Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae

Abstract

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

Figure 1. A colistin-resistant subpopulation increases in frequency during in vivo infection

a, Testing of E. cloacae clinical isolate R/S by colistin Etest, with drug concentration indicated in μg ml⁻¹. Colonies within the zone of inhibition indicate a colistin-resistant subpopulation. Data are representative of more than ten Etests. b, Population analysis profile of R/S as well as colistin-susceptible and -resistant E. cloacae clinical isolates (n = 3). c, Percentage of the colistin-resistant subpopulation in R/S in antibiotic-free media, after 24 h treatment with 100 μg ml⁻¹ colistin and after 8 h subculture of the colistin-treated culture in antibiotic-free media. ‘Colistin resistant (%)’ represents the number of c.f.u. in each culture that can grow on media containing 100 μg ml⁻¹ colistin, as a percentage of the total c.f.u. in the culture (n = 3). d, Colistin-resistant c.f.u. and total c.f.u. of R/S during 7 h treatment with 100 μg ml⁻¹ colistin in liquid culture (n = 3). e, Pre-infection inoculum (black bar) was used to infect mice and peritoneal lavage was performed and collected 24 h later and plated to calculate per cent colistin-resistant c.f.u. (n = 5). Mice were treated at 8, 14 and 20 h with colistin (grey bar) or PBS (red bar). Error bars represent standard error of the mean (s.e.m.) (Mann–Whitney test, *P < 0.05, **P < 0.01).

Figure 2. Innate immune host defences are required for the increased frequency of the colistin-resistant subpopulation during infection

a, Mice pretreated with PBS liposomes (control, grey) or clodronate liposomes (to deplete macrophages, red) were infected with R/S (pre-infection, black). After 8 h, peritoneal lavage fluid was collected and plated to calculate per cent colistin resistance (n = 5). b, Murine bone-marrow-derived macrophages were untreated or pretreated with cytochalasin D, infected with R/S, and per cent colistin resistance was calculated at the indicated time points (n = 6). c–e, R/S was either untreated or treated with the indicated amounts of H₂O₂ (c), lysozyme (d) or CRAMP (e) for 5 h, and per cent colistin resistance was calculated (n = 3). f, Wild-type (WT, grey) or triple knockout (TKO, red) mice lacking the gp91 subunit of the NADPH oxidase, lysozyme and CRAMP were infected with R/S (pre-infection, black). At 8 h post-infection, peritoneal lavage fluid was collected and plated to calculate per cent colistin resistance (n = 5). Data are compiled from two independent experiments. Error bars represent s.e.m. Mann–Whitney test (**P < 0.01) in a and f; Student’s two-tailed t-test (*P < 0.05) in b.

Figure 3. R/S is refractory to colistin during infection and leads to colistin treatment failure

a,b, Mice infected with R/S or the susceptible isolate were treated with colistin at 8, 14 and 20 h. Numbers of c.f.u. were quantified at 24 h in the peritoneal lavage fluid (a) and liver (b) (n = 5). c, Mice pretreated with PBS (left and middle panels) or clodronate (right panel) liposomes were infected with R/S and treated with colistin at 0 h (left panel) or 4 h (middle and right panels). A second dose of colistin was administered 2 h after the first; 2 h later, peritoneal lavage fluid was plated to enumerate c.f.u. (n = 5). d,e, Survival of mice infected with R/S or the colistin-susceptible isolate. Mice were treated with colistin or PBS starting at 12 h post-infection, with additional doses given every 6 h thereafter. Surviving mice were monitored until day 24 (n = 5). Error bars represent s.e.m.; centre values represent the median. Mann–Whitney test: *P < 0.05, **P < 0.01, NS = not significant.

Figure 4. PhoQ is required for the presence of the colistin-resistant subpopulation

a, Colistin Etest of R/S, ΔphoQ and the complement (phoQ-comp) strains, with drug concentration indicated in μg ml⁻¹. Colonies within the zone of inhibition indicate a colistin-resistant subpopulation. Data are representative of two Etests. b, Population analysis profile of R/S, ΔphoQ and colistin-susceptible and -resistant E. cloacae strains (n = 3). c, Mice infected with R/S or ΔphoQ were treated with colistin at 8, 14 and 20 h. Values of c.f.u. were quantified at 24 h in the peritoneal lavage fluid (n = 5). d, Survival of mice infected with R/S (top) or ΔphoQ (bottom). Mice were treated with colistin or PBS starting at 12 h post-infection, with additional doses given every 6 h thereafter (n = 5). Error bars represent s.e.m.; centre values represent the median. Mann–Whitney test: *P < 0.05, NS = not significant.

Figure 5. Clinical isolate harbouring an undetected colistin-resistant subpopulation causes a lethal, antibiotic-resistant infection

a, PAP of E. cloacae clinical isolate R/S-lo compared to R/S and the colistin-susceptible and -resistant isolates (n = 3). b,c, Infection of mice with the colistin-susceptible isolate (b) or R/S-lo with or without colistin treatment (c) every 6 h and initiated 12 h post-infection (n = 5). Surviving mice were monitored until day 24. d–f, Colistin Etest results, with drug concentration indicated in μg ml⁻¹, of R/S-lo from pre-infection inoculum (d), peritoneal lavage sample from a mouse infected for 8 h (e) and the peritoneal lavage sample subcultured overnight in drug-free media (f) (n = 5). Colonies in the zone of inhibition (e, red arrows) indicate resistant bacteria. Images are representative of five individual samples. g, Samples from d–f were plated to determine per cent colistin resistance (n = 5). Error bars represent s.e.m.