1

Drug Resistant Gene Distribution in P.aeruginosa (PANGL-1)

isolated from Urinary Tract Infected Cases
Vigila Christy. R a, C.Padmalatha d1 P.Dhasarathan d2 and A J A Ranjitsingh d3

( a - Microbiologist,Vivek Laboratory and Research Centre ,Nagercoil, b

d1-d3 Professors ,

Dept of Biotechnology Prathyusha Engineering College, Chennai

(a
vigila07christy@gmail.com , d2
hod.biotech@prathyusha.edu.in

d1latharac@gmail.com

d3
corresponding author – ajargnr@gmail.com)

ABSTRACT

Microbes have evolved several molecular strategies to overcome the bactericidal or

bacteriostatic effect of antibiotics. As most of the antibiotics work against the pathogens by

their β lactam ring (Penicillin, Cephalosporins, Carbapenems and monobactams), the bacteria

have developed a molecular change to secrete an enzyme β lactamase to erase the

killing/inhibition effect of β lactam. Further the microbes have developed mechanism to

produce different types of β lactamase including extended spectrum β lactamase (ESBL) to

face the antibiotics. .It has been identified that the genes, PER, VEB ,GES, TBC, TEM,

CTXm, OXA, and SHV genes express β lactamase synthesis. The enzyme bla-TEM is

synthesized mostly by Gram negative bacteria. Multi drug resistant gene development in

bacteria indicates its molecular adaptability to survive in the struggle for existence. In the

present observation P.aeruginosa isolates showed the presence of genes that are responsible

for the production of ESBLs and its new derivatives and it includes SHV(85%), CTX
(96.0%), TEM (89.0%), mec A (97.8%), Van B (72.2%), papC(98.0%), NDM(61.0%). In

the present study mec A gene was also reported. The mec A gene offer methicillin resistance

2.

in S.aureus. The presence of New Delhi Metallo – β lactamase-1 -NDM-1 gene – was also

observed. The development of drug resistance may be due to many factors. So there must be

a strict antibiotic policy in all global nations to fight the rising drug resistance in

P.aeruginosa bacterial isolates that are on the process of becoming “Superbugs”.

KEY WORDS: Drug resistant genes, SHV, CTX, TEM, mec A, Van B, papC, NDM,

Superbugs, P.aeruginosa

INTRODUCTION

Urinary Tract Infection (UTI) is emerging as one of the major health problems in

males and females globally (Brusch 2019). Women in the reproductive age, uncircumcised

male children, and elderly adults, persons predisposed to diabetes, catheterization, and

prostrate problems are vulnerable to the invasion of UTI causing microbes (Thatill etal.2018).

In general Gram negative bacteria predominates UTI. Of the different G-ve members, the

bacterial species belonged to Enterobacteriaceae group are the major causative agents. There

are many reports on drug resistance bacterial isolates in UTI patients ( Basu etal.2013,

Yacoub etal 2017 and Karishetti etal.2019). As most of the antibiotics inhibits the pathogens

by their β lactam ring (Penicillin, Cephalosporin, Carbapenems and monobactams).β lactam

drugs interfere with the functional mechanism of cell wall in bacteria . The enzyme β

lactamase hydrolyze the β lactam ring in drugs to collapse chemical structure of antibiotics

.The development of drug resistance is not because of intrinsic impermeability but also

involves molecular factors like plasmids, transposons and mutation. The drug resistant
microbes have evolved different molecular strategies to overpower the effect of antibiotics.

This may include intrinsic, acquired and adaptive resistance mechanism. Pseudomonas

aeruginosa  has lost its sensitivity to antibiotics (ZhengPang et al. 2018) . In the

3.

intrinsic mechanism of drug resistance P. aeruginosa shows a reduced permeability in   its

outer membrane and ability to send out the drugs from cell by efflux pump and can also

produce enzymes to inactivate antibiotics. .In the acquired resistance mechanism

horizontal transfer of resistance genes or mutational changes occurs (Breidenstein et al.,

2011). In the adaptive resistance mechanism  P. aeruginosa  forms biofilm that prevents

the  diffusion  antibiotics into the bacterial cells  ( Drenkard 2003 ) . In biofilm barrier,

the presence of multidrug-tolerant cells is reported (Mulcahay2010). Pseudomonas

aeruginosa was found to produce AmpC -lactamase to degrade β -lactam antibiotics and also

to eliminate aminoglycoside by protein efflux pump (MexXYOprM,) .The development of

antibiotic resistance gene is the key factor for drug resistance (Martin etal.2018). Multi drug

resistant gene development in bacteria indicates its molecular adaptability to survive in the

struggle for existence. So new antibiotics or alternative medicine are needed for treatment of

P. aeruginosa infections in the patients whose infections are drug resistant . Hence the

development of new antibiotics or alternative therapeutic strategies for treatment of P.

aeruginosa  infections is urgently required for the patients whose infections are resistant to

conventional antibiotics. P. aeruginosa has developed resistance to ceftazidime

carboxypenicillins, ,aztreonam; and to a third generation cephalosporins by the

hyperproduction of AmpC b-lactamase by the ampC gene expression (Lodge et al.,

1993).The blaCARB-4 gene encode b-lactamases in Pseudomonas aeruginosa so as to

produce resistance to carboxypenicillins , ureidopenicillins, and to different

cephalosporins derivatives . The SHV-2a ,SHV-5 and SHV-12 genes in Pseudomonas

aeruginosa produce enzymes to hydrolyze even the recent type cephalosporins

(Weldhagen et al., 2003). P. aeruginosa strains were reported to synthesis beta lactamase

encoded by GES-1 and TEM genes (Marchandin et al. 2000). Carbapenem-hydrolyzing

4.

β-lactamases, ( carbapenemases) are acquired through plasmids, transposons and

integrons to carry resistance determinants . The carbapenemases or New Delhi metalloβ-

lactamases-type enzymes in inactivated all known β-lactams, and making K. pneumoniae

resistant to almost all currently used antimicrobials (Sahuquillo-Arce et al.2015). The

development of carbapenemases limits all treatment options and pose a risk to bacterial

infected patients leading to high mortality (Sahuquillo-Arce et al.2015). The OXA genes

and its derivatives in plasmids produce class D β-lactamases (Antunes and Fisher 2014)

.According to recent thoughts proteins (OprM, OprJ, OprN) play an important roles in active

efflux systems with wide substrate specificity Hirakata et al. ( 2002). . Effusing antibiotics

from the cell also help to develop resistances to all drugs and it is mediated by four

genes ( Li et al 2000). As antibiotic resistance is encoded by genes, in the present study the

drug resistant P.aeruginosa (PNGAL 1) isolates were tested for the presence of the drug

resistant genes, SHV, CTX, TEM, mecA, Van-A, Van-B, AmpC, papC, NDM1 and VIM

and their frequencies in south Indian population.

MATERIAL AND METHODS

In the current study the urinary tract infected patients identified were used to collect

urine samples and from the midstream urine samples bacterial colonies present were

isolated. The inoculum of UTI bacterial isolates identified was grown overnight in the

Mueller-Hinton medium at 37°C and again sub-cultured in that medium overnight. From the
samples the bacteria were isolated, identified and sensitivity study was made using 16

antibiotics. Pseudomonas aeruginosa strain (PNGAL 1) isolated was further subjected to

specific medium culture to get pure colonies. PNGAL 1 isolates were swabbed in Mueller-

Hinton agar plates for anti biogram assay. The antibiotics were loaded into sterile disks and

5.

incubated in the swab for 24 h at 37°C. The antibacterial activity was measured by using the

diameter of zone of the inhibition. From resistant strains 15 P.aerugenosa isolates were

further subjected to drug resistance genes isolation.(Plate.1)

Bacterial genomic DNA extraction

The DNA of the 15 representative P. aeruginosa was extracted for genomic

DNA extraction assay .

Molecular identification of Antibiotic Resistance Gene

. Material & Methods:

The genomic DNA was extracted from P. aeruginosa by using (PureFast®

Bacterial DNA mini spin kit obtained from HELINI Biomolecules, Chennai, India). One ml

of overnight culture was transferred BHI broth in a 1.5ml micro centrifuge tubes and it was

centrifuged at 10000 rpm for 1 minute. The supernatant was removed and the bacterial

pellets were used for genomic DNA extraction. The extraction was made according to the

manufacturer instructions. The extracted g DNA was confirmed by "Nano drop"

spectrophotometer, and stored at -20C in till PCR assay .

2X Master Mix preparation and Agarose gel electrophoresis:

 It contained 2U of Taq DNA polymerase, 10X Taq reaction buffer, 2mM MgCl2, 1μl

of 10mM d NTPs mix and Red Dye PCR additives.

Agarose, Ethidium bromide, 50X TAE buffer, and 6X gel loading buffer from

HELINI Biomolecules, Chennai were used.

6.

PCR products used:

HELINI Ready to use CTX gene Primer mix - 5μl/reaction

PCR Product: 295bp;HELINI Ready to use VIM gene Primer mix - 5μl/reaction

PCR Product: 264bp ;HELINI Ready to use SHV gene Primer mix - 5μl/reaction

PCR Product: 275bp ;HELINI Ready to use mecA gene Primer mix - 5μl/reaction

PCR Product: 220bp ;HELINI Ready to use Van-A gene Primer mix - 5μl/reaction

PCR Product: 270bp ;HELINI Ready to use Van-B gene Primer mix - 5μl/reaction

PCR Product: 164bp ;HELINI Ready to use ampC gene Primer mix - 5μl/reaction

PCR Product: 150bp ;HELINI Ready to use papC gene Primer mix - 5μl/reaction

PCR Product: 150bp ;HELINI Ready to use NDM1 gene Primer mix - 5μl/reaction

PCR Product: 202bp andHELINI Ready to use TEM gene Primer mix - 5μl/reaction

PCR Product: 295bp

Bacterial DNA Purification

One ml of overnight PNGAL 1 culture was centrifuged at 6000rpm for 5min. After

discarding the supernatant the pellets were kept in in 0.2ml PBS and 180μl of

Lysozyme digestion buffer. After that Lysozyme-20μl [10mg/ml] was transferred

and incubated at 37 C for 15min. Further 400μl of binding buffer, 20μl of Proteinase

K and 5μl of internal control template were added and mixed well by inverting

several times. Incubated at 56ºC for 15min and added 300μl of ethanol .After

mixing well the whole sample was put into the PureFast® spin column for
 centrifugation for 1 min. After discarding the supernatant it was placed back into

the collection tube .After adding 500μl Wash buffer-1 to the PureFast® spin

column, the content was centrifuged for 30-60 seconds and flow-through was

discarded. The column was placed back into the same collection tube and added

7.

500μl Wash buffer-2 to the PureFast® spin column and centrifuged for 30-60 seconds

.After discarding the flow-through column was placed back into the same collection

tube and and centrifuged for an additional 1 min to avoid residual ethanol. The

PureFast® spin column was transferred into a fresh 1.5 ml micro-centrifuge tube and

to the center of PureFast® spin column membrane 100μl of Elution Buffer was

added. After incubating for 1 min at room temperature it was again centrifuged for 2

min. The column was discarded and the purified DNA was collected and stored at

-20°C. The quality and quantity of the extracted DNA was checked by using 1%

agarose gel .Further 5μl of the extracted DNA was used for PCR amplification.

PCR Procedure:

1. Reactions set up

Components Quantity

Red Dye HELINI PCR Master mix 10μl

Ready to use HELINI– Primer Mix 5μl

Bacterial DNA Purified 10μl

Total volume 25μl

The reaction components were mixed gently and spined down briefly. Then

placed into PCR machine and the program was followed as follows;

Timing for Initial Denaturation: 95ºC for 5 min

Timing for denaturation: 94ºC for 30sec

 Timing for annealing: 58ºC for 30sec 35 cycles

Timing for extension: 72ºC for 30sec

Timing for final extension: 72º C for 5 min

8.

Loading:Two 2gm of agarose was mixed with in 100ml of 1X TAE buffer to prepare

2percent agarose gel and it was electrophoresed at 50V untill the stain run three fourth

distance. Thebands were observed in UV trans illuminator.

Agarose gel electrophoresis:

To prepare two percent agarose gel 2gm of agarose was mixed in 100ml of 1X

TAE buffer and melted using microoven .When the agarose gel reached 60ºC, 5μl

of ethidium bromide was added. Then warm agarose solution was poured slowly into

the gel platform. The gel set was allowed to solidify .Again IXTAE buffer was

transferred to the submarine gel tank. The gel platform was placed into tank and the

tank buffer level 0.5cm was maintained above the gel. Gel loading dye was mixed

with along with 10μl HELINI 100bp DNA Ladder with different base pair. The

PCR samples were loaded and electrophoresed at 50V until the stain reaches 3/4

distance of the gel. The gel was observed in UV Trans illuminator and the bands were

documented.

Results and Discussion

For the molecular characterization of drug resistant gene in P. aeruginosa , ten

antibiotic gene primers were used against the DNA extracts of the 15 isolates . The resistant

genes present in the isolates were amplified in the gel. The examinations of the agarose gel in

which the isolated genes are present are given in (Fig1 & 2).

Drug Resistant Genes in P.aeruginosa

 P.aeruginosa is an opportunistic pathogens. It is a causative agent for nosocomial

septicemia ( Peymani et al., 2017). ESBLs in P.aeruginosa were reported to be associated

9.

with over production of chromosomal AMP-C ,Cephalosporinase or with non-enzymatic

mechanisms like outer membrane impermeability or drug efflux (Shakibaie et al., 2008).

The plasmid acquired ESBLs viz; PER, VEB, GES, IBC, TEM and SHV gene

reported in Enterobacteriaceae were also seen in PNGAL 1 . TEM and SHV genes observed

in the present study are reported to hydrolyze ampicillin at a greater degree than other

antibiotics like Oxacillin, Carbenicillin or Cephalothin (Farqad et al 2018). TEM – types

that are rarely reported in P.aeruginosa is detected in the present study . In the tested

isolates amp C gene is not represented. Bharti et al., (2017) reported that ESBLs genes that

are predominant in P.aeruginosa include TEM (Temoneira), SHY (Sulfhydroyl variable),

CTX-M (Cefataximase), PER (Pseudomonas extended beta lactamase), VEB (Vietnamese

ESBLs) and GES (Guiana ESBLs). Antibiotics like piperaacillin, tazabactum and

carbapenems etc. are able to inhibit the production of ESBLs and AmpC in P.aeruginosa

( Pragasam etal.2018). The bacteria that produce metallo-β lacatamase (MBLs) to hydrolyse

all class of β-lactum drugs except monobactum such as aztreonam are not noticed in the

study.

In the present observation P.aeruginosa isolates showed the presence of genes

causing resistance include SHV(85%), CTX (96.0%), TEM (89.0%), Mec A (97.8%), Van B

(72.2%), papC(98.0%), NDM(61.0%). These genes were reported to be responsible for the

production of ESBLs and its new derivatives (Pragasam etal.2018). Farqad et al. (2019)
isolated four extended spectrum β-lactamases genes from wound in Iraq samples . Farqad

et al. (2019) further stated that TEM, SHV and CTX genotypes are predominant within

members of the family Enterobacteriaceae in different Asian countries. Bokaeian et al.,

(2015) reported the presence of different classes of ESBLs in P. aeruginosa. As PNGAL 1

10.

isolates showed resistance genes to more than three classes of antibiotics (carbapenems,

penicillin, cephalosporins, monobactams, aminoglycosides etc.) MDR is prominently seen

in the present isolates.

In the present study mec A gene is also reported (97.8%) (Table 2). The mec A gene

offer methicillin resistance in S.aureus. The mec A genes that code for PBP2a (Penicillin

Binding Protein Family - PBP) the β-lactam drugs cannot inactivate or promotes

resistance in P.aeruginosa ( Le et al. 2013). In the present study NDM-1 gene – New Delhi

Metallo – β lactamase-1 was also reported (61.0%). Extended spectrum β-lactamases are

also mediated by plasmid and derived from mutations in several genes by one or more

amino acid substitution in the active site. These genes are responsible for resistance to beta

lactams in Pseudomonas aeruginosa. ((Farqad et al 2018 and Jovcic etal.2011) . According

toTanya (2009) the genes TEM- and SHV-type ESBLs in P. aeruginosa develop from other

Enterobacteriaceae member by gene transfer . The extended-spectrum oxacillinases genes in

plasmid- or integron- are reported to increase the presence of class D ESBLs in P. aeruginosa

isolates (Tanya 2009).

Findings of the present study shows that the P.aeruginosa isolates (PNGAL 1) have

made a molecular revolution to develop several new types of drug resistant proteins that are

coded in new genes. A molecular evolution to develop drug resistant gene is on progress in
P.aeruginosa. So a high level attention is needed to prevent P. aeruginosa to become

superbugs.

11.

Table 1. List of genes tested with base pair (bp)

PCR:

HELINI Ready to use CTX gene Primer mix - 5μl/reaction

PCR Product: 295bp

HELINI Ready to use VIM gene Primer mix - 5μl/reaction

PCR Product: 264bp

HELINI Ready to use SHV gene Primer mix - 5μl/reaction

PCR Product: 275bp

HELINI Ready to use mecA gene Primer mix - 5μl/reaction

PCR Product: 220bp

HELINI Ready to use Van-A gene Primer mix - 5μl/reaction

PCR Product: 270bp

HELINI Ready to use Van-B gene Primer mix - 5μl/reaction

PCR Product: 164bp

HELINI Ready to use ampC gene Primer mix - 5μl/reaction

PCR Product: 150bp

HELINI Ready to use papC gene Primer mix - 5μl/reaction

 PCR Product: 150bp

HELINI Ready to use NDM1 gene Primer mix - 5μl/reaction

PCR Product: 202bp

HELINI Ready to use TEM gene Primer mix - 5μl/reaction

PCR Product: 295bp

12.

Table 2. Table showing the distribution of drug resistant genes

Gene Drug resistant genes Gene Distribution

Percentage in 15 isolates
(sul)
SHV ✓ 85.6
ESBL – Plasmid

CTX ✓ 96.3

TEM ✓ 89.6
ESBL – Plasmid
✓
Mec A 97.8

Van A NTC X NIL

✓
Van B 72.1

amp C X NIL

PapC ✓ 98.4

NDM ✓ 61.5

VIM-NTC X NIL
Fig. 1 Multiplex – PCR profiles specific for P.aeruginosa isolates showing drug

resistant genes

Van B DNA14 KB NDM – 1 Pap C VIM –NTC AmpC

LANE SHOWING THE PRESENCE OF DRUG RESISTANT GENES IN

P. aeruginosa ISOLATED FROM UTI
Fig. 2. Multiplex – PCR profiles specific for P.aeruginosa isolates showing drug resistant
genes

DNA SHV CTX TEM Mec A Van-A-NTC

14KB

LANE SHOWING THE PRESENCE OF DRUG RESISTANT GENES

IN P. aeruginosa ISOLATED FROM UTI

Plate 1. Drug resistant P. aeruginosa showing poor inhibition zone
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