A long-standing E. coli infection of liver cysts

5.2. A long-standing E. coli infection of liver cysts

More complex scenarios were seen with a study of a patient with infected cysts (Low, 2002; Low et al., 2001). A patient suffering Caroli syndrome, which is a congenital disease frequently associated with the formation of liver cysts, was treated for frequent episodes of bacteraemia associated with E. coli colonisation of the cysts. Via surgical examination, it was estimated that the patient had ⬎100 cysts, many of which were infected. Cell densities in two

cysts, sampled surgically, were found to be 2 ⫻ 10 5 and 3.5 ⫻ 10 7 viable cells per millilitre. Over a 2-year period, five E. coli samples were recovered from

380 Ian R. Booth the blood and a further two from infected cysts during surgery. The isolates

showed unique patterns of antibiotic resistance of increasing severity and the final isolates were resistant to meropenem, imipenem, ceftazidime, and cefo- taxime as well as ciprofloxacin. Variable resistance was observed to trimetho- prim, but the isolates were sensitive to gentamicin throughout (Low et al., 2001). PFGE analysis suggested isolates that derived from a single infecting clone which then diversified. The clonal nature of the isolates was confirmed by the discovery that the ompC gene sequence had diverged significantly from that observed in E. coli K-12 and pathogenic E. coli (an observation subse- quently extended to other clinical isolates; J. Park, F. MacKenzie, I. M. Gould, and I. R. Booth, unpublished data). Sequencing the ompC gene demonstrated that it had diverged more than 9% from the E. coli K-12 sequence and that this was more than double the rate of change of other genes in the same strains and higher than the observed rate of change in E. coli O157 and in two commensal

E. coli obtained from the faeces of healthy volunteers (Low et al., 2001). All seven isolates from the patient had the same basic ompC gene sequence (Low et al., 2001). However, the strains progressively accumulated mutations in the ompC gene but did not at any stage express OmpF despite the presence of a functional structural gene. In the later isolates, the mutations rendered the OmpC protein unstable and consequently lowered levels of the protein were observed in the outer membrane.

A complex pattern of evolution of the ␤-lactamases was observed in these clinical isolates. Each isolate possessed the same dominant TEM-class enzyme (pI ⬃5), but possessed several other ␤-lactamase activities with more acidic (strains 1–4) or slightly more alkaline (strain 5) pI values. At least two different TEM-class proteins were detected in the first isolate by proteomic analysis of the periplasmic fractions. The first isolates had three large plasmids and this reduced to one by the final isolates. No attempt was made to study their transmission to other E. coli. The isolation of the E. coli in the individual liver cysts makes it unlikely that the variants arose by plasmid acquisition and would be consistent with at least two bla TEM genes in the first isolate and their divergence and selec- tion during subsequent antibiotic regimes. In this regard, it was notable that while the first two isolates were non-mutators, the later isolates displayed a very strong mutator phenotype (J. Park, F. MacKenzie, I. M. Gould, and I. R. Booth, unpublished data). Not only did the isolates alter their plasmid-encoded ␤-lactamase pattern, but they also evolved increased expression of chromosomal AmpC due to mutations in the promoter and regulatory regions. This evolution was most marked over the period after the isolate had acquired mutator proper- ties (J. Park, F. MacKenzie, I. M. Gould, and I. R. Booth, unpublished data).

The importance of the mutator status is also supported by the analysis of ciprofloxacin resistance (Figure 3). Ciprofloxacin had been used in treatment prior to the storage of the first blood culture for analysis. The MICs of the first

Evolution of Antibiotic Resistance within Patients 381

Caz, Ctx, Cxm*

Figure 3. The evolution of drug resistance in a patient with infected liver cysts. The patient first presented at the end of 1994 and was monitored for 3 years with bacterial samples from the blood and cysts being acquired at intervals. The figure shows the time line for the infec- tion, a summary of the antibiotic treatment (see Low et al., 2001 for further details) and the times at which samples were obtained and analysed. The lower half of the diagram indicates

a Split Tree Decomposition (STD) analysis of all the molecular data obtained from the patient samples. Isolates 3–7 were subsequently discovered to be mutators. Abbreviations: Cip, ciprofloxacin with MIC indicated as a superscript; 2 L and 5 L , the superscript indicates the isolate was obtained directly from a cyst; Leu and Pur (as subscripts) indicate the two isolates with auxotrophies. The STD analysis was performed by Dr Martin Maiden, University of Oxford.

two strains were in the sensitive range, but analysis of the gyrA gene sequence revealed one of the commonest mutations that gives rise to low-level resistance (D87Y) in the first two isolates and in all the subsequent strains. In the third isolate, the MIC rose to 128 mg/L but fell in subsequent isolates to 8 (isolate

4) and 16 (isolates 5, 6, and 7) mg/ml. Four of these strains were found to have additional mutations in gyrA (S83L) and parC (E84G). These mutations alone cannot explain the MIC values since isolate 1, 2, and 4 have the same muta- tions in gyrA, but have quite different MIC values and, similarly, isolates 3, 5,

6, and 7 have the same mutations in both gyrA and parC but have MIC values ranging from 128 to 8 mg/L. Mutations affecting efflux systems must have arisen in some of the strains, but to date none have been detected. The data illu- minate two points. First, the multiple mutations within the components that

382 Ian R. Booth can give rise to ciprofloxacin resistance point to the importance of the evolution

of the mutator status in these strains. Second, some mutations are present in the first and in all subsequent isolates, but others are present only transiently in the isolated strains. This pattern of the mutations in sequential isolates points to the separate and independent evolution of the antibiotic resistance. This is graphically illustrated in Split decomposition analysis of all the sequence data, suggesting that the infected cysts provided unique environments in which different evolutionary paths were followed.

Analysis of the total number of changes that took place during the evolution from a core infecting population led to the conclusion that the strains evolved in parallel (Low et al., 2001) (Figure 3). In this patient, we believe that sepa- rate cysts created unique environments in which mutation and selection took place at different rates and with separate outcomes. At intervals, the patient presented with a bacteraemia as a result of one, or more, of the cysts leaking their microbial load. The presence of the same mutations in some isolates but not in others that were isolated subsequently suggests that at different stages, some cysts became infected with organisms that originated in another cyst possibly during the bacteraemia. The mutator status of the later isolates from the liver cysts is interesting and is consistent with the prevalence of mutators in the antibiotic-resistant P. aeruginosa flora of CF patients and in uropathogenic

E. coli (Denamur et al., 2002; Oliver et al., 2000). This is possibly the first notification of mutator organisms from a tissue within the body cavity since previous isolates have been from lung and bladder. It may also explain the observation that two of the isolates had demonstrable separate auxotrophies while others had a more general impairment of growth on minimal medium (Low et al., 2001).