Biofilm Formation and Antimicrobial Susceptibility of Non-Diphtheria Corynebacterium Strains Isolated from Blood Cultures: First Report from Turkey.

OBJECTIVE: Non-diphtheriae Corynebacterium strains have been recognized as important pathogens after decades of confusion regarding their microbiological classification and clinical significance. The aim of this study was to identify non-diphtheriae Corynebacterium strains and the prevalence of biofilm formation and antimicrobial resistance.
METHOD: In total, 126 non-diphtheriae Corynebacterium strains were isolated from blood cultures of inpatients with bacteremia in our hospital between January 2015 and January 2020. Blood cultures were analyzed with the Bactec-9120 system. Strains were identified using MALDI-TOF MS (Bruker Daltonics, Germany). Antimicrobial susceptibilities were determined using the Kirby-Bauer disk diffusion method on a Mueller-Hinton agar and evaluated according to EUCAST standards. Biofilm formation was assessed with the Congo Red Agar method.
RESULTS: Corynebacterium striatum and Corynebacterium matruchotii were the most prevalent with 29 and 26 isolates, respectively. Biofilm production was detected in 62.06% (18/29) of C. striatum, in 53.8% (14/26) of C. matruchotii, in 50% (9/18) of Corynebacterium afermentans, 50% (6/12) of Corynebacterium amycolatum, and in 46% (7/15) of Corynebacterium jeikeium strains. Among the five most prevalent strains, we found a high biofilm rate of 54%. The resistance rates to penicillin, clindamycin, ciprofloxacin, rifampicin, tetracycline, and gentamicin were 91.2%, 87.3%, 79.3%, 56.3%, 45.2%, and 39.6%, respectively. All 126 strains were susceptible to vancomycin and linezolid.
CONCLUSION: Non-diphtheriae Corynebacterium strains isolated from blood cultures of hospitalized patients with bacteremia may have multidrug resistance and the ability to produce biofilm. These results emphasize the importance of identifying strains and determining their antimicrobial susceptibility and biofilm production potential. © Copyright Istanbul Medeniyet University Faculty of Medicine.

Introduction

Corynebacterium is a genus of aerobic, Gram-positive, non-motile bacteria called “diphtheroids” or “coryneform”. Corynebacterium spp. are typically club-shaped and nonsporulating rods. Corynebacterium spp. are divided into two groups: Corynebacterium diphtheriae and non-diphtheriae Corynebacterium[1,2].

Previously, C. diphtheriae was considered an agent that caused serious infections, whereas other non-diphtheriae Corynebacterium strains were dismissed as contaminating bacteria when isolated from clinical specimens. Recently, non-diphtheriae Corynebacterium spp. have been recognized as important pathogens after decades of confusion regarding their microbiological classification and clinical significance. Non-diphtheriae Corynebacterium spp. are normal flora bacteria in human skin and mucous membranes. When isolated from clinical specimens they cause serious infections and nosocomial outbreaks in critically ill immunocompromised patients, for example in those with end-stage cancer, hematologic malignancy, or who have prosthetic devices or stayed for prolonged periods in a hospital or nursing homes[3-5]. Nosocomial outbreaks, especially due to Corynebacterium striatum, are increasing in both industrialized and developing countries. C. striatum is associated with pulmonary infections, sepsis, endocarditis, meningitis, osteomyelitis, arthritis, sinusitis, skin wounds, and intrauterine infection[6].

The ability to form biofilm plays a pivotal role in the pathogenesis of nosocomial infections, whether or not they are associated with devices. Biofilm makes it easier for opportunistic pathogens to adhere to catheters, implanted medical devices, and build multidrug resistance. Biofilm-associated infections are increasingly reported due to the growing elderly population and the use of implantable medical devices[6-8]. The aim of this study was to identify non-diphtheriae Corynebacterium strains and the prevalence of biofilm formation and antimicrobial resistance.

Material and Method

The study was conducted according to the principles of the World Medical Association Declaration of Helsinki “Ethical Principles for Medical Research Involving Human Subjects” (amended in October 2013). Informed consent was obtained from the patients who participated in the study with their clinical specimens.

In total, 126 non-diphtheriae Corynebacterium strains were isolated from patients’ blood cultures between January 2015 and January 2020. The strains were isolated from routine clinical samples of inpatients with bacteremia in intensive care units and in other departments of İstanbul University-Cerrahpaşa, Cerrahpaşa School of Medicine Hospital. Only patients with at least two positive blood cultures were included. Corynebacterium diphtheriae and contaminated strains were excluded.

Blood cultures of 8-10 mL samples obtained from each patient were inoculated into BD BACTEC vials and incubated in the Bactec 9120 (Becton Dickinson, MD, USA) automated blood culture system. When Gram-positive pleomorphic bacilli were seen, colonies were identified using the matrix-assisted laser desorption/ionization time-of-flight method with MALDI-TOF MS (Bruker Daltonics, Germany). Antimicrobial susceptibilities were determined by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar and evaluated according to the criteria of EUCAST (The European Committee on Antimicrobial Susceptibility Testing)[9].

To qualitatively assess biofilm formation, we used the Congo Red Agar method following Ramos et al.[10] (2019). Black colonies were considered strong biofilm producers and red colonies as non-biofilm producers.

Results

Eighty-two (65%) patients were from internal medicine wards, and 44 (35%) were from intensive care units (ICUs; Table 1).

Table 1.

The distribution of the 126 non-diphtheriae Corynebacterium strains by hospital wards.

	2015	2016	2017	2018	2019	Total (%)
Internal Medicine	7	8	19	21	17	72 (57.1)
Pediatric Internal Medicine	1	1	5	2	1	10 (8)
Intensive Care Units	6	5	15	6	6	38 (30.1)
Pediatric Intensive Care Units	-	2	3	1	-	6 (4.8)
Total	14	16	42	30	24	126 (100)

Among non-diphtheriae Corynebacterium strains, Corynebacterium striatum was the most prevalent isolate (n:29; 23.01%), followed by C. matruchotii (n:26; 20.63%), C. afermentans (n:18; 14.28%), C. jeikeium (n:15; 11.9%), C. amycolatum (n:12; 9.52%), C. mucifaciens (n:6; 4.76%), C. kutscheri (n:5; 3.96%), C. pseudodiphtheriticum (n:3; 2.38%, C. xerosis (n:3; 2.38%), C. imitans 1 (n:2; 59%), C. minutissimum (n:2; 1.59%), and C. singulare, C. aguaticum, C. aurimucosum, C. propinquum, C. bovis (for each n:1; 0.8%).

Biofilm formation by non-diphtheriae Corynebacterium strains were determined as 50.8%. Biofilm production was detected in 62.06% (18/29) of C. striatum, in 53.8% (14/26) of C. matruchotii, in 50% (9/18) of C. afermentans, in 50% (6/12) of C. amycolatum, and in 46% (7/15) of C. jeikeium strains (Table 2). C. singulare, C. aguaticum, C. aurimucosum, C. propinquum, and C. bovis were identified as non-biofilm producing isolates. Biofilm production rates of the five most prevalent strains (more than 10 isolates) are shown in Figure 1. Among these strains, we found a high biofilm forming rate of 54%.

Table 2.

Distribution of biofilm-forming non-diphtheria Corynebacterium spp.

Bofilm production	N	%
C. striatum	18/29	62.0
C. matruchotii	14/26	53.8
C. afermentans	9/18	50.0
C. jeikeium	7/15	46.0
C. amycolatum	6/12	50.0
C. mucifaciens	3/6	50.0
C. kutscheri	3/5	60.0
C. pseudodiphtheriticum	1/3	33.3
C. xerosis	1/3	33.3
C. imitans	1/2	50.0
C. minutissimum	1/2	50.0
C. singulare	0/1	-
C. aguaticum	0/1	-
C. aurimucosum	0/1	-
C. propinquum	0/1	-
C. bovis	0/1	-
Total	64/126	50.8

Figure 1.

Biofilm production rates of top-five prevalent strains in the study.

The resistance rates to penicillin, clindamycin, ciprofloxacin, rifampicin, tetracycline, and gentamicin were 91.2%, 87.3%, 79.3%, 56.3%, 45.2%, and 39.6%, respectively. All 126 strains were susceptible to vancomycin and linezolid (Table 3). According to our results, there is an increasing rate of resistance to clindamycin, ciprofloxacin, rifampicin, gentamicin, tetracycline, and especially penicillin (Figure 2).

Table 3.

Distribution of antimicrobial resistance among non-diphtheriae Corynebacterium strains by years.

	2015		2016		2017		2018		2019		Total
	N	%	N	%	N	%	N	%	N	%	N	%
Penicillin	11/14	78.5	14/16	87.5	39/42	92.8	28/30	93.3	23/24	95.8	115/126	91.2
Clindamycin	11/14	78.5	13/16	81.2	39/42	92.8	27/30	90	20/24	83.3	110/126	87.3
Ciprofloxacin	9/14	64.2	12/16	75	35/42	83.3	26/30	86.6	20/24	83.3	100/126	79.3
Rifampicin	9/14	64.2	10/16	62.5	23/42	54.7	18/30	60	11/24	45.8	71/126	56.3
Gentamicin	6/14	42.8	6/16	37.5	19/42	45.2	14/30	46.6	12/24	50	50/126	39.6
Tetracycline	4/14	28.5	6/16	37.5	19/42	45.2	14/30	46.6	11/24	45.8	57/126	45.2
Linezolid	0/14	0	0/16	0	0/42	0	0/30	0	0/24	0	0/126	0
Vancomycin	0/14	0	0/16	0	0/42	0	0/30	0	0/24	0	0/126	0

Figure 2.

Trends of antimicrobial resistance to penicillin, clindamycin, ciprofloxacin, rifampicin, gentamicin, and tetracycline between 2015-2019.

Discussion

Corynebacterium spp. are common in the environment and as part of the normal skin flora and mucous membranes. The pathogenic potential of coryneform bacteria has long been underestimated. Long considered as a contaminant, they were ignored as a cause of infection when isolated from clinical specimens in microbiology laboratories[11]. However, owing to the increasing number of immunocompromised patients, Corynebacterium spp., which are usually found as opportunistic pathogens in patients with immune deficiency, have become clinically relevant. In recent years, various non-diphtheriae Corynebacterium spp. have been increasingly reported to be infectious agents in inpatients and have caused outbreaks in ICUs[3,5,10]. Yoldas et al.[12] showed that more than 10% of the microbial growth in clinical cultures were Gram-positive bacteria cultured from ICU patients.

Recent studies have increasingly reported that the following strains are important causes of infection: C. striatum, C. jeikeum, C. amycolatum, C. urealyticum, C. afermentans, C. ulcerans, C. minitissimum, C. propinquum, and C. pseudodiphtheriticum[3]. Studies investigating various clinical specimens have indicated that between 44% and 71% of patients with Corynebacterium bacteremia have true infections[13-15]. When coryneform bacteria are isolated from sterile samples such as blood, it is difficult for microbiologists to identify their clinical significance[11].

However, the lack of single definition of true infection and contamination hinders accurate conclusions regarding the incidence of true Corynebacterium infections[15]. For diagnosing true infection, some researchers use only one bacteriological criterion based on the presence of two or more positive blood cultures, while many studies have reported that time to positivity can be used to distinguish between contamination and bacteremia[13,14,16-18]. Zhang et al.[16] reported that the time to positivity was <36 h in 98% of bacteremia caused by Gram-positive bacteria. In the present study, positivity was detected within 24 h for all bacteremia cases. In other studies, patients’ clinical conditions were taken into consideration and intravascular catheters were accepted as risk factors for true Corynebacterium infection[15,19,20].

In the present study, non-diphtheriae Corynebacterium strains that grew in the blood cultures between 2015-2020 (with at least two positive blood cultures) were included if they were compatible with the patients’ clinical features. The most common isolated strain was C. striatum (23%) followed by C. matruchotii (21%).

C. striatum strains are rarely isolated from the blood, however they have often been reported as a cause of catheter-associated bloodstream infections and endocarditis[3,4,21,22]. In the present study, we saw that our strains were isolated mainly from samples sent from internal medicine and ICUs.

Although opportunistic infections caused by these microorganisms are mostly endogenous, epidemiological studies have revealed that bacterial transition from patient to patient is possible in ICUs. Hospital staffs play an important role as carriers with their contaminated hands in this transition, according to some studies[11,21].

A retrospective study conducted by Yanai et al.[14] demonstrated that C. striatum was the most common strain detected in bacteremia patients and infections of more than 50% of bacteremic patients were catheter-associated. This route of infection has been reported in many studies[18,23,24].

Forty-four (35%) strains were isolated from inpatients who used catheters or other foreign bodies in ICUs. Traditionally, skin commensal bacteria have relatively low virulence. However, biofilm-forming ability can be a high-virulence factor for multidrug-resistant C. striatum. Previous studies reported that C. striatum causes nosocomial outbreaks associated with biofilm formation[4,8].

During a nosocomial outbreak in a hospital in Rio de Janeiro, Brazil, the ability of various clones of multidrug-resistant and multidrug-sensitive C. striatum strains to form biofilm on the surfaces of foreign materials was investigated. The C. striatum type I-multidrug-resistant strain was shown to have the greatest ability to adhere to biotic and abiotic surfaces. This clarified the relationship between biofilm-forming ability, antimicrobial multi-resistance, and clonality[6,10]. All isolates were multidrug-resistant. Since biofilm-forming isolates are limited, we could not assess it statistically.

Biofilm production in non-diphtheriae Corynebacterium strains isolated in blood cultures was first reported by Qin et al.[19]. To the best of our knowledge, this is the first report on biofilm-forming non-diphtheriae Corynebacterium in blood culture isolates in Turkey. In our study, 51% of the strains were biofilm producers. Biofilm-forming ability was above average for C. striatum strains at a rate of 62%. Similarly, Qin et al.[19] reported a biofilm production rate of 64.3% in C. striatum strains isolated from blood cultures in Japan.

Previously, non-diphtheriae Corynebacterium strains were susceptible to many antibiotics, but recent studies have reported they are multidrug-resistant. Given the increasing use of broad-spectrum antibiotics, multidrug resistance also occurs in non-diphtheriae Corynebacterium strains[4,18,25]. Some studies have shown that antimicrobial resistance rates in biofilm-forming isolates were higher than in non-biofilm producers[26].

The present study assessed antimicrobial resistance rates over five years, which showed that strains have become more resistant to penicillin, clindamycin, ciprofloxacin, rifampicin, and tetracycline. We found the highest resistance to penicillin in 91.2%, followed by clindamycin in 87.3% of our isolates Asgin et al.[25] also reported a resistance rate of 87.7% to clindamycin in 81 C. striatum strains in Turkey. All isolated strains were susceptible to vancomycin and linezolid. So far, no vancomycin or linezolid resistance has been reported.

The resistance rates are in line with some recent studies[11,20]. Especially high penicillin resistance rates were detected in many studies[4,16,19]. In both present and previous reports, vancomycin has been proposed as an empirical therapy for severe infections caused by non-diphtheriae-Corynebacterium species[3,11]. However, the management of antimicrobial treatment for these infections is still fraught with controversies. In vitro susceptibility tests have shown that linezolid and tigecycline are effective against coryneform bacteria[3].

Many studies have recommended vancomycin as the first treatment option when invasive C. striatum infection was suspected, because none of the Corynebacterium strains have been reported to have in vitro resistance to vancomycin. If the patient was allergic to vancomycin, linezolid or daptomycin has been recommended[4,11,20,27]. Successful treatment in these cases depends on long-term, high-dose antimicrobial therapy and the removal of foreign body material[8]. These results suggest that non-diphtheriae Corynebacterium strains from inpatients with bacteremia are multidrug-resistant and increasingly have the ability to form biofilms.

Non-diphtheriae Corynebacterium spp. should not be overlooked when isolated from blood cultures, as it may actually be the cause of infection, especially considering their virulent biofilm-forming abilities.

To implement advanced control strategies to reduce non-diphtheriae Corynebacterium-associated infections in hospitals, it is important to perform effective infection control measures focused on non-diphtheriae-Corynebacterium colonization. It is especially important that healthcare workers comply with hand hygiene and cleaning medical equipment and hospital surfaces with appropriate disinfectants.

Surveillance studies on non-diphtheriae Corynebacterium should also be performed in hospitals to increase awareness of C. striatum and other non-diphtheriae Corynebacterium that cause bloodstream infections and to prevent biofilm-related infections. The results of this study are based on a small sample, so further studies need to be done with greater number of patients.

Conclusion

Corynebacterium striatum and Corynebac-terium matruchotii were the most prevalent non-diphtheriae Corynebacterium strains. Among the five most prevalent strains of the study, there was a high biofilm rate of 54%. An increasing resistance rate was found to clindamycin, ciprofloxacin, rifampicin, gentamicin, tetracycline, and especially penicillin. The highest resistance was to penicillin in 91.2%, followed by clindamycin in 87.3% of the strains. To the best of our knowledge, this is the first report on biofilm-forming non-diphtheriae Corynebacterium blood culture isolates in Turkey.