Search

Escherichia coli can harbor a broad repertoire of virulence and antimicrobial resistance (AMR) genes, which can be exchanged across the human gastrointestinal microflora, thus posing a public health risk. In this study, 6 batches of artisanal soft cheese and a 6-month ripened fermented dried sausage were investigated to assess the occurrence, phylogeny, and genomic traits (AMR, virulence, and mobilome) of E. coli. 30 and 3 strains isolated from salami and cheese food chains, respectively, were confirmed as E. coli by whole genome sequencing. The accumulation of single nucleotide polymorphism differences within small clusters of strains encompassing batches or processing stages, combined with high serotype and phylogroup diversity, suggested the occurrence of different contamination phenomena among the facilities. A total of 8 isolates harbored plasmid-mediated resistance genes, including one cheese strain that carried an IncQ1 plasmid carrying AMR determinants to macrolides [mph(B)], sulfonamides (sul1, sul2), trimethoprim (dfrA1), and aminoglycosides [aph(3")-Ib and aph(6)-Id]. A pool of virulence-associated genes in the class of adhesion, colonization, iron uptake, and toxins, putative ColV-positive iron uptake systems sit, iro, or iuc (8 salami and 2 cheese), plasmid-encoded hemolysin operon hlyABCD (one salami), and potential atypical enteropathogenic E. coli (3 salami environment) were reported. Overall, our findings underscore the importance of routine surveillance of E. coli in the artisanal food chain to prevent the dissemination of AMR and virulence. [ABSTRACT FROM AUTHOR]

Artisanal salami is produced in small-scale production plants, where the lack of full automation might result in higher variability in food intrinsic properties. The aim of the present study was to evaluate the inter- and intra-batch variability in physicochemical parameters and its impact on microbial quality and occurrence of foodborne pathogens on 480 samples collected from six batches of an artisanal Italian production of organic salami. Relatively high total bacterial counts (TBC) were found on the surface of the table in the stuffing room (4.29 ± 0.40 log cfu/cm2). High loads of Enterobacteriaceae in the meat mixture of batch 2 and TBC in batch 5 were associated with a higher occurrence of bacterial pathogens. During ripening, water activity (aw) and pH failed to reach values lower than 0.86 and 5.3, respectively. Six Staphylococcus aureus and four Listeria monocytogenes isolates were collected from the salami meat mixture during ripening and the processing environment. A total of 126 isolates of Enterobacteriaceae were characterized at a species level, with Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Citrobacter freundii isolated from the final products. Results suggest the relevance of first steps of production in terms of the hygiene of raw materials and handling during stuffing procedures, especially when the physicochemical parameters of the final products do not reach values that represent hurdles for foodborne pathogens. [ABSTRACT FROM AUTHOR]

Simple Summary: Although the use of antimicrobials and the related selection of antimicrobial-resistant (AMR) pathogens are recognized worldwide, limited or no information is available on the effect of the antibiotic treatment on the genetic structure as well as the dissemination and persistence of plasmids carrying AMR genetic determinants. This is of particular interest for bacterial pathogens like Avian Pathogenic Escherica coli (APEC), which have the potential to persist and be transmitted from broilers to humans through the food chain as well as for their AMR genetic determinants, which have the potential to be mobilized and spread. With a genomic approach, results of the present study revealed that during the enrofloxacin treatment of broilers, initial strains of extraintestinal E. coli disappeared, being substituted by new clones. Plasmid-mediated fluoroquinolone resistance did not appear to disseminate or persist among observed genomes, confirming that QRDR mutations rather than plasmids are the main drivers of quinolone resistance. Interestingly, plasmids carrying other AMR genes than quinolone-determinant ones were disseminated and persistent since they were found in different clones both before and after the treatment. The persistence of plasmids without a direct antimicrobial selective pressure, if confirmed with further studies, might give insights on the so-called plasmid paradox. The aim of the present study was to investigate the genetic diversity and antimicrobial resistance (AMR) of E. coli during enrofloxacin therapy in broilers affected by colisepticemia. Three unrelated farms with ongoing colibacillosis outbreaks were sampled at day 1 before treatment and at days 5, 10 and 24 post-treatment. A total of 179 E. coli isolates were collected from extraintestinal organs and submitted to serotyping, PFGE and the minimum inhibitory concentration (MIC) against enrofloxacin. PFGE clusters shifted from 3–6 at D1 to 10–16 at D5, D10 and D24, suggesting an increased population diversity after the treatment. The majority of strains belonged to NT or O78 and to ST117 or ST23. PFGE results were confirmed with SNP calling: no persistent isolates were identified. An increase in resistance to fluoroquinolones in E. coli isolates was observed along the treatment. Resistome analyses revealed qnrB19 and qnrS1 genes along with mutations in the gyrA, parC and parE genes. Interestingly, despite a fluoroquinolone selective pressure, qnr-carrying plasmids did not persist. On the contrary, two conjugative AMR plasmid clusters (AB233 and AA474) harboring AMR genes other than qnr were persistent since they were identified in both D1 and D10 genomes in two farms. Further studies should be performed in order to confirm plasmid persistence not associated (in vivo) to antimicrobial selective pressure. [ABSTRACT FROM AUTHOR]

In the present study, the occurrence of Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Escherichia coli VTEC was investigated in two batches of artisanal Italian salami tested in winter and summer. Moreover, enumerations of total bacterial count, lactic acid bacteria and Enterobacteriaceae were performed as well as monitoring of water activity and pH. Samples were taken from raw materials, production process environment, semi-finished product and finished products. The results revealed an overall increase of total bacterial count and lactic acid bacteria during the ripening period, along with a decrease of Enterobacteriaceae, pH and water activity. No significant difference was observed between the two batches. The enterobacterial load appeared to decrease during the maturation period mainly due to a decrease in pH and water activity below the limits that allow the growth of these bacteria. E. coli VTEC, Salmonella spp. or L. monocytogenes were not detected in both winter and summer batches. However, Klebsiella pneumoniae was detected in both summer and winter products. Except for one isolate, no biological hazards were detected in the finished salami, proving the efficacy of the ripening period in controlling the occurrence of microbiological hazard in ripened salami. Further studies are required to assess the virulence potential of the Klebsiella pneumoniae isolates. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the resistome and virulome diversity of 43 isolates of Listeria monocytogenes, Salmonella enterica and S. aureus collected from artisanal fermented meat and dairy products and their production environments in Portugal, Spain, Italy and Morocco. After DNA extraction, genomes were sequenced, and de novo assembled. Genetic relationships among genomes were investigated by SNP calling and in silico 7- loci MLST. Genomes of the same species belonged to different ST-types demonstrating the circulation of different clones in in the same artisanal production plant. One specific clone included genomes of S. Paratyphi B belonging to ST43 and repeatedly isolated for more than a year in an artisanal sausage production plant. No genomes but three (belonging to Salmonella enterica), were predicted as multiresistant to different antimicrobials classes. Regarding virulence, genomes of L. monocytogenes belonging to ST1, ST3 and ST489, as well as genomes of S.enterica enterica (ST43, ST33, ST314, ST3667, ST1818, ST198) and ST121 S. aureus were predicted as virulent and hypervirulent. The occurrence of virulent and hypervirulent L. monocytogenes, Salmonella enterica and S. aureus strains in artisanal fermented meat and dairy productions as well as in their finished products suggests the need for a specific focus on prevention and control measures able to reduce the risk of these biological hazards in artisanal food productions. [ABSTRACT FROM AUTHOR]

Artisanal cheeses are produced in small-scale production plants, where the lack of full automation and control of environmental and processing parameters suggests a potential risk of microbial contamination. The aim of this study was to perform a longitudinal survey in an Italian artisanal factory producing a spreadable soft cheese with no rind to evaluate the inter- and intra-batch variability of physicochemical and microbial parameters on a total of 720 environmental and cheese samples. Specifically on cheese samples, the evaluation was additionally performed on physicochemical parameters. Cheese samples were additionally collected during 15 days of storage at constant temperatures of 2 and 8 °C, as well as a dynamic profile of 2 °C for 5 days and 8 °C for 10 days. Furthermore, Enterobacteriaceae isolates were identified at species level to have a better knowledge of the environmental and cheese microbiota potentially harboring human pathogens. High interbatch variability was observed for lactic acid bacteria (LAB) and total bacteria count (TBC) in cheese at the end of production but not for pH and water activity. A temperature of 8 °C was associated with a significantly higher load of Enterobacteriaceae in cheeses belonging to batch 6 at the end of storage, and this temperature also corresponded with the highest increase in LAB and TBC loads over cheese shelf life. Results from generalized linear mixed models (GLMMs) indicated that drains in the warm room and the packaging area were associated with higher levels of TBC and Enterobacteriaceae in cheese. Regarding foodborne pathogens, no sample was positive for verotoxigenic Escherichia coli (VTEC) or Listeria monocytogenes, whereas six Staphylococcus aureus and one Salmonella pullorum isolates were collected in cheese samples during storage and processing, respectively. Regarding Enterobacteriaceae, 166 isolates were identified at species level from all batches, with most isolates belonging to Klebsiella oxytoca and pneumoniae, Enterobacter cloacae, Hafnia alvei, and Citrobacter freundii evidencing the need to focus on standardizing the microbial quality of cow milk and on hygienic procedures for cleaning and disinfection especially in warm and maturation rooms. Further studies should be performed to investigate the potential pathogenicity and antimicrobial resistance of the identified Enterobacteriaceae species in artisanal cheeses. [ABSTRACT FROM AUTHOR]

The present study aimed at assessing the occurrence of microbiological hazards (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Escherichia coli O157) in an artisanal soft cheese produced in northern Italy. In the same product total bacterial count, lactic acid bacteria and Enterobacteriaceae were enumerated, and pH and water activity measured in two batches sampled in summer and winter. Samples of raw materials, environmental swabs from the production processes and cheese during 15 days of storage at 2 and 8°C as well as dynamic temperature of 2°C for 5 days and 8°C for 10 days were collected and tested. The load of total bacterial count was significantly higher in the winter batch in comparison to the summer one, with a significant increase at the end of the storage period also noticed for lactic acid bacteria. Statistical higher values of pH were registered in raw materials and end of storage in winter batch. S. aureus was confirmed only in the winter batch within samples (n=4) of stored cheese. On plates used for E. coli O157 detection, colonies of Klebsiella pneumoniae and Klebsiella oxytoca were isolated. The results suggest that the highest bacterial population in the winter batch was associated to a higher pH in stored cheese and a higher number of biological hazards identified. Their isolation started in the maturation room suggesting this step as relevant for possible cheese contamination. [ABSTRACT FROM AUTHOR]

This study is focused on resistance to carbapenems and third-generation cephalosporins in Gram-negative microorganisms isolated from swine, whose transmission to humans via pork consumption cannot be excluded. In addition, the common carriage of carbapenem-resistant (CR) bacteria between humans and pigs was evaluated. Sampling involved 300 faecal samples collected from slaughtered pigs and 300 urine samples collected from 187 hospitalised patients in Parma Province (Italy). In swine, MIC testing confirmed resistance to meropenem for isolates of Pseudomonas aeruginosa and Pseudomonas oryzihabitans and resistance to cefotaxime and ceftazidime for Escherichia coli, Ewingella americana, Enterobacter agglomerans, and Citrobacter freundii. For Acinetobacter lwoffii, Aeromonas hydrofila, Burkolderia cepacia, Corynebacterium indologenes, Flavobacterium odoratum, and Stenotrophomonas maltophilia, no EUCAST MIC breakpoints were available. However, ESBL genes (blaCTXM-1, blaCTX-M-2, blaTEM-1, and blaSHV) and AmpC genes (blaCIT, blaACC, and blaEBC) were found in 38 and 16 isolates, respectively. P. aeruginosa was the only CR species shared by pigs (4/300 pigs; 1.3%) and patients (2/187; 1.1%). P. aeruginosa ST938 carrying blaPAO and blaOXA396 was detected in one pig as well as an 83-year-old patient. Although no direct epidemiological link was demonstrable, SNP calling and cgMLST showed a genetic relationship of the isolates (86 SNPs and 661 allele difference), thus suggesting possible circulation of CR bacteria between swine and humans. [ABSTRACT FROM AUTHOR]