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

Authors: El sayed Hamdy Mohammed; Ahmed Elsadek Fakhr; Hanan Mohammed El sayed; Said abd Elmohsen Al Johery; Wesam Abdel Ghani Hassanein
Journal: International Journal of Microbiology
 (2016)

---

## Abstract

Carbapenem-resistant Gram-negative bacilli resulting fromβ-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 b
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, b
l
a
C
T
X
-
M
-
1, and b
l
a
C
T
X
-
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 b
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, and b
l
a
C
T
X
-
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 b
l
a
VIM, 24(72.7%) had b
l
a
TEM, and 5(15%) showed b
l
a
S
H
V, while 12(36%), 6(18.2%), and 0(0.00%) harbored b
l
a
C
T
X
-
M
-
1, b
l
a
C
T
X
-
M
-
9, and b
l
a
C
T
X
-
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.


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## Body

## 1. Introduction

Gram-negative bacilli are a heterogeneous group of Gram-negative 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 theβ-lactam family, has a broad spectrum of activity and is stable to most β-lactamases. These properties make carbapenem an important therapeutic option for treating serious infections involving resistant strains of Enterobacteriaceae, anaerobes,Pseudomonas aeruginosa, andAcinetobacterspp. [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].This resistance may be attributed to presence of metallo-β-lactamase in bacteria such as IMP (Imipenemase), VIM (Verona-Integron metallo-β-lactamase) [6], and extended spectrum β-lactamases (ESBLs) such as SHV, TEM, and CTX-M [7].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 imipenem-resistant Gram-negative isolates and shed focused light on some genes encoding beta-lactamase enzymes responsible for such resistance in Zagazig University Hospitals in Egypt.


## 2. Material and Methods

### 2.1. Bacterial Isolates

Clinical isolates of Gram-negative bacilli includingEscherichia coli (n
=
30),Klebsiella pneumoniae (n
=
17),Pseudomonas aeruginosa (n
=
7),Proteus mirabilis (n
=
2),Citrobacter freundii (n
=
1),Acinetobacter baumanii (n
=
3), andEnterobacter cloacae (n
=
4) were collected from blood, urine, pus, and sputum specimens from hospitalized patients in Zagazig University Hospitals in Egypt from January 2013 to March 2014. These clinical samples were processed by plating on blood agar and MacConkey agar [9]. A growth temperature of 44°C was used sometimes to confirm the identity of these isolates and the identified strains were stored in glycerol (20% V/V) at 70°C and subcultured several times to be viable. All isolates were identified by standard biochemical tests [10] and confirmed by API 20E (BioMérieux, Marcy l’Étoile, France).


### 2.2. Antibiotic Susceptibility Testing

The susceptibility testing of studied isolates was performed by disc diffusion method (modified Kirby-Bauer method) using Muller-Hinton agar (Becton Dickinson, MA, USA) and interpreted according to the Clinical Laboratory Standard Institute (CLSI) guidelines [11]. The antibiotic disks used imipenem (IPM, 10 μg), amikacin (AK, 30 μg), ciprofloxacin (CIP, 5 μg), piperacillin (PRL, 100 μg), cefoperazone/sulbactam (CES, 10
+
5 μg), cefoxitin (FOX, 30 μg), and cefotaxime (CTX, 30 μg) which were placed 15 mm away from the central disc and the plates were incubated for about 18–24 hrs at 37°C.


### 2.3. Molecular Detection ofβ-Lactamase Genes by PCR

For detection ofβ-lactamase genes responsible for imipenem-resistance, 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 b
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, b
l
a
C
T
X
-
M
-
1, b
l
a
C
T
X
-
M
-
9, and b
l
a
C
T
X
-
M
-
8
/
25 (Table 1), in a 50 μL volume containing 10x PCR buffer, 2 mM deoxynucleoside triphosphates, 3.4 pmol of each primer, 2.5 mM MgCl2, 1 U Taq DNA polymerase, and 1 μL 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 b
l
a
V
I
M. 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.Table 1
Primer name, primer  sequences, and expected amplicon size of amplified DNA products.
PCR name
β-lactamase targeted
Primer name
Sequence (5′-3′)
Amplicon size (bp)
VIM
VIM variants including VIM-1 and VIM-2
VIM-for
GATGGTGTTTGGTCGCATA
390
VIM-rev
CGAATGCGCAGCACCAG
TEM
TEM variants including TEM-1 and TEM-2
TSO-T-for
CATTTCCGTGTCGCCCTTATTC
800
TSO-T-rev
CGTTCATCCATAGTTGCCTGAC
SHV
SHV variants including SHV-1
TSO-S-for
AGCCGCTTGAGCAAATTAAAC
713
TSO-S-rev
ATCCCGCAGATAAATCACCAC
CTX-M group 1
Variants of CTX-M group 1 including CTX-M-1, CTX-M-3, and CTX-M-15
CTXMGp 1-for
TTAGGAARTGTGCCGCTGYAb
688
CTXMGp 1.2 rev
CGATATCGTTGGTGGTRCCATb
CTX-M group 9
Variants of CTX-M-9 including CTX-M-9 and CTX-M-14
CTX-9-F
TCAAGCCTGCCGATCTGGT
561
CTX-9-R
TGATTCTCGCCGCTGAAG
CTX-M group 8/25
CTX-M-8, CTX-M-25, CTX-M-26, and CTX-M-39 to CTX-M-41
CTX-8/25-F
AACRCRCAGACGCTCTACb
326
CTX-8/25-R
TCGAGCCGGAASGTGTYATb
bY = T or C; R = A or G; S = G or C.


### 2.4. 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, χ
2 = chi-square test, and P values < 0.05 were considered significant.


## 2.1. Bacterial Isolates

Clinical isolates of Gram-negative bacilli includingEscherichia coli (n
=
30),Klebsiella pneumoniae (n
=
17),Pseudomonas aeruginosa (n
=
7),Proteus mirabilis (n
=
2),Citrobacter freundii (n
=
1),Acinetobacter baumanii (n
=
3), andEnterobacter cloacae (n
=
4) were collected from blood, urine, pus, and sputum specimens from hospitalized patients in Zagazig University Hospitals in Egypt from January 2013 to March 2014. These clinical samples were processed by plating on blood agar and MacConkey agar [9]. A growth temperature of 44°C was used sometimes to confirm the identity of these isolates and the identified strains were stored in glycerol (20% V/V) at 70°C and subcultured several times to be viable. All isolates were identified by standard biochemical tests [10] and confirmed by API 20E (BioMérieux, Marcy l’Étoile, France).


## 2.2. Antibiotic Susceptibility Testing

The susceptibility testing of studied isolates was performed by disc diffusion method (modified Kirby-Bauer method) using Muller-Hinton agar (Becton Dickinson, MA, USA) and interpreted according to the Clinical Laboratory Standard Institute (CLSI) guidelines [11]. The antibiotic disks used imipenem (IPM, 10 μg), amikacin (AK, 30 μg), ciprofloxacin (CIP, 5 μg), piperacillin (PRL, 100 μg), cefoperazone/sulbactam (CES, 10
+
5 μg), cefoxitin (FOX, 30 μg), and cefotaxime (CTX, 30 μg) which were placed 15 mm away from the central disc and the plates were incubated for about 18–24 hrs at 37°C.


## 2.3. Molecular Detection ofβ-Lactamase Genes by PCR

For detection ofβ-lactamase genes responsible for imipenem-resistance, 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 b
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, b
l
a
C
T
X
-
M
-
1, b
l
a
C
T
X
-
M
-
9, and b
l
a
C
T
X
-
M
-
8
/
25 (Table 1), in a 50 μL volume containing 10x PCR buffer, 2 mM deoxynucleoside triphosphates, 3.4 pmol of each primer, 2.5 mM MgCl2, 1 U Taq DNA polymerase, and 1 μL 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 b
l
a
V
I
M. 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.Table 1
Primer name, primer  sequences, and expected amplicon size of amplified DNA products.
PCR name
β-lactamase targeted
Primer name
Sequence (5′-3′)
Amplicon size (bp)
VIM
VIM variants including VIM-1 and VIM-2
VIM-for
GATGGTGTTTGGTCGCATA
390
VIM-rev
CGAATGCGCAGCACCAG
TEM
TEM variants including TEM-1 and TEM-2
TSO-T-for
CATTTCCGTGTCGCCCTTATTC
800
TSO-T-rev
CGTTCATCCATAGTTGCCTGAC
SHV
SHV variants including SHV-1
TSO-S-for
AGCCGCTTGAGCAAATTAAAC
713
TSO-S-rev
ATCCCGCAGATAAATCACCAC
CTX-M group 1
Variants of CTX-M group 1 including CTX-M-1, CTX-M-3, and CTX-M-15
CTXMGp 1-for
TTAGGAARTGTGCCGCTGYAb
688
CTXMGp 1.2 rev
CGATATCGTTGGTGGTRCCATb
CTX-M group 9
Variants of CTX-M-9 including CTX-M-9 and CTX-M-14
CTX-9-F
TCAAGCCTGCCGATCTGGT
561
CTX-9-R
TGATTCTCGCCGCTGAAG
CTX-M group 8/25
CTX-M-8, CTX-M-25, CTX-M-26, and CTX-M-39 to CTX-M-41
CTX-8/25-F
AACRCRCAGACGCTCTACb
326
CTX-8/25-R
TCGAGCCGGAASGTGTYATb
bY = T or C; R = A or G; S = G or C.


## 2.4. 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, χ
2 = chi-square test, and P values < 0.05 were considered significant.


## 3. Results

### 3.1. Isolation and Identification

The present study was conducted on 65 screened isolates of Gram-negative bacilli, obtained from 108 various clinical samples such as urine, blood, pus, and sputum, where 41 (78.8%) were isolated from urine, 12 (66.6%) from blood, 14 (48.2%) from respiratory secretions, and 2 (22.2%) from pus.Escherichia coli (46.1%) was the most commonly isolated organism among Gram-negative bacilli, followed byKlebsiella pneumoniae (26.2%),Pseudomonas aeruginosa (10.7%),Enterobacter cloacae (6.1%),Proteus mirabilis (3.07%), andAcinetobacter baumanii (4.6%) whileCitrobacter freundii andProteus vulgaris gave 1.5%. The isolation of Gram-negative bacilli isolates was significantly higher in patients with trauma (P
<
0.001), those hospitalized for more than 7 days (P
<
0.001), and those with ICU admission (P
<
0.001) harboring risk factors for acquiring Gram-negative bacilli infection.


### 3.2. The Antibiotic Susceptibility Testing

It was shown in Figure1, as observed, that 40 (61.5%) were susceptible to amikacin, 35 (53.8%) were susceptible to ciprofloxacin, 31 (47.7%) were susceptible to imipenem, and 21 (32.3%) were susceptible to cefoperazone/sulbactam. However, all isolates were resistant to cefotaxime (100%), 53 (81.5%) of isolates were resistant to cefoxitin, 44 (67.7%) were resistant to piperacillin, 40 (61.5%) were resistant to cefoperazone/sulbactam, 33 (50.8%) were resistant to imipenem followed by 24 (44.6%) resistant to amikacin, and 27 (41.5%) were resistant to ciprofloxacin.Figure 1
Antimicrobial susceptibility patterns of all 65 Gram-negative bacilli isolates.


### 3.3. PCR Assay Results

As shown in Table6, of 33 imipenem-resistant Gram-negative bacillistrains, the results of PCR amplification products of β-lactamases genes showed that 66.7% of isolates carried b
l
a
V
I
M at 390 bp (Figure 2(a)), 72.7% had b
l
a
T
E
M at 800 bp (Figure 2(b)), 36.0% harbored b
l
a
C
T
X
-
M
-
1 at 688 bp (Figure 2(c)), and 15.0% showed b
l
a
S
H
V (Figure 2(d)), while 18.2% (Figure 2(e)), 0.00% (Figure 2(f)) harbored b
l
a
C
T
X
-
M
-
9, b
l
a
C
T
X
-
M
-
8
/
25, respectively. Sequencing confirmed presence of these β-lactamase genes; the GenBank nucleotide sequence accession numbers for the sequences studied are detailed. Aligning of the obtained sequences with those of reference strains in GenBank confirmed the correct identification of b
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, and b
l
a
C
T
X
-
M genes by PCR.Figure 2
Presence and absence ofβ-lactamase genes by PCR amplification in some isolated samples. (a) The existence of VIM amplification fragment (390 bp). (b) The amplification of TEM fragments (800 bp). (c) The amplification of CTX-M-1 (688 bp). (d) The presence of SHV (713 bp) in some samples. (e) The presence of CTX-M-9 (561 bp). (f) No amplification was shown with CTX-M-8/25 primer at 326 bp.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.4. Sequences Analysis and Polymorphism

#### 3.4.1. The Analysis of VIM Gene (b
l
a
V
I
M
1,2)

The sequence of the purified product of VIM gene (b
l
a
V
I
M
1,2) was compared with homologous GenBank sequences using BLAST program and resulted in significant similarity to many metallo-β-lactamases genes of different bacterial strains.(1) VIM Gene (bla
VIM1,2
) in Escherichia coli Strains. The pairwise sequences alignments of resulting VIM gene (b
l
a
V
I
M
1,2) inE. coli strains, isolated from Zagazig University (ZU) Hospitals, in comparison with published VIM gene inE. coli strains from GenBank, for example (E. coliKC417377.1), 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 inE. 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.Figure 3
The multiple sequences alignments and trees of VIM gene in different species. (a) VIM gene inE. colistrains isolated from Zagazig University (ZU) Hospitals in comparison with published VIM gene ofE. colistrains from GenBank. (b) A tree of VIM sequence ofE. coliisolated from ZU Hospitals and published homologs in GenBank. (c) The multiple sequences alignments of VIM gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published VIM gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of VIM sequence ofK. pneumoniaeisolated from ZU Hospitals and published sequences in GenBank. (e) The multiple sequences alignments of VIM gene inA. baumaniistrains isolated from ZU Hospitals in comparison with published VIM gene inA. baumaniistrains from GenBank. (f) Phylogenetic tree of VIM sequence ofA. baumaniiisolated from ZU Hospitals and published sequences in GenBank.
(a)
(b)
(c)
(d)
(e)
(f)(2) VIM Gene (bla
VIM1,2
) in Klebsiella pneumoniae Strains. The VIM gene isolated fromK. pneumoniae strains in Zagazig University Hospitals was compared with published VIM gene inK. 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 inK. 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 (b
l
a
V
I
M
1,2) fromAcinetobacter 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 different common SNPs between the different strains. These positions were indicated in 246, 284, 306, 309, 330, 363, 378, 382, and 383 (Figure 3(e)). The phylogenetic tree of VIM sequence ofA. baumanii isolated from ZU Hospitals and published ones in GenBank showed dissimilarity between Egyptian strains and others (Figure 3(f)).


#### 3.4.2. Sequence Analysis of TEM Gene (b
l
a
V
I
M
1,2)

Sequences of the purified product of VIM gene (b
l
a
V
I
M
1,2) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo-β-lactamases genes of different bacterial strains.(1) TEM Gene (bla
TEM1,2
) of Escherichia coli. The TEM gene sequences ofE. coli strains, isolated from ZU Hospitals, were aligned with sequences of published TEM genes inE. coli strains from GenBank (e.g.,E. coli KM598665.1). The resulting alignments showed 4 different common SNP sites between the different strains. The SNPs positions were indicated commonly in 216, 232, 385, and 433 (Figure 4(a)). Phylogenetic tree was constructed from TEM gene (b
l
a
T
E
M
1,2) sequence ofEscherichia coli strains isolated from ZU Hospitals and published homologous sequences in GenBank (Figure 4(b)) showing similarity degree between Egyptian and Indian strains.Figure 4
The multiple sequences alignments and trees of TEM gene (b
l
a
T
E
M
1,2) in different bacterial species. (a) TEM gene inE. colistrains isolated from ZU Hospitals in comparison with published TEM gene ofE. colistrains from GenBank. (b) A phylogenetic tree of TEM gene ofE. coliisolated from ZU Hospitals and published homologous ones in GenBank. (c) The multiple sequences alignments of gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published TEM gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of sequence ofK. pneumoniae isolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)
(c)
(d)(2) TEM Gene (bla
TEM1,2
) of Klebsiella pneumonia. Multiple sequences alignments of TEM gene inK. pneumoniae strains, isolated from ZU Hospitals, were compared with other published TEM genes inK. 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 ofK.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)).


#### 3.4.3. The Analysis of SHV Gene (b
l
a
S
H
V
1) inKlebsiella pneumoniae Strains

The pairwise sequences alignments of resulting SHV gene (b
l
a
S
H
V
1) inK. pneumoniae strains, isolated from Zagazig University Hospitals, in comparison with published SHV gene inK. 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)).Figure 5
The multiple sequences alignments and trees of SHV gene in different species. (a) SHV gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published SHV gene ofK. pneumoniaestrains from GenBank. (b) Phylogenetic tree of SHV sequence ofK. pneumoniaeisolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)


#### 3.4.4. The Analysis of CTX-M-1 Gene (b
l
a
C
T
X
-
M
-
1)

Sequences of the purified product of CTX-M-1 gene (b
l
a
C
T
X
-
M
-
1) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo-β-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 fromEscherichia 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 ofE. 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 6
The multiple sequences alignments and trees of CTX-M-1 gene in different species. (a) CTX-M-1 gene inE. colistrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene ofE. colistrains from GenBank. (b) A tree of CTX-M-1 sequence ofE. coliisolated from ZU Hospitals and published homologs in GenBank. (c) The multiple sequences alignments of CTX-M-1 gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of CTX-M-1 sequence ofK. pneumoniaeisolated from ZU Hospitals and published homologous sequences in GenBank. (e) Multiple sequences alignments of CTX-M-1 gene inP. aeruginosastrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inP. aeruginosastrains from GenBank. (f) Phylogenetic tree of CTX-M-1 sequence ofP. aeruginosaisolated from ZU Hospitals and published homologs in GenBank.
(a)
(b)
(c)
(d)
(e)
(f)(2) CTX-M-1 Gene (bla
CTX-M-1
) of Klebsiella pneumoniae Strains. The alignment in this case showed 39 different common SNPs between the different strains. The SNPs positions were indicated in positions 1 → 39 (Figure 6(c)). A phylogenetic tree of CTX-M-1 gene sequence inK. 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 Egyptian strain (Figure 6(d)).(3) CTX-M-1 Gene (bla
CTX-M-1
) of Pseudomonas aeruginosa Strains. The CTX-M-1 gene isolated fromP. aeruginosastrains in Zagazig University Hospitals was compared using BLAST program with published CTX-M-1 gene inP. aeruginosastrains from GenBank (e.g.,P.aeruginosa KC571255.1). The results showed 4 different common SNPs between the different strains. The SNPs positions were indicated in 117, 206, 228, and 283 (Figure 6(e)). A phylogenetic tree showed similarity degree between Egyptian and Russian strains (Figure 6(f)).


#### 3.4.5. The Analysis of CTX-M-9 Gene (b
l
a
C
T
X
-
M
-
9) inEscherichia coli Strains

The CTX-M-9 gene isolated fromE. coli strains in Zagazig University Hospitals was compared with published CTX-M-1 gene inP. aeruginosa strains from GenBank using BLAST program (e.g.,E. coli JN676843.1). The multiple sequences alignments showed 2 different common SNPs in positions 74 and 272 (Figure 7(a)). The phylogenetic tree of CTX-M-9 gene sequence inE. 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)).Figure 7
The multiple sequences alignments and tree of CTX-M-9 gene in different species. (a) CTX-M-9 gene inE. colistrains isolated from ZU Hospitals in comparison with published CTX-M-9 gene ofE. colistrains from GenBank. (b) Phylogenetic tree of CTX-M-9 sequence ofE. coliisolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)


## 3.1. Isolation and Identification

The present study was conducted on 65 screened isolates of Gram-negative bacilli, obtained from 108 various clinical samples such as urine, blood, pus, and sputum, where 41 (78.8%) were isolated from urine, 12 (66.6%) from blood, 14 (48.2%) from respiratory secretions, and 2 (22.2%) from pus.Escherichia coli (46.1%) was the most commonly isolated organism among Gram-negative bacilli, followed byKlebsiella pneumoniae (26.2%),Pseudomonas aeruginosa (10.7%),Enterobacter cloacae (6.1%),Proteus mirabilis (3.07%), andAcinetobacter baumanii (4.6%) whileCitrobacter freundii andProteus vulgaris gave 1.5%. The isolation of Gram-negative bacilli isolates was significantly higher in patients with trauma (P
<
0.001), those hospitalized for more than 7 days (P
<
0.001), and those with ICU admission (P
<
0.001) harboring risk factors for acquiring Gram-negative bacilli infection.


## 3.2. The Antibiotic Susceptibility Testing

It was shown in Figure1, as observed, that 40 (61.5%) were susceptible to amikacin, 35 (53.8%) were susceptible to ciprofloxacin, 31 (47.7%) were susceptible to imipenem, and 21 (32.3%) were susceptible to cefoperazone/sulbactam. However, all isolates were resistant to cefotaxime (100%), 53 (81.5%) of isolates were resistant to cefoxitin, 44 (67.7%) were resistant to piperacillin, 40 (61.5%) were resistant to cefoperazone/sulbactam, 33 (50.8%) were resistant to imipenem followed by 24 (44.6%) resistant to amikacin, and 27 (41.5%) were resistant to ciprofloxacin.Figure 1
Antimicrobial susceptibility patterns of all 65 Gram-negative bacilli isolates.


## 3.3. PCR Assay Results

As shown in Table6, of 33 imipenem-resistant Gram-negative bacillistrains, the results of PCR amplification products of β-lactamases genes showed that 66.7% of isolates carried b
l
a
V
I
M at 390 bp (Figure 2(a)), 72.7% had b
l
a
T
E
M at 800 bp (Figure 2(b)), 36.0% harbored b
l
a
C
T
X
-
M
-
1 at 688 bp (Figure 2(c)), and 15.0% showed b
l
a
S
H
V (Figure 2(d)), while 18.2% (Figure 2(e)), 0.00% (Figure 2(f)) harbored b
l
a
C
T
X
-
M
-
9, b
l
a
C
T
X
-
M
-
8
/
25, respectively. Sequencing confirmed presence of these β-lactamase genes; the GenBank nucleotide sequence accession numbers for the sequences studied are detailed. Aligning of the obtained sequences with those of reference strains in GenBank confirmed the correct identification of b
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, and b
l
a
C
T
X
-
M genes by PCR.Figure 2
Presence and absence ofβ-lactamase genes by PCR amplification in some isolated samples. (a) The existence of VIM amplification fragment (390 bp). (b) The amplification of TEM fragments (800 bp). (c) The amplification of CTX-M-1 (688 bp). (d) The presence of SHV (713 bp) in some samples. (e) The presence of CTX-M-9 (561 bp). (f) No amplification was shown with CTX-M-8/25 primer at 326 bp.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.4. Sequences Analysis and Polymorphism

### 3.4.1. The Analysis of VIM Gene (b
l
a
V
I
M
1,2)

The sequence of the purified product of VIM gene (b
l
a
V
I
M
1,2) was compared with homologous GenBank sequences using BLAST program and resulted in significant similarity to many metallo-β-lactamases genes of different bacterial strains.(1) VIM Gene (bla
VIM1,2
) in Escherichia coli Strains. The pairwise sequences alignments of resulting VIM gene (b
l
a
V
I
M
1,2) inE. coli strains, isolated from Zagazig University (ZU) Hospitals, in comparison with published VIM gene inE. coli strains from GenBank, for example (E. coliKC417377.1), 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 inE. 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.Figure 3
The multiple sequences alignments and trees of VIM gene in different species. (a) VIM gene inE. colistrains isolated from Zagazig University (ZU) Hospitals in comparison with published VIM gene ofE. colistrains from GenBank. (b) A tree of VIM sequence ofE. coliisolated from ZU Hospitals and published homologs in GenBank. (c) The multiple sequences alignments of VIM gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published VIM gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of VIM sequence ofK. pneumoniaeisolated from ZU Hospitals and published sequences in GenBank. (e) The multiple sequences alignments of VIM gene inA. baumaniistrains isolated from ZU Hospitals in comparison with published VIM gene inA. baumaniistrains from GenBank. (f) Phylogenetic tree of VIM sequence ofA. baumaniiisolated from ZU Hospitals and published sequences in GenBank.
(a)
(b)
(c)
(d)
(e)
(f)(2) VIM Gene (bla
VIM1,2
) in Klebsiella pneumoniae Strains. The VIM gene isolated fromK. pneumoniae strains in Zagazig University Hospitals was compared with published VIM gene inK. 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 inK. 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 (b
l
a
V
I
M
1,2) fromAcinetobacter 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 different common SNPs between the different strains. These positions were indicated in 246, 284, 306, 309, 330, 363, 378, 382, and 383 (Figure 3(e)). The phylogenetic tree of VIM sequence ofA. baumanii isolated from ZU Hospitals and published ones in GenBank showed dissimilarity between Egyptian strains and others (Figure 3(f)).


### 3.4.2. Sequence Analysis of TEM Gene (b
l
a
V
I
M
1,2)

Sequences of the purified product of VIM gene (b
l
a
V
I
M
1,2) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo-β-lactamases genes of different bacterial strains.(1) TEM Gene (bla
TEM1,2
) of Escherichia coli. The TEM gene sequences ofE. coli strains, isolated from ZU Hospitals, were aligned with sequences of published TEM genes inE. coli strains from GenBank (e.g.,E. coli KM598665.1). The resulting alignments showed 4 different common SNP sites between the different strains. The SNPs positions were indicated commonly in 216, 232, 385, and 433 (Figure 4(a)). Phylogenetic tree was constructed from TEM gene (b
l
a
T
E
M
1,2) sequence ofEscherichia coli strains isolated from ZU Hospitals and published homologous sequences in GenBank (Figure 4(b)) showing similarity degree between Egyptian and Indian strains.Figure 4
The multiple sequences alignments and trees of TEM gene (b
l
a
T
E
M
1,2) in different bacterial species. (a) TEM gene inE. colistrains isolated from ZU Hospitals in comparison with published TEM gene ofE. colistrains from GenBank. (b) A phylogenetic tree of TEM gene ofE. coliisolated from ZU Hospitals and published homologous ones in GenBank. (c) The multiple sequences alignments of gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published TEM gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of sequence ofK. pneumoniae isolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)
(c)
(d)(2) TEM Gene (bla
TEM1,2
) of Klebsiella pneumonia. Multiple sequences alignments of TEM gene inK. pneumoniae strains, isolated from ZU Hospitals, were compared with other published TEM genes inK. 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 ofK.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)).


### 3.4.3. The Analysis of SHV Gene (b
l
a
S
H
V
1) inKlebsiella pneumoniae Strains

The pairwise sequences alignments of resulting SHV gene (b
l
a
S
H
V
1) inK. pneumoniae strains, isolated from Zagazig University Hospitals, in comparison with published SHV gene inK. 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)).Figure 5
The multiple sequences alignments and trees of SHV gene in different species. (a) SHV gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published SHV gene ofK. pneumoniaestrains from GenBank. (b) Phylogenetic tree of SHV sequence ofK. pneumoniaeisolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)


### 3.4.4. The Analysis of CTX-M-1 Gene (b
l
a
C
T
X
-
M
-
1)

Sequences of the purified product of CTX-M-1 gene (b
l
a
C
T
X
-
M
-
1) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo-β-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 fromEscherichia 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 ofE. 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 6
The multiple sequences alignments and trees of CTX-M-1 gene in different species. (a) CTX-M-1 gene inE. colistrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene ofE. colistrains from GenBank. (b) A tree of CTX-M-1 sequence ofE. coliisolated from ZU Hospitals and published homologs in GenBank. (c) The multiple sequences alignments of CTX-M-1 gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of CTX-M-1 sequence ofK. pneumoniaeisolated from ZU Hospitals and published homologous sequences in GenBank. (e) Multiple sequences alignments of CTX-M-1 gene inP. aeruginosastrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inP. aeruginosastrains from GenBank. (f) Phylogenetic tree of CTX-M-1 sequence ofP. aeruginosaisolated from ZU Hospitals and published homologs in GenBank.
(a)
(b)
(c)
(d)
(e)
(f)(2) CTX-M-1 Gene (bla
CTX-M-1
) of Klebsiella pneumoniae Strains. The alignment in this case showed 39 different common SNPs between the different strains. The SNPs positions were indicated in positions 1 → 39 (Figure 6(c)). A phylogenetic tree of CTX-M-1 gene sequence inK. 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 Egyptian strain (Figure 6(d)).(3) CTX-M-1 Gene (bla
CTX-M-1
) of Pseudomonas aeruginosa Strains. The CTX-M-1 gene isolated fromP. aeruginosastrains in Zagazig University Hospitals was compared using BLAST program with published CTX-M-1 gene inP. aeruginosastrains from GenBank (e.g.,P.aeruginosa KC571255.1). The results showed 4 different common SNPs between the different strains. The SNPs positions were indicated in 117, 206, 228, and 283 (Figure 6(e)). A phylogenetic tree showed similarity degree between Egyptian and Russian strains (Figure 6(f)).


### 3.4.5. The Analysis of CTX-M-9 Gene (b
l
a
C
T
X
-
M
-
9) inEscherichia coli Strains

The CTX-M-9 gene isolated fromE. coli strains in Zagazig University Hospitals was compared with published CTX-M-1 gene inP. aeruginosa strains from GenBank using BLAST program (e.g.,E. coli JN676843.1). The multiple sequences alignments showed 2 different common SNPs in positions 74 and 272 (Figure 7(a)). The phylogenetic tree of CTX-M-9 gene sequence inE. 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)).Figure 7
The multiple sequences alignments and tree of CTX-M-9 gene in different species. (a) CTX-M-9 gene inE. colistrains isolated from ZU Hospitals in comparison with published CTX-M-9 gene ofE. colistrains from GenBank. (b) Phylogenetic tree of CTX-M-9 sequence ofE. coliisolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)


## 3.4.1. The Analysis of VIM Gene (b
l
a
V
I
M
1,2)

The sequence of the purified product of VIM gene (b
l
a
V
I
M
1,2) was compared with homologous GenBank sequences using BLAST program and resulted in significant similarity to many metallo-β-lactamases genes of different bacterial strains.(1) VIM Gene (bla
VIM1,2
) in Escherichia coli Strains. The pairwise sequences alignments of resulting VIM gene (b
l
a
V
I
M
1,2) inE. coli strains, isolated from Zagazig University (ZU) Hospitals, in comparison with published VIM gene inE. coli strains from GenBank, for example (E. coliKC417377.1), 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 inE. 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.Figure 3
The multiple sequences alignments and trees of VIM gene in different species. (a) VIM gene inE. colistrains isolated from Zagazig University (ZU) Hospitals in comparison with published VIM gene ofE. colistrains from GenBank. (b) A tree of VIM sequence ofE. coliisolated from ZU Hospitals and published homologs in GenBank. (c) The multiple sequences alignments of VIM gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published VIM gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of VIM sequence ofK. pneumoniaeisolated from ZU Hospitals and published sequences in GenBank. (e) The multiple sequences alignments of VIM gene inA. baumaniistrains isolated from ZU Hospitals in comparison with published VIM gene inA. baumaniistrains from GenBank. (f) Phylogenetic tree of VIM sequence ofA. baumaniiisolated from ZU Hospitals and published sequences in GenBank.
(a)
(b)
(c)
(d)
(e)
(f)(2) VIM Gene (bla
VIM1,2
) in Klebsiella pneumoniae Strains. The VIM gene isolated fromK. pneumoniae strains in Zagazig University Hospitals was compared with published VIM gene inK. 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 inK. 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 (b
l
a
V
I
M
1,2) fromAcinetobacter 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 different common SNPs between the different strains. These positions were indicated in 246, 284, 306, 309, 330, 363, 378, 382, and 383 (Figure 3(e)). The phylogenetic tree of VIM sequence ofA. baumanii isolated from ZU Hospitals and published ones in GenBank showed dissimilarity between Egyptian strains and others (Figure 3(f)).


## 3.4.2. Sequence Analysis of TEM Gene (b
l
a
V
I
M
1,2)

Sequences of the purified product of VIM gene (b
l
a
V
I
M
1,2) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo-β-lactamases genes of different bacterial strains.(1) TEM Gene (bla
TEM1,2
) of Escherichia coli. The TEM gene sequences ofE. coli strains, isolated from ZU Hospitals, were aligned with sequences of published TEM genes inE. coli strains from GenBank (e.g.,E. coli KM598665.1). The resulting alignments showed 4 different common SNP sites between the different strains. The SNPs positions were indicated commonly in 216, 232, 385, and 433 (Figure 4(a)). Phylogenetic tree was constructed from TEM gene (b
l
a
T
E
M
1,2) sequence ofEscherichia coli strains isolated from ZU Hospitals and published homologous sequences in GenBank (Figure 4(b)) showing similarity degree between Egyptian and Indian strains.Figure 4
The multiple sequences alignments and trees of TEM gene (b
l
a
T
E
M
1,2) in different bacterial species. (a) TEM gene inE. colistrains isolated from ZU Hospitals in comparison with published TEM gene ofE. colistrains from GenBank. (b) A phylogenetic tree of TEM gene ofE. coliisolated from ZU Hospitals and published homologous ones in GenBank. (c) The multiple sequences alignments of gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published TEM gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of sequence ofK. pneumoniae isolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)
(c)
(d)(2) TEM Gene (bla
TEM1,2
) of Klebsiella pneumonia. Multiple sequences alignments of TEM gene inK. pneumoniae strains, isolated from ZU Hospitals, were compared with other published TEM genes inK. 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 ofK.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)).


## 3.4.3. The Analysis of SHV Gene (b
l
a
S
H
V
1) inKlebsiella pneumoniae Strains

The pairwise sequences alignments of resulting SHV gene (b
l
a
S
H
V
1) inK. pneumoniae strains, isolated from Zagazig University Hospitals, in comparison with published SHV gene inK. 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)).Figure 5
The multiple sequences alignments and trees of SHV gene in different species. (a) SHV gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published SHV gene ofK. pneumoniaestrains from GenBank. (b) Phylogenetic tree of SHV sequence ofK. pneumoniaeisolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)


## 3.4.4. The Analysis of CTX-M-1 Gene (b
l
a
C
T
X
-
M
-
1)

Sequences of the purified product of CTX-M-1 gene (b
l
a
C
T
X
-
M
-
1) were compared with homologous counterpart GenBank database using BLAST program and resulted in significant similarity to many metallo-β-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 fromEscherichia 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 ofE. 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 6
The multiple sequences alignments and trees of CTX-M-1 gene in different species. (a) CTX-M-1 gene inE. colistrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene ofE. colistrains from GenBank. (b) A tree of CTX-M-1 sequence ofE. coliisolated from ZU Hospitals and published homologs in GenBank. (c) The multiple sequences alignments of CTX-M-1 gene inK. pneumoniaestrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inK. pneumoniaestrains from GenBank. (d) Phylogenetic tree of CTX-M-1 sequence ofK. pneumoniaeisolated from ZU Hospitals and published homologous sequences in GenBank. (e) Multiple sequences alignments of CTX-M-1 gene inP. aeruginosastrains isolated from ZU Hospitals in comparison with published CTX-M-1 gene inP. aeruginosastrains from GenBank. (f) Phylogenetic tree of CTX-M-1 sequence ofP. aeruginosaisolated from ZU Hospitals and published homologs in GenBank.
(a)
(b)
(c)
(d)
(e)
(f)(2) CTX-M-1 Gene (bla
CTX-M-1
) of Klebsiella pneumoniae Strains. The alignment in this case showed 39 different common SNPs between the different strains. The SNPs positions were indicated in positions 1 → 39 (Figure 6(c)). A phylogenetic tree of CTX-M-1 gene sequence inK. 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 Egyptian strain (Figure 6(d)).(3) CTX-M-1 Gene (bla
CTX-M-1
) of Pseudomonas aeruginosa Strains. The CTX-M-1 gene isolated fromP. aeruginosastrains in Zagazig University Hospitals was compared using BLAST program with published CTX-M-1 gene inP. aeruginosastrains from GenBank (e.g.,P.aeruginosa KC571255.1). The results showed 4 different common SNPs between the different strains. The SNPs positions were indicated in 117, 206, 228, and 283 (Figure 6(e)). A phylogenetic tree showed similarity degree between Egyptian and Russian strains (Figure 6(f)).


## 3.4.5. The Analysis of CTX-M-9 Gene (b
l
a
C
T
X
-
M
-
9) inEscherichia coli Strains

The CTX-M-9 gene isolated fromE. coli strains in Zagazig University Hospitals was compared with published CTX-M-1 gene inP. aeruginosa strains from GenBank using BLAST program (e.g.,E. coli JN676843.1). The multiple sequences alignments showed 2 different common SNPs in positions 74 and 272 (Figure 7(a)). The phylogenetic tree of CTX-M-9 gene sequence inE. 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)).Figure 7
The multiple sequences alignments and tree of CTX-M-9 gene in different species. (a) CTX-M-9 gene inE. colistrains isolated from ZU Hospitals in comparison with published CTX-M-9 gene ofE. colistrains from GenBank. (b) Phylogenetic tree of CTX-M-9 sequence ofE. coliisolated from ZU Hospitals and published homologous ones in GenBank.
(a)
(b)


## 4. 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 β-lactamase OXA-48 [15].In the present study, prevalence ofβ-lactamases-producing 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%) (P
<
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].Table 2
The distribution of Gram-negative bacilli isolates in each clinical specimen.
Clinical  specimens
Gram-negative bacilli isolates
Type
Number
Number
(%)
Urine
52
41
78.8
Blood
18
12
66.6
Sputum
29
10
34.5
Pus
9
2
22.2
Total
108
65
(60.2%)
χ
2
=
21.28,  P
<
0.001 (statistically significant).Various risk factors ofβ-lactamases have been implicated in selection and spread producing strains from various clinical samples.In accordance with distribution of Gram-negative bacilli, (46.1%)Escherichia coli and (26.2%)Klebsiella pneumoniae isolates followed by (10.7%)P.aeruginosa were identified as the commonest isolates among Gram-negative bacilli (Table 3) and this coincided with that concluded by Sahu et al. [19] who found that 58% were identified asEscherichia coli followed by 27.7%Klebsiella pneumoniae and 15%Pseudomonas aeruginosain Udaipur, Rajasthan. In a study by Vipin et al. [20] 52 (58.42%) isolates ofEscherichia coliwere found to be the most common organisms in Allahabad followed byKlebsiella pneumoniae(20.22%),Pseudomonas aeruginosa(12.35%),Proteus vulgaris(3.37%),Proteus mirabilis (2.24%), andEnterobacter cloacae(2.24%). Also, in India, Sankarankutty and Kaup [21] documented the same result in that 58.42% isolates ofEscherichia coliwere found to be the most common organisms followed byKlebsiella pneumoniae(20.22%),Pseudomonas aeruginosa(12.35%),Proteus vulgaris(3.37%),Proteus mirabilis(2.24%), andEnterobacter aerogenes(2.24%).Table 3
API 20E identification of Gram-negative  bacilli isolates among positive  clinical specimens.
Organism
Total = 65
Number
(%)
Escherichia coli
30
46.1
Klebsiella pneumoniae
17
26.2
Enterobacter cloacae
4
6.1
Pseudomonas aeruginosa
7
10.7
Proteus mirabilis
2
3.07
Acinetobacter baumanii
3
4.6
Citrobacter freundii
1
1.5
Proteus vulgaris
1
1.5
P
<
0.001 (statistically significant).But our study disagreed with that published by Aboderin et al. [22] who reported thatPseudomonas aeruginosarecorded the highest prevalence followed byKlebsiella pneumoniae and ESBL producers, whereas frequency amongE. coli isolates was much lower thanKlebsiella 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 Table4. 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.Table 4
Risk factors associated with isolation of Gram-negative bacilli isolates.
Risk factor
Gram-negative bacilli isolates
Total number of samples
Relative risk (95% CI)∗
P value significance
Age
<40
26
45
1.07 (0.78–1.47)
0.66
>40
39
63
Sex
Male
29
49
1.09 (0.8–1.47)
0.59
Female
36
59
Trauma
Yes
48
59
2.34 (1.57–3.51)
<0.001
No
17
49
Hospitalization length other than ICU
<7 days
15
51
2.98 (1.33–4.61)
<0.001
>7 days
50
57
ICU admission
Yes
28
46
1.02 (0.45–2.47)
<0.001
No
37
62
Urinary catheter
Yes
18
32
0.91 (0.64–1.29)
0.58
No
47
76
Ventilator support
Yes
10
18
0.83 (0.51–1.35)
0.44
No
20
30
Central venous catheter
Yes
4
9
0.67 (0.31–1.43)
0.26
No
26
39
∗ = 95% confidence interval.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 ofKlebsiella pneumoniae isolates according to age (P
=
0.005) and mechanical ventilation (P
=
0.003), while trauma (P
=
0.87) and ICU stay (P
=
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 imipenem-resistance (50.8%) isolates with MICs ranging from 15.6 to 250μg/mL (Table 5) (P
<
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 84Klebsiella pneumoniae isolates exhibited resistance to imipenem with MICs ranging from 4 to >32 μg/mL in a Greek hospital [28].Table 5
Antimicrobial susceptibility patterns by disc diffusion method.
Antibiotic
Antimicrobial susceptibility patterns of all Gram-negative bacilli isolates
Susceptible
Intermediate
Resistant
Number
(%)
Number
(%)
Number
(%)
Imipenem
31
47.7
1
1.5
33
50.8
Amikacin
40
∗61.5
1
1.5
24
44.6
Ciprofloxacin
35
53.8
3
4.6
27
41.5
Piperacillin
15
23.1
6
∗9.2
44
67.7
Cefoperazone/sulbactam
21
32.3
4
6.1
40
61.5
Cefoxitin
12
18.4
0
0.00
53
81.5
Cefotaxime
0
0.00
0
0.00
65
∗100.00
P
∗
<
0.001 (statistically significant).Table 6
The percentage ofbla
V
I
M, bla
T
E
M,bla
S
H
V, and bla
C
T
X
-
M genes among Gram-negative bacilli isolates.
Isolates
β-lactamases genes
Organism
Number
VIM
TEM
SHV
CTX-M-1
CTX-M-9
CTX-M-8/25
Number
(%)
Number
(%)
Number
(%)
Number
(%)
Number
(%)
Number
(%)
Escherichia coli
15
10
66.6
13
87.0
0
0.0
3
20.0
4
26.6
0
0.00
Klebsiella pneumonia
10
8
80.0
9
90.0
5
50
6
60.0
1
10.0
0
0.00
Enterobacter  cloaca
2
1
50.0
1
50.0
0
0.0
0
0.00
0
0.00
0
0.00
Pseudomonas aeruginosa
4
2
50.0
1
25.0
0
0.0
3
75.0
1
25.0
0
0.00
Proteus mirabilis
1
0
0.00
0
0.00
0
0.0
0
0.00
0
0.00
0
0.00
Acinetobacter baumanii
1
1
100
0
0.00
0
0.0
0
0.00
0
0.00
0
0.00
Total
33
22
66.7
24
72.7
5
15.0
12
36.0
6
18.2
0
0.00Another 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 thatKlebsiella pneumoniaeandEscherichia coliisolates were found to be susceptible to imipenem in routine susceptibility disk diffusion tests. Other authors observed that all beta-lactamases producers ofEscherichia coli,Klebsiella pneumoniae, andPseudomonas 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 ofβ-lactamases has a potential for rapid dissemination, since it is often plasmid-mediated [2]. Consequently, rapid detection of β-lactamases is necessary to initiate effective infection control measures to prevent their uncontrolled spread in clinical settings. In Egyptian hospitals the β-lactamases presence was confirmed by PCR amplification.In our study, the percentage ofb
l
a
V
I
M, b
l
a
T
E
M, b
l
a
S
H
V, and b
l
a
C
T
X
-
M genes among Gram-negative bacilli isolates was shown in Table 6. As regards b
l
a
V
I
M gene detected in our results it is similar to that b
l
a
V
I
M gene encoding MBL among the isolates ofP. aeruginosa (61.3%) in Tehran hospitals [31]. On the contrary, b
l
a
V
I
M 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 andP. aeruginosa [33]. In Iran, Eftekhar et al. [34] showed 69.3%b
l
a
T
E
M and 31.37%b
l
a
C
T
X
-
M
-
1 genes among Gram-negative isolates and this agreed with our study. Also, presence of b
l
a
C
T
X
-
M
1,9, a
S
H
V, and b
l
a
T
E
M 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.In Switzerland, Zurfluh et al. [37] mentioned that the isolates of Enterobacteriaceae were further screened forbla genes encoding SHV, TEM, CTX-M group 1, CTX-M group 2, or CTX-M group 9 enzymes with (23%), (71.3%), (39.2%), (20.1%), and (15.6%), respectively.Other authors noted different observations in that clinical isolates ofEscherichia coli andPseudomonas aeruginosa carried b
l
a
C
T
X
-
M
-
t
y
p
e
s which was the most common (95.8%) followed by b
l
a
T
E
M (29.2%), b
l
a
S
H
V (7.3%), and b
l
a
V
I
M (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].


## 5. Conclusion

There is a high prevalence ofβ-lactamase genes in our clinical isolates that are responsible for such resistance. Hence, it is essential to report β-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 β-lactamases producing strains for the effective treatment of infections which are caused by them.



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*Source: 8382605-2016-03-31.xml*