Isolation of Listeria ivanovii from Bulk-Tank Milk of Sheep and Goat Farms—From Clinical Work to Bioinformatics Studies: Prevalence, Association with Milk Quality, Antibiotic Susceptibility, Predictors, Whole Genome Sequence and Phylogenetic Relationships

Abstract

A cross-sectional study was performed in 325 sheep and 119 goat dairy farms in Greece. Samples of bulk-tank milk were examined by standard microbiological techniques for Listeria spp. Listeria monocytogenes was isolated from one (0.3%) and Listeria ivanovii from three (0.9%) sheep farms. No associations between the isolation of L. monocytogenes or L. ivanovii and milk quality were found. No resistance to antibiotics was identified. Three variables emerged as significant predictors of isolation of the organism: the presence of pigs, low average relative humidity and a high number of ewes on the farm. The three L. ivanovii isolates were assessed in silico for identification of plasmids, prophages, antibiotic resistance genes, virulence factors, CRISPRs and CAS genes. Phylogenetic analysis using the core genome revealed that the three strains belonged to the L. ivanovii subsp. ivanovii branch and were especially close to the PAM 55 strain. All strains of the branch appeared to be very similar, with the distance between them being small.

Virulence Characteristics and Distribution of the Pathogen Listeria ivanovii in the Environment and in Food

“…A previous study highlighted that L. ivanovii did not grow in Fraser broth after 24 h [161] and in a recent investigation it was found that three L. ivanovii isolates from ovine bulk tank milk did not form colonies on agar Listeria Ottavani & Agosti (ALOA) after the first enrichment in half-Fraser and formed a few colonies after the second enrichment step in Fraser broth on this medium. A better growth was obtained on modified Oxford agar (MOX) after both enrichment steps [127].…”

“…Raw buffalo milk 1 (1.6) Egypt [115], 2 (0.8) Egypt [116] Raw cow milk 2 (0.4) Portugal [51], 82 (6.3) Mexico [117], 7 (35.0) Jordan [118], 8 (4.2) Nigeria [119], 7 (0.9) Syria [120], 1 (0.5) Egypt [93], 1 (0.4) Iran [121], 1 (0.5) Turkey [122], 1 (1.6) Egypt [115], 12 (6) Egypt [123], 2 (0.8) Egypt [116], 3 (4.4) Turkey [124] Raw ewe milk 2 (0.2) Spain [125], 5 (0.9) Spain [126], 5 (0.6) Syria [120], 1 (0.4) Iran [121], 1 (1.6) Egypt [115], 1 (0.4) Egypt [116], 3 (0.9) Greece [127] Raw goat milk 3 (0.2) Spain [128], 4 (20) Egypt [129] Raw zebu milk 2 (1.9) Tanzania [130] Raw milk (animal species not detailed) 1 (0.6) Turkey [53], 1 (0.4) Italy [56], 1 (0.9) India [127], 1 (0.4) Turkey [60], 2 (0.8) Egypt [50], 27 (4.9) Nigeria [131], 5 (1.6) Turkey [51], 1 (0.9) Turkey [66], 3 (1.5) Turkey [67], 7 (3.5) Libya [68], 2 (1.7) Sudan [132], 6 (1.2) Ethiopia [133] Raw buffalo milk 1 (1.6) Egypt [115], 2 (0.8) Egypt [116] Ready to eat (RTE) 1 food products Meat (animal species not detailed) 1 (0.2) Portugal [52], 3 (0.7) Iran [104], 1 (0.4) Egypt [134], 12 (3...…”

Virulence Characteristics and Distribution of the Pathogen <em>Listeria</em> <em>Ivanovii</em> in the Environment and in Food

“…A previous study highlighted that L. ivanovii did not grow in Fraser broth after 24 h [153] and in a recent investigation it was found that three L. ivanovii isolates from ovine bulk tank milk did not form colonies on agar Listeria Ottavani & Agosti (ALOA) after the first enrichment in half-Fraser and formed a few colonies after the second enrichment step in Fraser broth on this medium. A better growth was obtained on modified Oxford agar (MOX) after both enrichment steps [117].…”

“…Nigeria [90], 2 (0.8) Reda Egypt [91], 5 (4.0) Turkey [92], 7 (2.9) Nigeria [93], 1 (0.5) Lybia [62], 12 (11.5) Nigeria [94], 4 (0.9) Ethiopia [95], 4 (0.9) ) Nigeria [96] Raw buffalo meat (0.9) Syria [110], 1 (0.5) Egypt [88], 1 (0.4) Iran [111], 1 (0.5) Turkey [112], 1 (1.6) Egypt [105], 12 (6) Egypt [113], 2 (0.8) Egypt [106], 3 (4.4) Turkey [114] Raw ewe milk 2 (0.2) Spain [115], 5 (0.9) Spain [116], 5 (0.6) Syria [110], 1 (0.4) Iran [111], 1 (1.6) Egypt [105], 1 (0.4) Egypt [106], 3 (0.9) Greece [117] Raw goat milk 3 (0.2) Spain [118], 4 (20) Egypt [119] Raw zebu milk 2 (1.9) Tanzania [120] Raw milk (animal species not detailed) 1 (0.6) Turkey [47], 1 (0.4) Italy [50], 1 (0.9) India [121], 1 (0.4) Turkey [54],…”

Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota

“…In Greece and particularly in Epirus, sheep milk of native breeds, Boutsiko, Karamaniko, Frisarta, Karagouniko and others, is the primary milk used for traditional cheese production ( Fotou et al., 2011 ; Kondyli et al., 2012 ; Skoufos et al., 2017 ), most often after mixing with 10–30 % goat milk, pasteurization or mild thermization, and then addition of natural or commercial starters, depending on the cheese variety and the processor's practical experience ( Samelis et al., 2009 ; Voidarou et al., 2011 ; Vandera et al., 2019 ). So far numerous surveys have been conducted to evaluate the hygienic quality of bulk/tank Greek/Epirus raw sheep and goat milks, with emphasis on total microbial and somatic cell counts, the presence of bacterial pathogens, primarily S. au reus ( Morgan et al., 2003 ; Solomakos et al., 2009 ; Fotou et al., 2011 ; Alexopoulos et al., 2011 ; Zdragas et al., 2015 ; Pexara et al., 2016 ; Malissiova et al., 2017 ; Angelidis et al., 2020 ; Lianou et al., 2021a , 2021b , 2022 ), and occasionally on microbiological differences between sheep breeds ( Kondyli et al., 2012 ; Skoufos et al., 2017 ). In contrast, published studies on the microbial ecology of raw sheep or goat milks with emphasis on the isolation, identification and biotechnological characterization of indigenous LAB strains are still limited in Greece ( Samelis et al., 2009 ; Chanos and Williams, 2011 ; Parapouli et al., 2013 ), inclusively of few recent studies that have characterized some LAB isolates from raw sheep milk as the first processing step during artisanal Feta, Kefalograviera or Graviera raw milk cheese production ( Tsigkrimani et al., 2022a , 2022b ; Apostolakos et al., 2023 ; Psomas et al., 2023 ).…”