Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
435
Detection of OXA-23 among Carbapenem
Resistant Clinical Isolates of
Klebsiella pneumoniae in Hilla.
Fatima Moeen Abbas Eman Mohammad Jarallah
College of science for women College of science
Babylon university Babylon university
Fatima.abas99@yahoo.com Emanm.Jarallah@yahoo.com
Abstract
This study aimed to assess the presence of blaOXA-23 in clinical isolates of Klebsiella pneumoniae.
During the period from April to August 2011, a total of 801 various clinical samples were collected
from different hospitals in Hilla city. One hundred and seventy seven (22%) isolates were diagnosed as
Klebsiella spp.,117 isolates belonged to K.pneumoniae . High prevalence of K.pneumoniae was
detected in Babylon Teaching Hospital for Maternity and Pediatric with 65 isolates. Regarding clinical
samples, highest rate were observed for stool samples 38 (27%) followed by sputum 19 (15%). All 117
K.pneumoniae isolates were primarily screened for
? - lactams resistance, 91 (78%) showed positive
results for beta lactams . ?- lactam resistance isolates were underwent antimicrobial susceptibility to 26
antibiotics by Kirby-Bauer disk diffusion methods. High resistance rate was recorded for penicillins
(carbenicillin and ampicillin) 99% and 94.5%,respectively .Carbapenem resistance was reported in 17
(18.7%) of K. pneumoniae isolates. The presence of blaOXA-23 gene was checked by Polymerase Chain
reaction (PCR) and confirmed in 15 (88.2%) of isolates.
Keywords: Klebsiella pneumoniae, carbapenem resistance ,OXA-23 ?-lactamase, Polymerase Chain
Reaction.
الخالصة
هدت ا درت دس دح درة رلدح درد در ةدس د وجدوت مدوسوا درتا تا د و دو 23-OXA تدا دبتا ري اسلد درا ار دلة درس ولدح دو
درةصدوع د 177 بردح ري اسلدح دتا درد د دود مم دح مد جد 117Klebsiella بردح ر دح ر دو pneumoniae. K مد
مجمو 801 ا دح دسلسلو ومد مم دت م تد ل ا متا دح درة دح ور دسا مد تتدلدح ل د برد لدح 2011 أظ دسا در د إ ب أا دس
دبتا pneumoniae. K دو بر د مد م تد ر تدا در لمدل ر د لح ودت د ع تودقد 65 بردح دمد ة د مصدتس در ا دح دة دا
ا ا درتسدب38%( 27 )د رح ا ا درقت 19%( 15 )وقت دظ دس درم دا دلوردل رة دلح در دبتا رم د تدا درتا تا د و
د 91 برح )78 )%ي ا مق ومح رم ت دتمر ا ا ودتموي ل ا
دم تدسا ة دلح در دبتا درمق ومدح رم د تدا درتا تا د و ر م د تدا درةاولدح ر سلقدح د تد س درقدس )ياسبدل- رد وس( وأظ دسا
در دددد إ ب أ دددد ددددرح ر مق ومددددح ي ددددا رم دددد تدا درت دددد ا 99 %رم دددد ت درا سب دددد ا و9445 %رم دددد ت دتمر دددد ا لمدددد أتددددتا
17(1847 )%بردددح مددد ري اسلددد pneumoniae. K مق ومدددح رم ددد تدا درا سبددد ت لو وم ددد ا هدددن در دددبتا ر اتدددت ددد مدددوس ا
23-blaOXA ت ق لدح د ح د ةا درت مدسا )Reaction Chain Polymerase )ويتد ا در د إ ب 15( 8242 )% مد در دبتا
ي ا ة م ح ر موسا 23-blaOXA
الكممات المفتاحية: ري اسل درا ار لة درس ولح رم تدا درا سب ت لو موسوا درتا تا و و 23-OXA ح ةا درت مسا
Introduction
Klebsiella pneumoniae is an opportunistic and major hospital –acquired pathogen
that has the potential to cause sever morbidity and mortality, particularly in intensive
care units and amongst pediatrics patients but also in medical and surgical wards
(Branger et al.,1998; Decre et al.,1998; Podschun and Ullmann,1998). Klebsiella is
the cause of 5-7.5 % of all nosocomial infections and the third most–common
bacterial cause of hospital -acquired pneumonia (Jones,2010; Khorshidi et al.,2011).
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
436
Carbapenems are a group of
? -lactam antibiotics which considered as the last
line of therapy for multidrug-resistant isolates that are prevalent in many Gramnegative bacterial species ,especially those producing ESBLs or/ derepressed AmpC
? -lactamase (Oueenan and Bush,2007; Peirano et al.,2009). However , more recently
carbapenem –resistant K.pneumoniae have emerged in United States and various parts
of the world (;Woodford et al.,2004; Peirano et al.,2009 ;Aktas et al .,2012;Sacha et
al.,2012). This species is resistant to almost all available antimicrobial agents, and
infections with this organism have been associated with high rates of morbidity and
mortality, particularly among persons with prolonged hospitalization and those who
are critically ill and exposed to invasive devices (e.g., ventilators or central venous
catheters) (Wachino et al.,2004; Schwaber and Carmeli.,2008). The main mechanism
of resistance to carbapenems in K.pneumoniae is through the production of a
carbapenemase , those that hudrolyse imipenem and /or meropenem are classified in
either Ambler classes A, B or D (genetic differences) or in Bush –Jacoby- Medieros
groups 2f,3a or 3b (substrate preference and molecular structure). Although resistance
is not limited to this mechanism solely , another method of resistance includes ESBLs
and or / AmpC production coupled with outer membrane porin (OMP) alterations
(Thomson, 2001) .
Class D enzymes are OXA (for oxacillin hydrolyzing ) enzymes, which are
penicillinases capable of hydrolyzing oxacillin and cloxacillin. These serine betalactamases are plasmid encoded and are found primarily in Pseudomonas aeruginosa,
Acinetobacter baumannii ,and rarely in isolates of Enterobacteriaceae from the
United States. The major concern with OXA carbapenemases is their ability to rapidly
mutate and expand their spectrum of activity (Marsik and Nambiar.,2011).
The first OXA enzyme with carbapenemase activity was found in an isolate of A.
baumannii in 1985 from a patient in Scotland. This enzyme was called ARI-1(for
Acinetobacter resistant to imipenem ) which was later renamed OXA-23 (Paton et
al.,1993) .Among the carbapenem hydrolyzing OXA enzymes, there is 40-70% amino
acid homology within groups and within the group the homology is 92.5% or higher.
These enzymes have measurable activity against penicillins, early cephalosporins and
imipenem (Walther-Rasmussen and Hoiby.,2006 ; Oueenan and Bush,2007).
The present study was conducted to evaluate the prevalence of K.pneumoniae
isolated from various clinical specimen in Hilla city, determine carbapenem resistant
isolates and to detect blaOXA-23 gene by Polymerase Chain Reaction (PCR) method.
Materials and Methods
Bacterial isolates
In the present study, a total of 801 clinical samples were collected during the
period of five months from April to the end of August 2011 ,from patients
hospitalized / or attended to different hospitals in Hilla city / Babylon Province,
included: Babylon Teaching Hospital for Maternity and Pediatric, AL- Hilla Teaching
Hospital, Merjan Teaching Hospital and Chest Diseases Center. All samples were
cultured on MacConkey s agar (Himedia) and incubated at 37 C? for 24 hrs. Bacterial
isolates of K. pneumoniae were identified to the level of species by using the standard
biochemical tests according to methods described by Collee et al., (2006) and
MacFaddin (2000), confirmatory identification was carried out by VITEK 2 system
following manufacturers instructions.
Screening Test for
? -lactam Resistance
Preliminary screening of K.pnenmoniae isolates being resistant to
? -lactam
antibiotics was carried out using pick and patch method (NCCLS,2003) .Results were
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
437
compared with E.coli ATCC 25922(College of Medicine ,University of Kufa) as a
negative control.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing of
? -lactam resistant K.pneumoniae isolates
was performed on Mueller-Hinton agar (Oxoid) plates by using Kirby-Bauer disk
diffusion method (Bauer et al.,1966). The isolates were tested against the following
antibiotics: Ampicillin (10µg), Carbenicillin (100 µg), Piperacillin (100 µg),
Amoxicillin-clavulanic acid (30 µg), Cefotaxime (30 µg), Ceftazidime (30 µg),
Ceftriaxone (30 µg), Cefepime (30 µg), Cefoxitin (30 µg), Aztreonam (30 µg),
Cefaclor (10µg), Cefprozil (30µg), Imipenem (10 µg), Meropenem (10 µg),
Ertapenam (10µg), Gentamicin (10 µg), Amikacin (30µg); Kanamycin (30µg) ,
Nalidixic acid (30 µg) , Ciprofloxacin (5µg) ; Levofloxacin (5µg) ; TrimethoprimSulfamethoxazole (25µg) Nitrofurantion (30µg), Chloramphenicol (30µg)
,Tetracycline (30µg) and Doxycycline (30µg). The cultures were incubated at 37 Co
for 18 hrs under aerobic conditions and bacterial growth inhibition zones diameter
were measured and interpreted in accordance with the Clinical and Laboratory
Standards Institute (CLSI) guidelines (CLSI,2010).E. coli ATCC 25922 was used as
the standard strain for antibiotic susceptibility testing.
Molecular detection of blaOXA-23 gene
DNA preparation
DNA preparation from bacterial cells was performed by salting out method as
described by Pospiech and Neuman (1995) with some modification and used as a
template for PCR reaction .
PCR amplification of blaOXA-23 gene.
Polymerase chain reaction was used to amplify the entire sequence of blaOXA-23
gene. The primer (Bioneer) used for the amplification of this gene was : OXA-23/F
(5-
-TCTGGTTGTACGGTTTCAGC-3
-
) and OXA-23/R (5—AGT CTTT CCA AAA
ATTTTG -3
-
).Amplification reaction mixture was carried out in a 25 µl reaction
volume using 12.5 µl Go Taq Green Master Mix 2X (Promega), 5 µl DNA template,
2.5 µl of 10 pmol/ µl of specific up stream primers and, 2.5 µl of 10 pmol/ µl of
specific down stream primers, 2.5 µl nuclease-free water. Cycling parameters of
blaOXA-23 were as follows: an initial denaturation at 95 C? for 30sec, followed by 30
cycles of denaturation at 95 C? for 30 sec, anneling at 51 C? for1 min, extension at 72
C? for1 min. and a final extension step of 72 at 10 min.(Hujer et al.,2006;Srinvasan et
al.,2009).The resulting PCR product was run in 1.5 % agarose gels and electric
current was allowed at 70 volts for 2 hr. DNA bands were observed using UVTransilluminator and photographed with Gel documentation system. 100 bp DNA
Ladder (Bioneer) was used to assess PCR product size.
Results and Discussion
Results of the present study revealed the presence of 177 (22%) isolates
belonged to Klebsiella spp. (Table -1) .In a previous study in Hilla by Al- Charrakh
(2005), 29(13.8%) Klebsiella spp. isolates were obtained from 209 clinical samples.
Another study by Al-Muhannak (2010) reported that Klebsiella spp. was the second
frequently 62 (30.5%) isolated organisms in Najaf hospitals. Podchun and Ullmann
(1998) ,documented that the principle pathogenic reservoirs for transmission of
Klebsiella are the gastrointestinal tract and the hands of hospital personnel which
increase the likelihood of person – to -person transmission and contaminated
equipments are also important factors promoting the spread of Klebsiella spp . Also
table (1) showed that 117 (14.6%) isolates were identified as K. pneumoniae. This
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
438
result is similar to findings of local study in Hilla by Al-Saedi (2000) who stated that
K. pneumoniae isolates comprised (15.3%) from 725 clinical samples .
However, prevalence and distribution of K.pneumoniae varied among Hilla
hospitals , Table (1) shows distribution of Klebsiella pneumoniae in Hilla hospitals:
Babylon Teaching (23%) , Chest diseases centers (14.8%), Al-Hilla Teaching (11.1%)
and Merjan Teaching (3%).This variation among hospitals related to specification of
each hospital for receiving patients suffering from specific diseases related to those
hospitals .High prevalence rate (23%) was detected in Babylon Teaching Hospital,
this range may be related to that most patients received were infants and premature
babies suffering from diarrhea, vomiting ,meningitis and premature neonates in ICU
for long period ,followed by Chest Diseases Center (14.8%), which specified for
examining outpatients sputum with respiratory problem most of them for diagnosis of
tuberculosis. High prevalence rate may be related to that most patients were
debilitated by other diseases like diabetes mellitus and bronchoplumonary diseases .It
was mentioned that the oragansim is carried on respiratory tracts of about 10% of
normal people ,who are borne to pneumonia if host defense are impaired (Levinson
and Jawetz,2000).
Results of the present study revealed that 569/801(71%) of other bacterial spp.
isolates were recovered from clinical samples, (Table -1).There is no doubt that
hospitals are typical environments for the presence of pathogens such as
S.aureus E. coli, Klebsiella, Proteus, Morganella, Enterobacter, Citrobacter,
Serratia, Acinetobacter and Pseudomonas spp. (Kucukates and Kocazeybek, 2002;
Azimi et al., 2011 ). However, the dissemination of bacterial isolates in clinical
samples may be due to their ability to cause different nosocomial infections and
resistance to a wide range of antibiotics.
Table (1): Distribution of bacterial isolates recovered from clinical samples
among different hospitals in Hilla city.
Hospital s name No. of
sample
No. (%) of
Klebsiella
spp.
isolates
No.(%) of
K.
pneumoniae
isolates
No. (%) of
other
bacterial
spp.
isolates
No. (%)
of no
growth
cultures
Babylon Teaching
Hospital for
Maternity and
Pediatric
282 103 (36.5%) 65(23%) 179 (63.5%) 0
(0%)
Al- Hilla Teaching
Hospital
261 51
(19 %)
29 (11.1%) 195
(75%)
15
(6%)
Merjan Teaching
Hospital
130 4
(3%)
4(3%) 86
(66%)
40 (31%)
Chest Diseases
Center
128 19 (14.8%) 19(14.8%) 109 (85.2%) 0
(0%)
Total 801 177(22%) 117(14.6%) 569(71%) 55(7%)
The results of Table (2) showed that the majority of K. pneumoniae isolates
38/141(27%) were obtained from stool samples. High prevalence of K. pneumoniae in
stool samples was demonstrated by other researchers , Al-Saedi (2000) in Hilla ,
(14%) , Ali et al.(2010) in Jordon, (20%) .In sputum , K.pneumoniae was detected in
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
439
19/128 (15%) of samples .Increasing prevalence of K.pneumoniae in sputum was
observed by other researchers ,Al- Muhannak (2010) ( (15.7%) and Al-Sehlawi
(2012) ,(16%) .
In this study, VITEK 2 system was used to confirm identification of Klebsiella to
species and subspecies levels and to avoid variability in findings of biochemical tests.
Results of the present study indicated that K.pneumoniae subsp. pneumoniae was the
most frequent subspecies 77/117(9.6%), followed by K.pneumoniae subsp.ozaenae
34/117(4%) and K.pneumoniae subsp. rhinoscleromatis 6/117 (1%) (Table -2).
Dominance of K.pneumoniae subsp. pneumoniae among all other subspecies was
supported with a report documented by Al-Sehlawi (2012) ,who stated that
K.pneumoniae subsp. pneumoniae was the most frequent occurring subspecies.,
accounting for 88.9% .
Results showed that 26/141 (18%) of K.pneumoniae subsp. pneumoniae ,8/141
(6%) of K.pneumoniae subsp.ozaenae and 4/141(3%) of K.pneumoniae subsp.
rhinoscleromatis were obtained from stool samples .Similar findings were recorded
by Al-Charrakh (2005) who found that most Klebsiella spp. were recovered from
stool samples and K.pneumoniae subsp pneumoniae was the most frequently 87%
occurring subspecies followed by K.pneumoniae subsp.ozaenea (9.5%) and
K.pneumoniae subsp. rhinoscleromatis (3.5%) .
In sputum samples, the prevalence rate was 8/128 (6%) for K.pneumoniae
subsp.pneumoniae , 9/128 (7%) for K.pneumoniae subsp. ozaenae and 2/128 (2%) for
K.pneumoniae subsp. rhinoscleromatis. One study pointed out that the detection rates
of K.pneumoniae subsp. pneumoniae, K.pneumoniae subsp. ozaenae and
K.pneumoniae subsp. rhinoscleromatis in patients with lower respiratory tract
infections in Najaf were (4.3%),(8.6%),and (8.6%), respectively (AlMuhannak,2010).
Table (2): Numbers and percentages of K. pneumoniae subspecies among
different clinical samples.
Clinical
sample
No. of
sample
No. (%) of K.
pneumoniae
isolates
No. (%) of K . pneumoniae
subspecies
K.
pneumoniae
subsp.
pneumoniae
K.
pneumouiae
subsp.
ozaenae
K.
pneumoniae
subsp.
rhinoscleromatis
Stool 141 38 (27%) 26(18%) 8(6%) 4(3%)
Sputum 128 19 (15%) 8(6%) 9(7%) 2(2%)
Vagina 116 18 (15.5%) 18(15.5%) 0(0%) 0(0%)
Burn 153 18 (11.7%) 12(7.8%) 6(3.9%) 0(0%)
Urine 97 10 (10%) 3(3%) 7(7%) 0(0%)
Wound 60 8 (13.3%) 5(8.3%) 3(5%) 0(0%)
Blood 58 3 (5%) 2(3%) 1(2%) 0(0%)
Ear 30 2 (6.6%) 2(6.6%) 0 (0%) 0(0%)
Eye 8 1 (12.5%) 1(12.5%) 0(0%) 0(0%)
Throat 10 0(0%) 0(0%) 0(0%) 0(0%)
Total 801 117(14.6%) 77(9.6%) 34(4%) 6(1%)
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440
As shown in table (3) , 91/117 (78%) of K. pneumoniae isolates were resistant to
ampicillin and amoxicillin. This result is in accordance with a study in Hilla by AlCharrakh (2005) who stated that 73.8% Klebsiella isolates obtained from clinical
samples were resistant to both ampicillin and amoxicillin . Higher resistant to these
antibiotics could be attributed not only to the production of
? - lactamases, but also
other resistance mechanisms like decrease the affinity of target PBPs or decrease
permeability of the drug into the cell (Jacoby and Munoz-Price,2005).There are three
further resistance mechanisms include conformational changes in PBPs, permeability
changes in the outer membrane, and active efflux of the antibiotic(Amyes,2003).
Other studies reported that qnr genes (integron-associated) are associated with
resistance to several classes of antibiotics including ?-lactam ( Paterson,2006)
Table (3):
? - lactam resistant Klebsiella pneumoniae isolates recovered from
different clinical samples.
No. of K. pneumoniae
isolates
Susceptibility to ampicillin and amoxicillin
No. (%) of resistant
isolates
No. (%) of
sensitive isolates
117 91 (78%) 26 (22%)

All 117 K. pneumoniae isolates were screened for their antibiotic resistance
against selected antibiotic agents of different classes (Fig.1).
In the present study a high resistance was observed for penicillins (carbenicillin
and ampicillin) with rates of resistance 90(99%) and 86(94.5%),respectively, whereas
75(82.4%) of isolates were resistance to piperacillin. This result is in agreement with
a pervious study in Hilla by Al- Asady (2009) who found that all 15 (100%) ?-lactam
resistant Enterobacteriaceae isolates were resistant to ampicillin , piperacillin and
carbencillin . High resistance to this class of antibiotics may be due to widespread use
of these antibiotics in Hilla hospitals.
The lower resistance rate was observed to carbapenem antibiotics when
imipenem displayed (10 %) resistance rate. In spite of the low level of resistance, this
result is higher than that reported by other local studies contacted in Iraq which
reported that the susceptibility of K.pneumoniae isolates collected from clinical and
environmental samples to imipenem was (100%) (Hadi,2008; Al-Asady,2009;AlMuhannak,2010). Pathak et al(2012) demonstrated 2% resistance to imipenem by
K.pneumoniae in a surveillance study in two hospitals in India .Reasons behind
resistance may be due to inappropriate duration of antibiotic therapy and
subtherapeutic concentrations of the drug (Paquero et al.,1997;Philippe et al.,1999).
Meropenem antibiotics showed (17.6%) resistant rate. Meropenem is well –
tolerated and offers several potential advantages , including greater in vitro activity
against Gram –negative pathogens and the option of bolus administration (Verwaest
et al.,2000) Beside these , problem of renal metabolism of imipenem , and risk of
seizures (Prakash,2006)and availability of meropenem only in Hilla hospitals might
be the reasons behind possible greater use of meropenem over imipenem and hence
the high prevalence of resistance.
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
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Figure (1): Antibiotics susceptibility profile of Klebsiella pneumoniae isolates by disk diffusion method (n=91).
AMP,Ampicillin;PRL,Piperacillin;PY,Carbenicillin;AMC,Amoxi-clav;CTX,Cefotaxime;CAZ,Ceftazidime;CRO,Ceftraiaxone;FEP
Cefepime; Fox,Cefoxitin; ATM,Aztreonam; CF,Cefaclor; CPR,Cefprozil; IMP,Imipenem; MEM,Meropenem; ETP,Ertapenem; CN,
Gantamicin ; AK, Amikacin ; K, Kanamycin ; NA , Nalidixic acid ; CIP, Ciprofloxacin ; LE5
,Levofloxacin ; SXT, Trimethoprim-
Sulfamethoxazole;C,Chloramphenicol;F,Nitrofurantion;TE, Tetracycline;DO,Doxycycline.

Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
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All carbapenem resistant isolates were screened by PCR for the presence of
blaOXA-23. Results confirmed that 15 (88.2%) isolates carrying blaOXA-23 (Fig.2). This
represents the first report of OXA-23 among clinical isolates of K.pneumoniae in
Hilla. Recently , blaOXA-48 mediated resistance to carbepenem has been reported in
K.pneumoniae in Turkey [9].The present study found that the prevalence rate of
blaOXA-23 in carbapenem –resistant K.pneumoniae isolates from Hilla hospitals is
undoubtedly high.
Figure (2): Agarose gel electrophoresis (1.5%agarose,70 % volt for 2-3 hrs) for
blaOXA-23 gene product (ampilified size 606 bp) using DNA template of
carbapenem-resistant K. pneumoniae isolates extracted by using salting out
method. Lane (L), DNA moleculer size marker (100- bp Ladder).Lanes (K1,2,3,
4, 5, 7, 8, 10,11,12,13,14,15,16 and 17) of K. pneumoniae isolates show positive
results with blaOXA-23 gene , lanes (K 6 and 9) show negative results with blaOXA-23
gene.
L K1 K2 K3 K4 K5 K6 K7 K8 L
606 bp
L K10 K11 K12 K13 K14 K15 K16 K17
2000
1600 1200
1000 900
800 700
600 500
400
300
200
100

K9
606 bp
2000
1600
1200
1000 900
800
700
600
500
400
300
200
100
bp
bp
2000
1600
1200
1000
900
800
700
600
500
400
300
200
100
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(25): 2017
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