Collateral Sensitivity Strongly Connected Components in Real-World Clinical Surveillance Data Are Confounded by Clonal Lineage

Abstract

Collateral sensitivity (CS) - acquisition of resistance to one antibiotic inducing hypersensitivity to another - offers an evolutionary trap for multidrug-resistant pathogens. A strongly connected component (SCC) in the directed CS graph represents a closed cycle in which every drug is reachable from every other via successive CS edges. Prior evidence for CS SCCs derives from in vitro experiments; whether such structures exist in clinical surveillance data has not been tested. We mined 104,337 antibiotic susceptibility records from BV-BRC spanning four WHO critical-priority pathogens (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa; 18,821 unique isolates), applying Fisher's exact test with Benjamini-Hochberg FDR correction to all ordered antibiotic pairs, Tarjan's algorithm for SCC detection, and permutation testing (n = 1,000). A 3-node SCC in K. pneumoniae - imipenem, meropenem, tetracycline - met every criterion: empirical p = 0.001, bidirectional carbapenem-tetracycline edges at OR = 1.81-1.82 (q 850 per edge), tetracycline-specific relative to tigecycline, and stable across independent year bands. It does not survive stratification by clonal lineage. Assigning MLST sequence types to 5,276 of 5,382 genomes and pooling the same contingency tables by Cochran-Mantel-Haenszel gives within-lineage odds ratios of 0.93 and 0.95 (CMH p = 0.73 and 0.80), with confidence intervals excluding the unadjusted estimate. The two dominant strata pull in opposite directions (ST307, OR = 1.26; ST258, OR = 0.67). Permuting sequence-type labels 300 times while preserving stratum sizes leaves the odds ratio at a median of 1.76, so the collapse is attributable to lineage specifically rather than to stratification; de novo re-typing from genome sequence agreed with the assignments used in 32 of 33 isolates. The crude association is therefore confounded by clonal structure: carbapenem-resistant and tetracycline-resistant phenotypes co-occur because they are carried by different successful lineages, not because resistance to one induces susceptibility to the other. We report this as a negative result with a reusable control. Two larger clinical collateral-sensitivity analyses have been published without lineage adjustment, and the permutation test used here distinguishes genuine confounding from stratification artefact at negligible cost.