Lactic acid bacteria secreted proteins as potential Listeria monocytogenes quorum sensing inhibitors.

Listeria monocytogenes is an important human and animal pathogen able to cause an infection named listeriosis and is mainly transmitted through contaminated food. Among its virulence traits, the ability to form biofilms and to survive in harsh environments stand out and lead to the persistence of L. monocytogenes for long periods in food processing environments. Virulence and biofilm formation are phenotypes regulated by quorum sensing (QS) and, therefore, the control of L. monocytogenes through an anti-QS strategy is promising. This study aimed to identify, by in silico approaches, proteins secreted by lactic acid bacteria (LAB) potentially able to interfere with the agr QS system of L. monocytogenes. The genome mining of Lacticaseibacillus rhamnosus GG and Lactobacillus acidophilus NCFM revealed 151 predicted secreted proteins. Concomitantly, the three-dimensional (3D) structures of AgrB and AgrC proteins of L. monocytogenes were modeled and validated, and their active sites were predicted. Through protein-protein docking and molecular dynamic, Serine-type D-Ala-D-Ala carboxypeptidase and L,D-transpeptidase, potentially secreted by L. rhamnosus GG and L. acidophilus NCFM, respectively, were identified with high affinity to AgrB and AgrC proteins, respectively. By inhibiting the translocation of the cyclic autoinducer peptide (cyclic AIP) via AgrB, and its recognition in the active site of AgrC, these LAB proteins could disrupt L. monocytogenes communication by impairing the agr QS system. The application of the QS inhibitors predicted in this study can emerge as a promising strategy in controlling L. monocytogenes in food processing environment and as an adjunct to antibiotic therapy for the treatment of listeriosis.

Microbiology of street food: understanding risks to improve safety.

Street foods play important socioeconomic and cultural roles and are popular worldwide. In addition to providing convenient and low-cost meals for urban populations, street food offers an essential source of income for vendors, especially women, and it can reflect traditional local culture, which is an important attraction for tourists. Despite these benefits, the microbiological safety of street food has become a worldwide concern because it is often prepared and sold under inadequate safety conditions, without legal control and sanitary surveillance. Consequently, high counts of fecal indicator bacteria and several foodborne pathogens have been detected in street foods. This review provides insight into the microbiology of street food, focus on the associated microbiological safety aspects and main pathogens, and the global status of this important economic activity. Furthermore, the need to apply molecular detection rather than traditional culture-based methods is discussed to better understand the actual risks of microbial infection associated with street foods. Recognition is always the first step toward addressing a problem.

Effect of sub-inhibitory concentrations of antibiotics on biofilm formation and expression of virulence genes in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis.

Biofilm formation is a key factor in the pathogenesis of enterococcal infections. Thus, the biofilm-forming ability and frequency of biofilm-related genes in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) compared to penicillin- and ampicillin-susceptible E. faecalis (PSASEF) were assessed in the present study. In addition, the effect of sub-inhibitory concentrations (sub-MICs) of antibiotics on biofilm formation and expression of virulence genes was evaluated. Twenty PRASEF and 21 PSASEF clinical isolates were used to determine the effect of sub-MICs of antibiotics (ampicillin, penicillin, and gentamicin) on biofilm formation, and ten selected isolates were subjected to RT-qPCR to detect the transcript levels of virulence genes (efaA, asa1, esp, and ace). Antibiotic susceptibility was evaluated by the microdilution broth method. Biofilm formation assay was performed using the microtiter plate method. All PSASEF and PRASEF isolates produced biofilms in vitro. Most isolates had three or four virulence genes. Sub-MICs of ampicillin significantly decreased biofilm production and expression of ace and asa1 genes, although the transcript levels were significantly lower (-350% and -606.2%, respectively) among the PSASEF isolates only. Sub-MICs of gentamicin did not have any significant effect on biofilm formation, but slightly increased the transcript levels of efaA. In conclusion, this study showed that the biofilm-forming ability and frequency of the evaluated virulence genes were similar among the PRASEF and PSASEF isolates. Further, in vitro antibiotic sub-MICs were confirmed to interfere with the expression pattern of virulence genes and biofilm formation by E. faecalis. However, further studies are required to clarify the role of sublethal doses of antibiotics on enterococcal biofilms.

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6')Ie-aph(2″)Ia and aph(3')-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6')-Ie-aph(2″)-Ia or aph(3')-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis isolates are uncommon in non-clinical sources.

This study aimed to investigate whether penicillin-resistant, ampicillin-susceptible E. faecalis (PRASEF) isolates are disseminated in non-clinical sources, and to compare the molecular characteristics and antimicrobial resistance (AMR) profile of clinical and non-clinical E. faecalis isolates. Non-clinical samples (n = 280) were collected and 101 E. faecalis isolates were recovered from food (n = 18), faeces of healthy animals (n = 24), water (n = 28) and sewage (n = 31). PRASEF (n = 68) and penicillin-susceptible, ampicillin-susceptible E. faecalis (n = 77) isolates of clinical origin were also evaluated. A significant variety of AMR profiles was observed among non-clinical isolates according to the source. No food isolate exhibited a multidrug resistance (MDR) phenotype different from those of isolates from animal faeces (50.0%) and sewage (38.7%). Overall, the MDR phenotype was more frequent among clinical (56.6%) than non-clinical isolates (22.8%) (p < 0.01). Non-clinical PRASEF isolates (n = 3) were only recovered from hospital sewage. Note that representative clinical and non-clinical PRASEF isolates were grouped in pulsotype A, and belonged to CC9 (clonal complex). In conclusion, E. faecalis isolates exhibiting the unusual penicillin-resistant but ampicillin-susceptible phenotype appeared to be restricted to the hospital environment. Our findings highlight the ability of PRASEF isolates to survive in sewage, which could enable these hospital-adapted lineages to spread to new ecological niches.

Estratégias para controle de infeccção hospitalar causada por Enterococcus Vancomicina-resistentes: uma revisão integrativa / Strategies for the control of hospital infections caused by Vancomycin-resistant Enterococcus: an integrative review / Estrategias para controlar la infección del hospital causada por Enterococcus resistente a la Vancomicina: una revisión integrativa

Objetivo: avaliar a importância dos Enterococcus resistentes à Vancomicina (VRE) como agentes de infecções relacionadas à assistência à saúde (IRAS) e identificar as estratégias nacionais e internacionais usadas no controle de IRAS por VREs. Método: revisão integrativa da literatura. Seleção de 18 artigos que tratavam da importância de IRAS por VREs e das estratégias de controle. Resultados: observou-se concordância entre os artigos em relação às maneiras de minimizar os índices de IRAS por VREs. A ocorrência de IRAS é permissiva quando medidas para higiene são inadequadas. Ademais, treinamentos acerca do uso de antimicrobianos, culturas de vigilância e estruturação de uma comissão especializada são estratégias para redução de casos e que devem ser seguidas para contingência de VREs como agentes de IRAS. Conclusão: ações preventivas são a maneira mais eficiente de reduzir os casos de IRAS por VREs. É imprescindível padronizar e controlar o uso de antimicrobianos visando à prevenção da seleção de linhagens resistentes. Ainda, faz-se necessário manter ativas estratégias administrativas para controle de IRAS e atualização dos profissionais técnicos e da limpeza que atuam nos serviços de saúde, possibilitando educação continuada acerca de todos os tópicos que envolvam o controle dos casos.(AU)

Objective: to evaluate the importance of Vancomycin-resistant Enterococcus (VREs) as agents of Health care-associated infections (HAIs) and to identify national and international strategies used in the control of HAIs by VREs. Method: this is an integrative literature review. We selected 18 articles that dealt with the importance of HAIs by VREs and the control strategies. Results: we noted an agreement among the articles regarding ways to minimize the rates of HAIs by VREs. The occurrence of HAIs is permissive when hygiene measures are inadequate. Furthermore, training on the use of antimicrobials, surveillance cultures, and the structuring of a specialized committee are strategies to reduce cases and should be followed for the contingency of VREs as agents of HAIs. Conclusion: preventive actions are the most efficient way to reduce cases of HAIs by VREs. It is essential to standardize and control the use of antimicrobials, in order to prevent the selection of resistant strains. Furthermore, it is necessary to keep active administrative strategies for the control of HAIs and to update the technical and cleaning professionals who work in health services, enabling continuing education on all topics involving case control.(AU)

Objetivo: evaluar la importancia de los Enterococcus resistentes a la Vancomicina (VREs) como agentes de infecciones asociadas a la atención sanitaria (IAAS) e identificar las estrategias nacionales e internacionales utilizadas para controlar las IAAS causadas por los VREs. Método: revisión integradora de la literatura. Se seleccionaron 18 artículos que trataban sobre la importancia de las IAAS por los VREs y las estrategias de control. Resultados: se observó una concordancia entre los artículos sobre las formas de minimizar las tasas de IAAS por VREs. La aparición de IAAS es permisible cuando las medidas de higiene son inadecuadas. Además, los tratamientos sobre el uso de antimicrobianos, las culturas de vigilancia y la estructuración de una comisión especializada son estrategias para reducir los casos y que deben seguirse para la contingencia de los VREs como agentes de IAAS. Conclusión: las acciones preventivas son la forma más eficiente de reducir los casos de IAAS por VREs. Es imprescindible programar y controlar el uso de antimicrobianos para prevenir la aparición de líneas resistentes. Asimismo, es necesario mantener activas las estrategias administrativas para el control de las IAAS y la actualización de los profesionales técnicos y de limpieza que trabajan en los servicios sanitarios, permitiendo la formación continua en todos los temas que involucran el control de casos.(AU)

Infecção e fatores preditivos de óbito em pacientes queimados atendidos em um hospital universitário na cidade de Uberaba/MG / Infection and predicting factors of death in burnt patients attended in a university hospital in the city of Uberaba/MG

OBJETIVO: Avaliar a ocorrência de infecções e as variáveis associadas a óbito entre pacientes queimados atendidos em um hospital universitário na cidade de Uberaba/MG. MÉTODO: Estudo retrospectivo realizado por análise dos prontuários de pacientes queimados (2° e/ou 3° graus) internados entre janeiro de 2013 e janeiro de 2019. Foram avaliados tempo de internação, internação em unidade de terapia intensiva (UTI), uso de antibióticos, superfície corporal queimada (SCQ), infecções, vias de acesso e comorbidades anteriores. Os dados foram tabulados no Microsoft® Excel e analisados por IBM SPSS statistics 21 e GraphPad Prism 7.0 (p<0.05). RESULTADOS: Foram avaliados 168 prontuários de pacientes com idades entre 0,12 e 84 anos (p=0,58). A ocorrência de infecção foi correlacionada a um risco relativo (RR) de quase seis vezes maior de óbito (RR=5,96; p=0,003). Pacientes com SCQ superior a 50% apresentaram RR cerca de 18 vezes maior de óbito (RR=18,33; p<0,0001). Demonstrou-se também aumento do número de óbitos em pacientes com mais de três acessos (RR=35,53; p <0,0001) e associação de óbitos com os custos hospitalares de internação (p=0,0018). Raça, sexo, tempo de hospitalização, hospitalização em UTI, comorbidades, uso de antibiótico e grau de queimadura não interferiram na sobrevida dos pacientes avaliados. CONCLUSÕES: A incidência de infecções entre os pacientes queimados avaliados apresentou relação positiva com óbito, bem como a SCQ, o número de acessos utilizados e os gastos hospitalares. Atenção especial deve ser dada ao controle de infecções, visto ser esta uma variável possível de alteração mediante esforços e cuidados da equipe multiprofissional responsável por esses pacientes.

OBJECTIVE: To evaluate the occurrence of infections and the variables associated with death among burn patients treated at a university hospital in the city of Uberaba/MG. METHODS: Retrospective study performed by analyzing the medical records of burned patients (2nd and/or 3rd degrees) hospitalized between January 2013 and January 2019. Length of stay, intensive care unit (ICU) stay, use of antibiotics, burned body surface (BBS), infections, access routes and previous comorbidities were evaluated. The data were tabulated in Microsoft® Excel and analyzed by IBM SPSS statistics 21 and GraphPad Prism 7.0 (p<0.05). RESULTS: 168 medical records of patients aged 0.12 to 84 years (p=0.58) were evaluated. The occurrence of infection was correlated with a relative risk (RR) of almost six times higher of death (RR=5.96; p=0.003). Patients with BBS greater than 50% had about 18 times higher risk of death (RR=18.33; p <0.0001). There was also an increase in the number of deaths in patients with more than three accesses (RR=35.53; p <0.0001) and an association of deaths with hospitalization costs (p=0.0018). Race, sex, length of hospital stay, ICU, stay comorbidities, use of antibiotics and degree of burn did not interfere with the survival of the evaluated patients. CONCLUSIONS: The incidence of infections among the burn patients evaluated showed a positive relationship with death, as well as BBS, the number of accesses used and hospital expenses. Special attention should be given to infection control, as this is a variable that can be controlled through the efforts and care of the multidisciplinary team responsible for these patients.

Safety evaluation of the antimicrobial peptide bovicin HC5 orally administered to a murine model.

BACKGROUND: Bovicin HC5 is an antimicrobial peptide that shows a broad spectrum of activity and potential for biotechnological and therapeutic applications. To gain insight about the safety of bovicin HC5 application, the histological and immunostimulatory effects of orally administrated bovicin HC5 to BALB/c mice were evaluated. BALB/c mice were divided into three groups: negative control (NC group); mice given purified bovicin HC5 (Bov group); mice given ovalbumin (positive control, PC group; a murine model of enteropathy). The mice were initially pre-sensitized, and PBS, bovicin HC5 or ovalbumin were administered for 30 days by daily gavages. Histological and morphometric analysis were performed and the relative expression of cytokines was analyzed by real-time RT-PCR. RESULTS: The oral administration of bovicin HC5 to BALB/c mice reduced weight gain and caused alterations in the small intestine, although absorptive changes have not been detected. The number of total goblet cells and the mucopolysaccharides production were not affected by bovicin HC5 administration. A hypertrophy of Paneth cells and an increase in the number of mitotic cells were observed in Bov group, while the number of mast cells remained unaltered. Increased expression of TNF-α, INF-γ and IL-12 was observed in the small intestine upon bovicin HC5 administration. CONCLUSION: Bovicin HC5 has only minor effects on intestinal permeability and did not elicit an allergenic response upon oral administration to animal models. Considering the low in vivo toxicity of bovicin HC5, it might be a good candidate for enteral applications.

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

Interaction with lipid II induces conformational changes in bovicin HC5 structure.

Bovicin HC5 is a lantibiotic produced by Streptococcus bovis HC5 that targets the cell wall precursor lipid II. An understanding of the modes of action against target bacteria can help broadening the clinical applicability of lantibiotics in human and veterinary medicine. In this study, the interaction of bovicin HC5 with lipid II was examined using tryptophan fluorescence and circular dichroism spectroscopy with model membrane systems that do or do not allow pore formation by bovicin HC5. In the presence of lipid II, a blue-shift of 12 nm could be observed for the fluorescence emission maximum of the tryptophan residue for all of the membrane systems tested. This change in fluorescence emission was paralleled by a decrease in accessibility toward acrylamide and phospholipids carrying a spin-label at the acyl chain; the tryptophan residue of bovicin HC5 was located near the twelfth position of the membrane phospholipid acyl chains. Moreover, the binding of lipid II by bovicin HC5 induced remarkable conformational changes in the bovicin HC5 structure. The interaction of bovicin HC5 with lipid II was highly stable even at pH 2.0. These results indicate that bovicin HC5 interacts directly with lipid II and that the topology of this interaction changes under different conditions, which is relevant for the biological activity of the peptide. To our knowledge, bovicin HC5 is the only bacteriocin described thus far that is able to interact with its target in extreme pH values, and this fact might be related to its unique structure and stability.

Role of lipid II and membrane thickness in the mechanism of action of the lantibiotic bovicin HC5.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria, nisin being the most well-known member. Nisin inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon interaction with lipid II, the essential bacterial cell wall precursor. Bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II binding motif, and we investigated the mode of action of bovicin HC5 using both living bacteria and model membranes, with special emphasis on the role of lipid II. Bovicin HC5 showed activity against Staphylococcus cohnii and Staphylococcus warneri, but bovicin HC5 hardly interfered with the membrane potential of S. cohnii. In model membranes, bovicin HC5 was not able to cause carboxyfluorescein release or proton influx from DOPC vesicles containing lipid II. Bovicin HC5 blocked lipid II-dependent pore formation activity of nisin, and a high-affinity interaction with lipid II was observed (apparent binding constant [K(a)] = 3.1 × 10(6) M(-1)), with a 1:1 stoichiometry. In DOPC vesicles containing lipid II, bovicin HC5 was able to assemble with lipid II into a prepore-like structure. Furthermore, we observed pore formation activity of bovicin HC5, which was stimulated by the presence of lipid II, in thin membranes. Moreover, bovicin HC5 induced the segregation of lipid II into domains in giant model membrane vesicles. In conclusion, bovicin HC5 has a primary mode of action similar to that of nisin, but some differences regarding the pore-forming capacity were demonstrated.