Assessment of polymicrobial interactions in bacterial isolates from transfused platelet units associated with sepsis

Abstract

Background: In 2019 the Centers for Disease Control and Prevention (CDC) reported a series of 4 transfusion reactions that resulted from contamination of apheresis platelet products. Products involved in all 4 cases were contaminated with Acinetobacter calcoaceticus-baumannii complex (ACBC) and in 3 products Staphylococcus saprophyticus was found as well. CDC investigation found that bacterial isolates from the cases were genetically related and suggested a common source of contamination. The contamination of blood products with ACBC is rare and polymicrobial contamination of blood products even less common. ACBC and S. saprophyticus have been observed to adhere to one another and sediment out of suspension in vitro, a process referred to as coaggregation, and we hypothesized that there was an interaction between the strains from these cases that contributed to their co-contamination of blood products.

Study design and methods: To test the hypothesis of bacterial interaction, we performed coaggregation experiments and observed the growth characteristics of ACBC and S. saprophyticus strains recovered from contaminated blood products involved in a subset of the CDC cases.

Results: An increase in S. saprophyticus CFU concentration was observed after several days of co-culture with ACBC in LB and plasma; however, no other findings suggested coaggregation or augmentative growth interaction between the bacterial strains.

Conclusion: Ultimately, an interaction between ACBC and S. saprophyticus that could help explain their co-occurrence and growth in contaminated platelet units was not found; however future studies of potential interactions may be warranted.

FIGURE 1

Coaggregation of bacterial mixtures. Stationary phase cultures of representative bacteria were washed and diluted in PBS, then incubated at room temperature individually or in combination with other bacteria for 0, 2, and 24 h. The aggregation control was incubated in LB with arabinose to induce aggregation and compared to a non-induced culture without arabinose. OD600 values were obtained as a measure of turbidity and percent coaggregation calculated from the difference in OD600 of a given mixture and its constituent bacteria (100[Average of Constituents OD600 – Sample OD600]/Average of Constituents OD600). 30% represents the cut-off for evidence of coaggregation. Error bars are SD of biological replicates.

FIGURE 2

Cross-streak analysis of bacterial interaction. Horizontal lawns of A. calcoaceticus/baumanii complex (A), S. saprophyticus (B), S. aureus (C), S. epidermidis (D), P. aeruginosa (E), and E. coli (F) were grown over night on CBA (left) or LB agar (right). Vertical streaks of indicator organisms were inoculated with a loop and plates were again incubated overnight. CBA, Columbia Blood Agar; LB, Luria-Bertani.

FIGURE 3

Comparison of growth between monomicrobial and polymicrobial cultures in LB, plasma, and apheresis PAS platelets. Stationary phase cultures of ACBC and S. saprophyticus grown in LB broth, apheresis plasma, or apheresis PAS platelets were inoculated into identical growth media in triplicate (LB broth, A; apheresis plasma, B; apheresis PAS platelets, C). Monomicrobial cultures were prepared by inoculating platelets with either ACBC or S. saprophyticus; mixed subcultures were prepared by inoculating with both organisms. CFU were enumerated by serial dilution and spot plating. Error bars are SD of biological replicates. Dashed lines represent the lower limit of quantitation (LLOQ) of 100 CFU/ml that results from spotting 10 μl of undiluted sample. Values below LLOQ were assigned a value of 1.