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  .
hospital acquired infections due to gram - negative bacilli are a leading cause of morbidity and mortality worldwide .
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 . , 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 .
this resistance may be attributed to presence of metallo--lactamase in bacteria such as imp ( imipenemase ) , vim ( verona - integron metallo--lactamase ) , and extended spectrum -lactamases ( esbls ) such as shv , tem , and ctx - m . 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 .
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 .
clinical isolates of gram - negative bacilli including escherichia coli ( n = 30 ) , klebsiella pneumoniae ( n = 17 ) , pseudomonas aeruginosa ( n = 7 ) , proteus mirabilis ( n = 2 ) , citrobacter freundii ( n = 1 ) , acinetobacter baumanii ( n = 3 ) , and enterobacter 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 . a growth temperature of 44c
was used sometimes to confirm the identity of these isolates and the identified strains were stored in glycerol ( 20% v / v ) at 70c and subcultured several times to be viable .
all isolates were identified by standard biochemical tests   and confirmed by api 20e ( biomrieux , marcy l'toile , france ) .
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 .
the antibiotic disks used imipenem ( ipm , 10  g ) , amikacin ( ak , 30  g ) , ciprofloxacin ( cip , 5  g ) , piperacillin ( prl , 100  g ) , cefoperazone / sulbactam ( ces , 10 + 5  g ) , cefoxitin ( fox , 30  g ) , and cefotaxime ( ctx , 30  g ) which were placed 15  mm away from the central disc and the plates were incubated for about 1824  hrs at 37c . for detection of -lactamase genes responsible for imipenem - resistance , rapid genomic dna was prepared from about five colonies heated in 100  ml distilled water ( 95c 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 blavim , blatem , blashv , blactx - m-1 , blactx - m-9 , and blactx - 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 mgcl2 , 1  u taq dna polymerase , and 1  l of genomic dna .
amplification was carried out as follows : initial denaturation at 94c for 10 minutes , followed by 40 cycles of dna denaturation at 94c for 40 seconds , primer annealing at 60c for 40 seconds and primer extension at 72c for 1 minute , and a final elongation step at 72c for 7 minutes .
amplicons were then visualized after running in 2% agarose gel at 100  v for 30  mins . a 501000  bp dna ladder ( usa )
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 .
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 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 .
the statistical analysis was performed using spss version 20.0 ,  = chi - square test , and p values < 0.05 were considered significant .
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 by klebsiella pneumoniae ( 26.2% ) , pseudomonas aeruginosa ( 10.7% ) , enterobacter cloacae ( 6.1% ) , proteus mirabilis ( 3.07% ) , and acinetobacter baumanii ( 4.6% ) while citrobacter freundii and proteus 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 .
it was shown in figure 1 , 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 .
as shown in table 6 , of 33 imipenem - resistant gram - negative bacillistrains , the results of pcr amplification products of -lactamases genes showed that 66.7% of isolates carried blavim at 390  bp ( figure 2(a ) ) , 72.7% had blatem at 800  bp ( figure 2(b ) ) , 36.0% harbored blactx - m-1 at 688  bp ( figure 2(c ) ) , and 15.0% showed blashv ( figure 2(d ) ) , while 18.2% ( figure 2(e ) ) , 0.00% ( figure 2(f ) ) harbored blactx - m-9 , blactx - m-8/25 , respectively .
sequencing confirmed presence of these -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 blavim , blatem , blashv , and blactx - m genes by pcr .
the sequence of the purified product of vim gene ( blavim1,2 ) 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 ( blavim1,2 ) 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 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 ( blavim1,2 ) 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 ) .
these positions were indicated in 246 , 284 , 306 , 309 , 330 , 363 , 378 , 382 , and 383 ( figure 3(e ) ) .
the phylogenetic tree of vim sequence of a. baumanii isolated from zu hospitals and published ones in genbank showed dissimilarity between egyptian strains and others ( figure 3(f ) ) .
sequences of the purified product of vim gene ( blavim1,2 ) were compared with homologous counterpart genbank database using blast program and resulted in significant similarity to many metallo--lactamases genes of different bacterial strains . 

the tem gene sequences of e. coli strains , isolated from zu hospitals , were aligned with sequences of published tem genes in e. coli strains from genbank ( e.g. , e. coli km598665.1 ) .
the snps positions were indicated commonly in 216 , 232 , 385 , and 433 ( figure 4(a ) ) .
phylogenetic tree was constructed from tem gene ( blatem1,2 ) sequence of escherichia coli strains isolated from zu hospitals and published homologous sequences in genbank ( figure 4(b ) ) showing similarity degree between egyptian and indian strains . 

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 ) .
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 pairwise sequences alignments of resulting shv gene ( blashv1 ) 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 .
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 ) ) .
sequences of the purified product of ctx - m-1 gene ( blactx - 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 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 ) ) . 

( 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 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 egyptian strain ( figure 6(d ) ) . 

( 3 ) ctx - m-1 gene ( bla 
 ctx - m-1 
 ) of pseudomonas aeruginosa strains .
the ctx - m-1 gene isolated from p. aeruginosa strains in zagazig university hospitals was compared using blast program with published ctx - m-1 gene in p. aeruginosa strains from genbank ( e.g. ,
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 ) ) .
the ctx - m-9 gene isolated from e. coli strains in zagazig university hospitals was compared with published ctx - m-1 gene in p. 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 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 ) ) .
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 .
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 . in the present study ,
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 ) . and
by contrast , shanthi and sekar   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% ) .
various risk factors of -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 .   who found that 58% were identified as escherichia coli followed by 27.7% klebsiella pneumoniae and 15% pseudomonas aeruginosa in udaipur , rajasthan .
52 ( 58.42% ) isolates of escherichia coli were found to be the most common organisms in allahabad followed by klebsiella pneumoniae ( 20.22% ) , pseudomonas aeruginosa ( 12.35% ) , proteus vulgaris ( 3.37% ) , proteus mirabilis ( 2.24% ) , and enterobacter cloacae ( 2.24% ) .
also , in india , sankarankutty and kaup   documented the same result in that 58.42% isolates of escherichia coli were found to be the most common organisms followed by klebsiella pneumoniae ( 20.22% ) , pseudomonas aeruginosa ( 12.35% ) , proteus vulgaris ( 3.37% ) , proteus mirabilis ( 2.24% ) , and enterobacter aerogenes ( 2.24% ) .
but our study disagreed with that published by aboderin et al .   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 .   who exhibited major risk factors such as prolonged hospitalization > 8 days , previous antibiotic use , trauma , and mechanical ventilation which may contribute to the mortality .
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 .
documented that there was statistical significance in isolation of klebsiella 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 . 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 250  g / ml ( table 5 ) ( p < 0.001 ) . in egypt
, this was parallel to that reported by mohamed and raafat   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
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 .
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% .
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 .
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 of -lactamases has a potential for rapid dissemination , since it is often plasmid - mediated .
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 blavim , blatem , blashv , and blactx - m genes among gram - negative bacilli isolates was shown in table 6 .
as regards blavim gene detected in our results it is similar to that blavim gene encoding mbl among the isolates of p. aeruginosa ( 61.3% ) in tehran hospitals . on the contrary ,
blavim genes were not detected among the studied gram - negative isolates in other tehran hospitals .
tem , shv , and ctx - m genes are the most common plasmid - mediated lactamases often found in enterobacteriaceae and p. aeruginosa . in iran ,
eftekhar et al .   showed 69.3%  blatem and 31.37%  blactx - m-1 genes among gram - negative isolates and this agreed with our study .
also , presence of blactx - m1,9 , ashv , and blatem genes among tested rods of the enterobacteriaceae family was revealed by ojdana et al . .
another study reported by cuzon et al .   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 .  
mentioned that the isolates of enterobacteriaceae were further screened for bla 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 of escherichia coli and pseudomonas aeruginosa carried blactx - m - types which was the most common ( 95.8% ) followed by blatem ( 29.2% ) , blashv ( 7.3% ) , and blavim ( 12.5% ) in nepal .
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 ) .
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 .
 carbapenem - resistant gram - negative bacilli resulting from -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 blavim , blatem , blashv , blactx - m-1 , and blactx - 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 blavim , blatem , blashv , and blactx - m genes was done by polymerase chain reaction ( pcr ) . direct sequencing from pcr products 
 was subsequently carried out to identify and confirm these -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 blavim , 24(72.7% ) had blatem , and 5(15% ) showed blashv , while 12(36% ) , 6(18.2% ) , and 0(0.00% ) harbored blactx - m-1 , blactx - m-9 , and blactx - m-8/25 , respectively . 
 there is a high occurrence of -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 . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion