Emerging Bacterial Infection: Identification and Clinical Significance of Kocuria Species

Recently there have been reports of gram-positive cocci which are morphologically similar to both Staphylococci and the Micrococci. These bacteria have been identified as Kocuria species with the help of automated identification system and other molecular methods including 16S rRNA (ribosomal ribonucleic acid) evaluation. Kocuria belongs to the family Micrococcaceae which also includes Staphylococcus species and Micrococcus species. Isolation and clinical significance of these bacteria from human specimens warrant great caution as it does not necessarily confirm infection due to their ubiquitous presence, and as a normal flora of skin and mucous membranes in human and animals. Most clinical microbiology laboratories ignore such bacteria as laboratory and specimen contaminants. With increasing reports of infections associated with these bacteria, it is now important for clinical microbiologists to identify and enumerate the virulence and antibiotic susceptibility patterns of such bacteria and assist clinicians in improving the patient care and management. We review the occurrence and clinical significance of Kocuria species.


Introduction And Background
Actinobacteria are a unique and large group of bacterial species containing five classes, nineteen orders, 50 families and around 220 genera as revealed by the 16S rRNA studies. They have a rigid cell wall and appear Gram-positive or Gram-variable in Gram's staining. These bacteria are aerobic/facultatively anaerobic and grow better at neutral pH. Many species of Actinobacteria are saprophytic and are present in the environment growing under varied conditions as acidophiles, alkaliphiles, halophiles, and thermophiles. Several other actinobacterial species infect plants and animals. Morphologically Actinobacteria show different shapes ranging from cocci, coccobacilli, bacilli, and long filamentous bacteria.
Kocuria is a Gram-positive cocci arranged in pairs, short chains, tetrads, cubical packets of eight and irregular clusters. Kocuria belongs to the phylum Actinobacteria, class Actinobacteria, order Actinomycetales, sub order Micrococcinae and family Micrococcaceae. This bacterium was first identified and described by Miroslav Kosur, a Slovakian microbiologist. Currently, there are more than 18 [1]. Kocuria was also isolated from various environmental and ecological niches [2]. These are usually considered as non-pathogenic bacteria which are rarely associated with human infections. Recently there has been a rise in the incidence of infections caused by Kocuria spp causing both superficial infections and deep-seated/invasive infections. The cause of concern is that this bacterium appears to have a broad host range involving both immunocompromised as well as immunocompetent individuals. This review attempts to update the morphology, cultural characteristics, pathophysiological properties, and laboratory diagnosis of Kocuria spp.

Review Cultural characteristics of Kocuria
Kocuria spp do not produce hemolysis on blood agar, unlike most clinical isolates of Staphylococci. They usually form 2-3 mm whitish, small, round, raised, convex colonies on initial isolation and might develop non-diffusible yellowish pigmentation after prolonged incubation, as shown in Figure 1.

tetrads, clusters and deeply stained very large cocci
Biochemically these bacteria show great variability by reacting differently towards conventional laboratory identification tests including the catalase, urease, and citrate utilization test. These bacteria are normally negative for mannitol fermentation and coagulase enzyme (both bound and free coagulase).
Susceptibility towards bacitracin and lysozyme and resistance to nitrofurantoin, furazolidone and lysostaphin can be used to separate this bacterium from Staphylococci. Modified oxidase test results differentiate between Kicuria spp (negative) and Micrococci.

Clinical profile of Kocuria spp
Kocuria spp have been reported to be normal flora of human skin and oral cavity and are usually regarded as laboratory contaminants and ignored when isolated in the clinical specimens undermining its pathogenic potential. Kocuria was first identified as a causative agent of urinary tract infection way back in 1974, and it was named as Micrococcus kristinae [3]. Reports of infection with Kocuria species have gained prominence in the late twentieth century and are showing an increased trend, signifying its pathogenic potential. Infections associated with isolation of Kocuria include urinary tract infections, cholecystitis, catheter-associated bacteremia, dacryocystitis, canaliculitis, keratitis, native valve endocarditis, peritonitis, descending necrotizing mediastinitis, brain abscess and meningitis [4][5][6][7][8][9][10][11][12][13][14][15]. The predisposing factors associated with infections related to Kocuria spp include congenital deformities (short bowel syndrome), chronic catheterization (in cases of total parenteral nutrition), malignancies (ovarian cancer, gastric cancer, myelodysplastic syndrome, acute myelogenous leukaemia, non-Hodgkin's disease) and patients with end-stage renal disease undergoing continuous ambulatory peritoneal dialysis. Other underlying conditions associated with Kocuria infection include diabetes mellitus, tuberculosis, stem cell transplant patients, patients suffering from gallstones, methylmalonic aciduria and pancreatic pseudocyst [16][17][18][19][20][21]. This bacterium is normally misidentified in the clinical microbiology laboratories as coagulasenegative Staphylococci (CoNS) based on its gram reaction, catalase positive and coagulase negative properties. Other physiological and biochemical properties of Kocuria are the formations of non-hemolytic colonies on blood agar, non-capsulated, non-spore forming, non-motile, non-acid fast and positive for Voges-Proskauer test (VP). It has also been observed that various species of Kocuria react differently to routine biochemical tests like the oxidase, amylase, urease, citrate utilization test, gelatinase, phosphatase tests, utilization of inulin, arabinose, N-acetyl-L-glutamic acid, and nitrate reduction tests [22]. This could be attributed to the reason behind the inaccurate identification by both conventional and an automated bacterial identification systems.  [23]. Previous research has highlighted that in the case of non-availability of molecular and advanced laboratory methods, Kocuria can still be identified and differentiated from Staphylococci and Micrococci using morphological, cultural characteristics and differential antibiotic discs. Kocuria spp are sensitive to bacitracin, lysozyme and resistant to nitrofurantoin, furazolidone and lysostaphin [18,24].

Antimicrobial susceptibility profile of Kocuria spp
Currently, there are only a few studies that have evaluated the susceptibility profile of Kocuria spp. Antimicrobial susceptibility testing results of random case reports are available, although they are still insufficient to establish the exact susceptibility results of Kocuria [25]. Kocuria spp isolated from a case of peritonitis in a 57 year-old patient suffering from end-stage renal disease revealed sensitivity to ampicillin, cloxacillin, cefotaxime, ciprofloxacin, ofloxacin, levofloxacin, gentamicin, erythromycin, clindamycin, tetracycline, amikacin, linezolid, teicoplanin, vancomycin, imipenem, quinupristin, dalfopristin, rifampicin and was found to be moderately sensitive to ceftazidime [11]. Becker et al. have reported that the Kocuria rhizophila isolated in blood from a case of sepsis in pediatric age patient revealed resistance only to norfloxacin [21]. Studies by Lee et al., who reported multiple cases involving both extreme age groups noted that Kocuria marina isolated from peritoneal fluid was resistant only to tetracycline. Other observations by the same authors showed that K. kristinae isolated from bacteremia cases revealed resistance to oxacillin, cefazolin and intermediately sensitive to cefotaxime [11]. Resistance to ciprofloxacin and erythromycin was observed in K. rhizophila isolated from blood of a 3-year-old catheterized patient as reported by Moissenet et al [26]. K varians isolated in peritoneal fluid resistant only to levofloxacin was reported by Meletis et al in a patient undergoing continuous ambulatory peritoneal dialysis (CAPD) [27].

Recent advances
A recent research report has highlighted the significance of Kocuria in causing hospitalacquired infections [13]. The same study has also noted that although Kocuria spp are commensals of humans, animals and are present in the environment, they should be considered as potential pathogens in patients who are immunocompromised, undergoing critical care treatment and neonates. A study which included 12 pediatric age patients suffering from underlying debilitating conditions like premature birth and cancer had noted that more than 50% of patients suffered from invasive infections with Kocuria spp [28]. Reports of infections caused by Kocuria spp among previously healthy and immunocompetent individuals are showing an increased trend. Kocuria rosea was isolated from a case of descending necrotizing mediastinitis in a 58-year-old woman who was taking medications for gout and hypertension [13]. Another very recent report has observed endocarditis caused by Kocuria rosea in a 10year-old female patient. Although the patient was healthy before suffering from the infection, a history of surgery to correct congenital heart disease was present [9]. Evaluation of biofilm production by Kocuria spp isolated from a case of peritonitis showed that the strain was negative for biofilm production [18]. Isolation of K. marina showing tolerance to severe alkaline conditions in a 7-year-old patient receiving epoprostenol therapy should be considered as an alarming signal regarding the potential of Kocuria spp in causing both opportunistic and nosocomial infections [29][30].

Conclusions
Identification of Kocuria spp remains elusive because most clinical microbiology laboratories have limited or no access to advanced molecular techniques. Laboratory identification of Kocuria spp can be made conventionally only after high laboratory suspicion. Properties such as morphological variability between these bacteria and other similar gram-positive cocci, as well as biochemical properties including the antimicrobial susceptibility patterns against selective antibiotics could be used to presumptively identify Kocuria spp. Infections of Kocuria spp normally involve patients with various debilitated conditions. In the era of drug resistance, and prevalence of multi-drug resistant bacteria, occurrence of Kocuria spp in hospitalized patients should not always be ignored as contaminants. Further studies emphasizing the determination of the virulence, pathogenic potential, predisposing factors and antimicrobial susceptibility patterns of Kocuria spp are warranted.

Conflicts of interest:
The authors have declared that no conflicts of interest exist.