One-year monitoring of potentially pathogenic microorganisms in the waters and sediments of the Lesina and Varano lagoons (South-East Italy).

In this study, two Mediterranean coastal lagoons (Lesina and Varano) of southern Italy, located in the north of the Apulia region, were investigated for the presence of Shiga toxin Escherichia coli (STEC) and potentially enteropathogenic Vibrio species in parallel with norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the presence of potentially pathogenic bacteria and viruses in the water and sediments of these ecosystems. From March 2022 to February 2023, a total of 98 samples were collected: 49 water samples and 49 sediment samples. STEC strains were isolated in three samples (3.1%), of which one (2%) was water (stx1 and stx2 positive) and two (4.1%) were sediment (both stx2 positive) samples. Vibrio spp. were detected in twenty samples (20.4%), of which nine were water (18.4%) and eleven were sediment (22.4%) samples. The species detected included V. parahaemolyticus, V. cholerae, and V. vulnificus. NoV was detected in 25 (25.5%) samples, while none of the water or sediment samples were positive for HAV, HEV, and SARS-CoV-2. The results of this study provide an overview of the presence of potentially pathogenic microorganisms in areas influenced by anthropogenic pressure. Monitoring the circulation of these pathogens could be useful to evaluate the water flowing into the lagoons, in particular discharge waters (i.e., urban, agricultural, and livestock runoff), considering the presence of fish and shellfish farms in these sites.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Evidence of Circulation and Phylogenetic Analysis of Hepatitis E Virus (HEV) in Wild Boar in South-East Italy.

Hepatitis E virus (HEV) is an important cause of acute viral hepatitis in humans worldwide. The food-borne transmission of HEV appears to be a major route in Europe through the consumption of pork and wild boar meat. HEV epidemiology in wild boars has been investigated mainly in Northern and Central Italian regions, whilst information from Southern Italy is limited. We investigated the occurrence of HEV in wild boar in the Apulia and Basilicata regions (Southern Italy). Thirteen (10.4%) out of one hundred and twenty-five wild boar samples tested positive for HEV using a quantitative reverse transcription PCR. HEV prevalence was 12% in Apulia and 9.3% in Basilicata. Seven samples were genotyped, and different subtypes (c, f, m) of genotype 3 were identified. The complete genome of a 3m strain was determined, and the virus showed the highest nucleotide identity to a human HEV strain identified in France in 2017. These findings demonstrate the substantial circulation of HEV in the wild boar population in Italian Southern regions. Gathering information on the HEV strains circulating in different geographical areas is useful for tracking the origin of HEV outbreaks and assessing the epidemiological role of wild boar as a potential virus reservoir for domestic pigs.

Occurrence of a New Variant of Salmonella Infantis Lacking Somatic Antigen.

Salmonella Infantis is one of the most frequent serovars reported in broilers and is also regularly identified in human salmonellosis cases, representing a relevant public health problem. In the laboratories of the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZSPB), six Salmonella Infantis strains with antigenic formula -:r:1,5 have been isolated from the litter and carcass of broilers between 2018 and 2022. The strains were investigated to evaluate their phenotype, antibiotic resistance and genomic profiles. Genomic analysis confirmed that the isolates belonged to the Infantis serotype and to the sequence type ST32. Moreover, all strains showed a multidrug-resistant (MDR) profile and were characterised by the presence of the IncFIB plasmid incompatibility group. Three strains had the blaCTX-M-1 gene, and one of them carried IncX1. The presence of this new variant of S. Infantis is particularly relevant because it could expand the landscape of the S. Infantis population. The absence of the somatic antigen could pose a problem in both isolation and serotyping and a consequent public health concern due to the spread of Salmonella infection.

In vitro activity of ceftazidime/avibactam against carbapenem-nonsusceptible Klebsiella penumoniae isolates collected during the first wave of the SARS-CoV-2 pandemic: a Southern Italy, multicenter, surveillance study.

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) represent a serious threat for human health being frequently resistant to most of available antibiotics classes. Recently, ceftazidime/avibactam (CAZ/AVI) has been approved for treatment of infections by Gram-negative bacteria, including class A CPE (including KPC-producing K. pneumoniae). Following CAZ/AVI commercialization, resistance to this combination has been reported. The aim of this study was to evaluate the prevalence of CAZ/AVI resistance among carbapenem-resistant K. pneumoniae(CR-Kp) isolates recovered from bloodstream infections (BSI) and hospital-acquired pneumonia (HAP), representative of the contemporary southern Italy epidemiology, during the first pandemic wave of SARS-CoV-2. METHODS: From Jan...20-Jun...20, 4 Laboratories, collected all consecutive, non-replicated CR-Kp from BSIs and HAPs. All isolates were subjected to i) MALDI-ToF identification; ii) antimicrobial susceptibility testing by microdilution method. CAZ/AVI resistant (CAZ/AVI-R) isolates were screened for presence of most common carbapenemase genes and subjected to whole genome sequencing for characterization. RESULTS: A total of 89 isolates were collected. The majority of strains retained susceptibility to colistin, gentamicin and amikacin. Three strains (3/89, 3,4%) were CAZ/AVI-R (MIC range 16/4-64/4 mg/L). Among CAZ/AVI-R, one was KPC-type producer (an ST101) while the remaining where NDM-type and VIM-type producers and belonged to ST147, and ST45, respectively. CONCLUSION: During the pandemic period, in southern Italy, CAZ/AVI resistance remained infrequent but high-risk Klebsiella pneumoniae epidemic clones, producing the KPC-31 variant and class B carbapenamases were reported from some of the included centers.

Occurrence and Characterisation of Colistin-Resistant Escherichia coli in Raw Meat in Southern Italy in 2018-2020.

Colistin is a last-resort drug for the treatment of infections by carbapenem-resistant Enterobacteriaceae, and the emergence of colistin resistance poses a serious clinical challenge. The aim of this study was to investigate the occurrence of colistin-resistant Escherichia coli in retail meat in Southern Italy in 2018-2020. Of 570 samples, 147 contained E. coli. Two out of 147 (1.4%) E. coli showed a non-wild-type phenotype to colistin and harboured mcr-1. mcr-1 was also detected in a wild-type isolate, resulting in a 2% mcr prevalence. mcr-1-positive isolates originated from turkey meat collected in Apulia (n = 2) and Basilicata (n = 1). A whole-genome sequencing analysis confirmed mcr-1.2 and mcr-1.1 in two and one isolate, respectively. The strains were diverse, belonging to three multi-locus sequence types (ST354, ST410, SLV of ST10) and harbouring genes mediating resistance to antimicrobials in two, six and seven classes. mcr-1 was carried by IncX4 plasmids with high nucleotide similarity to IncX4 plasmids harbouring mcr-1.2 and mcr-1.1 in Enterobacterales from different sources and geographical regions. This is the first study reporting updates on E. coli non-wild-type to colistin from retail meat in Southern Italy, highlighting the importance of phenotypic and genotypic antimicrobial resistance surveillance to contain the dissemination of mcr among E. coli.

Detection of Coxiella burnetii DNA in sheep and goat milk and dairy products by droplet digital PCR in south Italy.

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that is responsible for Q fever, a common zoonosis which is present virtually worldwide. This microorganism infects a wide range of wild and domestic mammals, but the main reservoirs are cattle, goats and sheep, which also represent sources of human infection. A potential route of transmission of this pathogen to humans is the consumption of C. burnetii-contaminated raw milk or dairy products derived from contaminated raw milk, although the role of these foods as possible infection sources is controversial. The aims of this study were (i) to apply two ddPCR based assays targeting the C. burnetii IS1111 and icd genes for the detection and quantification of C. burnetii DNA, and (ii) to evaluate the occurrence of C. burnetii DNA in raw milk and raw milk products from sheep and goats in Apulia and Basilicata regions of Southern Italy. Of 413 milk and cheese samples tested, 78 were positive for the presence of C. burnetii DNA (18.9%), specifically, 68 of 285 milk samples (23.9%) and 10 of 128 cheese samples (7.8%) The presence of both IS1111 and icd genes was detected in only 2 (2.6%) of the 78 positive samples, while the remaining 76 (97.4%) were positive only for IS1111. C. burnetii DNA was specifically detected by the ddPCR method, whereas no cross-amplification was observed with the DNA of other foodborne bacterial pathogens. The sensitivity of the ddPCR method was determined as 0.35 and 0.56 copies/µL for IS1111 and icd genes, respectively. The findings of this study demonstrate the presence of C. burnetii DNA in a significant proportion of raw milk and dairy products. Although there is no conclusive epidemiological evidence that C. burnetii infection occurs via food, the presence of this organism in raw milk and dairy products made of raw milk should be considered a potential hazard. ddPCR is a useful tool to investigate the quality and safety of food products due to its sensitivity and precision, and could be applied to routine testing.

Airborne concentrations of SARS-CoV-2 in indoor community environments in Italy.

COVID-19 pandemic raised a debate regarding the role of airborne transmission. Information regarding virus-laden aerosol concentrations is still scarce in community indoors and what are the risks for general public and the efficiency of restriction policies. This work investigates, for the first time in Italy, the presence of SARS-CoV-2 RNA in air samples collected in different community indoors (one train station, two food markets, one canteen, one shopping centre, one hair salon, and one pharmacy) in three Italian cities: metropolitan city of Venice (NE of Italy), Bologna (central Italy), and Lecce (SE of Italy). Air samples were collected during the maximum spread of the second wave of pandemic in Italy (November and December 2020). All collected samples tested negative for the presence of SARS-CoV-2, using both real-time RT-PCR and ddPCR, and no significant differences were observed comparing samples taken with and without customers. Modelling average concentrations, using influx of customers' data and local epidemiological information, indicated low values (i.e. < 0.8 copies m-3 when cotton facemasks are used and even lower for surgical facemasks). The results, even if with some limitations, suggest that the restrictive policies enforced could effectively reduce the risk of airborne transmissions in the community indoor investigated, providing that physical distance is respected.

A review on measurements of SARS-CoV-2 genetic material in air in outdoor and indoor environments: Implication for airborne transmission.

Airborne transmission of SARS-CoV-2 has been object of debate in the scientific community since the beginning of COVID-19 pandemic. This mechanism of transmission could arise from virus-laden aerosol released by infected individuals and it is influenced by several factors. Among these, the concentration and size distribution of virus-laden particles play an important role. The knowledge regarding aerosol transmission increases as new evidence is collected in different studies, even if it is not yet available a standard protocol regarding air sampling and analysis, which can create difficulties in the interpretation and application of results. This work reports a systematic review of current knowledge gained by 73 published papers on experimental determination of SARS-CoV-2 RNA in air comparing different environments: outdoors, indoor hospitals and healthcare settings, and public community indoors. Selected papers furnished 77 datasets: outdoor studies (9/77, 11.7%) and indoor studies (68/77. 88.3%). The indoor datasets in hospitals were the vast majority (58/68, 85.3%), and the remaining (10/68, 14.7%) were classified as community indoors. The fraction of studies having positive samples, as well as positivity rates (i.e. ratios between positive and total samples) are significantly larger in hospitals compared to the other typologies of sites. Contamination of surfaces was more frequent (in indoor datasets) compared to contamination of air samples; however, the average positivity rate was lower compared to that of air. Concentrations of SARS-CoV-2 RNA in air were highly variables and, on average, lower in outdoors compared to indoors. Among indoors, concentrations in community indoors appear to be lower than those in hospitals and healthcare settings.

Case Report: Molecular Detection of Dirofilaria repens in an Italian Patient after a Stay in Tanzania.

A 35-year-old man was admitted to a hospital in the south of Italy because of a periocular nodule and subpalpebral edema. The patient reported having been stayed in Tanzania five months before. Hematologic parameters were within the normality range, the Acanthocheilonema viteae ELISA did not detect significant levels of antifilarial IgG, and no further symptoms were described. The surgical inspection of the nodule led to the isolation of two filarioid parasites, identified as Dirofilaria repens by scanning electron microscope (SEM), and then by molecular assays. Knott's test did not reveal microfilaremia, whereas loop-mediated isothermal amplification and PCR detected D. repens DNA. The patient was treated with doxycycline, and he was found no more positive at the follow-up.

Prevalence of Verocytotoxigenic Escherichia coli strains isolated from raw beef in southern Italy.

Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a significant foodborne public health hazard, where most human infections are associated with six serogroups (O157, O26, O103, O145, O111 and O104). VTEC was the fourth most commonly reported zoonosis in the EU in 2015, with 5901 confirmed human cases. Ruminant animals, including cattle, are a major reservoir of VTEC. The consumption of VTEC-contaminated animal-derived foodstuffs, especially undercooked ground beef, is an important transmission route. To the best of our knowledge, there are few data available on the contamination of VTEC in meat products in Italy. During 2015 and 2016, 250 raw meat samples were collected from retail markets in southern Italy (Apulia) and analysed for the occurrence of vtx genes (vtx1/vtx2) at the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB, Italy). In addition, the isolates were characterized by determining the presence of VTEC main virulence factors, the antimicrobial resistance profiles and the genetic relatedness by pulsed-field gel electrophoresis (PFGE). The results have shown that 8.4% (21/250) of the samples were positive for vtx genes in the preliminary screening step but VTEC strains were isolated from only 2% (5/250) of overall meat analysed samples, including raw ground beef, beef hamburger and beef carpaccio. 5 isolates displayed a multi-drug resistance phenotype. All VTEC strains were analysed by XbaI-PFGE and dendrogram revealed 5 distinct restriction profiles, indicating their relatively high genetic diversity. Although this study demonstrates a low prevalence of VTEC in raw beef marketed in southern Italy, the presence of potentially pathogenic E. coli strains points to the need for proper hygiene during meat production to reduce the risk of foodborne illness and transmission of multi-drug resistant organisms via foods to humans.

Verocytotoxin-producing Escherichia coli O26 in raw water buffalo (Bubalus bubalis) milk products in Italy.

Escherichia coli 026 is known as a verocytotoxin-producing E. coli (VTEC) organism that causes severe foodborne diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Although cattle are the most important reservoir of VTEC, only a few reports on the role of water buffalo (Bubalus bubalis) as a reservoir of VTEC and on the presence of these organisms in their milk are available. However, in Southern Italy, where water buffalo are intensively reared, an outbreak of hemolytic uremic syndrome due to E. coli 026 has recently been reported, in which the consumption of typical dairy products was considered to be a common risk factor. The aims of this work were to assess the prevalence of E. coli O26 in raw water buffalo milk, to characterize the virulence gene profiles of the isolates, and to evaluate their phenotypic antimicrobial resistance pattern. Of 160 analyzed samples, 1 (0.6%) tested positive for E. coli O26, and the isolate showed the stx1+/stx2+/eae-/hlyA+ genotypic profile. The strain showed resistance against glycopeptides, macrolides, and penicillins. The presence of VTEC organisms in raw water buffalo milk could be considered to be a potential threat to consumers; however, the strict adherence to the processes used in the preparation of the most common buffalo dairy products could strongly mitigate the foodborne risk. To our knowledge, this article reports the first isolation and characterization of E. coli O26 VTEC in raw water buffalo milk.