Detection of Aminoglycoside Modifying Enzymes and β-lactamases in Acinetobacter baumannii Isolated from Hospitalized Iraqi Patients

ISRAA Mohamed Safi Al-kadmy¹,

MARWAH Hasan Al-Kaabi², HADEEL Kareem Musafer¹

Greener Journal of Biological Sciences, vol. 5, no. 1, pp. 010-019, March, 2015

¹Department of Biology, College of Science, University of Mustansiriya, Baghdad 14022, Iraq.

²National center of Hematology, University of Mustansiriya, Baghdad, Iraq.

INTRODUCTION

Acinetobacter baumannii is an emerging pathogen responsible for causing a wide range of nosocomial infections, including pneumonia, urinary tract infections, and septicemia particularly in immunocompromied patients (Karlowsky et al., 2003). Due to its outstanding ability to upregulate or attain resistance determinants and its environmental flexibility, it’s considered one of the most microbial threat to the antibiotic treatment strategy (Nordmann, 2004).

Recently multi-drug resistant A. baumannii strains have now been reported, signifying a sentinel event that should be acted on promptly by the international health care community (Hussein et al., 2013). Extensive use of antimicrobial chemotherapy within hospitals has contributed to the emergence and procreation of A. baumannii strains which are resistant to a wide range of antibiotics, including broad spectrum β-lactams, aminoglycosides, and fluoroquinolones (Asadollahi et al., 2012). Resistance to β-lactams appears to be primarily caused by β-lactamase production, including the extended spectrum β-lactamases (bla_TEM, bla_SHV, bla_VEB, bla_PER), metallo-β-lactamases (bla_IMP, bla_VIM, bla_SIM, bla_GIM), and most commonly, oxacillinases (bla_OXA51, 23, 24 and 58), while the resistance to aminoglycosides may be through many known mechanisms but the enzymatic modification is the most prevalent in the clinical setting (Asadollahi et al., 2012).

Aminoglycoside modifying enzymes (AMEs) catalyze the modification at different −OH or −NH₂ groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltranferases, phosphotransferases, or acetyltransferases (Ramirez and Tolmasky, 2010). Thus, there are three types of these enzymes: Aminoglycoside N-Acetyltransferases (AACs), Aminoglycoside O-Nucleotidyltransferases (ANTs), Aminoglycoside O-Phosphotransferases (APHs). The AAC family of enzymes is composed of four major subclasses based on region specificity of aminoglycoside acetyltransfer: AAC(6ʹ), AAC(2ʹ), AAC(1), and AAC(3), while there are five members of the ANT family of enzymes, ANT(2ʹʹ), ANT(4ʹ), ANT(3ʹ), ANT(6ʹ) and ANT(9) (Arya, 2007). The third mechanism of aminoglycoside modification is ATP-dependent phosphorylation of key hydroxyl groups, a reaction catalyzed by the APH family of kinases including APH(2ʹʹ), APH(3ʹ), APH(3ʹʹ), APH(7ʹʹ), APH(4), APH(6), and APH(9) (Arya, 2007). Aminoglycoside-modifying enzymes have become widespread throughout bacterial communities because many of the encoding genes are found on mobile genetic elements such as integrons, transposons, and plasmids, thus facilitating horizontal gene transfer (Arya, 2007). The emergence of aminoglycoside-inactivating enzymes has contributed to the diminished use of several aminoglycoside antibiotics and the presence of these types of enzymes have been found in A. baumannii, often occurring in combination (Arya, 2007, Seward  et al., 1998).

Some reports referred to the presence of these types of enzymes in Iraq, especially the studies that were done on the military and civilian casualties from the Iraqi conflict and some genes responsible for Aminoglycoside modifying enzymes were detected, e.g: aacC1, aadA1a, aadB, aacA4, and aadA1 (Turton  et al., 2005). So this study aimed for the detection of other types of AMEs genes besides some of β-lactamases resistance genes  in some of  A. baumannii that have been isolated from hospitalized patients in Iraq.

MATERIAL AND METHODS

Bacterial strains and identification

A total of 36 A. baumannii isolates were recovered form hospitalized patients during the period from May to September 2012. The isolates were collected from different sites including: sputum (one isolate), cerebrospinal fluid (one isolate), urine (17 isolates), wounds (17 isolates), and blood (6 isolates). All these isolates were identified according to morphological features and biochemical tests. What’s more, identification was confirmed using the api20E system.

Antibiotic susceptibility test

Antibiotic susceptibility for  A. baumannii isolates was performed against  aminoglycosides  antibiotics including: amikacin (30 μg), gentamicin (30 μg), netilmicin (30 μg), tobramycin (10 μg), and sisomycine )10 μg( (Bioanalyse, Turkey) using disc diffusion method  in accordance with the guidelines established by the Clinical and Laboratory Standard Institute (CLSI, 2013) . Escherichia coli (E. coli ATCC 25922) was used as a quality control in susceptibility determination.

Rapid detection of ESβL production via ESβL strip:

The detection of β-lactamases production was performed using Rapid ESβL Detection kit (MAST Group (UK). This kit includes four tests: Preliminary screening kit, Metallo β lactamases, ESβLs confirmation and AmpC detection. The test was performed according to the procedure suggested by the manufacturing company: first, primary screening test to detect β-lactams resistance was performed by culturing bacterial isolates on appropriate medium with cefotaxime 30 µg/disc. The resisted isolates were submitted to ESβL production test. One drop of test substrate (approximately 20 μl) was dispensed on the filter pad of the strip. The test substrate was added immediately to the strip before testing. Using a loop, several identical colonies were picked up and spread on the filter pad of the test strip. Any change in color (from yellow to red) observed around the streaked line was considered as a positive result. The tested strips were observed after 2 to 15 minutes at room temperature, and the result was read after 15 minutes.

DNA Preparation and Polymerase Chain Reaction (PCR) amplification:

Total genomic DNA was extracted by boiling according to the method described by Ruppé et al. (2009). Briefly, few isolated colonies of overnight growth bacteria were suspended thoroughly in 1 ml distilled water and boiled in a water bath for 10 min. After centrifugation, supernatant was used as template DNA for the PCR (Ruppé et al., 2009). PCR was performed using primers specific for β-lactamases enzymes families (bla_SHV, bla_TEM , bla_CTX) and aminoglycosides modifying enzymes ( aac(6)-Ib, ant(4')-IIb, aa(c) 3′ and aph(3′)-VI). The oligonucleotide PCR primers specific for the target genes, annealing temperature and sizes of the expected amplification product were listed in Table 1.

The PCR reaction mixture was accomplished according to the procedure suggested by the manufacture company (KAPA, south Africa) within a total volume of 25 μl containing 5 μl of DNA template, 12.5 μl of Go Taq® Green Master Mix, 2X (KAPA, south Africa), 0.5 μl (0.6 pmol) of each of the forward and the reverse of the specific primers. The final volume (25 µl) was completed with 6.5 µl of nuclease free water. The conditions of PCR amplification steps was carried out using the thermal cycler (C 1000 thermal cycler, BIO-RAD, USA) and the amplified PCR products were submitted to electrophoresis using  1% agarose gel with ethidium bromide (0.5 μg/ml) for 7 V/cm for 90 min. DNA ladder (100bp) was employed for assessing PCR product size. Subsequently, PCR products were visualized by UV light at 336 nm, and photographs were taken by an aid of digital camera.

Table (1): The oligonucleotide PCR primers specific for the target genes, annealing temperature and sizes of the expected amplification product

RESULTS

Morphological and biochemical characterization

All clinical isolates obtained from various clinical specimens identified as A. baumannii based on their morphological and biochemical characterization.

Antibiotic resistance pattern

The aminoglycosides susceptibility results are depicted in Figure 1. Accordingly, 15 (41.66%) A. baumannii isolates were found to be resistant to the tested antibiotics.

Regarding to the source of the infection, high resistance rate was observed among the wound isolates which were completely resistance (100%) to gentamicin and sisomycine. Furthermore, they showed high resistance rate (72.72%) to tobramycin and intermediate resistance to netilmicin (54.54%) and amikacin (45.45%). On contrary, all urine isolates were sensitive to all antibiotic under investigation. For the blood isolates, results revealed a high sensitivity  to  aminoglycosides;  amikacin   and netilmicin  (100%)  and  83.44%  for  gentamicin,  tobramycin,  and sisomycine. While the sputum and the Cerebrospinal fluid isolates were completely sensitive to all the antibiotics except for the amikacin in which the sputum isolates were sensitive to this antibiotic.

Figure 1: Antibiotic resistance of the A. baumannii isolates towards aminoglycoside antibiotic

Detection of ESβL Production

The investigation of β-lactamases production was carried out using ESβL strips. According to this test, 17 (47.22%) isolates gave positive results in the preliminary screening of β-lactamases, the ESβLs type and AmpC enzyme while 13 (36.11%) isolates showed a positive result in Metallo-β- lactamases production test. Figure 2 illustrates the results for one isolates, A. baumannii (no. 10) in which red color appeared with the first strip (A no.1) for preliminary screening after addition of specific inhibitors, while the second stripe (A no. 2) represents a positive result for ESβLs after the addition of Clavulanic Acid, the third positive result (A no.3) for MβL strip with red color appeared after the addition of  EDTA and Mercaptoacetic acid and finally the 4^th strip (A no.4) signifies AmpC strip which appeared red color after the addition of  Boronic Acid. No change occurred with the negative control represented by the standard strain E. coli ATCC 25922 in the Figure (2) which clearly appeared as a yellow color.

Figure 2: Detection of β-lactemase types using easy strips test. A-1:positive result for primary screening test of E.coli no. 10. A-2: positive result for ESβLs  of A. baumannii no.10. A-3: positive result for MβL of A. baumannii no.10.  A-4: positive result for AmpC of A. baumannii no.10. B 1-4: negative result for all above tests of the standard strain E. coli ATCC 25922

PCR analysis

The prevalence of β-lactamas and  aminoglycoside resistance genes in 15 resistant A. baumannii  isolates (as determined by the disk diffusion method) was as follows: bla_TEM was detected in 3 (20%) isolates, bla_SHV was detected in 9 (60%) isolates, bla_CTX-M was detected in 11 (73.33%) isolates, aac (6)-Ib in 7 (46.66%) isolates, ant(4')-IIb in 5 (33.33%) isolates, aph(3′)-VI in 2 (13.33%) isolates, and aa(c) 3′ was found in 10 (66.66%) isolates (Figure 3,4,5,6,7,8,9). Interestingly, individual aminoglycoside-resistant isolates carried multiple (two to six) β-lactamases and modifying enzyme genes. Table 2 demonstrates the distribution of these genes in the A. baumannii isolates.

Table (2): the distribution of these genes among the A. baumannii isolates

Aw: A. baumannii  isolated from wound , Ab: A. baumannii  isolated from blood , As: A. baumannii  isolated from  sputum Ac : A. baumannii  isolated from , AKN : amikacin, GEN : gentamicin,  TOB : tobramycin, NTil : netilmicin, SIS: sisomycine.

Figure 3: Gel electrophoresis (1% agarose, 7 V/cm for  90 min) for bla TEM  gene for some A. baumannii  isolates. Line M 100bp DNA ladder,  lines (1, 4) positive results with 800bp amplicon

Figure 4: Gel electrophoresis (1% agarose, 7 V/cm for  90 min) for bla SHV  gene for some A. baumannii  isolates. line M 100bp DNA ladder, lines (1,2,3,4,5,7,8,9) positive results  with 200bp amplicon

Figure 5: Gel electrophoresis (1% agarose, 7 V/cm for  90 min) for  blaCTX-M  gene for some A. baumannii  isolates. Line M 100bp DNA ladder, lines (1, 2,3,4 ,5,6,7,8) positive  results  with 550bp amplicon

Figure 6: Gel electrophoresis (1% agarose, 7 V/cm for  90 min) for aa(c) 3′   gene for some A. baumannii  isolates. Line M 100bp DNA ladder, lines (1, 2, 4 ,5,6,7) positive results  with 227 bp amplicon

Figure 7: Gel electrophoresis (1% agarose, 7 V/cm for 90 min) for ant(4')-IIb gene for some A. baumannii  isolates. Line M 100bp DNA ladder, lines (3,5,6,7) positive results  with 364 bp amplicon

Figure 8: Gel electrophoresis (1% agarose, 7 V/cm for  90 min) for aph(3′)-VI gene for some A. baumannii  isolates. Line M 100bp DNA ladder, lines (1, 3,4) positive results  with 800bp amplicon

DISCUSSION

The present study describes the emergence and spread of β-lactamases and aminoglycoside resistant A. baumannii isolates in Baghdad, Iraq. Antibiotic resistance and genetic features were investigated. Remarkably, most isolates of A. baumannii were obtained from urine and wound specimens. The result of susceptibility for all A. baumannii isolates to aminoglycosides group revealed that Amikacin had the best efficacy against these isolates, given that only 16.66% of total isolates developed resistance. Dent et al. (2010) and Xu et al. (2012) found that resistance rate to Amikacin reached 56%, 58% and 53.7%, respectively. The resistance rate of gentamicin  antibiotic was 38.88%, this results was in accordance with the result of Wei-feug et al. (2005) in which they found that 48.6% of the isolates were resistance. Hassan et al. (2010) reported that 28% of A. baumannii Pakistani isolates were resistant to tobramycin. Such finding goes with ours. Nonetheless, Moniri et al. (2010) stated that the resistance rate to tobramycin was 68.3%. Whereas, the resistance rate of netilmicin reached 22.22% which is lower than what Jaggi et al. (2012) indicated, as they recorded 90.3% of the isolates were resistance.

Rapid detection of ESβL production via ESβL strip

Since there are no standard guidelines for the detection of MBL, different studies have reported the use of various methods (Jaggi  et al., 2012). In the current study four types of β-lactamases were detected including: preliminary screening for β-lactemase, ESβLs type, Metallo-β- lactamases and AmpC enzyme. About half of the A. baumannii isolates (47.22%) were able to produce β-lactamases, this result was in a good agreement with the result of Trivedi et al., (2012) in which they found that 60% of the isolates were β-lactemase producer. In our study, all of the β-lactemase producing A. baumannii isolates were found to be ESβLs and AmpC producer (47.22%). However, a disagreement was noticed, as they found that only 10% of the isolates were ESβLs producer and 20% of the isolates were AmpC producer.

In our study, Metallo-β-lactamases was detected in 36.11% of the A.baumannii isolates. Many previously studies reported Metallo-β-lactamases production in A. baumannii (Gupta et al., 2006; Ahir et al., 2012; Daef et al., 2012). An Indian study on the A. baumanii species stated a significant increase (70.9%) in Metallo-β- lactamases production which is much higher than our findings (Uma et al., 2009), while another study reported from Kerala, India, states that 21% of the A. baumannii isolates were found to be metallo-β-lactamase producers which is similar to our finding (Kumar et al., 2011). Another study from India demonstrated that 66.8% of the isolates were harboring Metallo-β-lactamases enzymes (Chaudhary and Payasi, 2012).

Although there are many methods for the phenotypic detection of β-lactamases enzymes (Drieux et al., 2008), we found that ESβL strip is a new advanced method for detecting the types of β-lactemase. It is easy to perform highly specific besides; different types of enzymes could be detected in the same run.

PCR analysis:

In this study two types of resistance genes were investigated in the A. baumanii isolates using PCR technique which they are specific for β-lactam and aminoglycoside resistance. Among these resistance genes, the bla_CTX was the most frequent resistance genes and detected in 13 (73.33%) of the A. baumanii isolates, while the aph(3′)-VI was detected in only 2 (13.33%) isolates.

The other genes (TEM, SHV, aac (6)-Ib, ant(4')-IIb, and aa(c) 3′ ) were detected in 20%, 60%, 46.66%, 33.33% and 66.66% respectively. This finding was not similar to the finding of Huang et al. (2012) in which they found that aac (6)-Ib, aph(3′)-VI and aa(c) 3′ genes were detected in  20%, 1.25% and 47.50% respectively.

By observing the result in Table 2, we find that most of the resistance A. baumanii isolates were harboring more than one gene (40% were harboring 2 genes, 20% were harboring 4 genes, 13.33% were harboring 3 and 5 genes; while 6.66% were harboring 6 and one gene). In 80% of the A. baumanii resistance isolates, most of these genes belonging to both β-lactamases and aminoglycoside modifying genes and this indicates the prevalence of multidrug resistance patterns among the A. baumanii isolates.

Multidrug resistance patterns in A. baumanii have been described previously in many studies done in different hospitals worldwide (Koo  et al., 2010), this increase in MDR A. baumanii is a frightening reality (Livermore, 2003), and has given rise to significant therapeutic challenges in the treatment of nosocomial infections (Koo  et al., 2010). In a recent study which was done by Taitt et al. (2014) on clinical A. baumannii isolates collected from military treatment facilities (MTFs) they found that 80% of the isolates were found to be MDR and this is similar to our finding. Among these resistance isolates, 2 isolates that were highly resistance to aminoglycoside antibiotic discs were harboring only the β-lactamases resistance genes (TEM, CTX and SHV) and no one of the aminoglycoside modifying enzymes were detected in spite of they were resistance to aminoglycoside, this is maybe due to that these isolates harbor another types of modification enzymes which were out of this study.

As a conclusion, A. baumannii isolates with AMEs and β-lactamases resistance genes represent a powerful nosocomial pathogen that threat the antibiotic area and life’s of immunocompromied and hospitalizes  patients and this should be take in to account to find a new ways for restriction of these powerful pathogens.

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