RESUMO
Akkermansia muciniphila is a mucin-degrading bacterium found in the human gut and is often associated with positive human health. However, despite being detected by as early as 1 month of age, little is known about the role of Akkermansia in the infant gut. Human milk oligosaccharides (HMOs) are abundant components of human milk and are structurally similar to the oligosaccharides that comprise mucin, the preferred growth substrate of human-associated Akkermansia. A limited subset of intestinal bacteria has been shown to grow well on HMOs and mucin. We therefore examined the ability of genomically diverse strains of Akkermansia to grow on HMOs. First, we screened 85 genomes representing the four known Akkermansia phylogroups to examine their metabolic potential to degrade HMOs. Furthermore, we examined the ability of representative isolates to grow on individual HMOs in a mucin background and analyzed the resulting metabolites. All Akkermansia genomes were equipped with an array of glycoside hydrolases associated with HMO deconstruction. Representative strains were all able to grow on HMOs with various efficiencies and growth yields. Strain CSUN-19, belonging to the AmIV phylogroup, grew to the highest level in the presence of fucosylated and sialylated HMOs. This activity may be partially related to the increased copy numbers and/or the enzyme activities of the α-fucosidases, α-sialidases, and ß-galactosidases. This study examines the utilization of individual purified HMOs by Akkermansia strains representing all known phylogroups. Further studies are required to examine how HMO ingestion influences gut microbial ecology in infants harboring different Akkermansia phylogroups. IMPORTANCE Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk and provide several benefits to developing infants, including the recruitment of beneficial bacteria to the human gut. Akkermansia strains are largely considered beneficial bacteria and have been detected in colostrum, breast milk, and young infants. A. muciniphila MucT, belonging to the AmI phylogroup, contributes to the HMO deconstruction capacity of the infant. Here, using phylogenomics, we examined the genomic capacities of four Akkermansia phylogroups to deconstruct HMOs. Indeed, each phylogroup contained differences in their genomic capacities to deconstruct HMOs, and representative strains of each phylogroup were able to grow using HMOs. These Akkermansia-HMO interactions potentially influence gut microbial ecology in early life, a critical time for the development of the gut microbiome and infant health.
Assuntos
Microbioma Gastrointestinal , Leite Humano , Akkermansia , Feminino , Humanos , Lactente , Oligossacarídeos , VerrucomicrobiaRESUMO
We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Infecções por Escherichia coli/microbiologia , Prófagos/enzimologia , Escherichia coli Shiga Toxigênica/enzimologia , Proteínas Virais/metabolismo , Fatores de Virulência/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Prófagos/genética , Toxina Shiga II/genética , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/virologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Proteínas Virais/genética , Virulência , Fatores de Virulência/metabolismoRESUMO
Campylobacter jejuni is a prevalent enteric pathogen that changes morphology from helical to coccoid under unfavorable conditions. Bacterial peptidoglycan maintains cell shape. As C. jejuni transformed from helical to coccoid, peptidoglycan dipeptides increased and tri- and tetrapeptides decreased. The DL-carboxypeptidase Pgp1 important for C. jejuni helical morphology and putative N-acetylmuramoyl-L-alanyl amidase AmiA were both involved in the coccoid transition. Mutants in pgp1 and amiA showed reduced coccoid formation, with ∆pgp1∆amiA producing minimal coccoids. Both ∆amiA and ∆amiA∆pgp1 lacked flagella and formed unseparated chains of cells consistent with a role for AmiA in cell separation. All strains accumulated peptidoglycan dipeptides over time, but only strains capable of becoming coccoid displayed tripeptide changes. C. jejuni helical shape and corresponding peptidoglycan structure are important for pathogenesis-related attributes. Concomitantly, changing to a coccoid morphology resulted in differences in pathogenic properties; coccoid C. jejuni were non-motile and non-infectious, with minimal adherence and invasion of epithelial cells and an inability to stimulate IL-8. Coccoid peptidoglycan exhibited reduced activation of innate immune receptors Nod1 and Nod2 versus helical peptidoglycan. C. jejuni also transitioned to coccoid within epithelial cells, so the inability of the immune system to detect coccoid C. jejuni may be significant in its pathogenesis.
Assuntos
Campylobacter jejuni/metabolismo , Forma Celular/fisiologia , Peptidoglicano/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/patogenicidade , Campylobacter jejuni/fisiologia , Carboxipeptidases/metabolismo , Parede Celular/metabolismo , Peptidoglicano/química , Peptidoglicano/imunologiaRESUMO
Campylobacter jejuni helical shape is important for colonization and host interactions with straight mutants having altered biological properties. Passage on calcofluor white (CFW) resulted in C. jejuni 81-176 isolates with morphology changes: either a straight morphology from frameshift mutations and single nucleotide polymorphisms in peptidoglycan hydrolase genes pgp1 or pgp2 or a reduction in curvature due a frameshift mutation in cjj81176_1105, a putative peptidoglycan endopeptidase. Shape defects were restored by complementation. Whole genome sequencing of CFW-passaged strains showed no specific changes correlating to CFW exposure. The cjj81176_1279 (recR; recombinational DNA repair) and cjj81176_1449 (unknown function) genes were highly variable in all 81-176 strains sequenced. A frameshift mutation in pgp1 of our laboratory isolate of the straight genome sequenced variant of 11168 (11168-GS) was also identified. The PG muropeptide profile of 11168-GS was identical to that of Δpgp1 in the original minimally passaged 11168 strain (11168-O). Introduction of wild type pgp1 into 11168-GS did not restore helical morphology. The recR gene was also highly variable in 11168 strains. Microbial cell-to-cell heterogeneity is proposed as a mechanism of ensuring bacterial survival in sub-optimal conditions. In certain environments, changes in C. jejuni morphology due to genetic heterogeneity may promote C. jejuni survival.
Assuntos
Campylobacter jejuni/citologia , Campylobacter jejuni/genética , Proteínas de Bactérias/metabolismo , Benzenossulfonatos , Infecções por Campylobacter/microbiologia , Células Clonais , Regulação Bacteriana da Expressão Gênica/genética , Peptidoglicano/metabolismoRESUMO
The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.
Assuntos
Campylobacter jejuni/metabolismo , Força Próton-Motriz/fisiologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Formiatos/metabolismo , Hidrogênio , Potenciais da Membrana , Oxirredução , Oxigênio , FosforilaçãoRESUMO
Bacteria have evolved different mechanisms to catabolize carbon sources from nutrient mixtures. They first consume their preferred carbon source, before others are used. Regulatory mechanisms adapt the metabolism accordingly to maximize growth and to outcompete other organisms. The human pathogen Campylobacter jejuni is an asaccharolytic Gram-negative bacterium that catabolizes amino acids and organic acids for growth. It prefers serine and aspartate as carbon sources, however it lacks all regulators known to be involved in regulating carbon source utilization in other organisms. In which manner C. jejuni adapts its metabolism towards the presence or absence of preferred carbon sources is unknown. In this study, we show with transcriptomic analysis and enzyme assays how C. jejuni adapts its metabolism in response to its preferred carbon sources. In the presence of serine as well as lactate and pyruvate C. jejuni inhibits the utilization of other carbon sources, by repressing the expression of a number of central metabolic enzymes. The regulatory proteins RacR, Cj1000 and CsrA play a role in the regulation of these metabolic enzymes. This metabolism dependent transcriptional repression correlates with an accumulation of intracellular succinate. Hence, we propose a demand-based catabolite repression mechanism in C. jejuni, depended on intracellular succinate levels.
Assuntos
Campylobacter jejuni/metabolismo , Repressão Catabólica/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácido Succínico/metabolismo , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/genética , Carbono/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Serina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The genetic structure of bacterial populations can be related to geographical locations of isolation. In some species, there is a strong correlation between geographical distance and genetic distance, which can be caused by different evolutionary mechanisms. Patterns of ancient admixture in Helicobacter pylori can be reconstructed in concordance with past human migration, whereas in Mycobacterium tuberculosis it is the lack of recombination that causes allopatric clusters. In Campylobacter, analyses of genomic data and molecular typing have been successful in determining the reservoir host species, but not geographical origin. We investigated biogeographical variation in highly recombining genes to determine the extent of clustering between genomes from geographically distinct Campylobacter populations. Whole-genome sequences from 294 Campylobacter isolates from North America and the UK were analysed. Isolates from within the same country shared more recently recombined DNA than isolates from different countries. Using 15 UK/American closely matched pairs of isolates that shared ancestors, we identify regions that have frequently and recently recombined to test their correlation with geographical origin. The seven genes that demonstrated the greatest clustering by geography were used in an attribution model to infer geographical origin which was tested using a further 383 UK clinical isolates to detect signatures of recent foreign travel. Patient records indicated that in 46 cases, travel abroad had occurred <2 weeks prior to sampling, and genomic analysis identified that 34 (74%) of these isolates were of a non-UK origin. Identification of biogeographical markers in Campylobacter genomes will contribute to improved source attribution of clinical Campylobacter infection and inform intervention strategies to reduce campylobacteriosis.
Assuntos
Campylobacter/genética , Genética Populacional , Genoma Bacteriano , Infecções por Campylobacter/microbiologia , Geografia , Humanos , América do Norte , Recombinação Genética , Reino UnidoRESUMO
Campylobacter jejuni is a leading cause of food-borne gastroenteritis in humans. It lives commensally in the gastrointestinal tract of animals, and tolerates variable conditions during transit/colonization of susceptible hosts. The C. jejuniâ CprRS two-component system contains an essential response regulator (CprR), and deletion of the cprS sensor kinase enhances biofilms. We sought to identify CprRS-regulated genes and better understand how the system affects survival. Expression from the cprR promoter was highest during logarithmic growth and dependent on CprS. CprR(D52A) did not support viability, indicating that CprR phosphorylation is essential despite the dispensability of CprS. We identified a GTAAAC consensus bound by the CprR C-terminus; the Asp52 residue of full-length CprR was required for binding, suggesting phosphorylation is required. Transcripts differing in expression in ΔcprS compared with wildtype (WT) contained a putative CprR binding site upstream of their promoter region and encoded htrA (periplasmic protease upstream of cprRS) and peb4 (SurA-like chaperone). Consistent with direct regulation, the CprR consensus in the htrA promoter was bound by CprR(CTD). Finally, ΔhtrA formed enhanced biofilms, and ΔcprS biofilms were suppressed by Mg(2+). CprRS is the first C. jejuni regulatory system shown to control genes related to the cell envelope, the first line of interaction between pathogen and changing environments.
Assuntos
Campylobacter jejuni/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Análise em Microsséries , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Óperon , Fenótipo , Fosforilação/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido NucleicoRESUMO
UNLABELLED: Campylobacter jejuni is a leading cause of bacterial diarrheal disease and a frequent commensal of the intestinal tract in poultry and other animals. For optimal growth and colonization of hosts, C. jejuni employs two-component regulatory systems (TCSs) to monitor environmental conditions and promote proper expression of specific genes. We analyzed the potential of C. jejuni Cjj81176_1484 (Cjj1484) and Cjj81176_1483 (Cjj1483) to encode proteins of a cognate TCS that influences expression of genes possibly important for C. jejuni growth and colonization. Transcriptome analysis revealed that the regulons of the Cjj81176_1484 (Cjj1484) histidine kinase and the Cjj81176_1483 (Cjj1483) response regulator contain many common genes, suggesting that these proteins likely form a cognate TCS. We found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression of Cjj81176_0438 and Cjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. We observed that the Cjj1483 response regulator binds target promoters in both unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. This work further expands the signaling mechanisms of C. jejuni and provides additional insights regarding the complex and multifactorial regulation of many genes involved in basic metabolism, respiration, and nutrient acquisition that the bacterium requires for optimal growth in different environments. IMPORTANCE: Bacterial two-component regulatory systems (TCSs) link environmental cues to expression of specific genes that enable optimal bacterial growth or colonization of hosts. We found that the Campylobacter jejuni Cjj1484 histidine kinase and Cjj1483 response regulator function as a cognate TCS to largely repress expression of target genes encoding a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract, as well as other genes encoding proteins for heme or iron acquisition, metabolism, and respiration. We also discovered different modes by which Cjj1483 may mediate repression with and without Cjj1484. This work provides insight into the signal transduction mechanisms of a leading cause of bacterial diarrheal disease and emphasizes the multifactorial and complex regulation of specific biological processes in C. jejuni.
Assuntos
Campylobacter jejuni/enzimologia , Campylobacter jejuni/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Regulon , Fatores de Transcrição/metabolismo , Perfilação da Expressão Gênica , Histidina Quinase , Transdução de SinaisRESUMO
BACKGROUND: Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases. RESULTS: We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains. CONCLUSIONS: Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.
Assuntos
Evolução Biológica , Escherichia coli O157/classificação , Escherichia coli/classificação , Escherichia coli/genética , Genoma Bacteriano , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/virologia , Escherichia coli/virologia , Escherichia coli O157/genética , Escherichia coli O157/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genômica , Metiltransferases/genética , Metiltransferases/metabolismo , Filogenia , Prófagos/metabolismo , Sorotipagem , Toxina Shiga/genética , Shigella/classificação , Shigella/genética , Fatores de Virulência/genéticaRESUMO
Salmonella enterica has the ability to form biofilms and large aggregates on produce surfaces, including on cilantro leaves. Aggregates of S. enterica serovar Thompson that remained attached to cilantro leaves after rigorous washing and that were present free or bound to dislodged leaf tissue in the wash suspension were observed by confocal microscopy. Measurement of S. Thompson population sizes in the leaf washes by plate counts failed to show an effect of 0.05% Tween 80 on the removal of the pathogen from cilantro leaves 2 and 6 days after inoculation. On the contrary, digital image analysis of micrographs of single cells and aggregates of green fluorescent protein (GFP)-S. Thompson present in cilantro leaf washes revealed that single cells represented 13.7% of the cell assemblages in leaf washes containing Tween 80, versus 9.3% in those without the surfactant. Moreover, Tween 80 decreased the percentage of the total S. Thompson cell population located in aggregates equal to or larger than 64 cells from 9.8% to 4.4% (P < 0.05). Regression analysis of the frequency distribution of aggregate size in leaf washes with and without Tween 80 showed that the surfactant promoted the dispersal of cells from large aggregates into smaller ones and into single cells (P < 0.05). Our study underlines the importance of investigating bacterial behavior at the scale of single cells in order to uncover trends undetectable at the population level by bacterial plate counts. Such an approach may provide valuable information to devise strategies aimed at enhancing the efficacy of produce sanitization treatments.
Assuntos
Coriandrum/microbiologia , Folhas de Planta/microbiologia , Polissorbatos/farmacologia , Salmonella enterica/efeitos dos fármacos , Tensoativos/farmacologia , Manipulação de Alimentos , Salmonella enterica/citologia , Salmonella enterica/fisiologiaRESUMO
We previously reported significantly different acid resistance between curli variants derived from the same Escherichia coli O157:H7 strain, although the curli fimbriae were not associated with this phenotypic divergence. Here we investigated the underlying molecular mechanism by examining the genes encoding the common transcriptional regulators of curli biogenesis and acid resistance. rpoS null mutations were detected in all curli-expressing variants of the 2006 spinach-associated outbreak strains, whereas a wild-type rpoS was present in all curli-deficient variants. Consequently curli-expressing variants were much more sensitive to various stress challenges than curli-deficient variants. This loss of general stress fitness appeared solely to be the result of rpoS mutation since the stress resistances could be restored in curli-expressing variants by a functional rpoS. Comparative transcriptomic analyses between the curli variants revealed a large number of differentially expressed genes, characterized by the enhanced expression of metabolic genes in curli-expressing variants, but a marked decrease in transcription of genes related to stress resistances. Unlike the curli-expressing variants of the 1993 US hamburger-associated outbreak strains (Applied Environmental Microbiology 78: 7706-7719), all curli-expressing variants of the 2006 spinach-associated outbreak strains carry a functional rcsB gene, suggesting an alternative mechanism governing intra-strain phenotypic divergence in E. coli O157:H7.
Assuntos
Proteínas de Bactérias/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Doenças Transmitidas por Alimentos/microbiologia , Fator sigma/genética , Spinacia oleracea/microbiologia , Proteínas de Bactérias/metabolismo , Surtos de Doenças , Infecções por Escherichia coli/epidemiologia , Escherichia coli O157/classificação , Escherichia coli O157/genética , Contaminação de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Regulação Bacteriana da Expressão Gênica , Variação Genética , Mutação , Fator sigma/metabolismo , Estados Unidos/epidemiologiaRESUMO
Campylobacter jejuni and Campylobacter coli are well known for their natural competence, i.e., their capacity for the uptake of naked DNA with subsequent transformation. This study identifies non-transformable C. jejuni and C. coli strains from domestic animals and employs genomic analysis to investigate the strain genotypes and their associated genetic mechanisms. The results reveal genetic associations leading to a non-transformable state, including functional DNase genes from bacteriophages and mutations within the cts-encoded DNA-uptake system, which impact the initial steps of the DNA uptake during natural transformation. Interestingly, all 38 tested C. jejuni ST-50 strains from the United States exhibit a high prevalence of non-transformability, and the strains harbor a variety of these genetic markers. This research emphasizes the role of these genetic markers in hindering the transfer of antimicrobial resistance (AMR) determinants, providing valuable insights into the genetic diversity of Campylobacter. As ST-50 is a major clone of C. jejuni globally, we additionally determined the prevalence of the genetic markers for non-transformability among C. jejuni ST-50 from different regions of the world, revealing distinct patterns of evolution and a strong selective pressure on the loss of competence in ST-50 strains, particularly in the agricultural environment in the United States. Our findings contribute to a comprehensive understanding of genetic exchange mechanisms within Campylobacter strains, and their implications for antimicrobial resistance dissemination and evolutionary pathways within specific lineages.
RESUMO
Assessing the microbes present on tree fruit carpospheres as the fruit enters postharvest processing could have useful applications, as these microbes could have a major influence on spoilage, food safety, verification of packing process controls, or other aspects of processing. The goal of this study was to establish a baseline profile of bacterial communities associated with apple (pome fruit), peach (stone fruit), and Navel orange (citrus fruit) at harvest. We found that commercial peaches had the greatest bacterial richness followed by oranges then apples. Time of harvest significantly changed bacterial diversity in oranges and peaches, but not apples. Shifts in diversity varied by fruit type, where 70% of the variability in beta diversity on the apple carposphere was driven by the gain and loss of species (i.e., nestedness). The peach and orange carposphere bacterial community shifts were driven by nearly an even split between turnover (species replacement) and nestedness. We identified a small core microbiome for apples across and between growing seasons that included only Methylobacteriaceae and Sphingomonadaceae among the samples, while peaches had a larger core microbiome composed of five bacterial families: Bacillaceae, Geodermtophilaceae, Nocardioidaceae, Micrococcaeceae, and Trueperaceae. There was a relatively diverse core microbiome for oranges that shared all the families present on apples and peaches, except for Trueperaceae, but also included an additional nine bacterial families not shared including Oxalobacteraceae, Cytophagaceae, and Comamonadaceae. Overall, our findings illustrate the important temporal dynamics of bacterial communities found on major commercial tree fruit, but also the core bacterial families that constantly remain with both implications being important entering postharvest packing and processing.
Assuntos
Citrus sinensis , Prunus persica , Humanos , Estações do Ano , Bactérias , Citrus sinensis/microbiologia , Frutas/microbiologiaRESUMO
Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.
Assuntos
Infecções por Campylobacter , Campylobacter coli , Campylobacter jejuni , Campylobacter , Criança , Humanos , Campylobacter coli/genética , Reação em Cadeia da Polimerase , Infecções por Campylobacter/diagnóstico , Infecções por Campylobacter/microbiologia , Fezes/microbiologiaRESUMO
OBJECTIVES: Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. METHODS: Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. RESULTS: The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. CONCLUSIONS: Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.
Assuntos
Antibacterianos , Campylobacter , Humanos , Criança , Antibacterianos/farmacologia , Peru , RNA Ribossômico 23S/genética , Farmacorresistência Bacteriana/genética , Macrolídeos , Campylobacter/genética , GenômicaRESUMO
The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of ≥ 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers' health.
Assuntos
Bacillaceae , Cucumis melo , Cucurbitaceae , Estados Unidos , Cucurbitaceae/microbiologia , Cucumis melo/microbiologia , RNA Ribossômico 16S/genética , Bactérias/genética , EnterobacteriaceaeRESUMO
Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. Deletion of fdhT or fdhU in C. jejuni resulted in apparent defects in adherence and/or invasion of Caco-2 epithelial cells when assessed by CFU enumeration on standard Mueller-Hinton agar. However, fluorescence microscopy indicated that each mutant invaded cells at wild-type levels, instead suggesting roles for FdhTU in either intracellular survival or postinvasion recovery. The loss of fdhU caused reduced mRNA levels of formate dehydrogenase (FDH) genes and a severe defect in FDH activity. Cell infection phenotypes of a mutant deleted for the FdhA subunit of FDH and an ΔfdhU ΔfdhA double mutant were similar to those of a ΔfdhU mutant, which likewise suggested that FdhU and FdhA function in the same pathway. Cell infection assays followed by CFU enumeration on plates supplemented with sodium sulfite abolished the ΔfdhU and ΔfdhA mutant defects and resulted in significantly enhanced recovery of all strains, including wild type, at the invasion and intracellular survival time points. Collectively, our data indicate that FdhTU and FDH are required for optimal recovery following cell infection and suggest that C. jejuni alters its metabolic potential in the intracellular environment.
Assuntos
Aderência Bacteriana , Campylobacter jejuni/enzimologia , Campylobacter jejuni/fisiologia , Células Epiteliais/microbiologia , Formiato Desidrogenases/metabolismo , Fatores de Virulência/metabolismo , Células CACO-2 , Contagem de Colônia Microbiana , Formiato Desidrogenases/genética , Deleção de Genes , Humanos , Viabilidade Microbiana , Fatores de Virulência/genéticaRESUMO
The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter(-1). C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely "bet-hedging" survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.
Assuntos
Campylobacter jejuni/fisiologia , Estresse Fisiológico/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Galinhas , Regulação Bacteriana da Expressão Gênica/fisiologia , Glucose/farmacologia , Humanos , Intestinos/microbiologia , Cloreto de Magnésio/farmacologia , Pressão Osmótica , Cloreto de Potássio/farmacologia , Cloreto de Sódio , Fatores de Tempo , TranscriptomaRESUMO
Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.