The Detection of Transposable ampicillin-resistance in Klebsiella pneumonia

Rabab OmranBiology department, College of Science, Babylon University

E-mail: omranradi@yahoo.com

Summary

Genetically instable clinical strain of Klebsiella pneumonia No.10 was isolated from respiratory tract infection. The isolate harbors large single non-conjugative plasmid pKP (120kbp) which encoding multiresistance for antibiotics. Successive subculturing of the isolate for about480 generations yielded several plasmid derivatives. Allmaintained ampicillin resistance, while they appeared variable deleted or abolished to the other antibiotic resistance markers. One of these derived plasmids (pKP2) encoding ampicillin (Apr) and trimethoprim (Tpr) resistance was transformed into E.coli MM294 which already harbors a non-conjugative plasmid pACYC184 (Cm, Tc). The transformed cells(pKP2 and pACYC184) were subjected to successive subculturing for about 480 generations in ampicillin medium at 37ºC with selection for ampicillin and tetracycline and chloramphenicol. Subsequently screeningfor plasmid content revealed that the presence (on agarose) of approximately seven bands of plasmid DNA ranging between pKP2 and pACYC184 in molecular size. Each of these bands was eluted from the gel, purified and re-transformed intro E.coli MM294 competent cells. The transformed colonies resistant to ampicillin, chloramphenicol and tetracycline were selected. The plasmid content of these cells revealed the presence of extra size (approximately 5kb) to the

1

pre-transformed DNA band (pACYC184) as indicated by gel electrophoresis. The added DNA could represent a transposable DNA fragment encoding ampicillin resistance.

Key word: Klebsiella pneumonia, transposable element, ampicillin resistance

لاصة� الخ�

لت� السلالة� عدمKlebsiella pneumonia No.10 عز� ت� ب� مي�ز� سي� وت� ف� ن� مج* المج�زى الت# من* خ�

ق�زارها الو راث/ ي�زي�اسن� ز مق� ي� ق�زد و غ� ز من� ي� د كب> مي� لار� ها علي ب� Dوائ مة ن* واحت# ا120 ) حج� ق�ز هد� ( وي�ش/ اعدى� وج* ق� لو ر� كي�

. عددة� اومة� المن� د الى المق� مي� لار� الي�كرر ) رع المي� م الحصول من* الز� ت�480 ت� jظ ف� عها احن� من� ة� خ� دي� مي� لار� ات� ب� ق� ن� لة� علي عدة� مش/ ة العز� ل( لهد� ي� ح�

. ة� ي� �uت ا ادات� الحي� اومة� للمض� زات� المق� ش/ Dؤة� م ي� ف� ف� ب� دان* او حد� ق� ي� ف� ن�ت� ف� �uت ا ي� ها ت� ن* ائ� ي� حي� ن* ف� سلي� ي� ها للامي� اومت� مق� ت�

2

حول الو راث/ زاء الت# ج� م ا� ة� ) ي� ت� ق� ن� دات� المش/ مي� لار� ة الي� ن*pKP2 لاحد هد� سلي� اومة� الامي� ق�زة� لمق� ( المش/ا� ب�� كي�ز هلة� من* ب� Dموا ال لاب� م الى الخ� زت� ي� مب/ ي�زن*E.coli MM294والي�زات� ز المق� ي� د غ� مي� لار� ا علي الي� ق� ة� مشن� وي� المحت#

pACYC184 (Cm, Tc)*ن ي�� د مي� لار� ة� علي كلا الي� وي� حولة� والمحت# ا المت# لاب� اع الخ� ض� خ� م ا� عدها ت� و pKP2 . ب�pACYC184 ( كرر رع المي� ة� جزارة� 480 الى الز� درج� ن* وب� سلي� ي� وسط الامي� ل ( ف� ي� ار 37 ح� ي� ت� م اح� م ت� م , ت/

وى� جزى� عن* المحت# م الت# عد ت� ما ب� ي� كول . وف� ي� ت� ن�* والكلورامق¥ كلي� ي�زاسي� ن* و الب# سلي� ادات� الامي� اومة� للمض� ا المق� لاب� الخ�مها ي� هلام الاكارور� وت�راوج حج� ة� ف� دي� مي� لار� م ب� ع جز� ك�ي/ز من* سن� Dود ا هرت� وج� jظ Dي� ا ة� الت� ج� ات� ا الي� لاب� دى� للخ� مي� لار� الي�

ن* ي�� د مي� لار� ي�ن* الي� مpACYC184 و pKP2ث� ها ت/ ت� ت� ق� ن� م كل علي حدة� من* الهلام وت# ة الجز� م اسي�زداد كل هد� . ت�ا ب�� كي�ز هلة� من* ب� Dموا ال لاب� الها كل علي حدة� الى الخ� دح� عادة� ا� عدE.coli MM294ا� ما ب� ي� . وف� /ى� uوراثحول ال ة� الت# عملي� ب�

سلي� ي� اومة� للامي� حولة� المق� ا المت# لاب� ار الخ� ي� ت� م اح� /ى�ن*ت� uوراثوى ال هر المحت# jد اظ .� وق� ن* كلي� ي�زاسي� كول و الب# ي� ت� و الكلورامق�ا ي� �ºت ق�ز ي� ب� اف� ض� م ا� و حج� د د� مي� لار� ها علي ب� Dوائ ا احت# لاب� ة الخ� ضلي�5لهد� Dد الا مي� لار� الي� ة� ب� اري� اعدى� مق� وج* ق� لو ر� كي�

pACYC184 طعة� ل ق� مي/ مكن* ان* ت� . وت� ي� هلام الاكارور� سلي�DNA ف� ي� ق�ز للامي� �ز� المش/ اف صز الق� ة� العن� ي� اف� .ن* الاض�

3

Introduction

Klebsiella pneumoniae is an important nosocomial

pathogen. Antibiotic resistant strains of Klebsiella

pneumoniae are frequently found in the hospital

environment (Podschun and Ullman, 1998). Resistance to

β-lactams, chloramphenicol and aminoglycosides is by

production of enzymes that degrade antibiotics, such as

β-lactamases (Branger, 1998). However, chromosome and

plasmid –mediated resistance to β-lactam has already

detected in various gram-negative bacteria, including

Klebsiella pneumoniae (Tschape, 1994). Klebsiella pneumoniae

expressing more than ten type of extended spectrum β-

lactamases (TEM and SHV) conferring resistance to

pincillins and cephalosporins (Branger, 1998). An SHV-5-

encoding plasmid has spread among several strains of

Klebsiella pneumoniae, which had caused three out breaks in

different area, were reported (Legaskis et al., 1995;

Branger, 1998). SHV-4, TEM-15 and TEM-16 are encoded by

conjugative plasmids ( 70 – 160 kbp) in Klebsiella pneumoniae

4

(Williams and Tomas, 1990;Shannan et al.,1998). Furthermore,

a multiresistance transposon, Tn1331, of 7.5 kbp which

harbors the amikacin and ampicillin resistance genes of

Klebsiella pneumoniae plasmid pJHCMW1 was isolated and

characterized by Tolmasky and Crosa (1987). Also Tn3

encoding ampicillin resistance was characterized which

is a 4957bp class II transposon. At each end it has

identical 38 bp inverted repeats . The Tn contain

transposase gene ( trpA) and resolvase gene ( tnpR) and

β–lactamase (bla). Tn3 also exhibits transposition

immunity, which appears to be a product of the resolvase

and its recognition of a terminal inverted repeat on a

target molecule. Therefore, the intramolecular movement

of Tn3 were reduced. (Kleckner, 1981; Kleckner, 1990)

Plasmid-mediated gene exchange between bacteria

plays an important role in bacterial adaptation and

flexibility. Plasmids have greatly contributed to the

rapid spread of antibiotic resistance genes in bacterial

populations due to antibiotic selective pressures

resulting from the intensive use of antibiotics in human

therapy and agriculture (Tschape, 1994; Witte, 1998).

We examined a multiresistant Klebsiella pneumoniae

No.10 strain in which resistance to ampicillin (Apr) ,

kanamycin (Kmr) , trimethoprim (Tpr) , tetracycline (Tcr)

, nalidixic acid (Nalr), streptomycin (Smr) ,

5

chloramphenicol ( Cmr) and amoxicillin (Axr) , were

encoded by an 120 kbp plasmid, pKP10. Molecular cloning

and partial deletion by salysilic acid of the pKP10

suggested that Kmr, Tpr, Tcr, Nalr, Smr and Cmr are

encoded by different genes while the Apr gene is

localized in a different region of pKP10 (Al-Jalawi,

2000).

This paper describes the transposition of ampicillin

resistance in K.pneumoniae.

Material and Methods:

Bacterial strains :

A mucoviscous K.pneumoniae No.10 strain previously

isolated from human respiratory tract infection used in

this study. It was re-identified and maintained on

regular media.

The standard strains and plasmid were obtained

from Institute of Genetic Engineering and Biotechnology

for Post-Graduate Studies in Baghdad University.

Echerichia coli MM 294 (hsd R, hsd M, thi-, end A, rif r) was used as

recipient in genetic analysis. Echerichia coli MM 294

harboring non-conjugative plasmid pACYC184 (Tcr and Cmr)

Antibiotics:

6

Antibiotics used as selective agents were added

to tryptic soy agar plates and Mueller Hinton agar

plates at the following concentrations (µg/ml) :

Ampicillin (Ap) , 100 to 1000 ; Tetracycline (Tc ) ,

20 : Kanamycin (Km) ,50 ; Nalidixic acid (Nal) ,20 ;

Streptomycin (Sm) , 100 ; Chloramphenicol (Cm) , 50 ;

Trimethoprime (Tp) , 50 ; Rifampicin ( Rif) , 100 .

DNA analysis :

DNA preparations were preformed by the method of

Kado and Liu (1981). Total DNA was prepared by the

method of salting out of Pospiech and Neuman (1995) with

some modification: Bacterial cells of 30 ml culture were

precipitated by centrifugation (10000 xg for 10 min).

The pellet was re-suspend at 5ml SET buffer (75mM NaCl,

25 mM EDTA pH8, 20 mM Tris pH 7.5). A volume of 600µl

25% SDS was added , mixed by inversion and incubated 5

min at 55ºC. Then 2ml of 5M NaCl was added to the

lysate, mixed thoroughly by inversion and let cool to 37

ºC. 5ml of phenol-chloroform-isoamyl alcohol (25:24:1

v/v) was added to the lysate and mixed by inversion for

30 min at 20ºC. After that, it was spin by Centrifuge

15, 4500xg at 20 ºC for 10 min, then the aqueous phase

transferred to a fresh tube, which contain nucleic acid.

Isopropanol (0.6 vol) was added to the extract and mixed

7

by inversion, after 3 min DNA spooled onto a sealed

Pasteur pipette, DNA rinsed in 5ml 70% ethanol , air

dried and dissolved in 1-2 ml TE at 55ºC.Then DNA

extract was kept in -20ºC.

Plasmid DNA was subjected to agarose (0.7 %) gel

electrophoresis in Tris – borat buffer (89 mM Tris base,

2.5 mM sodium EDTA, 89 mM boric acid) at 4 vol./ cm

(Pharmacia gel electrophoresis apparatuses) for 12 h.

Transformation of K.pneumoniae plasmid DNA into

competent culture of E.coli or K.pneumoniae was carried out

as described by Sambrook et al. (1989).

To recovery of the plasmid DNA from low melting

agarose gel, the plasmid DNA was electrophoresed on low

melting gel electrophoresis (0.8 %) at 6 vol./cm for

three hours and examined by UV transluminator. The DNA

plasmid bands were eluted from the gel according the

method of Sambrook et al.( 1989). The eluted plasmid

DNA was transformed into plasmidless E.coli MM294

competent cells.

Detection of genetic stability of K.pneumoniae

K.pneumoniae No.10 was subjected to successive

subculturing for about 480 generation (30 min /

generation) on nutrient agar at 37ºC. Antibiotic

resistance markers of the last subcultures (20th)

8

detected on Muller- Hinton agar supplemented with the

studied antibiotics and DNA content of the resulting

cells were analyzed.

Detection of transposon of ampicillin resistance

The detection of transposon of ampicillin

resistance was performed according to method Rubens et al.

(1976) with some modifications. Plasmid carrying Ap gene

(pKP2) was introduced into E.coli MM294 competent cells

which is already harboring the non-conjugative plasmid

pACYC184 (Tcr and Cmr) by transformation. The

transformants resistant to both antibiotics (Apr and

Tcr) were selected on Mueller- Hinton agar supplemented

with the 100µg /ml of ampicillin and 20 µg /ml of

tetetracyclin . The transformants that contain both

plasmid (pKP2 &pACYC184) were subjected to successive

subculturing for about 480 generation (30 min /

generation) on Mueller- Hinton agar supplemented with

the 1000 µg /ml of ampicillin at 37ºC. After that,

antibiotic resistance markers of the last subculture

(20th) detected on Mueller- Hinton agar supplemented

with the both antibiotics (Ap & Tc) and the DNA content

of the resulting cells were analyzed.

9

Results:

In previous study we isolated K.pneumoniae strain

from upper respiratory tract infections and found the

isolate harbors a single large plasmid (120kp) encoding

multiple resistance to mucoid phenotype and antibiotics

including Ampicillin, Tetracycline, Kanamycin,

Streptomycin, Chloramphenicol, Nalidixic acid and

Trimethoprim (Al-Jalawi, 2000; Al-Zaag and Omran, 2002).

In the present study, genetic stability of

K.pneumoniae No.10 was detected by subjected the isolate

to successive subculturing for about 480 generations on

nutrient agar at 37ºC. Three Klebsiella variants were

obtained which designated I, II and III (Table-1). They

varied in antibiotic resistance profile. Plasmid content

among the three derivative differed in comparison with

wild type strain, which contain one single large plasmid

(Fig-1).

The plasmid DNA from Klebsiella variants were

extracted and transformed into E.coli MM294 competent

cell. Results showed variable frequency of transformed

antibiotic resistance profile. Plasmids designated pKP1

t0 pKP10; all of the derived plasmids maintained

ampicillin resistance and appeared variable antibiotic

resistance profile to other antibiotics resistance

(Table-2). Each of them has lower molecular weight on

10

agarose gel electrophoresis in comparison with wild

plasmid (pKP). The results suggested that the variation

might due to deletion or insertion inactivation events.

On the other hand, K.pneumoniae No.10 could resist high

concentration of ampicillin until 1000 µg/ ml with

comparison to other Klebsiella isolates that resisted to

ampicillin until 100 µg/ ml .While the isolate resist to

chloramphenicol and tetracycline until 100 µg/ ml that

resemble to other isolates. This suggested that the DNA

content of K.pneumoniae No.10 might be contain more than

one copy of the Ap gene. From the over results we

suggested that the plasmid pKP may be carrying

transposable element encoding ampicillin resistance

which is responsible to genetic stability of the

isolate.

Table. 1: The variants derived from successive subculturing of

Klebsiella pneumoniae No.10

Variants

group of

Antibiotic resistance

(µg/ml)The

frequency

11

K.

pneumoniae

of the

variation

) (%

Ap

10

0

Tc

20

Sm

10

0

Cm

50

Km

50

Na

l

20

Tp

50

Wild

strain+ + + + + + + 100

Variants I + + - + + + + 35Variants II + - - + + + + 12Variants III + + + + + + + 53

* (+) Resistant or (-) sensitive to antibiotics. Ap:

Ampicillin; Tc: tetracycline; Sm: streptomycin; Cm:

Chloramphenicol; Km: Kanamycin; Nal: Nalidixic acid; Tp:

Trimethoprim.

12

devireD sdimsalp

dimsalP PKp

emosomorhC

3 2 14

Fig .1. Plasmid patterns of K.pneumoniae variants. Lane 1: Wild isolate , 2: Variant I, 2: Variant II,

3: Variant III

The Plasmids were electrophoresed in 0.6% gel in Tris-borate

buffer at 4 V/cm for 12h.

Another evidence to prove the ampicillin

resistance encoded by a jumping gene is the

transposition experiment, which was performed to induce

Ap gene to jump from the derived plasmid pKP2 onto the

non-conjugative plasmid pACYC184 (Tcr & Cm r). E.coli KP2O

derivative selected to prepare the derived plasmid pKP2;

subsequently it transformed into the competent E.coli

MM294 already harboring pACYC184. The transformants

strain that harbor both plasmid (pKP2 and pACYC184)

designated E.coli R1 (Fig.2). After the successive

subcultures ( 480 generation) on Mueller Hinton agar

supplemented with 1000µg ampicillin at 37ºC the

resulting cells (designated E.coli R2) from the last

13

generation (20th) appeared resistance to ampicillin ,

tetracycline and chloramphenicol and contain more than

five plasmid bands on the gel electrophoresis ( Fig.2).

The plasmid bands were ranging between pPK2 and pACYC184

in molecular weight. This result may be due to co-

integrate formation or deletion events by transposable

element. Each of these plasmid bands were eluted,

introduced into plasmidless E.coli MM294 competent cells

by transformation and selected the transformed cells

that appeared resistance to ampicillin, tetracycline and

chloramphenicol. The resulting cells E.coli R3 harboring

single plasmid band designated pR3 that had a molecular

size higher than pACYC184 and less than pKP2 the extra

size approximately 5kb to the pre-transformed DNA band

as indicated by gel electrophoresis. This result may be

due to transpose the Ap gene from pKP2 to pACYC184 which

is form pACYC184:: Tn-Ap structure.

Table.2: Klebsiella pneumoniae variants and

derived plasmids.Transform

ants of

Antibiotic resistance

(µg/ml)

The

frequency

14

The

derive

d

Plasmi

d

E.col MM294 of

transforma

tion

Ap

100

Tc

20

Sm

10

0

Cm

50

Km

50

Na

l

20

Tp

50

pKP1 KP1O + - - - - - - 4.6 x10-8

pKP2 KP2O + - - - - - + 4.5x10-8

pKP4 KP4O + - - + - - + 4x10-8

pKP6 KP6O + - - + + + - 3.6x10-8

pKP8 KP8O + - - + - + + 3.8x10-8

pKP9 KP9O + + + + + - - 2.8x10-8

pKP10 KP10O + + - + + + - 2.6x10-8

pKP KP + + + + + + + 2.4x10-8

- Recipient - - - - - - - -------pBR322 Control + + - - - - - 2.9x10-3

*(+) Resistant; (-) sensitive to antibiotic. Ap: Ampicillin;

Tc: tetracycline; Sm: streptomycin; Cm: chloramphenicol; Km:

kanamycin; Nal: Nalidixic acid; Tp: trimethoprim.

15

FIG. 2. Detection transposition of ampicillin resistance.

The cleared lysate prepared from each strain was electrophoresed

in a 0.8% agarose gel. Lane 1, lysate from E.coli PK2 ; lane 2,

lysate from E.coli R1 ( pACYC184 & pKP2); lane 3, lysate from E.coli R2;

lane 4, lysate from E.coli R3( pACYC184:: Tn-Ap). oc, open circular form;

ccc, covalently closed circular.

Discussion:

In this study, Klebsiella pneumonia No.10 isolate

exhibited a genetic instability became sensitive to

streptomycin (35%) and both streptomycin and

tetracycline ( 12%) when subjected to successive

subculturing for about 480 generation at 37ºC. The DNA

content of the resulting cells revealed the presence of

16

1 2 3 4

pACYC184::Tn

pKP2

Chromosome

pACYC184(oc)

pACYC184 (ccc)

several plasmid bands in comparison with original

isolate that contain single plasmid band as indicated by

gel electrophoresis. The derived plasmids appeared

variable loosing of antibiotic resistances marker while

all of them maintains ampicillin resistance may be due

to partial deletion or insertion inactivation. In

addition, these results agreeed with the previous

results when the same isolate treated with salicylic

acid yielded several plasmid species. All maintained

ampicillin resistance, while the other antibiotic

resistance markers deleted or abolished (Al-Jalawi,

2000). Over all may be speculated the presence of

transposable element confers ampicillin resistance.

Therefore, we try to detect the presence of Ap-

transposable element within the genome of Klebsiella

pneumonia No.10 by transposition assay by induced the Ap-

gene to jump from the derived plasmid pKP2 to the non-

conjunctive vector pACYC184. The resulting cells (E.coli

RIII) resist to ampicillin, tetracycline and

chloramphenicol. The plasmid content of these cells

revealed the presence of extra size (5kb) to the pre-

transformed DNA band as indicated by gel

electrophoresis. The added DNA could represent a

transposable DNA fragment encoding ampicillin

resistance.

17

Simon ( 1984) showed that transposition of Tn5

from chromosome onto the vector plasmid led to higher

level of Km/Nm resistance expression from multicopy

plasmid than a single copy inserted in the chromosome .

The presence of transposable elements (Tn A family)

including Tn3, Tn1331 and Tn1000 could be screened in a

bacterial cells by using growth media containing high

concentrations of ampicillin (Heffron et al., 1979;

Heffron et al., 1981). High concentration of ampicillin

may be made a stress on the host cell that leads to

induce the SOS response in bacterial cell which

consequently stimulate the excision of Tn and jumping

(Rusina et al.,1992; Miller et al., 2004). Tn3

transposition is always via a cointgrate intermediate

formation. Resolution occurs under the action of

resolvase at the IRS (the cross over point between two

copies of transposon). Finally, two replicons separated

each of them carrying a complete copy of the element

(Lewin, 1997). Transposition is a rare events, depending

on the physicochemical conditions within the cell and

element involved, in general occurring at around 10-4 –

10-7 times per element per generation (Kleckner, 1981).

K.pneumoniae No.10 became sensitive to tetracycline and

streptomycin indicated that the transposon might be

inserted into tet gene and str which encoding tetracycline

18

resistance and streptomycin in the bacteria

respectively. These results agree with previous studies

that found the transposable elements can randomly insert

into DNA (chromosome or plasmid) and can also promote

other type of DNA rearrangements such as inversion,

deletion, and replicon fusion, if the element inserted

into a functional gene, this lead to disrupt the

expression of the gene (Muzuuchi, 1992; Reznikoff,

1993;Lewin, 1997). Also Heffron et al. (1977) showed that

TnA (encoding ampicillin resistance) insertion appears

resembles the insertion sequences (IS) of E.coli in both

its mutagenic effects and in its ability to transpose as

a discrete unit. The size of pACYC184 plasmid vector was

increased about 5kb to form pACYC184:: Ap-Tn plasmid as

indicated by gel electrophoresis. This result agree

with both Meyers et al. (1976) and Hansen and Olsen (1978)

when they characterized Tn5 that conferring resistance

to certain aminoglycosid ( neomycin and kanamycin) on

IncP plasmid R751 and they found the size of plasmid

R751 was increased by 5.4kbp on agarose gel

electrophoresis.

The present research is the first step to future

studies will care for the transposable elements

isolation and characterization by using new approaches,

such as the exogenous plasmid isolation techniques

19

(Go¨tz et al., 1996) or the PCR-based detection of

mobile genetic elements in total community DNA (Hill et

al., 1992). Furthermore, PCR and DNA hybridization were

shown to be extremely valuable tools for

characterization and classification of transposable

elements and plasmid (Chang et al., 1992; Go¨tz et al.,

1996 ).

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