Staphylococcus hominis subsp. novobiosepticus, an emerging multidrug-resistant bacterium, as a causative agent of septicaemia in cancer patients.

Staphylococcus hominis subsp. novobiosepticus is a new sub-species of S. hominis, thus dividing S. hominis into subsp. hominis and novobiosepticus. This study was designed to identify subsp. novobiosepticus isolates amongst the S. hominis isolated from blood samples of patients with malignancy and septicaemia and to study their resistance profile. The identification was performed by using three simple tests which differentiated between the two sub-species. It was found that 22.8 per cent of S. hominis isolates belonged to subsp. novobiosepticus.

Staphylococcus hominis is one of the three most frequently identified isolates recovered from bloodstream infections (BSIs)1. In 1998, characterization of subsp. novobiosepticus divided S. hominis into two sub-species namely S. hominis subsp. hominis (SHH) and S. hominis subsp. novobiosepticus (SHN)2. SHN is reported to be exhibiting multidrug resistance, thus leaving narrow therapeutic options3. SHN has been reported from the USA, Spain and Brazil as a causative agent of invasive infections and outbreaks2345. This study was undertaken in the Microbiology division of Laboratory Medicine department in Delhi State Cancer Institute, a tertiary care cancer centre in New Delhi, India, over a period of 10 months (September 2013- July 2014) to look for the presence of subsp. novobiosepticus in the S. hominis isolates from patients with septicaemia and also to characterize their resistance profile to various antimicrobial agents. The study was approved by the institutional ethics committee and written informed consent was obtained from all participants.

Blood samples were collected from all inpatients suspected of having BSI. As far as possible, blood was collected from two different sites at the same time. From adults, 10-20 ml of blood, and from paediatric patients, 5-10 ml of blood was collected and added to BacT/ALERT® FA Plus Blood Culture Bottles and incubated in BacT/ALERT® 3D system (BioMe’rieux, Durham, North Carolina, USA)6. Sub-cultures were performed from the culture bottles flagged as positive. The colony characteristics of sub-cultured organisms were examined and Gram staining was done. Gram-positive cocci were identified in VITEK® 2 Compact System (BioMe’rieux, Durham, North Carolina, USA) using Gram-positive GP REF 21342 identification card. The pathogenicity of all coagulase-negative staphylococci (CoNS) isolates was confirmed. They were considered to be pathogenic if (i) the same isolate was obtained from blood drawn from two or more peripheral sites, and/or (ii) the clinical presentation correlated with the isolate and condition improved on administration of specific antimicrobial therapy. In cases where a single blood specimen was received, or organism was isolated in one out of two samples received, a repeat blood specimen was obtained and similarly cultured. The patients were examined and their records were studied for clinical correlation. Those pathogenic isolates which were identified as S. hominis were preserved in nutrient agar slopes at 4°C for later confirmation of sub-species6. The pathogenic isolates of CoNS which were not identified as S. hominis and those isolates of S. hominis which were not considered to be pathogens were excluded from the study. Antimicrobial susceptibility testing was done using VITEK® 2 AST card P-628 (BioMe’rieux). Antibiogram results were expressed as susceptible, intermediate or resistant according to the criteria of the Clinical Laboratory Standards Institute (CLSI)7.

For confirmation of sub-species, all S. hominis isolates were subjected to testing for novobiocin susceptibility and the production of acid aerobically from D-trehalose and N-acetyl-D-glucosamine (NAG)8. Novobiocin susceptibility was tested by Kirby-Bauer disc diffusion method8 using 5 μg novobiocin disc (HiMedia® Laboratories, Mumbai) in Mueller-Hinton agar. A zone size of <16 mm after incubation at 35°C for 24 h was considered indicative of resistance. The quality of novobiocin disc was checked using laboratory strains of Staphylococcus saprophyticus (resistant) and S. epidermidis (sensitive)8. Aerobic fermentation of D-trehalose and NAG was tested using peptone water fermentation medium with Andrade's indicator (HiMedia® Laboratories). After overnight incubation of the inoculated medium at 37°C, a change in colour to dark pink was interpreted as positive and to light pink was considered as weak positive. Positive controls (Klebsiella pneumoniae for D-trehalose and Lactobacillus for NAG) and un-inoculated controls were used with each run9.

Independently, the bio-profile and antimicrobial susceptibility pattern given by VITEK® 2 compact of all the isolates was re-analyzed and the findings were noted.

A total of 815 blood samples were received in 10 months duration from patients suspected of having BSI. A total of 249 were found positive by BacT/ALERT® 3D system and 172 grew clinically relevant pathogenic bacteria. In all, 102 staphylococci isolates were obtained, of which 92 were CoNS. The most commonly isolated CoNS were S. hominis (57/92 isolates, 61.9%). These 57 isolates identified as S. hominis by VITEK® 2 compact were further processed for sub-species identification.

Of the 57 isolates, 11 were resistant to novobiocin. Acid production from D-trehalose was positive in 31 isolates, weakly positive in six and negative in 20 isolates. Acid production from NAG was positive in 32 isolates, weakly positive in seven and negative in 18 isolates. Those isolates which were oxacillin (based on MIC given by VITEK® 2 Compact AST P-628 card) and novobiocin resistant were interpreted as belonging to sub-species novobiosepticus7. All the isolates thus identified as SHN failed to produce acid aerobically from D-trehalose, all except one isolates failed to produce acid aerobically from NAG also (Table I). On re-analysis of VITEK® 2 Compact bio-profile and MIC, it was found that there were five isolates which were both oxacillin resistant (based on MIC given by VITEK® 2 Compact AST P-628 card) and novobiocin resistant (based on the VITEK® 2 Compact bio-pattern). They were identified as S. hominis with low discrimination by VITEK® 2 Compact and the contraindicating biotype was shown to be SHN. These five isolates based on VITEK® 2 Compact bio-patterns were also interpreted as SHN. On comparing the two independent results, it was found that there were three isolates which were identified as SHN both by manual methods and with re-analysis of VITEK® 2 Compact bio-profile. Table I shows the demographic, clinical and laboratory characteristics of the patients identified to be having SHN bacteraemia.

Table I

Demographic, clinical and laboratory characteristics of the patients identified to be having Staphylococcus hominis subspecies novobiosepticus septicaemia

All the isolates identified as SHN (by both methods) were uniformly resistant to penicillin, oxacillin and ciprofloxacin. All isolates except one (intermediate resistant) were resistant to erythromycin (92.3% resistance). Resistance to trimethoprim/sulphamethoxazole was 61.5 per cent (eight out of 13 isolates being resistant) and that to clindamycin was 23.1 per cent (three out of 13 isolates being resistant). All isolates except one were susceptible to gentamicin (7.7% resistance). Susceptibility was maintained in all isolates against vancomycin, linezolid, daptomycin and tigecycline. The minimum inhibitory concentration of vancomycin (as shown by VITEK® 2 Compact AST report) was ≤0.5 μg/ml in two isolates and 1 μg/ml in the remaining 11 isolates (Table II). The difference in resistance patterns of SHH and SHN was not found to be significant using Pearson's Chi square test, except for ciprofloxacin, against which SHN was significantly more resistant than SHH (P<0.05).

Table II

Antimicrobial susceptibility results of the Staphylococcus hominis subspecies novobiosepticus isolates

In view of CoNS being one of the most important nosocomial pathogens related to BSIs and substantial increase in the frequency of methicillin resistance among CoNS isolates, accurate detection of pathogenic methicillin-resistant CoNS isolates by clinical microbiology laboratories is of crucial importance in guiding therapy and promoting the correct use of glycopeptides310. Manual identification of CoNS is labour-intensive and requires long incubation (24-72 h). VITEK® 2 automated detection system allows identification in lesser time (6-24 h) and provides high discrimination between species. However, the system identifies only the known pathogens which are fed into the software, and in case the bio-pattern obtained is not matching any organism, it gives a probable identification. Hence, if there is a doubt of a novel pathogen, the bio-pattern needs to be analyzed and interpreted by microbiologists, and also, the confirmation needs to be done by standard manual methods11.

The most frequently isolated CoNS in our set-up were S. hominis, of which 22.8 per cent were SHN. After first described by Kloos et al2 in 1998, SHN has been reported by authors from Europe, Spain and Brazil (Table III)34512. The highest number of isolates (67) has been reported by Balejova12 from different clinical specimens. Palazzo et al5 have reported six isolates from BSI patients, of which one identified as SHN by pulsed-field gel electrophoresis was found to be oxacillin susceptible. They have suggested that novobiocin resistance is intrinsic to SHN and oxacillin resistance has been acquired later on. SHN isolates causing BSIs are up till now described as vancomycin susceptible. However, increased MIC of SHN strains has been reported in an outbreak of BSI from the Intensive Care Unit in Brazil4. There are no reports of SHN from India, however, we have earlier reported probable presence of SHN in septicaemia cases, based on retrospective analysis of VITEK® 2 bio-patterns13.

Table III

Studies from all over the world reporting Staphylococcus hominis subspecies novobiosepticus as causative agents of invasive infections

In the present study, no significant difference was seen in resistance pattern of SHH and SHN isolates (except in ciprofloxacin). All SHN isolates had vancomycin MICs ≤1 μg/ml; four SHH isolates had vancomycin MICs of 2 μg/ml. However, the MICs against vancomycin need to be confirmed using the gold standard microbroth dilution method to know the actual trend towards increasing MIC.

Small sample size was the major limitation of our study. Studies with large sample size and those involving molecular tests are required to know the actual load, susceptibility pattern and clinical prognosis of this multidrug-resistant CoNS.