Wide distribution of carbapenem resistant Acinetobacter baumannii in burns patients in Iran

Antimicrobial resistance in carbapenem non-susceptible Acinetobacter baumannii (CNSAb) is a major public health concern globally. This study determined the antibiotic resistance and molecular epidemiology of CNSAb isolates from a referral burn center in Tehran, Iran. Sixty-nine CNSAb isolates were tested for susceptibility to antimicrobial agents using the E test methodology. Multiple locus variable number tandem repeat analysis (MLVA), Multilocus sequence typing (MLST) and multiplex PCR were performed. PCR assays tested for ambler classes A, B, and D β-lactamases. Detection of ISAba1, characterization of integrons, and biofilm formation were investigated. Fifty-three (77%) isolates revealed XDR phenotypes. High prevalence of blaOXA-23-like (88%) and blaPER-1 (54%) were detected. ISAba1 was detected upstream of blaADC, blaOXA-23-like and blaOXA51-like genes in, 97, 42, and 26% of isolates, respectively. Thirty-one (45%) isolates were assigned to international clone (IC) variants. MLVA identified 56 distinct types with six clusters and 53 singleton genotypes. Forty previously known MLST sequence types forming 5 clonal complexes were identified. The Class 1 integron (class 1 integrons) gene was identified in 84% of the isolates. The most prevalent (33%) cassette combination was aacA4-catB8-aadA1. The IC variants were predominant in the A. baumannii lineage with the ability to form strong biofilms. The XDR-CNSAb from burned patients in Iran is resistant to various antimicrobials, including tigecycline. This study shows wide genetic diversity in CNSAb. Integrating the new Iranian A. baumannii IC variants into the epidemiologic clonal and susceptibility profile databases can help effective global control measures against the XDR-CNSAb pandemic.


INTRODUCTION
Acinetobacter baumannii is an important healthcare-associated pathogen that can cause of life threatening infections (Öncül et al., 2014;Girerd-Genessay et al., 2015). A. baumannii is the second most common multidrug-resistant cause of nosocomial infection in burn patients in Iran (Alaghehbandan et al., 2012;Azimi et al., 2015). Its infections have become critical challenge to health care systems due to increasing levels of resistance to antimicrobial agents in nosocomial isolates of A. baumannii (Cai et al., 2012;Moradi et al., 2015). In developing countries, including Iran, the occurrence of carbapenem non-susceptinle A. baumannii (CNSAb) infections is a growing problem in hospitalized burn patients (Mahdian et al., 2015;Zowawi et al., 2015). Inadequate management of the antibiotic therapy of CNSAb infections often leads to the emergence of extensive and pandrug resistant (XDR and PDR) CNSAb strains, which present significant health challenges by prolonging hospitalization, treatment failures, and increased mortality (Liu et al., 2014b). The most important mechanism of carbapenem resistance in A. baumannii is the production of carbapenem-hydrolyzing β-lactamases of Ambler classes A, B, and D. In addition to being resistant to all β-lactams available, carbapenemases have a high capacity to spread, since their genes have been commonly found in transferable plasmids containing integrons and insertion sequence (IS) elements (Karah et al., 2012).
Worldwide surveillance has shown that the global population structure of CNSAb isolates is diverse, but a small number of widespread clones, including the international clonal (IC) lineage I-III, also defined based on multilocus sequence typing (MLST) as clonal complexes (CC), may be predominant in healthcare settings (Jeannot et al., 2014). Multilocus variablenumber tandem-repeat (VNTR) analysis (MLVA) has been considered as a rapid and cost-effective method for fine-scale typing of A. baumannii, enabling inter-laboratory comparison of typing data (Karah et al., 2012). A recent study found the high discriminatory power of MLVA, sufficient to detect diversity among isolates showing identical MLST types (Almeida and Araujo, 2013).
Up-to-date surveillance information regarding genotypic spread, plus local antimicrobial susceptibility patterns of clinical isolates of A. baumannii, are necessary for effective drug therapy and control of XDR and PDR A. baumannii hospital outbreaks (Fishbain and Peleg, 2010). Considering the points mentioned above, the aim of this study was to determine molecular epidemiology, to identify the dissemination of the most common resistance genes, to investigate the prevalence of integrons and arrangement of integron gene cassettes among CNSAb isolates from a referral burn center in Tehran, Iran.

Bacterial Isolates
A total of 92 non-repetitive A. baumannii strains were collected between January 2012 to May 2013, from the burn wound infections of hospitalized patients in Shahid Motahari hospital, the only referral burn center in Tehran, Iran. Species of these isolates were initially characterized using the API20NE system (bioMérieux, Marcy-l'Etoile, France) and then final identification of isolates were performed by multiplex PCR using gyrBdirected primers according to the study of Higgins et al. (2010b).  (Magiorakos et al., 2012). The formation assay and quantitative analysis of biofilm were performed according to a previous study (Zhang et al., 2014).

Genetic Diversity and Population Structure
The IC lineage so-called PCR-based group were identified using three-locus dual assay multiplex PCR as described previously (Turton et al., 2007). Isolates pertaining to the novel variant of the PCR-based group, according to the new combination of amplified products obtained from the two separate multiplex PCRs, did not correspond to those previously defined. Moreover, the CNSAb isolates were genotyped using the MLVA-8 scheme method developed by Pourcel et al. (2011). For clustering analysis, the allele strings were entered into the BioNumerics software v.7.0 (Applied Maths, Sint-Martens-Latem, Belgium) as character values. A cut-off value of 90% similarity was applied to define clusters (Nhu et al., 2014). MLST was performed according to the Bartual method described previously (Bartual et al., 2005). Allele sequences, sequence types (STs), primer sequences and other details are available from the MLST website at http://pubmlst.org. The minimum spanning tree (MSTree) algorithm was used to predict the clonal relationship between all CNSAb isolates. MSTree algorithm was constructed with the BioNumerics software v.7.0 (Applied Maths, Sint-Martens-Latem, Belgium) using a categorical coefficient according to a previous report (Ruan et al., 2013).

Statistical Analysis
Statistical analyses were performed by Student's t-test, Fisher's exact test and the chi-square test with the SPSS software package (version 16). P-values <0.05 in all experiments were considered as significant.
By the MLVA typing method, the 69 CNSAb isolates were grouped into 56 distinct MLVA types with 6 clusters and 53 singleton genotypes. Loci MLVA-AB_0845 was absent (N) in all studied isolates. In this study, the VNTR loci MLVA-AB_3002 and MLVA-AB_2240 displayed lower diversity, whereas VNTR loci MLVA-AB_2396 and MLVA-AB_3468 showed higher level of diversity.
Thirty STs corresponded to 30 MLVA types; other STs (118, 95, 75, 227, 405, 231, 788, 225, 227, and 133) included heterogeneous MLVA profiles (Figure 1). In this study, IC variants were classified into 23 STs belonging to CC92, CC405, CC227, CC225 and one singleton (Figure 1). Table 3 shows the frequency of antibiotics resistance in two major epidemic lineages and IC variants. Susceptibility to all antimicrobial agents, except tobramicin, in isolates belonged to IC II was higher than the IC I and IC variants ( Table 3). It is noteworthy that the IC variants had the highest resistance rate to tigecycline (7; 22.5%) with MIC 50 and MIC 90 of 0.25 and 3 mg/L, respectively (Tables 1 and 3). Among the 13 carbapenem   non-susceptible IC I, 12 (92%) were XDR while this rate was 14 (56%) and 27 (87%) in the isolates belonging to the IC variants and IC II, respectively. The resistance to beta lactam antibiotics in IC I and IC variant isolates was higher than that in IC II. Thirty-one (100%) IC variants and 22 (88%) of the IC II population formed strong biofilm but only 3 (23%) of IC I had this characteristic. In the present study, resistance to antimicrobials was comparatively higher among IC variant than IC I and IC II CNSAb isolates (P = 0.0012 and P = 0.0001, respectively). A slightly higher susceptibility to antimicrobials was observed in IC II than IC variants and IC I (P > 0.05). Our analysis revealed a significant correlation (P = 0.003) between the presence of bla PER1 and biofilm formation in the CNSAb isolates. The CNSAb isolates belonging to all clones had the bla OXA−23 -like gene. All CNSAb isolates classified in IC I, were positive for the bla OXA−23 -like gene ( Table 4). According to Table 4, the frequency of ISAba1/bla OXA−23 -like and bla PER−1 genes in a population belonging to IC variants were significantly higher than IC I and IC II (P = 0.027 and P = 0.003, respectively). Table 4 shows that the distribution of different types of class 1 integrons is related to major epidemic lineages with predominance of class 1 integrons belonging to type 2.

DISCUSSION
Nosocomial infections caused by CNSAb are currently among the most difficult to treat. The control of CNSAb infections among hospitalized patients continues to present serious challenges to patient management in developing countries like Iran (Diancourt et al., 2010;Giannouli et al., 2010;Bahador et al., 2013). The main challenge remains choosing the most likely effective antibiotics as determined by in vitro testing of antibiotics, based on the local susceptibility patterns (Fishbain and Peleg, 2010). Overall, the findings of this study are consistent with recent studies from Iran that show an alarming trend of CNSAb increase and in MDR A. baumannii resistance against a wide spectrum of antimicrobial agents (Moradi et al., 2015). However, in the present study colistin, tigecycline, and tetracyclines exhibited a potent activity against CNSAb isolates. Our data revealed a decrease (10%) of resistance in CNSAb isolates to tigecycline (Bahador et al., 2013), likely due to the implementation of restriction policy on the empirical prescription of tigecycline. Decrease in antibiotic resistance after implementation of restriction policies have been shown in several reports (Altunsoy et al., 2011;Sistanizad et al., 2013). Our finding of positive association in strong biofilm formation and resistance to antimicrobial is consistent with a recent report (Badave and Kulkarni, 2015). This could be due to inadequate penetration of the antimicrobials into the biofilms, and the emergence of persister cells in the biofilms (Penesyan et al., 2015). The findings of our study on ISAba1 elements upstream of bla OXA genes and their role as a promoter to enhance OXAenzymes expression and MIC of imipenem confirms previous reports (Liu et al., 2014a); however, this finding are in contrast to a recent report by Peymani et al. (2012b) from Iran. Not only did our research find that 42% of CNSAb isolates were the ISAba1-bla OXA−23 -like gene; but also, a significant correlation was observed between the presence of ISAba1 upstream of bla OXA−23 -like gene and increasing imipenem MICs (≥16 mg/L). This dissimilarity might be due to differences in the A. baumannii strains available for study. Additionally, our finding of high prevalence of ISAba1 sequences upstream of the bla ADC among 97% of CNSAb isolates is consistent with previous report from Iran; and indicate that presence of ISAba1 sequences upstream of bla ADC played an important role in increasing resistance to cephalosporins (MIC 50 = 128 mg/L).
study of Karah et al. (2012), 14 CNSAb isolates showed a new combination of amplicons and were considered in this article as the PCR-based group (G) 15-17. Three of the 6 PCR-based groups in the present study, namely G6, G9, and G10, have been documented in previous studies (Karah et al., 2012;Bahador et al., 2014). Although there are differences among the IC variant types in this study and previous studies in Iran (Peymani et al., 2012b;Rezaee et al., 2013;Hojabri et al., 2014), the IC variants are highly resistant to antibiotics, which suggests that it is conceivable that the MDR phenotype has substantially contributed to their spread in Iran (Bahador et al., 2014). The results of this study indicate that, in spite of the high rate of antibiotic resistance determinants in the IC I stains in comparison with the IC variant strains, the IC I strains had lower prevalence. This dissimilarity might be due to the higher frequency of strong biofilm formers in the IC variant strains than in the IC I strains. Several studies proved that, cell adhesiveness and ability to form biofilm were higher in the bla PER−1 producing isolates (Longo et al., 2014), and this is in agreement with the result of this study. The capability to form biofilm and presence of the bla PER gene were significantly higher in IC variants. In this study, the IC II had slightly higher susceptibility to antimicrobial agents than other clonal lineage population. This might be due to low-frequency of carbapenem-hydrolyzing class D β-lactamase (CHDL) genes and ISAba1 elements, as well as low ability of biofilm formation in IC II strain as compared to IC I and IC variant stains.
It has been clearly shown that, the high prevalence of class 1 integrons among MDR A. baumannii clinical isolates is responsible for several nosocomial outbreaks globally (Liu et al., 2014b). The resistance rates to all the tested antibiotics, except colistin, tigecycline, minocycline, and tetracycline, of the collected CNSAb isolates were >65%. However, the presence of integron cassette arrays cannot be responsible for all these resistance phenotypes. In Iran, class 1 integrons have been reported previously in >11% of A. baumannii isolates (Peymani et al., 2012a;Japoni-Nejad et al., 2013). In the current study, 84% of isolates contain class 1 integrons, but no class 2 and class 3 integrons was detected. This result shows that class 2 and class 3 integrons are not the major resistant determinants in CNSAb isolates in this study, which is consistent with previous studies (Peymani et al., 2012a;Japoni-Nejad et al., 2013).
Only six categories of gene cassettes, including aminoglycoside-modifying enzymes, chloramphenicol-resistant, trimethoprim/sulfamethoxazole-resistant, and rifampicinresistant were detected in spite of previous studies reporting integrons carrying carbepenemase genes (Japoni-Nejad et al., 2013). This result indicates that acquisitions of carbapenemhydrolyzing β-lactamase genes plays more important role than the class 1 integrons in carbapenem resistance in CNSAb. The class 1 integrons rate (84%) here defined for a referral burn center in central Iran is higher than the rates found in other geographical regions, including Western Iran (11%) (Salimizand et al., 2014), China (74%) (Wu et al., 2012) and European countries in general (43%) (Castanheira et al., 2014). This result is similar to that of previous studies from the Northwest and center of Iran (Peymani et al., 2012a;Japoni-Nejad et al., 2013).
From literature survey, this is the first study to report carriage of class 1 integrons and associated arrays in A. baumannii isolates from burn patients in Iran.
Analysis of the integron cassette arrays shows that most class 1 integrons include the globally distributed aacA4-catB8-aadA1 array (Liu et al., 2014a), and the cassette array aacC1-orfX-orfX'-aadA1 has been documented in previous studies in Iran (Japoni-Nejad et al., 2013). Moreover, gene cassettes encoding aminoglycoside-resistance are present in the majority of CNSAb isolates in this study, suggesting a close relationship between high-level resistance rate to aminoglycoside compounds such as gentamicin and amikacin and the A. baumannii isolates. The different types of gene cassette array in this study and previous studies in Iran (Japoni-Nejad et al., 2013;Salimizand et al., 2014), suggests that a geographical feature plays a major role in MDR isolates formation, and it could be due to different volumes and patterns of antibiotic consumption in distinct areas. In addition, the results of this study indicate that strains indistinguishable from each other by MLST and MLVA typing can have different cassette arrays, indicating that some of these may be transmitted very efficiently. In this study, amikacin resistance recorded in 10% of the CNSAb isolates was not cassette-encoded. Analysis of the integron cassette arrays show that 50% of these isolates harbor an integron without aminoglycoside resistance genes. In the cases where the class 1 integrons could not be amplified, there were no integrons and alteration of the primer binding site or a large size of the Integron are the most likely explanations for the negative result of PCRs.
According to the findings of this research, MLVA could identify closely epidemiologically related isolates clustered by MLST in some cases (Figure 1). In this study, isolates with the same ST, such as ST118, have closely related MLVA type (MTs) (Figure 1). This result suggests that the evaluation of MLVA in CNSAb isolates shows its great ability for discrimination of genetically closely related isolates.
In agreement with previous studies (Hojabri et al., 2014), the present results substantiated that CC92 represented the recent widely distributed clone in Iran, accounting for 52.2% of CNSAb isolates examined in this study. This most likely indicates that the isolates belonging to CC92 might benefit with respect to acquiring resistance determinants and surviving in the health care environment, when encountering selection by antibiotics such as carbapenems. The international spread of the CC92 isolates has been reported from all around the world (Karah et al., 2012). This study revealed that the 28 isolates belonging to this lineage were shown to be heterogeneous by MLVA (22 MTs), followed by MLST (13 STs). The 69 CNSAb isolates were shown to be heterogeneous by MLVA (56 MTs) and MLST (40 STs). Taken together, genetic diversity and wide dissemination clones among CNSAb isolates is consistent with recent studies from Iran that show high diversity of STs in A. baumannii isolates from burned patients (Hojabri et al., 2014), which are the evidence of dynamic population structure and evolution in progress. The referral of patients from other medical centers of Iran to the hospital in Tehran could be among the possible reasons for this diversity and failure to colonize a specific type. Spread of specific clonally related isolates could have been prevented by the success of infection control programs in the teaching hospital in Iran.

CONCLUSION
Evidence that XDR-CNSAb from burned patients in Iran is rapidly changing toward growing resistance to various antimicrobials, including tigecycline was presented. Despite the increasing resistance to several first-line antimicrobials, all resistant CNSAb isolates remain sensitive to the antimicrobial colistin, a viable agent in controlling XDR-CNSAb outbreaks, especially in developing countries. This is the first study to perform IC analysis and gene cassette mapping of class integrons of A. baumannii, obtained from burned patients in Iran. It can be confirmed that the dominant local IC is an IC variant, with the likelihood that G9 is the predominant member. In the current study, the aacA4-catB8-aadA1 gene cassette array which confers resistance to aminoglycoside and chloramphenicol was identified as the predominant cassette array of class 1 integrons in CNSAb. This study described the widest distribution of bla OXA−23like-horbering IC ST118 (CC92) and ST405 (CC405) were the principal reason for the rapid increase in the carbapenem resistance rate in burned patients in Iran. The findings highlight the importance of an international drug resistance and molecular epidemiology monitoring network of A. baumannii isolates to effect global control measures against XDR-CNSAb.

FUNDING
This research has been supported by Tehran University of Medical Sciences and health Services grant No. 92-03-30-23186.