Genomic insights into the evolution, pathogenicity, and extensively drug-resistance of emerging pathogens Kluyvera and Phytobacter.

Introduction: Kluyvera is a Gram-negative, flagellated, motile bacillus within the Enterobacteriaceae. The case reports of clinical infections shed light on the importance of this organism as an emerging opportunistic pathogen. The genus Phytobacter, which often be misidentified with Kluyvera, is also an important clinically relevant member of the Enterobacteriaceae. However, the identification of Kluyvera and Phytobacter is problematic, and their phylogenetic relationship remains unclear. Methods: Here, 81 strains of Kluyvera and 16 strains of Phytobacter were collected. A series of comparative genomics approaches were applied to the phylogenetic relationship reconstruction, virulence related genes profiles description, and antibiotic resistance genes prediction. Results: Using average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH), we offered reliable species designations of 97 strains, in which 40 (41.24%) strains were incorrectly labeled. A new Phytobacter genomospecies-1 were defined. Phytobacter and Kluyvera show great genome plasticity and inclusiveness, which may be related to their diverse ecological niches. An intergenomic distances threshold of 0.15875 was used for taxonomy reassignments at the phylogenomic-group level. Further principal coordinates analysis (PCoA) revealed 11 core genes of Kluyvera (pelX, mdtL, bglC, pcak-1, uhpB, ddpA-2, pdxY, oppD-1, cptA, yidZ, csbX) that could be served as potential identification targets. Meanwhile, the Phytobacter specific virulence genes clbS, csgA-C, fliS, hsiB1_vipA and hsiC1_vipB, were found to differentiate from Kluyvera. We concluded that the evolution rate of Kluyvera was 5.25E-6, approximately three times higher than that of Phytobacter. Additionally, the co-existence of ESBLs and carbapenem resistance genes were present in approximately 40% strains, suggesting the potential development of extensively drug-resistant or even fully drug-resistant strains. Discussion: This work provided a better understanding of the differences between closely related species Kluyvera and Phytobacter. Their genomes exhibited great genome plasticity and inclusiveness. They not only possess a potential pathogenicity threat, but also a risk of multi-drug resistance. The emerging pathogens Kluyvera and Phytobacter warrant close attention.

Epidemiological and molecular evidence of foodborne poisoning outbreak caused by enterotoxin gene cluster-harboring Staphylococcus aureus of new sequence type 7591.

OBJECTIVES: This paper presented a detailed analysis of the epidemiology and molecular characteristics of staphylococcal food poisoning (SFP) that occurred in a hotel in Hangzhou. METHODS: A total of 46 guests at the hotel underwent an epidemiological survey. Samples of stool from patients, vomit, swabs from the kitchen, leftover food items, and anal swabs from food handlers were taken and investigated for the presence of potential pathogenic bacteria. Molecular techniques and whole genome sequencing were performed to track the evolution of Staphylococcus aureus associated with the outbreak of SFP. RESULTS: Forty-six individuals displayed gastrointestinal symptoms. Seventeen isolates of S. aureus were discovered to carry the seg, sei, sem, sen, seo, and selu genes found in a specific enterotoxin gene cluster (egc) operon, but without the presence of classical enterotoxins such as SEA ∼ SEE. All egc-positive isolates shared identical pulsed-field gel electrophoresis profiles and were classified under new ST7591 (Clonal Complex 72) with identical spa typing t148. In addition, some isolates of S. aureus obtained from food sources sold in Hangzhou over the past 3 years and carrying egc genes were grouped under the ST72 lineage (CC72). Through whole genome sequencing, a strong genetic connection was revealed between these egc-positive isolates and clinical ST72 S. aureus found in China. CONCLUSIONS: S. aureus with non-classical egc enterotoxins was suggested to be a potential cause of SFP in humans.

Epidemiological and Genomic Characterization of Campylobacter jejuni Isolates from a Foodborne Outbreak at Hangzhou, China.

BACKGROUND: Foodborne outbreaks caused by Campylobacter jejuni have become a significant public health problem worldwide. Applying genomic sequencing as a routine part of foodborne outbreak investigation remains in its infancy in China. We applied both traditional PFGE profiling and genomic investigation to understand the cause of a foodborne outbreak in Hangzhou in December 2018. METHOD: A total of 43 fecal samples, including 27 sick patients and 16 canteen employees from a high school in Hangzhou city in Zhejiang province, were recruited. Routine real-time fluorescent PCR assays were used for scanning the potential infectious agents, including viral pathogens (norovirus, rotavirus, adenovirus, and astrovirus), and bacterial pathogens (Salmonella, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus and Vibrio cholerae). Bacterial selection medium was used to isolate and identify the positive bacteria identified by molecular test. Pulsed field gel electrophoresis (PFGE), and next generation sequencing (NGS) were applied to fifteen recovered C. jejuni isolates to further understand the case linkage of this particular outbreak. Additionally, we retrieved reference genomes from the NCBI database and performed a comparative genomics analysis with the examined genomes produced in this study. RESULTS: The analyzed samples were found to be negative for the queried viruses. Additionally, Salmonella, Shigella, Vibrio parahaemolyticus and Vibrio cholera were not detected. Fifteen C. jejuni strains were identified by the real-time PCR assay and bacterial selection medium. These C. jejuni strains were classified into two genetic profiles defined by the PFGE. Out of fifteen C. jejuni strains, fourteen have a unified consistent genotype belonging to ST2988, and the other strain belongs to ST8149, with a 66.7% similarity in comparison with the rest of the strains. Moreover, all fifteen strains harbored blaOXA-61 and tet(O), in addition to a chromosomal mutation in gyrA (T86I). The examined fourteen strains of ST2988 from CC354 clone group have very minimal genetic difference (3~66 SNPs), demonstrated by the phylogenomic investigation. CONCLUSION: Both genomic investigation and PFGE profiling confirmed that C. jejuni ST2988, a new derivative from CC354, was responsible for the foodborne outbreak Illustrated in this study.

Biofilm formation and prevalence of adhesion genes among Staphylococcus aureus isolates from different food sources.

To assess biofilm formation ability and identify differences in the prevalence of genes involved in biofilm formation among Staphylococcus aureus strains isolated from different food samples, the ability of biofilm formation among 97 S. aureus strains was evaluated using a colorimetric microtiter plate assay. Thirteen genes encoding microbial surface components recognizing adhesive matrix molecules, and the intracellular adhesion genes were detected by PCR using specific primers. Approximately 72% of the isolates produced biofilms. Among these isolates, 54.64% were weak biofilm producers, while 14.43% and 3.09% produced moderate and strong biofilms, respectively. The icaADBC, clfA/B, cidA, and fib genes were detected in all the S. aureus strains, whereas the bap gene was not present in any of the strains. The occurrence of other adhesin genes varied greatly between biofilm-producing and nonbiofilm-producing strains. However, a significant difference was observed between these two groups with respect to the fnbpB, cna, ebps, and sdrC genes. No obvious evidence was found to support the link between PFGE strain typing and the capacity for biofilm formation. Considerable variation in biofilm formation ability was observed among S. aureus strains isolated from food samples. The prevalence of adhesin-encoding genes also varied greatly within strains. This study highlights the importance of biofilm formation and the adhesins of S. aureus strains in food samples.

The occurrence and distribution characteristics of Cronobacter in diverse cereal kernels, flour, and flour-based products.

Cronobacter was positive in cereals at a relatively high rate. In the present study, we investigated the occurrence and characteristics of this pathogen systematically in diverse cereals. All sampled food (N = 467) contained Cronobacter with a high positive rate of 54.0%. The enumeration experiment showed the concentration ranged from 0.3 to more than 110 MPN/100 g, and 87.9% of 127 samples were less than 10 MPN/100 g. There were significant differences (p < 0.05) in positive rates for Cronobacter among cereal kernels (40.2%), cereal flour (66.7%), cereal products made from raw cereal flour (87.6%), and cereal products made from flour (ready-to-eat) (17.4%). The dominant Cronobacter species was C. sakazakii and C. dublinensis, followed by C. malonaticus and C. turicensis. Two interesting clusters with more than 90% similarities were identified by pulsed-field gel electrophoresis (PFGE). The C1 cluster (four isolates) indicated these strains were derived from a common source and persisted in the food production environment for an extended time. The C2 cluster (six isolates) indicated the pathogen could be transmitted via cereal processing. Our research provided baseline data for Cronobacter in diverse cereals and was helpful for understanding Cronobacter transmission. The results also indicate that additional control measures should be developed to reduce the risk of infection by these opportunistic pathogenic bacteria.

Potential reservoirs and routes of Cronobacter transmission during cereal growing, processing and consumption.

Cronobacter are opportunistic bacterial pathogens of both infants and adults. We investigated the incidence and distribution of Cronobacter in 1245 samples of cereal and related environments. 39.1% (101/258) rice-related and 46.9% (98/209) wheat-related samples tested positive for Cronobacter, and the positive rate differed notably according to processing method. Cronobacter was found in rice and wheat plants at the tillering, filling and mature stages. Soil, water and swab samples from nearby milling plants were assayed, and results revealed that 6.3% (7/122) of water from paddy fields, 49.1% (28/57) and 62.1% (41/67) of swab samples from rice and wheat flour milling plants were Cronobacter positive. Pulsed-field gel electrophoresis (PFGE) subtyping indicated that some strains had a common profile, which suggested their persistence in the environment, potential transmission routes and cross-contamination in processing. Finally, we surveyed 18 families to evaluate potential risks. None of the families who primarily ate rice cooked with water tested positive for Cronobacter, though of 66.7% families (6/9) whose food staples were produced from wheat flour tested positive. Taken together, our results are important for understanding Cronobacter transmission and will aid in the development of additional control measures to reduce the risk of infection by these opportunistic pathogenic bacteria.

Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems.

Airborne bacteria are significantly affected by meteorological and environmental conditions. However, there is little quantitative data available on the effects of these factors on airborne bacteria in urban ecosystems. In the present study, we analyzed weather-dependent changes in the composition of airborne bacterial communities using high throughput sequencing. Samples were collected before and after a period of constant hot weather at four selected sampling sites (YRBS, ZJGUSJC, TJCR, and BLQG) in Hangzhou. Our results show that the average amount of bacterial 16S rRNA gene copy numbers per m³ of air decreased significantly after constant high temperature. In addition, the number of operational taxonomic units and the Shannon⁻Wiener diversity indexes of the samples at all four selected sampling sites were significantly decreased after the heat event, showing notable impact on bacterial diversity. We also detected a significant increase in the abundances of spore-forming bacteria. Firmicutes increased from 3.7% to 9.9%, Bacillales increased from 2.6% to 7.6%, and Bacillaceae increased from 1.5% to 5.9%. In addition, we observed an increase in beta-Proteobacteria (18.2% to 50.3%), Rhodocyclaceae (6.9% to 29.9%), and Burkholderiaceae (8.1% to 15.2%). On the other hand, the abundance of alpha-Proteobacteria (39.6% to 9.8%), Caulobacteraceae (17.9% to 0.5%), Sphingomonadaceae (7.2% to 3.3%), and Xanthomonadaceae (3.0% to 0.5%) was significantly lower. Taken together, our data suggest that the composition of airborne bacterial communities varies greatly dependent on heat events, and that such communities include several species that are highly susceptible to high-temperature related stressors such as high air temperature, low relative humidity, and high intensity of solar radiation.

[Carbon metabolic characteristics of airborne microbes in Hangzhou].

Carbon metabolic characteristics in four sampling sites including Yan'an Road Business Street (YRBS), Tianmushan Jiaogong Cross Road (TJCR), Zhejiang Gongshang University Jiaogong Campus (ZJGSUJC) and Breeze-ruffled Lotus at Quyuan Garden (BLQG) in Hangzhou were analyzed using Biolog technology in this study. Results showed that average well color development (AWCD) values were at stationary phase after 240 h cultivation in all four selected sampling sites. Significant differences in sole carbon utilization characterized as AWCD value were found among the four sampling sites, and the highest carbon utilization capacity was observed at YRBS, followed by TJCR and ZJGSUJC, and the lowest at BLQG. The species abundance and diversity of airborne microbes deceased in the order of YRBS, TJCR, ZJGSUJC, and BLQG. In addition, sugar and amino acid utilization capacity of airborne microbes was significantly higher that other carbons sources in all four sampling sites. Significant differences in different sole carbon utilization of airborne microbes in the same sampling site were found. The sugar utilization capacity was the highest, and polymer utilization capacity was the lowest at YRBS. At BLQG, highest sugar and lowest amine utilization capacity was detected. Principal component analysis showed that the contribution of PC1 and PC2 was 43.8% and 23.4%, respectively, in different sampling sites. Significant differences in carbon metabolic characteristics of microbial community in the air were found among YRBS, BLQG and TJCR, and no differences were observed between TJCR and ZJGSUJC.

Inhibition of the met receptor tyrosine kinase signaling enhances the chemosensitivity of glioma cell lines to CDDP through activation of p38 MAPK pathway.

The Met receptor tyrosine kinase is known to be overexpressed in many solid tumors and plays a crucial role in tumor invasive growth and metastasis. In this study, we showed that hepatocyte growth factor-induced Met activation as well as Met-dependent downstream signaling of AKT and p44/42 mitogen-activated protein kinase (MAPK) could be efficiently blocked by TAT-coupled carboxyl-terminal tail peptide of Met receptor (TCTP), and inactivation of Met signaling significantly enhanced the sensitivity of T98G and U251 glioma cells to cis-diaminedichloroplatinum (CDDP, cisplatin). However, neither phosphoinositide 3-kinase/AKT inhibitor LY294002 nor p44/42 MAPK inhibitor PD98059 alone or combined could imitate the effect of TCTP on chemosensitivity enhancement of T98G cells to CDDP, indicating that Met-dependent inactivation of AKT and p44/42 MAPK signaling was not the main cause for the increased chemosensitivity to CDDP. Further studies revealed that TCTP significantly activated p38 MAPK in T98G and U251 cell lines. Activation of p38 MAPK by sorbitol pretreatment resembled the sensitization effects, whereas inhibition of p38 MAPK activation by its inhibitor SB202190 counteracted the sensitization effects induced by TCTP. Therefore, p38 MAPK activation was one of the major causes for the increased chemosensitivity to CDDP induced by Met inactivation. Taken together, the study indicated that Met receptor played an important role in regulating cell response to chemotherapy and suggested that inhibition of Met signaling could be used in combination with other chemotherapeutic regimens in treatment of tumor patients.