Is Haemophilus influenzae better satellite for Enterococcus faecalis?

Introduction

Haemophilus influenzae is a non-motile and non-spore-forming gram-negative coccobacillus and can lead sinusitis, otitis, conjunctivitis and meningitis as well as upper and lower respiratory tract infections [1-4].

H. influenzae needs factor X (Hemin) and factor V (Nicotin adenin dinucleotide) for growing of in laboratory conditions [2].  H. influenzae can grow on chocolate agar plate prepared with heat-lysed horse blood or rabbit blood, or on blood agar media with some bacteria such as Staphylococcus aureus, coagulase-negative staphylococci (CNS) or enterococci which are capable of beta-haemolysis and which can provide factor V; this cooperation is called “Satellite phenomenon” [1, 2, 5]. In the guidelines, the standard strain of S. aureus ATCC 25923 is proposed to be used for satellite test in order to identify the haemophilus-like colony as a conventional method in lack of factor  X, V or XV discs [1, 2, 5].

In this study, it was aimed to test and compare beta-haemolytic bacteria for conventional identification of H. influenzae.

Materials and Methods

Bacterial isolates

In the study, two different H. influenzae strains isolated from clinical specimens and identificated with API NH (BioMeriéux, France) and three S. aureus (Including S. aureus ATCC 25923), three CNS, and two Enterococcus faecalis strains all of which are beta-haemolytic isolates obtained from clinical specimens were used for satellite tests.

Satelite tests

To provide an objective and standard examination, H. influenzae strains were suspended in both 0.5 and 4 McFarland tribudity and inoculated onto blood agar media in duplicates. Paper discs in 6mm in-diameter were placed onto the media in order to standardize the colony sizes of the test bacteria by absorbing the suspensions. Each of the test bacteria was suspended in 1 McFarland and 5µL of each was added onto the discs. As a result, it was provided that each H. influenzae strain was tested for a total of four times with each beta-haemolytic strain. The media were incubated at 37⁰ C in CO₂-enriched incubator for 24h.

Reviewers       

At the end of incubation, the examinations were done by five reviewers constituting of two microbiology specialists, two research assistants and one master student all of that were experienced in microbiology for at least two years. The media cultures were examined by the reviewers for satellite tests as totally blinded of the row of the test bacteria and of the other reviewers’ test results.

Scoring

For each satellite test, the reviewers scored both the colony sizes of H. influenzae and the diameter of the growth-zone. The scores were determined as 0 (No growth), 1 (Low), 2 (Media) and 3 (High). The sum of the scores for the colony sizes and the growth-zones were determined as “total diagnostic score” (TDS). As a summary, each reviewer scored as a total of 64 satellite tests constituting of two H. influenzae strains in two distinct densities and in duplicates for 8 test bacteria.

Statistical analysis

Descriptive variables were presented as numbers and percentages and diagnostic scores were expressed as mean ± standard deviation (SD).

An independent sample T test or a Mann-Whitney U test was used for comparison between the two groups. An independent sample T test or a Mann-Whitney U test was used for comparison between the two groups, and analysis of variance test was used for comparison amongst all groups. The results were interpreted within a confidence interval of 95%, and a p value of <0.05 was considered as significant.

Results

At the end of the test, a total of 320 test scores of five reviewers were analysed. The mean total diagnostic score (MTDS) of S. aureus ATCC 25923 strain was 2.20±0.99. MDSs of the groups of S. aureus and E. faecalis were 2.01±0.97 and 5.38±0.95, respectively (Table 1). The MTDS of E. faecalis group was significantly higher than MTDS of S. aureus (p<0.001). MTDS of enterococci was also significantly higher than the other groups (p<0.001 for all). No significant difference was found between the scores of S. aureus and CNS groups (p>0.05) (Table  1).

In the S. aureus group, 23 (19.2%) tests had 0 points as TDS; but in enterococci group no isolates had lower scores than 3 points. In enterococci group, the rate of isolates which had 5 or 6 points was 77.5% (62/80); but in S. aureus group no isolate had higher than 4 points (Table 2).

Table 1. Mean total diagnostic scores (MTDS) of the isolate groups used in the satellite test

*MTDS calculated by addition of the points of colony size and growth-zone scored by the reviewers

**Coagulase-negative staphylococci

Table 2. Distribution of the number of isolates according to “Total diagnostic scores”

*Coagulase-negative staphylococci

Discussion

Despite latex particle agglutination test (LAT) or polymerase chain reaction (PCR) assay are more sensitive methods than culture to detect H. influenzae,  they are not widely used routinely in clinical settings [6, 7]. However, antibiotic susceptibility testing is not possible with LAT or PCR alone, so a parallel culture is necessary for large-sized laboratories [6, 7].

Culture of H. influenzae is not highly sensitive due to antibiotic use prior to sample collection, poor collection or transport techniques or poor examination of culture by low-experienced technicians [6, 8]. For these reasons, culture examination or conventional test methods for isolation of H. influenzae, importance of diagnosis increases particulary for small-sized laboratories [6, 8].

In the early 20^th century, the “satellite test” was found for presumptive laboratory diagnosis of H. influenzae and this test has been accepted as a conventional method for about a hundred years [1, 2]. Satellite test shows the dependence of the isolate to factor X and V for growing [1, 2]. Factor X and V dependence is needed to be tested for most clinical specimens [9, 10]. Guidelines state that a beta-haemolytic S. aureus strain is needed to be used in the test [2, 5]. However, some species of beta-haemolytic microorganisms other than S. aureus may provide positive results when tested with a heamophilus-like suspected-colony [2]. The question is, which bacteria can be the best for accurate identification.

Centers for Disease Control and Prevention advise S. aureus or enterococci to be used in satellite test but in our study enterococci was shown to be better to be preferred [5].

In this study, we have found that beta-haemolytic E. faecalis strains showed significantly more accurate results among all tested isolates as well as S. aureus. E. faecalis strains had significantly higher mean diagnostic scores as well as none of those caused false-negative results.  

Conclusion

Our study shows that using a beta-haemolytic E. faecalis strain will provide significantly more accurate results and will significantly reduce false-negative results for satellite test instead of S. aureus, which is particularly suggested to be used. According to our results, we consider that H. influenzae will be a “better satellite” for beta-haemolytic E. faecalis.

Acknowledgements

As the authors, we would like to thank Aysun Özüdogru, a specialist of  English literature, for her help in redaction of our manuscript.

Conflict of interest: none declared.