Estelle Jumas-Bilak

Dynamics and Clinical Evolution of Bacterial Gut Microflora in Extremely Premature Patients

OBJECTIVE To determine baseline clinical characteristics that influence bacterial gut flora dynamics in very preterm infants and the relationship between gut flora dynamics and clinical evolution. STUDY DESIGN Prospective, monocentric study enrolling 29 consecutive very preterm infants. We collected data about growth, digestive tolerance, nutrition, and antibiotic use. Microflora in stool samples, collected between 3 and 56 days of life, was identified with direct molecular fingerprinting. RESULTS Median (interquartile range) body weight and gestational age at birth were 950 g (760-1060 g) and 27 weeks (27-29 weeks), respectively. The diversity score (number of operational taxonomic units) increased 0.45 units/week (P < .0001), with staphylococci as the major group. Bifidobacterium was poorly represented. Gestational age (≥ 28 weeks) and caesarean delivery independently correlated with better diversity scores during follow-up (P < .05). The 6-week diversity score inversely correlated with the duration of antibiotherapy (P = .0184) and parenteral feeding (P = .013). The microflora dynamics was associated with the digestive tolerance profile. Weight gain increased with increasing diversity score (P = .0428). CONCLUSION Microflora diversity settled progressively in very preterm infants. Staphylococci were the major group, and few infants were colonized with Bifidobacterium spp. Measures that may improve microflora could have beneficial effects on digestive tolerance and growth.

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the SpeI, PacI and I‐CeuI restriction maps of the four biovars of the pathogenic bacterium Brucella suis. B. suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B. melitensis, B. abortus, B. ovis and B. neotomae. Two chromosomes were also observed in the genome of B. suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B. suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome‐containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B. suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.

Atypical 16S rRNA gene copies in Ochrobactrum intermedium strains reveal a large genomic rearrangement by recombination between rrn copies.

Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria. The 16S rDNA sequences of nine O. intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp. available in data banks. Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA. The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule. Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion. Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone. Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion. In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone. Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies. The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies.

Anaeroglobus geminatus gen. nov., sp. nov., a novel member of the family Veillonellaceae.

A hitherto unknown anaerobic coccus isolated from a post-operative fluid collection was characterized by phenotypic and phylogenetic methods. 16S rDNA sequence analysis revealed an affiliation of this isolate to the family Veillonellaceae. Also, a high level of sequence similarity was observed to some oral clone sequences of Megasphaera spp. contained in the GenBank database under designations BB166, CS025 and BS073. These clones and the unknown bacterium form a well-separated phylogenetic branch that may represent a novel lineage within the family Veillonellaceae. Based on phenotypic and phylogenetic evidence, a new genus, Anaeroglobus gen. nov., is proposed for the unknown bacterium, with one species, Anaeroglobus geminatus gen. nov., sp. nov. The type strain of Anaeroglobus geminatus is strain AIP 313.00T (= CIP 106856T = CCUG 44773T). It is also suggested that the oral clones BB166, CS025 and BS073 belong to the genus Anaeroglobus.

Antianaerobic activity of moxifloxacin compared with that of ofloxacin, ciprofloxacin, clindamycin, metronidazole and β-lactams

Minimal inhibitory concentrations of moxifloxacin were compared with those of ofloxacin, ciprofloxacin, clindamycin, metronidazole and six beta-lactams for 159 anaerobes isolated from human clinical samples. Unlike other fluoroquinolones, moxifloxacin demonstrated high activity against the 76 strains of the Bacteroides fragilis group as the minimal inhibitory concentration(50) was 0.5 mg/l. Porphyromonas, Prevotella, Fusobacterium and Gram-positive anaerobic cocci were inhibited by 1 mg/l or less of moxifloxacin. It inhibited 93.7, 94.9 and 98% of the 159 strains investigated at concentrations of 1, 2 and 4 mg/l, respectively. Moxifloxacin was more potent than ofloxacin and ciprofloxacin against Gram-positive rods and anaerobic cocci. Its broad anaerobic spectrum in vitro is promising for the treatment of intra-abdominal and respiratory infections.

Prosthetic joint infection due to Veillonella dispar

Abstract Described here is the first report of a monomicrobial joint infection caused by Veillonella dispar that resulted in loosening of a prosthesis. This clinical case shows that Veillonella spp. should not be disregarded as contaminant organisms, particularly when they are isolated in pure culture from clinical specimens involving cases of septic arthritis.

Multilocus genetics to reconstruct aeromonad evolution

BackgroundAeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes.ResultsThe MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas.ConclusionsEvolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or “disease specialized” while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters.

Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella.

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5-2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

Clinical and microbiological features of Inquilinus sp. isolates from five patients with cystic fibrosis.

ABSTRACT Patients with cystic fibrosis (CF) may be colonized with unusual gram-negative bacilli whose identification is difficult and clinical impact unclear. We describe the clinical and microbiological features of five colonizations with organisms belonging to the recently described genus Inquilinus in CF patients. Isolates were identified from Burkholderia cepacia selective medium by means of 16S rRNA analysis. All of them were resistant to colistin, penicillins, cephalosporins, and monobactams but exhibited a remarkable susceptibility to imipenem. One of the five patients was transiently colonized with a nonmucoid isolate, whereas the four other patients were persistently colonized over the period of follow-up (8 to 21 months) with mucoid isolates. Pulsed-field gel electrophoresis of SpeI-digested genomic DNA was powerful for strain genotyping and demonstrated the clonality of Inquilinus sp. colonization for the two patients tested. Clinical evolution after the onset of Inquilinus was heterogeneous, but for at least one patient the lung function worsened and eradication of Inquilinus sp. was unsuccessful despite several imipenem courses. Finally, Inquilinus spp. may represent a new threat for CF patients due to their mucoid characteristic, their multiresistant pattern to antibiotics, and their ability to persist in the respiratory tract.

Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site.

Tn5Map, a Tn5 derivative containing the 18 bp I-SceI site, was delivered from a RP4-mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-SceI sites in their genomes. Digestion of the DNA from Tn5Map-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion. These digests also gave direct and independent proof for the single circular chromosome of E. coli, and for the presence of two circular chromosomes in B. melitensis and of a circular and a linear chromosome in A. tumefaciens C58 (which also contains two large circular plasmids). This rapid and versatile technique is potentially applicable to the study of the genomic organization in all Gram-negative bacteria which support Tn5 transposition. Moreover, linearization of circular replicons could be the first step for a rapid method of physical mapping.