The expanding spectrum of human infections caused by Kocuria species: a case report and literature review
A 57-year-old man, a previously known case of diabetic nephropathy with end-stage renal disease undergoing continuous ambulatory peritoneal dialysis (CAPD) for the last four years, was admitted to a tertiary-care hospital in Puducherry, with complaints of abdominal pain, pedal edema and loose stool for three days. Upon initial physical examination, he was not febrile and had a normal-appearing catheter exit site. However, the peritoneal dialysate fluid was turbid. Subsequently, he became febrile, and a peripheral blood examination showed a total cell count of white blood cells 8700 cells/cu mm, neutrophils 84%, lymphocytes 12% and eosinophils 4%. The hemoglobin concentration was 9.0 g/dL, and the blood urea and creatinine levels were 161 mg/dL and 8.4 mg/dL, respectively. Random blood sugar was 170 mg/dL, total protein 5.9 mg/dL, serum albumin 1.8 mg/dL and globulin 4.1 mg/dL; the C-reactive protein level was raised to 10 mg/dL.\nThe peritoneal dialysis fluid (7 mL) inoculated into a BacT-alert FA bottle showed growth within 12 h (BacT-alert 240; bioMérieux, France). Direct microscopy of the sample showed gram-positive cocci in pairs and clusters. We performed all culturing procedures according to the 2005 update of the International Society for Peritoneal Dialysis recommendations and guidelines using specimens collected prior to antibiotic treatment. Subcultures of the peritoneal fluid were performed using sheep blood agar, MacConkey agar and chocolate agar; the plates were incubated at 35 °C for 48 h. After incubation, sheep blood agar yielded the pure luxuriant growth of pale-pink non-hemolytic colonies that were 1–2 mm size (). Subculture on nutrient agar yielded smooth, small, pale-cream to pale-pink colonies (). Gram staining of the culture revealed the cells to be gram-positive cocci in pairs or clusters (). The organism was preliminarily identified as Kocuria based on phenotypic test results, such as positive reactions for catalase, oxidase, nitrate reduction and growth in 5% NaCl and motility test negativity.\nSubsequent additional tests, such as bacitracin susceptibility, lysozyme sensitivity at 200 μg, resistance to furazolidone and lysostaphin, helped to discriminate micrococci from Staphylococci. The culture (designated as PKS1409) was later identified with a 97% probability as Kocuria rosea using a Vitek-2 system (bioMérieux) of 64 tests; the ID-GPC card panel tested positive only for α-glucosidase, leucine arylamidase, α-galactosidase, alanine arylamidase and tyrosine arylamidase. To verify the culture results, a second sample from the peritoneal fluid was evaluated, which also grew the same organism. The second isolate obtained from the second peritoneal dialysate fluid taken after 3 days showed a colony morphology, growth characteristics, biochemical test results and antibiogram identical to that of first isolate. However, an examination of the removed CAPD catheter and subsequent blood culture results showed no growth. Additionally, repeat blood samples collected from the patient who was already on antibiotics showed no growth.\nWe performed 16S rRNA gene sequencing, as previously described and compared the sequence results using the basic local alignment search tool with the EzTaxon database. The 16S rRNA gene amplified using a polymerase chain reaction Mastermix (Fermentas, Thermo Scientific, MA, USA) with the conserved primers PKRF (5′-ATC CTG GCT CAG AGC GAA CG-3′) and PKRR (5′-CCC TAC GGC TAC CTT GTT ACG-3′) generated a nearly complete sequence of the 16S rRNA gene (∼1365 bp) (Veriti-PCR; Applied Biosystems Inc., CA, USA). The polymerase chain reaction product was purified (Hi-Media, Mumbai, India) and sequenced using ABI technology (Eurofins, Bangalore, India). The basic local alignment search tool nucleotide sequence analysis revealed a high degree of homology (99.78%) with K. rosea (GenBank accession number JN084143); and the second closest match was K. polaris, with 99.56% homology (accession number AJ278868). This isolate was precisely identified and confirmed as K. rosea by a phylogenetic analysis of the 16S rRNA gene sequences by EzTaxon, a web-based tool used for the identification, using 16S rRNA gene sequences. Similar results were obtained when we constructed a neighbor-joining phylogenetic tree with the 16S rRNA gene sequences of all Kocuria species using MEGA v5.1 program ().\nSusceptibility testing through a modified Kirby-Bauer disc-diffusion technique and minimum inhibitory concentration (MIC) by microbroth dilution method was performed according to the Clinical and Laboratory Standards Institute guidelines for Staphylococcus. The disc-diffusion method revealed that PKS1409 was susceptible to ampicillin, cefotaxime, ciprofloxacin, cloxacillin, gentamicin, erythromycin, amikacin, imipenem, linezolid, teicoplanin and vancomycin, but showed intermediate resistance to ceftazidime. The MIC values for co-trimoxazole (10 mg/L), tetracycline (2 mg/L) and ceftazidime (2 mg/L) were relatively high when compared to other antimicrobials, such as benzylpenicillin (0.06 mg/L), clindamycin (0.5 mg/L) and levofloxacin (0.5 mg/L). The MIC value was less than 0.5 mg/L MIC for all the other antibiotics tested, including ciprofloxacin, erythromycin, gentamicin, linezolid, rifampicin, vancomycin and quinupristin–dalfopristin. The disc-diffusion results correlated well with the MIC values for all the common antibiotics evaluated. Biofilm production was assessed by the microtitre plate assay, and PKS1409 did not produce a biofilm. Biofilm production was further checked by growing the bacterium under a different set of conditions with different media, namely, brain–heart infusion, nutrient and Luria-Bertani broth, for different incubation time intervals of 6, 12 and 24 h. American Type Culture Collection reference strain Staphylococcus epidermidis 35984 was used as a positive control. All the experimental conditions reconfirmed that PKS1409 was not able to produce biofilm.\nA chest X-ray of the patient was normal, and an abdominal ultrasound showed findings suggestive of chronic renal failure. The intraperitoneal administration of amikacin and cefazolin was started for the empirical treatment of CAPD peritonitis. In addition, intravenous injections of vancomycin (1 g) and amikacin were given for 5 days. Improvement of the patient was visible after the initiation of antibiotic treatment, with fever subsiding and a decrease in the C-reactive protein level. Two methods of susceptibility testing showed that PKS1409 was susceptible to vancomycin. The patient responded well to the vancomycin treatment and did not develop any other complications. The patient was symptomless after 14 days of antibiotic therapy and catheter removal and was discharged.\nThe nearly complete sequence of the 16S rRNA gene of the Kocuria rosea isolate from this study has been deposited in GenBank under accession number HQ830206.