Mechanisms of Colonisation and Colonisation Resistance of the Digestive Tract Part 1: Bacteria/host Interactions

The digestive microflora is characterized by a high degree of complexity and diversity. Variation in the composition of the digestive flora of an individual occurs in space and time. Two kinds of s..

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or ‘classical’ carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits ‘non-classical’ carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.publishedVersio

Outbreak of OXA-48-producing Enterobacteriaceae in a neonatal intensive care unit in Western Sweden

In 2015, an outbreak caused by OXA-48-producing Enterobacteriaceae affected a neonatal intensive care unit at a Swedish University Hospital. The aim was to explore the transmission of OXA-48-producing strains between infants and the transfer of resistance plasmids between strains during the outbreak. Twenty-four outbreak isolates from ten suspected cases were whole-genome sequenced. A complete assembly was created for the index isolate (Enterobacter cloacae) and used as a mapping reference to detect its plasmids in the remaining isolates (17 Klebsiella pneumoniae, 4 Klebsiella aerogenes, and 2 Escherichia coli). Strain typing was performed using core genome MLST and SNP analysis. As judged from sequencing and clinical epidemiological data, the outbreak involved nine cases (two developed sepsis) and four OXA-48-producing strains: E. cloacae ST1584 (index case), K. pneumoniae ST25 (eight cases), K. aerogenes ST93 (two cases), and E. coli ST453 (2 cases). Two plasmids from the index strain, pEclA2 and pEclA4, carrying blaOXA48 and blaCMY-4, respectively, were traced to all K. pneumoniae ST25 isolates. Klebsiella aerogenes ST93 and E. coli ST453 harboured either only pEclA2, or both pEclA2 and pEclA4. One suspected case harbouring OXA-162-producing K. pneumoniae ST37 could be excluded from the outbreak. Once initiated by an E. cloacae strain, the outbreak was caused by the dissemination of a K. pneumoniae ST25 strain and involved inter-species horizontal transfer of two resistance plasmids, one of which carried blaOXA-48. To our knowledge, this is the first description of an outbreak of OXA-48-producing Enterobacteriaceae in a neonatal setting in northern Europe

Fecal short chain fatty acids in children living on farms and a link between valeric acid and protection from eczema

Children growing up on farms have low rates of allergy, but the mechanism for this protective effect has not been fully elucidated. Short chain fatty acids (SCFAs) produced by the gut microbiota may play a role in protection from allergy. We measured fecal SCFA levels in samples collected from 28 farming and 37 control children over the first 3\ua0years of life using gas chromatography. Data on diet and other host factors were recorded and allergy was diagnosed at 8\ua0years of age. Among all children, median propionic and butyric acid concentration increased over the first 3\ua0years, and longer SCFAs typically appeared by 1\ua0year of age. Farm children had higher levels of iso-butyric, iso-valeric and valeric acid at 3\ua0years of age than rural controls. In addition, children with elder siblings had higher levels of valeric acid at 3\ua0years of age, and dietary factors also affected SCFA pattern. High levels of valeric acid at 3\ua0years of age were associated with low rate of eczema at 8\ua0years of age. The fecal SCFA pattern in farm children suggests a more rapid maturation of the gut microbiota. Valeric acid or associated microbes may have protective potential against eczema

Genome Dynamics of Escherichia coli during Antibiotic Treatment: Transfer, Loss, and Persistence of Genetic Elements In situ of the Infant Gut

Elucidating the adaptive strategies and plasticity of bacterial genomes in situ is crucial for understanding the epidemiology and evolution of pathogens threatening human health. While much is known about the evolution of Escherichia coli in controlled laboratory environments, less effort has been made to elucidate the genome dynamics of E. coli in its native settings. Here, we follow the genome dynamics of co-existing E. coli lineages in situ of the infant gut during the first year of life. One E. coli lineage causes a urinary tract infection (UTI) and experiences several alterations of its genomic content during subsequent antibiotic treatment. Interestingly, all isolates of this uropathogenic E. coli strain carried a highly stable plasmid implicated in virulence of diverse pathogenic strains from all over the world. While virulence elements are certainly beneficial during infection scenarios, their role in gut colonization and pathogen persistence is poorly understood. We performed in vivo competitive fitness experiments to assess the role of this highly disseminated virulence plasmid in gut colonization, but found no evidence for a direct benefit of plasmid carriage. Through plasmid stability assays, we demonstrate that this plasmid is maintained in a parasitic manner, by strong first-line inheritance mechanisms, acting on the single-cell level, rather than providing a direct survival advantage in the gut. Investigating the ecology of endemic accessory genetic elements, in their pathogenic hosts and native environment, is of vital importance if we want to understand the evolution and persistence of highly virulent and drug resistant bacterial isolates

Reduced Phase Switch Capacity and Functional Adhesin Expression of Type 1-Fimbriated Escherichia coli from Immunoglobulin A-Deficient Individuals

The mannose-specific adhesin of type 1 fimbriae is the most common adhesin in Escherichia coli. One receptor for this adhesin is the carbohydrate chains of secretory immunoglobulin A (S-IgA), and intestinal E. coli from IgA-deficient individuals has a reduced capacity to adhere to mannose-containing receptors. Here, we investigated the expression of the mannose-specific adhesin and its capacity to switch to the fimbriated phenotype in colonic resident and transient E. coli strains isolated from control (n = 16) and IgA-deficient (n = 17) persons. Resident E. coli strains from IgA-deficient individuals displayed weaker mannose-specific adherence to colonic cells than resident strains from control individuals (21 versus 44 bacteria/cell, P = 0.0009) due to three mechanisms: a lower carriage rate of the fimH gene (90% versus 97%, not significant), more frequent failure to switch on the fim genes (30% versus 6%, P = 0.02), and the reduced adhesive potential of fimH(+) isolates capable of phase switch (26 versus 46 bacteria/cell, P = 0.02). On the other hand, resident strains from IgA-deficient individuals displayed stronger mannose-resistant adherence than resident strains from control individuals (P = 0.04) and transient strains from IgA-deficient individuals (P = 0.01). The presence of S-IgA appears to favor the establishment of E. coli clones which readily express mannose-specific adhesins in the bowel microbiota

Oral and faecal lactobacilli and their expression of mannose-specific adhesins in individuals with and without IgA deficiency

Lactobacilli are present in the intestine and oral cavity of most adults. Secretory IgA in mucosal secretions may provide carbohydrate receptors for bacterial adhesins. Here, oral and faecal samples from 33 IgA-deficient individuals and 34 controls were cultured for lactobacilli, which were identified using species-specific PCR or partial 165 rDNA sequencing and tested for expression of mannose-specific adhesins. Lactobacilli were found in the oral cavity of 76% of IgA-deficient and 85% of control individuals. Lactobacillus paracasei and Lactobacillus gasseri dominated in both groups. Lactobacillus fermentum was less common in IgA-deficient individuals than in controls (p = 0.0055) and Lactobacillus salivarius was less common in symptomatic than in healthy IgA-deficient individuals (p = 0.0051). Faecal samples yielded lactobacilli in most individuals. L. paracasei was most frequent, followed by L. gasseri and Lactobacillus plantarum. Mannose-specific adhesins were expressed more frequently by oral than by faecal isolates (p = 0.032) and oral isolates adhered in higher numbers than faecal isolates (46 vs. 14 bacteria/cell, p = 0.0038). Faecal isolates from IgA-deficient individuals more frequently expressed mannose-specific adhesins than faecal isolates from controls (p = 0.039). Mannose-specific adhesins may be a colonisation factor in the oral cavity, and the presence of secretory IgA may modify adhesin expression. However, secretory IgA seems to have little influence on Lactobacillus species distribution. (C) 2011 Elsevier GmbH. All rights reserved

Are all faecal bacteria detected with equal efficiency? A study using next-generation sequencing and quantitative culture of infants\u27 faecal samples

Background: Many species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota. Methods: Twenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1–V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains. Results: NGS yielded one read per 106 CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 108 CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >106 CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria. Conclusions: NGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy