Detection of outbreak caused by multi-drug resistant Acinetobacter baumannii in Assiut University Hospitals

There is mounting evidence that Acinetobacter baumannii has a naturally occurring carbapenemase gene intrinsic in this species. Presence of class 1 integrase gene in Acinetobacter isolates is a useful marker for causing outbreaks in hospitals and for being epidemic strains of A. baumannii. The goal of the present study was to detect the resistance and outbreak marker genes by multiplex polymerase chain reaction (PCR) (blaOXA-51-like gene and class 1 integrase gene). Also to detect the correlation between imipenem susceptibility and detection of blaOXA-51-like gene. For these purposes, 51 consecutive, non-duplicate, A. baumanii strains were isolated from various clinical and environmental specimens from the Intensive Care Units (ICUs) of Assiut University Hospitals, Egypt. All the isolates were identified by conventional standard methods. The antibiotic sensitivity pattern was determined by Kirby Bauer disc diffusion method. For imipenem, the minimum inhibitory concentrations (MICs) were determined using Epsilometer (E test). Multiplex PCR was performed for the detection of the blaOXA-51like and Class I integrase genes. The blaOXA-51-like gene was detected in (95.8%) and (96.3%) in clinical and environmental isolates, respectively. Class I integrase gene was detected in (75%) and (70.3%) in clinical and environmental isolates, respectively with statistically significant difference (P value of clinical samples = 0.041 and P value of environmental samples =0.011). This means that these strains have metallo-beta-lactamase (MBL) gene (cause outbreak in hospital at any time). Also (67.35%) of A. baumanii isolates are imipenem sensitive and positive for blaOXA-51-like gene and this means that these isolates contain hidden metallo beta lactamase MBL gene.


INTRODUCTION
Acinetobacter baumannii is an important opportunistic pathogen responsible for severe nosocomial infections, especially in intensive-care-unit patients (Takagi et al., 2009).The majority of infections are of epidemic origin, and treatment has become difficult because many strains are resistant to a wide range of antibiotics, including broad-spectrum β-lactams, aminoglycosides, and fluoroquinolones (Renu et al., 2010).
Carbapenems are the drugs of choice for A. baumannii infections and are often used as a last resort.However, decreased susceptibility to carbapenems has been recently observed worldwide (Peleg et al., 2008;Valenza et al., 2010).
There are several carbapenem resistance mechanisms described in Acinetobacter species (Peleg et al., 2008).Many carbapenem hydrolyzing beta-lactamases have been identified, amongst which are the metallo-betalactamases (MBLs).Most of the MBL-encoding genes reside on integrons and plasmids which in turn allows for the widespread dissemination of these genetic elements (Walsh et al., 2005;Perez et al., 2007).
The blaOXA-51-like genes are unique to the species, and then their detection could provide a simple and convenient method of identifying A. baumannii.This method could more easily be carried out than current definitive methods, such as amplified rRNA gene restriction analysis and biochemical identification which is most commonly used (Vaneechoutte et al., 1995;Woodford et al., 2006).
In recent years, a novel mechanism of resistance gene dissemination among bacteria has been described (Stokes and Hall, 1989).This mechanism is based on the location of these genes on integrons.The majority of integrons belongs to class 1 and has been found predominantly in clinical isolates of Gram-negative bacteria, including Acinetobacter species (Martinez-Freijo et al., 1998).Presence of class 1 integrase gene in Acinetobacter isolates is a useful marker for causing outbreaks in hospitals and being epidemic strains of A. baumannii (Koeleman et al., 2001;Turton et al., 2005).
Identifying MBL carrying isolates has been challenging due to the emergence of carbapenem-susceptible MBL carrying organisms which may be missed in daily laboratory practice, compromising the sensitivity of detection methods.These carbapenem susceptible organisms with hidden MBL genes can spread unnoticed in hospitals if such isolates are reported as sensitive without screening for the presence of MBLs.The treatment of these organisms poses a serious therapeutic challenge as these strains are most often resistant to multiple drugs (Walsh et al., 2005).
The present study aimed to detect theblaOXA-51-like gene which can be carried out as part of a multiplex PCR, which detects both blaOXA-51-like gene (resistance gene) and class 1 integrase gene (marker for outbreak).Also to detect the correlation between imipenem susceptibility and detection of bla OXA-51 like gene.

Bacterial strains
A total of 51 consecutive, non-duplicate, A. baumanii strains were isolated from various clinical and environmental specimens from the ICUs of Assiut University Hospitals during period of February 2011 to February 2012.Regarding the clinical specimens, A. baumanii strains (24 strains) were isolated from urine (n= 5), sputum (n= 8), swabs from endotracheal tubes (n= 6), blood cultures (n=1), throat swabs (n=3) and wound swabs (n=1) that were submitted for bacteriological testing from patients admitted to the ICUs.A total of 27 isolates were obtained from environmental swabs from the ICUs.Swabs were taken from call bells, bedrails, and bedside tables, bedside equipments, commodes, doorknobs and faucet handles.

Identification of strains
Using MacConkey agar and Herellea agar (Dijkshoorn et al., 2005), also using simple biochemical reactions as oxidase test, nitrate test, growth on TSI, Citrate test, Urease test, motility test and growth at 44°C (Collee et al., 1996).

Antimicrobial susceptibility testing
Antibiotic susceptibility testing was done for all isolates using commercially available discs (HiMedia, Mumbai, India) by Kirby Bauer disk diffusion method and interpreted as recommended by Clinical Laboratory Standards Institute (CLSI, 2010).

Determination of imipenem minimal inhibitory concentration (MIC) by IPM E-Test
AB Biodisk, Solna and Sweden E-test strips were placed overculture streaked over Muller Hinton agar.After overnight incubation in incubator at 35°C, the MIC was read as intersect where the ellipse of growth inhibition intersects the strip.It was used at a cutoff point of ≥16 μg/ml to define imipenem resistance and a cut-off point of ≤ 4 μg/ml to define imipenem susceptibility (CLSI, 2006).

Multiplex PCR for detection of blaOXA-51-like gene & Class 1 intgrase gene (Turton et al., 2005): a-DNA extraction
The boiling method was used to extract the DNA from the bacteria (Vaneechoutte et al., 1995).Briefly, one colony of a pure culture was suspended in 50 μl of sterile water and heated at 100°C for 15 min.After centrifugation in a micro centrifuge (6,000 x g for 3 min), the supernatant was stored at -20°C for further use.

b-PCR Amplification and detection
This was carried out in 25 μl reaction volumes with 3 μl of extracted DNA, 12.5 pmol of each primer as shown in Table 1 and 1.5 U of Taq DNA polymerase in 1X PCR buffer containing 1.5 mM MgCl 2 (QIAGEN) and 200 μM of each deoxynucleoside triphosphate.Conditions for the multiplex PCR were as following: 94°C for 3 min, Table 1.Primer sequences of blaOXA-51-like gene and Class 1 integrase gene (Koeleman et al., 2001;Woodford et al., 2006).

Statistical analysis
All data were analyzed using the computerized statistical analysis (SPSS, version 16).Descriptive statistics was used.The P value <0.05 was considered statistically significant.The percent differrence of each antimicrobial agent versus Imipenem was calculated at 99% confidence interval.

RESULT
Fifty one strains of Acinetobacter sp. were isolated in infection control laboratory at Assiut University Hospitals from February 2011 to February 2012 .They comprised of two sets of isolates, The first set consisted of 24 isolates recovered from clinical samples, the second set consisted of 27 isolates isolated from environmental samples .All Acinetobacter strains were described as Gram negative cocco-bacilli, non-motile, non-spore forming, capsulated, oxidase negative, not reduce nitrate to nitrite, not ferment sugar and citrate positive bacilli.
Acinetobacter grow on blood agar showing mucoid colonies, on MacConkey agar showed non-lactose fermenting colonies, on Herellea agar showed purple colonies.API20NE showed that these strains belong to Acinetobacter baumannii/calcoaceticus complex, and isolates identified as Acinetobacter baumannii by its ability to grow at 44°C.
The susceptibility of A. baumannii to different antimicrobial agents was compared to imipenem.Imipenem resistant A. baumannii are not susceptible to penicillin derivative or cephlosporine, but these strains are susceptible to tetracycline (more active), chloamphenicol (moderate active) and aminoglycosides (less active) in descending manner.But susceptibility of A. baumannii to quinolones is variable (Figure 1).
Detection of blaoxa-51-like gene and Class I intgrase gene showed that (95.8%) and (96.3%) of A. baumannii isolated from clinical and environmental samples respectively gave positive result for blaoxa-51-like gene (intrinsic carbapenamase gene) while (75%) and (70.3%) of A. baumannii isolated from clinical and environmental samples respectively gave positive result for Class I intgrase gene (Table 4 and Figure 2).
Relation between imipenem susceptibility and detection of blaOXA-51-like gene showed that 67.5% of A. baumannii isolates are imipenem sensitive and positive for blaOXA-51-like gene and this means that these isolates contain hidden MBL gene (Table 5).

DISCUSSION
A. baumannii infections present a global medical challenge.The interest in this organism has been growing rapidly because of the emergence of multi-drug resistant strains (MDR), some of which are pan resistant to antimicrobial agents (Muthusamy and Boppe, 2012).In the present study the majority of A. baumannii isolates were MDR showing resistance to three or more classes of antibiotics.There has been a lot of debate concerning the definition of multidrug resistance (MDR).Renu et al. (2010) defined MDR as resistance to 4 or more classes of antimicrobials.Others defined MDR as resistance to two or more drugs or drug classes of therapeutic relevance (Navon-Venezia et al., 2005).Resistance against carbapenemsis, in itself, considered sufficient to define an isolate of A. baumannii as highly resistant (Poirel and Nordmann, 2006).
In our study, the results of antimicrobial susceptibility test shown resistance to penicillin derivatives (61.82%),Cephalosporine derivatives (61.82%),Quinolones (64.18%),Monobactam (60.6%),Aminoglycosides (56.48%) and Chloramphenicol (53.01%).The lowest rate of resistance   was toImipenem (31.24%) and tetracyclines (25.2%).This agreed with Hashem et al. (2011) who showed that tetracycline was the most effective antimicrobial agent against A. baumannii derivatives and cephalosporins were the least active agents against Acinetobacter when compared with Impinem.Similar results reported by Hanaa et al. (2010) who found that sensitivity of Acinetobacter to imipenem.The findings of the present study confirm that penicillin was 64.2% while susceptibility to penicillin derivatives and cephalosporines was 0%.The predominant Acinetobacter sp. in clinical settings are the members of the A. calcoaceticus-baumannii complex which are multi drug resistant and are responsible for causing outbreaks.Carbapenem resistance in A. calcoaceticus-baumannii complex is very high and is predominantly due to carbapenemase production, metalloβ-lactamases, oxacillinases, mobile genetic elements, and reduced expression of outer membrane proteins (Limansky et al., 2002;Poirel et al., 2003;and Anil et al., 2011).
E-test results were showed that (34/51 or 66.67%) of Acintobactersp isolates were imipenem susceptibile (MIC below 4 μg/ml), while (17/51 or 33.33%) were imipenem resistant (MIC above 8 μg/ml).This agreed with (Livermore, 2002) who found that the high levels of imipenem MIC (16-256 mg/L) observed in these A. baumannii isolates suggested the presence of a metallo-βlactamase (MBL) or an oxacillinase, since these carbapenemases were considered the major mechanism of carbapenem resistance in these organisms.
The resistance of A. baumannii to antimicrobial agents is mediated by all of the major resistance mechanisms that are known to occur in bacteria.β-Lactamases are the most diverse group of enzymes that are associated with resistance, and more than 50 different enzymes, have been identified so far in A. baumannii.OXA-51-like carbapenemases are class D β-lactamases which are intrinsic to A. baumannii and confer resistance to carbapenems (Turton et al., 2006a;Brown et al., 2005).
In this study, (96%) of A. baumannii were showed band of blaOXA-51-like genes.This agreed with Turton et al. (2006b) who found that all A. baumannii gave a band in the blaOXA-51-like PCR, but they remain alert to the possibility of non-detection of some variants.A further potential problem is that these genes are sometimes associated with ISAba1, which may render them mobile.We currently encounter also results of Hanna et al. (2010) that showed that detection of blaOXA-51-like genes is the most specific, simple and reliable method for detection of A. baumannii as carbapenamse gene is intrinsic to this species.
Among imipenem-susceptible and resistant A. baumannii which were screened by PCR for different βlactamases.The blaOXA-51-like gene was the only one detected, even in imipenem-susceptible strain (Takagi et al., 2009).It has been reported that among A. baumannii isolates with blaOXA-51-like as sole carbapenemase gene, imipenem and/or meropenem resistance was associated only with isolates in which ISAba1 was upstream of blaOXA-51-like, suggests that ISAba1 is providing the promoter for this gene (Turton et al., 2006a).
In this study, (72.5%) of A. baumannii showed positive bands for class I integrase gene (gene responsible for outbreaks in hospitals).The analysis of A. baumannii strains with known epidemic behavior demonstrates that early identification of epidemic strains may be possible by detection of integrons or multiple antibiotic resistances.The integrase gene PCR identified almost 75% of the epidemic A. baumannii strains.Multiple antibiotic resistances, defined as resistance to five or more antibiotics, showed good correlation with the presence of integrons and epidemic behavior of the strains.
This result agree with (Dijkshoorn et al., 1996) who showed that strains may vary considerably in their epidemiological potential, and those strains that have been known to spread widely and rapidly among hospitalized patients have been designated epidemic A. baumannii strains.Antibiotic resistance has been shown to be one of the factors which can influence the nosocomial dissemination of A. baumannii.Few reports credit outbreak control to reduced prescribing of broad spectrum antibiotics, such as fluoroquinolones or carbapenems (Villegas and Hartstein, 2003).
Among imipenem-susceptible and resistant A. baumannii which were screened by PCR for different β-lactamases.The blaOXA-51-like gene was the only one detected, even in imipenem-susceptible strain (Takagi et al., 2009).It has been reported that among A. baumannii isolates with blaOXA-51-like as sole carbapenemase gene, imipenem and/or meropenem resistance was associated only with isolates in which ISAba1 was upstream of blaOXA-51-like, suggests that ISAba1 is providing the promoter for this gene (Turton et al., 2006a).tection of class I integrase gene is very important in the rapid epidemiologic investigation of an outbreak.

Table 2 .
Resistance patterns of A. baumannii to different antibiotics.
Figure 1.Forest representation of the 99% confidence interval comparing imipenem activity against other antimicrobials.

Table 3 .
Detection of Metallo-B-lactamase in A. baumannii by IPM E-test.

Table 4 .
Detection of blaoxa-51-like gene & Class 1 integrase gene by Multiplex PCR value of clinical samples = 0.041, P value of environmental samples = 0.011, P value of < 0.05 indicates significant results.No = number. P