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taq1 digestion showed two patterns with 150 bp and 200 bp bands
in contrary in a study in yazd 20 on molecular identification using taq1 restriction enzymes four patterns were produced
this situation must be related to special genotypes in some regions
it should be emphasized that leishmaniasis in chabahar is new and genetic variation is limited
the results of the present study showed that cl infections were more common in males 48 4 than in females 24 2 table 2 probably because males spend more time outdoors than females do in this region
our results showed relatively good concordance between parasitological results cultures and pcr
some studies have shown that pcr methods achieved positive results in more than 90 of cl cases 13
the crithidias are able to endosymbiotically live within some parasites related to the trypanosomatidae family and sub families of leishmania and move to different hosts along with them
some researchers believe that the only way to isolate crithidia from leishmania is through kdna and or ribosomal rna genes
it has been suggested that by extracting kdna and or rdna then amplifying them with pcr using the pcr rflp technique with the haeiii enzyme and isolation of bands at 350 bp for leishmania and at 450 bp for crithidia these two parasites can be isolated from each other 21
however its1 rflp pcr showed good sensitivity and two its1 analyses showed the same results as in a recent study by doudi et al
21
thus the results of the present study confirm that in the border area of sistan va baluchestan on the peninsula of chabahar l major is the causative agent of cl with genotypic variation in this area
background carbapenem resistant acinetobacter baumannii is an important nosocomial pathogen associated with a variety of infections
objectives the current study aimed to characterize the antimicrobial susceptibility analyze the prevalence of oxacillinase and metallo β lactamase mbl genes and molecular typing of clinical isolates of a baumannii
materials and methods a total of 124 non repetitive isolates of a baumannii were collected from various clinical specimens in two teaching hospitals in ahvaz south west of iran
antimicrobial susceptibility test was carried out by disk diffusion method
the minimum inhibitory concentrations mics of imipenem meropenem colistin and tigecycline were determined using e test
to screen for mbl production double disk synergy dds test and mbl e test were performed
the presence of bla oxa 23 like bla oxa 24 like bla oxa 51 like bla oxa 58 like bla vim bla imp and bla spm genes was assessed by polymerase chain reaction pcr
to identify clonal relatedness all isolates were subjected to repetitive sequence based pcr rep pcr results based on disk diffusion results the highest rate of resistance was observed in rifampin 96 8
colistin and polymyxin b were the most effective agents in vitro
according to the mic results the rate of resistance to imipenem meropenem colistin and tigecycline were 78 2 73 4 0 8 and 0 respectively
metallo β lactamase production was positive in 42 3 and 79 4 of the isolates by dds test and e test respectively
all isolates 100 carried bla oxa 51 like gene
according to the results of multiplex pcr bla oxa 23 like and bla oxa 24 like genes were detected in 85 6 and 6 2 of carbapenem resistant isolates respectively
no bla oxa 58 like bla vim bla imp and bla spm were detected
by rep pcr carbapenem resistant isolates were separated into six genotypes a to f
genotype a 30 9 was the most prevalent p value 0 001
genotypes b and c were found in 28 9 and 26 8 of the isolates respectively
conclusions the rate of carbapenem resistant a baumannii isolates were high in this study
since bla oxa 58 like or mbl genes were not detected it seems that resistance to carbapenems is related to bla oxa 23 like and bla oxa 24 like
moreover bla oxa 23 like was the most prevalent oxacillinase oxa gene
most of the isolates belonged to one of the four dominant genotypes indicating clonal dissemination in the hospitals under study
in order to control the spread of carbapenem resistant a baumannii infection control strategies are needed
1
background in recent years acinetobacter baumannii has emerged as an important pathogen in nosocomial infections and especially infects critically ill patients admitted to the intensive care units icus 1 2
septicemia pneumonia urinary tract infection wound infection and meningitis are among the infections caused by this pathogen 3
in the hospital environment resistance of a baumannii to antimicrobial agents raises concerns 4
carbapenem resistant a baumannii are great concerns for physicians because carbapenems are common choice to treat infections caused by this pathogen 4 5
in addition therapeutic efficacy of carbapenems is limited due to spread of carbapenem resistant a baumannii 4 6
carbapenem resistance is now observed worldwide in a baumannii and these isolates are usually resistant to all classes of antimicrobial agents
a plenty of outbreak due to carbapenem resistant a baumannii are reported from different countries and this situation had a worrying trend 4
carbapenem resistance in a baumannii is mediated by combined different mechanisms including reduced permeability changes in penicillin binding protein ampc stable derepression efflux pumps and mostly by production of oxacillinases oxas and less common by metallo β lactamase mbls genes 7 9
clonal transmission of drug resistant a baumannii is reported globally 10
inter hospital transmission of carbapenem resistant a baumannii is demonstrated 4
it is well documented that in the nosocomial outbreaks in most of the cases one or two epidemic clones are involved in a given hospital 4
for epidemiological purposes and to control the spread of resistant iaolates rapid diferrentiation of epidemic strains from the numerous incidental strains is necessary 11 12
molecular typing such as repetitive sequence based polymerase chain reaction rep pcr is required to determine the clonal relatedness of a baumannii 12
the rep pcr is beneficial for the molecular typing of a baumannii 11
2
objectives the current study aimed to determine the antimicrobial susceptibility pattern prevalence and types of oxacillinase and metallo β lactamase genes and molecular typing by rep pcr in the clinical isolates of a baumannii
3 1
collection and identification of acinetobacter baumannii isolates from july 2011 to january 2013 a total of 124 non duplicated a baumannii isolates were collected from various clinical specimens in two teaching hospitals in ahvaz south est of iran
bacterial isolates were initially identified as a baumannii by biochemical tests 13
suspected isolates were confirmed by pcr to identify blaoxa 51 like gene with specific primers listed in table 1 to amplify a 353 base pair sequence 14
dna template for pcr was obtained by boiling method 15
each reaction was carried out in a final volume of 25 µl containing 1x pcr buffer 1 u taq polymerase 1 5 mm mgcl2 200 µm of dntp sinaclon iran 10 pmol of each primer eurofins mwg operon germany and 1 µl of the extracted dna
pcr conditions were programmed in mastercycler eppendorf eppendorf germany as follows initial denaturation at 94 c for 3 minutes 35 cycles of 94 c for 45 seconds annealing 57 c for 45 seconds extension 72 c for 1 minute and final extension 72 c for 5 minutes
pcr products were separated on 1 5 agarose gel sinaclon iran by electrophoresis stained with ethidium bromide sinaclon iran and then visualized under uv illumination syngene genegenius gel documentation system
acinetobacter baumannii atcc 19606 was used as positive control 14
3 2
antimicrobial susceptibility testing antimicrobial susceptibility testing of all isolates was performed using kirby bauer method according to the clinical and laboratory standard institute clsi 2011 guidelines
the following antimicrobial agents were tested imipenem 10 µg meropenem 10 µg polymyxin b 300 u gentamicin 10 µg ceftriaxone 30 µg colistin 10 µg piperacillin 100 µg piperacillin tazobactam 100 10 µg cefepime 30 µg tobramycin 10 µg amikacin 30 µg tetracycline 30 µg ciprofloxacin 5 µg trimethoprim sulfamethoxazole 1 25 23 75 µg ceftazidime 30 µg rifampin 5 µg tigecycline 15 µg aztreonam 30 µg and ampicillin sulbactam 10 10 µg mast group ltd merseyside uk
escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as control strains 20
minimum inhibitory concentration mic of imipenem meropenem colistin and tigecycline were determined by e test strips liofilchem italy
measures were obtained according to the clsi guidelines
the us food and rug drug administration approved criteria and jones criteria were used for enterobacteriacea and tigecycline breakpoint respectively 21 22
3 3
screening the metallo β lactamase producing isolates all isolates were screened for mbl production by an imipenem edta ethylene diamine tetra acetic acid double disk synergy and e test mbl
briefly an overnight culture suspension of each sample was adjusted to a turbidity equivalent to 0 5 mcfarland and inoculated on the surface of a mueller hinton agar plate
two 10 µg of imipenem disk mast group ltd merseyside uk were placed on the plate 10 mm apart from edge to edge
then 10 µl of 0 5 m edta solution sinaclon iran was directly added to one of them to obtain the desired concentration 750 µg
the plates were incubated at 35 c for 18 hours
after incubation inhibition zones of the imipenem and imipenem edta disks were measured and compared
if enlarged zone with imipenem edta was 7 mm greater than the imipenem disk alone it was considered as mbl positive revealing the inactivation of metallo β lactamase class b activity by edta 23 24
also an e test mbl strip containing a double sided seven dilution range of imipenem 4 to 256 µg ml and imipenem 1 to 64 µg ml in combination with a fixed concentration of edta liofilchem italy was used
the results were interpreted according to the manufacturer s instruction
3 4
pcr amplification of oxa and metallo β lactamase genes multiplex pcr was performed to detect blaoxa 23 like blaoxa 24 like and blaoxa 58 like using specific primers as previously described 16
dna template was obtained by boiling method 15
each pcr reaction was performed in a final volume of 25 µl with 1x pcr buffer 1 u taq polymerase 2 mm mgcl2 200 µm of dntp sinaclon iran 0 2 µm of each primer tag copenhagen a s denmark and 1 µl of template dna
pcr conditions were programmed in mastercycler eppendorf eppendorf germany as follows initial denaturation at 94 c for 5 minutes followed by 30 cycles at 94 c for 30 seconds 53 c for 40 seconds and 72 c for 50 seconds and final extension at 72 c for 6 minutes
pcr products were separated by electrophoresis on 1 5 agarose gel sinaclon iran and after staining with ethidium bromide visualized under uv gel documentation system acinetobacter baumannii reference strains including nctc 13304 nctc 13302 nctc 13305 were used as positive control for blaoxa 23 like blaoxa 24 like and blaoxa 58 like respectively 16
for each gene one amplicon was sequenced bioneer south korea blavim blaimp and blaspm were sought by singleplex pcr and primers previously described 17 18
two clinical isolates of p aeruginosa harbored blaimp and blavim were sequenced using automated sequence analyzer bioneer south korea and used as positive control to identify the genes
the dna of blaspm positive p aeruginosa was purchased from pasteur institute of iran and used as positive control in pcr reactions
3 5
the rep pcr to investigate genotyping and identification of various clones all isolates were subjected to rep pcr with emphasis on carbapenem resistant isolates
specific primers were used according to the previously described bou et al
protocol 19
template for pcr was extracted by phenol chloroform method
each reaction mixture was done in the total volume of 25 µl with 1x pcr buffer 3 5 mm of mgcl2 300 µm of dntp 3 dimethyl sulfoxide dmso sinaclon iran 0 5 µm of each primer tag copenhagen a s denmark and 1u of taq polymerase and 1 µl of genomic dna
amplification conditions were as follows 94 c for 10 minutes 30 cycles of 94 c for 1 minute annealing temperatures 45 c for 1 minute 72 c for 2 minutes and 72 c for 16 minutes
products were separated by electrophoresis on 1 2 agarose gel sinaclon iran after staining with ethidium bromide they were visualized under uv gel documentation system then they were photographed and compared together by visual inspection 19
all fingerprints were observed by one observer
snelling et al
protocol was used for classified various clones 11
3 6
nucleotide sequence accession number the nucleotide sequences obtained in this study were submitted to the genbank nucleotide sequence database under the accession numbers hg937619 for blaoxa 23 like hg937620 for blaoxa 24 like and hg937621 for blaoxa 51 like

taq1 digestion showed two patterns with 150 bp and 200 bp bands

in contrary in a study in yazd 20 on molecular identification using taq1 restriction enzymes four patterns were produced

this situation must be related to special genotypes in some regions

it should be emphasized that leishmaniasis in chabahar is new and genetic variation is limited

the results of the present study showed that cl infections were more common in males 48 4 than in females 24 2 table 2 probably because males spend more time outdoors than females do in this region

our results showed relatively good concordance between parasitological results cultures and pcr

some studies have shown that pcr methods achieved positive results in more than 90 of cl cases 13

the crithidias are able to endosymbiotically live within some parasites related to the trypanosomatidae family and sub families of leishmania and move to different hosts along with them

some researchers believe that the only way to isolate crithidia from leishmania is through kdna and or ribosomal rna genes

it has been suggested that by extracting kdna and or rdna then amplifying them with pcr using the pcr rflp technique with the haeiii enzyme and isolation of bands at 350 bp for leishmania and at 450 bp for crithidia these two parasites can be isolated from each other 21

however its1 rflp pcr showed good sensitivity and two its1 analyses showed the same results as in a recent study by doudi et al

21

thus the results of the present study confirm that in the border area of sistan va baluchestan on the peninsula of chabahar l major is the causative agent of cl with genotypic variation in this area

background carbapenem resistant acinetobacter baumannii is an important nosocomial pathogen associated with a variety of infections

objectives the current study aimed to characterize the antimicrobial susceptibility analyze the prevalence of oxacillinase and metallo β lactamase mbl genes and molecular typing of clinical isolates of a baumannii

materials and methods a total of 124 non repetitive isolates of a baumannii were collected from various clinical specimens in two teaching hospitals in ahvaz south west of iran

antimicrobial susceptibility test was carried out by disk diffusion method

the minimum inhibitory concentrations mics of imipenem meropenem colistin and tigecycline were determined using e test

to screen for mbl production double disk synergy dds test and mbl e test were performed

the presence of bla oxa 23 like bla oxa 24 like bla oxa 51 like bla oxa 58 like bla vim bla imp and bla spm genes was assessed by polymerase chain reaction pcr

to identify clonal relatedness all isolates were subjected to repetitive sequence based pcr rep pcr results based on disk diffusion results the highest rate of resistance was observed in rifampin 96 8

colistin and polymyxin b were the most effective agents in vitro

according to the mic results the rate of resistance to imipenem meropenem colistin and tigecycline were 78 2 73 4 0 8 and 0 respectively

metallo β lactamase production was positive in 42 3 and 79 4 of the isolates by dds test and e test respectively

all isolates 100 carried bla oxa 51 like gene

according to the results of multiplex pcr bla oxa 23 like and bla oxa 24 like genes were detected in 85 6 and 6 2 of carbapenem resistant isolates respectively

no bla oxa 58 like bla vim bla imp and bla spm were detected

by rep pcr carbapenem resistant isolates were separated into six genotypes a to f

genotype a 30 9 was the most prevalent p value 0 001

genotypes b and c were found in 28 9 and 26 8 of the isolates respectively

conclusions the rate of carbapenem resistant a baumannii isolates were high in this study

since bla oxa 58 like or mbl genes were not detected it seems that resistance to carbapenems is related to bla oxa 23 like and bla oxa 24 like

moreover bla oxa 23 like was the most prevalent oxacillinase oxa gene

most of the isolates belonged to one of the four dominant genotypes indicating clonal dissemination in the hospitals under study

in order to control the spread of carbapenem resistant a baumannii infection control strategies are needed

1

background in recent years acinetobacter baumannii has emerged as an important pathogen in nosocomial infections and especially infects critically ill patients admitted to the intensive care units icus 1 2

septicemia pneumonia urinary tract infection wound infection and meningitis are among the infections caused by this pathogen 3

in the hospital environment resistance of a baumannii to antimicrobial agents raises concerns 4

carbapenem resistant a baumannii are great concerns for physicians because carbapenems are common choice to treat infections caused by this pathogen 4 5

in addition therapeutic efficacy of carbapenems is limited due to spread of carbapenem resistant a baumannii 4 6

carbapenem resistance is now observed worldwide in a baumannii and these isolates are usually resistant to all classes of antimicrobial agents

a plenty of outbreak due to carbapenem resistant a baumannii are reported from different countries and this situation had a worrying trend 4

carbapenem resistance in a baumannii is mediated by combined different mechanisms including reduced permeability changes in penicillin binding protein ampc stable derepression efflux pumps and mostly by production of oxacillinases oxas and less common by metallo β lactamase mbls genes 7 9

clonal transmission of drug resistant a baumannii is reported globally 10

inter hospital transmission of carbapenem resistant a baumannii is demonstrated 4

it is well documented that in the nosocomial outbreaks in most of the cases one or two epidemic clones are involved in a given hospital 4

for epidemiological purposes and to control the spread of resistant iaolates rapid diferrentiation of epidemic strains from the numerous incidental strains is necessary 11 12

molecular typing such as repetitive sequence based polymerase chain reaction rep pcr is required to determine the clonal relatedness of a baumannii 12

the rep pcr is beneficial for the molecular typing of a baumannii 11

2

objectives the current study aimed to determine the antimicrobial susceptibility pattern prevalence and types of oxacillinase and metallo β lactamase genes and molecular typing by rep pcr in the clinical isolates of a baumannii

3 1

collection and identification of acinetobacter baumannii isolates from july 2011 to january 2013 a total of 124 non duplicated a baumannii isolates were collected from various clinical specimens in two teaching hospitals in ahvaz south est of iran

bacterial isolates were initially identified as a baumannii by biochemical tests 13

suspected isolates were confirmed by pcr to identify blaoxa 51 like gene with specific primers listed in table 1 to amplify a 353 base pair sequence 14

dna template for pcr was obtained by boiling method 15

each reaction was carried out in a final volume of 25 µl containing 1x pcr buffer 1 u taq polymerase 1 5 mm mgcl2 200 µm of dntp sinaclon iran 10 pmol of each primer eurofins mwg operon germany and 1 µl of the extracted dna

pcr conditions were programmed in mastercycler eppendorf eppendorf germany as follows initial denaturation at 94 c for 3 minutes 35 cycles of 94 c for 45 seconds annealing 57 c for 45 seconds extension 72 c for 1 minute and final extension 72 c for 5 minutes

pcr products were separated on 1 5 agarose gel sinaclon iran by electrophoresis stained with ethidium bromide sinaclon iran and then visualized under uv illumination syngene genegenius gel documentation system

acinetobacter baumannii atcc 19606 was used as positive control 14

3 2

antimicrobial susceptibility testing antimicrobial susceptibility testing of all isolates was performed using kirby bauer method according to the clinical and laboratory standard institute clsi 2011 guidelines

the following antimicrobial agents were tested imipenem 10 µg meropenem 10 µg polymyxin b 300 u gentamicin 10 µg ceftriaxone 30 µg colistin 10 µg piperacillin 100 µg piperacillin tazobactam 100 10 µg cefepime 30 µg tobramycin 10 µg amikacin 30 µg tetracycline 30 µg ciprofloxacin 5 µg trimethoprim sulfamethoxazole 1 25 23 75 µg ceftazidime 30 µg rifampin 5 µg tigecycline 15 µg aztreonam 30 µg and ampicillin sulbactam 10 10 µg mast group ltd merseyside uk

escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as control strains 20

minimum inhibitory concentration mic of imipenem meropenem colistin and tigecycline were determined by e test strips liofilchem italy

measures were obtained according to the clsi guidelines

the us food and rug drug administration approved criteria and jones criteria were used for enterobacteriacea and tigecycline breakpoint respectively 21 22

3 3

screening the metallo β lactamase producing isolates all isolates were screened for mbl production by an imipenem edta ethylene diamine tetra acetic acid double disk synergy and e test mbl

briefly an overnight culture suspension of each sample was adjusted to a turbidity equivalent to 0 5 mcfarland and inoculated on the surface of a mueller hinton agar plate

two 10 µg of imipenem disk mast group ltd merseyside uk were placed on the plate 10 mm apart from edge to edge

then 10 µl of 0 5 m edta solution sinaclon iran was directly added to one of them to obtain the desired concentration 750 µg

the plates were incubated at 35 c for 18 hours

after incubation inhibition zones of the imipenem and imipenem edta disks were measured and compared

if enlarged zone with imipenem edta was 7 mm greater than the imipenem disk alone it was considered as mbl positive revealing the inactivation of metallo β lactamase class b activity by edta 23 24

also an e test mbl strip containing a double sided seven dilution range of imipenem 4 to 256 µg ml and imipenem 1 to 64 µg ml in combination with a fixed concentration of edta liofilchem italy was used

the results were interpreted according to the manufacturer s instruction

3 4

pcr amplification of oxa and metallo β lactamase genes multiplex pcr was performed to detect blaoxa 23 like blaoxa 24 like and blaoxa 58 like using specific primers as previously described 16

dna template was obtained by boiling method 15

each pcr reaction was performed in a final volume of 25 µl with 1x pcr buffer 1 u taq polymerase 2 mm mgcl2 200 µm of dntp sinaclon iran 0 2 µm of each primer tag copenhagen a s denmark and 1 µl of template dna

pcr conditions were programmed in mastercycler eppendorf eppendorf germany as follows initial denaturation at 94 c for 5 minutes followed by 30 cycles at 94 c for 30 seconds 53 c for 40 seconds and 72 c for 50 seconds and final extension at 72 c for 6 minutes

pcr products were separated by electrophoresis on 1 5 agarose gel sinaclon iran and after staining with ethidium bromide visualized under uv gel documentation system acinetobacter baumannii reference strains including nctc 13304 nctc 13302 nctc 13305 were used as positive control for blaoxa 23 like blaoxa 24 like and blaoxa 58 like respectively 16

for each gene one amplicon was sequenced bioneer south korea blavim blaimp and blaspm were sought by singleplex pcr and primers previously described 17 18

two clinical isolates of p aeruginosa harbored blaimp and blavim were sequenced using automated sequence analyzer bioneer south korea and used as positive control to identify the genes

the dna of blaspm positive p aeruginosa was purchased from pasteur institute of iran and used as positive control in pcr reactions

3 5

the rep pcr to investigate genotyping and identification of various clones all isolates were subjected to rep pcr with emphasis on carbapenem resistant isolates

specific primers were used according to the previously described bou et al

protocol 19

template for pcr was extracted by phenol chloroform method

each reaction mixture was done in the total volume of 25 µl with 1x pcr buffer 3 5 mm of mgcl2 300 µm of dntp 3 dimethyl sulfoxide dmso sinaclon iran 0 5 µm of each primer tag copenhagen a s denmark and 1u of taq polymerase and 1 µl of genomic dna

amplification conditions were as follows 94 c for 10 minutes 30 cycles of 94 c for 1 minute annealing temperatures 45 c for 1 minute 72 c for 2 minutes and 72 c for 16 minutes

products were separated by electrophoresis on 1 2 agarose gel sinaclon iran after staining with ethidium bromide they were visualized under uv gel documentation system then they were photographed and compared together by visual inspection 19

all fingerprints were observed by one observer

snelling et al

protocol was used for classified various clones 11

3 6

nucleotide sequence accession number the nucleotide sequences obtained in this study were submitted to the genbank nucleotide sequence database under the accession numbers hg937619 for blaoxa 23 like hg937620 for blaoxa 24 like and hg937621 for blaoxa 51 like