Prevalence of AmpC ß-lactamase producing carbapenem resistant clinical isolates of Klebsiella pneumoniae among different hospitals in Hilla City.

This study was performed to detect the presence of bla AmpC among carbapenem resistant Klebsiella pneumoniae isolates.A total of 117 clinical isolates of K. pneumoniae were collected from 801 clinical sample from different hospitals in Hilla City,during the period from April to August 2011.High prevalence of K. pneumoniae isolates were detected in 38 (32.5%)of stool samples, followed by sputum 19(16,2%),All isolates were tested for antimicrobial susceptibility by Kirby-Bauer disk diffusion methods .High resistance rate was recorded for Carbencillin (98%),followed by Amox-clavulanic acid (95%).Carbapenem resistance was reported in 17 of K. pneumoniae isolates.These were screened for cefoxitin resistance.Results revealed that all these isolates were found to be cefoxitin resistant, among 17 cefoxitin resistant K. pneumoniae isolates,AmpC ß-lactamases production were phenotypically detected in 2(11.8%),3(17.6%) by AmpC disk and MTDT respectively.None of the isolates were positive for inducible AmpC ß-lactamase. AmpC ß-lactamases were dectectable in 13(76.4%) of isolates by PCR method.


Introduction
The predominant mechanism for resistance to ß -lactam antibiotics in Gram-negative bacteria is by the synthesis of ß-lactamases (Suranjana and Manjusiri., 2005). Among the ßlactamases the production of extended spectrum beta Lactamases (ESBLs) and AmpC ßlactamases are the most common (Coudron et al., 2000).
AmpC ß -lactamases are of two types, chromosomal inducible and plasmid mediated noninducible, chromosome-mediated Ampc ß -Lactamases have been described in a wide variety of Gram-negative bacilli (Mohamudha et al., 2012). Overproduction of their chromosomal Ampc ß -lactamses by mutation is probably responsible for the resistance in these organisms (Yan et al., 2002). In most genera of the family Entrobacteriaceae, Ampc is inducible (Bush et al., 1995).
Plasmid mediated AmpC ß -lactamases (PMABLs) have evolved by the movement of chromosomal genes on to plasmids and are found in Escherichia coil, Klebsiella pneumoniae, Salmonella spp., Proteus mirabilis, Citrobacter freundii and Enterobacter aerogenes which confer resistance similar to their chromosomal counterparts (Mohamudha et al., 2012). Unlike chromosome-mediated AmpC, most plasmid-mediated AmpC genes, such as MIR-1, are expressed constitutively even in the presence of a complete system for induction (Phillipon et al., 2002).
Organisms producing PMABLs are often associated with multidrug resistance, leaving a few therapeutic options and represent a new threat since they confer resistance to cephamycins and are not affected by ß -Lactamase inhibitors, this resistance mechanism has been found 94 around the world, can cause nosocomial outbreak, appears to be increasing in prevalence. (Subha et al., 2003;Mohamudha et al., 2012).
Thus the present study was conducted to evaluate the prevalence of AmpC ß -lactamase producing K. pneumoniae in Hilla hospitals,the objectives of the study are: 1-Determine the antibiotic susceptibility pattern of clinical isolates of K. pneumoniae among different hospitals in Hilla City.
2-Detect the prevalence of AmpC ß -Lactamase gene by phenotypic and genotypic (PCR) methods.

Bacterial isolates
A total of 801clinical samples were collected during the period of five months from April 2011 to August 2011 from different Hospitals in Hilla City included: Merjan Medical City, AL-Hilla Teaching Hospital, Babylon Teaching Hospital for Pediatric and Maternity and Chest Diseases Center. All samples were cultured on MacConkey agar (Himedia) and incubated at 37 C˚ for 24 hr.Bacterial isolates of K. pneumoniae were identified using the standarad biochemical tests according to Holt et al (1994), Baron and Finegold (1994) and MacFaddin (2000).
The cultures were incubated at 37 C˚ for 18 hr under aerobic condition and the diameter of the zones of inhibition of bacterial growth were measured and interpreted as recommended by the National Committee for Clinical Laboratory Standard guidelines. E. coli ATCC 25922(College of medicine,University of Kufa) was used as the reference strain for antimicrobial susceptibility testing (CISI, 2010).

b. Confirmatory Tests of AmpC ß -Lactamase Modified Three-Dimensional Test (MTDT).
Fresh overnight growth from Mueller-Hinton agar plate was transferred to a pre-weighed sterile eppendrof tube. The tube was weighed again to ascertain the weight of the bacterial mass. The technique was standardized so as to obtain 15 mg of bacterial wet weight for each sample. The growth was suspended in peptone water and pelleted by centrifugation at 3000 rpm for 15 minutes. Bacterial growth washed with normal saline 2 to 3 times, crude enzyme extract was prepared by repeated freeze-thawing (approximately 10 cycles). Lawn cultures of E. coil ATCC 25922 were prepared on Mueller-Hinton agar plates and cefoxitin (30 µg) disks 95 were placed on the plate. Linear slits (3 cm) were cut using a sterile surgical blade 3 mm a way from the cefoxitin disk. 30 µl of extract was added in the wells made at outer edge of the slit. The plates were kept upright for 5-10 minutes until the solution dried, and then incubated at 37 C˚ overnight.Clear distortion of the zone of inhibition of cefoxitin were taken as AmpC producers (Coudron et al., 2003).

Ampc Disk Test
A lawn culture of E. coil ATCC 25922 was prepared on Mueller-Hinton agar plate. Sterile disks (6 mm) were moistened with sterile saline (20 µl) and inoculated with several colonies of each test organism. The inoculated disk was then placed beside a cefoxitin disk (almost touching) on the inoculated plate. The plates were incubated overnight at 37 C˚. A positive test appeared as flattening or indentation of the cefoxitin inhibition zone in the vicinity of the test disk. A negative test had an undistorted zone (Parveen et al., 2010).

Ceftazidime-Imipenem Antagonism Test (CIAT)
This test consist of an imipenem disk (10 µg) placed 20 mm a part ( edge to edge) from a ceftazidime disk (30 µg) on a Mueller-Hinton agar plate previously inoculated with a 0.5 McFarland bacterial suspension, and incubated for 24 hr at 35 C˚. For comparision a cefoxitin disk was also placed 20 mm a part from the ceftazidime disk. Antagonism indicated by a visible reduction in the inhibition zone around the ceflazidime disk adjacent to the imipenem or cefoxitin disks, was regarded as positive for the inducible AmpC ß -lactamase production. (Cantarelli et al., 2007).

Genotypic detection of bla AmpC 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 bla AmpC gene.
-Polymerase chain reaction was used to amplify the entire sequence of bla AmpC gene. The primer (Bioneer) used for the amplification of this gene was : bla AmpC /F (5 --ATCAAAACATGGCACCCA -3 -) and AmpC /R (5 --GAGCCCTTTTATGCACCCA-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. PCR conditions as follows : an initial denaturation at 94 C˚ for 30 sec, followed by 35 cycles of dematuration at 94 C˚ for 30 sec, anneling at 60 C˚ for 1 min, extension at 72 C˚ for 1 min, and a find extension step of 72 at 10 min. 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 UV-Transilluminator and photographed with Gel documentation system. 100 bp DNA Ladder (Bioneer) was used to assess PCR product size.

Antibiotic susceptibility test
All 117 K. pneumoniae isolates were screened for their antibiotic resistance against selected antibiotic agents of different classes (Fig.1).

-Cefoxitin Susceptibility
Cefoxitin susceptibility of 17 carbapenem resistant K. pneumoniae isolates was tested by standard Kirby-Bauer disk diffusion method. Results revealed that all carbapenem resistant K. pneumoniae isolates yielded cefoxitin zone diameter less than 18 mm, these isolates may be AmpC ß -lactamase producers.

Inducible AmpC ß -Lactamase Production -
Results of the present study revealed no blunting of the ceftazidime zone adjacent to imipenem disk among the 17 cefoxitin resistant isolates. (Table-2).

Discussion
Results of the present study revealed the presence of 117 (14.6%) of K. pneumoniae isolates obtained from 801 clinical samples. This result is in agreement with a previous study in Hilla by Al-Saedi (2000) who found that K. pneumoniae isolates comprised (15.3%) from 711 clinical samples. In another study, Al-Sehlawi (2012) reported that the detection rate of K. pneumoniae was (14%) of all pathogen isolated from clinical samples in Najaf hospitals.
K. pneumoniae predominantly isolated from stool samples, since it's a common member of the human intestinal flora. High prevalence of K. pneumoniae in stool samples was demonstrated by other researchers, Al-Gharakh (2005) in Hilla, Ali et al (2010) in Jorden. In sputum, prevalence rate of K. pneumoniae was ( 16.2 %), this in accordance with the result of AL-Sehlawi (2012) who found that K. pneumoniae comprised (16%) in sputa of 450 patients in Najaf hospitals.
Results of the present study revealed that K. pneumoniae isolates confer high level of resistance against antibiotics tested (Fig.1). This may be due to in appropriate and incorrect administration of antimicrobial agents and lack of appropriate infection control strategies (Zakaria, 2005).
The results revealed that higher resistance rate was found for carbenicillin (98 %), piperacillin (82%), this result is in agreement with a pervious study in Hilla by AL-Hilli (2010) who stated that all K. pneumoniae isolates were resistance to carbenicillin (100%) and (81%) to piperacillin,high resistance to carbenicillin and piperacillin may be due to widespread use of these antibiotics in Hilla hospitals.
Higher resistance was noted for Amox-clav (95 %). Similar finding also was recorded by Lim et al.,( 2009) who found that out of 51 K. pneumoniae isolates, 49 (96%) were resistance for Amox-clav in Malaysia.There is a higher resistance to cefoxitin (91.4%) in clinical isolates of K. pneumoniae. Resistance to cefoxitin may be due to high level expression of plasmid mediated AmpC ß-Lactamase and / or development of porinedeficient mutants (Tan et al.,2009).
However, levels of resistance were (86 %) for cefotazidime and (84 %) for cefotaxime .In a study conducted by Aminizadeh and Kashi (2011) in Iran (87%) of K. pneumoniae isolates were found to be resistant to ceftazidime. For cefotaxime our result was higher than that reported in Jorden by Al-Shara ( 2011) who showed that resistance rate for cefotaxime was (42.9%).
High level of resistance could be attributed to the presence of ESBLs, since it mediate resistance to broadspectrum cephalosporins (e.g., ceftazidime, ceftriaxone, cefotuxime) and aztreonam (Umadevi et al., 2011).
Resistance to meropenem was ( 14%), this result is higher than that reported by AL-Sehlawi (2012) in Najaf who found that only four isolates (3.9%) of K. pneumoniae were resistance to meropenem. Resistance to impienem was (8%), but this resulti s higher than that reported by Aminizadeh and Kashi (2011) in Iran who found that resistance rate of K. pneumoniae to imipenem was (2%). Other study showed that the susceptibility of 150 clinical isolates of K. pneumoniae to imipenem was (100%) in Nigeria (Iroha et al., 2011).
Result also revealed that all 17 (100 %) carbapenem resistant K. pneumoniae isolates yielded cefoxitin zone diameter less than 18 mm, these isolates may be AmpC ß -Lactamase producers. In a study from India, Mohamudha et al (2012) showed that out of 109 clinical isolates of K. pneumoniae collected from hospitalized patients 83 (76.1 %)of isolates were found to be resistant to cefoxitin. Other study characterized that 84 (72 %) of Klebsiella isolates were found to be resistant to cefoxitin in Chennai (Subha et al., 2003). Of the 17 cefoxitin resistant K. pneumoniae isolates, 2 (11.8 %) isolate revealed positive result by AmpC disk test (Table-2). Other study by Mohamudha et al (2012) reported that AmpC ß -Lactamase was confirmed in 137 (73.2 %) of cefoxitin resistant E. coli and K. pneumoniae isolates.In this study there is low prevalence in AmpC ß-lactamase in comparison with cefoxitin susceptibility result ,this may be due to a lack of permeation of porin or that some isolates may have AmpC genes ,but not expressed in all the isolates.They might have silent genes or there might be low level expression of AmpC genes that was not detected (Jacoby,2009).
Many Gram -negative bacteria harbor chromosomal AmpC beta -lactamase genes,which are constitutively expressed at low level, these bacteria can acquire plasmid-encoded AmpC genes resulting in a stably derepressed resistance phenotype (Thomas,2007;Polsfuss et al .,2011). In this study, screening for inducible AmpC ß -Lactamase was done by the disk antagonism test, results showed that none of the isolates were positive for inducible AmpC ß -100 lactamase (Table-2). This result in agreement with previous study in Hilla by Al-Hilli (2010) who found that among 7 (28 %) cefoxitin resistant isolates, no K. pneumoniae isolates was positive for inducible AmpC ß -Lactamase production.
Using PCR method, result demoustrated that 13 (76.4 %) isolates were amplified with bla AmpC primers (Fig. -2). These isolates showed cefoxitin resistant in cefoxitin susceptibility test. Cefoxitin resistant in AmpC non-producers could be due to some other resistance mechanism. Hernandez-Alles et al (2000) demonstrated that the interruption of a porin gene by insertion sequences is a common type of mutation that causes loss or decrease of outer membranes porin expression and increased cefoxitin resistance in E. coli and Klebsiella spp.
It is pertinent to note that in this study only 2 (11.8 %) of cefoxitin resistant isolates were detected by AmpC disk test and 3 (17.6 %) by. MTDT .Phenotypic tests alone may not reflect the true number of AmpC ß -Lactamase producers, hence molecular studies, although not possible routinely in clinical laboratories, need to be employed in surveillance studies.