F. Jauréguy

Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains

BackgroundExtraintestinal pathogenic Escherichia coli (ExPEC) strains represent a huge public health burden. Knowledge of their clonal diversity and of the association of clones with genomic content and clinical features is a prerequisite to recognize strains with a high invasive potential. In order to provide an unbiased view of the diversity of E. coli strains responsible for bacteremia, we studied 161 consecutive isolates from patients with positive blood culture obtained during one year in two French university hospitals. We collected precise clinical information, multilocus sequence typing (MLST) data and virulence gene content for all isolates. A subset representative of the clonal diversity was subjected to comparative genomic hybridization (CGH) using 2,324 amplicons from the flexible gene pool of E. coli.ResultsRecombination-insensitive phylogenetic analysis of MLST data in combination with the ECOR collection revealed that bacteremic E. coli isolates were highly diverse and distributed into five major lineages, corresponding to the classical E. coli phylogroups (A+B1, B2, D and E) and group F, which comprises strains previously assigned to D. Compared to other strains of phylogenetic group B2, strains belonging to MLST-derived clonal complexes (CCs) CC1 and CC4 were associated (P < 0.05) with a urinary origin. In contrast, no CC appeared associated with severe sepsis or unfavorable outcome of the bacteremia. CGH analysis revealed genomic characteristics of the distinct CCs and identified genomic regions associated with CC1 and/or CC4.ConclusionOur results demonstrate that human bacteremia strains distribute over the entire span of E. coli phylogenetic diversity and that CCs represent important phylogenetic units for pathogenesis and comparative genomics.

Real-Time PCR for Quantitative Analysis of Human Commensal Escherichia coli Populations Reveals a High Frequency of Subdominant Phylogroups

ABSTRACT Escherichia coli is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of E. coli in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the E. coli population, i.e., 105 CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the E. coli microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.

A mosaic transposon encoding OXA-48 and CTX-M-15: towards pan-resistance

Sir, Reports of OXA-48-producing Enterobacteriaceae have recently dramatically increased worldwide, in particular in the Middle East and Europe. The dissemination of the blaOXA-48 gene among various enterobacterial species is mainly related to the dissemination of a single 62 kb IncL/M conjugative plasmid, which carries the blaOXA-48 gene as the only resistance determinant. The co-production of an extended-spectrum b-lactamase (ESBL) in 80% of the OXA-48 producers (L. Poirel and P. Nordmann, unpublished results) is therefore due to the acquisition of additional plasmids. The blaOXA-48 gene was first identified as part of Tn1999, a composite transposon made of two copies of the insertion sequence IS1999 (Figure 1). Two other Tn1999-derivative structures have been reported. Transposon Tn1999.2, identified in various enterobacterial isolates from Turkey, differs from Tn1999 by the insertion of insertion sequence IS1R into the left-hand IS1999 copy (Figure 1). Transposon Tn1999.3 has been identified from a single Escherichia coli isolate recovered in Italy, being similar to Tn1999.2 but harbouring a second copy of IS1R (Figure 1). We report here the identification of two enterobacterial isolates harbouring the blaOXA-48 gene encoded by a novel Tn1999 transposon derivative that additionally contains the ESBL gene blaCTX-M-15. E. coli ESS-1 and Enterobacter cloacae ESS-2 were isolated in France in September 2011 from a rectal swab of a 70-year-old patient previously hospitalized in Algeria. Identification of those two isolates was performed using the API 20E system (bioMérieux, La Balme-les-Grottes, France). Susceptibility testing done by the disc diffusion method and MICs determined by Etest (bioMérieux) and interpreted according to the CLSI guidelines revealed that the two isolates were resistant to penicillins, penicillin/b-lactamase inhibitor combinations and broad-spectrum cephalosporins, and had decreased susceptibility to carbapenems. The MICs of imipenem, ertapenem and meropenem were 0.5, 0.5 and 0.12 mg/L, respectively, for E. coli ESS-1 and 0.75, 3 and 0.38 mg/L, respectively, for E. cloacae ESS-2. The patient did not develop any clinical infection and these organisms were considered to be colonizers. PCR analysis and sequencing, performed as described previously, identified the blaOXA-48 carbapenemase gene and the blaCTX-M-15 ESBL gene in both isolates. E. coli ESS-1 additionally harboured the blaTEM-1 gene. Conjugation experiments using E. coli ESS-1 and E. cloacae ESS-2 as donors and E. coli J53 as recipient were performed as described, using ertapenem (0.5 mg/L) and azide (100 mg/L) for selection. A single type of transconjugant was obtained for both donor strains. The E. coli transconjugants displayed the exact same resistance profile and were resistant to penicillins, penicillin/b-lactamase inhibitor combinations and broadspectrum cephalosporins, and were of decreased susceptibility to carbapenems. For these two transconjugants, the MICs of imipenem, ertapenem and meropenem were 0.5, 0.38 and 0.12 mg/L, respectively. PCR experiments showed that both transconjugants co-harboured the blaOXA-48 and blaCTX-M-15 genes. Plasmid analysis showed that both E. coli transconjugants harboured a single 66 kb plasmid. By using specific primers designed from the blaOXA-48-positive reference plasmid pOXA-48a from Klebsiella pneumoniae 11978, we showed that the single plasmid of both E. coli transconjugants belonged to the IncL/M group, as did most of the OXA-48-positive isolates. Mapping of the blaOXA-48-flanking regions by PCR revealed a peculiar genetic structure. The blaOXA-48 gene was part of a novel transposon named Tn1999.4, consisting of Tn1999.2 truncated by another transposon. This transposon, named Tn2015, was 4603 bp long and consisted of ISEcp1, blaCTX-M-15 and a truncated Tn2-type transposase gene (Figure 1). Transposon Tn2015 was bracketed by two imperfect 14 bp inverted-repeat sequences, the inverted repeat left (IRL) of ISEcp1 and an inverted repeat right (IRR)-like sequence sharing 6 out of 14 bp with the IRL of ISEcp1 (Figure 1). Tn2015 was inserted into the LysR-encoding gene of Tn1999 and a 5 bp duplication of the target site (TTTAA) was identified as a signature of the ISEcp1mediated transposition event (Figure 1). Mapping of the Tn1999.4-flanking regions revealed that this transposon was inserted into the tir gene, as observed for Tn1999 in plasmid pOXA-48a. Since the insertion of Tn1999.4 was identified at the same position as Tn1999, this suggested that Tn1999.4 likely evolved from Tn1999.2 by the insertion of another transposon element into Tn1999.2, once it had been incorporated onto the plasmid backbone. We describe here a novel Tn1999 derivative, which actually corresponds to a mosaic structure in which the transposon carrying the carbapenemase gene blaOXA-48 is associated with that carrying the ESBL gene blaCTX-M-15. This association gave rise to a complex genetic structure capable of compromising the efficiency of all available b-lactams. To our knowledge, this is the first identification of a transposon carrying both an ESBL and a carbapenemase gene, being a significant evolutionary step towards pan-resistance. Considering that at least one copy of IS1999 and subsequently its transposase gene is still intact, we may speculate that the whole structure might still be functional and might mobilize the two b-lactamase genes as a whole. Research letters

Gram-negative bacteremia: Which empirical antibiotic therapy? ☆

PURPOSE Given the increasing frequency of cefotaxime-resistant strains, third-generation cephalosporins (3GC e.g. cefotaxime, ceftriaxone) might not be recommended any longer as empirical antibiotic therapy for community-acquired Gram-negative bacteremia (CA-GNB). PATIENTS AND METHODS We conducted a multicenter prospective descriptive study including patients with CA-GNB. RESULTS Two hundred and nineteen patients were included. Escherichia coli and Pseudomonas aeruginosa were the most frequently isolated species in 63% (n=138) and 11% (n=24) of the cases, respectively. The prevalence of cefotaxime-resistance reached 18% (n=39) mostly due to intrinsic resistance (27 cases, 12%). The presence of invasive material (P<0.001), the origin of the patient (Paris region or West of France) (P=0.006), and home health care (P<0.001) were variables predicting resistant GNB. The negative predictive value for resistance in patients with invasive material coming from the West of France, or without invasive material and with home health care was 94%. The positive predictive value for patients with invasive material living in Paris, or without invasive material and with home health care only reached 58 and 54%, respectively. CONCLUSIONS Using 3GC for CA-GNB due to cefotaxime-resistant strains was relatively frequent, ESBL-producing Enterobacteriaceae being rarely involved. Our study highlights the role of local epidemiology; before any changes to first-line antibiotic therapy, local epidemiological data should be taken into account.

Diagnosis of a non-toxigenic tox gene-bearing strain of Corynebacterium diphtheriae in a young male back from Senegal to France

Abstract Cutaneous diphtheria is uncommon in Europe. In this study, we report a case of imported cutaneous infection due to a non-toxigenic but tox gene-bearing (NTTB) strain of Corynebacterium diphtheriae. The NTTB strains are recognized as emerging pathogens across Europe, and physicians and bacteriologists should be aware of the circulation of these strains.

MALDI-TOF MS contribution to diagnosis of melioidosis in a nonendemic country in three French travellers.

Melioidosis is an endemic disease in Southeast Asia and northern Australia. An increasing number of cases are being reported in nonendemic countries, making the diagnosis less obvious. We discuss the identification of Burkholderia pseudomallei using matrix-assisted desorption ionization–time of flight mass spectrometry on the occasion of recent cases of imported melioidosis in French travellers.