Bioinformatic survey of CRISPR loci across 15 Serratia species

The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) system of prokaryotes is an adaptative immune defense mechanism to protect themselves from invading genetic elements (e.g., phages and plasmids). Studies that describe the genetic organization of these prokaryotic systems have mainly reported on the Enterobacteriaceae family (now reorganized within the order of Enterobacterales). For some genera, data on CRISPR-Cas systems remain poor, as in the case of Serratia (now part of the Yersiniaceae family) where data are limited to a few genomes of the species marcescens. This study describes the detection, in silico, of CRISPR loci in 146 Serratia complete genomes and 336 high-quality assemblies available for the species ficaria, fonticola, grimesii, inhibens, liquefaciens, marcescens, nematodiphila, odorifera, oryzae, plymuthica, proteomaculans, quinivorans, rubidaea, symbiotica, and ureilytica. Apart from subtypes I-E and I-F1 which had previously been identified in marcescens, we report that of I-C and the I-E unique locus 1, I-E*, and I-F1 unique locus 1. Analysis of the genomic contexts for CRISPR loci revealed mdtN-phnP as the region mostly shared (grimesii, inhibens, marcescens, nematodiphila, plymuthica, rubidaea, and Serratia sp.). Three new contexts detected in genomes of rubidaea and fonticola (puu genes-mnmA) and rubidaea (osmE-soxG and ampC-yebZ) were also found. The plasmid and/or phage origin of spacers was also established

Legionella and legionellosis in touristic-recreational facilities. Influence of climate factors and geostatistical analysis in Southern Italy (2001-2017)

Legionella is the causative agent of Legionnaires' disease, a flu-like illness normally acquired following inhalation or aspiration of contaminated water aerosols. Our recent studies revealed that climatic parameters can increase the number of reported cases of community-acquired Legionnaires' disease. Here, we evaluated the presence of Legionella in water networks and the distribution of Legionnaires' disease cases associated with touristic-recreational facilities in the Apulia region (southern Italy) during the period 2001-2017 using geostatistical and climatic analyses. Geostatistical analysis data revealed that the area with the highest concentration of Legionella in water systems also had the greatest number of cases of Legionnaires' disease associated with touristic-recreational facilities. Climatic analysis showed that higher daily temperature excursion (difference between maximum and minimum temperature) on the day of sampling was more often associated with Legionella-positive samples than Legionella-negative samples. In addition, our data highlighted an increased risk of Legionnaires' disease with increases in precipitation and average temperature and with decreases in daily temperature excursion (difference between maximum and minimum temperature over the course of 24 h in the days of incubation period of disease) and minimum temperature. Healthcare professionals should be aware of this phenomenon and be particularly vigilant for cases of community-acquired pneumonia during such climatic conditions and among the tourist population. The innovative geo-statistical approach used in this study could be applied in other contexts when evaluating the effects of climatic conditions on the incidence of Legionella infections

Identification and Characterisation of pST1023 A Mosaic, Multidrug-Resistant and Mobilisable IncR Plasmid

We report the identification and characterisation of a mosaic, multidrug-resistant and mobilisable IncR plasmid (pST1023) detected in Salmonella ST1023, a monophasic variant 4,[5],12:i: strain of widespread pandemic lineage, reported as a Southern European clone. pST1023 contains exogenous DNA regions, principally gained from pSLT-derivatives and IncI1 plasmids. Acquisition from IncI1 included oriT and nikAB and these conferred the ability to be mobilisable in the presence of a helper plasmid, as we demonstrated with the conjugative plasmids pST1007-1D (IncFII) or pVC1035 (IncC). A sul3-associated class 1 integron, conferring resistance to aminoglycosides, chloramphenicol and trimethoprim-sulphonamides, was also embedded in the acquired IncI1 DNA segment. pST1023 also harboured an additional site-specific recombination system (rfsF/rsdB) and IS elements of the IS1, IS5 (IS903 group) and IS6 families. Four of the six IS26 elements present constituted two pseudo-compound-transposons, named PCT-sil and PCT-Tn10 (identified here for the first time). The study further highlighted the mosaic genetic architecture and the clinical importance of IncR plasmids. Moreover, it provides the first experimental data on the ability of IncR plasmids to be mobilised and their potential role in the horizontal spread of antimicrobial-resistant genes

Occurrence of Legionella in groundwater used for sprinkler irrigation in Southern Italy

Legionellae are opportunistic bacteria that cause various conditions after exposure to contaminated aerosols, ranging from a serious type of pneumonia to a mild case of an influenza-like illness. Despite the risks of exposure, little is known about the occurrence of Legionella in natural environments and, even though studies have shown that there is a potential risk of transmission via inhalation, it does not have to be detected in groundwater that is used for irrigation. The culture methods traditionally used to detect Legionella have several limits that can be partly solved by applying molecular techniques. Samples from 177 wells in Apulia, Southern Italy, were collected twice, in winter and in summer, and analyzed. When compared with the guidelines, 145 (81.9%) of the sampled wells were suitable for irrigation use. The culture-based method highlighted the presence of different species and serogroups of Legionella in 31 (21.2%) of the 145 wells that were shown to be suitable for irrigation use. A greater number of wells returned positive results for Legionella in summer than in winter (p = 0.023), and the median concentrations were mostly higher in summer (500 CFU/L) than in winter (300 CFU/L). The median temperature in the Legionella positive well waters was significantly higher than that in the negative ones, both in winter and in summer (p < 0.001). Using molecular techniques, Legionella non-pneumophila was found in 37 of the 114 wells earlier detected as suitable for irrigation use but negative for Legionella by the culture-based methods. The distribution of Legionella differ significantly in porous aquifers compared to the karst-fissured ones both with quantitative polymerase chain reaction (qPCR) (p = 0.0004) and viable cells by propidium monoazide (PMA-qPCR) (p = 0.0000). Legionella concentrations were weakly correlated with temperature of water both with qPCR (ρ = 0.47, p = 0.0033) and PMA-qPCR (ρ = 0.41, p = 0.0126). Our data suggest that water that aerosolizes when sprinkled on plants represents a potential source of Legionellosis, with a higher risk from exposure in summer. On a practical level, this finding is important for workers (farmers and gardeners) who are in contact with waters used for irrigation

Food Hygiene Surveillance in Italy: Is Food Ice a Public Health Risk?

Food ice is used as an ingredient or as a coolant in drinks and in the storage of food, especially fishery products. Studies show that ice can be polluted both by chemical substances and by bacteria and fungi. In particular, the presence of fungi in these food matrices has acquired an important role in Public Health, as it can represent a risk factor for fungal complications in immunocompromised subjects. In the present study we evaluated the hygiene–sanitary quality of food ice from public and collective catering establishments in a large area of Southern Italy, investigating the mandatory parameters (Escherichia coli, coliform and Enterococci) and some accessory parameters (Staphylococcus aureus, Pseudomonas aeruginosa and fungi) provided for Italian Legislative Decree 31/01. Although 54.5% of samples were compliant, the results highlight a vast contamination of food ice by bacteria and fungi. In particular, 95.8% of samples were contaminated by fungi, stressing no difference between compliant and non-compliant samples. Their presence is generally attributable to the poor sanitation conditions in the production and/or administration phase and to the incorrect sanitization and ordinary maintenance procedures. It seems appropriate to suggest the need to carry out a specific risk assessment with respect to the self-control plan

Evaluation of Air Contamination in Orthopaedic Operating Theatres in Hospitals in Southern Italy: The IMPACT Project

Postoperative infections are a concern, especially in total knee and total hip arthroplasty. We evaluated the air quality in orthopaedic operating theatres in southeastern Italy to determine the level of bacterial contamination as a risk factor for postoperative infection. Thirty-five hospitals with operating theatres focused on total knee and total hip arthroplasty participated. We sampled the air passively and actively before surgeries began for the day (at rest) and 15 min after the surgical incision (in operation). We evaluated bacterial counts, particle size, mixed vs turbulent airflow systems, the number of doors, number of door openings during procedures and number of people in the operating theatre. We found no bacterial contamination at rest for all sampling methods, and significantly different contamination levels at rest vs in operation. We found no association between the number of people in the surgical team and bacteria counts for both mixed and turbulent airflow systems, and low bacterial loads, even when doors were always open. Overall, the air quality sampling method and type of ventilation system did not affect air quality

Potential Use of Untreated Wastewater for Assessing COVID-19 Trends in Southern Italy

As a complement to clinical disease surveillance, the monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in wastewater can be used as an early warning system for impending epidemics. This study investigated the dynamics of SARS-CoV-2 in untreated wastewater with respect to the trend of coronavirus disease 2019 (COVID-19) prevalence in Southern Italy. A total of 210 wastewater samples were collected between May and November 2020 from 15 Apulian wastewater treatment plants (WWTP). The samples were concentrated in accordance with the standard of World Health Organization (WHO, Geneva, Switzerland) procedure for Poliovirus sewage surveillance, and molecular analysis was undertaken with real-time reverse-transcription quantitative PCR (RT-(q) PCR). Viral ribonucleic acid (RNA) was found in 12.4% (26/210) of the samples. The virus concentration in the positive samples ranged from 8.8 × 102 to 6.5 × 104 genome copies/L. The receiver operating characteristic (ROC) curve modeling showed that at least 11 cases/100,000 inhabitants would occur after a wastewater sample was found to be positive for SARS-CoV-2 (sensitivity = 80%, specificity = 80.9%). To our knowledge, this is the first study in Italy that has applied wastewater-based epidemiology to predict COVID-19 prevalence. Further studies regarding methods that include all variables (meteorological phenomena, characteristics of the WWTP, etc.) affecting this type of wastewater surveillance data would be useful to improve data interpretation

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)