Zona de influencia del Colegio de la Real Legión Británica

Historically, AMRGs have not been widely associated with macrolide resistance in NTHi, and studies reporting on macrolide resistance mechanisms in NTHi have typically been unable to detect AMRGs among macrolide-resistant isolates. In a study by Peric et al. (2004) involving 31 macrolide-resistant NTHi isolates, no isolate was found to carry mef(A),

erm(A), erm(B), or ere(A).253 In a follow-up to that study including many of the same isolates and some new isolates, Bogdanovich et al. (2006) also failed to detect the presence of

mef(A), erm(A), erm(B), or ere(A).252 More recently, Seyama et al. (2016) were not able to detect mef(A) or erm(B) among a smaller collection of 7 clarithromycin-resistant isolates, with resistance instead being attributed to increased transcription of the acrB gene as a result of alterations of the acrR regulatory gene,251 and Pettigrew et al. did not detect erm(A),

†_{FOOTNOTE: The content of the literature review is based on publications up to December 16}th _{2016 in} account of a January 6th 2017 thesis submission date. However, a very recent study – available in advance access online from December 20th 2016 – has reported the detection of an isolate of H. influenzae with

mef(A).254 This will not be discussed in the literature review or Chapters 3-7, but is briefly discussed in the general discussion of Chapter 8.

85 erm(B), erm(C), erm(F) or mef(A) in any isolates (n = 200, or 100 persistent strain pairs) included in their study (MIC increases were either attributed to L22 alterations or the mechanism was not determined).29

In recent years, there has been one report where AMRGs have been widely reported among NTHi isolates. In a study by Roberts et al. (2011) regarding the prevalence of particular macrolide resistance genes among CF isolates of NTHi, the authors aimed to characterise the macrolide resistance mechanisms in NTHi isolates taken from CF patients enrolled in a placebo-controlled azithromycin trial. Each isolate was tested for phenotypic macrolide resistance by E-test MIC, as well as for the presence of L4 and L22 mutations (23S rRNA was not investigated), and the erm(A), erm(B), erm(C), erm(F) and (undifferentiated) mef(A) genes. The authors found that 25.5% of their strains were resistant to azithromycin and erythromycin (using BSAC interpretive criteria; erythromycin MIC > 8 µg/mL; azithromycin MIC > 4 µg/mL), and 73.6% were intermediate to both macrolides (using BSAC interpretive criteria; 1 µg/mL ≤ erythromycin MIC ≤ 8 µg/mL; 0.5 µg/mL ≤ azithromycin MIC ≤ 4 µg/mL). None of these resistant strains carried L4 or L22 mutations, but 93% of all the strains included in the study (regardless of phenotypic macrolide resistance profile) carried at least one of erm(B) (31% prevalence), erm(F) (29% prevalence) and mef(A) (74% prevalence). The remaining isolates that were negative for these 3 genes were additionally tested for and found to carry at least one of erm(A) and erm(C), resulting in 100% of the isolates included in the study carrying at least one AMRG. The author also note that erm(A) and erm(C) prevalence may have been underestimated as only a small number of the isolates in the study were tested for these genes.93

Furthermore, the authors demonstrated that select donors were able to transfer these genes to

H. influenzae Rd KW20 as well as E. faecalis JH2-2 via conjugation. The H. influenzae

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24-fold increase in azithromycin MIC compared to the recipient, while the E. faecalis

transconjugants demonstrated a 16-fold increase in erythromycin MIC compared to the recipient.93 These findings suggested that these genes were associated with mobile genetic elements, and that NTHi could act as a donor and recipient of these genes. Given that these genes are detectable among respiratory bacteria and P. aeruginosa, the authors speculated that selective pressure via continuous antibiotic exposure associated with CF therapy might facilitate the exchange of these AMRGs in vivo.93

The ability to successfully transfer AMRGs to H. influenzae by conjugation was not a novel finding. Chung et al. (1999) were able to transfer erm(F) to H. influenzae RD8 (erythromycin MIC = 1 µg/mL) from both Prevotella bivia and Haemophilus aphrophilus (since re- classified as Aggregatibacter aphrophilus), with transconjugants exhibiting increased erythromycin MICs (64 µg/mL),158 while Luna et al. (2000) have previously described the transfer of mef from S. pneumoniae to H. influenzae RD8 (phenotype changes were not reported).255 However, this study represented the first report of erm(A), erm(C) and erm(F) among clinical NTHi, and the authors also state that this is the second report of mef(A) and

erm(B) among NTHi isolated from CF patients.93 Furthermore, the authors of the study go on to state that there may be some correlation between the acquisition of these genes by NTHi and the development of macrolide resistance in NTHi. This was based on the observation that 56% of the macrolide-resistant NTHi isolates in the study carried at least 2 macrolide genes. By comparison, 23% of the isolates that were defined as intermediate to macrolides carried at least 2 genes.93 However, it is worth noting that the MIC values of many of the isolates included in the study did not appear to be highly raised compared to a typical wild-type strain despite carrying at least one AMRG. Many isolates carrying an erm

gene were defined at “intermediate” according to the MIC results and did not demonstrate the high level resistance that would be typically be produced by these genes. Out of 7 isolates in

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the study that carried all of mef(A), erm(B) and erm(F), only 2 had MIC values falling above the resistance breakpoint, with the remaining defined as “intermediate”, and there was 1 particular isolate that carried erm(B) and mef(A) simultaneously but was susceptible to erythromycin.93 The reason for these particular findings is not clear but suggests that there may be other factors involved that allow for these genes to grant macrolide resistance in NTHi.