Background
Acinetobacter baumannii is a frequent cause of nosocomial infections in burn patients and has remained an important opportunistic pathogen responsible for increasing rates of morbidity and mortality due to infection in developing countries [
1,
2]. Dissemination of MDR
A. baumannii causes a global public health challenge. Antibiotic treatment of
Acinetobacter baumannii (
A. baumannii) infections plays an essential role in reducing prevalence and death rates, but about half of strains of
A. baumannii in many parts of the world are now resistant to multiple drugs [
3]. Prevalence of carbapenemases, cephalosporinase (AmpCs), Extended-Spectrum β-Lactamases (ESBLs), and Metallo-β-Lactamases (MBLs) producing
A. baumannii is rapidly increasing and conventional antibiotic therapeutics have become increasingly inefficient [
3,
4]. Coexistence of various antibiotic resistance mechanisms including extrusion of drugs by active efflux pumps (encoded by various
tet genes), Carbapenem-Hydrolyzing Class D β-Lactamases (CHDLs) (OXA-23, OXA-24/40, OXA-58, OXA-143, and OXA-235), MBLs (IMP, VIM, NDM, SPM, GIM, and SIM), ESBLs (PER, TEM, SHV,and CTX), contributes to the increase in the number of MDR- AB strains [
3,
5]. Multidrug resistance conferred by ESBLs and MBLs genes has recently been raised around the world among Carbapenem-Non-Susceptible
A. baumannii (CNSAB) strains, and also these resistance genes can be harbored by a transferable plasmid containing integrons or Insertion Sequence (IS) elements into chromosome becoming widely disseminated among other strains, and conferring resistance to almost all β-lactam antibiotics [
3,
6]. Recently, much attention has been focused on biofilm formation in
A. baumannii, because microbial cells grown in biofilms are less sensitive to antimicrobial agents and more persistent to environmental conditions such as intubation tubes, catheters, and cleaning instruments [
7,
8].
Drug resistance in CNSAB can result from many mechanisms such as extrusion of drugs by active efflux pumps, decrease in cellular permeability, biofilm formation, and overexpression of drug-modifying and -inactivating enzymes or target modification by mutation [
9]. There are few studies conducted in Iran on coexistence of different antibiotic resistance mechanisms with the ability to form biofilms in multidrug-resistant
Acinetobacter baumannii isolated from burn wound infections, and there is relatively few information on diversity of these strains. Nowadays, understanding molecular characteristics of various antibiotic resistance mechanisms along with molecular epidemiology analysis in regions with high prevalence rates of MDR
A. baumannii infection plays an essential role in developing therapeutic strategies and controlling MDR-AB outbreaks, both in community and hospitals [
1,
7,
10]. Among molecular typing methods, Pulsed-Field Gel Electrophoresis (PFGE) is usually considered gold standard for epidemiological typing of
A. baumannii [
6]. Therefore, this study was carried out to determine antimicrobial susceptibility profiles, biofilm-forming ability, resistance determinants, and clonal relatedness among MDR
A. baumannii strains recovered from patients with burns in Iran.
Discussion
More than 93% of
A. baumannii strains obtained from three major hospital centers in Iran were found to be resistant to six antimicrobial categories (Tables
3 and
4). Our study results demonstrated high prevalence rates of CNSAB, MDR-AB, and XDR-AB isolates and emergence of them in respective hospitals as a worrying tendency. In line with other studies conducted in Iran, colistin, and polymyxin B exhibited excellent activity against CNSAB strains [
6,
21]. In the present study, high MIC50 and MIC90 values for carbapenems were detected and indicated a markedly reduced efficacy of these agents that could be due to their overuse. In this study, 23 isolates showed resistance to colistin (MIC = 4 ≥ μg/ml), indicating rise of this phenomenon worldwide [
22]. This resistance could result from modification of Lipopolysaccharide (LPS) at outer cell envelope, but identification of mechanism of colistin resistance in these isolates was beyond scope of our study [
23]. Similar to other studies, our findings showed very high rates of biofilm formation in XDR
A. baumannii strains [
24], and this phenomenon is associated with protective properties of persistent cells in the biofilms,and could be a result of inadequate penetration of antimicrobial agents into the biofilms [
24,
25]. Our study results showed a statistically significant association between antimicrobial resistance phenotypes and biofilm formation, which was in line with other findings [
9,
24], but it was in contrast to results of the study by Baniya et al. [
25].
A. baumannii strains formed strong biofilm were endemic in respective hospitals, indicating that microbial cells grown in biofilms are more resistant to different environmental stress conditions [
24]. Results of the present study showed that a high distribution of multiple genes, mainly genes of
blaOXA-23-like /
blaOXA-40-like/
blaOXA-51-like carbapenemase,
blaPER-1/
blaVEB-1 extended-spectrum-β- lactamase,
blaIMP- and
blaVIM-family metallo-β-lactamase, and
tetB efflux pump is responsible for detection of drug-resistance in burn patients.
blaOXA-23-like and
blaOXA-40-like are common in CNSAB isolates. Similar to other studies carried out in Iran,
blaOXA-23-like was the most prevalent OXA-type carbapenemases among carbapenem-resistant strains, [
6] and it has reported frequently in many countries including Pakistan, South Korea, and China [
26]. According to the data represented in Table
5, increased MIC values observed for imipenem and meropenem in studied
blaOXA-23-like and
blaOXA-40-like carrying isolates. The increased MICs indicate that presence of these genes play an important role in creating resistance to carbapenems. Notably, most of the XDR
A. baumannii isolates co-harbored
blaOXA-23-like and
blaOXA-40-like genes, could contribute to clonal dissemination. However, it remains unclear that co-existing carbapenemase genes can cause resistance and it needs further investigation.
However, other carbapenemase genes such as
blaOXA-58-like,
blaOXA-143-like, and
blaOXA-253-like were not detected in any of the isolates, which was in agreement with previous reports [
27]. Results of this study revealed that IS
Aba1 + blaOXA-51-like and IS
Aba1 +
blaOXA-23-like -carrying
A. baumannii isolates had high MIC values for both imipenem and meropenem, and also most resistant patterns were observed to be associated with them (Table
5),and our findings indicate that, this IS element influences expression of antibiotic resistance
blaOXA-23-like and
blaOXA-51-like genes, providing higher levels of MIC values to carbapenems,which was in line with results of previous studies [
10,
27]. After PFGE analysis, dissemination of clonally related strains was determined in three major hospital centers. Interestingly, all isolates belonging to clusters 24, 29, 32, and 33 were found to be associated with
blaOXA genes adjacent to IS
Aba1 element, appearing to be predominant clones in monitored hospitals with high similarity in their molecular typing.
Results revealed that 90.1% (
n = 147) and 85% (52.2%) of isolates were MBL and ESBL producers, respectively, and 43.5% of them were positive for both. It was also found that, ESBL- and MBL- producing
A. baumannii isolates were genetically related and belonged to the same clone. In the present study,
blaPER-1 genes (55.3%) were predominant followed by
blaVEB-1 (42.3%)
, and
blaTEM (33%) among ESBL-
A. baumannii isolates,which was in accordance with previous studies conducted in Iran and India [
28,
29]. On the contrary, Safari et al. reported that 58 and 20% of ESBL-
A. baumannii isolates harbored SHV, and CTX-M genes, respectively [
30]. The
blaCTX-M gene was not detected in any ESBL producing isolates; higher result was reported (76%) by Abrar et al., 2019 [
31]. Also, three (3.5%) ESBLs producing isolates were negative for ESBL encoding genes; however, other mechanisms must be involved in antibiotic resistance of these strains. The
blaVIM- family genes were the most common MBL genotype found in the present study, as reported previously in other studies [
32]. On the contrary,
blaVIM- family genes not detected in any MBL-producing isolates from Iran and Egypt [
21,
33]. The
blaGIM,
blaSIM, and
blaNDM-1 not identified in the present study; this is in accordance with another report conducted in Iran [
30]. There was not any significant correlation between phonotypic MBL and ESBL -producing and resistance genes. In addition, the results obtained by phenotypic identification of MBL production did not comply with genotypic results and indicating that false-positive results among these strains with respect to resistance phenotype. This phenomenon may appear due to effect of the EDTA on cell wall of
A. baumannii or as chelator to inhibit OXA enzymes [
26,
34].
In the current study, 107 (65.6%) and 56 (34.4%) of isolates had class 1 and class 2 integrons, but class 3 integron was not detected. Prevalence of carbapenemases, MBL, and ESBL genes was higher in integron-positive
A. baumannii strains than in negative strains, and a correlation was found between
blaVIM gene and class 1 integron (
P < 0.05) revealing an association of the genes encoding MBL with presence of class 1 integron among MDR-AB isolates. Accordingly, horizontal transfer of this integrons by plasmid promotes spread of multiple resistance genes in sporadic and outbreak isolates of
A. baumannii [
1].
MIC values of tetracycline decreased in presence of efflux pumps inhibitors suggesting involvement of efflux activity. Our findings showed that
tetA and
tetB genes were detected in clinical strains with resistance to tetracyclines and doxycycline, which was in agreement with other studies conducted in Spain and Iran [
8,
35].
TetB was found to be associated with resistance to minocycline, while
tetA was not found in any of the minocycline-nonsusceptible isolates. These results indicate that
tetB efflux system might be a potential cause of minocycline resistance, and our results are in concordance with another study conducted by wang et al. [
5]. Both of over- expression and lake of
tetB gene may lead to minocycline-nonsusceptible phenotype [
5], but we should consider that other factors and mechanisms could involve in resistance occurrence. All isolates belonging to 29, 32, and 33 PFGE genotypes were found to contain
tetB gene, possibly leading to clonal dissemination of
tet(B) positive
A. baumannii isolates in monitored hospitals. Finally, 15, 17, 24, 29, 32, and 33 PFGE genotypes contained more drug-resistant genes, with the ability to form strong biofilms, and were found to be predominant epidemic clusters, and it is noteworthy that, colistin-resistant
A. baumannii isolates were widespread in genotypes as mentioned above. Our data obtained from molecular epidemiology analysis is of a significant value for the clinicians to make therapeutic decisions and manage infection control.
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