Cryopreservation of virulent Acinetobacter baumannii to reduce variability of in vivo studies

Abstract

BackgroundMicrobiological assays require accurate and reproducible preparation of bacterial inocula. Inocula prepared on different days by different individuals can vary significantly from experiment to experiment. This variance is particularly problematic for Gram-negative bacterial infections, for which threshold effects can result in marked variations in host outcome with minor differences in inocula.ResultsWe compared the accuracy of traditional methods versus using frozen stocks for preparing Acinetobacter baumannii inocula for infection in mice. Standard inoculum preparation resulted in substantial variability, both with respect to the actual inocula achieved compared to the targeted inocula, and with respect to the in vivo outcome resulting from similar inocula. Cryopreservation of the bacteria resulted in no significant decrement in growth of the bacteria. Furthermore, preparation of multiple infectious inocula from a frozen stock significantly improved the accuracy of the achieved inocula, and resulted in more reproducible in vivo outcomes from infection. Frozen stocks reduced inter-experiment variability associated with inoculum preparation, displayed no significant loss of growth capacity, and maintained virulence, increasing the reliability of infection.ConclusionsFrozen stocks require considerably less time to prepare and enhance reproducibility of in vivo experimental results when infecting with A. baumannii.

Natural history of Acinetobacter baumannii infection in mice

“…Bacterial inocula, made from fresh or frozen bacteria, was prepared as described in previous work. [15] Frozen stocks of HUMC1, VA-AB41, and LAC-4 Col R were thawed and diluted in PBS to adjust the bacterial density as needed for infection. 8-week old male and 10-week old female C3HeB/FeJ mice were infected via tail vein injection and the inoculum bacterial density was confirmed by plating serial dilutions on TSA plates and incubating overnight at 37°C.…”

Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges

“…In contrast, examination showed that A/J and C3HeB/FeJ strains of mice are intrinsically susceptible to lethal intravenous and lung infections by some clinical isolates of A. baumannii at inocula commensurate to (or lower than) those required for other commonly recognized virulent pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus (54,(69)(70)(71)(72). A/J mice had delayed neutrophil recruitment to the lungs due to diminished CXC chemokine responses to the bacteria, possibly explaining their susceptibility to pulmonary infection (54).…”

Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

“…E. coli DH5α, A. baumannii HUMC1 ( 15 , 16 , 33 , 34 ), and A. baumannii ATCC 17978 were cultured using aseptic technique. Single colonies were first streaked out on tryptic soy agar (TSA) from frozen glycerol stocks.…”

“…The cells were pelleted by centrifugation (10 min at 10,000 × g ) and washed five times with 1 ml of 10% glycerol. The cells were separated into 100-µl aliquots and stored at −80°C for later use as we have previously described ( 33 ).…”

“…We have previously published that HUMC1, an XDR A. baumannii clinical blood and lung isolate resistant to all clinically reported antibiotics except colistin, is hypervirulent in murine models of infection ( 15 , 16 , 33 , 34 ). Given its virulence and near-pan-drug-resistant status, intentional induction of colistin resistance in this strain, for example by inserting the MCR gene, would raise ethical concerns.…”