Study of antibiogram and virulence factors of Moraxella catarrhalis from a tertiary care hospital

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Study of Antibiogram and Virulence Facto rs of Moraxella Catarrhalis From a
Tertiary Care Hospital
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All India Institute of Hygiene and Public Health
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Original Research Article
236 | P a g e Int J Med Res Prof.2017; 3(2); 236-38. www.ijmrp.com
Study of Antibiogram and Virulence Factors of Moraxella Catarrhalis
From a Tertiary Care Hospital
Bhattacharyya S
1*
, Singh S
2
, Sarfraz A
3
, Jaiswal NK
4
, Kumar R
4
, Kumar A
4
, Sengupta A
3
,
Kumar D
3
, Singh S
4
1*
Assistant Professor,
3
Senior Resident,
4
Tutor,
Department of Microbiology, All India Institute of Medical Sciences, Patna, Bihar, India.
2
Senior Resident,
Department of General Medicine, All India Institute of Medical Sciences, Patna, Bihar, India.
ABSTRACT
Background:
Moraxella catarrhalis is a known secondary
pathogen in COPD. Its antibiogram has not been studied much
but is important for empiric chemotherapy. Also, studying its
virulence factors is important.
Objectives:
This study was planned to see antibiogram,
virulence factors and biofilm formation.
Methods:
Over 16 months, M. catarrhalis was isolated from
sputum and other samples by inoculating on suitable media
and identified using staining and standard biochemicals.
Virulence factors studied were lecithinase, lipase and protease,
serum resistance assay, and biofilm formation by test tube
method. Antibiogram was done by Disk diffusion method using
11 different antibiotics.
Results:
A total of 60 M. catarrhalis isolates were recovered;
51 from sputum and 9 from other samples. Mean age and male
to female ratio of patients with respiratory isolates were 38.4
and 1.8:1 respectively. In 3 cases, M. catarrhalis was found to
coexist with M. tuberculosis and S. aureus. All respiratory
isolates were susceptible to Ceftazidime and Piperacillin-
Tazobactum. Resistance to Prulifloxacin and Cotrimoxazole
were considerable, and that against Amoxiclav and
Levofloxacin were low. Four isolates were Multi-drug resistant,
3 from sputum and 1 from urine. Lipase was found in 38.4%
isolates; lecithinase and protease were absent. Biofilm was
shown by 67% isolates. All were serum resistant.
Conclusion:
M. catarrhalis is a smart pathogen, having array
of virulence factors and can cause disseminated infection.
Prulifloxacin and Cotrimoxazole are not warranted for empiric
therapy, at least in our area.
Keywords:
Moraxella, LRTI, MDR.
*Correspondence to:
Dr Sayan Bhattacharyya,
M.D., Assistant Professor,
Department of Microbiology,
AIIMS, Patna, Bihar, India.
Article History:
Received:
26-02-2017,
Revised:
16-03-2017,
Accepted:
28-03-2017
Access this article online
Website:
www.ijmrp.com
Quick Response code
DOI:
10.21276/ijmrp.2017.3.2.047
INTRODUCTION
Moraxella catarrhalis, a Gram negative coccobacillus, known as
secondary pathogen in COPD.1 It is generally susceptible to most
antibiotics, but due to Beta-lactamases (BRO-beta lactamases), it
is prone to develop resistance to empirically given Penicillins but
usually not to cephalosporins.2 Hence knowing its antibiogram is
very important. Very few such studies are available in the
repository. Study of virulence traits of the pathogen is also
important since it can predict success of antibiotic therapy.3
Keeping these things in mind, we aimed to (i) isolate and Identify
M. catarrhalis from the various clinical samples and observe its
antibiogram and (ii) see virulence factors like lecithinase, lipase,
protease, serum resistance and biofilm formation. Our objectives
were to identify M. catarrhalis by biochemicals, perform
antibiogram by disk diffusion and study virulence factors on egg
yolk agar (lecithinase, lipase, protease) and standard methods.
MATERIALS AND METHODS
Type of study: This was a laboratory-based observational study.
Place of study: The study was carried out in the Department of
Microbiology of the institute.
Time of study: From February 2014 to June 2015 (16 months).
This was a pure observational study using lab isolates and
patients' identity was not revealed.
Methodology: Moraxella catarrhalis was isolated and identified
from clinical samples routinely received in the lab., like sputum,
urine and blood.
Identification: M. catarrhalis isolates were identified by NLF, Dry
colonies that were wholly movable over surface of agar (hockey-
puck colonies) on CLED/Chocolate agar.
From colonies, Gram stain was done. Gram negative coccobacilli,
Oxidase positive (using 1% TMPPD disks, Himedia labs, Delhi),

Bhattacharyya S et al. Antibiogram and Virulence Factors of Moraxella Catarrhalis
237 | P a g e Int J Med Res Prof.2017; 3(2); 236-38. www.ijmrp.com
which were positive for Nitrate reduction (KNO3 broth), with no
sugar fermentation (on sugar broth with phenol red), and having
no growth on MacConkey agar but good growth on CLED/ NHA
(Serum-free media), were taken as Moraxella catarrhalis,
according to literature references.4
Isolates that were positive for all these were considered as M.
catarrhalis.4
Sputum sample: Growth was considered significant only when
growth pure/almost pure. Sixty (60) such isolates from various
samples were studied.
Antibiotic susceptibility of the isolates were tested by Kirby-Bauer
disk diffusion method against 11 different antibiotics, e.g.
Ceftazidime (30 µg), Cotrimoxazole, Amikacin (30 µg),
Azithromycin (15 µg), Piperacillin-Tazobactum (110 µg),
Amoxiclav (30 µg), Levofloxacin (5 µg), Prulifloxacin (5 µg),
Gentamicin (30 µg), Cefotaxime (30 µg), and Cefixime (5 µg ) as
per CLSI protocol, and for Prulifloxacin, new interpretive data.5,6
Susceptibility and resistance was calculated by SENTRY
interpretiver criteria of disk diffusion.7 All disks were supplied by
HiMedia labs, Delhi, India. Patients were also followed up for
clinical recovery to match data with in-vivo response.
Virulence Traits Tested: The following virulence factors were
tested, like lecithinase, Lipase, Protease (all on Egg-yolk agar),
serum resistance and biofilm formation (by test-tube method).
Positive lecithinase (phospholipase) was indicated by zone of
haziness around colonies on Egg yolk agar. Lipase was indicated
by pearly shine on colonies, and protease by zone of clearing
observed around colonies.8 Biofilm formation was assessed
visually using test tube method.9
Serum resistance test: In 2 separate test tubes, 100 µl of 1
MacFarland bacterial suspension was mixed with (a) 300 µl of
peptone water, and (b) 300 µl of human serum, and incubated for
90 minutes at 37°C.
Then 10 µl of each was streaked on Chocolate agar plates and
incubated overnight at 37°C.
The isolate was taken as serum sensitive, when there was >90%
inhibition of colony count in culture done from tube where serum
was added. When there was <90% inhibition of growth, it was
serum resistant.10
RESULTS
Out of a total of 60 M. catarrhalis isolates which were recovered,
51 were from sputum and 9 from other samples. These other
samples were synovial tissue, blood, swab and urine.
Mean age of patients growing it from sputum, was 38.4 years.
For respiratory isolates, male to female ratio was 1.8:1. (Thus
male-dominant)
M. catarrhalis formed 43% of all respiratory bacterial isolates. (All
were OPD isolates)
Other isolates (in decreasing order) were Pseudomonas
aeruginosa, K. pneumoniae, Staphylococcus aureus etc.
In 3 cases, M. catarrhalis was found to coexist in sputum with
other pathogens (M. tuberculosis in 2 and S. aureus in 1 case). All
respiratory isolates were universally susceptible to Ceftazidime
and Piperacillin-Tazobactum. Resistance to Prulifloxacin and
Cotrimoxazole was exorbitant (78.5% and 77.7% respectively).
The same figures against Amoxiclav and Levofloxacin were lower
(4.7% and 10.7% respectively).
MDR (Multi-drug resistance) was found in 3 respiratory isolates
(MDR: Resistance to 3 or more different groups of antibiotics as
depicted by Gupta et al).11
Study of virulence factors: Lipase enzyme was found in 38.4%
isolates; lecithinase and protease could be found in no isolates.
Biofilm formation was shown by 67% isolates. All these isolates
were serum resistant.
Results of antibiogram are shown in Table 1.
Apart from sputum, 9 other samples grew M. catarrhalis. (Urine: 4,
Synovial fluid/tissue: 2, Wound swab: 1, Blood: 1, Ascitic fluid: 1)
All of the urinary isolates were susceptible to Piperacillin-
Tazobactum. One urinary isolate was MDR.
Table 1: Resistance to some antibiotics other than Prulifloxacin and Cotrimoxazole
Antibiotic
Resistance (%)
Amikacin
25%
Azithromycin
27.8%
Cefixime
15.38%
Amoxicillin-Clavulinic Acid
4.76%
Levofloxacin
10.71%
Ceftazidime
0
Piperacillin-Tazobactum
0
Gentamicin
11.1%
DISCUSSION
M. catarrhalis, earlier called Branhamella catarrhalis, is a smart
pathogen, gaining significance as a pathogen over few decades,
and having an array of virulence factors, of which biofilm formation
and lipase and serum or complement resistance are important.12,13
This bacterium is an exclusively human pathogen causing Lower
Respiratory Tract Infection, Otitis media and other infections.1
Earlier thought only to be a respiratory tract commensal, since the
late 1970s, it has been clearly shown that M. catarrhalis is an
important and common human respiratory tract pathogen and it
has been shown to cause LRTI specially after COPD.1 However,
Isolation in children, is mostly regarded as colonisation.14 In other
Indian studies also, M. catarrhalis was found to be third most
common cause of airway pneumonia, and was more commonly
found in male patients and in age group above 60 years.15 In
another study from Karnataka, South India, also, M. catarrhalis
respiratory isolates were highly susceptible to Amoxicillin-
Clavulinic acid but mostly refractory to Ampicillin.16
According to our findings, as also from other reports, M.
catarrhalis is often serum resistant and has a high propensity to

Bhattacharyya S et al. Antibiogram and Virulence Factors of Moraxella Catarrhalis
238 | P a g e Int J Med Res Prof.2017; 3(2); 236-38. www.ijmrp.com
cause disseminated infection, as also reported by Attia et al.17 We
here report that it can also coexist with other pathogens, and can
not only cause pneumonia but are also implicated in Urinary tract
infections (UTI), arthritis, wound infection and bloodstream
infection. More infection in males, found in our study, was
probably due to more active smoking and consequent COPD in
male subjects. Another interesting observation was that
Prulifloxacin and Cotrimoxazole are not warranted for empiric
therapy, at least in our area. A study from Italy shows his figure in
Prulifloxacin to be about 14% in acute exacerbation of COPD.18
Further studies regarding this are awaited.
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[
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cited.
Cite this article as: Bhattacharyya S, Singh S, Sarfraz A, Jaiswal
NK, Kumar R, Kumar A, Sengupta A, Kumar D, Singh S. Study of
Antibiogram and Virulence Factors of Moraxella Catarrhalis From
a Tertiary Care Hospital. Int J Med Res Prof. 2017; 3(2):236-38.
DOI:10.21276/ijmrp.2017.3.2.047
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