Key Steps in Culture and Susceptibility Testing
For decades, conventional culture and susceptibility testing (C&ST) has been the standard diagnostic test to guide the diagnosis and treatment of bacterial infections in veterinary medicine and consists of a few serial steps in which different stakeholders are involved. For example, the owner of a diseased pet is the first to detect signs of disease, can take certain samples, might have a role in the treatment, and will pay the bill. The veterinarian, however, is the central stakeholder gathering different kinds of information from the owner (e.g., anamnesis, sometimes samples), the animals (e.g., clinical history and examination, point-of-care testing), and from diagnostic laboratories (e.g., C&ST results). This means that in the end, it is the veterinarian that is making the diagnosis and choosing the treatment protocol, taking the C&ST results into account but interpreting it under specific circumstances.
Taking a sample?
Sampling for C&ST is mainly performed when this can result in important information to guide treatment or control of an infectious disease, for example, when there is no clue on which agents may be involved or if acquired resistance is plausible. The mode of sampling and subsequent storage during transport to the lab has a dual goal: on the one hand it aims at holding a large number of clinically relevant microbial agents and on the other hand, the number of non-relevant contaminants should be kept to a minimum. Since the way of sampling can affect obtained results substantially (e.g., cystocentesis vs. midstream urine for diagnosing cystitis), this should be taken into account when interpreting C&ST results. When sampling sterile organs or body fluids, an aseptic technique is necessary to obtain relevant results, especially when an enrichment (e.g., hemocultures) is involved.
When is a bacterial culture result clinically relevant?
Veterinary samples often contain bacteria, whether they are clinically relevant or not, which can complicate the interpretation of obtained results. Considering the effect C&ST results can have on the appropriateness of the chosen treatment, it is of utmost importance that only clinically relevant results are taken into account. Understanding the importance of bacterial growth is, however, not always clear-cut and should be interpreted taking into account the moment (e.g., before treatment, acute or chronic stage of infection?) and the way the samples were taken (e.g., was perfect asepsis reached for hemocultures?) and stored during transport to the lab (e.g., cooling, transport medium), the presence of microflora at the location of sampling (e.g., nose versus lungs), the abundance and purity of the obtained culture, and the perceived pathogenicity of the cultured organism in the specific sample. On the other hand, false-negative results may also be the result of conditions discussed above, next to the fact that some bacterial pathogens need very specific conditions and/or longer incubation periods to allow growth (e.g., Mycoplasma spp.), or cannot be grown under routine conditions at all (e.g., spirochetes, obligatory intracellular pathogens, etc.). Other diagnostic options, such as for example molecular testing or histopathology, should be considered in that case. Close communication between veterinarian and C&ST provider is therefore of utmost importance for correct interpretation.
In the last few years, bacterial identification has undergone a revolution; MALDI-TOF MS and third-generation sequencing have led or will lead to the fastest, most comprehensive, and most reliable results ever in veterinary microbiology, but also hold the risk of over-interpretation of obtained results (see lecture Advanced Microbiology Techniques: What Are the Possibilities?).
Susceptibility Testing Is No Golden Bullet
Reporting and susceptibility testing of a non-causative bacterium is the number one reason for C&ST results to misguide the antimicrobial treatment (cfr previous paragraph). However, when a relevant bacterial isolate is obtained, appropriate susceptibility testing and subsequent interpretation can be an important tool in selecting the most appropriate treatment protocol. While bacterial identification is probably highly reliable in most veterinary diagnostic labs, for example, due to the use of MALDI-TOF MS, susceptibility testing results seem somewhat less reliable in general. Less reliable susceptibility testing reports can be the result of not testing relevant antimicrobial agents (e.g., oxacillin should be used to detect methicillin-resistant Staphylococcus pseudintermedius), not using the correct testing method (e.g., disk diffusion test is not reliable for testing polymyxins), incorrect reading of the results (e.g., not taking growth edge into account when reading penicillin resistance in staphylococci), not applying expert rules (e.g., on natural resistance in Pseudomonas aeruginosa), or due to the fact that often relevant clinical breakpoints are absent (should be ideally animal-, organ-, and bacterium-antimicrobial agent specific; cats are not small dogs; antimicrobial concentration differs between organs). For example, even though there are quite some canine-specific clinical breakpoints described by CLSI, for cats, this is limited to mainly beta-lactam antibiotics and fluoroquinolones (mainly highest priority critically important antimicrobials; Lhermie et al. 2020) and interpretive criteria for other antimicrobial classes are often based on human clinical breakpoints. In addition, if treatment protocols are used that divert from the treatment regimens as described to establish the clinical breakpoints, used clinical breakpoints may not accurately predict clinical outcomes. A special case in that matter is the use of topical treatment with antimicrobial agents (e.g., skin, ears, eyes): since there are no breakpoints for topically applied agents, susceptibility testing results do not necessarily predict clinical efficacy. However, because local concentrations due to topical treatment or regional infusion can reach concentrations of 10–1,000 times those obtained in plasma, local treatments are generally expected to be clinically effective. On the other hand, clinical breakpoints based on systemic concentrations may underestimate resistance in certain sites (e.g., eye, joints) because therapeutic concentrations may not be reached using systemic treatment (CLSI 2019).
Taking the above into account, it is clear that ideally, next to the susceptibility testing result, also other information should be available in the C&ST report or should be easily obtained from the involved microbiology lab or an infectious disease specialist (Guardabassi, Prescott 2015). Some examples of important interpretive information are: the treatment regimens linked with the clinical breakpoints, reliability of used clinical breakpoints, and advice on whether specific antimicrobial agents are first choice or rather last resort agents according to (inter)national guidelines. Considering the fact that in certain circumstances, specific susceptibility results may not accurately predict treatment efficacy, clinical progress and response to treatment remain important features for (re)evaluation of antimicrobial management, next to C&ST results.
Speeding Up the Process
Especially in emergency and critical care patients, sample-to-result time can be vital (Kollef et al. 2021). There are currently various technical possibilities to obtain information on specific pathogens or resistance mechanisms within a few hours, but the lack of affordable veterinary applications and both logistic and organizational issues may prohibit significant time savings can be achieved (see lecture Advanced Microbiology Techniques: What Are the Possibilities?).
References
1. CLSI. Understanding susceptibility test data as a component of antimicrobial stewardship in veterinary settings. CLSI Report VET09. 1st ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2019.
2. Guardabassi L, Prescott JF. Antimicrobial stewardship in small animal veterinary practice: from theory to practice. Vet Clin North Am Small Anim Pract. 2015;45(2):361–376.
3. Lhermie G, La Ragione RM, Weese JS, Olsen JE, Christensen JP, Guardabassi L. Indications for the use of highest priority critically important antimicrobials in the veterinary sector. J Antimicrob Chemother. 2020;75(7):1671–1680.
4. Kollef MH, Shorr AF, Bassetti M, Timsit JF, Micek ST, Michelson AP, Garnacho-Montero J. Timing of antibiotic therapy in the ICU. Crit Care. 2021;25(1):360.