Genetic diversity, antibiotic resistance, and virulence profiles of Listeria monocytogenes from retail meat and meat processing.

Human listeriosis outbreaks are often associated with consumption of contaminated food, especially meat products. To better understand meat contamination of L. monocytogenes, whole genome sequencing(WGS) was performed on all detected isolates to investigate genetic relationships between retail markets and slaughterhouses. 110 and 13 isolates were isolated from 1914 food samples and 67 food and environmental samples, respectively. IIa (51/123,41.5%) and IIc (7/123,5.7%) were detected as the dominant serogroups of 123 L. monocytogenes isolates.Most isolates were penicillin-resistant (22/123,17.9%) in the phenotypic test, and all isolates were also found to be susceptible to ampicillin, meropenem, and vancomycin. All of them harbored virulence-associated genes and premature stop codons (PMSCs) in inlA genes were occurred in 35 strains. 22 multilocus sequence types and 19 clonal complexes were identified with ST9 being most common. This study also showed the prevalence and uniqueness of strains from Jilin, China compared with worldwide epidemic international strains. The findings of this study will contribute to the epidemiological understanding of transmission of L. monocytogenes from production and circulation in the region of northern China.

Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities.

Listeria species (spp.) are contaminants that can survive in food, on equipment, and on food processing premises if appropriate hygiene measures are not used. Homologous stress tolerance genes, virulence gene clusters such as the prfA cluster, and clusters of internalin genes that contribute to the pathogenic potential of the strains can be carried by both pathogenic and nonpathogenic Listeria spp. To enhance understanding of the genome evolution of virulence and virulence-associated properties, a comparative genome approach was used to analyze 41 genome sequences belonging to L. innocua and L. welshimeri isolated from food and food processing facilities. Genetic determinants responsible for disinfectant and stress tolerance were identified, including the efflux cassette bcrABC and Tn6188_qac_1 disinfectant resistance determinant, and stress survival islets. These disinfectant-resistant genes were more frequently found in L. innocua (12%) than in L. welshimeri (2%). Several isolates representing the presumed nonpathogenic L. innocua still carried virulence-associated genes, including LGI2, LGI3, LIPI-3, and LIPI-4 which were absent in all L. welshimeri isolates. The mobile genetic elements identified were plasmids (pLGUG1 and J1776) and prophages (PHAGE_Lister_vB_LmoS_188, PHAGE_Lister_LP_030_3, PHAGE_Lister_A118, PHAGE_Lister_B054, and PHAGE_Lister_vB_LmoS_293). The results suggest that the presumed nonpathogenic isolates especially L. innocua can carry genes relevant to the strain's virulence and stress tolerance in the food and food processing facilities. IMPORTANCE This study provides genomic insights into the recently expanded genus in order to gain valuable information about the evolution of the virulence and stress tolerance properties of the genus Listeria and the distribution of these genetic elements pertinent to the pathogenic potential across Listeria spp. and clonal lineages in South Africa (SA).

Whole Genome-Based Characterization of Listeria monocytogenes Isolates Recovered From the Food Chain in South Africa.

Listeria monocytogenes is an important foodborne pathogen which has the ability to adapt and survive in food and food processing facilities where it can persist for years. In this study, a total of 143 L. monocytogenes isolates in South Africa (SA) were characterized for their strain's genetic relatedness, virulence profiles, stress tolerance and resistance genes associated with L. monocytogenes. The Core Genome Multilocus Sequence Typing (cgMLST) analysis revealed that the most frequent serogroups were IVb and IIa; Sequence Types (ST) were ST204, ST2, and ST1; and Clonal Complexes (CC) were CC204, CC1, and CC2. Examination of genes involved in adaptation and survival of L. monocytogenes in SA showed that ST1, ST2, ST121, ST204, and ST321 are well adapted in food processing environments due to the significant over-representation of Benzalkonium chloride (BC) resistance genes (bcrABC cassette, ermC, mdrL and Ide), stress tolerance genes (SSI-1 and SSI-2), Prophage (φ) profiles (LP_101, vB LmoS 188, vB_LmoS_293, and B054 phage), plasmids profiles (N1-011A, J1776, and pLM5578) and biofilm formation associated genes. Furthermore, the L. monocytogenes strains that showed hyper-virulent potential were ST1, ST2 and ST204, and hypo-virulent were ST121 and ST321 because of the presence and absence of major virulence factors such as LIPI-1, LIPI-3, LIPI-4 and the internalin gene family members including inlABCEFJ. The information provided in this study revealed that hyper-virulent strains ST1, ST2, and ST204 could present a major public health risk due to their association with meat products and food processing environments in SA.

Virulence genes and antimicrobial resistance of pathogenic Escherichia coli isolated from diarrheic weaned piglets in Korea.

For efficient prevention and treatment of enteric colibacillosis, understanding about latest virulence factors and antimicrobial resistance of Escherichia coli is essentially needed. The aim of this study was to survey antimicrobial resistance and determine the prevalence of fimbriae and enterotoxin genes among 118 pathogenic E. coli isolates obtained from Korean pigs with diarrhea between 2016 and 2017. The genes for the toxins and adhesins were amplified by polymerase chain reaction (PCR). The susceptibility of the E. coli isolates to antimicrobials were tested using the standard Kirby-Bauer disk diffusion method. The most prevalent fimbrial antigen was F18 (40.7%), followed by F4 (16.9%), and the most prevalent combinations of toxin genes were Stx2e (21.2%), STb:EAST-1 (19.5%), and STa:STb (16.9%), respectively. Among the pathotypes, enterotoxigenic E. coli (ETEC) was the most predominant (67.8%), followed by Shiga-toxin producing E. coli (STEC, 23.7%). We confirmed high resistance rates to chloramphenicol (88.1%), tetracycline (86.4%), streptomycin (86.4%), and ampicillin (86.4%). And the majorities of isolates (90.7%) showed multi-drug resistance which means having resistance to 3 or more subclasses of antimicrobials. Results of this study can be a source of valuable data for investigating the epidemiology of and control measures for enteric colibacillosis in Korean piggeries.

Comparative Genomic Analysis of Capsule-Producing Black Yeasts Exophiala dermatitidis and Exophiala spinifera, Potential Agents of Disseminated Mycoses.

The two black yeasts Exophiala dermatitidis and Exophiala spinifera that are clinically considered as the most virulent species potentially causing disseminated infections are both producing extracellular capsule-like material, are compared. In this study, 10 genomes of E. spinifera and E. dermatitidis strains, including both clinical and environmental isolates, were selected based on phylogenetic analysis, physiology tests and virulence tests, sequenced on the Illumina MiSeq sequencer and annotated. Comparison of genome data were performed between intraspecific and interspecific strains. We found capsule-associated genes were however not consistently present in both species by the comparative genomics. The prevalent clinical species, E. dermatitidis, has small genomes containing significantly less virulence-associated genes than E. spinifera, and also than saprobic relatives. Gene OG0012246 and Myb-like DNA-binding domain and SANT/Myb domain, restricted to two strains from human brain, was shared with the neurotropic species Rhinocladiella mackenziei. This study indicated that different virulence profiles existed in the two capsule-producing black yeasts, and the absence of consistent virulence-associated profiles supports the hypothesis that black yeasts are opportunists rather than primary pathogens. The results also provide the key virulence genes and drive the continuing research forward pathogen-host interactions to explore the pathogenesis.

Prevalence of O-serogroups, virulence genes, and F18 antigenic variants in Escherichia coli isolated from weaned piglets with diarrhea in Korea during 2008-2016.

To diagnose colibacillosis, detection of O-serogroups and virulence genes has been recommended worldwide. The prevalence of virulence factors can fluctuate over time. The objectives of this study were to determine the prevalence of O-serogroups, virulence genes, and F18 subtypes among pathogenic Escherichia coli isolated from weaned piglets with diarrhea in Korea. Between 2008 and 2016, 362 E. coli were isolated from weaned piglets with diarrhea. Hemolysis was determined in blood agar, and O-serogroups were identified using the slide agglutination technique. The genes for the toxins and fimbriae were amplified by polymerase chain reaction (PCR). Real-time PCR was conducted to discriminate between F18 subtypes. Although the most prevalent serogroup was O149 (11.3%) in the last 9 years, O139 (19.1%) became the most prevalent in recent years (2015-2016). The most predominant pathotype was enterotoxigenic E. coli (61.3%). The frequencies of Shiga-like toxin-producing E. coli (STEC) (23.4%), O139 (19.1%), Stx2e (35.1%), and F18ab (48.7%) increased over the most recent years. Although enterotoxigenic E. coli was the most predominant pathotype, the frequencies of O139, Stx2e, STEC, and F18ab have increased in recent years. These results demonstrate that there have been temporal changes in the predominant O-serogroups and virulence genes over the last decade in Korea. These findings can be practicable for use in epidemiology and control measures for enteric colibacillosis in Korean piggeries.

Prevalence of O-serogroups, virulence genes, and F18 antigenic variants in Escherichia coli isolated from weaned piglets with diarrhea in Korea during 2008–2016

To diagnose colibacillosis, detection of O-serogroups and virulence genes has been recommended worldwide. The prevalence of virulence factors can fluctuate over time. The objectives of this study were to determine the prevalence of O-serogroups, virulence genes, and F18 subtypes among pathogenic Escherichia coli isolated from weaned piglets with diarrhea in Korea. Between 2008 and 2016, 362 E. coli were isolated from weaned piglets with diarrhea. Hemolysis was determined in blood agar, and O-serogroups were identified using the slide agglutination technique. The genes for the toxins and fimbriae were amplified by polymerase chain reaction (PCR). Real-time PCR was conducted to discriminate between F18 subtypes. Although the most prevalent serogroup was O149 (11.3%) in the last 9 years, O139 (19.1%) became the most prevalent in recent years (2015–2016). The most predominant pathotype was enterotoxigenic E. coli (61.3%). The frequencies of Shiga-like toxin-producing E. coli (STEC) (23.4%), O139 (19.1%), Stx2e (35.1%), and F18ab (48.7%) increased over the most recent years. Although enterotoxigenic E. coli was the most predominant pathotype, the frequencies of O139, Stx2e, STEC, and F18ab have increased in recent years. These results demonstrate that there have been temporal changes in the predominant O-serogroups and virulence genes over the last decade in Korea. These findings can be practicable for use in epidemiology and control measures for enteric colibacillosis in Korean piggeries.

Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency "Ospedale dei Colli," Naples, Italy.

Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency "Ospedale dei Colli," Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD50 and LD90) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.

Prevalence, Potential Virulence, and Genetic Diversity of Listeria monocytogenes Isolates From Edible Mushrooms in Chinese Markets.

Listeria monocytogenes, an intracellular foodborne pathogen, is capable of causing listeriosis, such as meningitis, meningoencephalitis, and abortion. In recent years, the occurrence of Listeria monocytogenes in edible mushroom products has been reported in several countries. There are no guidelines for qualitative and quantitative detection of L. monocytogenes in mushroom products in China. Therefore, this study aimed to investigate the prevalence and contamination level of L. monocytogenes in edible mushrooms in Chinese markets and to determine the antibiotic resistance and sequence types (STs) of these isolates to provide data for risk assessments. Approximately 21.20% (141/665) of edible mushroom samples were positive for L. monocytogenes, while 57.44% (81/141) of positive samples contained contamination levels of less than 10 MPN/g. The 180 isolates derived from positive samples belonged to serogroup I.1 (1/2a-3a, n = 111), followed by serogroup II.2 (1/2b-3b-7, n = 66), and serogroup III (4a-4c, n = 3). Antibiotic susceptibility testing showed that over 95% of L. monocytogenes isolates were resistant to penicillin, ampicillin, oxacillin, and clindamycin, while over 90% were susceptible to 16 antibiotic agents, the mechanisms of resistance remain to be elucidated. According to multilocus sequencing typing, the 180 isolates represented 21 STs, one of which was identified for the first time. Interestingly, ST8 and ST87 were predominant in edible mushroom products, indicating that specific STs may have distinct ecological niches. Potential virulence profiles showed that most of the isolates contained full-length inlA genes, with novel premature stop codons found in isolate 2035-1LM (position 1380, TGG→TGA) and 3419-1LM (position 1474, CAG→TAG). Five isolates belonging to serogroup II.2 carried the llsX gene from Listeria pathogenicity island (LIPI)-3, present in ST224, ST3, and ST619; 53 (29.44%) harbored the ptsA gene from LIPI-4, presenting in ST3, ST5, ST87, ST310, ST1166, and ST619. Five potential hypervirulent isolates carrying all three of these virulence factors were identified, suggesting edible mushrooms may serve as possible transmission routes of potential hypervirulent L. monocytogenes, which may be of great public health concern to consumers. Based on our findings, the exploration of novel approaches to control L. monocytogenes contamination is necessary to ensure the microbiological safety of edible mushroom products.

Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland.

Staphylococcus aureus is arguably the most important pathogen involved in bovine mastitis. The aim of this study was to determine the virulence gene profiles of 124 Staph. aureus isolates from subclinical mastitis in cows in eastern Poland. The presence of 30 virulence genes encoding adhesins, proteases and superantigenic toxins was investigated by PCR. The 17 different combinations of adhesin genes were identified. Occurrence of eno (91·1%) and fib (82·3%) genes was found to be common. The frequency of other adhesion genes fnbA, fnbB, ebps were 14·5, 50, 25%, respectively, and for cna and bbp were 1·6%. The etA and etD genes, encoding exfoliative toxins, were present in genomes of 5·6 and 8·9% isolates, respectively. The splA and sspA, encoding serine protease, were detected in above 90% isolates. The most frequent enterotoxin genes were sei (21%), sem (19·4%), sen (19·4%), seg (18·5%) and seo (13·7%). The tst gene was harboured by 2·4% isolates. The 19 combinations of the superantigenic toxin genes were obtained and found in 35·5% of isolates. Three of them (seg, sei, sem, sen, seo; sec, seg, sei, sem, sen, seo and seg, sei, sem, sen) were the most frequent and found in 16·1% of the isolates. The most common virulotype, present in 17·7% of the isolates, was fib, eno, fnbB, splA, splE, sspA. The results indicate the variation in the presence of virulence genes in Staph. aureus isolates and considerable diversity of isolates that are able to cause mastitis in cows.