Moutong Chen

Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.

Listeria monocytogenes Prevalence and Characteristics in Retail Raw Foods in China

The prevalence and levels of Listeria monocytogenes in retail raw foods covering most provincial capitals in China were studied with testing of 1036 samples of vegetables, edible mushrooms, raw meat, aquatic products and quick-frozen products from September 2012 to January 2014. The total prevalence of Listeria monocytogenes was 20.0% (207/1036), and the most probable number (MPN) values of 65.7% of the positive samples ranged from 0.3 to 110 MPN/g. Geographical differences were observed in this survey, and the results of both qualitative and quantitative methods indicated that the levels in the samples from North China were higher than those in the samples from South China. A total of 248 isolates were analyzed, of which approximately half belonged to molecular serogroup 1/2a-3a (45.2%), followed by 1/2b-3b-7 (30.6%), 1/2c-3c (16.1%), 4b-4d-4e (5.2%) and 4a-4c (2.8%). Most of the isolates carried hly (100%), inlB (98.8%), inlA (99.6%), inlC (98.0%) and inlJ (99.2%), and 44.8% of the isolates were llsX-positive. Seventeen epidemic clones (ECs) were detected, with 7 strains belonging to ECI (2.8%) and 10 belonging to ECIII (4.03%). Resistance to clindamycin (46.8%) was commonly observed, and 59 strains (23.8%) were susceptible to all 14 tested antibiotics, whereas 84 (33.9%) showed an intermediate level of resistance or were resistant to two or more antibiotics, including 7 multi-resistant strains that exhibited resistance to more than 10 antibiotics. The data obtained in the present study provides useful information for assessment of the possible risk posed to Chinese consumers, and this information will have a significant public health impact in China. Furthermore, the presence of virulence markers, epidemic clones, as well as the antibiotic resistance amongst the isolates strongly implies that many of these strains might be capable of causing listeriosis, and more accurate treatment of human listeriosis with effective antibiotics should be considered. This research represents a more full-scale and systematical investigation of the prevalence of L. monocytogenes in retail raw foods in China, and it provides baseline information for Chinese regulatory authorities that will aid in the formulation of a regulatory framework for controlling L. monocytogenes with the aim of improving the microbiological safety of raw foods.

Prevalence, enumeration, and pheno- and genotypic characteristics of Listeria monocytogenes isolated from raw foods in South China

Listeria monocytogenes is an important foodborne pathogen that can cause serious illness in immunocompromised individuals, pregnant women, the elderly, and newborns. The aim of this study was to: (i) evaluate the prevalence and contamination level [most probable number (MPN)] of L. monocytogenes in 567 retail raw foods (fishery products, n = 154; raw/fresh meat, n = 123; frozen foods, n = 110; edible fungi, n = 108; vegetables, n = 72) collected from South China and (ii) to gain further knowledge on the phenotype and genotype distributions of this important foodborne pathogen. Approximately 22% of the samples were positive for L. monocytogenes. The contamination levels were between 0.3 and 10 MPN/g in 75.0%, between 10 and 100 MPN/g in 11.0% and less than 100 MPN/g in 14.0% of the countable samples. Five serogroups were identified among the 177 foodborne L. monocytogenes isolates, with 1/2a-3a (42.4%) and 1/2b-3b (26.0%) serogroups being the most dominant. Serogroups I.1 and II.2 were only found in the edible mushrooms, while serogroup III was dominant in the fishery products, suggesting that specific serogroups of L. monocytogenes may have distinct ecological niches. Ten (5.6%) L. monocytogenes isolates exhibited multidrug resistance. Genetic relatedness analysis revealed the absence of distinct associations between specific food types, antibiotic resistance, serogroups, and genetic diversity. The present study provided the first baseline data on the prevalence, contamination level, and characteristics of L. monocytogenes isolated from raw foods in South China. Some multidrug resistant strains belonged to the epidemiologically important serogroups (I.1 and II.1), implying a potential public health risk. In addition, these findings also provide basic information for the Chinese food safety associated authorities to draft appropriate standards to control L. monocytogenes contamination and improve microbiological safety of raw foods.

Prevalence and contamination patterns of Listeria monocytogenes in Flammulina velutipes plants.

Four mushroom (Flammulina velutipes) production plants were sampled to investigate the prevalence and contamination source of Listeria monocytogenes. Among 295 samples, the prevalence of L. monocytogenes was 18.6%; the contamination appeared to originate from the mycelium-scraping machinery, contaminating both the product and upstream packaging equipment. Of 55 L. monocytogenes isolates, lineages I.1 (1/2a-3a) and II.2 (1/2b-3b-7) accounted for 65.5% and 34.5%, respectively. In addition, lineage I.1 formed significantly thicker biofilms than those within lineage II.2, as determined by crystal violet staining and scanning electron microscopy. Genotype analyses of L. monocytogenes isolates using enterobacteria repetitive intergenic consensus-polymerase chain reaction, and random amplified polymorphic DNA revealed that the surfaces of mycelium-scraping machinery may serve as the main source of L. monocytogenes contamination in three of the four plants. This study was the first report to explore the potential contamination sources of L. monocytogenes in the mushroom production chain, thereby providing baseline information for adopting prophylactic measures for critical control points during production in mushroom plants to avoid L. monocytogenes contamination.

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.

Reconstituting the History of Cronobacter Evolution Driven by Differentiated CRISPR Activity

ABSTRACT Cronobacter strains harboring the CRISPR-Cas system are important foodborne pathogens causing serious neonatal infections. However, the specific role of the CRISPR-Cas system in bacterial evolution remains relatively unexplored. In this study, we investigated the impact of the CRISPR-Cas system on Cronobacter evolution and obtained 137 new whole-genome Cronobacter sequences by next-generation sequencing technology. Among the strains examined (n = 240), 90.6% (193/213) of prevalent species Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter dublinensis strains had intact CRISPR-Cas systems. Two rare species, Cronobacter condimenti (n = 2) and Cronobacter universalis (n = 6), lacked and preserved the CRISPR-Cas system at a low frequency (1/6), respectively. These results suggest that the presence of one CRISPR-Cas system is important for a Cronobacter species to maintain genome homeostasis for survival. The Cronobacter ancestral strain is likely to have harbored both subtype I-E and I-F CRISPR-Cas systems; during the long evolutionary process, subtype I-E was retained while subtype I-F selectively degenerated in Cronobacter species and was even lost by the major Cronobacter pathovars. Moreover, significantly higher CRISPR activity was observed in the plant-associated species C. dublinensis than in the virulence-related species C. sakazakii and C. malonaticus. Similar spacers of CRISPR arrays were rarely found among species, suggesting intensive change through adaptive acquisition and loss. Differentiated CRISPR activity appears to be the product of environmental selective pressure and might contribute to the bidirectional divergence and speciation of Cronobacter. IMPORTANCE This study reports the evolutionary history of Cronobacter under the selective pressure of the CRISPR-Cas system. One CRISPR-Cas system in Cronobacter is important for maintaining genome homeostasis, whereas two types of systems may be redundant and not conducive to acquiring beneficial DNA for environmental adaptation and pathogenicity. Differentiated CRISPR activity has contributed to the bidirectional divergence and genetic diversity of Cronobacter. This perspective makes a significant contribution to the literature by providing new insights into CRISPR-Cas systems in general, while further expanding the roles of CRISPR beyond conferring adaptive immunity and demonstrating a link to adaptation and species divergence in a genus. Moreover, our study provides new insights into the balance between genome homeostasis and the uptake of beneficial DNA related to CRISPR-based activity in the evolution of Cronobacter.

Prevalence and Genetic Diversity of Enterococcus faecalis Isolates from Mineral Water and Spring Water in China

Enterococcus faecalis is an important opportunistic pathogen which is frequently detected in mineral water and spring water for human consumption and causes human urinary tract infections, endocarditis and neonatal sepsis. The aim of this study was to determine the prevalence, virulence genes, antimicrobial resistance and genetic diversity of E. faecalis from mineral water and spring water in China. Of 314 water samples collected from January 2013 to January 2014, 48 samples (15.3%) were contaminated E. faecalis. The highest contamination rate occurred in activated carbon filtered water of spring water (34.5%), followed by source water of spring water (32.3%) and source water of mineral water (6.4%). The virulence gene test of 58 E. faecalis isolates showed that the detection rates of asa1, ace, cylA, gelE and hyl were 79.3, 39.7, 0, 100, 0%, respectively. All 58 E. faecalis isolates were not resistant to 12 kinds of antibiotics (penicillin, ampicillin, linezolid, quinupristin/dalfopristin, vancomycin, gentamicin, streptomycin, ciprofloxacin, levofloxacin, norfloxacin, nitrofurantoin, and tetracycline). Enterobacterial repetitive intergenic consensus-PCR classified 58 isolates and three reference strains into nine clusters with a similarity of 75%. This study is the first to investigate the prevalence of E. faecalis in mineral water and spring water in China. The results of this study suggested that spring water could be potential vehicles for transmission of E. faecalis.

Prevalence and Characterization of Staphylococcus aureus Isolated From Retail Vegetables in China

Staphylococcus aureus is a pathogen associated with serious community and hospital-acquired diseases. The aim of this study was to investigate the prevalence of S. aureus from retail vegetables in China and then characterized S. aureus isolates by antibiotic resistance, staphylococcal enterotoxin genes, spa-typing and multi-locus sequence typing. Of 419 retail vegetable samples from 39 cities in China during 2011–2016, 24 (5.73%) samples were positive for S. aureus and the geometric mean was 3.85 MPN/g. The prevalence of S. aureus was highest in lettuce (13/84, 15.48%) followed by tomato (7/110, 6.36%), caraway (2/87, 2.30%), and cucumber (2/128, 1.56%), whereas other vegetables were free of S. aureus. A total of 30 isolates were analyzed. For antibiotics susceptibility test, most isolates (93.3%) were resistant to ampicillin and penicillin, whereas all isolates were susceptible to linezolid, trimethoprim/sulphamethoxazole 1:19, nitrofurantoin, rifampicin, and teicoplanin. All isolates (30/30, 100%) were resistant or intermediate resistant to more than three tested antibiotics, including 9 isolates (30%) were resisted more than 10 antibiotics. Five isolates were resistant to cefoxitin and carried mecA genes which confirmed as MRSA. Of the 18 investigated SE genes, the sem gene was the most frequently detected (86.7%) followed by the sec (83.3%), sep (70.0%), seg (56.7%), sel (53.3%), seh (50.0%), seq (50.0%), sej (46.7%), seb (36.7%), sen (36.7%), and ser (33.3%) genes were harbored by more than one third of the isolates, whereas the seo and seu were detected in only 6.75% of the isolates. MLST and spa typing observed high genetic diversity in S. aureus isolated from retail vegetable in China. ST59-t437 was the predominant types (3/5, 60%) of MRSA isolates, whereas ST188-t189 was the predominant types (7/25, 28%) of MSSA isolates. Our study reflects that the retail vegetable in China could be contaminated with S. aureus but the levels of S. aureus were not very excessive. In addition, these isolates had virulence potential, most of them were enterotoxigenic and multiple antimicrobial resistance, should be draw public attention. These data have signification implications for epidemiological and public health studies of this pathogen.

Occurrence, Antibiotic Resistance, and Population Diversity of Listeria monocytogenes Isolated From Fresh Aquatic Products in China

Listeria monocytogenes is an important Gram-positive foodborne pathogen. However, limited information is available on the comprehensive investigation and potential risk of L. monocytogenes in fresh aquatic products, which are popular to consumers in China. This study aimed to determine the occurrence, virulence profiles, and population diversity of L. monocytogenes isolated from aquatic products in China. In total, 846 aquatic product samples were collected between July 2011 and April 2016 from 43 cities in China. Approximately 7.92% (67/846) aquatic product samples were positive for L. monocytogenes, 86.57% positive samples ranged from 0.3 to 10 MPN/g, whereas 5.97% showed over 110 MPN/g by the Most Probable Number method, which included two samples of products intended to be eaten raw. Serogroups I.1 (serotype 1/2a), I.2 (serotype 1/2b), and III (serotype 4c) were the predominant serogroups isolated, whereas serogroup II.1 (serotype 4b) was detected at much lower frequencies. Examination of antibacterial resistance showed that nine antibacterial resistance profiles were exhibited in 72 isolates, a high level susceptibility of 16 tested antibiotics against L. monocytogenes were observed, indicating these common antibacterial agents are still effective for treating L. monocytogenes infection. Multilocus sequence typing revealed that ST299, ST87, and ST8 are predominant in aquatic products, indicating that the rare ST299 (serotype 4c) may have a special ecological niche in aquatic products and associated environments. Except llsX and ptsA, the 72 isolates harbor nine virulence genes (prfA, actA, hly, plcA, plcB, iap, mpl, inlA, and inlB), premature stop codons (PMSCs) in inlA were found in four isolates, three of which belonged to ST9. A novel PMSC was found in 2929-1LM with a nonsense mutation at position 1605 (TGG→TGA). All ST87 isolates harbored the ptsA gene, whereas 8 isolates (11.11%) carried the llsX gene, and mainly belonged to ST1, ST3, ST308, ST323, ST330, and ST619. Taken together, these results first reported potential virulent L. monocytogenes isolates (ST8 and ST87) were predominant in aquatic products which may have implications for public health in China. It is thus necessary to perform continuous surveillance for L. monocytogenes in aquatic products in China.

Characterization of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae From Retail Food in China

In this study, we characterized the β-lactamase genes and phenotypic resistance of cephalosporin-resistant Enterobacteriaceae isolated from retail foods in China. Of 1,024 Enterobacteriaceae isolates recovered from raw meat products, aquatic products, raw vegetables, retail-level ready-to-eat (RTE) foods, frozen foods, and mushrooms from 2011 to 2014, 164 (16.0%) showed cefotaxime (CTX) and/or ceftazidime (CAZ) cephalosporin resistance, and 96 (9.4%) showed the extended-spectrum β-lactamase (ESBL) phenotype. More than 30% isolates were resistant to all antimicrobial agents except carbapenems (MEM 3.1% and IPM 5.2%), cefoxitin (FOX 6.3%), and amoxicillin/clavulanic acid (AMC 26%), and 94.8% of the strains were resistant to up to seven antibiotics. Polymerase chain reaction analysis showed that blaTEM (81.9%) was the most common gene, followed by blaCTX-M (68.1%) and blaSHV (38.9%). Moreover, 16.8% (72/429) of food samples contained ESBL-positive Enterobacteriaceae, with the following patterns: 32.9% (23/70) in frozen foods, 27.2% (5/29) in mushrooms, 17.6% (24/131) in raw meats, 13.3% (4/30) in fresh vegetables, 11.1% (8/72) in RTE foods, and 9.3% (9/97) in aquatic products. In addition, 24 of 217 foods collected in South China (11.1%), 25 of 131 foods collected in North of the Yangtze River region (19.1%), and 23 of 81 foods collected in South of the Yangtze River region (28.4%) were positive for ESBL- Enterobacteriaceae. Conjugation experiments demonstrated that the 22 of 72 isolates were transconjugants that had received the β-lactamase gene and were resistant to β-lactam antibiotics as well as some non-β-lactam antibiotics. These findings demonstrated that retail foods may be reservoirs for the dissemination of β-lactam antibiotics and that resistance genes could be transmitted to humans through the food chain; and the predominant ESBL-producing Enterobacteriaceae in China was isolated from in frozen chicken-meat, followed by frozen pork, cold noodles in sauce, cucumber, raw chicken meat, frozen pasta, brine-soaked chicken and tomato.