In this study, we have identified that the FLC sign observed in the blood cultures, in which Gram staining showed GPC in cluster, could be an identifiable feature of CoNS. To the best of our knowledge, this is the first study suggesting that the FLC sign could help in the rapid identification of CoNS from SA, in aerobic bottles. This Gram stain-based approach is a convenient, cost-effective, and time-effective method to rapidly identify the causative pathogens. In Japan, for many decades, infectious diseases clinicians and primary care providers have been using Gram staining in the emergency room or ward to determine the appropriate antimicrobial regimen.12, 13 Traditional use of Gram stain is not prevalent in many countries,14 but this method could be useful for low-cost and rapid estimation of the causative agent in such countries where Gram staining is used as a traditional method immediately after a positive blood culture as well as sputum or urine samples.15 Gram stain has contributed immensely towards curbing the overuse of broad-spectrum antimicrobials without affecting the effectiveness of the treatment 13.
It has been reported previously that the GPCs tend to differ in sizes between aerobic and anaerobic bottles.16 The size of CoNS was reported to be < 1 µm in size in aerobic and > 1 µm in size in anaerobic bottles. These findings are consistent with the current study showing that the FLC signs in aerobic bottles are of a relatively smaller size than those in anaerobic bottles; however, the reason behind this observation is still unknown.
No statistically significant associations were observed between SA and grapes sign, although the fraction of grapes sign in SA group was higher than that in the CoNS group. This might be because of the small sample size. Further clinical studies including larger sample size are therefore required. By combining this study along with the other useful finding i.e. the “Oozing sign” to identify SA in Gram staining,15 more contributable identification between SA and CoNS might be possible by observing Gram staining. This will be a more promising and cost-effective option.
It is true that the sensitivity of this method is lower than the tube coagulase test, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH), and real-time PCR 7, 8, 9, 10. Based on the current study only, clinicians cannot rule out CoNS in the absence of the FLC sign with low sensitivity. However, the high specificity of FLC signs could help clinicians predict the presence of CoNS with higher confidence while awaiting the culture results. Furthermore, we believe that this cost-effective and easy to perform a method, which uses only Gram staining, could be an effective way in the facilities where PNA-FISH or real-time PCR are not available. Most importantly, by using this methodology, inappropriate empirical antimicrobial therapy used for SA could be minimized.
This study has several limitations. First, this was a relatively small observational study performed with samples taken from a single institution. For further validation, it is essential to compare the results with blood cultures taken from other hospitals. Second, this study did not include non-Staphylococcal GPC in cluster such as Micrococcus spp., or Aerococcus spp.8, 15 Hence, the characterization of specific signs in Gram staining for these microorganisms could not be done. Third, this study did not assess the comparison between methicillin-susceptible SA (MSSA) and methicillin-resistant SA (MRSA). It remains unknown whether there are differences in size or number of FLS signs between MSSA and MRSA. Although the treatment varies between the two, this finding could help clinicians predict whether they should start the empirical treatment for SA with consideration about whether MSSA or MRSA could be the causative pathogen based on its regional prevalence and risk factors. Fourth, the respondents analyzing Gram stain slides in this study were fellows under training, which has the potential for less accurate responses compared to those from experienced microbiologits or laboratory technicians. However, it is noteworthy that this highly specific result was obtained by a non-skilled microbiologist. Finally, this study did not compare by species among CoNS. It also remains unknown whether there are differences between S. epidermidis and methicillin-susceptible CoNS such as S. lugdunensis or S. schleiferi. Although the application of this method should be carefully based on the clinical status of patients, we believe that the presence of the FLC sign could contribute to the identification of CoNS by clinicians. Further studies with larger sample size and diverse blood culture systems are also warranted.