Spread of TEM, VIM, SHV, and CTX-M β-Lactamases in Imipenem-Resistant Gram-Negative Bacilli Isolated from Egyptian Hospitals

Carbapenem-resistant Gram-negative bacilli resulting from β-lactamases have been reported to be an important cause of nosocomial infections and are a critical therapeutic problem worldwide. This study aimed to describe the prevalence of imipenem-resistant Gram-negative bacilli isolates and detection of bla VIM, bla TEM, bla SHV, bla CTX-M-1, and bla CTX-M-9 genes in these clinical isolates in Egyptian hospitals. The isolates were collected from various clinical samples, identified by conventional methods and confirmed by API 20E. Antibiotic susceptibility testing was determined by Kirby-Bauer technique and interpreted according to CLSI. Production of bla VIM, bla TEM, bla SHV, and bla CTX-M genes was done by polymerase chain reaction (PCR). Direct sequencing from PCR products was subsequently carried out to identify and confirm these β-lactamases genes. Out of 65 isolates, (46.1%) Escherichia coli, (26.2%) Klebsiella pneumoniae, and (10.7%) Pseudomonas aeruginosa were identified as the commonest Gram-negative bacilli. 33(50.8%) were imipenem-resistant isolates. 22 isolates (66.7%) carried bla VIM, 24(72.7%) had bla TEM, and 5(15%) showed bla SHV, while 12(36%), 6(18.2%), and 0(0.00%) harbored bla CTX-M-1, bla CTX-M-9, and bla CTX-M-8/25, respectively. There is a high occurrence of β-lactamase genes in clinical isolates and sequence analysis of amplified genes showed differences between multiple SNPs (single nucleotide polymorphism) sites in the same gene among local isolates in relation to published sequences.


Introduction
Gram-negative bacilli are a heterogeneous group of Gramnegative bacteria that are common commensals, infectious agents and also sometimes referred to as "nightmare bacteria" [1]. Hospital acquired infections due to Gram-negative bacilli are a leading cause of morbidity and mortality worldwide [2].
Carbapenem, a member of the -lactam family, has a broad spectrum of activity and is stable to most -lactamases. These properties make carbapenem an important therapeutic option for treating serious infections involving resistant strains of Enterobacteriaceae, anaerobes, Pseudomonas aeruginosa, and Acinetobacter spp. [3], although carbapenems, including imipenem and meropenem, are often used as "antibiotics of last resort" when patients with infections become severely ill or are suspected of harboring resistant bacteria [4]. However, carbapenem-resistant Gram-negative bacilli isolates were increasingly reported worldwide [5].
For establishment of appropriate antimicrobial therapy and control of the spread of drug resistant Gram-negative bacilli, the PCR-based detection methods of resistant genes show the bioinformatics analysis of their molecular diversity and evolution becoming increasingly important [8].
This work aimed to study distribution of imipenemresistant Gram-negative isolates and shed focused light on 2 International Journal of Microbiology  some genes encoding beta-lactamase enzymes responsible for such resistance in Zagazig University Hospitals in Egypt.

Molecular Detection of -Lactamase Genes by PCR.
For detection of -lactamase genes responsible for imipenemresistance, rapid genomic DNA was prepared from about five colonies heated in 100 mL distilled water (95 ∘ C for 10 min) followed by a centrifugation step of cell suspension at 12.000 rpm for 5 min; then supernatant was taken as a source of template DNA. PCR amplification was carried out by using DNA thermal cycler (Biometra, Singapore) using a specific primer for VIM , TEM , SHV , CTX-M-1 , CTX-M-9 , and CTX-M-8/25 (Table 1), in a 50 L volume containing 10x PCR buffer, 2 mM deoxynucleoside triphosphates, 3.4 pmol of each primer, 2.5 mM MgCl 2 , 1 U Taq DNA polymerase, and 1 L of genomic DNA [12]. Amplification was carried out as follows: initial denaturation at 94 ∘ C for 10 minutes, followed by 40 cycles of DNA denaturation at 94 ∘ C for 40 seconds, primer annealing at 60 ∘ C for 40 seconds and primer extension at 72 ∘ C for 1 minute, and a final elongation step at 72 ∘ C for 7 minutes. The annealing temperature was optimal at 55 ∘ C instead of 60 ∘ C for amplification of VIM . Amplicons were then visualized after running in 2% agarose gel at 100 V for 30 mins. A 50-1000 bp DNA ladder (USA) was used as a size marker. Finally, PCR products were purified with innuPREP PCRpure kit (Analytik Jena, Germany) and subjected to direct sequencing via GATC Company by use of ABI 3730xl DNA sequencer.

Bioinformatics and Sequences
Analysis. The obtained chromatogram sequencing files were inspected and corrected using the software application Chromas 2.3 (Technelysium, Helensvale, Australia) and JalView (2.8).
The sequences obtained from our samples were aligned with GenBank sequences. The phylogenetic tree for each sequence was obtained by performing neighbor-joining analysis of the alignment of sequences with reference strains (accession numbers/country of origin) that were retrieved from GenBank. The studied strains were marked by the sign [◼]. Meanwhile, the reference sequences were marked by the sign [ ].
The BLAST and FASTA programs of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih .gov/Blast.cgi) were used to search databases for similar nucleotide sequences [13]. Multiple sequence alignments of the nucleic acid were carried out using the ClustalW program. The statistical analysis was performed using SPSS version  20.0, 2 = chi-square test, and values < 0.05 were considered significant.

Sequences Analysis and Polymorphism
3.4.1. The Analysis of VIM Gene ( VIM1,2 ). The sequence of the purified product of VIM gene ( VIM1,2 ) was compared with homologous GenBank sequences using BLAST program and resulted in significant similarity to many metallo-lactamases genes of different bacterial strains.
(1) VIM Gene (bla VIM1,2 ) in Escherichia coli Strains. The pairwise sequences alignments of resulting VIM gene ( VIM1,2 ) in E. coli strains, isolated from Zagazig University (ZU) Hospitals, in comparison with published VIM gene in E. coli strains from GenBank, for example (E. coli KC417377.1), showed single common SNP (single nucleotide polymorphism) sites between the different strains. The SNPs position was indicated in position 382 in the Egyptian strains ( Figure 3(a)). The phylogenetic tree of VIM gene sequence in E. coli strains, isolated from Zagazig University Hospitals, and published homologous sequences in GenBank showed different degrees of dis/similarity between the different strains ( Figure 3(b)). It was interesting to detect that both strains, the most similar and most dissimilar strains, were from the same country, Greece, indicating biodiversity in the same geographical location.
(2) VIM Gene (bla VIM1,2 ) in Klebsiella pneumoniae Strains. The VIM gene isolated from K. pneumoniae strains in Zagazig University Hospitals was compared with published VIM gene in K. pneumoniae strains from GenBank (e.g. K. pneumoniae DQ143913.1). The results showed 6 different common SNPs between the different strains. The SNPs positions were indicated in 45, 150, 168, 284, 309, and 363 ( Figure 3(c)). The phylogenetic tree of VIM gene sequence in K. pneumoniae strains, isolated from ZU Hospitals, and published homologous sequences in GenBank showed different degrees of dis/similarity between the different strains with many unique sequences in the Egyptian strain ( Figure 3(d)).
(3) VIM Gene (bla VIM1,2 ) in Acinetobacter baumanii Strains. The sequence of purified product of VIM gene ( VIM1,2 ) from Acinetobacter baumanii strain was compared with the GenBank sequence using BLAST program. Interestingly, it was revealed that there was a single strain present in GenBank which is completely different from the studied strains and this is not matching with our study (e.g., Staphylococcus phage StB12, complete genome). The sequence alignment showed 9

Sequence
Analysis of TEM Gene ( VIM1,2 ). Sequences of the purified product of VIM gene ( VIM1,2 ) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo--lactamases genes of different bacterial strains.   (2) TEM Gene (bla TEM1,2 ) of Klebsiella pneumonia. Multiple sequences alignments of TEM gene in K. pneumoniae strains, isolated from ZU Hospitals, were compared with other published TEM genes in K. pneumoniae strains from GenBank (e.g., K. pneumoniae KF268357.1). (Figure 4(c)) showed 2 different common SNPs between different strains. The SNPs positions were indicated in 174 and 343. A phylogenetic tree of TEM sequence of K. pneumoniae isolated from Zagazig University Hospitals and other published ones in GenBank showed the degree of similarity between strains where Egyptian strain was dissimilar to that of Iranian and Indian strains (Figure 4(d)).

The Analysis of SHV Gene ( SHV1 ) in
Klebsiella pneumoniae Strains. The pairwise sequences alignments of resulting SHV gene ( SHV1 ) in K. pneumoniae strains, isolated from Zagazig University Hospitals, in comparison with published SHV gene in K. pneumoniae strains from GenBank using BLAST program (e,g., K. pneumoniae AF124984.1) showed five common SNPs between the different strains. The SNPs position was indicated in 454, 563, 631, 635, and 650 in Egyptian strains ( Figure 5(a)). The phylogenetic tree of SHV gene in this case showed that Egyptian strain was more similar to France strain ( Figure 5(b)).

The Analysis of CTX-M-1 Gene ( CTX-M-1 ).
Sequences of the purified product of CTX-M-1 gene ( CTX-M-1 ) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo--lactamases genes of different bacterial strains.
(1) CTX-M-1 Gene (bla CTX-M-1 ) of Escherichia coli Strains. The sequence of the purified product of CTX-M-1 gene from Escherichia coli strains was compared with the GenBank sequence using BLAST program. Interestingly, it was revealed that there were strains present in GenBank which are completely different from the studied strains and this is not matching with our study (e.g., Pseudomonas aeruginosa DNA, AP014646.1). The sequence alignment showed 81 different common SNPs between the different strains. The SNPs positions were indicated in 601, 604 → 615, 617 → 626, and 628 → 688 (Figure 6(a)). The phylogenetic tree of CTX-M-1    sequence of E. coli isolated from Zagazig University Hospitals and published homologous ones in GenBank showed dissimilarity degree between Egyptian and other universal strains (Figure 6(b)).     (Figure 7(a)). The phylogenetic tree of CTX-M-9 gene sequence in E. coli strains, isolated from ZU Hospitals, and published homologous sequences in GenBank showed different degrees of dis/similarity between the different strains and many unique sequences in the Egyptian strain similar to that of Russia and Australia and dissimilar to that of Japan (Figure 7(b)).

Discussion
The Gram-negative bacilli are among the most important causes of serious nosocomial and community-onset bacterial infections in humans and antimicrobial resistance has become a global threat to effective health care delivery [14]. However, carbapenem-resistant Gram-negative bacilli have been increasingly reported worldwide [4]. Various acquired carbapenemases have been identified in the last years, belonging to either acquired metallo-beta-lactamases (IMP, VIM, SPM, GIM, NDM, and DIM types) or class A (KPC and GES) and class D -lactamase OXA-48 [15].
In the present study, prevalence of -lactamasesproducing isolates was found in Table 2. Different studies carried out by other workers in various parts of the world show quite variable results. In a study carried out, the frequency of beta-lactamases-producing isolates was urine (61%), followed by blood cultures (38%), wound swabs (13%), and tracheal aspirates (5%) ( < 0.001) [16]. And this is similar to our study. By contrast, Shanthi and Sekar [17] in India reported that Gram-negative isolates were obtained from the respiratory tract (41.8%) followed by urinary tract (25.5%), wound (20%), and blood (12.7%). Also, pus was the most common specimen accounting for 21% followed by tracheal aspirate (17%), sputum (16%), urine (11%), and blood (7%) [18]. Various risk factors of -lactamases have been implicated in selection and spread producing strains from various clinical samples.
But our study disagreed with that published by Aboderin et al. [22] who reported that Pseudomonas aeruginosa recorded the highest prevalence followed by Klebsiella pneumoniae and ESBL producers, whereas frequency among E. coli isolates was much lower than Klebsiella pneumoniae. Hence, prevalence of pathogens often varies dramatically between communities according to geography, hospitals in the same community and among different patient populations in the same hospital.
In this study, risk factors associated with isolation of Gram-negative bacilli isolates were shown in Table 4. And this was matched with that reported by Kumar et al. [23] who exhibited major risk factors such as prolonged hospitalization > 8 days, previous antibiotic use, trauma, and mechanical ventilation which may contribute to the mortality.
In Turkey, Aktas et al. [24] reported risk factors for acquisition including prolonged hospitalization, an ICU stay, ventilator usage, previous use of carbapenem antibiotics, and the presence of underlying diseases and this is compatible with our research. But in Brazil, Tuon et al. [25] documented that there was statistical significance in isolation of Klebsiella pneumoniae isolates according to age ( = 0.005) and mechanical ventilation ( = 0.003), while trauma ( = 0.87) and ICU stay ( = 0.25) had a statistical significance as major risk factors.
The importance of these risk factors lies in the epidemiological implications at the hospital level because the results suggest a probable nosocomial transmission of the infection.
Resistance pattern among nosocomial bacterial pathogens may vary widely from country to country at any time and within the same country over time [26].
In our study, all the isolates were resistant to cefotaxime (100%) and displayed unusually high level of imipenemresistance (50.8%) isolates with MICs ranging from 15.6 to 250 g/mL (Table 5) ( < 0.001). In Egypt, this was parallel to that reported by Mohamed and Raafat [27] who reported (52.2%) imipenem-resistance among isolates, (100%) cefotaxime resistance, (55%) susceptible-ciprofloxacin, and (70%) susceptibility to amikacin. In the Middle East, the occurrence of imipenem-resistant Gram-negative bacilli is alarmingly elevated. Another similar study showed that all 84 Klebsiella pneumoniae isolates exhibited resistance to imipenem with MICs ranging from 4 to >32 g/mL in a Greek hospital [28].
Another dissimilar study was shown in Saudi Arabia, where the susceptibility rate of Gram-negative organisms isolated from a tertiary care hospital to imipenem was reported to be as low as 10% [29]. Galani et al. [30] reported that Klebsiella pneumoniae and Escherichia coli isolates were found to be susceptible to imipenem in routine susceptibility disk diffusion tests. Other authors observed that all beta-lactamases producers of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were susceptible to imipenem showing coresistance to other antibiotics of aminoglycosides, fluoroquinolones, and others [17]. The extensive use of carbapenems in some locations has likely created a selective antibiotic pressure which in turn has resulted in an increased prevalence of carbapenem-resistant Gram-negative isolates.
The carbapenem resistance due to production oflactamases has a potential for rapid dissemination, since it is often plasmid-mediated [2]. Consequently, rapid detection of -lactamases is necessary to initiate effective infection control measures to prevent their uncontrolled spread in clinical settings. In Egyptian hospitals the -lactamases presence was confirmed by PCR amplification.
In our study, the percentage of VIM , TEM , SHV , and CTX-M genes among Gram-negative bacilli isolates was shown in Table 6. As regards VIM gene detected in our results it is similar to that VIM gene encoding MBL among the isolates of P. aeruginosa (61.3%) in Tehran hospitals [31]. On the contrary, VIM genes were not detected among the studied Gram-negative isolates in other Tehran hospitals [32]. TEM, SHV, and CTX-M genes are the most common plasmid-mediated lactamases often found in  Enterobacteriaceae and P. aeruginosa [33]. In Iran, Eftekhar et al. [34] showed 69.3% TEM and 31.37% CTX-M-1 genes among Gram-negative isolates and this agreed with our study. Also, presence of CTX-M1,9 , SHV , and TEM genes among tested rods of the Enterobacteriaceae family was revealed by Ojdana et al. [35]. Another study reported by Cuzon et al. [36] harbored similar results regarding carbapenem-resistant Gram-negative isolates carrying TEM, SHV, CTX-M-1, and CTX-M-9 genes.
Other authors noted different observations in that clinical isolates of Escherichia coli and Pseudomonas aeruginosa carried CTX-M-types which was the most common (95.8%) followed by TEM (29.2%), SHV (7.3%), and VIM Table 6: The percentage of bla VIM , bla TEM , bla SHV , and bla CTX-M genes among Gram-negative bacilli isolates. (12.5%) in Nepal [38]. These observations contribute to the knowledge of the epidemiology of VIM, TEM, SHV, and CTX-M-producing Gram-negative isolates that have now become endemic in major hospitals in Egypt. Continuous monitoring, proper infection control, and surveillance and prevention practices will limit the further spread of these infections within these hospitals and clinical settings. Multiple sequences analysis is used in such biological studies to extract important phylogenetic and evolutionary information using different scoring matrices (BLOSUM62 for BLAST, BLOSUM50 for SEARCH and FASTA) [39].

Conclusion
There is a high prevalence of -lactamase genes in our clinical isolates that are responsible for such resistance. Hence, it is essential to report -lactamases production along with routine sensitivity reports, which will help the clinician in prescribing proper antibiotics. Also, the sequence analysis of amplified genes showed differences between multiple SNPs in the same gene among different local isolates and with internationally published sequences. In the end, it has been felt that there is a need to formulate strategies to detect and prevent the emergence of -lactamases producing strains for the effective treatment of infections which are caused by them.