Avaliação microbiológica, detecção e susceptibilidade a antimicrobianos de potenciais enteropatógenos das famílias Enterobacteriaceae e Vibrionaceae em mexilhões Perna perna da praia de Itaipu, Niterói-RJ

Por serem organismos filtradores, os mexilhões devem ser extraídos para consumo somente de águas com padrões microbiológicos regulamentados pelo Conselho Nacional do Meio Ambiente, uma vez que intoxicações alimentares de origem bacteriana são as consequências mais comuns relacionadas ao consumo destes moluscos. Após a retirada dos costões, estes organismos passam por processos de fervura, lavagem, acondicionamento em sacos plásticos e transporte até a chegada ao mercado onde são comercializados; etapas estas, realizadas sem cuidados assépticos. Objetivando avaliar a qualidade microbiológica de mexilhões Perna perna coletados na praia de Itaipu, Niterói, RJ; após a sua fervura; e comercializados no Mercado São Pedro, foram realizadas contagens de coliformes totais e termotolerantes e pesquisa de Escherichia coli, Salmonella spp. e Vibrio spp. por metodologia convencional e molecular em amostras obtidas destes locais. Pelo teste de disco-difusão foi observado o perfil de resistência das cepas isoladas. Do total de amostras analisadas (27) apenas 3 dos mexilhões que sofreram processo de aferventação, 1 do comercializado e 3 do in natura, se encontravam dentro dos padrões aceitáveis pela legislação. Não houve diferença significativa entre as contagens de coliformes termotolerantes dos diferentes mexilhões analisados, mas sim entre os períodos seco e chuvoso. Somente uma das nove coletas de água mostrou-se própria para o cultivo de mexilhões (até 14 CF/mL). Foram isoladas e caracterizadas fisiológicamente, 77 estirpes da espécie E. coli, sendo confirmadas molecularmente por PCR os sorotipos EPEC, STEC e EAEC; 4 cepas Salmonella spp. sendo apenas uma confirmada por PCR; e das 57 cepas caracterizadas como Vibrio spp., 51 foram confirmadas por PCR, sendo 46 Vibrio spp., 2 V. cholerae, 1 V. vulnificus, 1 V. parahaemolyticus e 1 V. mimicus. Entre as estirpes de E. coli, 13% apresentaram multirresistência e 15,6% apresentaram resistência múltipla. A estirpe de Salmonella spp. se mostrou sensível a todos os antimicrobiados testados. Das estirpes de Vibrio spp. testadas, 68,6% apresentaram multirresistência e 72,5% apresentaram resistência múltipla. A partir da pesquisa de genes direto do caldo de enriquecimento foi possível detectar todos os genes pesquisados, com exceção para os sorotipos de Salmonella e V. cholerae. Baseado nos resultados do presente trabalho pode-se inferir que os mexilhões, amplamente comercializados no município de Itaipú, podem se constituir em risco para a saúde pública dos consumidores no Rio de Janeiro, necessitando dos órgãos competentes uma eficiente fiscalização nos pontos de venda e cultivo destes moluscos.

Antibacterial effect of the Indian squid, Loligo duvauceli (d'Orbigny), ink

The ink of the Indian squid Loligo duvauceli (d'Orbigny) was tested for antibacterial activity. The antibacterial effect of bacteria present in the ink gland was also tested. Only one type of bacteria was found to be present in the ink gland of squid and was identified as Photobacterium leiognathi. Among the various forms of ink extracts, the precipitated and freeze-dried ink showed more pronounced antibacterial effect against Gram-negative bacteria, Salmonella, spp. Escherichia coli, Vibrio cholerae, V. parahaemolyticus and Pseudoinonas spp., and a less pronounced effect against Gram-positive bacteria, Staphylococcus spp. and Micrococcus spp., P. leiognathi did not inhibit any of the above bacteria. The antibacterial activity was associated with the compounds of the ink.

Bacterial quality and depuration of the green mussel Perna viridis from natural beds

Green mussel (Perna viridis) and sea water from their natural beds on the coastal areas of Porto Novo were studied between April and August 1996 for their bacterial quality. Water samples from the beds were also analysed for their physico-chemical parameters. The total bacterial count of mussels from natural beds as well as bed waters ranged 10³ organisms per gram of mussel meat suspension and per milliliter of sea water. The faecal coliforms were found to be within the permissible limits. Pathogenic bacteria such as Salmonella spp., Streptococcus spp. and Staphylococcus spp. were absent. The variations in pH, temperature, salinity and dissolved oxygen of the seawater samples were insignificant. The mussels were subjected to depuration by different methods among which chlorination was found to be most effective.

Studies on the occurrence of enteric bacteria in the estuarine waters along the Mangalore coast

Occurrence of enteric bacteria in water, sediment and shellfishes of Mulki, Pavanje, Gurpur and Netravathi estuaries of the Mangalore coast is reported. 70 water samples, 71 sediment samples and 37 shellfish samples were analysed in 18 months. Total bacterial load in sediment and shellfishes was found to be more than that in water samples. The total bacterial load was not very high. However, enterococci, particularly coliforms in sediments, water and shellfishes were found to be quite high, indicative of faecal pollution. The incidence of Salmonella spp. was recorded in all the estuaries except the Mulki estuary.

Microbiological and physicochemical aspects of “salpicão”, a traditional dry sausage produced in the northeast of Portugal

Counts of total viable mesophilic bacteria (TVC), lactic acid bacteria (LAB), Microccocaceae, Enterobacteriaceae, Salmonella spp. and Listeria monocytogenes, in traditional Portuguese dry sausages from two industrial producers, were compared in batter and final product. During the production process, the TVC increased significantly, most likely due to the multiplication of fermentative flora. Enterobacteriaceae decreased from batter to final product while the S. aureus increased. Great variability was verified in detection of L. monocytogenes both between batches and industrial producers

Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae

Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.

The biology of habitat dominance; can microbes behave as weeds?

Competition between microbial species is a product of, yet can lead to a reduction in, the microbial diversity of specific habitats. Microbial habitats can resemble ecological battlefields where microbial cells struggle to dominate and/or annihilate each other and we explore the hypothesis that (like plant weeds) some microbes are genetically hard-wired to behave in a vigorous and ecologically aggressive manner. These 'microbial weeds' are able to dominate the communities that develop in fertile but uncolonized - or at least partially vacant - habitats via traits enabling them to out-grow competitors; robust tolerances to habitat-relevant stress parameters and highly efficient energy-generation systems; avoidance of or resistance to viral infection, predation and grazers; potent antimicrobial systems; and exceptional abilities to sequester and store resources. In addition, those associated with nutritionally complex habitats are extraordinarily versatile in their utilization of diverse substrates. Weed species typically deploy multiple types of antimicrobial including toxins; volatile organic compounds that act as either hydrophobic or highly chaotropic stressors; biosurfactants; organic acids; and moderately chaotropic solutes that are produced in bulk quantities (e.g. acetone, ethanol). Whereas ability to dominate communities is habitat-specific we suggest that some microbial species are archetypal weeds including generalists such as: Pichia anomala, Acinetobacter spp. and Pseudomonas putida; specialists such as Dunaliella salina, Saccharomyces cerevisiae, Lactobacillus spp. and other lactic acid bacteria; freshwater autotrophs Gonyostomum semen and Microcystis aeruginosa; obligate anaerobes such as Clostridium acetobutylicum; facultative pathogens such as Rhodotorula mucilaginosa, Pantoea ananatis and Pseudomonas aeruginosa; and other extremotolerant and extremophilic microbes such as Aspergillus spp., Salinibacter ruber and Haloquadratum walsbyi. Some microbes, such as Escherichia coli, Mycobacterium smegmatis and Pseudoxylaria spp., exhibit characteristics of both weed and non-weed species. We propose that the concept of nonweeds represents a 'dustbin' group that includes species such as Synodropsis spp., Polypaecilum pisce, Metschnikowia orientalis, Salmonella spp., and Caulobacter crescentus. We show that microbial weeds are conceptually distinct from plant weeds, microbial copiotrophs, r-strategists, and other ecophysiological groups of microorganism. Microbial weed species are unlikely to emerge from stationary-phase or other types of closed communities; it is open habitats that select for weed phenotypes. Specific characteristics that are common to diverse types of open habitat are identified, and implications of weed biology and open-habitat ecology are discussed in the context of further studies needed in the fields of environmental and applied microbiology.

Phage Display-Derived Binders Able to Distinguish Listeria monocytogenes from Other Listeria Species

The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries. © 2013 Morton et al.

Validation of a high-throughput immunobead array technique for multiplex detection of three foodborne pathogens in chicken products

This study rigorously evaluated a previously developed immunobead array method to simultaneously detect three important foodborne pathogens, Campylobacter jejuni, Listeria monocytogenes, and Salmonella spp., for its actual application in routine food testing. Due to the limitation of the detection limit of the developed method, an enrichment step was included in this study by using Campylobacter Enrichment Broth for C. jejuni and Universal Pre-enrichment Broth for L. monocytogenes and Salmonella spp.. The findings show that the immunobead array method was capable of detecting as low as 1 CFU of the pathogens spiked in the culture media after being cultured for 24 hours for all three pathogens. The immunobead array method was further evaluated for its pathogen detection capabilities in ready-to-eat (RTE) and ready-to-cook (RTC) chicken samples and proven to be able to detect as low as 1 CFU of the pathogens spiked in the food samples after being cultured for 24 hours in the case of Salmonella spp., and L. monocytogenes and 48 hours in the case of C. jejuni. The method was subsequently validated with three types of chicken products (RTE, n=30; RTC, n=20; raw chicken, n=20) and was found to give the same results as the conventional plating method. Our findings demonstrated that the previously developed immunobead array method could be used for actual food testing with minimal enrichment period of only 52 hours, whereas the conventional ISO protocols for the same pathogens take 90-144 hours. The immunobead array was therefore an inexpensive, rapid and simple method for the food testing.

Development of infrared spectroscopy for assessing bacterial quality in foods

Rapid and specific detection of foodborne bacteria that can cause food spoilage or illness associated to its consumption is an increasingly important task in food industry. Bacterial detection, identification, and classification are generally performed using traditional methods based on biochemical or serological tests and the molecular methods based on DNA or RNA fingerprints. However, these methodologies are expensive, time consuming and laborious. Infrared spectroscopy is a reliable, rapid, and economic technique which could be explored as a tool for bacterial analysis in the food industry. In this thesis it was evaluated the potential of IR spectroscopy to study the bacterial quality of foods. In Chapter 2, it was developed a calibration model that successfully allowed to predict the bacterial concentration of naturally contaminated cooked ham samples kept at refrigeration temperature during 8 days. In this part, it was developed the methodology that allowed the best reproducibility of spectra from bacteria colonies with minimal sample preparation, which was used in the subsequent work. Several attempts trying different resolutions and number of scans in the IR were made. A spectral resolution of 4 cm-1, with 32 scans were the settings that allowed the best results. Subsequently, in Chapter 3, it was made an attempt to identify 22 different foodborne bacterial genera/species using IR spectroscopy coupled with multivariate analysis. The principal component analysis, used as an exploratory technique, allowed to form distinct groups, each one corresponding to a different genus, in most of the cases. Then, a hierarchical cluster analysis was performed to further analyse the group formation and the possibility of distinction between species of the same bacterial genus. It was observed that IR spectroscopy not only is suitable to the distinction of the different genera, but also to differentiate species of the same genus, with the simultaneous use of principal component analysis and cluster analysis techniques. The utilization of IR spectroscopy and multivariate statistical analysis were also investigated in Chapter 4, in order to confirm the presence of Listeria monocytogenes and Salmonella spp. isolated from contaminated foods, after growth in selective medium. This would allow to substitute the traditional biochemical and serological methods that are used to confirm these pathogens and that delay the obtainment of the results up to 2 days. The obtained results allowed the distinction of 3 different Listeria species and the distinction of Salmonella spp. from other bacteria that can be mistaken with them. Finally, in chapter 5, high pressure processing, an emerging methodology that permits to produce microbiologically safe foods and extend their shelf-life, was applied to 12 foodborne bacteria to determine their resistance and the effects of pressure in cells. A treatment of 300 MPa, during 15 minutes at room temperature was applied. Gram-negative bacteria were inactivated to undetectable levels and Gram-positive showed different resistances. Bacillus cereus and Staphylococcus aureus decreased only 2 logs and Listeria innocua decreased about 5 logs. IR spectroscopy was performed in bacterial colonies before and after HPP in order to investigate the alterations of the cellular compounds. It was found that high pressure alters bands assigned to some cellular components as proteins, lipids, oligopolysaccharides, phosphate groups from the cell wall and nucleic acids, suggesting disruption of the cell envelopes. In this work, bacterial quantification and classification, as well as assessment of cellular compounds modification with high pressure processing were successfully performed. Taking this into account, it was showed that IR spectroscopy is a very promising technique to analyse bacteria in a simple and inexpensive manner.

Avaliação da qualidade microbiológica e da atividade antimicrobiana de água-mel

A produção de água-mel é conhecida em alguns países do Mediterrâneo como Itália e Portugal como uma prática antiga por parte dos apicultores. A água-mel resulta do aproveitamento de mel, própolis e poléns das colmeias após a extração do mel ao qual é adicionado água e sujeita a tratamento térmico prolongado. Conhecida na doçaria regional como ingrediente para bolos ou simplesmente como doce, a água-mel também é retratada pelos populares como remédio para doenças do trato respiratório e tratamento de feridas. Neste trabalho de dissertação o principal objetivo foi verificar a qualidade microbiológica da água-mel assim como a existência de atividade antimicrobiana perante alguns microrganismos de interesse para a saúde humana. As 21 amostras de água-mel foram analisadas em relação aos parâmetros de qualidade microbiológica que incluíram a contagem de aeróbios totais, bolores e leveduras bactérias da família Enterobacteriaceae, a presença de Salmonella spp e esporos de clostrideos sulfito-redutores, no qual todas as amostras apresentaram valores negativos, à exceção de uma amostra que apresentou um valor de microrganismos aeróbios de 3,41±0,09 Log10 UFC/g e de esporos de clostrideos sulfito-redutores o valor foi de 4,05±0,11 Log10 UFC/g. Na análise da atividade antimicrobiana, foram selecionadas quatro amostras de água-mel (1B2010, 1B2011, 1F2011 e 1H2011) baseadas nos resultados obtidos na análise físico-química. Foram testadas diferentes concentrações de água-mel, nomeadamente 20%, 30%, 40% e 50% (p/v) no crescimento de três estirpes de bactérias Gram negativas, oito bactérias Gram positivas e duas leveduras. Após cálculo das percentagens de inibição observou-se inibição de todas as estirpes testadas à exceção da levedura Saccharomyces cerevisiae que se demonstrou menos suscetível (P<0,05). Podemos concluir que a água-mel é um produto seguro para consumo humano do ponto de vista microbiológico e apresenta benefícios para a saúde humana pela sua capacidade de inibir o crescimento de agentes patogénicos.

Antimicrobial activity of essential oils isolated from Portuguese endemic species of Thymus

Aims: Thymus species are wild species mostly found in the arid lands of Portugal. Possible antimicrobial properties of Thymus essential oils have been investigated. The chemical composition of the essential oils and the antimicrobial activity of Thymus mastichina (L) L. subsp. mastichina , T. camphoratus and T. lotocephalus from different regions of Portugal were analysed. Methods and Results: Hydrodistillation was used to isolate the essential oils and the chemical analyses were performed by gas chromatography (GC) and GC coupled to mass spectrometry. The antimicrobial activity was tested by the disc agar diffusion technique against Candida albicans , Escherichia coli , Listeria monocytogenes , Proteus mirabilis , Salmonella spp. and Staphylococcus aureus . Pure linalool, 1,8-cineole and a mixture (1:1) of these compounds were included. Linalool, 1,8-cineole or linalool/1,8-cineole and linalool/1,8-cineole/linalyl acetate were the major components of the essential oils, depending on the species or sampling place. The essential oils isolated from the Thymus species studied demonstrated antimicrobial activity but the micro-organisms tested had significantly different sensitivities. Conclusions: The antimicrobial activity of essential oils may be related to more than one component. Significance and Impact of the Study: Portuguese endemic species of Thymus can be used for essential oil production for food spoilage control, cosmetics and pharmaceutical use. Further studies will be required to elucidate the cell targets of the essential oil components.

Qualidade nutricional e microbiológica de linguiça produzida artesanalmente em Cavo Verde

Dissertação de mestrado, Gestão da Qualidade e Marketing Agro-Alimentar, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

La salmonellose chez les bovins laitiers : présentation clinique et culture bactériologique

Huit cent trente et un troupeaux de vaches laitières répartis dans 5 états américains ont été enrôlés dans une étude de cohorte prospective. Un modèle d’équations d'estimation généralisées a été utilisé pour étudier l'association entre les signes cliniques et la détection de salmonelles dans les fèces des animaux soupçonnés de salmonellose clinique. La sensibilité et la spécificité de la culture bactériologique ont été estimées à l’aide d’un modèle de classes latentes. Dix-huit pour cent des 874 échantillons provenant de veaux et 29% des 1479 échantillons de vaches adultes étaient positifs pour Salmonella spp. Il n’a pas été possible d’établir une association claire entre les différents signes cliniques observés et la détection de salmonelles. Les 2 sérotypes les plus fréquemment isolés étaient Typhimurium et Newport. La probabilité de détecter des salmonelles était plus élevée chez les veaux où un autre agent entéropathogène était également détecté. La proportion d’échantillons positifs était plus élevée parmi les vaches ayant reçu des antibiotiques dans les jours précédant l’échantillonnage. La sensibilité de la culture a été estimée à 0,48 (intervalle de crédibilité à 95% [ICr95%]: 0,22-0,95) pour les veaux et 0,78 (ICr95%: 0,55-0,99) pour les vaches. La spécificité de la culture était de 0,94 (ICr95%: 0,87-1,00) pour les veaux et de 0,96 (ICr95%: 0,90-1,00) pour les vaches. Malgré une sensibilité imparfaite, la culture bactériologique demeure utile pour obtenir une meilleure estimation de la probabilité post-test de salmonellose clinique chez un bovin laitier, par rapport à la probabilité estimée suite au seul examen clinique.

Horizontal nutrient fluxes and production efficiencies in urban and peri-urban crop and livestock husbandry of Niamey, Niger

Urban and peri-urban agriculture (UPA) increasingly supplies food and non-food values to the rapidly growing West African cities. However, little is known about the resource use efficiencies in West African small-scale UPA crop and livestock production systems, and about the benefits that urban producers and retailers obtain from the cultivation and sale of UPA products. To contribute to filling this gap of knowledge, the studies comprising this doctoral thesis determined nutrient use efficiencies in representative urban crop and livestock production system in Niamey, Niger, and investigated potential health risks for consumers. Also assessed was the economic efficiency of urban farming activities. The field study, which was conducted during November 2005 to January 2008, quantified management-related horizontal nutrient flows in 10 vegetable gardens, 9 millet fields and 13 cattle and small ruminant production units. These farms, selected on the basis of a preceding study, represented the diversity of UPA crop and livestock production systems in Niamey. Based on the management intensity, the market orientation and especially the nutrient input to individual gardens and fields, these were categorized as high or low input systems. In the livestock study, high and low input cattle and small ruminant units were differentiated based on the amounts of total feed dry matter offered daily to the animals at the homestead. Additionally, economic returns to gardeners and market retailers cultivating and selling amaranth, lettuce, cabbage and tomato - four highly appreciated vegetables in Niamey were determined during a 6-months survey in forty gardens and five markets. For vegetable gardens and millet fields, significant differences in partial horizontal nutrient balances were determined for both management intensities. Per hectare, average annual partial balances for carbon (C), nitrogen (N), phosphorus (P) and potassium (K) amounted to 9936 kg C, 1133 kg N, 223 kg P and 312 kg K in high input vegetable gardens as opposed to 9580 kg C, 290 kg N, 125 kg P and 351 kg K in low input gardens. These surpluses were mainly explained by heavy use of mineral fertilizers and animal manure to which irrigation with nutrient rich wastewater added. In high input millet fields, annual surpluses of 259 kg C ha-1, 126 kg N ha-1, 20 kg P ha-1 and 0.4 kg K ha-1 were determined. Surpluses of 12 kg C ha-1, 17 kg N ha-1, and deficits of -3 kg P ha-1 and -3 kg K ha-1 were determined for low input millet fields. Here, carbon and nutrient inputs predominantly originated from livestock manure application through corralling of sheep, goats and cattle. In the livestock enterprises, N, P and K supplied by forages offered at the farm exceeded the animals’ requirements for maintenance and growth in high and low input sheep/goat as well as cattle units. The highest average growth rate determined in high input sheep/goat units was 104 g d-1 during the cool dry season, while a maximum average gain of 70 g d-1 was determined for low input sheep/goat units during the hot dry season. In low as well as in high input cattle units, animals lost weight during the hot dry season, and gained weight during the cool dry season. In all livestock units, conversion efficiencies for feeds offered at the homestead were rather poor, ranging from 13 to 42 kg dry matter (DM) per kg live weight gain (LWG) in cattle and from 16 to 43 kg DM kg-1 LWG in sheep/goats, pointing to a substantial waste of feeds and nutrients. The economic assessment of the production of four high value vegetables pointed to a low efficiency of N and P use in amaranth and lettuce production, causing low economic returns for these crops compared to tomato and cabbage to which inexpensive animal manure was applied. The net profit of market retailers depended on the type of vegetable marketed. In addition it depended on marketplace for amaranth and lettuce, and on season and marketplace for cabbage and tomato. Analysis of faecal pathogens in lettuce irrigated with river water and fertilized with animal manure indicated a substantial contamination by Salmonella spp. with 7.2 x 104 colony forming units (CFU) per 25 g of produce fresh matter, while counts of Escherichia coli averaged 3.9 x 104 CFU g-1. In lettuce irrigated with wastewater, Salmonella counts averaged 9.8 x 104 CFU 25 g-1 and E. coli counts were 0.6 x 104 CFU g-1; these values exceeded the tolerable contamination levels in vegetables of 10 CFU g-1 for E. coli and of 0 CFU 25 g-1 for Salmonella. Taken together, the results of this study indicate that Niamey’s UPA enterprises put environmental safety at risk since excess inputs of N, P and K to crop and livestock production units favour N volatilisation and groundwater pollution by nutrient leaching. However, more detailed studies are needed to corroborate these indications. Farmers’ revenues could be significantly increased if nutrient use efficiency in the different production (sub)systems was improved by better matching nutrient supply through fertilizers and feeds with the actual nutrient demands of plants and animals.