Investigating the human jejunal microbiota

Descriptions of the small intestinal microbiota are deficient and conflicting. We aimed to get a reliable description of the jejunal bacterial microbiota by investigating samples from two separate jejunal segments collected from the luminal mucosa during surgery. Sixty patients with morbid obesity selected for elective gastric bypass surgery were included in this survey. Samples collected by rubbing a swab against the mucosa of proximal and mid jejunal segments were characterized both quantitatively and qualitatively using a combination of microbial culture, a universal quantitative PCR and 16S deep sequencing. Within the inherent limitations of partial 16S sequencing, bacteria were assigned to the species level. By microbial culture, 53 patients (88.3%) had an estimated bacterial density of < 1600 cfu/ml in both segments whereof 31 (51.7%) were culture negative in both segments corresponding to a bacterial density below 160 cfu/ml. By quantitative PCR, 46 patients (76.7%) had less than 104 bacterial genomes/ml in both segments. The most abundant and frequently identified species by 16S deep sequencing were associated with the oral cavity, most often from the Streptococcus mitis group, the Streptococcus sanguinis group, Granulicatella adiacens/para-adiacens, the Schaalia odontolytica complex and Gemella haemolysans/taiwanensis. In general, few bacterial species were identified per sample and there was a low consistency both between the two investigated segments in each patient and between patients. The jejunal mucosa of fasting obese patients contains relatively few microorganisms and a core microbiota could not be established. The identified microbes are likely representatives of a transient microbiota and there is a high degree of overlap between the most frequently identified species in the jejunum and the recently described ileum core microbiota.publishedVersio

Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae

A multidrug-resistant (MDR) strain of Haemophilus influenzae, Hi-228, with phenotypic resistance toward ampicillin, cefotaxime, chloramphenicol, gentamicin, and azithromycin, was isolated in Oslo, Norway. The strain was part of a clonal outbreak (2016–2017) comprising five ST143 strains with identical resistotypes. Hi-228 carries a novel integrative and conjugative element (ICE), Tn7100, contributing to this remarkable and previously unreported MDR profile. Tn7100 contains the following resistance genes: blaTEM−1B, catA2, aac(6′)-Im, aph(2″)-Ib, mef (E), and mel. The latter four are previously unreported or rarely reported in H. influenzae. In this study, we investigated the genetic environment, mechanisms of transfer, impact on phenotypic susceptibility, and fitness cost of this ICE. We found that Tn7100 has an overall structure similar to the previously described ICE Tn6686, with blaTEM−1B and catA2 carried by Tn3 and Tn10, respectively. The major difference between Tn7100 and Tn6686 is that Tn7100 lacks tet(B) but carries the resistance gene pairs aac(6′)-Im and aph(2″)-Ib and mef (E) and mel. The gene pairs are located on the novel transposable elements Tn7470 and Tn7471, which have high sequence identities to a plasmid in Enterobacterales and an ICE in streptococcal species, respectively. Tn7100 does circularize and is transferable, however, at a low frequency. Head-to-head competition experiments showed that uptake of Tn7100 reduces bacterial fitness. Our study shows that MDR strains are capable of clonal spread and that the H. influenzae supragenome comprises an increasingly wide range of transferable resistance genes, with evidence of transfer from unrelated genera. The findings offer a glimpse into the genome dynamics of H. influenzae, highlighting the importance of rational antibiotic usage to contain antimicrobial resistance and the emergence of MDR strains in this important pathogen

Emergence of clonally related multidrug resistant haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013

Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T?+?N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T?+?L389F?+?N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3 clone with TEM-1 beta-lactamase and co-resistance to ciprofloxacin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. Prior to this study, no multidrug resistant high-rPBP3 H. influenzae had been reported in Norway. Intensified surveillance of antimicrobial resistance is needed to guide empiric therapy. </jats:p

Investigating the human jejunal microbiota

Descriptions of the small intestinal microbiota are deficient and conflicting. We aimed to get a reliable description of the jejunal bacterial microbiota by investigating samples from two separate jejunal segments collected from the luminal mucosa during surgery. Sixty patients with morbid obesity selected for elective gastric bypass surgery were included in this survey. Samples collected by rubbing a swab against the mucosa of proximal and mid jejunal segments were characterized both quantitatively and qualitatively using a combination of microbial culture, a universal quantitative PCR and 16S deep sequencing. Within the inherent limitations of partial 16S sequencing, bacteria were assigned to the species level. By microbial culture, 53 patients (88.3%) had an estimated bacterial density of < 1600 cfu/ml in both segments whereof 31 (51.7%) were culture negative in both segments corresponding to a bacterial density below 160 cfu/ml. By quantitative PCR, 46 patients (76.7%) had less than 104 bacterial genomes/ml in both segments. The most abundant and frequently identified species by 16S deep sequencing were associated with the oral cavity, most often from the Streptococcus mitis group, the Streptococcus sanguinis group, Granulicatella adiacens/para-adiacens, the Schaalia odontolytica complex and Gemella haemolysans/taiwanensis. In general, few bacterial species were identified per sample and there was a low consistency both between the two investigated segments in each patient and between patients. The jejunal mucosa of fasting obese patients contains relatively few microorganisms and a core microbiota could not be established. The identified microbes are likely representatives of a transient microbiota and there is a high degree of overlap between the most frequently identified species in the jejunum and the recently described ileum core microbiota

Multilocus sequence typing and ftsI sequencing: a powerful tool for surveillance of penicillin-binding protein 3-mediated beta-lactam resistance in nontypeable Haemophilus influenzae

Background Beta-lactam resistance in Haemophilus influenzae due to ftsI mutations causing altered penicillin-binding protein 3 (PBP3) is increasing worldwide. Low-level resistant isolates with the N526K substitution (group II low-rPBP3) predominate in most geographical regions, while high-level resistant isolates with the additional S385T substitution (group III high-rPBP3) are common in Japan and South Korea. Knowledge about the molecular epidemiology of rPBP3 strains is limited. We combined multilocus sequence typing (MLST) and ftsI/PBP3 typing to study the emergence and spread of rPBP3 in nontypeable H. influenzae (NTHi) in Norway. Results The prevalence of rPBP3 in a population of 795 eye, ear and respiratory isolates (99% NTHi) from 2007 was 15%. The prevalence of clinical PBP3-mediated resistance to ampicillin was 9%, compared to 2.5% three years earlier. Group II low-rPBP3 predominated (96%), with significant proportions of isolates non-susceptible to cefotaxime (6%) and meropenem (20%). Group III high-rPBP3 was identified for the first time in Northern Europe. Four MLST sequence types (ST) with characteristic, highly diverging ftsI alleles accounted for 61% of the rPBP3 isolates. The most prevalent substitution pattern (PBP3 type A) was present in 41% of rPBP3 isolates, mainly carried by ST367 and ST14. Several unrelated STs possessed identical copies of the ftsI allele encoding PBP3 type A. Infection sites, age groups, hospitalization rates and rPBP3 frequencies differed between STs and phylogenetic groups. Conclusions This study is the first to link ftsI alleles to STs in H. influenzae. The results indicate that horizontal gene transfer contributes to the emergence of rPBP3 by phylogeny restricted transformation. Clonally related virulent rPBP3 strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. The need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rPBP3 strains is underlined. Combining MLST and ftsI/PBP3 typing is a powerful tool for this purpose