CamPype: an open-source workflow for automated bacterial whole-genome sequencing analysis focused on Campylobacter.

BACKGROUND: The rapid expansion of Whole-Genome Sequencing has revolutionized the fields of clinical and food microbiology. However, its implementation as a routine laboratory technique remains challenging due to the growth of data at a faster rate than can be effectively analyzed and critical gaps in bioinformatics knowledge. RESULTS: To address both issues, CamPype was developed as a new bioinformatics workflow for the genomics analysis of sequencing data of bacteria, especially Campylobacter, which is the main cause of gastroenteritis worldwide making a negative impact on the economy of the public health systems. CamPype allows fully customization of stages to run and tools to use, including read quality control filtering, read contamination, reads extension and assembly, bacterial typing, genome annotation, searching for antibiotic resistance genes, virulence genes and plasmids, pangenome construction and identification of nucleotide variants. All results are processed and resumed in an interactive HTML report for best data visualization and interpretation. CONCLUSIONS: The minimal user intervention of CamPype makes of this workflow an attractive resource for microbiology laboratories with no expertise in bioinformatics as a first line method for bacterial typing and epidemiological analyses, that would help to reduce the costs of disease outbreaks, or for comparative genomic analyses. CamPype is publicly available at https://github.com/JoseBarbero/CamPype .

Genomic Characterization of Campylobacter jejuni Associated with Perimyocarditis: A Family Case Report.

Campylobacter spp. is the leading cause of foodborne gastrointestinal infections in humans worldwide. This study reports the first case of four family members who had contact with the same source of Campylobacter jejuni contamination with different results. Only the little siblings were infected by the same C. jejuni strain, but with different symptoms. Whereas the daughter was slightly affected with mild enteritis, the son suffered a longer campylobacteriosis followed with a perimyocarditis. This is the first case of the youngest patient affected by C. jejuni-related perimyocarditis published to date. The genomes of both strains were characterized by whole-genome sequencing and compared with the C. jejuni NCTC 11168 genome to gain insights into the molecular features that may be associated with perimyocarditis. Various comparison tools were used for the comparative genomics analysis, including the identification of virulence and antimicrobial resistance genes, phase variable (PV) genes, and single nucleotide polymorphisms (SNPs) identification. Comparisons of the strains identified 16 SNPs between them, which constituted small but significant changes mainly affecting the ON/OFF state of PV genes after passing through both hosts. These results suggest that PV occurs during human colonization, which modulates bacteria virulence through human host adaptation, which ultimately is related to complications after a campylobacteriosis episode depending on the host status. The findings highlight the importance of the relation between host and pathogen in severe complications of Campylobacter infections.

Endoscopic ultrasound-guided fine-needle aspiration for splenomegaly and focal splenic lesion: is it safe, effective and necessary?

INTRODUCTION: splenomegaly and/or focal splenic lesions (FSL) have limited histopathologic studies due to the risk posed by splenic punctures. Percutaneous biopsies with a fine needle are difficult, especially due to interposition of gases, ascites, obesity or a history of abdominal surgery. On the other hand, endoscopic ultrasound (EUS) takes advantage of the proximity of the gastric wall to the spleen in order to puncture and visualize the needle and its movements in real time. OBJECTIVE: to describe the initial experience and results obtained with EUS-FNA in patients with splenomegaly or FSL. MATERIALS AND METHODS: this was a descriptive observational study. EUS-FNA of the spleen was performed with a slow-pull technique, which avoided fanning with an average of 3 needle passes. Biopsies were sent in Cytorich RedTM solution for analysis by cytology and cell block. RESULTS: punctures were performed in 15 patients (9 females) and the median age was 67 years (range 44-86). Patients studied due to an enlarged spleen or splenic FSL, in the context of fever of an unknown origin, adenopathies and abnormal weight loss were included. A conclusive diagnosis was achieved by EUS-FNA in 10 patients (66.7 %), 4 were large cell type B non-Hodgkin's lymphoma and one Hodgkin's lymphoma. There were no immediate or delayed complications related to the procedure. CONCLUSIONS: EUS-guided splenic punctures appear to be safe, effective and may be necessary in some clinical settings in order to complete the etiologic filiation of splenomegaly of an uncertain origin or FSL and to rule out malignancy.

Biofilm formation, virulence and antimicrobial resistance of different Campylobacter jejuni isolates from a poultry slaughterhouse.

The fastidious requirement of the zoonotic pathogen Campylobacter jejuni contrasts with its ability to overcome harsh conditions. Different strategies might be involved in the survival and persistence of C. jejuni through the poultry food chain. Therefore, the aims of this study were to get insights in the survival strategies in the poultry slaughterhouse environment by (i) characterizing factors such as biofilm formation, virulence and antimicrobial resistance in environmental isolates and (ii) understanding the possible link between the phenotypic and genetic characterization using whole genome sequencing (WGS). Results have shown that three STs: ST 443 (PFGE A), ST 904 (PFGE C) and ST 3769 (PFGE G), out of the six studied, formed biofilms with variable intensity according to different conditions (temperatures -37 °C, 30 °C, 25°C- and materials -stainless steel and plastic-). High levels of antimicrobial resistance were found in isolates to ciprofloxacin, nalidixic acid and tetracycline as well as to two common detergents used in the slaughterhouse. A combination of several changes in the genome of ST 904 (PFGE C) including mutations, insertions in antimicrobial resistance genes, the presence of T6SS and a set of genes related to virulence factors might explain its ability to form biofilm and persist longer in the environment. However, the complexity of the survival strategies adopted by the different strains of C. jejuni suggests that multiple mechanisms may exist that allow these organisms to persist and ultimately cause disease in humans.

Cat scratch esophagus.

The cat scratch esophagus is an uncommon entity. The first case described in the literature of this type of lesions was published in 2007 and was located in the colon. There are two cases described in the esophagus, this has been the first detected by endoscopic capsule. It is defined by the presence of linear, erythematous, shiny and superficial breaks of the mucosa without significant hemorrhage associated. The diagnosis is morphological. Even though the etiology is unknown, it has been postulated that the main pathogenic mechanism is barotrauma secondary to insufflation. It has also been described association with other processes that can affect the esophageal elasticity as well as the previous use of NSAIDs.

Characterization of Campylobacter species in Spanish retail from different fresh chicken products and their antimicrobial resistance.

Contaminated chicken products have been recognized as the primary vehicles of Campylobacter transmission to human. Pulsed-field gel electrophoresis (PFGE) and antimicrobial resistance of Campylobacter isolates from fresh chicken products at retail were studied. A total of 512 samples including: thigh, breast, marinated and minced chicken were purchased from different retail stores. Half of the samples were packed and the other half were unpacked. The 39.4% of the samples were Campylobacter positive; being unpacked chicken products (45.3%) more contaminated than packed chicken (33.6%). PFGE typing showed a high diversity among isolates; clustering 204 isolates into 76 PFGE types: 55 clusters of C. jejuni, 19 of C. coli and 2 of C. lari. C. coli genotypes showed higher resistance than other Campylobacter species. Although modified atmosphere packaging can reduce the prevalence of Campylobacter spp., it does not avoid their presence in at least 33.6% of packed chicken products analyzed. Some pulsotypes might persist in the processing plant or butcher shops environment for longer than previously thought. More stringent control measures are needed in previous steps of the chicken food chain, in order to avoid the presence of Campylobacter spp. strains at retail that can compromise consumer's safety.

Campylobacter jejuni survival in a poultry processing plant environment.

Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Consumption of poultry, especially chicken's meat is considered the most common route for human infection. The aim of this study was to determine if Campylobacter spp. might persist in the poultry plant environment before and after cleaning and disinfection procedures and the distribution and their genetic relatedness. During one month from a poultry plant were analyzed a total of 494 samples -defeathering machine, evisceration machine, floor, sink, conveyor belt, shackles and broiler meat- in order to isolate C. jejuni and C. coli. Results showed that C. jejuni and C. coli prevalence was 94.5% and 5.5% respectively. Different typing techniques as PFGE, MLST established seven C. jejuni genotypes. Whole genome MLST strongly suggest that highly clonal populations of C. jejuni can survive in adverse environmental conditions, even after cleaning and disinfection, and persist for longer periods than previous thought (at least 21 days) in the poultry plant environment. Even so, it might act as a source of contamination independently of the contamination level of the flock entering the slaughter line.

Characterization by culture-dependent and culture-independent methods of the bacterial population of suckling-lamb packaged in different atmospheres.

This study offers insight into the dynamics of bacterial populations in fresh cuts of suckling lamb under four different atmospheric conditions: air (A), and three Modified Atmosphere Packaging (MAP) environments, 15%O2/30%CO2/55%N2 (C, commercial), 70%O2/30%CO2 (O), and 15%O2/85%CO2 (H) for 18 days. Microbial analyses by both conventional methods and PCR-DGGE were performed. Controversial and surprising results emerged from comparing both methods in relation to the genus Pseudomonas. Thus, conventional methods detected the presence of high numbers of Pseudomonas colonies, although PCR-DGGE only detected this genus in air-packaged samples. PCR-DGGE detected higher microbial diversity in the control samples (A) than in the modified atmospheres (C, O, H), having atmosphere H the fewest number of species. Brochothrix thermosphacta, LAB (Carnobacterium divergens and Lactobacillus sakei), and Escherichia spp. were detected in all the atmospheres throughout storage. Moreover, previously undescribed bacteria from lamb meat such as Enterobacter hormaechei, Staphylococcus equorum and Jeotgalicoccus spp. were also isolated in this study by DGGE. Additionally, qPCR analysis was used to detect and characterize strains of Escherichia coli. Virulence genes (stx1, stx2 and eae) were detected throughout storage in 97% of the samples. A high CO2 atmosphere was the most effective packaging combination doubling storage time in comparison with commercial atmosphere.

Tracing Campylobacter jejuni strains along the poultry meat production chain from farm to retail by pulsed-field gel electrophoresis, and the antimicrobial resistance of isolates.

In this study Campylobacter jejuni isolates were recovered from birds, carcasses and carcass portions from two broiler chicken flocks and from equipment used for carcass and meat processing along the production chain from farms to retail stores. Isolates were subjected to pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes and their antimicrobial susceptibilities were determined. C. jejuni was recovered from product and equipment used with both flocks at each point in the production chain. The prevalence of C. jejuni in poultry products at retail stores was 58.97% (flock 1) and 69.23% (flock 2). SmaI divided 122 C. jejuni strains from flock 1 and 106 from flock 2 into 17 and 13 PFGE types, respectively. PFGE types H and F were present at all steps along the chain, from farms to retail products. Similarly, for both flocks PFGE type D was detected in crates, slaughterhouse and retail stores. Moreover, the PFGE types were highly diverse at the processing and retail steps. Most PFGE types were resistant to ciprofloxacin (95.45%) and tetracycline (81.82%); and multidrug resistant PFGE types were found in the final products. Our study showed that there were several points of cross-contamination of product along the chain, and a high diversity of PFGE types with antimicrobial resistance to ciprofloxacin and tetracycline in the retail products.

Antimicrobial Effect of Simira ecuadorensis Extracts and Their Impact on Improving Shelf Life in Chicken and Fish Products.

The objective of this work was to evaluate the antimicrobial potential of different extracts of Simira ecuadorensis, a characteristic plant of Ecuador, and to validate its potential as a food preservative. Four extracts referred to as ethanol, ethanol-water (50:50 v/v), spray-dried, and freeze-dried were obtained under different processes. Initially, their antimicrobial activities were evaluated against a wide group of microorganisms consisting of 20 pathogenic and spoilage microbial strains found in foods through the agar diffusion method. Then, the extracts with the best yields and antimicrobial properties against microorganisms of greatest interest were selected to determine their effect on model foods preserved under normal commercial conditions through challenge tests. Spray-dried and ethanol-water extracts were tested for their ability to inhibit C. jejuni in chicken model products, where is a common pathogen and Shew. putrefaciens in fish model products as it is a spoilage microorganism frequently found in fish. One solid and one liquid were chosen as model foods: burger and broth, respectively. Campylobacter jejuni and Shewanella putrefaciens were effectively inhibited by the four extracts with minimum inhibitory concentration (MIC) of 80 mg/mL. Bacillus cereus, Yersinia enterocolitica, Clostridium perfringens, and Leuconostoc mesenteroides were also inhibited by ethanolic extract. The ethanol-water extract showed greater antimicrobial activity in fish products, whereas spray-dried extract had low growth inhibition of C. jejuni in chicken burgers; however, it was quite effective on C. jejuni in broth. The spray-dried extract significantly decreased the pH of the chicken burgers, while the ethanolic extract had a slight impact on the pH of the fish burgers. The presence of antibacterial effects revealed that the S. ecuadorensis extracts could be potentially used in food preservation and as a natural antimicrobial.

Easy Nitrite Analysis of Processed Meat with Colorimetric Polymer Sensors and a Smartphone App.

We have developed an in situ methodology for determining nitrite concentration in processed meats that can also be used by unskilled personnel. It is based on a colorimetric film-shaped sensory polymer that changes its color upon contacting the meat and a mobile app that automatically calculates the manufacturing and residual nitrite concentration by only taking digital photographs of sensory films and analyzing digital color parameters. The film-shaped polymer sensor detects nitrite anions by an azo-coupling reaction, since they activate this reaction between two of the four monomers that the copolymer is based on. The sensory polymer is complemented with an app, which analyzes the color in two different digital color spaces (RGB and HSV) and performs a set of 32 data fittings representing the concentration of nitrite versus eight different variables, finally providing the nitrite concentration of the test samples using the best fitting curve. The calculated concentration of nitrite correlates with a validated method (ISO 2918: 1975) usually used to determine nitrite, and no statistically significant difference between these methods and our proposed one has been found in our study (26 meat samples, 8 prepared, and 18 commercial). Our method represents a great advance in terms of analysis time, simplicity, and orientation to use by average citizens.

Comparison between conventional and qPCR methods for enumerating Campylobacter jejuni in a poultry processing plant.

Campylobacter jejuni is worldwide recognized as a human foodborne pathogen. It is widely present in poultry meat and slaughterhouses, but little is known about its fate during the processing of poultry meat preparations. In stress conditions, this pathogen can enter into a viable but non-culturable state, where quantitative PCR (qPCR) becomes more convenient for its detection. In this study, two different pairs of primers, targeting the rpoB and the hipO genes, were compared for its detection and quantification by PCR. Two calibration curves were prepared: one for the meat samples and the other for the environmental samples. rpoB primers showed higher sensitivity with a quantification limit of 1 log cfu/g or ml. Microbial Assessment Scheme (MAS) was used to select the Critical Sampling Locations (CSLs) along the poultry processing line. Forty-six out of 48 samples were positive by qPCR after enrichment (t = 48 ) while only 6 samples were positive by ISO 10272-1:2006. Forty-three samples showed positive signal without enrichment (t = 0 h), however only 16 samples could be quantified. These results showed the high prevalence of C. jejuni in the poultry industry and the need for new, rapid and sensitive techniques, such as qPCR, for the detection and quantification of C. jejuni in meat and environmental samples.

Wine Pomace Product Inhibit Listeria monocytogenes Invasion of Intestinal Cell Lines Caco-2 and SW-480.

Red wine pomace products (WPP) have antimicrobial activities against human pathogens, and it was suggested that they have a probable anti-Listeria effect. This manuscript evaluates the intestinal cell monolayer invasive capacity of Listeria monocytogenes strains obtained from human, salmon, cheese, and L. innocua treated with two WPP (WPP-N and WPP-C) of different polyphenol contents using Caco-2 and SW480 cells. The invasion was dependent of the cell line, being higher in the SW480 than in the Caco-2 cell line. Human and salmon L. monocytogenes strains caused cell invasion in both cell lines, while cheese and L. innocua did not cause an invasion. The phenolic contents of WPP-N are characterized by high levels of anthocyanin and stilbenes and WPP-C by a high content of phenolic acids. The inhibitory effect of the WPPs was dependent of the strain and of the degree of differentiation of the intestinal cells line. The inhibition of Listeria invasion by WPPs in the SW480 cell line, especially with WPP-C, were higher than the Caco-2 cell line inhibited mainly by WPP-N. This effect is associated with the WPPs' ability to protect the integrity of the intestinal barrier by modification of the cell-cell junction protein expression. The gene expression of E-cadherin and occludin are involved in the L. monocytogenes invasion of both the Caco-2 and SW480 cell lines, while the gene expression of claudin is only involved in the invasion of SW480. These findings suggest that WPPs have an inhibitory L. monocytogenes invasion effect in gastrointestinal cells lines.

Genotyping, virulence genes and antimicrobial resistance of Campylobacter spp.isolated during two seasonal periods in Spanish poultry farms.

Campylobacter spp. are the leading causes of bacterial human gastroenteritis worldwide; being poultry farms the main source of infections. In order to obtain information on prevalence and diversity of Campylobacter-infected flocks in the North of Spain, fourteen farms were studied between autumn and spring in 2014 and 2015, respectively. Moreover, virulence genes involved in pathogenicity and antimicrobial resistance were investigated. A survey about preventive hygiene practices at farms was performed to determine the risky practices that could contribute to the presence of Campylobacter in this step of the poultry food chain. Testing the presence of Campylobacter spp. showed 43 % of the farms were positive during autumn, whereas only 31 % were positive in spring. A very high prevalence within-flock was observed (43.1 % to 88.6 %) and C. jejuni was the most prevalent species in both periods. Genotyping by pulsed field gel electrophoresis (PFGE) showed a high heterogeneity among farms (309 isolates clustered into 21 pulsotypes). Virulence genes were present in all C. jejuni isolates while cdtA and cdtC were absent in C. coli. On the contrary, the latter showed higher antimicrobial resistance than C. jejuni. This study suggests that environment might be one of the main sources for Campylobacter transmission, as water supply seemed to be a clear cause of the contamination in a specific farm. However, in other farms other environmental factors contributed to the contamination, confirming the multifactorial origin of Campylobacter colonization in broilers. Therefore, biosecurity measures in farms are crucial to reduce Campylobacter contamination, which may have important implications for human and animal health.

The Response to Oxidative Stress in Listeria monocytogenes Is Temperature Dependent.

The stress response of 11 strains of Listeria monocytogenes to oxidative stress was studied. The strains included ST1, ST5, ST7, ST6, ST9, ST87, ST199 and ST321 and were isolated from diverse food processing environments (a meat factory, a dairy plant and a seafood company) and sample types (floor, wall, drain, boxes, food products and water machine). Isolates were exposed to two oxidizing agents: 13.8 mM cumene hydroperoxide (CHP) and 100 mM hydrogen peroxide (H2O2) at 10 °C and 37 °C. Temperature affected the oxidative stress response as cells treated at 10 °C survived better than those treated at 37 °C. H2O2 at 37 °C was the condition tested resulting in poorest L. monocytogenes survival. Strains belonging to STs of Lineage I (ST5, ST6, ST87, ST1) were more resistant to oxidative stress than those of Lineage II (ST7, ST9, ST199 and ST321), with the exception of ST7 that showed tolerance to H2O2 at 10 °C. Isolates of each ST5 and ST9 from different food industry origins showed differences in oxidative stress response. The gene expression of two relevant virulence (hly) and stress (clpC) genes was studied in representative isolates in the stressful conditions. hly and clpC were upregulated during oxidative stress at low temperature. Our results indicate that conditions prevalent in food industries may allow L. monocytogenes to develop survival strategies: these include activating molecular mechanisms based on cross protection that can promote virulence, possibly increasing the risk of virulent strains persisting in food processing plants.

A mathematical modeling approach to the quantification of lactic acid bacteria in vacuum-packaged samples of cooked meat: Combining the TaqMan-based quantitative PCR method with the plate-count method.

The TaqMan-based quantitative Polymerase Chain Reaction (qPCR) method and the Plate Count (PC) method are both used in combination with primary and secondary mathematical modeling, to describe the growth curves of Leuconostoc mesenteroides and Weissella viridescens in vacuum-packaged meat products during storage under different isothermal conditions. Vacuum-Packaged Morcilla (VPM), a typical cooked blood sausage, is used as a representative meat product, with the aim of improving shelf-life prediction methods for those sorts of meat products. The standard curves constructed by qPCR showed good linearity between the cycle threshold (CT) and log10 CFU/g, demonstrating the high precision and the reproducible results of the qPCR method. The curves were used for the quantification of L. mesenteroides and W. viridescens in artificially inoculated VPM samples under isothermal storage (5, 8, 13 and 18 °C). Primally, both the qPCR and the PC methods were compared, and a linear regression analysis demonstrated a statistically significant linear correlation between the methods. Secondly, the Baranyi and Roberts model was fitted to the growth curve data to estimate the kinetic parameters of L. mesenteroides and W. viridescens under isothermal conditions, and secondary models were used to establish the dependence of the maximum specific growth rate on the temperature. The results proved that primary and secondary models were adequate for describing the growth curves of both methods in relation to both bacteria. In conclusion, the results of all the experiments proved that the qPCR method in combination with the PC method can be used to construct microbial growth kinetics and that primary and secondary mathematical modeling can be successfully applied to describe the growth of L. mesenteroides and W. viridescens in vacuum-packaged morcilla and, by extension, other cooked meat products with similar characteristics.

Characterization of Virulence and Persistence Abilities of Listeria monocytogenes Strains Isolated from Food Processing Premises.

We report the characterization of 15 Listeria monocytogenes strains isolated from various food processing plants by multivirulence locus sequence typing to determine virulence types (VTs) and epidemic clones. Molecular mechanisms involved in adaptation to food processing environments and related to virulence were also studied. Phenotypic behaviors associated with various antimicrobials, biofilm formations, and invasiveness were assessed. There were 11 VTs among the 15 L. monocytogenes strains. Strains belonging to six VTs were stress survival islet 1 (SSI-1) and one strain of VT94 was SSI-2. Tn6188 was found in VT6 and VT94 strains, and bcrABC cassette genes were identified in VT21, VT60, and VT63 strains. Only one strain, in VT20, showed llxS, whereas a full-size inlA was detected in strains belonging to VT8, VT20, VT21, and VT63. VT10, VT20, VT21, VT60, and VT63 strains were the most tolerant to studied disinfectants. A VT6 strain showed the strongest biofilm formation ability in polyvinyl chloride, and strains belonging to VT10, VT11, VT20, and VT94 had moderate abilities. Antimicrobial sensitivity tests showed that all the L. monocytogenes strains were multidrug resistant. F tests revealed that only strains of VT10, VT60, and VT94 were significantly noninvasive (P < 0.05) in Caco-2 cells. Our findings illustrate how L. monocytogenes isolates exploit diverse mechanisms to adapt to adverse conditions. Consequently, detailed characterization of L. monocytogenes isolates is required for comprehensive elimination of this pathogenic bacterium in food processing environments.

Distribution and Persistence of Listeria monocytogenes in a Heavily Contaminated Poultry Processing Facility.

We studied the colonization and distribution of Listeria monocytogenes in a heavily contaminated poultry processing plant over a 1-year period. A total of 180 nonfood contact surfaces, 70 food contact surfaces, 29 personnel, and 40 food samples were analyzed. L. monocytogenes isolates were subtyped by PCR serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. L. monocytogenes was detected in samples collected at every visit to the plant, and 43.8% (visit 4) to 65.6% (visit 7) of samples were positive, for an overall prevalence of 55.2%. The deboning area had the highest prevalence of positive samples (83.3%), and the processing area had the highest diversity of PFGE types. Ninety percent of the final products were positive for L. monocytogenes. Most of the isolates belonged to well-known persistent L. monocytogenes sequence types (ST9 and ST121). This study illustrates a well-established L. monocytogenes contamination problem in a poultry processing plant associated with a generalized failure of the food safety system as a whole. These findings reflect the potential for L. monocytogenes contamination when the food safety and quality management system is unsatisfactory, as described in the present study. It is essential to revise food safety and quality management systems to eliminate L. monocytogenes from food processing facilities, to control the entrance of sporadic sequence types, and to prevent L. monocytogenes spread within such facilities, especially in those premises with higher L. monocytogenes prevalence in the environment and final food products.