Role of milk and meat products as vehicles for antibiotic-resistant bacteria

This subject is reviewed under the following headings: Microbial contamination of raw meat and raw milk; Antibiotic resistance of food-borne pathogens; Antibiotic resistance of commensal and potentially pathogenic bacteria as a new threat in food microbiology; Antibiotic-resistant staphylococci in fermented meat and [in] milk products; Antibiotic-resistant Enterococcus sp. in fermented meat and [in] milk products; Enterococci in farm animals and meat; Enterococci in fermented food; Molecular characterization of resistance of food-borne enterococci; and Further ecological and epidemiological considerations of resistant live bacteria in food. It is concluded that further research is needed, particularly into the possible transfer of the resistance of bacteria consumed in meat or milk products to the indigenous bacteria of the human consumer.

Ascorbate recycling in human neutrophils: Induction by bacteria

Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen Candida albicans. Induction of recycling resulted in as high as a 30-fold increase in intracellular ascorbate compared with neutrophils not exposed to microorganisms. Recycling occurred at physiologic concentrations of extracellular ascorbate within 20 min, occurred over a 100-fold range of effector/target ratios, and depended on oxidation of extracellular ascorbate to dehydroascorbic acid. Ascorbate recycling did not occur in bacteria nor in C. albicans. Ascorbate did not enter microorganisms, and dehydroascorbic acid entry was less than could be accounted for by diffusion. Because microorganism lysates reduced dehydroascorbic acid to ascorbate, ascorbate recycling was absent because of negligible entry of the substrate dehydroascorbic acid. Because ascorbate recycling occurs in human neutrophils but not in microorganisms, it may represent a eukaryotic defense mechanism against oxidants with possible clinical implications.

Acid-fast bacteria and yeasts as indicators of disinfection efficiency /

Mode of access: Internet.

Pathogenic micro-organisms; a text-book of microbiology for physicians and students of medicine,

Mode of access: Internet.

Isolation and characterisation of bacteria associated with flying insects in hospitals, with particualr emphasis on Clostridum difficile

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies.

Evidences for an opportunistic and endophytic lifestyle of the Bursaphelenchus xylophilus -associated bacteria Serratia marcescens PWN146 isolated from wilting Pinus pinaster

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.

Quality of sausage elaborated using minced Nile Tilapia submmitted to cold storage

Filleting yield of Nile tilapia Oreochromis niloticus (L.) is low (30%) and generates large amount of wastes that may turn into environmental and economic problem. However, these wastes can be used for the extraction of minced fish (MF) which can be used in the preparation of sausages. The objective of this study was to assess the quality of sausages prepared with 0, 20, 40, 60, 80 and 100% of MF from Nile tilapia filleting waste during storage at 0±0.3ºC. Alterations in the instrumental color (L*, a* and b*), lipid oxidation (TBARS), total volatile nitrogenous bases (TVB-N), pH, microbiological condition (pathogenic bacteria and aerobic psychrotrophic bacteria), and sensory attributes (color, odor, flavor, texture and overall acceptability) were evaluated for up to 40 days. The addition of MF to sausages increased TBARS values and decreases TVB-N, L*, a* and b* values. Acceptability of color attribute decreased with increasing MF; best flavor, texture and overall acceptability scores were registered for sausages containing 40 and 60% MF; best odor was registered for 100% MF. Pathogenic microorganisms were not detected, but decrease in pH and proliferation of aerobic psychrotrophic bacteria which, however, did not compromise sensory evaluation of sausages were registered throughout storage. Sausages prepared with MF from tilapia filleting waste have a shelf-life of 40 days when stored at 0±0.3ºC, and the maximum recommended MF inclusion to maintain good sensory quality is 60%.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

New genes of Xanthomonas citri subsp citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background: Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. Results: Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands. Conclusion: The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Prevalência de resistência a antimicrobianos em isolados ambientais de Escherichia coli e enterococos

A água é um recurso escasso e indispensável à vida, podendo ser um importante veículo de microrganismos patogénicos com origem fecal. A matéria fecal é também uma fonte de microrganismos resistentes a antimicrobianos e contribui para a sua disseminação e dos seus genes de resistência no ambiente e entre as comunidades microbianas comensais e microrganismos patogénicos humanos e animais. A qualidade microbiológica da água é monitorizada recorrendo à utilização de bioindicadores como Escherichia coli, enterococos e microrganismos totais. O presente estudo apresentou como principal objetivo determinar a prevalência de ESBLs e AmpCs e ainda avaliar a prevalência de estirpes de enterococos com resistência à vancomicina (VRE) em águas do rio Douro e da orla costeira da cidade do Porto. As amostragens de água foram realizadas em quatro locais localizados no estuário do rio Douro e orla costeira da cidade do Porto entre o mês de Abril e Julho. A deteção e quantificação dos bioindicadores foram realizadas pelo método de filtração por membrana. A suscetibilidade das estirpes de E. coli e enterococos foi testada pelo método de difusão em disco relativamente a várias classes de antimicrobianos. As contagens microbianas mais elevadas foram determinadas entre Abril e Junho e em amostras de água doce. Foram isoladas 62 estirpes de E. coli e 49 estirpes de enterococos que apresentaram prevalências de resistência a antimicrobianos de 90,3% (56/62) e 83,7% (41/49), respetivamente. As estirpes de E. coli apresentaram altas frequências de resistência à ampicilina (74,2%) e tetraciclina (61,3%). Nestas estirpes verificou-se ainda fenótipos associados a multirresistência a um mínimo de três classes de antimicrobianos em 56,5% (35/62) dos isolados. Verificou-se que as estirpes de enterococos apresentaram altos níveis de resistência à rifampicina (34,7%) e azitromicina (40,8%), detetando-se ainda a manifestação de fenótipo de resistência à vancomicina em 26,5% das estirpes. Observou-se uma prevalência de 36,7% (18/49) de estirpes de enterococos associadas a fenómenos de multirresistência antimicrobiana. Ana Martins vi Os resultados obtidos sugerem que o rio Douro e orla costeira, bem como os ambientes aquáticos, constituem reservatórios de bactérias e genes de resistência a antimicrobianos e possuem um papel preponderante na sua disseminação.

Iron oxide/gold core/shell nanomagnetic probes and CdS biolabels for amplified electrochemical immunosensing of Salmonella typhimurium

There is an imminent need for rapid methods to detect and determine pathogenic bacteria in food products as alternatives to the laborious and time-consuming culture procedures. In this work, an electrochemical immunoassay using iron/gold core/shell nanoparticles (Fe@Au) conjugated with anti-Salmonella antibodies was developed. The chemical synthesis and functionalization of magnetic and gold-coated magnetic nanoparticles is reported. Fe@Au nanoparticles were functionalized with different self-assembled monolayers and characterized using ultraviolet-visible spectrometry, transmission electron microscopy, and voltammetric techniques. The determination of Salmonella typhimurium, on screen-printed carbon electrodes, was performed by square-wave anodic stripping voltammetry through the use of CdS nanocrystals. The calibration curve was established between 1×101 and 1×106 cells/mL and the limit of detection was 13 cells/mL. The developed method showed that it is possible to determine the bacteria in milk at low concentrations and is suitable for the rapid (less than 1 h) and sensitive detection of S. typhimurium in real samples. Therefore, the developed methodology could contribute to the improvement of the quality control of food samples.

Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.

Routine screening of harmful microorganisms in beach sands: implications to public health

Beaches worldwide provide recreational opportunities to hundreds of millions of people and serve as important components of coastal economies. Beach water is often monitored for microbiological quality to detect the presence of indicators of human sewage contamination so as to prevent public health outbreaks associated with water contact. However, growing evidence suggests that beach sand can harbor microbes harmful to human health, often in concentrations greater than the beach water. Currently, there are no standards for monitoring, sampling, analyzing, or managing beach sand quality. In addition to indicator microbes, growing evidence has identified pathogenic bacteria, viruses, and fungi in a variety of beach sands worldwide. The public health threat associated with these populations through direct and indirect contact is unknown because so little research has been conducted relating to health outcomes associated with sand quality. In this manuscript, we present the consensus findings of a workshop of experts convened in Lisbon, Portugal to discuss the current state of knowledge on beach sand microbiological quality and to develop suggestions for standardizing the evaluation of sand at coastal beaches. The expert group at the "Microareias 2012" workshop recommends that 1) beach sand should be screened for a variety of pathogens harmful to human health, and sand monitoring should then be initiated alongside regular water monitoring; 2) sampling and analysis protocols should be standardized to allow proper comparisons among beach locations; and 3) further studies are needed to estimate human health risk with exposure to contaminated beach sand. Much of the manuscript is focused on research specific to Portugal, but similar results have been found elsewhere, and the findings have worldwide implications.

Diversity and characterisation of coagulasenegative staphylococci isolated from healthy individuals in Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) has significantly increased in human medicine. CoNS are common commensal colonisers of the human skin, although now also recognised as major nosocomial pathogens. Over the last decades, several studies have been carried out in order to understand the pathogenicity mechanisms of CoNS. The well known determinants in the pathogenesis of CoNS infections are their ability to form biofilms and an exceptional resistance to several antibiotics. Nevertheless, there is a lack of studies regarding the commensal lifestyle of these microorganisms. Additionally, it is now hypothesised that commensal bacteria might be a reservoir of pathogenic determinants. Therefore, the work described throughout this thesis was aimed to perform a phenotypic and genotypic characterisation of different CoNS species isolated from healthy Portuguese individuals. A total of 61 CoNS isolates, comprising 7 different species, were obtained and characterised at the level of biofilm formation and antibiotic susceptibility profiles. According to the results, biofilm formation ability and presence of biofilm-associated genes were commonly found features, highlighting their pivotal role in the colonising lifestyle of CoNS. This study also addressed the correlation between phenotypic and genotypic characteristics of biofilm formation, corroborating and raising questions about the importance of some genes in this process. Moreover, it was observed a great proportion of isolates with decreased susceptibility and multiple resistances to some important antibiotics. A significant association between antibiotic resistance and biofilm formation was also demonstrated, and some hypotheses about the nature of such association were provided. Lastly, the expression patterns of two biofilm-associated genes at two distinct biofilm developmental stages were determined, confirming their importance in the accumulative stage of biofilm formation. Overall, the results presented in this thesis indicate that staphylococcal skin flora might be an important reservoir of potentially pathogenic bacteria and, simultaneously, bring to light new perceptions about the molecular basis of staphylococcal biofilm formation, and the nature of the association between antibiotic resistance and biofilm formation.