Since Staphylococcus nepalensis were reported for the first time from Nepalese animal specimen, and have been reported from human specimens elsewhere, this bug can be a threat in our part. Protocols must be designed aimed at their identification in our laboratory during microbiological analysis of clinical specimens.

1. Mini review
Staphylococcus nepalensis: From the lap of Himalayas
Mr. Shyam Kumar Mishra
Assistant Professor, Department of Microbiology
Institute of Medicine, Tribhuvan University
shyammishra@iom.edu.np
The genus Staphylococcus which belongs to the family Staphylococcaceae comprises 51 validly described
species (1). The genus includes not only a potent pathogen, Staphylococcus aureus, type species of the
genus, there are other bacteria which are regarded as commensals and also those which are regarded as
animal isolate only. Now, these "commensals" and the non-human isolates are no longer uncommon as
an etiological agent of human infections.
In early 2000s, Spergser et al conducted a study to determine the prevalence and significance of bacteria
in the respiratory tract of goats with pneumonia from different areas of Himalayan country Nepal. In
that study, the researchers isolated four unidentified staphylococci. These four strains were recovered
from three nasal swabs and were named PM34, RW78, CW1T
, and one strain, MM3, was isolated from
lung sample collected from four goats, with respiratory symptoms, kept in different areas on Nepal
(Pakhribas, Mangalbare, Rajarani, Chitwan). Strain CW1T
(Type species) showed highest 16S rDNA
sequence similarities to Staphylococcus cohnii subsp. urealyticus ATCC 49330T
, Staphylococcus
saprophyticus subsp. saprophyticus ATCC 15305T
, S. cohnii subsp. cohnii ATCC 29974T
, Staphylococcus
arlettae ATCC 43957T
, Staphylococcus gallinarum ATCC 35539T
, Staphylococcus succinus ATCC 700337T
and Staphylococcus xylosus ATCC 29971T
(99.0, 98.8, 98.8, 98.4, 98.2, 98.1 and 98.1 %, respectively),
indicating its classification within the genus Staphylococcus. The polar lipid composition, fatty acid
profiles, quinine systems and diagnostic cell-wall diamino acid were complying with the characteristics
of Staphylococcus (2).
On preliminary routine investigations to speciate the isolates, it was found that the strains were
coagulase-negative and resistant towards novobiocin. It is noteworthy that the Staphylococcus species
sharing these traits consists of Staphylococcus arlettae, Staphylococcus cohnii, Staphylococcus equorum,
Staphylococcus gallinarum, Staphylococcus kloosii, Staphylococcus saprophyticus, Staphylococcus
succinus and Staphylococcus xylosus. However, the four isolates could not be assigned to any of the
aforementioned species based on mere phenotypic studies. They were assigned to a single separate
species, Staphylococcus nepalensis, (N.L. masc. adj. nepalensis pertaining to Nepal, where clinical
samples for bacteriological examination were collected from local goats), on the basis of almost identical
biochemical and physiological traits, protein profiles obtained after sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) and identical genomic fingerprints generated after
enterobacterial repetitive intergenic consensus (ERIC)-PCR (2).

2. Coagulase-negative staphylococci (CoNS) are frequent inhabitants of mucocutaneous sites of humans,
mammals and birds, but they can be isolated from the environment (3). When CoNS are isolated from
nonsterile or sterile sites, most of the laboratories simply ignore them or report without further species
level identification (4,5). Nevertheless, there has been increase in concern regarding clinical significance
of CoNS infections in humans as strategies in medical practice lead to more invasive procedures (6).
Routine species level identification may better define the epidemiology and pathogenic potential of
individual species of CoNS (4,5). As we become more aware of the various strategies used by CoNS, we
will be in a better position to compromise their defense mechanisms and improve treatment (6).
Therefore, identification of staphylococcal species is necessary. However, the precise identification of
these bacteria to the species level is not an easy piece of cake.
Several manual and automated methods based on phenotypic characteristics have been developed for
identification of Staphylococcus species that are most often isolated from clinical samples.
Unfortunately, these systems have their limitations, mostly due to phenotypic differences between
strains from the same species (7). It has been shown that biochemical traits of S. nepalensis are hardly
distinguishable from those of S. xylosus or S. succinus or from S. equorum (3). Moreover, S. nepalensis is
not a common human clinical isolate, hence clinical microbiology laboratory do not give attention for its
identification and hence we might have been missing this strain.
Novakova et al tested five presumptive S. xylosus strains obtained from human urine (patient treated for
cystitis), gastrointestinal tract of squirrel monkeys, pig skin and from the environment for their precise
identification. They performed various molecular studies to characterize the strains. When looked for
different phenotypic tests, the hydrolysis of Tween 80 was found to be a helpful test for S. nepalensis
identification because hydrolysis of Tween 80 is generally a rare property among CoNS. It is noteworthy
that Novakova et al were the first to report S. nepalensis from human clinical material in 2006 (3).
Various molecular DNA-based methods for the identification of Staphylococcus species have been
developed. These methods typically require the use of several species-specific PCR primers,
hybridization probes, or multiple restriction enzymes and usually are not designed to differentiate all
known species simultaneously. 16S rRNA gene sequencing and PCR-restriction fragment length
polymorphism (PCR-RFLP) analysis, amplified fragment length polymorphism fingerprinting, Whole-
genome DNA-DNA hybridization analysis are not always suitable for routine use (8). Regarding S.
nepalensis, partial 16S rRNA gene sequencing, EcoRI and HindIII ribotyping and whole-cell protein
fingerprinting are suitable and reliable methods for the differenatiation of S. nepalensis strains from the other
novobiocin resistant staphylococci, whereas pulsed field gel electrophoresis (PFGE) analysis was found to be a
good tool for strain typing (3). Likewise, an oligonucleotide array targeting the manganese-dependent
superoxide dismutase (sodA) gene has also been found to be a bona fide candidate for S. nepalensis
typing (7).
Bacteriology of S. nepalensis (1,9)
Morphology
Cells are Gram-positive cocci, 1.1–1.6 micrometer in diameter, that occur singly, in pairs and in irregular
clusters. Motility is not observed.
Cultural characteristics

3. Colonies after 2 days on P agar are circular, low-convex, smooth, glossy, opaque white and 2–6 mm in
diameter. Growth occurs aerobically and anaerobically in the presence of 0–7.5 % NaCl (w/v). The type
strain grows well in the presence of 10 % NaCl (w/v), but growth is variable for other strains. No growth
is observed in the presence of 15 % NaCl. Growth occurs between 20 and 400
C, best growth at 300
C. No
growth at 15 or 450
C.
Biochemical properties
Catalase-positive, Oxidase-negative.
Produces urease, alkaline phosphatase, pyrrolidonyl arylamidase, beta-galactosidase and beta-
glucuronidase.
Hydrolyses aesculin and Tween 80.
Reduces nitrate to nitrite.
Positive for aerobic production of acid from D-glucose, D-fructose, D-mannose, maltose, lactose,
trehalose, mannitol, sucrose, L-arabinose, N-acetylglucosamine, galactose, glycerol, D-xylose, and
salicin.
Negative for oxidase activity, clumping factor, coagulase, hyaluronidase, arginine dihydrolase, ornithine
decarboxylase, acetoin, arginine arylamidase, alpha- and beta-haemolysins, heat-stable and heat-labile
nucleases, indole, hydrogen sulphide and lecithinase.
Acid is not formed aerobically from D-raffinose, ribose, D-cellobiose, D-arabinose, L-xylose, adonitol,
rhamnose, L-sorbose, dulcitol, starch, inositol, glycogen, inulin, cellobiose,.
Acid production from D-arabitol, sorbitol is variable; the type strain is positive.
Resistance- Resistant to novobiocin, bacitracin, vibriostatic agent O/129, lysozyme, metronidazole and
optochin.
G+C content of the type strain is 33 mol% (HPLC). The type strain is CW1 (DSM 15150; CCM 7045).
Table: Characteristics of S. nepalensis and some other novobiocin-resistant, oxidase-negative
Staphylococcus spp.
Characteri
stics
S.
nepale
nsis
S.
arlett
ae
S.
coh
nii
subs
p.
coh
nii
S.
gallinar
um
S.
kloo
sii
S.
saprophyt
icus
subsp.
saprophyt
icus
S.
saprophyt
icus
subsp.
bovis
S.
succin
us
subsp
.
succin
us
S.
xylos
us
S.
equor
um

4. Urease + + - + + + + + + +
Nitrate
reduction
+ - - + - - + - + +
Aesculin
hydrolysis
+ + - + d - - + d d
Tween 80
hydrolysis
+ - d - - - - - - -
Pyrrolidon
yl
arylamidas
e
+ - - - d d - - d -
Acid
productio
n from
Salicin
+ - - + - - - - - +
Note: +, > or =90% of the strains positive
d, 11-89% of strains positive
-, 0-10% of strains positive
Recently, S. nepalensis have also been isolated from guano of bats which indicate that guano
accumulated near or directly in human dwellings and buildings may represent a significant risk for
human health. However, their detailed virulence factors have not been identified except that they
possess capsule (10).
Since these bacteria were reported for the first time from Nepalese animal specimen, and have been
reported from human specimens elsewhere, this bug can be a threat in our part. Protocols must be
designed aimed at their identification in our laboratory during microbiological analysis of clinical
specimens.
References:
1. http://www.bacterio.net/staphylococcus.html, Accessed on July 21, 2015.
2. Spergser J, Wieser M, Taubel M, Rossello-Mora RA, Rosengarten R, Busse HJ. Staphylococcus
nepalensis sp. nov., isolated from goats of the Himalayan region. Int J Syst Evol Microbiol 2003;53:2007-
11.
3. Novakova D, Pantucek R, Petras P, Koukalova D, Sedlacek I. Occurrence of Staphylococcus nepalensis
strains in different sources including human clinical material. FEMS Microbiol Lett 2006;263:163-8.

5. 4. Tan TY, Ng SY, Ng WX. Clinical Significance of Coagulase-Negative Staphylococci Recovered from
Nonsterile Sites. J Clin Microbiol 2006;44:3413-4.
5. Weinstein MP, Mirrett S, Pelt LV, McKinnon M, Zimmer BL, Kloos W, Reller LB. Clinical Importance of
Identifying Coagulase-Negative Staphylococci Isolated from Blood Cultures: Evaluation of MicroScan
Rapid and Dried Overnight Gram-Positive Panels versus a Conventional Reference Method. J Clin
Microbiol 1998;36:2089-92.
6. Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative staphylococci. J Clin
Microbiol 1994;7:117-40.
7. Giammarinaro P, Leroy S, Chacornac JP, Delmas J, Talon R. Development of a new oligonucleotide
array to identify staphylococcal strains at species level. J Clin Microbiol 2005;43:3673-80.
8. Ghebremedhin B, Layer F, Konig W, Konig B. Genetic classification and distinguishing of
Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene
sequences. J Clin Microbiol 2008;46:1019-25.
9. Schleifer KH, Bell JA. Family VIII. Staphylococcaceae fam. nov.. In: (Eds.) Vos PD, Garrity G, Jones D,
Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB. Bergey's Manual of Systematic Bacteriology,
Volume 3: The Firmicutes, Springer, 392-426.
10. Vandzurova A, Backor P, Javorsky P, Pristas P. Staphylococcus nepalensis in the guano of bats
(Mammalia). Vet Microbiol 2013;164:116-21.