Assessing refrigerated preservation performance using Listeria predictive microbiology models and temperature data: Refrigerator performance indicator and time-temperature equivalent.

Time-temperature data for queso fresco (QF) cheese varieties stored in a residential refrigerator operating at 5°C and a predictive microbiology secondary model for Listeria monocytogenes in QF were used to estimate a refrigerator performance indicator (RPI) of microbial preservation. RPI values were used to assess how compressor technology (single [SS] and variable speed [VS]), ambient temperature (21.1°C [LT] and 32.2°C [HT]), and refrigerator load (22.5 kg regular load and 39 kg higher load) affected preservation performance. All deterministic and probabilistic RPI estimations slightly exceeded the desirable 1.0 value, i.e., the variable temperatures for the QF kept in the refrigerator were worse than keeping it constantly at the temperature recommended by food safety agencies for QF. Furthermore, the mean comparison of estimates of the time-temperature equivalent indicator previously developed by French researchers showed similar behavior to those observed for RPI. Finally, statistical analysis showed that Tambient was the factor with the highest impact on refrigerator performance because of its impact on the sample temperature increase during door openings and when exposed to ambient temperature during product use. This highlights the need to reduce the time for product temperature recovery by improving the compressor operation logic. Also important are consumer behavior changes such as a reduction in product exposure to ambient temperature and in the door opening duration and frequency. PRACTICAL APPLICATION: This study demonstrated how a quantitative tool (RPI) can assess refrigerator preservation performance. Although the findings presented can be applied to any cold chain segment, the data used was collected for its weakest link, the domestic refrigerator. Surveys show that 77% of them operate above the recommended 4°C. The RPI methodology is ready for use by refrigerator designers to assess performance improvements possible by modifications of the compressor operation logic. Moreover, it can be integrated into smart-hubs monitoring the frequency and duration of refrigerator door openings to inform consumers when their habits are compromising the preservation performance of the refrigerator.

Listeria monocytogenes in beef: a hidden risk.

Listeria monocytogenes in beef receives less attention compared to other pathogens such as Salmonella and Escherichia coli. To address this gap, we conducted a literature review focusing on the presence of L. monocytogenes in beef. This review encompasses the pathogenic mechanisms, routes of contamination, prevalence rates, and the laws and regulations employed in various countries. Our findings reveal a prevalence of L. monocytogenes in beef and beef products ranging from 2.5% to 59.4%. Notably, serotype 4b was most frequently isolated in cases of beef contamination during food processing, with the skinning and evisceration stages identified as critical points of contamination.

[Molecular characterization of Listeria monocytogenes isolates from human and food sources in Argentina, 2018-2023]. / Caracterización molecular de aislamientos de Listeria monocytogenes de origen humano y alimentario en Argentina, 2018-2023.

Human listeriosis is an infectious disease caused by Listeria monocytogenes. The invasive form of this disease leads to a high rate of hospitalizations and fatality. The main mode of transmission is through contaminated ready-to-eat foods such as dairy, vegetables and meat products. The knowledge of the diversity and population dynamics of isolates collected from human and food sources is essential for the detection of clusters and the identification of common sites of infection. The aim of this study was the molecular characterization of L. monocytogenes isolates in Argentina. We sequenced a total of 63 isolates, 35 from human and 28 from food sources, collected between 2018 and 2023. Our genomic study divided the isolates into two lineages, four serogroups, 17 sequence types and 15 clonal complexes (CCs). The hypervirulent clone CC1 (lineage I; serogroup IVb) predominated in human and food samples. The phylogenomic analysis showed a high and possible epidemiological relationship between isolates from human and/or food sources, suggesting the presence of transmission chains in our country. These findings highlight the need to strengthen genomic surveillance of L. monocytogenes in Argentina. The identification of geographic distribution and characteristics of predominant and emerging clones from human and food sources might help to focus action plans and public health policies better directed at the control and prevention of listeriosis.

Raw milk cheeses from Beira Baixa, Portugal-A contributive study for the microbiological hygiene and safety assessment.

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g-1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g-1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production.

Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples.

The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290-1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290-1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.

Meningitis por Listeria: serie de casos en un hospital de tercer nivel y utilidad de las nuevas técnicas de diagnóstico microbiológico / Listeria meningitis: case series in a tertiary level hospital and the usefulness of new microbiological diagnostic techniques

Introducción: La meningitis por Listeria monocytogenes (MLM) es una entidad grave con complicaciones a corto plazo. La reacción de polimerasa en cadena (RPC) puede ayudar a mejorar su diagnóstico y pronóstico. Objetivos: Conocer las características de los pacientes diagnosticados de meningitis por L. monocytogenes en los últimos años, a través de diferentes métodos microbiológicos. Pacientes y Métodos: Serie de casos de pacientes adultos ingresados con MLM en el Hospital Clínico San Carlos, Madrid, España, durante doce años (2009-2021). Se describieron variables epidemiológicas, clínicas, microbiológicas, radiológicas y terapéuticas. Resultados: Se registraron doce pacientes con MLM (edad media 67,5 años, 75% varones). En ocho se obtuvo un cultivo positivo a L. monocytogenes. La RPC en líquido cefalorraquídeo (LCR) fue positiva en los dos casos en los que se realizó la prueba. El tratamiento dirigido en todos los casos fue ampicilina durante 21 días. Se registraron complicaciones en un cuarto de los casos. Del total de pacientes uno falleció. Conclusiones: La MLM es una enfermedad poco frecuente y de difícil diagnóstico. En nuestra serie de casos los dos pacientes diagnosticados por RPC tuvieron resultado de cultivo de LCR negativo, y presentaron buena evolución. La determinación de RPC podría permitir diagnosticar un mayor número de casos y con mayor precocidad.

Background: Listeria monocytogenes meningitis (LMM) is a serious entity with short-term complications. Polymerase chain reaction (PCR) can help to improve its diagnosis and prognosis. Aim: To know the characteristics of patients diagnosed with meningitis by L. monocytogenes in recent years, through different microbiological methods. Methods: Case series of adult patients admitted with LMM at the Hospital Clínico San Carlos of Madrid, Spain, during twelve years (2009-2021). Epidemiological, clinical, microbiological, radiological and therapeutic variables were described. Results: Twelve patients with LMM were recorded (mean age 67.5 years, 75% male). Eight had a positive culture for L. monocytogenes. cerebrospinal fluid (CSF) PCR was positive in the two cases in which the test was performed. Treatment in all cases was ampicillin for 21 days. Complications were recorded in a quarter of the cases. One patient died. Conclusions: LMM is a rare and difficult to diagnose disease. In our series of cases, the two patients diagnosed by PCR had negative CSF culture results, and presented good evolution. PCR determination could allow a greater number of cases to be diagnosed earlier.

Listeria innocua and serotypes of Listeria monocytogenes isolated from clinical cases in small ruminants in the northwest of Uruguay / Listeria innocua e serotipos do Listeria monocytogenes isoladas dos casos clínicos em pequenos ruminantes no noroeste do Uruguai

Listeriosis is an infectious disease caused by bacteria of the genus Listeria, the neurological form being more common in ruminants. There are many reports of listeriosis in small ruminants in the region that includes Brazil, Argentina and Uruguay. However, these diagnoses were mainly based on histological lesions in the central nervous system (CNS) without the isolation and characterization of the involved Listeria strains. The aim of this study was to report sheep and goats listeriosis cases from 2016 to 2021 in northwestern Uruguay. The diagnosis was made according to lesions observed at histopathology, plus Listeria isolation in CNS, identifying it at specie and serotype level. Nine animals (n=9) of three outbreaks and five sporadic cases of listeriosis were studied. Sheep was the species with more cases in relation to goats, and adults were the category most affected. Cases occurred in spring and less frequently in winter. All presented neurological clinical signs and the lesions in the CNS were consistent with suppurative meningoencephalitis and micro-abscesses in the brainstem. In eight of nine CNS samples, Listeria strains were isolated (seven L. monocytogenes and one L. innocua). All the L. monocytogenes isolates carried the inlA gene; serotyping showed that four strains belonged to serotype 1/2b, two isolates belonged to serotype 4b, and one to serotype 1/2a. Considering that listeriosis is a common disease in this region and the fact that isolates are scarcely recovered from small ruminants, it would be important to emphasize the need for Listeria isolation to better characterize the strains that affect animals. Not only to improve knowledge about the epidemiology of disease but also with the objective of developing serotype specific vaccines for animal use.

Listeriose uma doença bacteriana causada pelo gênero Listeria, a forma nervosa é a mais comum em ruminantes. No Brasil, Argentina e Uruguai há vários relatos de listeriose em pequenos ruminantes com diagnóstico baseado na histopatologia do sistema nervoso central (SNC), sem o isolamento e a caracterização do agente. O objetivo deste trabalho foi relatar uma série de casos diagnosticados em ovinos e caprinos no período 2016-2021 no noroeste do Uruguai. O diagnóstico foi feito basado nas lesões observadas na histopatologia, e caracterização das cepas de Listeria recuperadas do SNC quanto à espécie e sorotipo. Nove animais (n=9) do três surtos e cinco casos isolados de listeriose foram estudados. Os ovinos foram a espécie com o maior número de casos em relação aos caprinos, sendo os animais adultos a categoria mais afetada em ambas espécies. A doença ocorreu principalmente na primavera com alguns casos observados no inverno. Todos os casos apresentavam sinais clínicos nervosos e as lesões no SNC caracterizavam-se por meningoencefalite supurativa com presença de microabscessos no tronco encefálico. Em oito de nove amostras do SNC foram isoladas cepas de Listeria (sete L. monocytogenes e uma L. innocua). Todos os isolados de L. monocytogenes continham o gene inlA; a sorotipagem apresentou quatro cepas do serotipo 1/2b, duas cepas serotipo 4b e uma cepa 1/2a. Levando em consideração que nesta região a listeriose é uma doença frequente e que existem poucos isolados recuperados de casos clínicos em pequeño ruminantes, torna-se relevante o isolamento deste agente para caracterização das cepas que afetam os animais. Não só para melhorar o conhecimento sobre a epidemiologia da doença, mas também com o objetivo de desenvolver vacinas sorotipo-especificas para uso animal.

Activity of rosemary (Rosmarinus officinalis - Family: Lamiaceae) essential oil compared to peracetic acid in Listeria monocytogenes biofilms

The objective of this study was to evaluate the activity of Rosmarinus officinalis essential oil (EO) compared to peracetic acid (PA) regarding formation and elimination of Listeria monocytogenes biofilms on polystyrene surface. The minimum inhibitory concentration (MIC) was determined according to standard protocol. Isolates were inoculated according to MIC standards polystyrene plate wells, which were then incubated at 37°C/96 hours for evaluation of biofilm formation. Regarding the evaluation of biofilm elimination, the biofilms were treated under MIC for 10 minutes. The MIC obtained were 2.0 and 3.0 mg mL-1 for EO and 0,015% for PA. Therefore, the results showed a reduction in the formation of biofilm with the presence of EO and PA, EO being more efficient (p < 0.05). Both compounds had a good capacity of eliminating biofilms, however the EO reduced the biofilm formation when compared to PA, highlighting its potential as an antibacterial agent and antibiofilm.(AU)

Perfil etiológico de las meningitis adquiridas en la comunidad en pacientes adultos en el Complejo Asistencial Sótero del Río, Santiago 2011-2017 / Community acquired meningitis in Santiago, Chile, 2011-2017

INTRODUCCIÓN: En Chile existe poca información sobre los microorganismos causantes de meningitis adquirida en la comunidad (MAC), la que es relevante a la hora de escoger el esquema antimicrobiano empírico. OBJETIVO: Describir la microbiología de MAC en pacientes mayores de 15 años atendidos en un hospital público de Santiago (Chile). METODOLOGÍA: Revisión de cultivos de líquido cefalorraquídeo positivos durante 2011-2017. Se recolectó la información clínica de los pacientes incluidos. Se excluyeron cultivos considerados como contaminación y las meningitis post-quirúrgicas. RESULTADOS: Se identificaron 20 episodios de meningitis bacteriana aguda (MBA) y seis episodios de meningitis criptocócica (MC) entre 2.720 cultivos. Los microorganismos causantes de MBA fueron: Streptococcus pneumoniae (50%), Listeria monocytogenes (25%) y otros cinco agentes (25%). Todos los pacientes con infección por L. monocytogenes presentaban alguna comorbilidad significativa. Cuatro de cinco casos de MC presentaban infección por VIH. CONCLUSIÓN: Streptococcus pneumoniae fue el microorganismo más frecuente de las MAC en esta serie, seguido por L. monocytogenes. Las recomendaciones actuales de esquemas empíricos de MAC consideran adecuadamente la cobertura de S. pneumoniae en todos los pacientes y de L. monocytogenes solo ante factores de riesgo. Además, es relevante considerar MC en casos en pacientes inmunocomprometidos.

BACKGROUND: In Chile, there is scarce information on the frequency of the causative microorganisms of community-acquired meningitis (CAM), which is relevant for the choice of empiric treatment. AIM: To describe the microbiology of CAM in patients over 15 years treated at a public hospital in Santiago (Chile). METHODS: Retrospective review of positive cerebrospinal fluid cultures during 2011-2017. Clinical information of the included patients was collected. Cultures considered as contamination and cases of post-surgical meningitis were excluded. RESULTS: We identified 20 episodes of bacterial meningitis (BM) and six episodes of cryptococcal meningitis (CM) in 2720 cultures. The microorganisms identified in BM cases were Streptococcus pneumoniae (50%), Listeria monocytogenes (25%) and five other agents (25%). All patients with L. monocytogenes infection had at least one well-known risk factor for this infection. Four of the five cases of CM had HIV infection. CONCLUSION: Streptococcus pneumoniae was the most frequent causative microorganism of CAM in this series, followed by L. monocytogenes. Current recommendations for empiric CAM regimens adequately consider coverage for S. pneumoniae in all patients and for L. monocytogenes only in those with risk factors. Furthermore, it is relevant to consider CM in cases involving immunocompromised patients.

Electrochemical nanobiosensors equipped with peptides: a review.

Recent research in the field of electrochemical biosensors equipped with peptides and nanomaterials have been categorized, reviewed, and critically analyzed. Indeed, using these innovative biosensors can revolutionize biomedical diagnostics in the future. Saving lives, time, and money in this field will be considered as some main benefits of this type of diagnosis. Here, these biosensors have been categorized and evaluated in four main sections. In the first section, the focus is on investigating the types of electrochemical peptide-based nanobiosensors applied to detect pathogenic microorganisms, microbial toxins, and viruses. In the second section, due to the importance of rapid diagnosis and prognosis of various cancers, the electrochemical peptide-based nanobiosensors designed to detect cancer biomarkers have been reviewed and analyzed. In the third section, the electrochemical peptide-based nanobiosensors, which were applied to detect the essential and effective biomolecules in the various diseases, and health control, including enzymes, hormones, biomarkers, and other biomolecules, have been considered. Finally, using a comprehensive analysis, all the used elements in these biosensors have been presented as conceptual diagrams that can effectively guide researchers in future developments. The essential factors in evaluating and analyzing these electrochemical peptide-based nanobiosensors such as analyte, peptide sequence, functional groups interacted between the peptide sequences and other biosensing components, the applied nanomaterials, diagnostic techniques, detection range, and limit of detection have also been included. Other analyzable items such as the type of used redox marker and the location of the peptide sequence against the signal transducer were also considered.

Listeriosis outbreak in sheep raised in feedlots in the Southern Region of Rio Grande do Sul state, Brazil

Background: A listeriosis outbreak in a sheep fattening feedlot in the Southern Region of Rio Grande do Sul State, Brazilis described. This disease is caused by Listeria monocytogenes and represents a risk to public health since it affects notonly ruminants but also humans. This agent is widely spread in the environment, such as in the soil and water. It is alsofound in decaying vegetable matter and the feces and fluids of domestic animals. The aim of this study was to describe alisteriosis outbreak in sheep raised in feedlots, its epidemiology, and to establish the importance of this disease in this typeof sheep management system, evaluate the possible sources of infection, and suggest ways to control it.Cases: Sheep were kept in a 2-sector shed, one with east solar orientation and the other with west solar orientation, thelatter with free access to domestic birds. Sheep were fed silage and concentrate. Seven sheep were affected, 5 died and 2recovered. Clinically, the sheep displayed loss of balance, excessive drooling, and tremors; one exhibited circling, headdeviation, apathy, nystagmus, lateral recumbency, paddling, and labored breathing. At necropsy, macroscopic lesions werenot found, and histologically several micro-abscesses and perivascular cuffs with lymphocytes, macrophages, and neutrophils were present in the brain stem. Listeria monocytogenes suspected colonies were observed in the microbiologicalculture, and the bacteria was identified by biochemical analysis. The immunohistochemistry test in brain stem sectionswas positive for the antibody BD DifcoTM Listeria O Antiserum Poly Serotypes 1 and 4.Discussion: A listeriosis outbreak in a feedlot sheep was confirmed through epidemiological findings, histological lesions,bacterial culture, and immunohistochemistry analysis. This infection is frequent...

Listeriosis outbreak in sheep raised in feedlots in the Southern Region of Rio Grande do Sul state, Brazil

Background: A listeriosis outbreak in a sheep fattening feedlot in the Southern Region of Rio Grande do Sul State, Brazilis described. This disease is caused by Listeria monocytogenes and represents a risk to public health since it affects notonly ruminants but also humans. This agent is widely spread in the environment, such as in the soil and water. It is alsofound in decaying vegetable matter and the feces and fluids of domestic animals. The aim of this study was to describe alisteriosis outbreak in sheep raised in feedlots, its epidemiology, and to establish the importance of this disease in this typeof sheep management system, evaluate the possible sources of infection, and suggest ways to control it.Cases: Sheep were kept in a 2-sector shed, one with east solar orientation and the other with west solar orientation, thelatter with free access to domestic birds. Sheep were fed silage and concentrate. Seven sheep were affected, 5 died and 2recovered. Clinically, the sheep displayed loss of balance, excessive drooling, and tremors; one exhibited circling, headdeviation, apathy, nystagmus, lateral recumbency, paddling, and labored breathing. At necropsy, macroscopic lesions werenot found, and histologically several micro-abscesses and perivascular cuffs with lymphocytes, macrophages, and neutrophils were present in the brain stem. Listeria monocytogenes suspected colonies were observed in the microbiologicalculture, and the bacteria was identified by biochemical analysis. The immunohistochemistry test in brain stem sectionswas positive for the antibody BD DifcoTM Listeria O Antiserum Poly Serotypes 1 and 4.Discussion: A listeriosis outbreak in a feedlot sheep was confirmed through epidemiological findings, histological lesions,bacterial culture, and immunohistochemistry analysis. This infection is frequent...(AU)

Comparison between immunofluorescence and immunohistochemistry for Listeria monocytogenes detection in formalin-fixed paraffin-embedded tissues / Comparação entre imunofluorescência e imuno-histoquímica para detecção de Listeria monocytogenes em tecidos fixados e parafinizados

Listeria monocytogenes is a bacterium that infect humans and animals and causes a zoonotic disease characterized by encephalitis, septicemia or abortion. In addition, listeriosis leads to significant economic losses due to animal death and sacrifice. This research compared the technique of immunofluorescence (IF) and immunohistochemistry (IHC) for the diagnosis of L. monocytogenes in formalin-fixed and paraffin-embedded (FFPE) tissues. A total of 30 tissue blocks from 15 animals with history and/or lesions compatible with listeriosis were selected. For both IHC and IF, the same diluted (1:200) polyclonal primary antibody was used against L. monocytogenes serotypes 1 and 4. For IHC, a polymer secondary antibody conjugated to peroxidase (HRP) was used. For IF, samples were incubated with a fluorescein-labeled anti-rabbit IgG secondary antibody. Each sample was classified according to the presence and percentage of immunolabeling area. From 30 samples, 10 were positive at least for one technique, whereas eight samples were positive for both IHC and IF with similar score. There was strong immunolabeling in tissue samples from bovines experimentally infected with L. monocytogenes ATCC 7644, as well as in nervous tissues from naturally infected ruminants. Additionally, IF did not show any difference in sensitivity when compared to IHC. Using processed biological materials for IF, instead of fresh tissues, is a quite unique technique, since there are few protocols described. Therefore, this study demonstrated that both techniques are efficient to detect L. monocytogenes in FFPE tissues.

Listeria monocytogenes é uma bactéria que infecta humanos e animais, podendo causar uma doença zoonótica caracterizada por encefalite, septicemia e abortos. Além disso, a listeriose resulta em perdas econômicas significativas pela morte e sacrifício de animais. O objetivo deste trabalho foi comparar a técnica de imunofluorescência (IF) e imuno-histoquímica (IHQ) para detecção de L. monocytogenes em tecidos fixados e parafinizados. Foram selecionados 30 blocos de tecidos de 15 animais com histórico e/ou lesões compatíveis com listeriose. Para ambas as técnicas, foi utilizado o mesmo anticorpo primário policlonal diluído (1:200) para detecção de L. monocytogenes sorotipos 1 e 4. Para a IHQ foi utilizado anticorpo secundário em polímero acoplado a peroxidase (HRP). Para a IF, as amostras foram incubadas com anticorpo secundário anti-IgG de coelho marcado com fluoresceína. Cada amostra foi classificada quanto à presença de imunomarcação e porcentagem de área positiva. Das 30 amostras, 10 foram positivas em pelo menos uma das técnicas, sendo oito amostras positivas em ambas IHQ e IF com o mesmo escore. Houve forte imunomarcação tanto em amostras de bovino experimentalmente infectado com L. monocytogenes ATCC 7644, como em amostras de tecido nervoso de ruminantes naturalmente infectados. Além disso, a IF não apresentou diferença na sensibilidade quando comparada com a IHQ. O uso de materiais biológicos processados para IF, ao invés de tecidos frescos, é algo inovador, uma vez que existem poucos protocolos descritos. Portanto, este estudo demonstrou que as duas técnicas foram eficientes para detectar L. monocytogenes em tecidos fixados em formol e parafinizados.

Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil.

Listeria monocytogenes and Salmonella spp. are considered important foodborne pathogens that are commonly associated with foods of animal origin. The aim of this study was to perform molecular characterization of L. monocytogenes and Salmonella spp. isolated from biofilms of cattle and poultry slaughterhouses located in the Federal District and State of Goiás, Brazil. Fourteen L. monocytogenes isolates and one Salmonella sp. were detected in poultry slaughterhouses. No isolates were detected in cattle slaughterhouses. All L. monocytogenes isolates belonged to lineage II, and 11 different pulsotypes were detected. Pulsed-field gel electrophoresis analysis revealed the dissemination of two strains within one plant, in addition to the regional dissemination of one of them. The Salmonella isolate was identified via whole genome sequencing as Salmonella enterica serovar Minnesota ST548. In the sequence analysis, no premature stop codons were detected in the inlA gene of Listeria. All isolates demonstrated the ability to adhere to Caco-2 cells, while 50% were capable of invading them. Antimicrobial resistance was detected in 57.1% of the L. monocytogenes isolates, and resistance to sulfonamide was the most common feature. The tetC, ermB, and tetM genes were detected, and four isolates were classified as multidrug-resistant. Salmonella sp. was resistant to nine antimicrobials and was classified as multidrug-resistant. Resistance genes qnrB19, blaCMY-2, aac(6')-Iaa, sul2, and tetA, and a mutation in the parC gene were detected. The majority (78.5%) of the L. monocytogenes isolates were capable of forming biofilms after incubation at 37°C for 24 h, and 64.3% were capable of forming biofilms after incubation at 12°C for 168 h. There was no statistical difference in the biofilm-forming capacity under the different evaluated conditions. Salmonella sp. was capable of forming biofilms at both tested temperatures. Biofilm characterization was confirmed by collecting the samples consistently, at the same sampling points, and by assessing biofilm formation in vitro. These results highlight the potential risk of cross-contamination in poultry slaughterhouses and the importance of surveillance and pathogen control maintenance programs within the meat production industry.

Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp, and Listeria monocytogenes DNA in milk / [Padronização de um método rápido de PCR quadriplex para a detecção simultânea de DNA de bovino, bubalino, Salmonella spp. e Listeria monocytogenes em leite]

The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.

Antibody- and nucleic acid-based lateral flow immunoassay for Listeria monocytogenes detection.

Listeria monocytogenes is an invasive opportunistic foodborne pathogen and its routine surveillance is critical for protecting the food supply and public health. The traditional detection methods are time-consuming and require trained personnel. Lateral flow immunoassay (LFIA), on the other hand, is an easy-to-perform, rapid point-of-care test and has been widely used as an inexpensive surveillance tool. In recent times, nucleic acid-based lateral flow immunoassays (NALFIA) are also developed to improve sensitivity and specificity. A significant improvement in lateral flow-based assays has been reported in recent years, especially the ligands (antibodies, nucleic acids, aptamers, bacteriophage), labeling molecules, and overall assay configurations to improve detection sensitivity, specificity, and automated interpretation of results. In most commercial applications, LFIA has been used with enriched food/environmental samples to ensure detection of live cells thus prolonging the assay time to 24-48 h; however, with the recent improvement in LFIA sensitivity, results can be obtained in less than 8 h with shortened and improved enrichment practices. Incorporation of surface-enhanced Raman spectroscopy and/or immunomagnetic separation could significantly improve LFIA sensitivity for near-real-time point-of-care detection of L. monocytogenes for food safety and public health applications.

Listeria monocytogenes: review of pathogenesis and virulence determinants-targeted immunological assays.

Listeria monocytogenes is one of the most invasive foodborne pathogens and is responsible for numerous outbreaks worldwide. Most of the methods to detect this bacterium in food require selective enrichment using traditional bacterial culture techniques that can be time-consuming and labour-intensive. Moreover, molecular methods are expensive and need specific technical knowledge. In contrast, immunological approaches are faster, simpler, and user-friendly alternatives and have been developed for the detection of L. monocytogenes in food, environmental, and clinical samples. These techniques are dependent on the constitutive expression of L. monocytogenes antigens and the specificity of the antibodies used. Here, updated knowledge on pathogenesis and the key immunogenic virulence determinants of L. monocytogenes that are used for the generation of monoclonal and polyclonal antibodies for the serological assay development are summarised. In addition, immunological approaches based on enzyme-linked immunosorbent assay, immunofluorescence, lateral flow immunochromatographic assays, and immunosensors with relevant improvements are highlighted. Though the sensitivity and specificity of the assays were improved significantly, methods still face many challenges that require further validation before use.

Ohmic heating processing of milk for probiotic fermented milk production: Survival kinetics of Listeria monocytogenes as contaminant post-fermentation, bioactive compounds retention and sensory acceptance.

The survival kinetics of Listeria monocytogenes (9 log CFU/mL) as a post-fermentation contaminant in probiotic fermented milk (Lactobacillus acidophilus La-5, 8-9 log CFU/mL) processed with milk subjected to ohmic heating (0, 4, 6, and 8 V/cm; CONV, OH4, OH6, OH8, 90-95 °C/5 min) was investigated using Weibull predictive model. Additionally, the presence of bioactive compounds (antioxidant activity, inhibition of the enzymes α-glucosidase, α-amylase, and angiotensin-converting) and sensory analysis (consumer test) of probiotic fermented milks were evaluated. Overall, OH provided a decrease in the viability of Listeria monocytogenes, suitable Lactobacillus acidophilus counts, and satisfactory results in the gastrointestinal tract survival. The Weibull model presented an excellent fit to the data of all conditions. Furthermore, lower δ values (217-298 against 665 h, CONV), and increased R2 values (0.99 against 0.98, CONV) were obtained for the OH-treated samples, emphasizing the best performance of OH data. In addition, OH improved the generation of bioactive compounds as well as the sensory acceptance. Indeed, considering functional and safety purposes, OH presented as an interesting technology to be used in milk for manufacturing probiotic fermented milk.

Favorable Conservative Management of a Rare Primary Aortitis Secondary to Listeria Monocytogenes: Case Report and Review of the Literature.

Primary aortitis (PA) secondary to Listeria monocytogenes is extremely rare with only a few cases reported in the literature. Presently, there is no consensus concerning the best treatment when no complications are found in the thoracic computed tomography (CT) imaging. This report illustrates the clinical presentation and favorable clinical course of a rare case of PA secondary to Listeria monocytogenes in an 82-year-old diabetic woman, successfully treated with conservative management with 18 months of follow up. Included in this article, we additionally present a review of the literature of this uncommon etiology of infectious aortitis.

Listeria monocytogenes inhibition by lactic acid bacteria and coliforms in Brazilian fresh white cheese.

INTRODUCTION: Minas fresh cheese (MFC), a Brazilian white cheese, is one of the most popular cheeses nationwide. Studies have shown that Listeria monocytogenes occurrence in this product is generally low, while high populations of coliforms can be found. This study aimed to evaluate the influence of coliforms in the behavior of L. monocytogenes in MFC. METHODS: Pasteurized milk was inoculated with L. monocytogenes and coliforms, and the acidification was made by lactic acid or by the addition of a starter culture. The cheeses of each production were divided into 3 groups and stored at 5 ºC, 12 ºC and cycles of 5 ºC followed by 25 ºC. In predetermined days, samples were taken and L. monocytogenes, coliforms and lactic acid bacteria populations were evaluated, besides the pH, water activity (aw), titratable acidity and NaCl concentration. RESULTS: The inhibition of L. monocytogenes in the presence of coliforms was observed (p < 0.05), except for those samples prepared with lactic acid and stored at temperature cycles. The values of pH and aw were not sufficiently low to cause inhibition; however, titratable acidity was higher in cheeses containing coliforms. In vitro tests containing lactic acid and L. monocytogenes showed that the bacterium is sensitive to concentration of lactic acid ≥ 0.3%, indicating that lactic acid produced by coliforms strongly influences the population of L. monocytogenes. CONCLUSIONS: Thus, it can be concluded that coliforms negatively impact populations of L. monocytogenes in MFC. We strongly recommend that producers of MFC adopt good hygiene practices to not only avoid contamination with L. monocytogenes, but also coliforms.