Cross-sectional analysis of the Spanish collection

About half of the patients which had had a positive sputum culture for P. aeruginosa were female (38/75), being their median age at time of sputum collection of 25.7 (7-51) years.

Most of these patients were chronically colonized by P. aeruginosa (n=64), whereas just 11 patients showed an intermittent infection-colonization pattern. Primary patients’

characteristics are summarized in Table 4.3.

Table 4.3. Primary characteristics of patients colonized by P. aeruginosa.

CF Unit No.

patients Age in years Colonization pattern (No. patients)

Considerable genetic diversity was documented among the P. aeruginosa Spanish CF isolates. By PFGE, 70 different restriction patterns were observed among the 79 typed isolates. As well, 72 different STs were detected, each grouping 1 to 3 isolates. Up to 48 (67%) had not been previously described (http://pubmlst.org/paeruginosa/) and most resulted from new allele combinations (83.3%), as just 8 new alleles sequences were defined in this work. The Array Tube genotyping technique enabled the detection of 51 new and 14 previously described Array Tube genotypes, containing one to three isolates each (Table 4.4.).

Table 4.4. Genotyping results obtained from the analysis of the Spanish CF collection.

CF

Unit Isolate^a PFGE^b Array

Tube ST^c MLST allelic profile^c

acsA aroE guaA nuoD mutL ppsA trpE

a Different morphotypes in the same CF patient (from 1 to 4).

b Isolates exhibiting similar or identical PFGE patterns have been highlighted in grey.

c Non-previously described alleles and STs are indicated in bold.

Table 4.4. Genotyping results obtained from the analysis of the Spanish CF collection. (Cont.) CF

Unit Isolate^a PFGE^b Array

Tube ST^c MLST allelic profile^c

acsA aroE guaA nuoD mutL ppsA trpE

a Different morphotypes in the same CF patient (from 1 to 4).

b Isolates exhibiting similar or identical PFGE patterns have been highlighted in grey.

c Non-previously described alleles and STs are indicated in bold.

Table 4.4. Genotyping results obtained from the analysis of the Spanish CF collection. (Cont.) CF

Unit Isolate^a PFGE^b Array

Tube ST^c MLST allelic profile^c

acsA aroE guaA nuoD mutL ppsA trpE

a Different morphotypes in the same CF patient (from 1 to 4).

b Isolates exhibiting similar or identical PFGE patterns have been highlighted in grey.

As collected in Table 4.4., identical PFGE band patterns, ST and Array Tube genotypes were detected in P. aeruginosa isolates recovered from a pair of siblings (isolates 78 and 79).

The same PFGE band pattern was also observed in unrelated isolates, some of which match with other genotyping methodologies. Isolates from PFGE clone types 3, 30, 35 and 59 were demonstrated to belong to the same CC by MLST (Figure 4.4.) and, with the exception of isolates 31 and 32 (PFGE-30), also to a related Array Tube genotype. The most relevant case concerned a possible intrahospital cross-transmission related to isolates 66 and 68 (PFGE-59), which exhibited an identical PFGE restriction pattern and Array Tube genotype but different MLST, being ascription to different STs due to a single point mutation within mutL nucleotide sequence (nt331AC) that provokes the switch of allele 48 by allele 3, and consequently the assignation of ST1881 instead of ST348. Similarly, isolates 38, 39 and 41 (PFGE-35) were ascribed to STs 1874 and 1875, just differing in their mutL sequence by two point mutations, and to Array Tube genotypes E428 and E42A, just differing in the presence or absence of exoS. As shown, discordances within mutL nucleotide sequences were frequently involved in ascription to different STs. By contrast, isolates from PFGE clone types 31 and 48 yielded a non-related MLST allelic profile but showed the same Array Tube genotype (Table 4.4. and Figure 4.4.).

Conversely, several isolates exhibiting different PFGE restriction patterns were ascribed to the same ST and/or Array Tube genotype (Table 4.4. and Figure 4.4.).

As above described (section 3.3.3.), the Array Tube genotyping method also includes several probes to explore the accessory genome. In this sense, just isolates 66 and 68 (PFGE-59) exhibited an identical repertoire of accessory genes (data not shown). As well, isolates 38, 39 and 41 (PFGE-35) just differ in the presence or absence of fpvB gene, coding for the alternative type-I ferripyoverdine receptor (data not shown).

Figure 4.4. MST based on the MLST allelic profiles. Isolates with the same Array Tube genotype are represented with the same colour. As well, isolates ascribed to the same PFGE clone type are indicated with red lines.

Overall, ferripyoverdine receptor genes (fpvA) type I, IIa, IIb and III were detected in 23, 22, 6 and 11 isolates respectively, and the alternative type-I ferripyoverdine receptor gene fpvB was present only in 45 isolates. Ferripyoverdine receptor genes were not detected in 15 isolates, and all but two were isolates from adult patients. On average, isolates possess 2.3 genome islets and 2.4 genome islands, ranging from 0 to 5. The flagellin glycosylation island was encountered to be the most prevalent one (n=53, 67%). Nevertheless, 2 of these isolates lacked the a-type flagellin and, on the contrary, in 3 isolates expressing the a-type flagellin the flagellin glycosylation island was not detected. PAGI-1 was detected in only 45 isolates (57%) and the genomic islands of the CLC family PAGI-2/3 in 23 isolates (29%).

Two isolates harboured both PAPI-1 and PAPI-2, and 43 isolates (54%) harboured only PAPI-2; other pKLC102-like islands were detected in 30 isolates (38%). Statistical differences within the global collection and the different subsets, adult or paediatric population, were not observed (Table 4.5.).

mutL 13/127 mutL 3/48

mutL 61/3 nuoD 27/4 brothers

mutL 61/3 nuoD 27/4 guaA 70/11

mutL 7/13 trpE 7/137

Table 4.5. Prevalence (%) of ferripyoverdine receptor genes, gene islands and genome islets among the Spanish collection.

fpvA/B Fla-glyc PAGI-1/2-3 PAPI-1/2 pKLC102-like Genome Islets

Global (n=79) 77/57 67 57/29 2/54 38 87

Adult (n=61) 83/59 64 59/26 3/54 36 93

Paediatric (n=18) 72/50 78 50/39 0/56 44 72

The Array Tube genotyping tool also includes probes for detecting T3SS effectors exoS and exoU genes. An unusual low prevalence, which did not correlate with obtained results by

Non-susceptibility rates, MIC50 and MIC90 values for the Balearic Islands CF collection (January 2003 to June 2013) are summarized in Table 4.6. Up to 726 P. aeruginosa CF isolates were included in the final data analysis.

Colistin was the compound for which a minor percentage of non-susceptibility was registered (5.5%) followed by ceftazidime and meropenem, for which about 20% of the isolates showed in vitro resistance. Conversely, aztreonam and ciprofloxacin were the antibiotics exhibiting major resistance rates (44% and 73%, respectively) (Table 4.6.). Overall, the MDR rate was set in 17% and, more worrisome, 1% of the isolates met the PDR criteria.

MIC50 values for all tested compounds but aztreonam were within the susceptibility range, whereas just colistin MIC90 fell within this range (Table 4.6.). Of note, 89% of the isolates exhibited a tobramycin MIC under 64 mg/L, which is the suggested breakpoint for inhaled therapy.

Table 4.6. Non-susceptibility rates, MIC50 and MIC90 obtained for the Balearic Islands collection.

Antibiotic

TZ IP MP CI TM CO AT^a

% (I+R)^b 20 33 21 44 28 5 73

MIC50 2 3 0.38 0.5 2 1 3

MIC90 64 32 24 4 96 2 256

a AT was tested just in P. aeruginosa isolates obtained in 2012 and 2013 (n=120).

b EUCAST clinical breakpoints version 3.1. was applied (Annex 3).

Results from the antibiotic resistance analysis of the Spanish collection are collected in Table 4.8. Colistin was also the most active compound, and only three isolates (4%) were classified as resistant. Conversely, around the 60% were non-susceptible to both fluoroquinolones tested. Considering co-resistances and excluding aztreonam, 15 isolates