The Gram stain: Implication in blood culture reporting

Abstract

Bloodstream infections are life-threatening. They are responsible for prolonged hospital stays and high healthcare costs. Clinical microbiology has an essential role to play in its management. Direct Gram-stain reporting from positive blood culture bottles has been found helpful. We aimed to evaluate the use of the automated blood culture system in adjunct to the conventional Gram-stain technique, with a direct antimicrobial susceptibility test on the second day. Concordance was highly satisfactory.

Keywords

Gram-stain bloodstream infections antimicrobial susceptibility test

Introduction

Mortality due to bloodstream infection (BSI) among hospitalised patients is approximately 20%.^1,2 Prompt and accurate identification of aetiological agents from positive blood culture bottles and their antimicrobial susceptibility test (AST) remains a critical function, which has revolutionised turnaround time and improved the accurate detection of microorganisms.³

Materials and method

We evaluated the use of automated methods in adjunct with the conventional Gram-stain technique for early blood culture reporting and correlated this with matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) identification (VITEK-MS system, BioMérieux, Marcy-l’Étoile, France). The study was carried out in a tertiary healthcare centre over two months. A workflow for automated blood culture (BacT/ALERT, BioMérieux, Marcy-l’Étoile, France) reporting was prepared as given in the flowchart (see Figure 1).

Figure 1.

A workflow for automated blood culture (BacT/ALERT, BioMérieux, Marcy-l’Étoile, France) reporting was prepared as given in the flowchart.

According to protocol, all blood specimens were incubated in the BacT/ALERT 3D instrument (BioMérieux) till flagged positive or for a maximum of five days, whichever is earlier. A total of 905 blood samples were included during the study period, of which 113 were flagged positive (rate = 12.5%). All flagged bottles were immediately subjected to Gram-stain, followed by culture on blood and MacConkey agar (Day 1).⁴ Three independent observations were made for each Gram-stain slide. Subsequent decisions on subculture and AST were based on the Gram-stain observation. If this revealed Gram-positive cocci (GPC) or Gram-negative bacilli (GNB), drug susceptibility testing (DST) was performed using the Kirby Bauer disk diffusion method with a panel of drugs against GPC or GNB following CLSI 2022 guidelines. If Gram-stain revealed ≥ 2 different organisms or organisms with doubtful morphology such as thin filamentous bacilli or Gram-positive bacilli (GPB), AST was performed following confirmation using MALDI-TOF on the second day. If Gram-stain revealed budding yeast cells (BYCs), the flagged blood culture bottle was immediately transferred to the mycology lab for further processing. If a flagged bottle revealed no organism on Gram-staining, its culture report was awaited on the second day. On a subsequent day, the culture plates were examined for the growth of organisms, whose identification was carried out by MALDI-TOF. A preliminary report was prepared, including MALDI-TOF results (final identification) and preliminary DST. On the same day, final AST was also carried out by the VITEK II (BioMérieux, Marcy-l’Étoile, France) system. On the third day, the results of AST and DST were compared, and the final report was prepared.

Results

A total of 113 (12.4%) flagged blood samples out of 905 received in automated blood culture bottles were examined, of which 64 (56.6%) were flagged within 24 h, 35 (30.9%) within 48 h, 14 (12.3%) within 72 h, and one (0.8%) at 96 h of incubation. Figure 2 shows the comparison of a direct Gram-stain with the identification by MALDI-TOF from culture. Commonly isolated organisms are shown in Table 1.

Figure 2.

Comparison of direct Gram-stain with matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF).

Table 1.

Commonly isolated organisms in blood culture.

GNB/GNCB	GPC	GPB
Escherichia coli	Staphylococcus aureus	Microbacterium paraoxydans
Klebsiella pneumoniae	Staphylococcus hominis	Microbacterium oxydans
Klebsiella oxytoca	Staphylococcus epidermidis	Bacillus altitudinis
Acinetobacter baumannii	Staphylococcus haemolyticus	Bacillus cereus
Pseudomonas stutzeri	Micrococcus luteus
Burkholderia cenocepacia	Staphylococcus kloosii
Ochromobactrum intermedium	Enterococcus faecium

GPC: Gram-positive cocci; GNB: Gram-negative bacilli; GNCB: Gram-negative coccobacilli; GPB: Gram-positive bacilli.

Gram-stain of 66 blood culture bottles (58%) revealed GPC, and the result was matched with the MALDI-TOF finding. Out of 66 samples, 62 revealed a pure growth of GPC on blood and MacConkey agar with a concordance rate of 93.9%. DST and AST by VITEK II were observed concordant on the third day. In the remaining four samples where only GPC was found on Gram-staining, growth of both GPC and GNB was found on culture and confirmed by MALDI-TOF.

Among the 113 samples, 29 (26%) were found positive for GNB, and 27 showed a pure growth of GNB on culture samples with a concordance rate of 93.1%. AST results revealed similar patterns by both methods on the third day. The other two samples showed GPB growth on culture and were identified as contaminants following the MALDI-TOF report.

GPB was observed in the Gram-staining of five samples and confirmed by MALDI-TOF the next day. Occasional GPB was observed (1–2/oil immersion field) in one instance. The culture revealed growth of GPC the next day and was identified to be Staphylococcus epidermidis. Pure BYCs were seen on Gram-stain in four samples. A mixture of BYC and GPC was seen on Gram-stain in one sample. The bottle was subcultured and transferred to the mycology laboratory for further processing. The GPC was identified as Staphylococcus haemolyticus using MALDI-TOF.

Gram-staining of eight samples (7%) revealed no organism. Seven of these showed concordant results on culture the next day (87.5%). One of these samples had a pure growth of GNB, which was subsequently identified as Ochromobactrum intermedium by MALDI-TOF. All the significant and concordant results had a time to positivity ranging from 24 to 72 h, and all samples found to have contaminants grown on culture were observed to be flagged at 96 h. All this led to the generation of a preliminary report a whole day earlier than normal.

Discussion

Gram stain is the most rapid and simplest test for any positive blood culture to characterise microorganisms. It will help assess the adequacy of antimicrobial therapy selected after collecting blood culture specimens before the final identification of the microorganism.⁵

In our study, 93.9% concordance was seen in the case of GPC and 93.1% in the case of GNB. However, there were a few discordant results. In four samples, GPC was seen on the first day but the culture grew a mixture of GPC and GNB on the second day. Two samples showed GNB on the first day but the culture grew GPB the next. This could have been due to low load or technical error in performing Gram-stain, possibly in the decolourisation step leading to GPB appearing as GNB. Hence, careful examination of smears and following the correct staining steps are extremely important.

In another sample, a mixture of BYCs and GPC was seen on Gram stain (Table 2) which was subsequently identified as Staphylococcus haemolyticus. Here, we want to highlight that, had the traditional workflow been followed, the presence of BYC might have been missed owing to similar colony morphology, viz. GPC without haemolysis on blood agar.

Table 2.

Special scenarios.

Sl no	Direct Gram-stain on day 1	Gram-stain from culture on day 2	Microscopic image (100 × objective) and culture plate images
1.	BYC + GPC	BYC + GPC	The arrow shows (A) a mixture of BYC & GPC on day 1, (B) a mixture of BYC & GPC from culture on day 2 and (C) colonies of GPC and BYC with similar morphology on blood agar
2.	Abundant pus cells without organism	No growth in culture	Abundant pus cells were seen in gram stain on day 1

GPC: Gram-positive cocci; BYC: budding yeast cell.

No organism was detected in the Gram-stain of eight samples. Seven of these showed concordant results on culture the next day. The Gram stain of two false-positive samples showed an abundance of pus cells (Table 2). It has been reported that leucocytes produce carbon dioxide as a part of their metabolic processes, causing false-positive growth signals in the automated systems that spectrophotometrically detect the increase in carbon dioxide.⁶

A major advantage of following this workflow is that a preliminary report was obtained on the second day including the identification of the organism and the sensitivity pattern, a day earlier than the traditional workflow. Therefore, targeted therapy can be initiated earlier and will contribute to better patient prognosis and antimicrobial stewardship in an era of rising antimicrobial resistance.

However, our study has a few limitations. The sample size analysed here is low. Since Gram stain is a critical part of the workflow, the technical and observational errors might lead to wrong interpretations. Detection of GPC or mixed bacteria in Gram stain may not conclusively differentiate true infection from contamination. Hence, practices of sample collection for blood culture should be properly followed and all results must be clinically correlated.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Sarita Mohapatra

Hitender Gautam

Neha Nityadarshini

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