An alternative penicillin-binding protein involved in Salmonella relapses following ceftriaxone therapy

Abstract

Background: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy.

Methods: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2_SAL or PBP3_SAL. Affinity of PBP2_SAL and PBP3_SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model.

Findings: S. Typhimurium does not express PBP2_SAL or PBP3_SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3_SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3_SAL was unable to cause relapse in mice following ceftriaxone therapy.

Interpretation: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3_SAL, remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3_SAL with novel drugs.

Keywords: Ceftriaxone; Intracellular; Penicillin-binding protein; Phagosome; Relapse; Salmonella enterica.

Fig. 1

S. Typhimurium produces the pathogen-specific PBP2_SAL and PBP3_SAL in acidic pH and during infection. (a) Levels of PBP2, PBP2_SAL, PBP3, and PBP3_SAL determined by western blot in bacteria grown in the indicated media and pH values. For these assays, bacteria expressing functional 3x-flag-tagged PBP2_SAL and PBP3_SAL, were used; (b) replacement of PBP2/PBP3 by PBP2_SAL/PBP3_SAL occurring in minimal medium PCN adjusted to pH 4.6. Note that such replacement is favoured at the lowest pH value tested; (c) production of PBP2_SAL/PBP3_SAL prevail over that of PBP2/PBP3 in intracellular bacteria; (d) PBP2_SAL and PBP3_SAL replace PBP2 and PBP3 in bacteria colonizing the spleen of susceptible mice used in a murine typhoid model. The assays were repeated in at least two independent biological replicates.

Fig. 2

PBP3_SAL has low affinity for binding of Bocillin-650, a fluorescent reagent that detects PBPs. The binding was performed at the indicated pH values. The upper panels refer to levels of the respective PBPs, the lower panels to the signal obtained with the fluorescent Bocillin-650. Note that PBP3_SAL binds Bocillin-650 with low efficiency at all pH values tested, despite acidic pH the optimal for its function. The assays were repeated in three independent biological replicates.

Fig. 3

PBP3_SAL decreases ceftriaxone susceptibility at acidic pH. (a) Bacterial viability was tested in the indicated strains in PCN minimal medium at pH values 7.4 and 4.6 in the presence/absence of ceftriaxone 5 µg/mL. While the antibiotic drastically reduces viability at neutral pH (condition of PBP3 production), it only affects bacterial viability in acidic pH in the presence of PBP3_SAL. (b) MIC values obtained by the E-test assay for different beta-lactams that bind with high affinity to PBP3: aztreonam (ATM), cefotaxime (CTX), ceftazidime (CAZ), cefuroxime (CXM) and cephalotin (KF). (c) Representative E-test assays showing a marked increase in the MIC value to aztreonam (ATM) in response to acid pH (4.6) that is further enhanced in the ΔPBP3 mutant, expressing only PBP3_SAL. The assays were repeated in three independent biological replicates.

Fig. 4

PBP3_SAL allows S. Typhimurium to resist antibiotic (ceftriaxone) therapy and facilitate relapses. One group of susceptible mice (lower panel) was treated with ceftriaxone preventing appearance of symptoms. All members of this group showed relapse seven days after discontinuing ceftriaxone treatment. Note that colonization of the liver in the group of mice with relapse was much lower, 3-log difference and statistically significant, **, P = 0.0021, (Kruskal-Wallis multiple comparison test), in bacteria lacking PBP3_SAL compared to the rest of strains used. No statistically significant differences were found in untreated mice (upper panels). n.s., not significant.