ABSTRACT
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: bla TEM-1B, catA2, aac(6')-Im, aph(2â³)-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 bla TEM-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(2â³)-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.
ABSTRACT
BACKGROUND: We evaluated if flowcytometry, using Sysmex UF-5000, could improve diagnosis of urinary tract infections by rapid identification of culture negative and contaminated samples prior to culture plating, thus reducing culture plating workload and response time. We also evaluated if it is possible to reduce the response time for antibiotic susceptibility profiles using the bacteria information flag on Sysmex UF-5000 to differentiate between Gram positive and negative bacteria, followed by direct Antibiotic Susceptibility Testing (dAST) on the positive urine samples. METHODS: One thousand urine samples were analyzed for bacteria, white blood cells and squamous cells by flowcytometry before culture plating. Results from flowcytometric analysis at different cut-off values were compared to results of culture plating. We evaluated dAST on 100 urine samples that were analyzed as positive by flowcytometry, containing either Gram positive or Gram negative bacteria. RESULTS: Using a cut-off value with bacterial count ≥100.000/mL and WBCs ≥10/µL, flowcytometry predicted 42,1% of samples with non-significant growth. We found that most contaminated samples contain few squamous cells. For 52/56 positive samples containing Gram negative bacteria dAST was identical to routine testing. Overall, there was concordance in 555/560 tested antibiotic combinations. CONCLUSION: Flowcytometry offers advantages for diagnosis of urinary tract infections. Screening for negative urine samples on the day of arrival reduces culture plating and workload, and results in shorter response time for the negative samples. The bacteria information flag predicts positive samples containing Gram negative bacteria for dAST with high accuracy, thus Antibiotic Susceptibility Profile can be reported the day after arrival. For the positive samples containing Gram negative bacteria the concordance was very good between dAST and Antibiotic Susceptibility Testing in routine. For positive samples containing Gram positive bacteria the results were not convincing. We did not find any correlation between epithelial cells and contamination.