Is Teicoplanin Resistance Detected By The Automated System In Staphylococcus spp Strains Reliable?

Summary

Here, a protocol is presented to evaluate the reliability of an automated system (VITEK 2) for determining teicoplanin susceptibility in Staphylococcus isolates by comparison with broth microdilution (BMD), with applications in confirming resistance and guiding appropriate antimicrobial therapy.

Abstract

A total of 467 Staphylococcus aureus (S. aureus) and 66 coagulase-negative Staphylococcus (CoNS) isolates collected between June 2022 and December 2024 were included in this study. Teicoplanin susceptibility testing was performed using the automated antimicrobial susceptibility testing system, broth microdilution (BMD), and a gradient test method. Methicillin resistance was detected in 71.7% of S. aureus isolates and in all CoNS isolates. In this study, 13 S. aureus isolates and 4 CoNS isolates were identified as teicoplanin-resistant by automated susceptibility testing system. All 13 S. aureus isolates were found to be susceptible to teicoplanin by both the gradient test and BMD. All four CoNS isolates were identified as susceptible by the gradient test, whereas BMD classified two isolates as susceptible and two as resistant. Teicoplanin resistance was detected in four strains [S. capitis (n = 1) and S. haemolyticus (n = 3)] among the CoNS isolates by BMD. The four teicoplanin-resistant CoNS strains identified by BMD were isolated from blood cultures; three were obtained from pediatric patients and one from an adult patient. Overall, the methicillin-resistant S. aureus (MRSA) rate was relatively high in the study, and all CoNS isolates were methicillin resistant. In addition, automated systems were found to be unreliable for determining teicoplanin resistance, and isolates identified as resistant should be confirmed by BMD. Given that teicoplanin is a last-resort antibiotic used to treat pathogens such as MRSA and methicillin-resistant CoNS, its susceptibility should be closely monitored.

Introduction

Staphylococcus aureus (S. aureus) is one of the most clinically significant Staphylococcus species because of its wide range of virulence factors and its ability to cause severe infections in various tissues and organs. It is responsible for skin and soft tissue infections, deep tissue infections, respiratory tract infections, urinary tract infections, and bloodstream infections, and remains one of the most important pathogens in hospital settings^1,2. In particular, methicillin-resistant S. aureus (MRSA) is a major cause of healthcare-associated infections worldwide and continues to be a leading cause of postoperative wound infections^2,3. Coagulase-negative staphylococci (CoNS), although part of the normal skin flora, have increasingly been implicated in both local and systemic infections in recent years. This increase has led to more frequent antimicrobial susceptibility testing and, consequently, increased detection of resistance to glycopeptide antibiotics such as teicoplanin⁴.

Because S. aureus and CoNS strains commonly exhibit resistance to β-lactam antibiotics, glycopeptide antibiotics, primarily vancomycin and teicoplanin, are widely used for the treatment of these infections5. The increasing prevalence of resistant gram-positive nosocomial pathogens has led to the expanded use of glycopeptides, which has contributed to declining bacterial susceptibility to these agents^5,6. Vancomycin and teicoplanin have comparable antimicrobial efficacy; however, multiple studies have demonstrated that teicoplanin is associated with fewer adverse effects, particularly reduced nephrotoxicity^7,8. Therefore, teicoplanin is often considered a suitable alternative to vancomycin in specific patient populations, including those with neutropenia, bacteremia, or impaired renal function^9,10.

Rapid and accurate determination of glycopeptide susceptibility is important because of the widespread use of these agents in methicillin-resistant Staphylococcus infections and the increasing reports of glycopeptide-resistant strains¹¹. Several laboratory methods are available for assessing glycopeptide susceptibility in Staphylococcus species, including agar dilution, gradient tests, broth microdilution (BMD), and automated antimicrobial susceptibility testing systems⁵. Previous studies have reported discrepancies between automated systems and reference methods in detecting reduced susceptibility or resistance to glycopeptides, particularly teicoplanin, in Staphylococcus species^4,11.

The present study aimed to evaluate the reliability of the automated antimicrobial susceptibility testing system for determining teicoplanin susceptibility in Staphylococcus isolates, compared with the broth microdilution method, the reference standard. Evaluation of the automated system's performance characteristics may help clinical laboratories assess its suitability for routine detection of glycopeptide susceptibility in clinical practice.

Protocol

The study was approved by the local ethics committee on January 27, 2025 (protocol number: HRU/25.02.03). The study was conducted in accordance with the principles of the Declaration of Helsinki.

1. Study design

This retrospective study evaluated the performance of the automated antimicrobial susceptibility testing system for determining teicoplanin susceptibility in Staphylococcus species, comparing results with the broth microdilution (BMD) reference method and a gradient test method.

2. Isolate selection

1. Collection of clinical isolates
   1. Include a total of 467 S. aureus and 66 coagulase-negative Staphylococcus (CoNS) isolates obtained from various clinical specimens between June 2022 and December 2024.
   2. Process all clinical specimens according to routine microbiological procedures in the diagnostic laboratory.
   3. Include only one isolate per patient in the analysis. Exclude repeated isolates obtained from different specimens from the same patient.
2. Initial identification and screening
   1. Identify all isolates at the species level using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).
   2. Test S. aureus and CoNS isolate for methicillin resistance and teicoplanin susceptibility using VITEK 2 AST-P664 cards with the automated susceptibility testing system.
   3. Record the minimum inhibitory concentration (MIC) values generated by the automated susceptibility testing system for subsequent comparison.
3. Isolate storage and subculture
   1. Store isolates at −80 °C in tryptic soy broth supplemented with 20% glycerol following automated susceptibility testing system.
   2. Before reference testing, thaw isolates at room temperature and subculture them twice on 5% sheep blood agar.
   3. Incubate plates at 37 °C for 24 h after each passage to ensure purity and viability.

3. Antimicrobial susceptibility testing

1. Broth microdilution (BMD)
   1. Preparation of Teicoplanin solutions
      1. Prepare a stock solution of teicoplanin powder at a concentration of 2560 mg/L in cation-adjusted Mueller-Hinton broth (CAMHB).
   2. Broth microdilution procedure
      1. Perform broth microdilution testing using sterile 96-well U-bottom microplates.
      2. Dispense 100 µL of CAMHB into each well. Dilute the teicoplanin stock solution tenfold and add 100 µL to the first well.
      3. Prepare twofold serial dilutions across wells 1–10 to obtain final antibiotic concentrations ranging from 64 to 0.125 µg/mL.
      4. Use well 11 as the growth control containing CAMHB and bacterial inoculum. Use well 12 as the negative control containing only CAMHB¹².
   3. Inoculum preparation and incubation
      1. Suspend pure bacterial colonies in sterile saline and adjust the suspension to a turbidity equivalent to a 0.5 McFarland standard.
      2. Prepare a 1:100 dilution in CAMHB and add 100 µL of the bacterial suspension to wells 1–11 to achieve a final inoculum concentration of approximately 5 × 10⁵ CFU/mL.
      3. Incubate the microplates at 37 °C for 24 h under ambient atmospheric conditions¹².
   4. MIC determination and interpretation
      1. Define the minimum inhibitory concentration (MIC) as the lowest concentration of teicoplanin that completely inhibits visible bacterial growth after incubation.
      2. Interpret MIC values according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) version 14.0 guidelines¹³.

4. Quality control

1. Use Staphylococcus aureus ATCC 29213, with a teicoplanin MIC reference range of 0.25–1 mg/L, as the quality control strain.

5. Statistical analysis

1. Method comparison
   1. Compare teicoplanin MIC results obtained using the automated susceptibility testing system with those obtained using the BMD reference method.
   2. Define categorical agreement (CA) as concordant susceptible or resistant results between the two methods.
2. Error classification
   1. Define very major errors (VME) as isolates categorized as susceptible by the automated susceptibility testing system but resistant by BMD.
   2. Define major errors (ME) as isolates categorized as resistant by the automated susceptibility testing system but susceptible by BMD.
3. Performance criteria
   1. Consider method performance acceptable when categorical agreement is ≥90% and the rates of VME and ME are ≤3%, in accordance with published evaluation criteria^14,15.

Results

A total of 533 isolates recovered from clinical samples over a 2.5-year period were included in the analysis. All methicillin-resistant CoNS (MR-CoNS) isolates, and 71.7% of S. aureus isolates, were found to be methicillin resistant. Wound swab samples were the most common clinical specimens yielding S. aureus isolates, whereas MR-CoNS isolates were most frequently recovered from blood cultures (Figure 1).

Discussion

Antimicrobial resistance is one of the most significant threats to global public health, contributing to increased healthcare costs, treatment failure, and mortality³. The incidence of both community- and hospital-acquired infections caused by Staphylococcus aureus continues to increase nationally and globally. Methicillin resistance, in particular, is associated with increased morbidity and mortality; therefore, resistance profiles are routinely monitored worldwide¹⁶

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cation-Adjusted Mueller Hinton Broth	Sigma-Aldrich, Darmstadt, Germany	CM0405B	For bacterial growth in the broth microdilution method
MALDI-TOF MS	VITEK MS, BioMérieux, France	410893	For bacterial identification
McFarland standard	bioMérieux, France	95060-958	Standardization of bacterial inoculum
Petri dishes	RTA, Türkiye	2102	For medium preparation and bacterial cultivation
Pippette tip 20 - 200 μL	Sigma-Aldrich, Darmstadt, Germany	Z740105	For the distribution of cation-adjusted Mueller–Hinton broth (CAMHB) into U-bottom microplates and preparation of serial dilutions
Sheep blood agar	RTA, Türkiye	2001	Isolation and cultivation of microorganisms
Staphylococcus aureus	American Type Culture Collection	29213	Reference strain used in this study
Teicoplanin powder	Sigma-Aldrich, Darmstadt, Germany	T0578	Antibiotics
Tryptic soy broth	Merck, Germany	1,054,590,500	Isolation and cultivation of microorganisms
U bottom well plate	Thermo Fisher Scientific	163320	For antimicrobial susceptibility testing by the broth microdilution method
VITEK 2 AST-P664 cards	bioMérieux, France	414197	For antimicrobial susceptibility testing
VITEK2 Compact	bioMérieux, France	4700030	For antimicrobial susceptibility testing