Journal of Clinical and Diagnostic Research. 2017 Jul, Vol-11(7): DC44-DC48 4444DOI: 10.7860JCDR201726009.10311Original Article [626290]

Journal of Clinical and Diagnostic Research. 2017 Jul, Vol-11(7): DC44-DC48 4444DOI: 10.7860/JCDR/2017/26009.10311Original Article
Microbiology SectionAnaerobic Bacteria in Clinical
Specimens – Frequent, But a
Neglected Lot: A Five Year Experience
at a Tertiary Care Hospital
INTRODUCTION
Anaerobic bacteria constitute a significant proportion of the normal
microbiota colonizing skin and various mucosal surfaces of human
body [1]. Anaerobes are more commonly found in polymicrobial
aerobic and anaerobic infections of endogenous origin. Breach in
mucosal barriers due to surgery, trauma, tumours, or ischemia lead
to infections by these microbes following entry of endogenous flora
into normally sterile sites [2,3]. Infections by Clostridium spp. are
mainly of exogenous origin [2]. The most commonly encountered
anaerobes in clinical specimens include Bacteroides fragilis group,
pigmented Prevotella spp. and Porphyromonas spp., Fusobacterium
spp., Peptostreptococcus spp., Clostridium spp. and Actinomyces
spp. [3]. The pathogenic anaerobes may cause variety of infections
ranging from mild to severe life threatening ones, involving various
anatomic sites [3,4]. Varying rates of anaerobic bacterial isolation
have been reported across the globe from different clinical infection
sites [5-8].
Whenever there is high suspicion of anaerobic aetiology, the
management of such infections is often dependent only on
empirical antibiotic therapy. The reason could be attributed to
tedious anaerobic culture techniques, cost and more importantly
longer turnaround time for intimation of anaerobic culture reports to
treating clinician. However, resistance to metronidazole, the empiric
drug of choice for anaerobic coverage is on the rise [9].
Anaerobes are the most overlooked microorganisms in many of the
clinical specimens. Failure to identify them and provide antimicrobial
coverage may result in therapeutic failure. Therefore, it is important to
know the microbial pathogen responsible for the infectious process.
A study was undertaken to determine the frequency of isolation
of anaerobes from various clinical specimens in the Microbiology laboratory attached to a tertiary care teaching hospital in coastal
Karnataka, India.
MATERIALS AND METHODS
A retrospective study was conducted over a period of five years from
January 2011 to December 2015 in the department of Microbiology
of Kasturba Medical College, Manipal, a tertiary care teaching
hospital in Southern India. For microbiological analysis, specimens
including tissue, pus aspirate, body fluids, corneal scrapings, wound
swabs and stool for C. difficile were aseptically inoculated into a
wide mouth sterile container and/or Robertson’s Cooked Meat
(RCM) medium soon after collection and transported immediately
to the laboratory. In cases where wound swabs were the only mode
of sampling, samples were collected and inoculated at bedside into
RCM broth. The specimens were processed for Gram stain and the
anaerobic cultures were done on 5% sheep blood agar, neomycin
blood agar and phenyl ethyl alcohol agar with metronidazole (5 µg,
Oxoid) disc. The specimens were inoculated into RCM if bedside
inoculation was not performed. The inoculated culture plates were
incubated in anaerobic Gaspak jars (BD Diagnostics, Sparks, MD,
USA) (from January 2011 to July 2013) or anaerobic workstation
(Whitley A35 Anaerobic workstation, Don Whitley Scientific, Shipley,
UK) (from August 2013 to December 2015). The Gaspak jars were
opened after 48-72 hours for inspection of plates, whereas plates
were inspected daily for anaerobic growth when anaerobic chamber
was used for incubation. The inoculated RCM broth was incubated
till seven days and subcultures were done on to 5% sheep blood
agar if any additional bacterial morphotypes were noted on Gram
stain from the broth. The specimens were also cultured aerobically
on 5% sheep blood agar and MacConkey agar and isolates were
identified following standard methods [10].PadMaja ananth Shenoy1, ShaShidhar ViShwanath2, aShwini Gawda3, SeeMa Shetty4,
renuka aneGundi5, Muralidhar VarMa6, Chiranjay MukhoP adhyay7, kiran Chawla8

Keywords: Abscess, Anaerobe, Gram-negative bacilli, Gram-positive cocci, Polymicrobial infectionsABSTRACT
Introduction: Anaerobic bacteria which constitute a significant
proportion of the normal microbiota also cause variety of
infections involving various anatomic sites. Considering
the tedious culture techniques with longer turnaround time,
anaerobic cultures are usually neglected by clinicians and
microbiologists.
Aim: To study the frequency of isolation of different anaerobic
bacteria from various clinical specimens.
Materials and Methods: A retrospective study to analyse
the frequency of isolation of different anaerobic bacteria, was
conducted over a period of five years from 2011 to 2015 including
various clinical specimens submitted to anaerobic division of
Microbiology laboratory. Anaerobic bacteria were isolated and
identified following standard bacteriological techniques.Results: Pathogenic anaerobes (n=336) were isolated from 278
(12.48%) of overall 2227 specimens processed with an average
yield of 1.2 isolates. Anaerobes were isolated as polymicrobial
flora with or without aerobic bacterial pathogens in 159 (57.2%)
patients. Anaerobic Gram-negative bacilli (140, 41.7%) were
the predominant isolates. B. fragilis group (67, 19.9%) were the
most commonly isolated anaerobic pathogens. Anaerobes were
predominantly isolated from deep seated abscess (23.9%).
Conclusion: Pathogenic anaerobes were isolated from
various infection sites. Unless culture and susceptibility tests
are performed as a routine, true magnitude of antimicrobial
resistance among anaerobic pathogens will not be known.
Knowledge of the distribution of these organisms may assist
in the selection of appropriate empirical therapy for anaerobic
infections.

www.jcdr.net Padmaja Ananth Shenoy et al., Anaerobic Bacteria in Clinical Specimens
Journal of Clinical and Diagnostic Research. 2017 Jul, Vol-11(7): DC44-DC48 4545
Keywords: Abscess, Anaerobe, Gram-negative bacilli, Gram-positive cocci, Polymicrobial infections
[Table/Fig-1]: Demographic and microbiological profile of study subjects (n=278).[Table/Fig-2]: Distribution of anaerobic bacterial pathogens isolated during the
study.Preliminary identification of the anaerobic isolates was done by
colony morphology and Gram stain, aerotolerance test on chocolate
agar, fluorescence under long-wave (365 nm) ultraviolet light (UVP ,
LLC), antibiotic identification discs (vancomycin 5 µg, kanamycin
1000 µg and colistin 10 µg), biochemical tests and susceptibility
to Sodium Polyanethol Sulfonate (SPS) [11]. Automated microbial
identification systems, VITEK 2 (ANC card, bioMerieux) (from
January 2011 to May 2015) or Matrix Assisted Laser Desorption/
Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry (VITEK
MS, bioMerieux) (from June 2015 to December 2015) were used for
species level identification.
RESULTS
A total of 2227 samples were received in anaerobic division of
Microbiology laboratory over a period of five years involving diverse
infections in our tertiary care hospital. Pathogenic anaerobes (n=336)
were isolated from 278 (12.48%) patients with an average yield of
1.2 isolates per specimen showing anaerobic growth [Table/Fig-1].
Anaerobic Gram-negative bacilli (140, 41.7%) were the predominant
isolates. Bacteroides fragilis group (67, 19.9%) were the most
commonly isolated anaerobic pathogens. Amongst Gram-positive
Anaerobic Cocci (GPAC), Finegoldia magna (43, 12.8%) was the
most frequently isolated pathogen followed by Peptostreptococcus
anaerobius (20, 6%) [Table/Fig-2]. The most commonly isolated
anaerobe in monomicrobial flora were Clostridium spp. (39, 32.8%)
[Table/Fig-3]. In 159 (57.2%) patients, anaerobes were isolated
as polymicrobial flora with or without aerobic bacterial pathogens
[Table/Fig-1].
Anaerobes were predominantly isolated from deep seated abscess
(23.9%), followed by diabetic foot infection (20%), necrotizing fasciitis
(15.6%), chronic osteomyelitis (7.8%), infected non healing ulcer
(7.3%), antibiotic associated diarrhea (6.8%), corneal ulcer (4.4%),
fournier’s gangrene (3.4%), gas gangrene (2.9%), empyema (2.4%),
cellulitis (2.4%), pyometra (1.9%) and endophthalmitis (0.9%).
Anaerobic Gram-negative bacilli (n=7) were the predominant
isolates found in polymicrobial anaerobic infections (n=28). Among
infections with mixed aerobic and anaerobic bacterial flora (131,
47.1%), anaerobes were mainly isolated in association with E. coli
in 43 (32.8%) patients followed by K. pneumoniae in 31 (23.7%)
patients. B. fragilis group (47, 35.9%) was the most common
anaerobic bacteria found in association with aerobic bacterial
pathogens.
DISCUSSION
Anaerobic bacteria constitute a large majority of commensal flora Characteristic no. of patients Percentage (%)
age (yrs)
0-20 16 5.75
21 to 40 91 32.7
41 to 60 116 41.7
61 to 80 52 18.7
> 80 3 1.0
Gender
Male 199 71.6
Female 79 28.4
Specimen
Tissue 130 46.8
Pus aspirate 88 31.7
Body fluids 22 7.9
Wound swabs 15 5.4
Stool for C. difficile culture 14 5.0
Corneal scrapings 9 3.2
nature of growth with anaerobic infections (n=278)
Pure anaerobic growth (n=147, 52.9%)•
Monomicrobial anaerobic growth 119 42.8
Polymicrobial anaerobic growth 28 10.1
Mixed anaerobic and aerobic growth (n=131, 47.1%)•
1 Aerobic + 1 Anaerobic growth 82 29.5
1 Aerobic + 2 Anaerobic growth 20 7.2
2 Aerobic + 1 Anaerobic growth 19 6.8
2 Aerobic + 2 Anaerobic growth 10 3.6isolates (n=336) number Percentage
anaerobic Gram-Positive Cocci (n=85, 25.3%)
Finegoldia magna 43 12.8
Peptostreptococcus anaerobius 20 6.0
Peptoniphilus asaccharolyticus 18 5.4
Parvimonas micra 3 0.9
Anaerococcus prevotii 1 0.3
anaerobic Gram-negative Cocci (n=24, 7.1%)
Veillonella parvula 24 7.1
anaerobic Gram-negative Bacilli (n=140, 41.7%)
Bacteroides fragilis subsp. fragilis 56 16.7
Bacteroides fragilis subsp. thetaiotaomicron 7 2.1
Bacteroides fragilis subsp. ovatus 3 0.9
Bacteroides fragilis subsp. vulgatus 1 0.3
Parabacteroides distasonis 1 0.3
Prevotella spp. 25 7.4
Prevotella bivia 8 2.4
Prevotella buccae 5 1.5
Prevotella disiens 5 1.5
Prevotella melaninogenica 2 0.6
Fusobacterium nucleatum 13 3.9
Fusobacterium necrophorum 5 1.5
Fusobacterium varium 3 0.9
Fusobacterium mortiferum 2 0.6
Porphyromonas asaccharolytica 4 1.2
anaerobic Gram-Positive Bacilli (n=87, 25.9%)
Clostridium spp. 17 5.1
Clostridium difficile 14 4.2
Clostridium bifermentans 9 2.7
Clostridium sporogenes 9 2.7
Clostridium perfringens 6 1.8
Clostridium clostridioforme 5 1.5
Clostridium ramosum 3 0.9
Clostridium baratii 3 0.9
Clostridium septicum 2 0.6
Clostridium cadaveris 1 0.3
Clostridium subterminale 1 0.3
Clostridium sordelli 1 0.3
Clostridium innocuum 1 0.3
Clostridium histolyticum 1 0.3
Propionibacterium acnes 11 3.3
Bifidobacterium spp. 1 0.3
Eggerthella lenta 1 0.3
Lactobacillus gasseri 1 0.3
total 336 100

Padmaja Ananth Shenoy et al., Anaerobic Bacteria in Clinical Specimens www.jcdr.net
Journal of Clinical and Diagnostic Research. 2017 Jul, Vol-11(7): DC44-DC48 4646which inhabit various body sites, including mucosal surfaces of oral
cavity, pharynx, gastrointestinal tract, genitourinary tract orifices and
skin. This microbiome serves as source for majority of infections
involving anaerobes [12]. Anaerobes as pathogens are isolated
from various anatomic sites with variable recovery rates. Anaerobic bacterial pathogens are isolated in high frequency (50-100%) from
gas gangrene, d iabetic foot infections, infections after colorectal
surgery and appendectomy, perianal abscess, non-clostridial
crepitant cellulitis, lung abscess, aspiration pneumonia, brain
abscess, intraperitoneal/pelvic abscess, soft tissue/subcutaneous
abscess, dental/oral infections, chronic sinusitis and mammary
abscess [13]. Our data shows isolation of various anaerobic
bacteria from diverse infections. We isolated anaerobes mainly from
abscesses (23.9%) and diabetic foot infection (20%). Some of the
reported isolation rates of anaerobic bacteria from different infection
sites are summarized in [Table/Fig-4] [5-8,14-33].
Successful isolation of anaerobes depends on specimen collection
and transportation procedures, anaerobic incubation system and
the quality and selection of the primary isolation media. For optimal
recovery, it is necessary that specimens are transported within 30
minutes after collection and if anaerobic transport media are used,
within 2-3 hours to the laboratory [34]. The commonly used culture
media for isolating anaerobes from clinical specimens include, 5%
sheep blood agar with hemin (5 µg/mL) and vitamin K1 (1 µg/mL),
kanamycin-vancomycin laked blood agar, phenyl ethyl alcohol
sheep blood agar, Columbia nalidixic acid agar, Bacteroides bile
esculin agar, cycloserine-cefoxitin fructose agar, egg yolk agar,
supplemented thioglycollate broth with hemin, vitamin K1 and
sodium bicarbonate and RCM broth [11].
In the recent years, major taxonomic changes of anaerobic bacteria
have occurred, more so among Gram-negative bacilli and Gram-
positive cocci. It is essential for both microbiologists and the clinician
to be updated with the changes in bacterial names for better
description and recognition of the bacterium-disease associations
[35]. Anaerobic Gram-negative bacilli were the predominant
pathogens in our study as also reported in various studies [6,15,36-
39]. However, GPAC are isolated as most frequent pathogens in
other reports [5,7,40,41].
B. fragilis which forms about only 0.5% of normal commensal flora
in the colon is the most commonly isolated anaerobic bacterial
pathogen as reported in literature by virtue of its virulence factors.
These factors include, tissue adherence by fimbriae and agglutinins;
polysaccharide capsule, lipopolysaccharide and a variety of
enzymes which help in evading oxygen toxicity and phagocytosis;
and histolytic enzymes which cause tissue destruction [42].
B. fragilis group (19.9%) were the predominant isolates, mirroring
the finding of other studies [6,37,39]. The capsule production by
B. fragilis helps in abscess formation [42]. Majority of our B. fragilis
strains (n=25) were isolated from deep seated abscess.
Infections caused by Veillonella parvula are seldom reported. They
are commonly found in head and neck infections, skin and soft
tissue infections, infections in the respiratory tract, peritoneal fluid,
blood and abdominal infections [11]. In our study, majority of V.
parvula were isolated from necrotising fasciitis (n=7).
The Gram-Positive Anaerobic Bacilli (GPAB) which are seen in
the laboratory include the spore forming Clostridium spp. and
the nonsporing, Actinomyces, Bifidobacterium, Eggerthella,
Eubacterium, Lactobacillus and Propionibacterium spp. Identification
of the non-sporing GPAB in the clinical microbiology laboratories is
difficult. Gas liquid chromatography is helpful in accurately identifying
these bacilli when the basic information of Gram stain reaction,
spore status, oxygen susceptibility, catalase and indole reactions
are available. These GPAB can also be missed due to their complex
transport and growth requirements and being often seen along with
non-fastidious aerobic bacteria as part of polymicrobial flora. Their
epidemiology, clinical significance and pathogenic potential needs
further understanding [43,44]. Among the spore forming GPAB,
Clostridium spp. was commonly found in association with diabetic
foot infection (n=20) and C. perfringens (1.9%) was isolated from
six cases of gas gangrene. C. difficile which is being increasingly
recognized and iso lated from patients with antibiotic associated anaerobes number (%)
Clostridium spp. including C. difficile 39 (32.8%)
Bacteroides fragilis group. 21 (17.6%)
Finegoldia magna 16 (13.4%)
Prevotella spp. 13 (10.9%)
Peptostreptococcus anaerobius 9 (7.6%)
Propionibacterium acnes 7 (5.9%)
Veillonella parvula 7 (5.9%)
Fusobacterium spp. 4 (3.4%)
Porphyromonas asaccharolytica 2 (1.7%)
Peptoniphilus asaccharolyticus 1 (0.8%)
[Table/Fig-3]: Anaerobic bacteria isolated as monomicrobial flora: (n=119).
[Table/Fig-4]: Isolation rates of anaerobic bacterial pathogens from different
infection sites.Study
investigatoryear Clinical profiletotal no. of
specimensisolation
rate
Brook I et al., [14] 1998 Retroperitoneal
abscesses 161 78.9%
De A et al., [6] 2001 Diverse clinical infections 2591 8%
De A et al., [15] 2002 Pleuropulmonary
infections100 72%
De A et al., [16] 2003 Gas gangrene 580 26.8%
Saini S et al., [17] 2004 Surgical infections 117 50.4%
Tanaka K et al.,
[18]2005 Bartholin’s gland abscess 224 53.1%
Boyanova L et
al., [19]2006 Deep-space head and
neck infections 118 74.6%
Gadepalli R et al.,
[20]2006 Diabetic foot ulcer 80 35%
Huang TT et al.,
[21]2006 Deep neck infections 128 59.3%
Singhal R et al.,
[22]2006 Anaerobic bacteremia 1743 1.14%
Citron D M et al., [5] 2007 Diabetic foot infections 454 45.2%
Ng LS et al., [8] 2008 Diabetic foot infections 38 78.9%
López VN et al.,
[23]2009 Iliopsoas abscess 124 15.1%
Mathew A et al.,
[24]2010 Necrotising fasciitis 50 18.5%
Al-Benwan K et
al., [25]2011 Breast abscess 114 28%
Ingle M et al., [26] 2011 Clostridium difficile
infection99 17%
Vishwanath S et
al., [27]2012 Chronic suppurative otitis
media94 19.14%
Urban E et al., [28] 2012 Anaerobic bacteremia 43992 0.69%
Vishwanath S et
al., [29]2013 Clostridium difficile
infection 25 16%
Kamble S et al.,
[30]2014 Cutaneous and
subcutaneous wound
infections50 18%
Garg R et al., [7] 2014 Diverse clinical infections 100 19%
Antony B et al.,
[31]2016 Surgical infections 393 39.9%
Sudhaharan S et
al., [32] 2016 Brain abscess 430 41.1%
Shenoy PA et al.,
[33]2016 Surgical infections 261 24.5%

www.jcdr.net Padmaja Ananth Shenoy et al., Anaerobic Bacteria in Clinical Specimens
Journal of Clinical and Diagnostic Research. 2017 Jul, Vol-11(7): DC44-DC48 4747colitis mainly in a nosocomial setting was isolated in 4.2% (n=14)
cases. Gorbach SL et al., i n their analysis found intra-abdominal
sepsis associated with trauma or prior intestinal surgery as a major
source for Clostridial infections [45]. Clostridium spp. have also
been reported to be predominantly isolated from wound infections,
abscesses, abdominal infections, and blood [30,36].
GPAC are frequently isolated from clinical specimens and account
for 24-31% of anaerobic isolates [1]. In our study, 25.3% (n=85)
isolates were found to be GPAC. Peptostreptococcus, Finegoldia,
Parvimonas, Anaerococcus and Peptoniphilus are the more
commonly reported GPAC [1]. Zone of inhibition of ≥15 mm around
a 5 µg metronidazole disc differentiates GPAC from microaerophilic
Gram-positive cocci. Infections involving GPAC are usually
polymicrobial and are isolated mainly from abscesses, infections of
oral cavity, skin and soft tissues, bone and joints, upper respiratory
and female genital tract [46]. However, F. magna is reported to be
isolated as monomicrobial flora from various infection sites [1,46].
F. magna is found in high frequency in chronic wounds like diabetic
ulcers and pressure ulcers [1]. Majority of F. magna which were the
most frequent GPAC in our study, were obtained from diabetic foot
infection (n=13) and necrotising fasciitis (n=8).
Anaerobic blood stream infections are relatively uncommon
and contribute to 0.5%-12% of all positive blood cultures which
corresponds to an occurrence of 0.5 – 1.0 cases per 1,000 hospital
admissions [22,47]. There are conflicting data on the incidence
and trends of anaerobic bacteremia over time, and the clinical
significance of isolating anaerobic bacteria from blood cultures
[22,48]. However, it is recommended that anaerobic blood cultures
are performed as a routine in all patients with suspected blood
stream infections [48]. Commercial automated blood culture bottles
can also be used for inoculation of non-blood specimens like sterile
body fluids for isolation of anaerobic bacteria.
Anaerobes were predominantly isolated as polymicrobial flora
involving aerobic and anaerobic pathogens from clinical specimens
(159, 57.2%). B. fragilis (n, 47) was the common anaerobe found in
association with facultative aerobes such as E. coli and Klebsiella
spp. Microbial synergy leads to enhanced pathogenicity and
severity of infection in polymicrobial infections with aerobic and
anaerobic bacterial pathogens . B. fragilis which is known to be the
most frequent anaerobic pathogen in polymicrobial infections is
associated with a mortality rate of more than 19% [42].
It is essential that the clinicians recognize the importance of anaerobic
bacteria as pathogens and utilize the expertise of the laboratories
having facilities for anaerobic culture and susceptibility testing for
infections with suspected anaerobic aetiology. Performance of
anaerobic cultures, along with aerobic cultures will provide complete
bacterial work-up of specimens from infectious sites. With increasing
instances of antimicrobial resistance amongst anaerobic bacteria
to commonly used antimicrobials and the inherent drug resistance
amongst some of these bacteria, knowledge of the distribution of
these organisms may assist in the selection of appropriate empirical
therapy for anaerobic infections.
LIMITATION
As we do not perform routine anaerobic blood cultures, incidence
of anaerobic bacteremia could not be obtained. We could not
obtain the follow-up clinical data on antibiotic prescription practices
based on anaerobic culture reports and therapeutic response of the
patients.
CONCLUSION
Anaerobes as pathogens are isolated from diverse infection sites.
Unless they are cultured and susceptibility tests are performed as a
routine, true magnitude of antimicrobial resistance among anaerobic
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PartiCularS oF ContriButorS:
1. Associate Professor, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
2. Associate Professor, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
3. Postgraduate Student, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
4. Senior Lecturer, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
5. Assistant Professor, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
6. Associate Professor, Department of Medicine, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
7. Professor, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
8. Professor and Head, Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
naMe, addreSS, e-Mail id oF the CorreSPondinG author:
Dr. Shashidhar Vishwanath,
Associate Professor, Department of Microbiology, Kasturba Medical College,
Madhavnagar, Manipal-576104, Karnataka, India.
E-mail: shashidhar.v@manipal.edu
FinanCial or other CoMPetinG intereStS: None.Date of Submission: dec 16, 2016
Date of Peer Review: Feb 08, 2017
Date of Acceptance: May 04, 2017
Date of Publishing: jul 01, 2017