Introduction:
Corynebacteria is an important part of the commensal flora of the skin, mucous membranes and secretions from the oropharynx, and eye. Corynebacterium other than C. diphtheria are referred to as Non- diphtheritic corynebacteria “diphtheroids”. Of the Corynebacterium species, C. jeikeium, C. urealyticum, C. minutissimum and more recently C. striatum have gained medical interest due to an evolving number of publications referring to community-acquired and nosocomial infections (1). C. jeikeium is well-characterized as a pathogen in neutropenic hosts and those with prosthetic devices and indwelling catheters (2).
Human skin flora is rich in coryneform bacteria. Hence, when isolated from clinical specimens these isolates are dismissed as mere contaminants in the laboratory setting, without going into the details of patient profile or repeat microbiological analysis. Non-diphtheria corynebacteria species cause infections in risk populations such as immunocompromised patients and patients with indwelling medical devices. They are also found associated with catheter-associated blood stream infections, meningitis, neurosurgical shunt infection, brain abscess, peritonitis, osteomyelitis, septic arthritis, urinary tract infections, empyema and pneumonia. The importance of these isolates in clinical settings is still underestimated as evidenced by the lack of comprehensive data with a large number of isolates (2). Currently, more than half of the infectious diseases affecting mildly immunocompromised patients involve commensals and that are commonly encountered in the environment (3).
Only recently the Clinical and Laboratory Standards Institute (CLSI) has proposed a reference microdilution method for testing these organisms. At present, there are no specific guidelines with regard to the disc susceptibility testing and antimicrobial susceptibility pattern of coryneform bacteria. Only a few studies have focused on particular organisms such as Corynebacterium jeikium and Corynebacterium urealyticum. Report on these group of organisms from India is sparse, except for a rare case report concerning a particular species. There is a total absence of the antimicrobial susceptibility pattern of the coryneform organisms in Indian settings, and we have to depend only on western data (1).
These opportunistic pathogens may be endowed with an array of virulence factors that facilitate their ability to survive within host tissues and confer resistance to clearance by host immune mechanisms. The ability to form biofilms may be a prerequisite for the pathogeneses of nosocomial diseases associated (or not) with the use of medical devices. Biofilm is responsible for the bacterial infection, infection on medical devices, deterioration of water quality and contamination of food. Biofilms have been previously described in Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium urealyticum and Corynebacterium jeikeium (4,5). Biofilms can act as physical diffusion barriers to prevent antibiotics from reaching it targets (4). In addition, the ability of bacterial cells to transfer genes horizontally is enhanced within biofilm communities, thereby facilitating the spread of antibiotic resistance.
This study was performed, to find out the spectrum of the species of NDCs belonging to the genus that actually cause clinical infections and to determine its antimicrobial susceptibility.
Materials and Methods
This cross section study (December 2016- June 2017) was conducted in the Division of Microbiology, Malabar cancer centre, Thalassery, Kannur. Clinical specimens like sputum, pus, exudates, bronco-alveolar aspirates, urine and blood submitted to the Division of Microbiology were analyzed with approval by Institutional Review Board (IRB), MCC.
Samples received in the microbiology lab were cultured on Blood agar, Chocolate agar and Mac Conkey agar plates and incubated at 37ºC for 24 - 48 hrs. Gram’s staining was performed on direct smears to assess the quality of specimens and presence of microorganisms. The diphtheroids were considered as clinically significant and further processed, when they were isolated in pure growth or their predominance when they are found in association with other microorganisms. The identification of isolates was done based on colony morphology, pigmentation, hemolysis, presence of metachromatic granules in Albert’s stain, motility and biochemical tests like Catalase test, Oxidase test, Nitrate reduction, Urease production, Esculin hydrolysis and fermentation of Glucose, Maltose, Sucrose, Lactose, Galactose, and Trehalose.
Antibiotic Sensitivity Testing
Antibiotic sensitivity testing was done using the Kirby-Bauer disc diffusion technique using Mueller Hinton broth enriched with 5% horse blood against ampicillin (10 μg, amoxicillin- clavulanic acid (20/ 10 μg), cefaperazone-sulbactum (75/10μg), ceftriaxone (30 μg), chloramphenicol (30 μg), clindamycin (2μg), ciprofloxacin (5μg), erythromycin (15μg), gentamicin (10μg), imipenem (10μg), linezolid (30μg), nitrofurantoin (300 μg), oxacillin (1μg), penicillin (10 μg), piperacillin-tazobactum (100/10 μg), tetracycline (30 μg), tigecycline (15μg), vancomycin (30μg). Antibiotics for which CLSI has not defined any susceptibility criteria were adopted from British Society for Antimicrobial Chemotherapy (BSAC) guidelines. Quality control was achieved by Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922.
Biofilm production test was performed by the microtitre plate method by following O’Toole and Kolter protocol to access the biofilm forming capacity of the isolates.
Results:
A total number of 1,662 specimens were received for culture during the study period. Of these, 560 specimens yielded meaningful growths – implying that a bacterial etiology could be established in only 33.7% of the cases. The rest of 902 (66.3%) of the samples either produced no growth or non-significant growths or were contaminated. Diphtheroids accounted for 100 of the 560 infections. Thus, the incidence of infections caused by coryneform bacteria among cancer patients in our study was estimated to be 17.9%. (Table 1).
| Table 1: Incidence of infections caused by Diphtheroids |
Specimens |
Numbers |
Total no. of specimens for culture |
1662 |
Culture positive specimens |
560 (33.7%) |
Specimens yielding pathogenically significant diphtheroids |
100 |
Incidence of diphtheroids among bacterial pathogens |
17.9% |
The overwhelming majority of infections due to coryneform bacteria were seen in the elderly age groups. 90% of the cases being >40 years old (Fig 1). As regards the gender distribution showed that, 59% isolated were in males while 41% were from female patients. The male predilection of Corynebacterium pseudodiphtheriticum was significantly high with 24 strains and 23 from females (Table 2).
 |
Fig 1: Age distribution of coryneform isolates in cancer patients. |
| Table 2: Number of NDCs obtained from clinical samples based on gender |
Isolate |
Male % |
Female % |
Total |
Corynebacterium pseudodiphtheriticum |
23% |
24% |
47 |
Corynebacterium ulcerans |
23% |
6% |
29 |
Corynebacterium straitum |
4% |
8% |
12 |
Corynebacterium minitissimum |
7% |
3% |
10 |
Corynebacterium xerosis |
2% |
0 |
2 |
Total |
59 |
41 |
100 |
Corynebacterium pseudodiphtheriticum was the predominant isolate in our study in that, this species was found to constitute 47 (47%) of all the diphtheroid across all specimens. This was followed by Corynebacterium ulcerans 29 (29%), Corynebacterium striatum 12 (12%), Corynebacterium minutissimum 10 (10%), and Corynebacterium xerosis 2 (2%).
Sputum along with other respiratory secretions (54%) and pus (32%) were the most prominent samples presenting diphtheroids as pathogenically significant isolates. Blood (6%), fluids and other exudates (7%) and urine were other specimens from which diphtheroids were isolated (Table 3). A majority of infections – 71 (71%) involving diptheroids that were studied by us were polymicrobial – diphtheroids contributing to the infections rather than causing them solely. However, these pathogens were also found to be primary pathogens –causing monomicrobial infections and isolated as pure culture from 29 (29%) of the samples.
| Table 3: Percentages of non-diphtheritic corynebacterial species from clinical samples |
Isolate |
Respiratory |
Pus |
Urine |
Blood |
Exudate |
Corynebacterium pseudodiphtheriticum |
24 |
17 |
1 |
3 |
2 |
Corynebacterium ulcerans |
20 |
6 |
- |
2 |
1 |
Corynebacterium Minutissimum |
4 |
5 |
- |
- |
1 |
Corynebacterium xerosis |
1 |
1 |
- |
- |
- |
Corynebacterium striatum |
5 |
4 |
- |
- |
3 |
Total |
54 |
33 |
1 |
5 |
7 |
Staphylococcus aureus was the most predominant co-pathogen (18%) followed by Moraxella catarrhalis (15%), Pseudomonas aeruginosa (12%), Streptococcus spp (10%), Klebsiella pneumonia (6%), Acinetobacter baumanii (6%), Escherichia coli (3%) and Enterococcus faecalis (1%) (Table 4).
| Table 4: Co-pathogens isolated along with diphtheroids |
Co-pathogen |
Respiratory |
Pus |
Urine |
Blood |
Exudates |
Total |
Staphylococcus aureus |
11 |
7 |
0 |
0 |
0 |
18 |
Moraxella catarrhalis |
9 |
4 |
0 |
0 |
2 |
15 |
Pseudomonas aeruginosa |
7 |
5 |
0 |
0 |
0 |
12 |
Streptococcus Spp |
6 |
4 |
0 |
0 |
0 |
10 |
Klebsiella pneumonia |
3 |
2 |
1 |
0 |
0 |
6 |
Acinetobacter baumanii |
3 |
3 |
0 |
0 |
0 |
6 |
Escherichia coli |
2 |
1 |
0 |
0 |
0 |
3 |
Enterococcus faecalis |
1 |
0 |
0 |
0 |
0 |
1 |
All |
42 |
26 |
1 |
0 |
2 |
71 |
More than one-half i.e., 42 (59.2%) of the polymicrobial infections involving Coryneform bacteria in this study were respiratory followed by skin and soft tissue (pus samples) 26 (36.6%), urinary (1) and other fluids (2). All the blood stream infections (6) caused by diphtheroids were monomicrobial.
Table 5 presents an alarming state of antibiotic resistance showed by these organisms. Most of the tested antibiotics showed poor effectiveness except imipenem (100% susceptibility), linezolid (100% susceptibility) and tigecycline (100% susceptibility). Few of the other antibiotics showed some in-vitro activity – ampicillin (36% sensitivity), amoxicillin (27%) and clindamycin (27%). A notable aberration was the poor in-vitro result of vancomycin susceptibility testing which showed only 8% susceptibility.
| Table 5: Antibiotic sensitivity pattern of clinical isolates from all clinical sample percentage |
Name of organism |
Total no. of isolates |
AMP |
AMC |
CFS |
CTR |
C |
CIP |
CD |
AZM |
GE N |
IPM |
LZ |
NIT |
CX |
P |
PIT |
TE |
TGC |
VAN |
C.pseudodiphtheriticum |
47 |
11 |
11 |
0 |
0 |
2 |
2 |
12 |
0 |
0 |
100 |
100 |
1 |
1 |
0 |
0 |
0 |
100 |
0 |
C. straitum |
29 |
100 |
50 |
25 |
0 |
0 |
50 |
66 |
0 |
0 |
100 |
100 |
25 |
8 |
0 |
0 |
8 |
100 |
25 |
C. ulcerans |
10 |
11 |
70 |
70 |
0 |
0 |
0 |
14 |
0 |
0 |
100 |
100 |
2 |
0 |
0 |
0 |
0 |
100 |
0 |
C
.minutissimum |
2 |
60 |
60 |
20 |
0 |
20 |
40 |
20 |
20 |
30 |
100 |
100 |
0 |
0 |
0 |
20 |
10 |
100 |
30 |
C.xerosis |
12 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
100 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
All isolates |
100 |
36 |
27 |
15 |
0 |
1.5 |
16 |
27 |
0.4 |
0.6 |
100 |
100 |
8 |
3 |
0 |
0.4 |
2.5 |
100 |
8 |
| AMP-ampicillin, IPM-imepenem, AMC-amoxicillin, LZ-linezolid, CFS-cefoperazone sulbactam, NIT-nitrofurantoin, CTR-ceftriaxone, CX-cefoxitin, C-chloramphenicol, P-penicillin, CIP-ciprofloxacin PIT-piperacillin-tazobactam, CD-clindamycin, TE-tetracycline, AZM-azithromycin, TGC-tygecycline, GEN-gentamicin, VAN-vancomycin. |
We tested our corynebacterium isolates for their ability to form biofilms. The ability to form biofilm production was obtained in pus and exudates. Isolates showed variable biofilm production (Table 6).
| Table 6: Biofilm production of diphtheroides in clinical samples |
Sample |
OD value at 640nm |
Blood |
0.476 |
Respiratory tract specimens |
0.497 |
Urine |
0.498 |
Pus |
0.544 |
Exudates |
0.678 |
Discussion
Studies have demonstrated that Corynebacterium species and various coryneforms, particularly those taxa found as part of normal skin flora, are prominent contaminants of clinical materials, although occasionally it is difficult to correctly decide in a timely fashion if recovery of such bacteria implies contamination or has clinical relevance (6).
It is agreed by the vast majority of experts that clinical relevance must be afforded to coryneforms if the organism is isolated (i) from normally sterile body sites, e.g., blood culture (with the exception if recovered from only one of multiple specimens taken becoming positive), (ii) from adequately collected clinical material where Corynebacterium species or coryneforms are the predominant organism, and (iii) if recovered from urine specimens, e.g., C. urealyticum, is the sole bacterium encountered with a bacterial count of >104/ml or if it is the predominant organism recovered and the total bacterial count is >105/ml (6). Going by these guidelines we sought to study the role of Coryneform bacteria in infections among cancer patients. During the study period, we cultured all the specimens that were submitted to the Microbiology laboratory with an aim to recover, isolate, identify and study the properties of clinically significant coryneform bacteria causing infections among cancer patients.
The incidence of diphtheroids among bacterial pathogens found by us is remarkably high (17.9%) as compared to that found in previous studies and found it to be 5.5% (7,8). There is a paucity of published data as not many studies have been conducted on infections caused by diphtheroids in patients with cancer. The high incidence seen in our study could be due to our patient population. Being debilitated and immunosuppressed, these group of patients are increasingly vulnerable to opportunistic infections by resident flora.
The overwhelming majority of our cases were elderly patients - >90% of them being >40years old. This was anticipated by us as our centre is a cancer institute. The commonly isolated NDC in our study are C.pseudodiphtheriticum followed by C.ulcerans, C.straitum, C.minutissimum and C.xerosis. C.pseudodiphtheriticum was also most commonly isolated non diphtheritic corynebacteria by Camello et.al (2008) wherein 94.7% was recovered in pure culture from clinical specimens (8). C.ulcerans was also most commonly isolated nondiphtheritic corynebacteria followed by 8% C.striatum, 6% C.minutissimum, 3% C.urealyticum and 0.7% C.xerosis
in naother study(9). Our percentage and pattern of different species of non-diphtheritic corynebacteria when compared by other
studies may be due to the infection caused by corynebacterial species in healthy subjects.
Among the non-diphtheritic corynebacteria isolated C.pseudodiphtheriticum (47%) were most commonly seen in males compared with females followed by C.ulcerans (29%), C.minutissimum (10%), C.striatum (12%) and C. xerosis (2%).
In the present study, among the 100 NDC, 20 isolates were found to be obtained as pure growth and remaining were obtained as mixed growth with diphtheroids as the predominant isolate. Staphylococcus aureus (18%) isolate was found to be commonest bacteria associated with diphtheroids followed by Moraxella catarrhalis (17%), Pseudomonas aeruginosa (12%), Streptococcus spp (10%), Klebsiella pneumoniae (6%), Acinetobacter baumanii (6%) and E.coli (3%). E.coli (24%) was the most commonly isolated bacteria followed by Klebsiella spp (12%), Pseudomonas (12%), Staphylococcus aureus (12%), Moraxella catarrhalis (8%), Enterococcus spp (4%) and group C Streptococcus spp (4%).(10)
The overall susceptibility pattern of our study revealed high susceptibility pattern against tigecyclin (100%), imepenem (100%) and linezolid (100%). The susceptibility pattern of the antibiotics in our study was near accordance with results presented by Reddy et.al (11), in which the percentage of their isolates which were sensitive to linezolid (96%), imepenem (92%). penicillin (0%) was the least active drug followed by chloramphenicol, gentamycin, vancomycin, azithromycin, cefoperazone etc (11). Similarly, Soriano et.al (4) also reported vancomycin resistance in C.pseudodiphtheriticum (4). Multidrug resistance
was found in all corynebacterial species. This may be due to the ability to form biofilm which may be prerequisite for the pathogenesis of nosocomial disease associated (or not) with the use of medical devices. In our study high biofilm production
was seen in C.ulcerans, C.psuedodiphtherirticum, C.striatumm C.minutissimum and C.xerosis which has been reported previously also in C.diphtheriae, C.pseudomonas, C.renale, C.urealyticum, and C.jeikeium. Thus the better recognition and understanding of the biology and virulence potential of Corynebacterium species strain may help to effectively prevent infection caused by them.
Conclusion
Corynebacteria are an important part of the commensal flora of the skin, mucous membranes, secretions from the oropharynx, pus and eye infections. Hence this study was performed to find out the spectrum of the species of the organism belonging to the genus Corynebacterium that actually cause clinical infections and to determine their antimicrobial susceptibility.
The incidents of infections caused by bacteria among cancer patients in our study were estimated to be 17.9%. Among the infected individuals 59% were male patients and 41% were female patients. The overwhelming majority of our cases were elderly patients (>90%) of them being more than 40 years old. C. pseudodiphthereticum was isolated from 47% of the specimens. Sputum along with other respiratory secretions (54%) and pus (32%) were the most prominent samples presenting diphtheroids as pathogenically significant isolates. Blood (6%) fluids and other exudates (7%) and urine were other specimens from which diphtheroids were isolated. However, these pathogens were also found to be primary pathogens causing mono-microbial infections and isolated as pure culture from 29 (29%) of the samples. There was an alarming state of resistance among the isolates. Most of the tested antibiotics showed poor effectiveness except imipenam (100% susceptibility), linezolid (100% susceptibility) and tigecycline (100% susceptibility). The isolates were also found capable of forming biofilms. To conclude from the findings, Corynebacterium sp. pathogens were found predominantly from the samples obtained from cancer patients. Isolates were found to possess biofilm forming capacity and are highly resistant to major class of antibiotics.
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