336 Indian Journal of Forensic Medicine & Toxicology, April-June 2020, Vol. 14, No.

Detection of Quorum Sensing Signal Molecules and

Identification of espB and Crt4 genes among Biofilm Forming
of Citrobacter freundii

Wathiq Abbas Hatite Al-Daraghi1, Mautham Khazaal Kadhim AL-Behadili1

1
Institute of Genetic Engineering and Biotechnology for Postgraduate Studies, University of Baghdad, Iraq

Abstract
150 samples from different clinical sources were collected from October 2018 to March 2019 from three
hospitals in Maysan: Al Sadr General Education Hospital, Al Zahrawi Surgical Hospital and Maternity and
Child Hospital to demonstrate the spread and distribution of Citrobacter freundii in several hospitals in
Maysan. All isolates were identified based on morphological characteristics and biochemical tests. Results
were confirmed by Api 20 E and Vitek 2 compact. A total of 14 isolates (9.3%) of 150 were found to be
Citrobacter freundii. The isolates were considered as acute diarrhea in children, UTI, burns, wounds and
most frequent ear swabs. PCR results showed that the LuxR 428bp genes were present in the Citrobacter
freundii bacteria identified by previous diagnostic methods and this confirms the accuracy of the tests and
methods used to determine this type.

Keywords: Citrobacter freundii; espB and Crt4 genes; Quorum sensing signal

Introduction Method and materials

Citrobacter, a genus of the Enterobacteriaceae Samples collection

family, Gram-negative, facultative anaerobic bacteria
that look as coccobacilli or rods (1) . Citrobacter spp. 150 samples from different clinical sources were
are motile using their peritrichous flagella, can ferment collected from October 2018 to March 2019 from three
mannitol with making of H2S, and can use citrate as hospitals in Maysan.
their single source of carbon (2),(3). Citrobacter spp. are
Isolation
uncommon opportunistic nosocomial bacteria can cause
urinary tract, hematologic, or neonatal infections (e.g. each sample was inoculated on the Salmonella
meningitis, sepsis, general bacteremia); intra-abdominal shigella (SS) agar medium, the plates were left to
sepsis; brain abscesses; or pneumonia (4) ,(5). Citrobacter solidify at room temperature, and then were incubated
spp. infections can be mortal with 33-48% overall death at 37 °C for 24-48 hours. Later the grown colonies were
rates being reported including 30% for children(6),(7). further investigated
Children and immune deficiency, elderly, or weakened
patients are at risk of infection (2), (9). Citrobacter spp. Identification
is prevailing worldwide, as it is a part of the normal The Citrobacter isolates were identified to the level
intestinal flora of humans (10),(11). Less well known of species using the traditional morphological and
species that have also been implicated in foodborne biochemical tests (13). The identification of isolates was
disease like some strains of Citrobacter spp. (notably C. confirmed by vitek2 compact system.
freundii), Klebsiella spp., Providencia spp. Enterobacter
spp. and Proteus spp., may occasionally cause what is Cultural characteristics on selective and
often described as opportunistic gastroenteritis (12), this differential media.
study aimed to isolation and identification of C. freundii
from chicken meat samples using cultural and molecular SS, MacConkey and Xylose lysine deoxycholate
techniques. (XLD) agar
 Indian Journal of Forensic Medicine & Toxicology, April-June 2020, Vol. 14, No. 2 337

The organisms were cultured on S.S agar media Identification of bacteria by Vitek 2 compact
and incubated overnight at 37°C. The colonies of C. system.
freundii appear with black center after 24hrs incubation
period, The suspected colonies of C. freundii cultured Vitek 2 compact was used to identify the bacterial
on MacConky media, the positive result appears pink isolates. It is a compact system of two parts, Instrument
(Lactose fermenters) after 24hrs incubation period, pale and computer. The reagent cards have 64 wells
colonies further incubated for 24hrs to identify the (late that can each contain an individual test substrate.
lactose fermenters). The selected colonies were cultured Substrates measure various metabolic activities such
on Xylose lysine deoxycholate agar, after 24hrs, the as acidification, alkalinisation, enzyme hydrolysis, and
positive result appeared as yellow colonies (13). growth in the presence of inhibitory substances.

Eosin Methylene Blue (EMB) agar Identification of Bacteria by PCR

In order to differentiate Citrobacter from E.coli, the DNA Extraction

lactose fermenter isolates were subcultured on EMB Genomic DNA was isolated from Bacteria according
for 24hr. at 37Co. Brown colonies were the positive to the protocol of Wizard Genomic DNA Purification Kit,
result(14). Intron. A PCR reaction with a specific primer (Table-1).

Table 1-Primers sequences

Primer Name Sequences Tm˚C Size (bp)

LuxR-F GCACGGATTACATCATTA 49.3

428
LuxR-R GCACGGATTACATCATTA 49.3

For LuxR gene was performed to identify C. freundii (Table-2).

Table 2-Reaction mixture

PCR master mix Volume

DNA template 5 µL

Green master mix 12.5 µL

Forward Primer10pmol 2.5 µL

Reverse Primer10pmol 2.5 µL

Free nuclease water 2.5 µL

Total 25 µL

(25μl) of PCR amplification mixture contained (12.5 Results and Discussion

μl) Master mix, (1 μl) forward primer, (1 μl) reverse
Bacterial Isolation and Identification
primer, (8.5 μl) nuclease free water, and (2 μl) DNA
template. The protocol for PCR condition was initial Twenty five chicken meat samples were collected
denaturation 95°C for 5 min. denaturation 95°C for 30 from local markets in Baghdad city. Citrobacter was
sec., annealing 60 °C for 40 sec., extension 72 °C for 1 detected in 3 samples, were all samples cultured on
min. and final extension 72 °C for 7min. S.S. agar for initial isolation, after incubation at 37°C
for 24 hr ; different types of bacterial isolates appeared
on S.S. agar, of them: small pale flattened colonies with
338 Indian Journal of Forensic Medicine & Toxicology, April-June 2020, Vol. 14, No. 2

black center due to their ability to produces H2S on S.S colour, these were depended as Citrobacter. To confirm
agar, then these colonies sub-cultured on MacConkey, the primary identification Gram stain was performed to
XLD and EMB to differentiate Citrobacter from examine the microscopic properties which were Gram
Salmonella because both of them are H2S, Citrobacter negative bacilli. The ability of Citrobacter to produce
is lactose fermenter on MacConkey agar appeared as urease enzyme was detected using urease test in order to
pink colonies while Salmonella is pale colonies (Non differentiate it from the genus Proteus which was urease
lactose fermenter) on XLD Citerobacter appeared as producer while Citrobacter isolates were non urease
yellow colonies while Salmonella appeared as red producers. Thus depending on colonial morphology;
colonies with black center . After incubation period; bacterial isolates were identified as Citrobacter Figure-1
lactose fermenter (pink) on MacConkey and yellow (A, B, C, D) and (Table-3) showed these biochemical
colonies on XLD while on EMB they were brown in tests used to identify Citrobacter as described by(15), (16).

Figure1-Different selective and differential media cultured with Citrobacter spp. after incubation at 37°C for 24 hr.
A. Pale colonies with black center on S.S. agar
B. Small pink (Lactose fermenter) colonies on MacConkey agar
C. Yellow colonies on XLD agar
D. Brown colonies on EMB.
 Indian Journal of Forensic Medicine & Toxicology, April-June 2020, Vol. 14, No. 2 339
Table 3-Result of biochemical tests

Test Result

Growing on MacConkey agar Dry Pink colonies

Growing on EMB Not forms green metallic sheen

Gram stain reaction Gram negative bacteria

Urease Non urease producer

S.S agar Pale colonies with black center

XLD agar Yellow colonies

To confirm the identification of Citerobacter spp. Vitek 2 compact system was depended and the result showed
that the isolated bacteria in this study was Citerobacter and the species freundii

In order to confirm the identification of Citrobacter to species level LuxR gene amplification was performed
using monoplex PCR technique, 1.5 % agarose gel electrophoresis was used to detect the positive result as shown in
Figure-2.

(Figure 2) Amplified PCR products of LuxR gene (428 bp): Agarose gel electrophoresis, ethedium bromide stained, 1.5 %
agarose, electrophoresed in 75 volt for 2 hrs and photographed under ultraviolet trans-illuminator. M: The DNA molecular
weight marker (100 bp ladder) and 1: the amplified PCR product of LuxR of C10 isolate of Citrobacter freundii.

One of the most gorgeous likely uses of 16Sr RNA • The prevalence and distribution of Citrobacter
gene sequence informatics is to offer genus and species spp. in some Maysan hospitals shows a relatively low
or tax identification for isolates (17). Although 16SrRNA percentage in its distribution.
gene sequencing is highly valuable in regards to
bacterial classification (18). PCR products were exposed • The dominance species of Citrobacter was
to direct sequencing, both strands of PCR products were Citrobacter freundii.
sequenced with an automatic sequencer. Sequences
• All isolates of Citrobacter freundii produced
were analyzed with the Basic Local Alignment Search
Bioflim formation by Congo red agar, Christensen
Tool (BLAST) in National Center for Biotechnology
method and micro-titer plate assay.
Information (NCBI).
• The effect of different temperature and pH values
Conclusion on Citrobacter freundii growth showed that best growth
The following conclusions were obtained from this temperature was 37°C and the best growth pH for growth
study: was 7.
340 Indian Journal of Forensic Medicine & Toxicology, April-June 2020, Vol. 14, No. 2

• These data are of great significance as the signal 8. Owoseni, M. and Okoh, A. Assessment of chlorine
molecules aid in biofilm formation which in turn confer tolerance profile of Citrobacter species recovered
various properties of pathogenicity to the clinical isolates from wastewater treatment plants in Eastern Cape,
including drug resistance. The use of quorum sensing South Africa. Environmental monitoring and
signal blockers to attenuate bacterial pathogenicity is assessment, 2017; 189(4): 201.
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the emergence of multi antibiotic resistant bacteria. Clinical features and antimicrobial susceptibility
trends in Citrobacter freundii bacteremia. Journal
Ethical Clearance: The Research Ethical
of microbiology, immunology, and infection, 2002.
Committee at scientific research by ethical approval of
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both environmental and health and higher education and
scientific research ministries in Iraq 10. Holmes, B. and Aucken, H. 1998. Citrobacter,
Enterobacter, Klebsiella, Serratia and other
Conflict of Interest: The authors declare that they members of the Enterobacteriaceae, in Collier L,
have no conflict of interest. Balows A, Sussman M (eds):Microbiology and
Microbial Infections: Systematic Bacteriology (ed
Funding: Self-funding
9). London, Arnold, (999-1033).
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