while plasmid analysis revealed the presence of two plasmids, one of which was ~100 kbp long and encoded a phage sequence. A core genome-based phylogeny was inferred to integrate outbreak strain genomes with background genomes from public databases and the local surveillance program. All strains belonged to the globally disseminated sequence type 2 (ST2) clone and were mainly divided into two clades. Isolates from the outbreak clustered with surveillance isolates from 2019, suggesting that the outbreak was caused by two strains that were already circulating in the hospital before the start of the pandemic. The intensive spread of A. baumannii in the hospital was enhanced by the extreme emergency situation of the first COVID-19 pandemic wave that resulted in reduced attention to infection prevention and control practices. IMPORTANCE The COVID-19 pandemic, especially during the first wave, posed a great challenge to the hospital management and generally promoted nosocomial pathogen dissemination. MDR A. baumannii can easily spread and persist for a long time on surfaces, causing outbreaks in health care settings. Infection prevention and control practices, epidemiological surveillance, and microbiological screening are fundamental in order to control such outbreaks. Here, we sequenced the genomes of 96 isolates from an outbreak of MDR A. baumannii strains using both short- and long-read technology in order to reconstruct the outbreak events in fine detail. The sequence data demonstrated that two endemic clones of MDR A. baumannii were the source of this large hospital outbreak during the first COVID-19 pandemic wave, confirming the effect of COVID-19 emergency disrupting the protection provided by the use of the standard prevention procedures.

Gut Microbiota and B Cell Receptor (BCR) Inhibitors for the Treatment of Chronic Lymphocytic Leukemia: Is Biodiversity Correlated with Clinical Response or Immune-Related Adverse Event Occurrence? A Cross-Sectional Study.

Several studies have strengthened the link between the gut microbiota (GM) and the response to immunotherapy in patients with tumors, highlighting the potential role of GM as a biomarker of response. Targeted therapies including B-cell receptor (BCR) inhibitors (BCRi) represent the newest approach to the treatment of chronic lymphocytic leukemia (CLL); however, not all patients achieve a satisfactory response, and immune-related adverse events (irAEs) can also impact the efficacy. The aim of the study was to compare GM biodiversity in patients with CLL, treated with BCRi for at least 12 months. Twelve patients were enrolled: 10 patients in the responder group (R) and 2 patients in the non-responder group (NR). We identified seven patients (58.3%) who experienced adverse reactions (AE). Although we did not observe a significant difference across the study population in terms of relative abundance and alpha and beta diversity, we found a differing distribution of bacterial taxa between the analyzed groups. We noted a higher level of the class Bacteroidia and the order Bacteroidales in the R group, and an inversion in the Firmicutes and Bacteroidetes ratio in the AE group. No prior studies have focused on linking GM and response to BCRi in these patients. Although the analyses are preliminary, they provide suggestions to guide future research.

Ventilator-Associated Pneumonia Due to MRSA vs. MSSA: What Should Guide Empiric Therapy?

The guidelines on ventilator-associated pneumonia (VAP) recommend an empiric therapy against methicillin-resistant Staphylococcus aureus (MRSA) according to its prevalence rate. Considering the MRSA and MSSA VAP prevalence over the last 9 years in our tertiary care hospital, we assessed the clinical value of the MRSA nasal-swab screening in either predicting or ruling out MRSA VAP. We extracted the data of 1461 patients with positive bronchoalveolar lavage (BAL). Regarding the MRSA nasal-swab screening, 170 patients were positive for MRSA or MSSA. Overall, MRSA had a high prevalence in our ICU. Despite the COVID-19 pandemic, there was a significant downward trend in MRSA prevalence, while MSSA remained steady over time. Having VAP due to MRSA did not have any impact on LOS and mortality. Finally, the MRSA nasal-swab testing demonstrated a very high negative predictive value for MRSA VAP. Our results suggested the potential value of a patient-centered approach to improve antibiotic stewardship.

The Bio-Diversity and the Role of Gut Microbiota in Postmenopausal Women with Luminal Breast Cancer Treated with Aromatase Inhibitors: An Observational Cohort Study.

The interactions between aromatase inhibitors (AI) in breast cancer (BC) and gut microbiota (GM) have not been completely established yet. The aim of the study is to evaluate the bio-diversity of GM and the relationship between GM, inflammation and tumor-infiltrating lymphocytes (TILs) in postmenopausal women with BC during adjuvant AI treatment compared to women with disease relapse during or after one year of AI therapy ("endocrine-resistant"). We conducted a monocenter observational case-control study. Eighty-four women with BC (8 cases, 76 controls) were enrolled from 2019 to 2021. We observed a significant difference in the mean microbial abundance between the two groups for the taxonomic rank of order (p 0.035) and family (p 0.029); specifically, the case group showed higher diversity than the control group. Veillonella reached its maximum abundance in cases (p 0.022). Cytokine levels were compared among the groups created considering the TILs levels. We obtained a statistically significant difference (p 0.045) in IL-17 levels among the groups, with patients with low TILs levels showing a higher median value for IL-17 (0.15 vs. 0.08 pg/mL). Further studies about the bio-diversity in women with BC may lead to the development of new biomarkers and targeted interventions.