Inuencing factors in the satellitism test of Haemophilus inuenzae and Haemophilus parainuenzae supplemented V factor by Staphylococcus aureus

Background: Many factors affecting satellitism test are not clear, and it is dicult to avoid misidentied even if the medium is properly selected. We investigated the factors that cause false positives of Haemophilus inuenzae and false negatives of Haemophilus parainuenzae in the satellitism test supplemented V factor by Staphylococcus aureus. Methods: H. inuenzae (four reference strains and 47 clinical isolates), H. parainuenzae (two reference strains and 67 clinical isolates), four different media, and two strains of S. aureus activated on two different media were involved in this study. Results: The type of medium used to activate S. aureus was the most common factor causing false positives of H. inuenzae, followed by the strain of S. aureus, and again the type of medium used for the experiment. The production of false negatives of H. parainuenzae was only related to the medium used in the experiment. Conclusions: Tryptic soy agar and S. aureus (ATCC 25923) activated with nutrient agar should be used in the satellitism test for Haemophilus spp. to improve the accuracy of the test.


Introduction
Haemophilus in uenzae is an important pathogen that causes community-acquired pneumonia, nosocomial pneumonia, chronic bronchitis in older adults, and upper respiratory tract infections in children. Encapsulated H. in uenza, such as type b, is more virulent than unencapsulated strains and can cause diseases such as meningitis, pneumonia, epiglottitis, and sepsis [1,2]. However, invasive infections are more often attributed to unencapsulated strains since the introduction of the H. in uenzae serotype b conjugate vaccines [3,4].
In contrast, Haemophilus parain uenzae may colonize the human upper respiratory tract and is usually considered to be a member of the normal oral micro ora [5][6][7]. It is less pathogenic but does cause occasional cases of bacterial endocarditis [8,9], sepsis [10], urethritis, upper respiratory tract infections, intracerebral abscess [11], etc.
Although a cryptic species of Haemophilus spp. is phylogenetically close to, but distinct from, H. parain uenzae, especially they both require V factor (NAD) but not X factor (heme) to grow [16], the satellitism test is the classic way to con rm Haemophilus spp. [17], and it is still commonly used in clinical laboratories because of its convenience. In practice, we found it was more often challenging to distinguish H. in uenzae from H. parain uenzae by the satellitism test supplemented NAD by Staphylococcus aureus than by discs impregnated with NAD, primarily due to false positives of H.  Satellitism test H. in uenzae and H. parain uenzae were both activated on CAPs, while S. aureus was activated on BAPs and NAPs. The satellitism test was carried out with reference to the literature [18] brie y described as follows: rst, a loopful of activated Haemophilus colonies was suspended in 2 ml of sterile physiologic saline; second, the bacterial suspension was spread evenly onto Muller Hinton agar plates (MHAPs), NAPs and tryptic soy agar plates (TSAPs) using sterile swabs; third, a pure culture of S. aureus was streaked across each of the inoculated plates; and nally, the inoculated plates were placed in a carbon dioxide incubator at 35-37 °C for 18-24 h and then examined for growth and satellite colonies. Satellite colonies were de ned as small colonies growing in the vicinity of the S. aureus, where the tested bacteria had been inoculated.

Satellitism test of reference strains
The results (Table 1) showed that the following three conditions were less likely to produce false positives: using S. aureus activated on NAPs rather than activated on BAPs, using ATCC 25923 rather than ATCC 29213, and using TSAPs rather than TSAPs supplemented with beef extract power. Strictly speaking, the media used in the satellitism test were free of false positives and false negatives only under three conditions: one was to use TSAPs (Huankai) and only ATCC 25923 activated on NAPs, while the other two were to use MHAPs from either manufacturer and only ATCC 29213 activated on NAPs. In fact, there were very few satellite colonies in the case of "±", which was very different from "+" and might be considered negative.
In this way, TSAPs (BD) and MHAPs from both manufacturers could be used in the satellitism test of six reference strains of Haemophilus spp. with ATCC 25923 activated on NAPs. Furthermore, only H. parain uenzae (ATCC 9796) did not grow on NAPs in all four cases, meaning false-negative ndings. In addition, the results also showed that the same type of media from different manufacturers had so little in uence on the satellitism test results that this factor could be ignored. The results (Table 2) of the clinical isolates were similar to those of the reference strains, except that some of the H. parain uenzae isolates did not grow or grew more poorly on MHAPs than on NAPs. The growth performance of H. parain uenzae isolates on different media was obviously different, especially for TSAPs, where its growth was the best. There was only one isolate that grew slightly worse than all of the other H. parain uenzae isolates on all four TSAPs (BD) conditions. In addition, the H. parain uenzae isolates grew better on TSAPs.
However, the growth performance of H. in uenzae was signi cantly different among the four conditions on each medium. Although one isolate of H. in uenzae was misidenti ed as H. parain uenzae on TSAPs (BD), overall, the use of TSAPs and ATCC 25923 activated on NAPs signi cantly reduced false positives of H. in uenzae in the satellitism test.

Discussion
There are two methods of the satellitism test of Haemophilus spp., one is to use discs containing V factor, X factor or both, and the other is to use S. aureus to provide V factor, and to use blood in the medium containing blood to provide X factor. The latter is more widely used because it is easy to use.
Improper use of media can easily lead to false identi cation. Trace amounts of hemin in the medium or in a heavy inoculum are responsible for false positives of H. in uenzae, and insu cient medium to maintain the growth of bacteria is the cause of false negatives of H. parain uenzae [19,20] Our research suggested that among the factors that caused positive results in the satellitism tests of Haemophilus, such as the type of medium, different strains of S. aureus, and type of medium used for activating S. aureus, the latter had a much greater impact than the other factors. The use of BAPs to activate S. aureus was most likely to produce false positives for H. in uenzae in the satellitism test. S. aureus uses the iron-regulated surface-determinant (Isd) system to acquire iron ions in blood cells. S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB [21][22][23]. Thus, hemoglobin that has not yet been transported through the peptidoglycan layer can be released due to binding instability. This may be why the satellitism test using BAPs-activated S. aureus is prone to false positives.
Furthermore, our research also found that the effect of different strains of S. aureus on H. in uenzae on MHAPs and TSAPs might be reversed, even if they were all activated on NAPs. This might be because the stability of the binding between hemoglobin and IsdB of ATCC 25923 was not as strong as that of ATCC 29213 on MHAPs, and the opposite was true for the satellitism test using TSAPs.
The beef extract powder test we performed showed that even if S. aureus was activated on NAPs, TSAPs supplemented with beef extract powder were more likely to grow H. in uenzae colonies than the ones not supplemented. This might be why the satellitism tests on MHAPs or NAPs were more likely to produce false positives for H. in uenzae, perhaps because both media formulations contained beef extract powder and trace heme remained in the beef extract powder due to incomplete cleansing of blood cells before beef processing. However, it is unknown why a few colonies of H. in uenzae grow on TSAPs under the same condition, and further research is needed.
Although in our experiments the reference strains of H. parain uenzae were more likely to produce false negatives on NAPs, the clinical isolates were more likely to produce false negatives on MHAPs, with some discrepancies. This suggests that neither MHAPs nor NAPs are su cient to sustain the growth of all strains of H. parain uenzae in the satellitism test. However, from another aspect, the amount of NAD required for the growth of H. parain uenzae was ve times that required for the growth of H. in uenzae (1-5 pg/ml vs. 0.2-1 pg/ml) [24], which can explain why some strains of H. parain uenzae fail to grow on MHAPs or NAPs. This might be because NAD produced from S. aureus metabolism on MHAPs or NAPs is insu cient to meet the needs of some strains of H. parain uenzae, and the growth of these strains on TSAPs might be due to the generation of su cient NAD.

Conclusions
False positives of H. in uenzae and false negatives of H. parain uenzae in the satellitism test are relatively common due to the use of improper media and application methods. To ensure the accuracy of the results, we recommend that TSAPs and S. aureus (ATCC 25923) activated on NAPs should be used in the satellitism test for Haemophilus spp.

Declarations
Ethics approval and consent to participate No formal ethics approval was required in the study and informed written consent was waived by the Ethics Committee of Autobio Diagnostics Co., because the study is only about six reference strains and 114 clinical isolates of in-depth study, which does not involve in the human body, human specimens, or personal information.

Consent for publication
Not applicable.

Availability of data and material
The datasets analysed during the current study available from the corresponding author on reasonable request.