Distribution-based maximum likelihood estimation methods are preferred for estimating Salmonella concentration in chicken when contamination data are highly left-censored.

Salmonella is a common chicken-borne pathogen that causes human infections. Data below the detection limit, referred to as left-censored data, are frequently encountered in the detection of pathogens. The approach of handling the censored data was regarded to affect the estimation accuracy of microbial concentration. In this study, a set of Salmonella contamination data was collected from chilled chicken samples using the most probable number (MPN) method, which consisted of 90.42% (217/240) non-detect values. Two simulated datasets with fixed censoring degrees of 73.60% and 90.00% were generated based on the real-sampling Salmonella dataset for comparison. Three methodologies were applied for handling left-censored data: (i) substitution with different alternatives, (ii) the distribution-based maximum likelihood estimation (MLE) method, and (iii) the multiple imputation (MI) method. For each dataset, the negative binomial (NB) distribution-based MLE and zero-modified NB distribution-based MLE were preferable for highly censored data and resulted in the least root mean square error (RMSE). Replacing the censored data with half the limit of quantification was the next best method. The mean concentration of Salmonella monitoring data estimated by the NB-MLE and zero-modified NB-MLE methods was 0.68 MPN/g. This study provided an available statistical method for handling bacterial highly left-censored data.

Machine learning approach for predicting single cell lag time of Salmonella Enteritidis after heat and chlorine treatment.

The importance of single-cell variability is increasingly prominent with the developments in foodborne pathogens modeling. Traditional predictive microbiology model cannot accurately describe the growth behavior of small numbers of cells due to individual cell heterogeneity. The objective of the present study was to develop predictive models for single cell lag times of Salmonella Enteritidis after heat and chlorine treatment. A time-lapse microscopy method was employed to evaluate the single cell lag time by monitoring cell divisions. Four supervised machine learning algorithms including gradient boosting regression tree (GBRT), artificial neural network (ANN), random forest (RF), and support vector regression (SVR) were applied and compared. Results show that all four machine learning models have good predictive capabilities without an overfitting of the data. The ANN approach demonstrated superior prediction performance over other machine learning models (RMSE: 0.209, MAE: 0.135 and R2: 0.989). Furthermore, the SHapley Additive exPlanation (SHAP) measures were used to capture the influence of each feature on the model output, and results revealed that population lag times and sublethal injury rate have dominant impacts on the single cell lag time. Consequently, the findings generated from this study may be useful in managing the potential food safety risk caused by single cells of foodborne pathogens.

Antibiotic Resistance of Salmonella Typhimurium Monophasic Variant 1,4,[5],12:i:-in China: A Systematic Review and Meta-Analysis.

Antibiotic resistance in Salmonella is a global public health problem. Salmonella enterica serovar 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), a monophasic variant of Salmonella Typhmurium, is one of the leading Salmonella serovars in several countries. This study aimed to assess the prevalence of antibiotic resistance to this serovar in China through a systematic review and meta-analysis. Nineteen eligible studies during 2011-2021 were included. A total of 4514 isolates from humans, animals, foods, and the environment were reported, which mainly concerned isolates found in Guangdong, Guangxi, Jiangsu, and Shanghai. A random-effects model was used to estimate the pooled resistance rate of S. 1,4,[5],12:i:-. Rates were found to be very high (values ≥ 75%) for tetracycline, ampicillin, sulfisoxazole, and streptomycin; high (50-75%) for nalidixic acid, amoxicillin-clavulanic acid, and chloramphenicol; and moderate (25-50%) for trimethoprim-sulfamethoxazole, kanamycin, trimethoprim, and gentamicin. The rates of resistance to ciprofloxacin, cefotaxime, ceftriaxone, cefepime, ceftazidime, and colistin were low (values ≤ 25%), but of great concern in terms of their current clinical importance. Furthermore, a high multidrug resistance rate (86%, 95% CI: 78-92%) was present in S. 1,4,[5],12:i:-, with the ASSuT pattern largely dominating. Subgroup analysis results showed that the high heterogeneity of resistance rates was not entirely dependent on isolated sources. Taken together, the severity of antibiotic resistance in S. 1,4,[5],12:i:- urgently requires the rational use of antibiotics in future infection control and antibiotic stewardship programs.

A Meta-Analysis and Systematic Review of Listeria monocytogenes Response to Sanitizer Treatments.

Listeria monocytogenes is a ubiquitous organism that can be found in food-related environments, and sanitizers commonly prevent and control it. The aim of this study is to perform a meta-analysis of L. monocytogenes response to sanitizer treatments. According to the principle of systematic review, we extracted 896 records on the mean log-reduction of L. monocytogenes from 84 publications as the dataset for this study. We applied a mixed-effects model to describe L. monocytogenes response to sanitizer treatment by considering sanitizer type, matrix type, biofilm status, sanitizer concentration, treatment time, and temperature. Based on the established model, we compared the response of L. monocytogenes under different hypothetical conditions using forest plots. The results showed that environmental factors (i.e., sanitizer concentration, temperature, and treatment time) affected the average log-reduction of L. monocytogenes (p < 0.05). L. monocytogenes generally exhibited strong resistance to citric acid and sodium hypochlorite but had low resistance to electrolyzed water. The planktonic cells of L. monocytogenes were less resistant to peracetic acid and sodium hypochlorite than the adherent and biofilm cells. Additionally, the physical and chemical properties of the contaminated or inoculated matrix or surface also influenced the sanitizer effectiveness. This review may contribute to increasing our knowledge of L. monocytogenes resistance to sanitizers and raising awareness of appropriate safety precautions.

The Prevalence and Epidemiology of Salmonella in Retail Raw Poultry Meat in China: A Systematic Review and Meta-Analysis.

Foodborne disease caused by Salmonella is an important public health concern worldwide. Animal-based food, especially poultry meat, is the main source of human salmonellosis. The objective of this study was to evaluate the prevalence and epidemiology of Salmonella contamination in raw poultry meat commercialized in China. Following the principle of systematic review, 98 sets of prevalence data were extracted from 74 publications conducted in 21 Chinese provincial regions. The random-effect model was constructed for subgrouping analysis by meat category, preservation type, and geographical location. The prevalence levels differed from high to low among raw poultry meat, including chicken, 26.4% (95% CI: 22.4-30.8%); pigeon, 22.6% (95% CI: 18.2-27.8%); duck, 10.1% (95% CI: 5.3-18.2%); and other poultry meat, 15.4% (95% CI: 12.0-19.5%). Prevalence data on the preservation type revealed that chilled poultry meat might be more likely to experience cross-contamination than non-chilled poultry meat in China. The distribution map of Salmonella for raw poultry meat showed that a higher prevalence level was found in the Shaanxi, Henan, Sichuan, and Beijing regions. All subgroups possessed high amounts of heterogeneity (I2 > 75%). The scientific data regarding the differences in prevalence levels between meat category, preservation method, and geographical region sources might be useful to improve specific interventions to effectively control the incidence of Salmonella in poultry meat.

Probabilistic model for estimating Listeria monocytogenes concentration in cooked meat products from presence/absence data.

A quantitative probabilistic model was developed to estimate the concentration of Listeria monocytogenes in cooked meat products based on presence/absence data and an assumed zero-inflated distribution, i.e. zero-inflated Poisson (ZIP) or zero-inflated Poisson lognormal (ZIPL) distribution. The performance of these two distributions was compared in two data sets (data set A and B), which represented L. monocytogenes prevalence and concentrations in cooked meat products. In this study, L. monocytogenes contamination data consisted of 4.23% (8/189) and 4.17% (5/120) non-zero counts for data set A and B, respectively. The contamination level of L. monocytogenes, determined by the most probable number (MPN) technique, ranged from 3 to 93 MPN/g among 13 positive samples. The goodness-of-fit test indicated that the ZIPL distribution was better than the simpler ZIP distribution, when L. monocytogenes contamination levels on positive cooked meat samples illustrated large heterogeneity. Results obtained from ZIPL distribution showed that the logarithmic mean value of L. monocytogenes positive samples was 1.5 log MPN/g (log σ = 0.4) for data set A and B. This study provides an alternative probabilistic method when only qualitative data is available in Quantitative microbial risk assessment (QMRA), in particular if pathogen concentrations consist of large numbers of zero counts and represent high variability.

The prevalence of Listeria monocytogenes in meat products in China: A systematic literature review and novel meta-analysis approach.

Meat products are commonly regarded as one of the main sources of human listeriosis caused by Listeria monocytogenes. The objective of this study was to estimate the prevalence of L. monocytogenes in a range of meat products from 24 different Chinese regions by using meta-analysis of literature data and a novel sensitivity analysis approach. A total of 112 publications from five databases, published between 1 January 2007 and 31 December 2017, were systematically selected for relevance and covered meat products sampled between 2000 and 2016. Estimated by the random-effects model, the pooled prevalence of L. monocytogenes was 8.5% (95% CI: 7.1%-10.3%) in raw meats and 3.2% (95% CI: 2.7%-3.9%) in ready-to-eat (RTE) meats. The prevalence differed from high to low among raw meats including prefabricated raw meats 12.6% (95% CI: 6.9%-21.7%), fresh pork 11.4% (95% CI: 8.6%-14.9%), fresh beef 9.1% (95% CI: 6.3%-13.0%), fresh poultry 7.2% (95% CI:4.9%-10.4%), frozen raw meats 7.2% (95% CI: 5.7%-9.0%), and fresh mutton 5.4% (95% CI: 2.5%-11.0%). A higher L. monocytogenes prevalence level was shown in the meat products from central and northeastern China provincial regions. The entropy-based sensitivity analysis utilized in the meta-analysis indicated that the sampling period and location were two critical factors influencing the prevalence level of L. monocytogenes in meat products. A better understanding of differences in prevalence levels per geographic region and between meat product sources may allow the competent authorities, industry, and other relevant stakeholders to tailor their interventions to control the occurrence of L. monocytogenes in meat products effectively.

[Progress in detection and modeling of quorum sensing molecules of foodborne pathogens].

Quorum sensing (QS) plays a major role in the outbreak mechanism of foodborne diseases caused by food poisoning and food spoilage. QS affects the formation of cell membrane and pathogenicity ofpathogenic bacteria. Through the in-depth understanding of QS molecules of food-borne pathogens, we describe here the types of signal molecules produced by Gram-negative and Gram-positive bacteria, and the differences in QS molecules. Meanwhile, we introduce the detection of QS molecules by different technologies. According to the influence of QS on food, we propose also future research needs for the control of foodborne pathogenic bacteria.

Role of keratinocyte growth factor in the pathogenesis of autosomal dominant polycystic kidney disease.

BACKGROUND: Previous studies have shown that the expression and distribution of keratinocyte growth factor (KGF), also known as FGF-7 (fibroblast growth factor-7) or HBGF-7 (heparin-binding growth factor-7), may be implicated in kidney cyst formation and expansion. However, there are no data on KGF expression in human autosomal dominant polycystic kidney disease (ADPKD) tissue, and it is unknown whether it affects ADPKD cyst-lining epithelial cell epithelial cell proliferation. METHODS: The expression and distribution of KGF and KGF receptor (KGFR) mRNA in ADPKD cystic and normal kidney tissues were examined using quantitative real-time polymerase chain reaction (PCR) and in situ hybridization. KGF and KGFR protein expression in the above tissues was analysed by immunohistochemistry and western blot. The effect of KGF on cyst-lining epithelial cell proliferation was assessed by MTT assay, and its effect on the cyst-lining epithelial cell cycle was analysed by flow cytometry. The effect of KGF on cyclin D1 and P21(wafl) gene expression in cyst-lining epithelial cells was also determined. RESULTS: KGF and KGFR mRNA expression in ADPKD cysts was higher than in normal kidney tissues. KGF and KGFR protein expression was also higher in ADPKD cysts and was localized to cyst-lining epithelial cells, tubular and interstitial cells. In vitro experiments revealed that KGF promoted cyst-lining epithelial cell proliferation, and decreased the ratio of G0/G1 phase but increased that of S phase. In response to KGF, the expression of the cyclin D1 gene in cyst-lining epithelial cells increased markedly while P21(wafl) expression decreased. CONCLUSIONS: KGF and KGFR expression was upregulated in ADPKD kidney tissues. KGF stimulated the proliferation of cyst-lining epithelial cell in vitro by regulating the expression of cyclin D1 and P21(wafl) genes. KGF may play a role in pathogenesis of ADPKD.

Mutation analysis of autosomal dominant polycystic kidney disease genes in Han Chinese.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2. The complexity of these genes, particularly PKD1, has complicated genetic screening, though recent advances have provided new opportunities for amplifying these genes. In the Han Chinese population, no complete mutational analysis has previously been conducted across the entire span of PKD1 and PKD2. Here, we used single-strand conformation polymorphism (SSCP) analysis to screen the entire coding sequence of PKD1 and PKD2 in 85 healthy controls and 72 Han Chinese from 24 ADPKD pedigrees. In addition to 11 normal variants, we identified 17 mutations (12 in PKD1 and 5 in PKD2), 15 of which were novel ones (11 for PKD1 and 4 for PKD2). We did not identify any seeming mutational hot spots in PKD1 and PKD2. Notably, we found several disease-associated C-T or G-A mutations that led to charge or hydrophobicity changes in the corresponding amino acids. This suggests that the mutations cause conformational alterations in the PKD1 and PKD2 protein products that may impact the normal protein functions. Our study is the first report of screenable mutations in the full-length PKD1 and PKD2 genes of the Han Chinese, and also offers a benchmark for comparisons between Caucasian and Han ADPKD pedigrees and patients.

[Expression of PKD1 and PKD2 transcripts and proteins and its significance in different types of kidney tissues and kidney lines].

OBJECTIVE: To investigate the expression and function of PKD1 and PKD2 in different kidney tissues and cell lines. METHODS: Immunoprecipitation, Western blotting, In situ hybridization and immunohistochemical staining methods were used to observe the expression of PKD1 mRNA and PKD2 mRNA and their protein abundance in different kidney tissues and cell lines. RESULTS: Coordinate expressions of PKD1 and PKD2 were found in all kidney tissues and cell lines. Distribution of PKD1 mRNA and PKD2 mRNA and their protein polycystin-1 and polycystin-2 in normal human adult kidney tissue were mainly expressed in the medullary collecting ducts and distal tubules. Positive staining was also found in the majority of cyst-lining epithelial cells of PKD1 cystic kidney tissue, PKD1 cyst-lining epithelia cell line and LLC-PK1. The expression level of them in cystic epithelia of ADPKD kidney tissue was much higher than that in adult renal tubules (P < 0.01). CONCLUSIONS: Similar expression pattern of PKD1 and PKD2 and their different tissue distribution in different kidney tissues show that the molecular mutuality of PC-1 and PC-2 might be the base of their functional correlation. Polycystins might play an important role in the maintenance of tubular architecture.

[Gene diagnosis of autosomal dominant polycystic kidney disease type 2 using microsatellite DNA tightly linked to polycystic kidney disease gene 2].

OBJECTIVE: To use microsatellite DNA tightly linked to polycystic kidney disease gene 2 in the gene diagnosis of autosomal dominant polycystic kidney disease type 2. METHODS: Microsatellite DNA of D4S1534, D4S1542, D4S1563,D4S2460 and D4S423 were amplified with PCR and the fragments of products were analyzed by capillary electrophoresis and Genescan and Genotyper software, and then gene diagnosis of the pedigrees was made by linkage analysis. RESULTS: Three families were found to be linked to PKD2 in 20 families. Two carriers of PKD2 mutation were revealed by linkage analysis. CONCLUSION: Gene diagnosis can be done for PKD2 mutation carriers prior to cytogenesis. Linkage analysis is a rapid, simple method for studying the heterogeneity of polycystic kidney disease and for diagnosing the disease at the molecular level.

[Mutation detection of PKD2 gene in Chinese by denaturing high-performance liquid chromatograph].

OBJECTIVE: To detect the mutations of autosomal dominant polycystic kidney disease gene 2(PKD2)in Chinese. METHODS: The white blood cell genomic DNA from patients of 94 Chinese autosomal dominant polycystic kidney disease(ADPKD) pedigrees was isolated and amplified by polymerase chain reaction(PCR). The PCR products were analyzed by denaturing high-performance liquid chromatography(DHPLC). The samples with abnormal profiles were sequenced. RESULTS: Eight mutations were identified, including 2 nonsense mutations, 2 deletion mutations,1 insertion mutation and 3 missense mutations. Two nonsense mutations occurred in exon 5(1249C-->T) and exon 13(2407C-->T),both resulted in a stop codon. The insertion was in exon 2(636-637 ins T),and the deletion mutations were in exons 12(2348-2351 del AGAA) and 13(2401 delete A),resulting in the reading frame shift. Three missense mutations were in exons 1(G568-->A),4(C964-->T),and 5(G1168-->A), which caused amino acid changes (190Ala-->Thr,322Arg-->Trp,390Gly-->Ser). CONCLUSION: The method of DHPLC was used in detecting mutations successfully and 8 mutations in PKD2 were identified. It will be useful in the molecular diagnosis of ADPKD in advance of the cysts formation and birth.

[Preparation and characterization of monoclonal antibody against N-terminal domain of polycystin 1].

AIM: To prepare and identify monoclonal antibody (mAb) against N-terminal domain of polycystin 1. METHODS: Total RNA was extracted from kidney tissue of a healthy man. Gene sequence encoding polycystin 1 N-terminal domain was amplified by one-step RT-PCR. The target gene was inserted into prokaryotic expression vector pQE30 and transformed into competent cells E. coli M15. The fusion protein was expressed under IPTG induction and purified by affinity chromatography. The purified fusion protein was then used to immunize BALB/c mice. The splenocytes from immunized mice were fused with myeloma cells Sp2/0 by PEG 4000 mediator method and the hybridomas were selected in HAT medium. The hybridoma clones secreting mAb against polycystin 1 amino-terminal domain were detected by ELISA and cloned by limiting dilution. The specificity of mAb against polycystin 1 N-terminal domain was verified by ELISA and Western blot. RESULTS: cDNA encoding polycystin 1 extracellular region was obtained. Fusion protein of polycystin 1 N-terminal domain were expressed in pQE30 expression system. The relative molecular masses (Mr) of the two fusion proteins were 19,800 and 18,900, respectively. One hybridoma cell 7B1 secreting specific mAb was obtained. Western blot analysis showed that the mAb reacted strongly and specifically to polycystin 1 N-terminal domain. CONCLUSION: polycystin 1 N-terminal fusion proteins have been expressed in E.coli M15. Anti-fusion protein mAb with antigen-binding activity has been prepared successfully.