Introduction

Typically found on skin and mucosal surfaces and considered normal flora in humans and animals, β-hemolytic Streptococcus (BHS) species cause opportunistic infections ranging from superficial wounds and pharyngitis to invasive, life-threatening infections. BHS are often referred to by their Lancefield grouping, a serologic classification based on cell wall differences, of which there are 20 designated by a letter. In humans, important BHS include Group A streptococci (Streptococcus pyogenes) and Group B (Streptococcus agalactiae). Significant BHS members affecting veterinary species include Group B (S. agalactiae), Group C (Streptococcus equi, Streptococcus dysgalactiae), and Group G (Streptococcus canis).

In human medicine, antimicrobial susceptibility testing (AST) is not typically performed for BHS infections because no significant resistance has been noted to the first line therapy, penicillin, despite decades of use. Resistance to β-lactams, such as penicillin or ampicillin, would be a cause of considerable alarm and further confirmation would be required at a reference laboratory. Conversely, reports of β-lactam resistant BHS are found in veterinary literature that have led to concern and confusion. Are we really observing a completely different pattern of resistance in veterinary-origin BHS or are there methodological differences that account for the discordance?

Inconsistent Test Method Performance

In early 2023, veterinary diagnostic laboratories (VDLs) participating in the National Animal Health Laboratory Network (NAHLN) Antimicrobial Resistance (AMR) Project were made aware of ambiguous manufacturer instructions resulting in varied test methodology for AST on Streptococcus species using Sensititre AST plates. The procedures described in the instructions for use (IFU) were correct; however, due to the ambiguous wording describing the full procedure, many different variations for preparing sample inoculum were employed across laboratories. In addition, instructions on the need for manual adjustment of the minimum inhibitory concentration (MIC) values were not clearly described, and it was noted that many laboratories were not performing these manual interpretations appropriately.

Antimicrobial Susceptibility of Streptococci

AST is performed when the susceptibility to recommended antimicrobials used for treatment cannot be predicted, usually due to the potential for acquired resistance. Antimicrobials selected for AST are dependent on the bacterial species with secondary considerations including animal host species, disease status, and body site.¹ To obtain accurate AST results, laboratories must use standardized procedures to minimize variability among testing personnel, produce accurate results that will appropriately guide clinical treatment, and ensure that data can be shared and reproduced by laboratories for surveillance purposes.^2–4

Human and veterinary medicine in the United States and internationally use Clinical and Laboratory Standards Institute (CLSI) standards to interpret results of AST through use of clinical breakpoints. CLSI standards are developed and regularly updated by experts in fields across the laboratory professions.⁴ In addition to interpretative criteria, CLSI documents describe standardized methods for test performance, genetic determinants of resistance, commonly seen resistance patterns, methods for confirmation of test results, results that require further investigation, and quality control parameters.^2–4 Because interpretative criteria change as test methods evolve and new evidence emerges, it is critically important for laboratories, manufacturers, and researchers to use the most up-to-date guidelines available.⁴

In human medicine, BHS are nearly universally susceptible to β-lactams, cephems, and carbapenem class antimicrobials.^3,5–7 Due to predictable AST patterns, current clinical practice guidelines recommend physicians treat non-invasive infections empirically with penicillin.⁸ AST is not recommended for BHS, except in the case of an invasive infection.³ Despite empiric treatment with β-lactams for decades, the occurrence of resistance in these organisms to β-lactams, cephems, and carbapenems is considered highly unusual and requires confirmation of organism identification and repeat AST by a second method or by a reference laboratory.³

In contrast, BHS of veterinary origin have been reported to be resistant to predictably susceptible β-lactams, raising significant concern in the microbiology community. Studies of BHS from horses have reported up to 8% resistance to β-lactams.^9–12 However, several of these studies referenced outdated CLSI documents when deriving AST interpretations while others did not report a specific edition.^9–12 These studies also had common errors found in published veterinary AST reports, such as not specifying the animal species or body site from which the interpretation was derived, inappropriately extrapolating interpretative criteria from different genera of bacteria, or listing percent resistant/susceptible rather than publishing the MIC range of isolates.⁴ These errors make analysis of study findings difficult and can lead to incorrect application of breakpoints and therefore, erroneous AST results.

Implications for Clinical Practice, Research, and Surveillance

A 2017 survey of VDLs found that 59% reported using Sensititre AST plates, making this platform one of the most widely used AST methods in veterinary microbiology laboratories in the United States.¹³ As a commonly used test method with unclear instructions, it is reasonable to think that there has been extreme variability in test method performance and interpretation. Inconsistencies in interpretation of the Sensititre IFU, variable use of CLSI standards, and challenges within the laboratory can contribute to the increased resistance observed in BHS resistance patterns of veterinary origin. Clinicians, researchers, and surveillance partners must be aware of the possible consequences to their respective fields.

Public Health Implications

Current published evidence regarding β-lactam resistance of veterinary BHS suggests significant resistance, but no genetic mechanism of resistance is yet described among BHS regardless of species of isolation, including human beings. Lack of evidence and the issues described above support the idea that the widely varied test method performance does not accurately reflect AMR in veterinary BHS. Personal communications from VDL colleagues suggest β-lactam resistance is virtually non-existent. If resistance to β-lactams and other antimicrobials is emerging in veterinary BHS, this would represent a significant public health concern.

A Call to Action for the Veterinary Profession

As veterinary professionals, we must be stringent about the methods we use for AST performance and interpretation, for both research and diagnostic medicine. AMR patterns, such as those observed with BHS, which vary from those widely recognized in human medicine must be thoroughly evaluated with a skeptical eye and through use of molecular methodologies to confirm these resistance mechanisms, if present. Further review of existing studies and new research evaluating resistance trends in veterinary BHS is necessary. Studies comparing broth microdilution methods to disk diffusion and gradient testing may shed light on discrepancies between AST methods or may further confirm or refute resistance patterns of concern in veterinary BHS.

When performing AST, we must ensure that our test methods and interpretations are truly reflective of CLSI standards and manufacturer IFUs, which are not open to procedural modification at the discretion of the test performer.⁴ When conducting retrospective studies, researchers must evaluate which test methods were used and whether divergence in test methods could have impact on test results. Where impact is unknown, it should be described as a possible limitation of study findings or alternatively, the information should be excluded from the analysis. When CLSI documents are cited in an article, reviewers should confirm that the most current standards have been used for method performance and interpretative criteria.⁴ While many CLSI standards are available for purchase, the most current editions of VET01S and M100 are available online for free; therefore, these documents are accessible to all researchers and VDLs.

Antimicrobial stewardship is ultimately a collaborative effort. Microbiologists, veterinarians, and when available, veterinary pharmacologists, must work together to determine which antimicrobials should be routinely tested and reported for BHS. Because routine AST performance does not lend additional information to guide treatment, AST should be reserved for invasive infections or surveillance. As a form of antimicrobial stewardship and veterinarian education, VDLs should consider following human medicine and not routinely perform AST on non-invasive, clinically significant BHS isolates. In our respective laboratories, the authors report BHS isolates with a comment indicating that isolates are predictably susceptible to β-lactams or recommended first line antimicrobials. Routine laboratory reporting of an antimicrobial increases the likelihood that an antimicrobial will be prescribed; therefore, reporting an inappropriate broad-spectrum antimicrobial will increase prescription frequency.^16,17 We must all understand that reporting MIC results in the absence of interpretative criteria or appropriate spectrum of action does not add value to treatment of infection and may in fact do more harm than good for patients if inappropriate antimicrobials are selected for treatment.^16,17

AMR in veterinary medicine is as much of a concern for public health as AMR in human medicine, and although veterinarians are often the face of antimicrobial stewardship, everyone in the veterinary profession is responsible and provides valuable contributions. For VDLs, stewardship is achieved when laboratories report only the most appropriate antimicrobials, use the most current CLSI standards, and perform AST with standardized methods per manufacturer IFU and CLSI standards. When patterns of resistance for veterinary isolates differ from those reported in human medicine, we must partner with our human counterparts for confirmatory testing and review. We are all partners in stewardship and must consider how our test methods, data, and interpretations will impact treatment outcomes and antimicrobial availability for our patients, their owners, and future generations.