High Prevalence of blaNDM-1, blaVIM, qacE, and qacEΔ1 Genes and Their Association with Decreased Susceptibility to Antibiotics and Common Hospital Biocides in Clinical Isolates of Acinetobacter baumannii

The objective of this study was to evaluate the susceptibility of metallo-β-lactamase (MBL)-producing Acinetobacter baumannii (A. baumannii) clinical isolates to biocides. We also determined the prevalence and correlation of efflux pump genes, class 1 integron and MBL encoding genes. In addition, blaVIM, blaNDM-1, qacE and qacEΔ1 nucleotide sequence analysis was performed and compared to sequences retrieved from GenBank at the National Center for Biotechnology Information database. A. baumannii had a resistance rate to carbapenem of 71.4% and 39.3% and was found to be a MBL producer. The minimum inhibitory concentrations (MICs) of chlorhexidine and cetrimide were higher than the recommended concentrations for disinfection in 54.5% and 77.3% of MBL-positive isolates respectively and their MICs were significantly higher among qac gene-positive isolates. Coexistence of qac genes was detected in 68.1% and 50% of the isolates with blaVIM and blaNDM-1 respectively. There was a significant correlation between the presence of qac genes and MBL-encoding blaVIM and blaNDM-1 genes. Each of the blaNDM-1, blaVIM, qacE and qacEΔ1 DNA sequences showed homology with each other and with similar sequences reported from other countries. The high incidence of Verona integron-encoded metallo-β-lactamases (VIM) and New-Delhi-metallo-β-lactamase (NDM) and qac genes in A. baumannii highlights emerging therapeutic challenges for being readily transferable between clinically relevant bacteria. In addition reduced susceptibility to chlorhexidine and cetrimide and the potential for cross resistance to some antibiotics necessitates the urgent need for healthcare facilities to periodically evaluate biocides efficacy, to address the issue of antiseptic resistance and to initiate a “biocidal stewardship”.


Phenotypic Detection of MBL
E-test MBL strips (AB Biodisk, Solna, Sweden) were used in accordance with the manufacturer's instructions to investigate MBL production. MIC ratio of imipenem to imipenem/EDTA of 8 or the presence of a phantom zone was taken as a positive result.

Determination of Disinfectants/Antiseptics Susceptibility by the Broth Macrodilution Method
The biocides selected for testing were the chemical disinfectants/antiseptics recommended for patient-care items and instruments (most widely used biocides). Chlorhexidine (0.3%, CLX), cetrimide (3%), and aqueous povidone-iodine (10% w/v, PVP-I 2 ) solution were included. The MICs of the disinfectants/antiseptics were determined by the broth macrodilution method according to [31]. Each disinfectant solution was diluted by a serial two-fold dilution method using Muller Hinton broth (MHB) ( Table 1). The positive control tube contained 0.1 mL inoculum in 1 mL MHB and should show evidence of bacterial growth (turbidity). The negative control tube contained MHB and the disinfectant to be tested and should not show bacterial growth (clear). Table 1. The chemical agents, their starting concentrations and dilutions used for minimum inhibitory concentration (MIC) test.

PCR and DNA Sequencing
Genomic DNA was extracted from A. baumannii isolates using Qiaamp Mini DNA kit (Qiagen, Hilden, Germany). bla OXA-51 -like gene, Intl1, bla VIM , bla NDM-1 , bla IMP , bla GIM , bla SIM , bla SPM , qacE, qac∆E1 and cepA were amplified for A. baumannii isolates using the primers listed in Table 2. The primers were prepared by Invitrogen (Grand Island, NY, USA). GoTaq Green Master Mix (Promega, Madison, WI, USA) was used in all PCR assays. The PCR products were separated through a 1.5% agarose gel by electrophoresis. Amplified products were cut and purified using Qiaquick gel purification kit (Qiagen, Hilden, Germany) and sequenced at DNA Biotechnology Facility at University of Connecticut, USA (using the Genetic analyzer 3030X1 (Applied Biosystems, CA, USA) with a big Dye terminator cycle sequencing kit). Sequences in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to identify and compare the genes detected in this study. The bla VIM , bla NDM-1 , qacE and qac∆E1 specified DNA amplicon sequences and similar sequences recovered from NCBI GenBank database were used to create the phylogenetic trees in order to understand the genetic relatedness of the study sequences. Alignment of these genes nucleotide sequence was performed using the DNASTAR program (DNASTAR Inc., Madison, WI, USA).

Statistical Analysis
Statistical analysis was done using SPSS version 20 (IBM ® , New York, NY, USA). Chi-square test was performed. A p-value of <0.05 was considered indicative of a statistically significant difference.

Characteristics of Isolates and Their Antibiotics Susceptibility
Out of 56 A. baumannii isolates, 22 were recovered from blood (39.3%), 18 from pus/wound swabs (32.1%), nine from sputum (25%), three from a chest tube (5.4%), two from throat swabs (3.6%), one from a urine specimen (1.8%) and one from a nasal swab (1.8%). Table 3 shows the antimicrobial suscebtipility patterns of 56 A. baumannii isolates. A. baumannii resistance rate to carbapenem (imipenem and meropenem) was 71.4% (40/56) and 39.3% (22/56) were MBL producers. Forty-nine isolates were MDR (87.5%) and seven were extensively drug resistant (XDR) (12.5%). "MDR Acinetobacter spp." is defined as isolate non-susceptible to at least one agent in three or more antimicrobial categories. "XDR Acinetobacter spp." is defined as an Acinetobacter spp. isolate that is resistant to all standard antimicrobial agents except colistin or tigecycline [38]. Colistin and tigecycline were totally effective against all tested isolates. All MBL-producing isolates were MDR and 31.8% of them were XDR. Antibiotic susceptibility results for MBL-producing A. baumannii are shown in Table 4.

Detection of MBL-Encoding Genes
The amplified products of bla OXA-51 -like gene, Intl1 gene and MBL-positive genes for the A. baumannii isolate were shown in Figure 1. The intrinsic β-lactamase gene, bla OXA-51 -like, was amplified from all A. baumannii isolates. Of the positive MBL isolates, MBL-encoding gene bla VIM was identified in 86.4% (19/22) of MBL-producing isolates and bla NDM-1 was identified in 13 (59.1%) isolates. Coexistence of bla VIM and bla NDM-1 was encountered in 13 isolates (all bla NDM-1 -positive isolates). In this study, MBL bla IMP , bla GIM , bla SIM , and bla SPM were not detected. All the MBL-producing isolates were positive for class 1 integron. study, MBL blaIMP, blaGIM, blaSIM, and blaSPM were not detected. All the MBL-producing isolates were positive for class 1 integron. Pus Pus - Pus

Correlation of Efflux Pump Genes with MIC of Tested Biocides
In the present study, MBL A. baumannii isolates were effectively inhibited by the user's defined concentrations of PVP-I 2 , whereas the MICs of 54.5% and 77.3% of the tested isolates for CLX and cetrimide, respectively, were higher than the actual concentrations recommended for disinfection. The amplified products of qac genes were shown in Figure 1E,F.
Among MBL-positive isolates, qacE and qac∆E1 efflux genes were present in 10 (45.5%) and 15 (68%) isolates respectively, whereas cepA gene was not detected. In general, for qac-positive isolates, the MICs ranged from 47 to 750 µg/mL and 470 to 7500 µg/mL for CLX and cetrimide, respectively. For qac-negative isolates, MIC ranged from 23.4 to 94µg/mL and 470 to 1875 µg/mL for CLX and cetrimide, respectively. The MICs of PVP-I 2 ranged from 390 to 12,500 µg/mL with both qac-positive and negative isolates (Table 4). There was a significant correlation between qac genes carriage and increased MICs of CLX and cetrimid (p = 0.0096 and 0.0085, respectively) as showed in Table 5.

Correlation of bla VIM and bla NDM-1 Genes with qac Genes
As indicated in Table 6, coexistence of bla VIM and qac genes was detected in 15 isolates, while bla NDM-1 was concomitant in 11 isolated with qac genes, and coexistence of the qacE and qacE∆1 genes was encountered in 10 of the MBL-positive isolates. There is significant correlation between qac and the carriage of both bla VIM and bla NDM-1 genes (p = 0.0064 and 0.0467, respectively).

Nucleotides Sequence and Phylogenetic Analysis
Sequencing of intrinsic bla VIM and bla NDM-1 confirmed that the nucleotide sequences obtained were identical to genes for VIM and NDM for A. baumannii. In the same way, the nucleotide sequences of class I integrin, qacE and qacE∆1 also agreed with the results expected by the PCR analysis.
The nucleotide sequences of the bla VIM gene-positive isolates were highly similar, with sequence identities of 90.8-100%. The genetic divergence and homogeneity of the bla VIM sequences are apparent in the phylogenetic tree Figure 2. Among the 19 bla VIM gene sequences, there were nine different clusters. Those from isolates number 14, 18 and 20 were found to form distinct clusters, while six clusters formed from the rest of isolates' shared similarity.
The nucleotide sequences of the bla NDM-1 gene in 13 out of 22 A. baumannii isolates were highly similar, with sequence identities of 88-100%. bla NDM-1 sequences of isolates number 3, 13 and 20 were found to form distinct clusters whilst the other isolates shared similarity in four diverse clusters. These sequences were closely related to bla NDM-1 sequences from other countries: one from China (GenBank accession number: KP772138), two from Iran (GenBank accession numbers: LC154934.1 and LC154949.1) and two from India (GenBank accession numbers: KU510390 and KU510393). In addition, two sequences reported from Iraq (GenBank accession numbers: KU378644.1 and KU378645.1) were also clearly apparent in the tree (Figure 3).

Discussion
The worldwide increased occurrence of carbapenem-resistant A. baumannii infections in healthcare settings has led to a greater alertness of the threat of hospital acquired infections. In the current study, there was high frequency of carbapenem-resistant A. baumannii strains that may be attributed to the extensive misuse of carbapenems. Resistance to carbapenem in clinical isolates of A. baumannii has been reported in Egypt [39][40][41][42][43]. At this time, there are limited selections of treatment options for carbapenem-resistant A. baumannii infection, according to our results; colistin and tigecycline are considered the last choice to control (100% sensitivity). This finding has been also reported in different studies [4,[42][43][44][45].
The high frequency of MBL A. baumannii detected in the study is not common, since two previous separate studies have shown 0% and 2.5% MBL activity among A. baumannii isolated in Egypt [42,43]. Resistance due to MBL production has a potential for rapid dissemination, since it is often plasmid-mediated [40]. The current study showed that all MBL-producing isolates were MDR that exhibited high resistance to beta-lactams, aminoglycosides, and quinolones.
Because of its ability to spread, carbapenem resistance related to VIM and NDM β-lactamase production has become a serious concern. MBL VIM has been reported intermittently in Egypt [40,42]. The nucleotide sequences of the blaVIM gene from the positive isolates were highly similar, with sequence identities of 90.8-100%. blaVIM originated from Greece and South Korea [46]. blaVIM nucleotide sequences showed high similarity with these two countries and other countries like India and Iran. Horizontal transfer of VIM-encoding genes can be inferred from the presence of the same integron in genetically non-related isolates in different species and genera [47]. The most recent MBL identified in A. baumannii are the emerging NDM enzymes. The blaNDM-1 genes have been reported in A. baumannii from India [48] and Germany [49]. Kaase et al. [50] reported the first NDMproducing A. baumannii in Egypt from a patient who transferred from Egypt to Germany without obvious link with the Indian subcontinent. After recent identification of NDM-producing isolates in Iraq [51] and the Sultanate of Oman, the clinical case suggests that NDM-producing bacteria disseminated in the Middle East countries [3]. These isolates were resistant to high levels of all βlactams, including carbapenems. In our study, 68% of the isolates carried blaNDM-1 gene. The nucleotide sequences of blaNDM-1 gene showed high similarity between isolates (with sequence identities of 88-100%) and with the sequence of blaNDM-1 gene identified from other countries (mainly including India, Iran and Iraq). blaVIM and blaNDM-1 genes are horizontally transferable as they are inserted in integrons, and some of these integrons are located on conjugative plasmids [46]. The large number of trips

Discussion
The worldwide increased occurrence of carbapenem-resistant A. baumannii infections in healthcare settings has led to a greater alertness of the threat of hospital acquired infections. In the current study, there was high frequency of carbapenem-resistant A. baumannii strains that may be attributed to the extensive misuse of carbapenems. Resistance to carbapenem in clinical isolates of A. baumannii has been reported in Egypt [39][40][41][42][43]. At this time, there are limited selections of treatment options for carbapenem-resistant A. baumannii infection, according to our results; colistin and tigecycline are considered the last choice to control (100% sensitivity). This finding has been also reported in different studies [4,[42][43][44][45].
The high frequency of MBL A. baumannii detected in the study is not common, since two previous separate studies have shown 0% and 2.5% MBL activity among A. baumannii isolated in Egypt [42,43]. Resistance due to MBL production has a potential for rapid dissemination, since it is often plasmid-mediated [40]. The current study showed that all MBL-producing isolates were MDR that exhibited high resistance to beta-lactams, aminoglycosides, and quinolones.
Because of its ability to spread, carbapenem resistance related to VIM and NDM β-lactamase production has become a serious concern. MBL VIM has been reported intermittently in Egypt [40,42]. The nucleotide sequences of the bla VIM gene from the positive isolates were highly similar, with sequence identities of 90.8-100%. bla VIM originated from Greece and South Korea [46]. bla VIM nucleotide sequences showed high similarity with these two countries and other countries like India and Iran. Horizontal transfer of VIM-encoding genes can be inferred from the presence of the same integron in genetically non-related isolates in different species and genera [47]. The most recent MBL identified in A. baumannii are the emerging NDM enzymes. The bla NDM-1 genes have been reported in A. baumannii from India [48] and Germany [49]. Kaase et al. [50] reported the first NDM-producing A. baumannii in Egypt from a patient who transferred from Egypt to Germany without obvious link with the Indian subcontinent. After recent identification of NDM-producing isolates in Iraq [51] and the Sultanate of Oman, the clinical case suggests that NDM-producing bacteria disseminated in the Middle East countries [3]. These isolates were resistant to high levels of all β-lactams, including carbapenems. In our study, 68% of the isolates carried bla NDM-1 gene. The nucleotide sequences of bla NDM-1 gene showed high similarity between isolates (with sequence identities of 88-100%) and with the sequence of bla NDM-1 gene identified from other countries (mainly including India, Iran and Iraq).
bla VIM and bla NDM-1 genes are horizontally transferable as they are inserted in integrons, and some of these integrons are located on conjugative plasmids [46]. The large number of trips between the countries combined with the ease of resistance transmission among bacteria led us to consider that our isolates may share genetic similarity of bla VIM and bla NDM-1 with other countries.
The increase in reduced susceptibility to antibiotics is paralleled by a similar trend in reduced susceptibility to biocides [15,52]. Antiseptic resistance has been reported for several agents including quaternary ammonium compounds and biguanides [53]. Interestingly, more than half of the tested isolates showed higher MICs of CLX and cetrimide than the actual concentrations recommended for disinfection. This may be attributed to the extensive use of these types of disinfectants in the routine infection control in Egypt. Moreover, broader and more indiscriminate use of biocides in healthcare facilities could drive the emergence of new genetic elements, with unpredictable consequences for human welfare.
Resistance to biocides is attributable to production of efflux proteins encoded by plasmid-mediated genes such as qac genes [54]. The MICs of CLX and cetrimid significantly correlated to qac genes carriage.
We found that the majority of MBL-positive A. baumannii isolates harbor at least one of the qac genes tested and qacE∆1 was the most prevalent, with high nucleotide sequence identities between isolates. High levels of qacE∆1 carriage have been reported in clinical isolates of Acinetobacter, and it has been suggested that the increase in both antibiotic and antiseptic resistance in this organism is related to the presence of this gene [55]. Resistance to several antibiotics has frequently been reported in different clinical isolates in association with qac genes [56,57]. The acquisition of efflux pumps and co-selection of antibiotic resistance genes through their linkage with biocide resistance determinants on the same mobile element are the mechanisms by which biocide resistance increases the distribution of antibiotic-resistant bacteria [58][59][60][61].
Increased frequency of biocide resistance genes in A. baumannii from clinical specimens points to the possibility that the hospital atmosphere could apply selective pressure for presence of these strains. Biocide resistance may allow persistence of organisms in the presence of small levels of biocide and contribute to the survival of MDR strains [62]. Different studies have shown that disinfectant-resistance gene expression can be induced by exposure to subinhibitory concentrations of biocides [53]. Reduction in effectiveness can allow more strains harboring qac genes to survive and spread these genes among A. baumannii, which may explain the higher incidence of these resistance genes.
The genetic similarity between isolates was apparent in the nucleotide sequences of bla VIM , bla NDM-1 , qacE and qacE∆1 and suggests a common genetic vehicle related dissemination of the β-lactamase genes and efflux pump genes. Furthermore, these sequences also showed similarities with those previously reported from different geographical areas. The class 1 integrons are the most common mechanism by which bacteria are able to move resistance gene cassettes from one bacterium to another. The importance of horizontal transfer of resistance genes through mobile genetic elements and its relationship with increased incidence of multidrug-resistant A. baumannii in hospitals is a critical issue. We evaluated intl1 gene which comprises the genetic platforms of class 1 integrons (genetic mobile elements). We found that all MBL-producing A. baumannii isolates harbor the intl1 gene. Since efflux genes are located on mobile genetic elements along with specific antibiotic resistance genes [63], cross-resistance and co-resistance may be acquired together [64]. The MBL genes are mostly harbored by class 1 integrons and the integrons may be targeted as epidemiological molecular markers for identifying and surveying MBL-producing Gram-negative bacilli [2].
In conclusion, a prevalence of MBL-producing A. baumannii isolates was observed in this study. The bla VIM -specific amplicons in these isolates were found to be genetically similar to each other. As well, the genetic similarity between isolates was obvious in the nucleotide sequences of bla NDM-1 genes. Furthermore, these sequences also showed similarities with those previously reported from other countries. Thus, this study argues for the urgent implementation of strict control measures to prevent the spread of resistance genes and that it warrants the need for constant surveillance. It is crucial to highlight the need for health care facilities to assess the antimicrobial effectiveness of biocides periodically to overcome dissemination of MBL-producing A. baumannii with reduced sensitivity to biocides. Reduced susceptibility to CHX and cetrimide and the potential for cross resistance to some antibiotics highlights the need to restrict the use of these biocides. Strains harboring qac genes may be more likely to survive the disinfection process and serve as a source of nosocomial outbreaks. The use of biocides may have driven the fixation and spread of the class 1 integrons, and now may contribute to antibiotic resistance.