Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Gradução em Aquicultura, Florianópolis, 2015. / Made available in DSpace on 2016-05-31T04:04:27Z (GMT). No. of bitstreams: 1 339430.pdf: 6599717 bytes, checksum: 189e1a199660bab2ec06da438ba5798e (MD5) Previous issue date: 2015 / Uma cepa de bactéria amilolítica potencialmente probiótica foi isolada do intestino do jundiá (Rhamdia quelen), visando melhorar a digestibilidade de carboidratos em peixes. Duas de 31 cepas isoladas do intestino anterior foram consideradas amilolíticas e a que produziu maior halo pela secreção da amilase, foi a escolhida. A cepa foi caracterizada fenotipicamente e analisada para determinação da sua tolerância frente à bile e pH, além da análise quantitativa da atividade enzimática da amilase. A candidata probiótica, identificada como Aeromonas veronii, mostrou a capacidade de sobreviver a diversos níveis de pH e sais biliares, além de secretar um interessante perfil enzimático. A utilização da bactéria amilolítica A. veronii como suplemento probiótico na dieta foi analisada com relação ao crescimento, digestibilidade de nutrientes e histologia do jundiá, peixe onívoro, com intestino curto. No ensaio de digestibilidade, os peixes (425,6 ± 159,94 g) foram alimentados em triplicata com dieta suplementada ou não com 107 unidades formadoras de colônia (UFC) de A. veronii por g-1 de dieta, por 79 dias. Foi utilizado o marcador inerte óxido de ítrio nas dietas experimentais, as fezes foram coletadas por sedimentação e os intestinos coletados para análise histológica. O coeficiente de digestibilidade aparente e nutriente digestível para fibra alimentar total foram maiores (P < 0,05) nos peixes que receberam a dieta controle, mas não houve efeito na digestibilidade do amido. Não foram observadas alterações histológicas intestinais significativas com a suplementação da bactéria probiótica. O crescimento dos peixes foi avaliado em outro ensaio, onde os peixes (26,19 0,32 g) foram alimentados com as mesmas dietas do ensaio de digestibilidade, por 45 dias. O peso final e ganho em peso foram maiores nos peixes alimentados com a dieta controle, sem probiótico. Apesar da suplementação dietética com a bactéria amilolítica não melhorar a digestibilidade do amido na dieta, há indícios que a A. veronii degrade o amido e este fique mais disponível para o jundiá aproveitá-lo, porém, talvez esta espécie de peixe não consiga utilizar de forma eficiente o carboidrato disponibilizado pela bactéria.<br> / Abstract : A strain of a potential probiotic amylolytic bacterium was isolated from the gut of jundiá catfish (Rhamdia quelen) aiming at improving carbohydrate digestibility in fish. Two of 31 strains isolated from the foregut were considered amylolytic and the strain selected was the one which presented the higher halo provided by the amylase secretion. The strain was phenotypically characterized and analysed to determine bile and pH tolerance as well extracellular quantitative amylase activity. The probiotic candidate, identified as Aeromonas veronii, showed the ability to survive a range of pH and bile salt conditions and secreted an interesting enzymatic profile. The use of A. veronii as a dietary probiotic supplement was analysed for growth, nutrient digestibility and histology of jundiá catfish. In the digestibility trial, fish (425.6 ± 159.94 g) were fed in triplicate with diets supplemented or not with 109 colony-forming units (CFU) of A. veronii per 100 g-1 on a diet, for 79 days. Feces were collected by sedimentation and the foreguts collected for histological analysis. The apparent digestibility coefficient and digestible nutrient of total dietary fiber were higher (P < 0.05) in fish fed the control diet, but there was no effect on starch digestibility. No significant changes were observed in intestinal histology with dietary bacterial probiotic supplementation. Fish growth was evaluated in another trial, where fish (26.19 ± 0.32 g) were fed the same diets used on the digestibility trial, for 45 days. The final weight and weight gain were higher in fish fed the control diet without probiotic supplementation. Although dietary supplementation with the amylolytic bacteria does not improve the digestibility of starch in the diet, there are indications that A. veronii degrade starch making it more available to the fish. However, maybe jundiá cannot efficiently use the carbohydrate provided by bacterium degradation.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Aeromonas salmonicida sous-espèce salmonicida, une bactérie pathogène infectant les poissons, cause une maladie nommée la furonculose qui est traitée par antibiotique. À cause des résistances aux antibiotiques, ceux-ci ne sont plus aussi efficaces. Comme les tests d'antibiogrammes nécessitent sept jours, une PCR multiplex ciblant les gènes de résistance aux antibiotiques homologués (sulfonamides et triméthoprime, tétracyclines, chloramphénicols) permettrait de détecter rapidement ces résistances. Suite à la caractérisation de séquences génomiques et l'optimisation de PCR multiplex, plusieurs souches d’A. salmonicida sous-espèce salmonicida ont été testées et leurs résultats ont été comparés avec ceux obtenus par les tests d'antibiogrammes. Il en ressort que la trousse développée est très performante voire plus que les tests d'antibiogrammes pour détecter les résistances aux sulfonamides, tétracyclines et chloramphénicols tout en étant plus rapide. Une connaissance plus approfondie sur les résistances aux sulfonamides et triméthoprime demeure d’actualité puis l'amélioration de la trousse diagnostique est à envisager. / Aeromonas salmonicida subspecies salmonicida, a pathogenic bacterium that infects fish, causes a disease named furunculosis, which is treated with antibiotics. The latters are not as effective because of antibiotic resistances. As the susceptibility tests require seven days, a multiplex PCR targeting the genes coding resistances against homologated antibiotics (sulfonamides and trimethoprim, tetracyclines, chloramphenicols) would detect resistance rapidly. Following the characterization of genomic sequences and optimization of multiplex PCR, several strains of A. salmonicida subspecies salmonicida were tested and the results were compared with those obtained by the susceptibility tests. It shows that the kit is highly efficient even more rapidly than the susceptibility tests to detect resistance to sulfonamides, tetracyclines and chloramphenicols while being faster. A deeper knowledge about sulfonamides and trimethoprim resistance and the improvement of the diagnostic kit should be considered.

Les éléments mobiles de l’ADN, des séquences capables de transfert intracellulaire ou extracellulaire, existent dans la plupart des génomes (eucaryotes ou procaryotes). Ce sont des vecteurs importants de variation génétique. Certains portent des gènes accessoires qui procurent un avantage sélectif à leur organisme-hôte. Les séquences d’insertion (IS) sont un cas spécial d’éléments mobiles bactériens. Elles sont exemptes de gènes accessoires, mais autonomes pour leur transposition. Elles peuvent donc causer des variations structurales, ce qui peut perturber les fonctions de la bactérie-hôte. Néanmoins, la plupart des génomes bactériens portent plusieurs copies de différentes familles d’IS. Plusieurs hypothèses, allant du parasitisme des IS à une forme de commensalisme IS-bactérie, ont été formulées pour expliquer la persistance de ces éléments mobiles dans les génomes. Aeromonas salmonicida ssp. salmonicida, l’agent étiologique de la furonculose chez les salmonidés, est un modèle intéressant pour étudier les IS. Son génome possède plusieurs familles d’IS présentes en différents nombres de copies. De plus, des travaux réalisés précédemment ont montré que ces éléments mobiles étaient responsables de variations structurales dans les plasmides de la bactérie et de l’inactivation de certains de ses facteurs de virulence. Bien que les évènements mentionnés ci-dessus semblent neutres ou désavantageux, il n’est pas exclu que certains évènements impliquant les IS puissent apporter un avantage adaptatif à la bactérie. L’objectif de cette thèse était donc d’explorer les éléments mobiles du génome d’A. salmonicida ssp. salmonicida, en portant une attention particulière aux IS, afin d’identifier des évènements à répercussion bénéfique pour la bactérie. La richesse d’information et l’accessibilité grandissante du séquençage à haut débit en ont fait une technique de choix pour étudier les IS et autres transposons en association avec un autre élément mobile essentiel à leur transmission intercellulaire, les plasmides. Une première étude a permis de découvrir des variants d’un grand plasmide d’A. salmonicida ssp. salmonicida, pAsa4, dont la forme de référence porte des gènes de résistance aux antibiotiques, des IS et un transposon. Les variants identifiés lors de l’étude, pAsa4b et pAsa4c, présentent des variations structurales par rapport à la référence. Elles ont des conséquences sur les gènes de résistance portés et sur la capacité de transfert des plasmides. La génomique comparative faite entre les pAsa4 suggère que les éléments mobiles tiennent un rôle primordial dans la création de certaines variations structurales, tant par leur capacité de transposition ou d’intégration que par leur propension à la recombinaison homologue. L’élargissement de la famille des pAsa4 à trois variants procure d’autres exemples de variabilité attribuable aux éléments mobiles de l’ADN. Dans une second temps, nous avons approfondi un évènement étudié précédemment : la perte du locus du système de sécrétion de type trois (SSTT). Cette région, située sur le grand plasmide pAsa5, peut être perdue lorsque A. salmonicida ssp. salmonicida est cultivée à 25°C. Dans certains cas, la recombinaison homologue d’IS flanquant les régions touchées avait été mise en cause. Dans le cadre de ce projet, le génome de la souche 01-B526, qui avait été utilisée pour générer des pAsa5 réarrangés, a été séquencé par une combinaison de technologies à courtes et à longues lectures. Un nouveau plasmide, pAsa9, ainsi qu’une nouvelle IS de pAsa5, ISAS5Z, ont été découverts. Ensemble, ces deux éléments ont permis de proposer un nouveau schéma de recombinaison homologue qui s’est avéré exact pour tous les réarrangements de pAsa5 générés lors de l’expérience précédente et considérés comme irréguliers. Ces nouveaux résultats permettent de regrouper tous les scénarios de réarrangement de pAsa5 sous un seul mécanisme, la recombinaison homologue entre IS, en plus d’ajouter une nouvelle entité au plasmidome d’A. salmonicida ssp. salmonicida. Plusieurs évènements ont donc été ajoutés au catalogue des variations structurales causées par les éléments mobiles de l’ADN d’A. salmonicida ssp. salmonicida. Dans les derniers chapitres de cette thèse, les conséquences de ces différents évènements sont discutées. Certaines variations sur pAsa4 apportent des gènes accessoires qui sont bénéfiques dans un environnement donné. Autrement, l’étude des éléments observés est limitée par le manque de connaissance sur les facteurs environnementaux qui causent ces variations et sur leur impact in vivo, chez le poisson. Quoi qu’il en soit, les IS du génome d’A. salmonicida ssp. salmonicida influencent certainement les relations hôte-pathogène de cette bactérie et participent intimement à son histoire évolutive. / Mobile genetic elements are sequences capable of intracellular and extracellular transfer. They exist in the majority of eukaryotic and prokaryotic genomes, and are important vectors of genetic variability. Some mobile genetic elements carry accessory genes that confer selective advantages to their host. Insertion sequences (IS), do not belong to the latter group, as they are exempt of accessory genes, yet they are autonomous in their transposition. ISs are thus important structural variation producers, which may endanger the host’s functions and its genomic integrity. However, most bacterial genomes carry ISs. Hypotheses ranging from parasitism to commensalism were formulated to explain how ISs persist in genomes. A. salmonicida subsp. salmonicida, the causative agent of furunculosis in salmonids, is a good model to study ISs. Its genome carries many ISs divided in different families. Moreover, previous work showed that these elements cause structural variations in plasmids and virulence factor inactivations. Even though the events observed to date seemed neutral or detrimental for the bacteria, we cannot exclude that ISs may procure an adaptative advantage in certain cases. This thesis’ objective was to explore the A. salmonicida subsp. salmonicida mobile genetic elements, especially ISs, to identify events with a positive outcome for the bacteria. ISs and transposons were studied in association with another mobile element accountable for their intercellular transfer, plasmids. High-throughput sequencing was used as a preferred method because of its growing availability and the richness of information it generates. In a first article, variants of a large plasmid that carries antibiotic resistance genes, pAsa4, were discovered. pAsa4b and pAsa4c display structural variations when compared to the reference plasmid that impact their antibiotic resistance genes and their conjugation capabilities. Comparative genomics between the three variants shows that mobile elements, either by transposition or recombination, have an important role in some variations. Adding two new members to the pAsa4 family of plasmids gave further examples of genetic diversity driven by mobile elements. In a second article, we investigated previous results concerning the type three secretion system (TTSS) loss. This is an essential virulence factor for A. salmonicida subsp. salmonicida, and its genes are mainly encoded in a single locus of the large plasmid pAsa5. The region can be lost when the bacterium is cultivated at 25°C or higher. In some cases, homologous recombinations between IS copies flanking the TTSS locus have been identified as causing the deletion. In this study, the genome of the strain 01-B526, which was used in a previous study to produce TTSS-negative mutants, was sequenced using Illumina and PacBio technologies. A new plasmid, named pAsa9, and a new pAsa5 IS, ISAS5Z, were discovered. Together, those elements permitted to build a new IS-driven homologous recombination model that was tested in the mutant strains. All TTSS-negative strains generated in previous study and that were not explained by other IS recombination patterns fitted with the new ISAS5 homologous recombination model. These results allowed to regroup all pAsa5 rearrangements under a consistent mechanism: recombination using IS copies as a template. In this thesis, more structural variation events involving ISs were uncovered. In the last chapters, their involvement in A. salmonicida subp. salmonicida fitness in vitro and in the fish host is discussed. Some variations in pAsa4 bring new resistance genes, which are beneficial for the bacteria in an aquaculture context. However, for most events, we lack situational information to properly conclude their fitness impact. Regardless, ISs of the A. salmonicida subp. salmonicida genome certainly influence host-pathogen relations and participate in the bacteria evolutive history.

Disease outbreaks have overwhelmed the aquaculture industry as a whole and have been catastrophic for many single operations. To minimize disease outbreaks, efforts are underway to enhance animal health and disease resistance to pathogens without the use of antibiotics. The overall purpose of this study was to explore a potential prophylactic, a novel antibody diet, for the bacterial pathogen, Aeromonas hydrophila, in tilapia. The tilapia were on an anti-interleukin-10 antibody diet as a neutralization of interleukin-10 (IL-10), an anti-inflammatory cytokine. The anti-inflammatory function of IL-10 has been shown to allow persistence of gastrointestinal pathogens. Tilapia were fed the novel diet and were challenged via bath immersion or oral gavage with A. hydrophila. Four trials of challenge studies were conducted. Clinical signs of the disease and survival were monitored post-challenge of the bacteria. Out of the 4 trials, one bath immersion trial showed significantly lower survival in the group fed the novel antibody diet (p=0.044) compared to the control fed group, after challenged with A. hydrophila. The other trials tested showed no significant differences in survival between diets. Among the survival percentages collected as a whole, it cannot be determined from in vivo results whether this anti-IL-10 diet is effective in preventing mortality from A. hydrophila in tilapia. Therefore, an in vitro study using an enzyme-linked immunosorbent assay (ELISA) was used to determine the neutralization capability of anti-IL-10 on IL-10 using tilapia splenocytes. Interferon-γ, a pro-inflammatory cytokine, was quantified in order to find a trend in expression of IL-10 in vitro in various tilapia cell treatments. The protocol for the ELISA study is under development being that the use of this antibody is novel and has never before been done in fish. / Master of Science in Life Sciences

La bactérie Aeromonas salmonicida utilise le système de sécrétion de type trois (SSTT) pour injecter des effecteurs à l'intérieur des cellules de l'hôte lors de l'infection. L'étude du génome d'A. salmonicida a révélé l'existence d'Ati2, un nouvel effecteur du SSTT. Ce projet avait pour but d'étudier la relation structure-fonction d'Ati2 afin de déterminer son rôle dans la virulence d'A. salmonicida. Des analyses biochimiques et en modélisation moléculaire ont permis de démontrer qu'Ati2 est une inositol polyphosphate 5-phosphatase qui hydrolyse les PtdIns(4,5)P₂ et les PtdIns(3,4,5)P₃. Divers mutants d'Ati2 ont été produits et clones dans des vecteurs d'expression chez l'amibe Dictyostelium discoideum. Les tests d'expression ont démontré qu'Ati2 est toxique pour l'amibe et que cela est lié à son activité catalytique. Ce projet de recherche a ainsi permis de démontrer l'existence d'un nouvel effecteur du SSTT et d'en spécifier la fonction dans la pathogénicité d'A. salmonicida.

Yes / Aeromonas hydrophila is a well-known waterborne pathogen that recently was found to infect humans. Here, we report the draft genome of a freshwater isolate from a Malaysian waterfall, A. hydrophila strain M023, which portrays N-acylhomoserine lactone-dependent quorum sensing. / University of Malaya via High Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, A-000001-50001, and UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Práticas nutricionais saudáveis e a globalização cultural popularizaram o consumo de pratos à base de peixe cru, anteriormente restritos aos países orientais. Estimativas mostram que doenças de origem alimentar causam aproximadamente 76 milhões de casos, 325 mil hospitalizações e 5 mil mortes a cada ano, somente nos Estados Unidos. Casos com etiologia desconhecida somam 62 milhões, com 265 mil hospitalizações e 3.200 mortes. O objetivo deste estudo foi investigar a presença de Escherichia coli, Salmonella spp. e Staphylococcus aureus e cepas potencialmente patogênicas de Aeromonas spp. e Vibrio spp. em peixes comercializados em feiras livres da cidade de São Paulo, Brasil. Vinte amostras de peixes, de diferentes espécies, foram adquiridas em feiras livres e analisadas utilizando metodologia convencional para investigação de patógenos em alimentos. Altos níveis de contaminação fecal foram detectados em 25% das amostras. Staphylococcus aureus foi isolado em 10% das amostras, entretanto em valores abaixo do permitido pela legislação brasileira. Todas as amostras estavam negativas para Salmonella spp. V. parahaemolyticus não foi isolado, 30% das amostras foram positivas para outras espécies de Vibrio, inclusive Vibrio cholerae não-O1/não-O139. Aeromonas spp. , incluindo A. hydrophila foi isolada em 50% das amostras de peixe. O isolamento de Vibrio cholerae não-O1/não-O139 e Aeromonas hydrophila, assim como Staphylococcus aureus e Escherichia coli, sugere que peixes comercializados em feiras livres da cidade de São Paulo podem representar um risco para os consumidores e ser um importante veículo de transmissão de espécies enteropatogênicas. / Healthier nutritional lifestyles and cultural globalization have popularized the consumption of raw fish dishes that were previously restricted to oriental countries. Estimates indicate that food-borne diseases cause approximately 76 million illnesses, 325,000 hospitalizations and 5,000 deaths each year in the United States alone. Cases with unknown etiology account for 62 million illnesses, 265,000 hospitalizations and 3,200 deaths. The purpose of this study was to investigate the presence of Escherichia coli, Salmonella spp., Staphylococcus aureus and potentially pathogenic strains of Aeromonas spp. and Vibrio spp. in fish commercialized at the retail level in the markets of São Paulo City, Brazil. Twenty fish of different species were analysed for foodborne pathogens using conventional methodologies. High levels of faecal contamination were detected in 25% of fish samples. Staphylococcus aureus was isolated from 10% of samples; however, in each case this was below the limits established by Brazilian legislation. All samples were negative for Salmonella and 30% tested positive for others Vibrio spp., including Vibrio cholerae non-O1/non-O139. Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, were isolated in 50% of fish samples. The isolation of Vibrio cholerae non-O1/non-O139 and Aeromonas hydrophila as well as Staphylococcus aureus and Escherichia coli suggests that fish commercialized in São Paulo City may represent a health risk to consumers and be an important vehicle for transmission of these enteropathogenic species.

Práticas nutricionais saudáveis e a globalização cultural popularizaram o consumo de pratos à base de peixe cru, anteriormente restritos aos países orientais. Estimativas mostram que doenças de origem alimentar causam aproximadamente 76 milhões de casos, 325 mil hospitalizações e 5 mil mortes a cada ano, somente nos Estados Unidos. Casos com etiologia desconhecida somam 62 milhões, com 265 mil hospitalizações e 3.200 mortes. O objetivo deste estudo foi investigar a presença de Escherichia coli, Salmonella spp. e Staphylococcus aureus e cepas potencialmente patogênicas de Aeromonas spp. e Vibrio spp. em peixes comercializados em feiras livres da cidade de São Paulo, Brasil. Vinte amostras de peixes, de diferentes espécies, foram adquiridas em feiras livres e analisadas utilizando metodologia convencional para investigação de patógenos em alimentos. Altos níveis de contaminação fecal foram detectados em 25% das amostras. Staphylococcus aureus foi isolado em 10% das amostras, entretanto em valores abaixo do permitido pela legislação brasileira. Todas as amostras estavam negativas para Salmonella spp. V. parahaemolyticus não foi isolado, 30% das amostras foram positivas para outras espécies de Vibrio, inclusive Vibrio cholerae não-O1/não-O139. Aeromonas spp. , incluindo A. hydrophila foi isolada em 50% das amostras de peixe. O isolamento de Vibrio cholerae não-O1/não-O139 e Aeromonas hydrophila, assim como Staphylococcus aureus e Escherichia coli, sugere que peixes comercializados em feiras livres da cidade de São Paulo podem representar um risco para os consumidores e ser um importante veículo de transmissão de espécies enteropatogênicas. / Healthier nutritional lifestyles and cultural globalization have popularized the consumption of raw fish dishes that were previously restricted to oriental countries. Estimates indicate that food-borne diseases cause approximately 76 million illnesses, 325,000 hospitalizations and 5,000 deaths each year in the United States alone. Cases with unknown etiology account for 62 million illnesses, 265,000 hospitalizations and 3,200 deaths. The purpose of this study was to investigate the presence of Escherichia coli, Salmonella spp., Staphylococcus aureus and potentially pathogenic strains of Aeromonas spp. and Vibrio spp. in fish commercialized at the retail level in the markets of São Paulo City, Brazil. Twenty fish of different species were analysed for foodborne pathogens using conventional methodologies. High levels of faecal contamination were detected in 25% of fish samples. Staphylococcus aureus was isolated from 10% of samples; however, in each case this was below the limits established by Brazilian legislation. All samples were negative for Salmonella and 30% tested positive for others Vibrio spp., including Vibrio cholerae non-O1/non-O139. Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, were isolated in 50% of fish samples. The isolation of Vibrio cholerae non-O1/non-O139 and Aeromonas hydrophila as well as Staphylococcus aureus and Escherichia coli suggests that fish commercialized in São Paulo City may represent a health risk to consumers and be an important vehicle for transmission of these enteropathogenic species.

O potencial de bactérias em produzir polihidroxialcanoatos (PHA) a partir de óleo de soja foi avaliado com ênfase em Aeromonas spp. Dez isolados apresentando características de Aeromonas spp. (colônias amarelas em GSP agar e produção de P3HB-co-3HHx) e perfis ARDRA similares foram obtidos. Dois isolados produzindo PHAMCL foram também obtidos e experimentos de identificação adicionais serão necessários para confirmar se pertencem ao gênero Aeromonas. P3HB-co-3HHx contendo diferentes frações molares de 3HHx foram produzidos. Isolados produzindo P3HB-co-3HHx a partir de óleo de soja ou ácido láurico foram incapazes de produzir P3HB a partir de glicose sugerindo sua incapacidade de gerar monômeros 3HB a partir de acetil-CoA. Introdução de plasmídeo abrigando genes de biossíntese de P3HB de Ralstonia eutropha em isolados capazes de utilizar eficientemente óleo de soja como fonte de carbono permitiu a produção de P3HB, mas não PHA contendo monômeros de cadeia média, sugerindo uma transferência ineficiente de intermediários a partir da <font face=\"Symbol\">&#946-oxidação para PHA sintase. / The potential of polyhydroxyalknoates (PHA) production from plant oils was evaluated especially in Aeromonas spp. Ten isolates presenting features of Aeromonas spp. (yellow colonies in GSP agar and production of P3HB-co-3HHx) and similar ARDRA profiles were obtained. Two isolates producing PHAMCL were also obtained and further identification experiments will be needed to confirm their position in Aeromonas genus. P3HB-co-3HHx containing different molar fractions of 3HHx were produced. Isolates producing P3HB-co-3HHx from soybean oil or lauric acid were unable to produce P3HB from glucose suggesting their incapability to generate 3HB monomers from acetyl-CoA. Introduction of a plasmid harboring P3HB biosynthesis genes from Ralstonia eutropha into isolates able to use efficiently soybean oil as carbon source allowed the production of P3HB but not PHA containing HAMCL suggesting an inefficient transfer of intermediates from &#946-oxidation to PHA synthase.