Comparison of Conventional Methods,Automated Systems,and DNA Sequence Analysis Methods in the Identification of Corynebacterium afermentans and Corynebacterium mucifaciens Bacteria Isolated from Blood and Catheter Culture Samples

Abstract

The aim of this study is to compare different methods due to the difficulties in identifying coryneform bacteria to species level and to determine antibiotic resistance profiles. Isolates identified as Turicella otitidis (n:45) by VITEK 2 Compact and Corynebacterium mucifaciens (n:1) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), isolated from blood and catheter cultures between 2015 and 2017 were included in the study. For identification of the isolates, conventional tests and 16S rDNA sequence analysis were performed. Antibiotic susceptibilities of the isolates were determined by Etest. The isolates identified as T. otitidis with VITEK 2 Compact could not be identified by MALDI-TOF MS and described as C. mucifaciens/Corynebacterium afermentans spp. by 16S rDNA sequence analysis. One isolate identified as C. mucifaciens by MALDI-TOF MS could not be identified with VITEK 2 Compact and described as C. mucifaciens by 16S rDNA sequence analysis and conventional methods. All isolates (n:45) described as C. mucifaciens/C. afermentans spp. by 16S rDNA sequence analysis were identified as C. afermentans subsp. afermentans with conventional methods. All 45 isolates identified as C. afermentans subsp. afermentans were resistant to penicillin, erythromycin, and clindamycin and were susceptible to vancomycin and daptomycin, whereas 31 (69%) were resistant to trimethoprim-sulfamethoxazole (TMP-SXT). The isolate identified as C. mucifaciens was susceptible to penicillin, vancomycin, daptomycin, and TMP-SXT; it was resistant to erythromycin and clindamycin. In this study, we reported 45 C. afermentans isolates misidentified as T. otitidis in routine laboratory processes. To our knowledge, this is the first study to include the highest number of C. afermentans blood isolates.

Introduction

Corynebacterium, one of the coryneform group bacteria, was first described by Lehmann and Neumann in 1896.¹ Corynebacterium species are aerobic or facultative anaerobic, immotile, spore-free, unencapsulated, irregularly shaped, catalase-positive, and oxidase-negative gram-positive bacilli, often with mycolic acid on the cell wall. These bacteria are commonly found in nature and are also elements of the skin and mucous membranes microbiota. Therefore, isolation of these bacteria from clinical specimens is often considered as contamination. However, there are many cases, in which diphtheroid bacilli are reported as pathogens.²

Corynebacterium is the genus that includes the largest number of species within coryneform bacteria and is divided into subgroups. Corynebacterium group absolute nonfermenter 1 (ANF-1) was first described by the CDC (Centers for Disease Control and Prevention) in 1981. The ANF-1 group of bacteria is pleomorphic gram-positive bacilli that do not produce acid from any sugar and do not have urease activities.³ Bacteria in this group have been isolated from various clinical samples, mainly blood, ear, and skin. Corynebacterium afermentans subsp. afermentans and C. afermentans subsp. lipophilum are bacteria in the ANF-1 group.⁴ Turicella otitidis has many features in common with Corynebacterium species phenotypically and biochemically and is often isolated from ear samples. It has also been called “ANF-1-like bacteria” due to its biochemical similarities with the ANF-1 group.⁵ Corynebacterium mucifaciens is the only Corynebacterium species with mucoid colonies. It is not included in the ANF-1 group, but it has 98.5% phylogenetic similarity with C. afermentans spp.⁶

Today, there are still difficulties in identifying coryneform bacteria. The aim of this study was to compare different methods for the identification of corynebacteria and to determine antibiotic susceptibilities.

Materials and Methods

Isolates

The study was conducted between August 2015 and January 2017 in Hacettepe University Medical Faculty Hospital Clinical Microbiology Laboratory. Between the specified dates, blood and venous catheter culture samples sent from various services of Hacettepe University Faculty of Medicine Hospital to the microbiology laboratory were examined. According to the services; 14 samples were sent from intensive care units, 10 from emergency services and 22 from other services. In Gram staining, 46 blood isolates with the appearance of coryneform bacteria were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (BioMerieux, France). The isolates that could not identified by MALDI-TOF MS were identified with VITEK 2 Compact (BioMerieux, France). The isolates identified as T. otitidis and C. mucifaciens with automated systems were included in the study.

16S rDNA sequence analysis

Partial 16S rDNA sequence of the isolates was performed using the specific p8FPL 5′-AGT TTG ATC ATG GCT CAG-3′ and p806R 5′-GGA CTA CCA GGG TAT CTA AT-3′ primers. The p8FPL and p806R sequences are the universal broad-range bacterial primers for amplification of ∼800 bp region of 16S rDNA.⁷ For this purpose, DNA extraction of strains has been made with the colon-based DNA isolation kit (DNA Mini Kit, Qiagen, Germany). Following isolation, ∼800 base farm 16S rDNA regions were duplicated in both directions using the GeneAmp PCR System 9700 (Applied Biosystems, USA) thermal cycler device. Amplification conditions were applied as 94°C/30 sec denaturation in 35 cycles, 60°C/30 sec binding, and 72°C/1 min elongation, following 3-min initial denaturation at 94°C. The tapes obtained in 1% agarose gel electrophoresis were purified using the QIAquick Gel Extraction kit (Hilden, Germany) for sequence analysis. Using the ABI Prism BigDyeTerminator v3.1 (Applied Biosystems) kit, a total of 35 cycles of dideoxynucleotide sequencing was performed in both directions at 96°C/10 sec, 50°C/5 sec, and 60°C/6 min (GeneAmp PCR System 9700; Applied Biosystems). Sequence products were loaded on the ABI Prism 310 Genetic Analyzer (Applied Biosystems) and obtained chromatograms were compared with other isolates registered in the database using the GenBank and BLAST (Basic Local Alignment Search Tool) server at NCBI (National Center for Biotechnology Information). To ensure high accuracy in type-level identification of the resulting sequences, data with an e-value of 0.0 and maximum similarity rates of over 99% were used for identification. A total of ∼1,200 nucleotides were analyzed and isolates were identified.

Identification by conventional methods

Catalase tests, DNase tests, and Christie–Atkins–Munch-Peterson (CAMP) tests were performed to all 46 isolates included in the study. For the CAMP test, observing increased beta-hemolysis at the junction of the two organisms as arrowhead-shaped was interpreted as a positive result. The absence of increased hemolysis was interpreted as a negative result. For the DNase test, a clear zone in methyl green DNAse agar was interpreted as a positive result. If the medium remained green, the result was interpreted as negative.⁸

Antibiotic susceptibility testing

Mueller-Hinton agar supplemented with 5% sheep blood was used. Susceptibility to clindamycin, erythromycin, vancomycin, daptomycin, penicillin, and trimethoprim-sulfamethoxazole (TMP-SXT) was investigated. To determine the minimal inhibitory concentrations of antimicrobial agents, the gradient strip method (Etest, BioMerieux, France) was used. The e-value cutoff was interpreted according to CLSI (Clinical and Laboratory Standards Institute) recommendations regarding Corynebacterium spp.⁹

Results

Colony morphology and microscopic features

Most of the colonies were 1–2 mm in diameter, gray-white, opaque, nonmucoid, gamma hemolytic, and smooth. One of the isolates was round, convex, bright, light yellowish, and had mucoid colonies. In the microscopic examination of Gram-stained smears made from colonies, short, nonbranching, spore-free, pleomorphic gram-positive bacilli have been observed to be arranged like Chinese letters.

Identification with automated system

Of the 46 isolates, only 1 isolate was identified as C. mucifaciens by MALDI-TOF MS. The isolate identified as C. mucifaciens by MALDI-TOF MS could not be identified by VITEK 2 Compact, while 45 isolates that could not be identified by MALDI-TOF MS were identified as T. otitidis.

16S rDNA sequence analysis

The isolate MALDI-TOF MS identified as C. mucifaciens is also defined as C. mucifaciens by sequence analysis. Thus, the isolate, which is macroscopically compatible with C. mucifaciens, was identified as C. mucifaciens by conventional methods, MALDI-TOF MS, and DNA sequence analysis. The other 45 isolates were defined as C. mucifaciens/C. afermentans spp. by sequence analysis. C. mucifaciens shows a phylogenetic similarity with C. afermentans spp. at 98.5% but can be separated conventionally from C. afermentans spp. by having mucoid colonies and a negative CAMP test.

Identification by conventional methods

The catalase test was positive for all isolates. The isolate identified as C. mucifaciens by MALDI-TOF MS was negative for the CAMP test and DNase test. The CAMP test was positive in 44 of 45 isolates identified as T. otitidis by VITEK 2 Compact. The DNase test was negative in all 45 isolates (Table 1). T. otitidis is called ANF-1-like bacteria because it is very similar to C. afermentans spp. (CDC coryneform group ANF-1) isolates. The colony structures of both bacteria do not differ from each other. The use of CAMP and DNase tests provides additional information for identification at the species level. Some strains of C. afermentans spp. and all strains of T. otitidis isolates are CAMP test positive. While the DNase test is positive in T. otitidis isolates, it is negative in C. afermentans spp. isolates. DNase tests of all 45 isolates were found to be negative.

Table 1.

Turicella otitidis, Corynebacterium mucifaciens, Corynebacterium afermentans subsp. afermentans, and Corynebacterium afermentans subsp. lipophilum Bacteria Tests for Species Level Separation and Phenotypic Properties

Bacteria name	Turicella otitidis	Corynebacterium mucifaciens	Corynebacterium afermentans subsp. afermentans	C. afermentans subsp. lipophilum
DNase test	+	−	−	−
CAMP test	+	−	V	V
Catalase test	+	+	+	+
Lipophilicity	−	−	−	+^a
Acid production from glucose	−	+	−	−
Colony structure	Creamy, white colonies	Bright, light yellow, mucoid colonies	Creamy, white, large (0.5–1.5 mm) colonies	Glassy, gray, small (<0.5 mm) colonies
Gram staining future	Long, nonbranching, irregular appearance gram-positive bacilli	Coccoid or club shaped gram-positive bacilli	Short, V-shaped gram-positive bacilli	Coccoid or club-shaped gram-positive bacilli
MALDI-TOF Database Availability (v2.0database)	−	+	−	−

When 1% Tween 80 is added to the 5% sheep blood agar, the colonies turn white, and the colony size increases.

CAMP, Christie–Atkins–Munch-Peterson; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; V, variable.

Subspecies distinction of 45 isolates identified as C. afermentans spp. by conventional tests and DNA sequence analysis were made according to colony morphology. After 24 hr of incubation at 37°C in 5% sheep blood agar, C. afermentans subsp. afermentans colonies were gamma hemolytic, smooth-edged, opaque, white-cream morphology, and 1–2 mm in size, while C. afermentans subsp. lipophilum colonies were bright, gray, glassy, and smaller than 0.5 mm. When colony morphology was evaluated, all 45 isolates were found to be compatible with C. afermentans subsp. afermentans.

Specimen types and properties of isolates

Of the 46 corynebacteria samples, 33 were isolated from blood and 13 from central venous catheter culture. Double blood/catheter culture was sent from 9 of 46 patients. Five patients had a single-positive blood culture, while four patients had two-positive blood cultures, which exhibited different bacterial growth.

Antibiotic susceptibility test

All 45 isolates identified as C. afermentans subsp. afermentans; were found resistant to penicillin, erythromycin, and clindamycin; susceptible to vancomycin and daptomycin; and, additionally, 31 (69%) were resistant to TMP-SXT. C. mucifaciens was found to be susceptible to penicillin, vancomycin, daptomycin, and TMP-SXT antibiotics and resistant to erythromycin and clindamycin (Table 2).

Table 2.

Evaluation of Antibiotic Susceptibility Profiles of Isolates

Antimicrobial agent	Corynebacterium mucifaciens (n = 1)			C. afermentans subsp. afermentans (n = 45)
	MIC value (μg/mL)	Category		MIC50/MIC90 (μg/mL)	MIC range (μg/mL)	Category
	MIC value (μg/mL)	S (%)	R (%)	MIC50/MIC90 (μg/mL)	MIC range (μg/mL)	S (%)	I (%)	R (%)
Penicillin	0.064	1 (100)		>32/>32	>32			45 (100)
Erythromycin	>32		1 (100)	>256/>256	24 to >256			45 (100)
Clindamycin	>256		1 (100)	>256/>256	>256			45 (100)
Vancomycin	0.5	1 (100)		0.5/1.5	0.19 to 1.5	45 (100)
Daptomycin	0.19	1 (100)		0.064/0.094	0.023 to 0.094	45 (100)
TMP-SXT	0.75	1 (100)		>32/ > 32	1 to >32	8 (18)	6 (13)	31 (69)

MIC, minimal inhibitory concentration; TMP-SXT, trimethoprim-sulfamethoxazole.

Discussion

In this study, we used MALDI-TOF MS, VITEK 2 Compact, 16S rDNA sequence analysis, and conventional methods for accurate identification of C. afermentans spp. isolates and also a C. mucifaciens isolate. Furthermore, antimicrobial susceptibility tests were performed for each isolate. To our knowledge, this is the first study to include such a high number of C. afermentans spp. blood isolates.

Corynebacterium species are found in humans as a member of the microbiota in the skin, mucocutaneous membranes, and gastrointestinal tract. They can, however, also lead to bacteremia, endocarditis, osteomyelitis, or lower respiratory tract, eye, or genitourinary system infections in humans. These bacteria are reported as an increasingly infectious agent due to the increase in the number of immunocompromised patients and the exposure of these patients to more invasive diagnostic methods and intensive treatment processes. Corynebacterium species often cause infections by acting as opportunistic pathogens in immunosuppressed patients with underlying serious disease, intravenous catheters, or those who are receiving broad spectrum antibiotic treatment.¹⁰

There are many studies that have isolated Corynebacterium species from clinical specimens in hospitals and identified them as causative agents of disease. C. afermentans spp. was isolated frequently in blood cultures. Also, antibiotic susceptibility results of C. afermentans spp. isolates in these studies were consistent with our study.^10,11 There are more than 40 cases, in which C. mucifaciens has been isolated from clinical specimens, and, in some of these cases, it has been accepted as an agent, frequently isolated from blood cultures as causative agent of bacteremia.^12,13

Corynebacteria can also be transferred from patient to patient through hospital staff, and hospital-derived isolates have often been reported more resistant to drugs. Chandra et al. performed identification and antibiotic susceptibility studies of 100 diphtheroid bacilli that grew in pure culture in the clinical samples. In this study, one reason for the high level of multidrug resistance was thought to be due to the fact that the isolates included in the study were nosocomial pathogens. They also concluded that the hospital staff could be colonized with multidrug-resistant coryneform isolates, and therefore, attention should be drawn to the risk of infection through hospital staff.¹⁴

In various studies, C. afermentans spp. isolates were found to be susceptible to tigecycline, gentamicin, rifampicin, doxycycline, teicoplanin, vancomycin, tetracycline, ciprofloxacin, fusidic acid, and linezolid and resistant to penicillin and ampicillin.^15,16 Different results were obtained against clindamycin, cefazolin, ceftriaxone, cefepime, cefotaxime, meropenem, quinopristine-dalfopristine in C. afermentans spp. isolates.¹⁷ C. mucifaciens is usually found to be susceptible to beta-lactam antibiotics, aminoglycosides, and glycopeptides, and resistant to macrolides.^6,12 In our study, in accordance with other studies, the isolate identified as C. mucifaciens was susceptible to penicillin, vancomycin, daptomycin, and TMP-SXT and resistant to erythromycin and clindamycin. All 45 isolates identified as C. afermentans subsp. afermentans were resistant to penicillin, erythromycin, and clindamycin and susceptible to vancomycin and daptomycin. TMP-SXT sensitivity was found to be different between C. afermentans isolates.

C. mucifaciens may cause pneumonia and fatal sepsis.^12,13 C. afermentans subsp. lipophilum has been reported in infections such as lung, liver, and brain abscesses and endocarditis, and C. afermentans subsp. afermentans has been reported in brain abscess as a causative agent.^18–21 Although Corynebacterium species are normal members of microbiota, they can also lead to life-threatening diseases. Therefore, the identification of coryneform bacteria at the species level and performing antibiotic susceptibility tests are important for establishing a database according to the hospital and patient population. Developing antibiotic resistance in Corynebacterium species has required identification of coryneform bacteria at the species level and monitoring of antimicrobial resistance patterns.

The results obtained in the identification of Corynebacterium using MALDI-TOF MS (Bruker Biotyper System; Bruker Daltonics, USA), conducted by Alatoom et al., were compared with 16S rRNA gene sequence analysis, and it has been reported that there were difficulties in distinguishing the Corynebacterium genus at the species level by MALDI-TOF.²² As Zasada and Mosiej stated in their study that there are still difficulties in identifying corynebacteria today. Identification of the species level with automated systems is limited. Insufficient databases can lead to failure in identification or false identification, and the isolate is matched with the closest pattern.²³ In our study, MALDI-TOF alone was found to be insufficient in the identification of C. afermentans spp. at the species level. The MALDI-TOF MS database (v2.0 database) did not contain C. afermentans spp. Also, the ANC card of VITEK 2 Compact used to identify the corineal bacteria contains T. otitidis but not C. afermentans. T. otitidis has many common features with C. afermentans spp. biochemically. Because of its similarities to C. afermentans, VITEK 2 Compact misidentified 45 C. afermentans isolate as T. otitidis. The MALDI-TOF MS database (v2.0 database) was including C. mucifaciens, so it correctly identified this isolate.

16S rDNA sequence analysis is the gold standard for identification of corynebacteria, but it is not practical for routine laboratories. Instead, it is often used for scientific studies. In our study, the isolate identified as C. mucifaciens by MALDI-TOF was also defined as C. mucifaciens by sequence analysis; the other isolates (n:45) were defined as C. mucifaciens/C. afermentans spp. by sequence analysis. C. mucifaciens shows a phylogenetic similarity with C. afermentans spp. at 98.5% and can be separated conventionally from C. afermentans by having mucoid colonies and a negative CAMP test. Thus, by conventional tests, 45 isolates were identified as C. afermentans. This type has two subspecies as C. afermentans subsp. afermentans and C. afermentans subsp. lipophilum. Differentiation of these two subspecies is also possible with conventional methods: 45 isolates were defined as C. afermentans subsp. afermentans by these methods.

It is important to determine if the blood culture comes as a set to distinguish whether the isolate is a contaminant or not. Most of the patients had only a single blood culture. If two blood cultures were obtained, a single blood culture was positive. Therefore, all of the isolates obtained in the study were evaluated as contaminants.

Rapid and accurate diagnosis of bacteria is very important in guiding the treatment to be administered to the patient. Currently, the use of automated systems in microbiology laboratories for rapid diagnosis has become widespread and has replaced conventional methods in many centers. However, automated systems also have deficiencies and sometimes erroneous results can be detected. Considering these factors, it is sometimes necessary to verify the identification detected by an automated system with conventional testing. For identification of some species of corynebacteria, automated systems are not enough. Therefore, we need to apply conventional tests with an automated system for accurate identification.

In summary, as a result of the study, it is concluded that the use of automated systems and conventional methods together is necessary for accurate identification. This study also aimed to draw attention to the fact that these bacteria, which can be easily considered contaminants, can be causative agents. Furthermore, various identification methods have been compared for fast and accurate diagnosis and antimicrobial susceptibility profiles of isolated bacteria were studied to contribute to species-specific antibiotic susceptibility profiles.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Research Fund of the Yüzüncü Yıl University Scientific Research Project as a project numbered TTU-2017-5729.

References

Funke

, and Bernard

K.A.

. 2012. Genus Corynebacterium, Lehmann and Neumann 1896. In W.B. Whitman, M. Goodfellow, P. Kämpfer, H.J. Busse, M.E. Trujillo, W. Ludwig, K. Suzuki (ed.), Bergey's manual of systematic bacteriology: Volume 5: The Actinobacteria. Springer New York Dordrecht Heidelberg,, London, pp. 245–289.

Koneman

E.W.

, Janda

, Winn

J.W

, Allen

, Procop

, Schreckenberger

, and Woods

. 2006. Aerobic and Facultative Gram-Positive Bacilli (Chapter 14). Koneman's Color Atlas and Textbook of Diagnostic Microbiology. Lippincott Williams & Wilkins, Philadelphia, PA.

Hollis

D.G.

, and Weaver

R.E.

. 1981. Gram-Positive Organisms: A Guide to Identification. Special Bacteriology Section, Centers for Disease Control, Atlanta.

Riegel

, De Briel

, Prévost

, Jehl

, Monteil

, and Minick

. 1993. Taxonomic Study of Corynebacterium Group ANF-1 Strains: proposal of Corynebacterium afermentans sp. nov. Containing the Subspecies C. afermentans subsp. afermentans subsp. nov. and C. afermentans subsp. lipophilum subsp. nov. Int. J. Syst. Evol. Microbiol. 43:287–292.

Renaud

F.N.

, Grégory

, Barreau

, Aubel

, and Freney

. 1996. Identification of Turicella otitidis isolated from a patient with otorrhea associated with surgery: differentiation from Corynebacterium afermentans and Corynebacterium auris. J. Clin. Microbiol. 34:2625–2627.

Funke

, Lawson

P.A

, and Collins

M.D.

. 1997. Corynebacterium mucifaciens sp. nov., an unusual species from human clinical material. Int. J. Syst. Evol. Microbiol. 47:952–957.

McCabe

K.M.

, Zhang

Y.H

, Huang

B.L

, Wagar

E.A

, and McCabe

E.R.

. 1999. Bacterial species identification after DNA amplification with a universal primer pair. Mol. Genet. Metab. 66:205–211.

Murray

P.R.

, Baron

E.J

, Jorgensen

J.J

, Pfaller

M.A

, and Yolken

R.H.

. 2003. Manuel of Clinical Microbiology. 8th ed. ASM Press, Washington, DC.

Clinical and Laboratory Standards Institute. 2010. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline-Second Edition. CLSI Document M45-A2. Clinical and Laboratory Standards Institute, Wayne, PA.

10.

Babay

H.A.

, and Kambal

A.M.

. 2004. Isolation of coryneform bacteria from blood cultures of patients at a University Hospital in Saudi Arabia. Saudi Med. J. 25:1073–1079.

11.

Riegel

, Ruimy

, Christen

, and Monteil

. 1996. Species identities and antimicrobial susceptibilities of corynebacteria isolated from various clinical sources. Eur. J. Clin. Microbiol. Infect. Dis. 15:657–662.

12.

Djossou

, Bézian

M.C

, Moynet

, Le Flèche-Matéos

, and Malvy

. 2010. Corynebacterium mucifaciens in an immunocompetent patient with cavitary pneumonia. BMC Infect. Dis. 10:355.

13.

Cantarelli

V.V.

, Brodt

T.C

, Secchi

, Inamine

, Pereira Fde

, and Pilger

D.A.

. 2006. Fatal case of bacteremia caused by an atypical strain of Corynebacterium mucifaciens. Braz. J. Infect. Dis. 10:416–418.

14.

Chandra

, Puthukkichal

D.R

, Suman

, and Mangalore

S.K.

. 2016. Diphtheroids-Important Nosocomial Pathogens. J. Clin. Diagn. Res. 12:DC28–DC31.

15.

von Graevenitz

, and Funke

. 2014. Turicella otitidis and Corynebacterium auris: 20 years on. Infection, 42:1–4.

16.

Funke

, Lawson

P.A

, and Collins

M.D

. 1995. Heterogeneity within human-derived centers for disease control and prevention (CDC) coryneform group ANF-1-like bacteria and description of Corynebacterium auris sp. nov. Int. J. Syst. Bacteriol. 45:735–739.

17.

Bernard

, and Pacheco

A.L.

. 2015. In vitro activity of 22 antimicrobial agents against Corynebacterium and Microbacterium species referred to the Canadian National Microbiology Laboratory. Clin. Microbiol. Newsl. 37:187–198.

18.

Dykhuizen

R.S.

, Douglas

, Weir

, and Gould

I.M.

. 1995. Corynebacterium afermentans subsp. lipophilum: multiple abscess formation in brain and liver. Scand. J. Infect. Dis. 27:637–639.

19.

Minkin

, and Shapiro

J.M.

. 2004. Corynebacterium afermentans lung abscess and empyema in a patient with human immunodeficiency virus infection. South. Med. J. 97:395–398.

20.

Sewell

D.L.

, Coyle

M.B

, and Funke

. 1995. Prosthetic valve endocarditis caused by Corynebacterium afermentans subsp. lipophilum (CDC coryneform group ANF-1). J. Clin. Microbiol. 33:759–761.

21.

Kumari

, Tyagi

, Marks

, and Kerr

. 1997. Corynebacterium afermentans spp. afermentans sepsis in a neurosurgical patient. J. Infect. 35:201–202.

22.

Alatoom

A.A.

, Cazanave

C.J

, Cunningham

S.A

, Ihde

S.M

, and Patel

. 2012. Identification of non-diphtheriae Corynebacterium by use of matrix-assisted laser desorption ionization–time of flight mass spectrometry. J. Clin. Microbiol. 50:160–163.

23.

Zasada

A.A.

, and Mosiej

. 2018. Contemporary microbiology and identification of Corynebacteria spp. causing infections in human. Lett. Appl. Microbiol. 66:472–483.