The majority of nonfermenting gram-negative bacilli are responsible for respiratory tract infections. The aims of this study are to identify the nonfermenting gram-negative bacilli species in respiratory tract infections, to describe their antibiotic resistance and to describe the factors associated with these bacteria. It is a prospective study of 54 respiratory samples for a period of six months from October 2019 to March 2020 in the laboratory of the University Hospital of Befelatanana Antananarivo. Among the 54 respiratory samples, the microbiological results showed 12 (22.2%) cases of nonfermenting gram-negative bacilli. These bacilli were represented by 7 (58.3%) cases of Acinetobacter baumannii, 4 (33.3%) cases of Pseudomonas aeruginosa and 1 (8.3%) case of Stenotrophomonas maltophilia. Concerning the associated factors, subjects under 40 years (24%) (p=0.77; NS), men (30.8%) (p=0.03) and subjects hospitalized in intensive care units (40.7%) (p=0.003) were the most affected by the nonfermenting gram-negative bacilli. Regarding antibiotic resistance, it varies from 71.4% to 100% for Acinetobacter baumannii and 0% to 100% for Pseudomonas aeruginosa. The Stenotrophomonas maltophilia isolate had a broad spectrum ß-lactamase but was sensitive to the other antibiotics. Nonfermenting gram-negative bacilli are responsible for severe nosocomial pneumonia and the majorities are multi-resistant bacteria.

1. Role of Nonfermenting Gram-Negative Bacilli Responsible for Respiratory Tract Infections seen in the Laboratory of the University Hospital of Befelatananana Antananarivo Madagascar
Role of Nonfermenting Gram-Negative Bacilli Responsible
for Respiratory Tract Infections seen in the Laboratory of
the University Hospital of Befelatananana Antananarivo
Madagascar
Zafindrasoa Domoina Rakotovao-Ravahatra*1
, Fidiniana Mamy Randriatsarafara2
, Andriamiadana Luc
Rakotovao3
, Andry Rasamindrakotroka4
1Biologist doctor, Medical Biology Department of the Faculty of Medicine, University of Antananarivo, Madagascar
2Public Health Specialist, Public Health Department of the Faculty of Medicine, University of Antananarivo, Madagascar
3Professor in biological hematology, Medical Biology Department of the Faculty of Medicine, University of Antananarivo,
Madagascar
4Professor of Immunology, Medical Biology Department of the Faculty of Medicine, University of Antananarivo,
Madagascar
The majority of nonfermenting gram-negative bacilli are responsible for respiratory tract infections. The
aims of this study are to identify the nonfermenting gram-negative bacilli species in respiratory tract
infections, to describe their antibiotic resistance and to describe the factors associated with these
bacteria. It is a prospective study of 54 respiratory samples for a period of six months from October 2019
to March 2020 in the laboratory of the University Hospital of Befelatanana Antananarivo. Among the 54
respiratory samples, the microbiological results showed 12 (22.2%) cases of nonfermenting gram-
negative bacilli. These bacilli were represented by 7 (58.3%) cases of Acinetobacter baumannii, 4 (33.3%)
cases of Pseudomonas aeruginosa and 1 (8.3%) case of Stenotrophomonas maltophilia. Concerning the
associated factors, subjects under 40 years (24%) (p=0.77; NS), men (30.8%) (p=0.03) and subjects
hospitalized in intensive care units (40.7%) (p=0.003) were the most affected by the nonfermenting gram-
negative bacilli. Regarding antibiotic resistance, it varies from 71.4% to 100% for Acinetobacter
baumannii and 0% to 100% for Pseudomonas aeruginosa. The Stenotrophomonas maltophilia isolate
had a broad spectrum ß-lactamase but was sensitive to the other antibiotics. Nonfermenting gram-
negative bacilli are responsible for severe nosocomial pneumonia and the majorities are multi-resistant
bacteria.
Keywords: Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, antibiotic resistance,
carbapenemase.
INTRODUCTION
Nonfermenting gram-negative bacilli (NFGNB) are a
taxonomically diverse group of aerobic, non-sporing, bacilli
that either do not utilize glucose as a source of energy or
utilize it oxidatively (Malini et al., 2009). NFGNB are
defined as strictly aerobic and non-spore forming group of
bacteria that do not ferment carbohydrates but generate
energy required for their metabolic activities by oxidative
pathway (Chiu et al., 2015). Intrinsic resistance of NFGNB
to the commonly used antiseptics and their ability to
*Corresponding Author: Zafindrasoa Domoina
Rakotovao-Ravahatra, Laboratory of Joseph Raseta
Befelatanana University Hospital Antananarivo,
Madagascar. E-mail: ravahatradomoina@yahoo.fr
Co-Author 2
Email: fidyrfm@yahoo.fr
3
Email: lucdina007@yahoo.fr
4
Email: andryrasamindrakotroka@gmail.com
Research Article
Vol. 5(1), pp. 155-159, June, 2020. © www.premierpublishers.org. ISSN: 0379-9160
World Journal of Microbiology

2. Role of Nonfermenting Gram-Negative Bacilli Responsible for Respiratory Tract Infections seen in the Laboratory of the University Hospital of Befelatananana Antananarivo Madagascar
Rakotovao-Ravahatra et al. 156
survive under a wide range of environmental conditions
have aided them to occupy different settings. They have
been isolated from soil, aquatic environment, and hospital
environments such as anesthesia equipment, sinks,
intravenous fluids, and even distilled water. Their survival
in different hospital settings facilitated them to transfer
from patient to patient through fomites or the hands of
medical staff causing hospital acquired infections (Rit et
al., 2013). NFGNB are taxonomically diverse group of
bacteria that have been considered as commensals or
contaminants for many years. However, numerous recent
studies revealed that NFGNB are important cause of
different types of nosocomial infections, including
ventilator-associated pneumonia, septicemia, urinary tract
infection, and surgical site infection (Bitew, 2019). NFGNB
are innately resistant to many antibiotics and are known to
produce extended spectrum ß-lactamases and metallo ß-
lactamases. Currently, they exhibit resistance not only to
ß-lactam and the other groups of antibiotics, but also to
carbapenems (Gniadek et al., 2016). It is important to
correctly identify all clinically significant NFGNB
considering the intrinsic multidrug resistance exhibited by
these bacteria. To better understand the role of NFGNB in
respiratory tract infections, the aims of this study are to
identify the NFGNB species in respiratory tract infections,
to describe their antibiotic resistance and to describe the
factors associated with these bacteria.
MATERIALS AND METHODS
It is a prospective study of 54 respiratory samples for a
period of six months from October 2019 to March 2020 in
the laboratory of the University Hospital of Befelatanana
Antananarivo Madagascar. This study includes all
respiratory samples of patients who have applied for
microbiological examination. These respiratory samples
are either sputum, bronchial aspiration fluids or broncho
alveolar fluids. This study excludes any non-compliant
respiratory samples such as salivary specimens and
respiratory samples contained in a transport medium other
than the laboratory vial (sterile red cap vial). In the
laboratory, the respiratory samples are cultured in petri
dishes, containing ordinary agar called Uriselect® and in
sheep blood agar prepared from a mixture of Columbia
agar® and sheep blood. Afterwards, the petri dishes are
incubated in the oven at 37 ° C for 48 hours. Blood agars
are incubated in an atmosphere rich in CO2 to facilitate the
culture of certain bacteria. Then, a microscope
examination is performed after gram staining to quantify
epithelial cells and leukocytes and to identify bacteria.
After 48 hours, the positivity criteria are the presence of
bacterial colonies ≥ 107 CFUs (colony-forming units) per
mL, the number of epithelial cells ≤ 25 and the number of
leukocytes ≥ 25. Then, the bacterial colonies are identified
by the various bacterial identification tests available in the
laboratory. Finally, the antibiogram corresponding to the
identified bacterial species is carried out. Resistance to
antibiotics was determined by the Mueller / Hinton agar
diffusion method, according to the recommendations of the
“comité de l'antibiogramme de la sociétéfrançaise de
Microbiologie” (2019).The antibiotics tested are
represented by penicillins (ticarcillin), 3rd generation
cephalosporins (3CG), penemes (imipenem),
aminoglycosides (gentamycin, tobramycin, amikacin),
quinolones (ciprofloxacin), and sulfonamides
(cotrimoxazole). The dependent variable is constituted by
the positivity of the microbiological culture showing
NFGNB and the results of antibiogram. The independent
variables consist of the type of respiratory sample, the
gender, the age, the clinical information, the departments,
the result of microbiological culture of the pulmonary
secretion samples and the results of antibiogram. This
study was authorized by the Director of Establishment of
the University Hospital of Befelatanana and the
Department Head of the laboratory before its
implementation. This study respected the notion of
anonymity and confidentiality. The data entry and
processing were performed on the software Epi-info 3.5.2.
The comparison of percentages used the Chi square tests.
The statistical significance threshold used was p = 0.05.
RESULTS
Microbiological results of the respiratory samples
Among the 54 respiratory samples, the microbiological
results showed 12 (22.2%) cases of NFGNB (Figure 1).
The negative results represent the 23 (42.6%) respiratory
samples in which no bacteria were found.
Figure 1: Microbiological results of the respiratory
samples
These bacilli were represented by 7 (58.3%) cases of
A.baumannii, 4 (33.3%) cases of P. aeruginosa and 1
(8.3%) case of S.maltophilia (Figure 2).
Figure 2: Nonfermenting gram-negative bacilli (NFGNB)
species

3. Role of Nonfermenting Gram-Negative Bacilli Responsible for Respiratory Tract Infections seen in the Laboratory of the University Hospital of Befelatananana Antananarivo Madagascar
Associated factors with NFGNB in respiratory tract
infections
Concerning the associated factors, the average age is
46.8 years and the sex ratio is 2.6. Subjects under 40
years (24%) (p=0.77; NS), men (30.8%) (p=0.03) and
subjects hospitalized in intensive care units (40.7%)
(p=0.003) were the most affected by NFGNB (Table 1).
Table 1: Associated factors with NFGNB in respiratory
tract infections
NFGNB
(N=12)
Other
results
(N=42)
Total p-value
n % N %
Age (years)
<40 6 24.0 19 76.0 25
0.77
≥40 6 20.7 23 79.3 29
Gender
Female 0 0.0 15 100.0 15
0.03
Male 12 30.8 27 69.2 39
Departments
Intensive care
units 11 40.7 16 59.3 27 0.003
Other departments 1 3.7 26 96.3 27
Antibiotic resistance of NFGNB strains
Regarding antibiotic resistance, they vary from 71.4% to
100% for A. baumannii (Figure 3) and 0% to 100% for P.
aeruginosa (Figure 4).
Figure 3: Antibiotic resistance of A.baumannii
Figure 4: Antibiotic resistance of P. aeruginosa
Among the isolates of A. baumannii, 5 (71%) have a
carbapenemase(resistant to penemes like imipenem) and
World J. Microbiol. 157
are resistant to all the antibiotics tested. Among the strains
of P. aeruginosa, an isolate has a carbapenemase and is
resistant to all antibiotics tested except to Amikacin.
S. maltophilia isolate has a broad-spectrum beta-
lactamase (resistant to ticarcillin and 3CG) but sensitive to
other antibiotics (Table 2).
Table 2: Antibiotic resistance of S. maltophilia
Antibiotics Isolate of S. maltophilia
Cotrimoxazole Sensitive
Ticarcillin Resistant
Ciprofloxacin Sensitive
Gentamycin Sensitive
3CG Resistant
Tobramycin Sensitive
Imipenem Sensitive
Amikacin Sensitive
DISCUSSION
The current study has shown the isolation of NFGNB in
22.2% of respiratory samples. This result is higher
compared to another study carried out in Coastal
Karnatakawhich found 5% of NFGBN (Chawla et al.,
2013). Similarly, Malini et al. (2009) from Kolar in India
have documented the isolation of 6.8% (25 of 365) of
NFGNB in respiratory samples. The importance of
isolation of non-fermenters has increased in last decade,
after more and more reports are correlating them with the
either infection outbreaks in hospitals, or healthcare-
associated infections (Chawla et al., 2013). Earlier the
identification of non-fermenters, based on biochemical
tests, was cumbersome and many non-fermenters were
misidentified. But, now with the availability of commercial
systems, the identification has become easier.
P. aeruginosa and A. baumannii are the most common
NFGNB isolated in our study. Other studies have also
found the same results (Malini et al., 2009; Hurley, 2016;
Kaier, 2019).
Among the isolates of A. baumannii, 100% are resistant to
the 3 families of antibiotics (β-lactams, quinolones,
sulfonamides,). Similarly, 71% have a carbapenemase
and are resistant to all the antibiotics tested. Any of them
did not produce extended spectrum β-lactamase
highlighting other resistance mechanisms (impermeability
of the wall, efflux pump, etc.). A similar study carried out in
Arabia Saudi Arabia has identified strains of A. baumannii
multi-resistant (94%) (Alyamani et al., 2015). The study
carried out by Guckan et al. (2015) also highlighted the
multi-resistant nature of the isolates of A. baumannii.
These isolates were resistant to ciprofloxacin (97.3%),
gentamicin (77.2%), trimethoprim-sulfamethoxazole
(68.9%), imipenem (89.1%), meropenem (90.3%).
However, colistin (5.5%) and netilmicin (19.5%) were
effective on these strains. Another recent study in Iran
showed resistance A. Baumannii with cefixime (99%),

4. Role of Nonfermenting Gram-Negative Bacilli Responsible for Respiratory Tract Infections seen in the Laboratory of the University Hospital of Befelatananana Antananarivo Madagascar
Rakotovao-Ravahatra et al. 158
ceftazidime (99%), ciprofloxacin (98%), meropenem
(99%), trimethoprim-sulfamethoxazole (99%), imipenem
(91.5%), ceftriaxone (99%), levofloxacin (96.5%),
amikacin (70 %) and gentamycin (85%) (Goudarzi et al.,
2013). In contrast, tigecycline (4%) and colistin (0%) are
effective (Goudarzi et al., 2013). According to these 2
studies, colistin remains the molecule of choice for the
treatment of infections caused by A. Baumannii (Guckan
et al., 2015; Goudarzi et al., 2013). The resistance rate
varies widely between different hospitals and different
types of hospital services. The mechanisms of resistance
to antibiotics in multidrug-resistant bacteria are numerous
and diverse. The resistance of A. baumannii strains to
these families of antibiotics could be due to the misuse and
inappropriate use of antibiotics. In addition, the resistance
of the strains could be due to two factors favoring the
emergence and dissemination of multidrug resistance
including the selection pressure exerted by antibiotics and
the spread of these bacteria by cross-transmission
(Guessennd et al., 2013). In view of the antimicrobial
resistance of A. baumannii, detection in a patient affected
by pneumonia must be the subject of an official declaration
of the circulation of this strain at Coordination center for
the fight against nosocomial infections (Vaux et al., 2013).
Concerning the isolates of P. aeruginosa, they are less
resistant than the strains of A. baumannii. However, this
study found 25% of P. Aeruginosa isolates produce
carbapenemase. Carbapenemase-producing bacteria are
among the highly resistant emerging bacteria (Maamar et
al., 2019). Infection with these microorganisms is
responsible therapeutic difficulties or even dead ends. Of
more, these germs represent a potential danger in as
sources, reservoirs, and vehicles of carbapenemases as
well as a risk of hospital epidemics which can quickly
become uncontrollable (Maamar et al., 2019). Fortunately,
amikacin is effective against P. aeruginosa strains. Indeed,
the aminoglycosides, in particular amikacin, can be
considered to be molecules of choice to integrate into the
treatment regimen of infections with carbapenemase-
producing bacteria (Maamar et al., 2019).
The current study has shown the isolation of one isolate of
S. maltophilia. S. maltophilia is considered now as a
common non-fermenter to cause infection in hospital
settings (Velázquez-Acosta et al., 2018).These authors
have reported 76 strains of Stenotrophomonas maltophilia
responsible for nosocomial pneumonias. These
nosocomial pneumonia are frequently associated with
mechanical ventilation, tracheostomy, previous exposure
to broad-spectrum antibiotics, the use of respiratory tract
equipment such as nebulizers and therapy with
aerosolized polymyxin (Chawla et al., 2013). In this study,
the isolate of S. maltophilia had broad spectrum β-
lactamase but was sensitive to the other antibiotics
especially ciprofloxacin and cotrimoxazole (trimethoprim-
sulfamethoxazole). Similarly, Malini et al. (2009) have
documented 100% sensitivity to ciprofloxacin and
trimethoprim-sulfamethoxazole. Despite trimethoprim-
sulfamethoxazole often being the mainstay of treatment,
resistance is widely encountered, and alternative
regimens, including combination therapy, are often used
(Abbott and Peleg, 2015).
Concerning the associated factors, men and subjects
hospitalized in intensive care units are the most affected
by NFGNB infections with significant differences. Indeed,
men are the most affected because they are more
numerous in the workplace than women and they are more
exposed to many infections. Similarly, other studies have
also highlighted the frequency of NFGNB infections in
intensive care units (Agarwal et al., 2017). Currently, the
emergence and spread of multi-drug resistant NFGNB in
Intensive Care Units and their genetic potential to transmit
diverse antibiotic resistance regardless of their ability to
ferment glucose poses a major threat in hospitals. Invasive
procedures such as mechanical ventilation, tracheostomy
or the use of catheters favor nosocomial pneumonia in
Intensive Care Units (Merzougui et al., 2018).
In brief, NFGNB are responsible for severe respiratory
tract infections and the majorities are multi-resistant
bacteria. These bacteria are common in Intensive Care
Units. Thus, in view of the antimicrobial resistance of
NFGNB, detection in a patient must be the subject of an
official declaration of the circulation of this strain at
Coordination center for the fight against nosocomial
infections.
CONCLUSION
Any NFGNB isolated from respiratory tract infection should
not be ignored as mere contaminant, but correlated
clinically for its pathogenic potential and identified using
standard methods so as to institute appropriate and timely
antibiotic coverage.
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Accepted 23 June 2020
Citation: Rakotovao-Ravahatra ZD, Randriatsarafara FM,
Rakotovao AL, Rasamindrakotroka A (2020). Role of
Nonfermenting Gram-Negative Bacilli Responsible for
Respiratory Tract Infections seen in the Laboratory of the
University Hospital of Befelatananana Antananarivo
Madagascar. World Journal of Microbiology, 5(1): 155-
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