Head-to-head comparison of CAMPYAIR aerobic culture medium versus standard microaerophilic culture for Campylobacter isolation from clinical samples

Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%–99.7%) and 100% (95% CI 98%–100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%–99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79–1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.

1 Introduction

The zoonotic pathogens, Campylobacter jejuni and Campylobacter coli, are considered the leading causes of acute bacterial gastroenteritis worldwide. During 2016 in the EU, the number of reported confirmed cases of human campylobacteriosis was 246,307, with a notification rate of 66.3 per 100,000 population being the most commonly reported zoonoses, and representing almost 70% of all the reported cases, followed by salmonellosis, yersiniosis, and STEC infections (EFSA and ECDC, 2017). In the United States, a total of 303,520 culture-confirmed campylobacteriosis cases were reported during 2004–2012, with an average annual incidence rate (IR) of 11.4 cases/100,000 persons, ranging by state from 3.1 to 47.6 cases/100,000 persons (Geissler et al., 2017).

Campylobacter spp. also causes other symptoms in humans including acute exacerbation of inflammatory bowel disease and acute appendicitis. Furthermore, extra-intestinal infections such as septic thrombophlebitis, bacteremia, endocarditis, neonatal sepsis, pneumonia, bloodstream infections, brain abscesses, and meningitis may also result from Campylobacter infections (Igwaran and Okoh, 2019). Moreover, post-infectious complications associated with campylobacteriosis include reactive arthritis, severe demyelinating neuropathy, Guillain–Barré syndrome, and Miller–Fisher syndrome as well as post-infectious irritable bowel syndrome (Scallan et al., 2015; Igwaran and Okoh, 2019).

Although the epidemiology of campylobacteriosis and its long-term sequelae have been well established in developed countries, the impact of campylobacteriosis in medium- and low-income countries remains underestimated mainly due to insensitive and inconsistently applied isolation methods (Platts-Mills et al., 2014; Porte et al., 2016). In developing countries, Campylobacter epidemiology might differ compared to developed regions in that it is usually endemic, asymptomatic, and without marked seasonality (Platts-Mills and Kosek, 2014).

Campylobacter gastroenteritis is generally self-limiting, and treatments with antibiotics are not generally recommended, except in prolonged cases, systemic infections, or infections in the elderly, very young, immuno-compromised, or pregnant individuals. Campylobacter has become increasingly resistant to certain drugs, however, especially to fluoroquinolones, which are widely used for the treatment of farm animals (Fernandez and Perez-Perez, 2016). Therefore, the World Health Organization (WHO) recently promoted studies addressing the incidence and antibiotic resistance of Campylobacter spp., especially in low- and middle-income countries (LMIC) (WHO, 2013). To accomplish this goal, the development and evaluation of isolation methods, which are suitable in environments with limited resources, are crucial (WHO, 2013). Since this is hampered by economic and technical requirements, culture methods that do not require the production of artificial microaerophilic atmospheres during incubation would serve as valuable tools to determine the incidence of Campylobacter in locations that are unable to afford traditional Campylobacter culturing techniques (Hinton, 2013; Hinton, 2016).

The newly developed selective solid chromogenic medium, CAMPYAIR, supports the growth of C. jejuni and C. coli. Colonies of the pathogen are usually shiny and purple colored with or without metallic sheen after 48–72 h of incubation under aerobic conditions (Levican and Hinton, 2022). The present study aims to evaluate the ability of this novel medium to recover Campylobacter from routine clinical fecal samples compared to a commercially available medium.

2 Materials and methods

2.1 Preparation of the medium

The CAMPYAIR medium was prepared as described by Levican and Hinton (2022) with the following components and concentrations: beef extract [Merck Millipore, USA; Catalog Number (CN) B4888], 50.0 g/L; tryptose (Sigma-Aldrich Co., St. Louis, MO, USA; CN 70937), 10.0 g/L; sodium lactate syrup, 60% w/v (Sigma-Aldrich Co., USA; CN L1375), 3.0 ml/L; soluble starch (Sigma-Aldrich Co., St. Louis, MO, USA; CN S9765), 1.0%; sodium bicarbonate (Merck Millipore, Burlington, MA, USA; CN 1.06329), 1.5 g/L; agar-agar (Liophilchem, Roseto degli Abruzzi, TE, Italy; CN 611001), 15 g/L; sodium deoxycholate (Merck Millipore, Burlington, MA, USA; CN 106504), 0.1%; 2,3,5-triphenyltetrazolium (TTC, Sigma-Aldrich Co., St. Louis, MO, USA; CN T8877), 200 mg/L; and 800 ml of distilled water. The basal medium was prepared by dissolving all components, which were then sterilized by an autoclave at 121°C for 15 min. The autoclaved media was allowed to cool to 55°C then supplemented with 100 ml of filter-sterilized (0.2 μm filter) 1.5% sodium bicarbonate, 100 ml of defibrinated sheep blood, and the antibiotic supplement containing 32 mg of cefoperazone, 10 mg of amphotericin B (CCDA supplement, Lyophilchem, Roseto degli Abruzzi, TE, Italy; CN 81037), and 10 mg of vancomycin hydrochloride (Sigma-Aldrich Co., St. Louis, MO, USA; CNs 75423). The reference strains C. jejuni ATCC 33560 and C. coli DSM 4689 were used as positive controls for each batch of the medium.

2.2 Campylobacter isolation

A correlation study was designed to evaluate the agreement between the results obtained by the new medium CAMPYAIR and the CASA™ (Biomerieux, France) medium. The sample size (n = 150) was calculated by using the Epi Info program (CDC; https://www.cdc.gov/epiinfo/esp/es_index.html) with a confidence interval of 99%, 5% of error, and an expected prevalence of 6%, based on a previous data from the same clinical setting (Porte et al., 2016). Fecal samples were obtained consecutively at the Clinical Laboratory of Clínica Alemana, Santiago, Chile. To compare Campylobacter isolation, the samples were cultured on CASA™ agar plates (Biomerieux, France) and incubated for 48 h at 42°C under microaerophilic atmospheres (i.e., 5%–10% O₂, 5%–10% CO₂, and 80%–90% N₂) into an anaerobic jar (Anaerocult C, Merck Millipore, USA). Next, the samples were also cultured in parallel on CAMPYAIR agar plates, which were sealed with plastic tape and then incubated for 48–72 h at 42°C under aerobic conditions. During the trials, the CAMPYAIR technique was performed by the laboratory personnel according to the instruction manual without additional instructions or interventions of the research team. Typical Campylobacter colonies (i.e., reddish on CASA medium and purple on CAMPYAIR medium) were confirmed by MALDI-TOF mass spectrometry (Vitek MS, Biomérieux).

2.3 Statistical analyses

The performance of CAMPYAIR was compared to CASA medium, which previously showed 95% sensitivity and 99.7 negative predictive value (NPV) (Le Bars et al., 2011). Kappa Cohen coefficient was calculated with GraphPad (www.graphpad.com/quickcalcs/kappa2) and used as a measure of agreement. Results were interpreted using the following criteria: 0.01–0.20 slight agreement, 0.21–0.40 fair agreement, 0.41–0.60 moderate agreement, 0.61–0.80 substantial agreement, and 0.81–1.00 almost perfect or perfect agreement. Other performance characteristics such as sensitivity, specificity, negative likelihood ratio, positive predictive value (PPV), negative predictive value (NPV), and accuracy as well as their 95% confidence intervals were calculated using MedCalc (www.medcalc.org/calc/diagnostic_test.php), using culture on CASA medium as reference standard.

3 Results

A total of 191 consecutive samples were analyzed in parallel by CASA and CAMPYAIR media, and the ability of the media to recover Campylobacter from the samples was compared. Campylobacter spp. were detected in 8 (4.2%) samples on CASA medium and in 7 (3.7%) samples on CAMPYAIR medium (Table 1). All isolates were confirmed as C. jejuni. Campylobacter strains grew on CAMPYAIR as large, purple, and partly confluent colonies, while on CASA, colonies were reddish, with variable sizes, but predominantly small pinpoint colonies. An almost perfect agreement was observed between both media, with a 0.93 Kappa Cohen coefficient. Compared to the reference standard, CAMPYAIR had 87.5% (95% CI 47.4–99.7%) sensitivity, 100% (95% CI 98100%) specificity, 100% PPV, and 99.45% NPV (Table 2).

TABLE 1

Table 1 Samples with growth on CAMPYAIR and/or CASA medium.

TABLE 2

Table 2 Diagnostic performance of CAMPYAIR medium compared to CASA medium in routine stool samples (n = 191).

Growth of non-Campylobacter bacterial isolates was observed in nine samples (4.7%, 95% CI 2.5–8.7) on CAMPYAIR medium and in three samples (1.6%, 95% CI 0.5–4.5) on CASA medium. None of these isolates exhibited typical Campylobacter morphology. These isolates recovered from both media were identified by MALDI-TOF MS as mostly Enterobacterales, which are rod-shaped, Gram-negative, non-spore-forming, facultative anaerobes. Additionally, Lactobacillus spp., rod-shaped, Gram-positive, aerotolerant anaerobes or microaerophilic bacteria were also recovered on CAMPYAIR medium (Table 1).

4 Discussion

To validate a novel simplified Campylobacter culture technique, the CAMPYAIR medium protocol was compared head-to-head to a standard culture medium for the ability to recover Campylobacter from clinical stool samples. Results indicated that CAMPYAIR had a 0.93 Kappa Cohen coefficient. The new medium also showed high agreement with the traditional, reference method, exhibiting a sensitivity of 87.5% and an NPV of 99.45%, which is in accordance with a previous study (Le Bars et al., 2011). Although there was one was false-negative sample recovered on CAMPYAIR medium, a better growth of Campylobacter was observed on this medium with larger colonies being produced. The only sample that was positive by CASA™ agar and negative by CAMPYAIR was the first one included in this study. Therefore, the possibility that this disagreement may be due to the lack of experience of the technician who performed this analysis at the beginning of the study cannot be ruled out.

C. jejuni was the only Campylobacter species isolated from the stool samples, but C. coli showed the same morphological characteristics when grown on CAMPYAIR in previous tests (Levican and Hinton, 2022). Campylobacter culture has shown a poor sensitivity compared to molecular and immunological detection, and this poor sensitivity has been explained by the loss of Campylobacter viability in clinical specimens stored in transport medium, the number of bacteria present in the sample, and the fact that the typical laboratory culture methods are optimized for C. jejuni and C. coli and are not set up to detect additional pathogenic Campylobacter species like C. lari and C. upsaliensis (Buss et al., 2019). However, the World Health Organization (WHO, 2013) has promoted studies addressing incidence and antibiotic resistance of Campylobacter spp., especially in LMIC, by the development and evaluation of isolation methods by culturing, which are suitable in environments with limited resources. Moreover, it has been stated that the uptake of molecular and immunological detection threatens the utility of the surveillance systems, and their incorporation should be done strategically to maintain and improve surveillance for antimicrobial resistance, outbreaks, and the role of various Campylobacter species in human illness (Geissler et al., 2017). In this line, future studies aimed to assess the performance of the medium CAMPYAIR in comparison to non-cultural methods and the growth of other species different to C. jejuni and C. coli are warranted.

Although both media compared in the present study were able to inhibit the growth of most non-Campylobacter bacteria from intestinal flora, CAMPYAIR medium did allow the growth isolates of Enterobacterales and Lactobacillus; these last isolates were probably related to probiotics prescription to the patients. However, those bacteria showed their typical morphology on blood agar, i.e., large, smooth, shiny, circular, and raised white colonies for Enterobacterales, and small to medium gray colonies that were alpha or non-hemolytic for Lactobacillus. In contrast, Campylobacter produced colonies with a very different morphology (large, purple, and partly confluent colonies, with or without metallic sheen). In a previous study, Le Bars et al. (2011) have also reported non-specific growth of non-fermenters Gram-negative bacteria and yeasts in 24 of 260 samples (9.2%) on CASA™ medium, while in the present study, less growth of non-Campylobacter bacterial isolates was observed, i.e., 4.7% and 1.6% on CAMPYAIR and CASA media, respectively. The selectiveness of CAMPYAIR requires further studies with higher sample numbers and different settings. However, due to the chromogenic properties of CAMPYAIR, Campylobacter colonies were easy to differentiate, which is crucial for routine microbiological diagnosis.

In this study, the typical Campylobacter colonies on CAMPYAIR obtained after 48–72 h of incubation under aerobic conditions were confirmed by MALDI-TOF mass spectrometry (Vitek MS, Biomérieux). However, in laboratories with limited resources, the isolates can be identified by submitting them to Gram staining, oxidase and hippuricase determination, and motility observation. The isolates of Campylobacter spp. are Gram-negative bacilli, curved, S or seagull shaped, positive for oxidase and motility. Moreover, those isolates that are positive for hippuricase can be certainly identified as C. jejuni, which account for 90% or more of the cases (Levican et al., 2019).

5 Conclusion

The novel CAMPYAIR medium had a very high agreement with the standard technique based on a commercial Campylobacter medium on the ability to isolate Campylobacter from fecal samples. CAMPYAIR showed a high degree of performance for the selective isolation of Campylobacter spp. from stool samples without the need of incubating containers under microaerophilic atmospheres. Because of these lower technical requirements, CAMPYAIR could be useful to implement Campylobacter culture in countries with limited resources.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

Author contributions

Conceptualization: AL, AH, LP, IB, and TW. Methodology: AL, CV, FG, and BM. Formal analysis: AL, AH, CV, and LP. Writing—original draft preparation: AL. Writing—review and editing: AL, AH, LP, IB, and TW. Funding acquisition: AL. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by grant Fondecyt Iniciación 11181262 by the Agencia Nacional de Investigación y Desarrollo (ANID, Chile) and by grant PUCV DIE 035.691-35/2018 by Pontificia Universidad Católica de Valparaíso (PUCV, Chile).

Acknowledgments

The authors thank TM Juana Escobar and the staff from Clinical Laboratory of the Hospital Gustavo Fricke from Viña del Mar, Chile, for their help in first testing the CAMPYAIR medium.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

Buss, J. E., Cresse, M., Doyle, S., Buchan, B., Craft, D., Young, S. (2019). Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods. Eur. J. Clin. Microbiol. Infect. Dis. 38, 1087–1093. doi: 10.1007/s10096-019-03499-x

PubMed Abstract | CrossRef Full Text | Google Scholar

EFSA, ECDC (2017). The European union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. EFSA J. 15 (12), 228. doi: 10.2903/j.efsa.2017.5077

CrossRef Full Text | Google Scholar

Fernandez, H., Perez-Perez, G. (2016). Campylobacter: fluoroquinolone resistance in Latin-American countries. Arch. Med. vet. Valdivia 48 (3), 255–259. doi: 10.4067/S0301-732X2016000300002

CrossRef Full Text | Google Scholar

Geissler, A. L., Bustos Carrillo, F., Swanson, K., Patrick, M. E., Fullerton, K. E., Bennett, C., et al. (2017). Increasing Campylobacter infections, outbreaks, and antimicrobial resistance in the united states 2004-2012. Clin. Infect. Dis. 65 (10), 1624–1631. doi: 10.1093/cid/cix624

PubMed Abstract | CrossRef Full Text | Google Scholar

Hinton, J. A. (2013). Aerobic growth of Campylobacter in media supplemented with C3-monocarboxylates and C4-dicarboxylates. J. Food Prot. 76, 685–690. doi: 10.4315/0362-028X.JFP-12-430

PubMed Abstract | CrossRef Full Text | Google Scholar

Hinton, J. A. (2016). Growth of Campylobacter incubated aerobically in fumarate-pyruvate media or media supplemented with dairy, meat, or soy extracts and peptones. Food Microbiol. 58, 23–28. doi: 10.1016/j.fm.2016.03.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Igwaran, A., Okoh, A. I. (2019). Human campylobacteriosis: a public health concern of global importance. Heliyon 5 (11), e02814. doi: 10.1016/j.heliyon.2019.e02814

PubMed Abstract | CrossRef Full Text | Google Scholar

Le Bars, H., Kayal, S., Bonnaure-Mallet, M., Minet, J. (2011). CASA chromogenic medium for enteric Campylobacter species. J. Clin. Microbiol. 49 (10), 3675–3677. doi: 10.1128/JCM.00899-11

PubMed Abstract | CrossRef Full Text | Google Scholar

Levican, A., Hinton, J. A. (2022). CAMPYAIR, a new selective, differential medium for Campylobacter spp. isolation without the need for microaerobic atmosphere. Microorganisms 10 (7), 1403. doi: 10.3390/microorganisms10071403

PubMed Abstract | CrossRef Full Text | Google Scholar

Levican, A., Ramos-Tapia, I., Briceño, I., Guerra, F., Mena, B., Varela, C., et al. (2019). Genomic analysis of Chilean strains of Campylobacter jejuni from human faeces. BioMed. Res. Int. 2019, 1902732. doi: 10.1155/2019/1902732

PubMed Abstract | CrossRef Full Text | Google Scholar

Platts-Mills, J. A., Kosek, M. (2014). . Curr. Opin. Infect. Dis. 27 (5), 444–450. doi: 10.1097/QCO.0000000000000091

PubMed Abstract | CrossRef Full Text | Google Scholar

Platts-Mills, J. A., Liu, J., Gratz, J., Mduma, E., Amour, C., Swai, N., et al. (2014). Detection of Campylobacter in stool and determination of significance by culture, enzyme immunoassay, and PCR in developing countries. J. Clin. Microbiol. 52 (4), 1074–1080. doi: 10.1128/JCM.02935-13

PubMed Abstract | CrossRef Full Text | Google Scholar

Porte, L., Varela, C., Haecker, T., Morales, S., Weitzel, T. (2016). Impact of changing from staining to culture techniques on detection rates of campylobacter spp. in routine stool samples in Chile. BMC Infect. Dis. 16, 196. doi: 10.1186/s12879-016-1546-7

PubMed Abstract | CrossRef Full Text | Google Scholar

Scallan, E., Hoekstra, R. M., Mahon, B. E., Jones, T. F., Griffin, P. M. (2015). An assessment of the human health impact of seven leading foodborne pathogens in the united states using disability adjusted life years. Epidemiol. Infect. 143 (13), 2795–2804. doi: 10.1017/S0950268814003185

PubMed Abstract | CrossRef Full Text | Google Scholar

WHO. (2013). The global view of campylobacteriosis: report of an expert consultation (Utrecht: World Health Organization). Available at: http://apps.who.int//iris/handle/10665/80751 (Accessed January 15, 2023).

Google Scholar