during recent years , infection of acinetobacter baumanii showed a rapid growth in hospitals and community .
baumanii infection mainly occurs in the lung and blood , the notable feature being that multi - drug resistant strains cause outbreaks in hospitals .
large outbreaks of a. baumanii infection have been reported in hospitals from usa , france , spain , belgium , britain and south korea , which brought great economic loss to the patients and hospitals ( 15 ) .
recently , a. baumanii infection increases the mortality rate by 7.823% through cohort and case - control studies ( 6 ) . in the past few years , reports about breaks of a. baumanii infection have also increased in china . since the outbreak among over 20 wards in beijing union medical college hospital in 2004 ,
similar cases have been reported in a number of cities , including chongqing and hangzhou ( 710 ) .
when pathogens invade the body , the natural immune system recognizes foreign pathogens through pattern recognition receptors .
so far , toll - like receptors ( tlrs ) , a member of the interleukin-1 receptor ( il-1r ) superfamily , are regarded as the most important pattern recognition receptors .
tlr-1 , tlr-6 and tlr-10 do not recognize the ligand directly , and play their roles by forming heterodimers with other tlrs .
although tlr-7 and tlr-8 were reported to interact with imidazoquinoline , the synthetic and anti - viral small molecule , their natural ligands have not been identified .
currently , researches about tlr-11 are few , and lipopolysaccharide ( lps ) , pgn or poly ( i : c ) can not stimulate nf-b activation in tlr-11-expressing cells .
therefore , tlr-2 , tlr-4 , tlr-5 and tlr-9 are the most important tlr members related to the immunal protection against bacteria in the body .
more and more studies indicate that single nucleotide polymorphisms ( snps ) in tlr-2 , tlr-4 , tlr-5 and tlr-9 are closely related to the susceptibility to diseases in humans .
tlr-2 has a molecular weight of 89.8 kda , forms heterodimers with tlr-1 or tlr-6 , and interacts with bacterial ligands such as lipopeptides , peptidoglycan and membrane fatty acid of gram - positive bacteria ( 11 ) .
one variant of tlr-2 ( g2477a / arg753gln ) , with an incidence of 2.79.4% in the population , is associated with the susceptibility to asthma , atopic dermatitis and the infection of mycobacterium tuberculosis , staphylococcus aureus and candida ( 12 , 13 ) .
another variant of tlr-2 ( c2029t / arg677trp ) is related to the pathogenesis of lepromatous leprosy in the asian population . furthermore , the levels of interleukin-12 ( il-12 ) are decreased in peoples carrying this polymorphism ( 14 ) .
tlr-4 , a protein with 839 amino acids and a molecular weight of 95.6kda , can form homodimers .
tlr-4 recognizes heat shock proteins and cilia of gram - positive bacteria , envelope glycoproteins of retroviruses , heat - labile compositions of mycobacteria , as well as muramic acids of spirochetes ( 15 ) .
furthermore , tlr-4 plays a definite and critial role during recognizing lps of escherichia coli , neisseria meningitidis , salmonella and legionella pneumophila , as well as subsequent signal transduction ( 14 ) .
two common snps in tlr-4 , a1287g / asp299gly and c13174t / thr399ile , change its extracellular structure , slow its response to lps and increase the incidence of septic shock ( 16 ) . besides , these snps are also closely related to the pathogenesis of severe respiratory syncytial virus ( rsv ) bronchiolitis ( 17 ) .
another two common snps in tlr-4 ( a896 g and c1196 t ) have protective effects against the infection of legionella pneumophila ( 18 ) .
tlr-5 contains 858 amino acids with a molecular weight of 97.7 kda , and can form homodimers or heterodimers with tlr-4 .
tlr-5 recognizes flagellin , and induces production of high levels of interleukin-8 ( il-8 ) .
one tlr-5 variant ( 1174c / t ) is related to the susceptibility to infection of l. pneumophila(19 ) .
it was reported that a polymorphism in the tlr9 promoter ( 1237t / c ) is related to the susceptibility to asthma , eczema , and crohn s disease ( 14 ) . despite the prevalence of a. baumanii infection in many hospitals and communities , the reason underlying a. baumanii outbreaks remains unclear .
previous studies mainly focus on the mechanism of drug resistance of bacteria , ignoring the role of the interaction between the susceptibility to infection and the baterial genotype . in this study , we investigated the mechanism of a. baumanii infection from the perspective of host genetics .
we screened snps in tlr-2 , tlr-4 , tlr-5 and tlr-9 , and studied whether polymorphisms in tlrs were associated with the susceptibility to a. baumanii infection .
the study was approved by the review board / ethics committees of the chinese pla general hospital .
cases of a. baumanii infection were collected in major cities of china using the national hospital infection surveillance network .
three hundred and eighty five uninfected patients from the same ward and with the same gender were selected as the controls .
besides , the ages of infected and uninfected patients from the same ward were less than 2 years . to reduce the interference of population stratification , all the patients in this study were han chinese .
genomic dna was extracted using qiagen blood mini kit ( qiagen , ca , usa ) , and stored at 80 c .
thirteen snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes were analyzed ( ta .
snps were selected according to their frequencies and positions , as well as their associations with infectious diseases reported previously .
snp genotyping was performed using an improved multiplex ligation detection reaction ( imldr ) technique ( genesky biotechnologies inc . ,
the genotyping success rates of rs3804099 , rs7656411 , rs76112010 , rs1927914 , rs10759932 , rs11536889 , rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 , rs17163737 and rs187084 were 99.5% , 99.3% , 99.5% , 99.4% , 99.5% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% and 99.5% , respectively .
the association of a. baumanii infection with gender and age were examined using the pearson chi - square test and the t test , respectively .
the chi - square tests were used to determine whether each polymorphism was in hardy  weinberg equilibrium .
a multiple logistic regression model was used to investigate the individual effect of tlr-2 , tlr-4 , tlr-5 and tlr-9 snps on a. baumanii infection . the analysis was based on additive , recessive and dominant models , and adjusted for age . the odd ratios ( ors ) with 95% confidence intervals ( cis ) were presented .
the statistical analysis was performed using snpalyze v4.0 ( dynacom , kanagawa , japan ) .
the study was approved by the review board / ethics committees of the chinese pla general hospital .
cases of a. baumanii infection were collected in major cities of china using the national hospital infection surveillance network .
three hundred and eighty five uninfected patients from the same ward and with the same gender were selected as the controls .
besides , the ages of infected and uninfected patients from the same ward were less than 2 years . to reduce the interference of population stratification , all the patients in this study were han chinese .
genomic dna was extracted using qiagen blood mini kit ( qiagen , ca , usa ) , and stored at 80 c .
thirteen snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes were analyzed ( ta .
snps were selected according to their frequencies and positions , as well as their associations with infectious diseases reported previously .
snp genotyping was performed using an improved multiplex ligation detection reaction ( imldr ) technique ( genesky biotechnologies inc . ,
the genotyping success rates of rs3804099 , rs7656411 , rs76112010 , rs1927914 , rs10759932 , rs11536889 , rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 , rs17163737 and rs187084 were 99.5% , 99.3% , 99.5% , 99.4% , 99.5% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% and 99.5% , respectively .
the association of a. baumanii infection with gender and age were examined using the pearson chi - square test and the t test , respectively .
the chi - square tests were used to determine whether each polymorphism was in hardy  weinberg equilibrium .
a multiple logistic regression model was used to investigate the individual effect of tlr-2 , tlr-4 , tlr-5 and tlr-9 snps on a. baumanii infection . the analysis was based on additive , recessive and dominant models , and adjusted for age . the odd ratios ( ors ) with 95% confidence intervals ( cis ) were presented .
the statistical analysis was performed using snpalyze v4.0 ( dynacom , kanagawa , japan ) .
we selected 13 snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes ( table 1 ) .
the pearson chi - square test showed that the susceptibility to a. baumanii infection was not associated with the gender significantly ( p = 0.98 ) .
however , a. baumanii - infection increased with ages , as indicated by the t test ( p = 8.6  10 ) .
characteristics of the studied snps at the tlr-2 , tlr-4 , tlr-5 and tlr-9 genes maf , minor allele frequency ; hwe , hardy  weinberg equilibrium ; statistically significant values are shown in bold . the positions and minor allele frequencies ( mafs ) of the snps
all snps are in hardy - weinberg equilibrium ( hwe ) , except for rs1341987 ( p = 0.046 in control patients ) and rs1640827 ( p = 0.021 in control patients ) of tlr-5 and rs187084 of tlr-9 ( p = 0.021 in control patients ) .
the associations of tlr2 , tlr4 , tlr5 and tlr9 gene polymorphisms with a. baumanii infection after logistic regression analysis are shown in table 2 .
the snp of tlr-9 , rs187084 , was related to a. baumanii - infection under the additive model ( 2a / a + g / a , to g / g , p = 0.05 , or = 0.82 , 95% ci = 0.671.00 ) and recessive model ( g / g , to a / a + g / a , p = 0.0064 , or = 0.59 , 95% ci = 0.400.86 ) after adjustment with age .
however , as mentioned above , rs187084 did not obey the hwe rule in control patients .
association of tlr-2 , tlr-4 , tlr-5 and tlr-9 gene polymorphisms with ab infection after logistic regression analysis or : odd ratio ; ci : confidence interval .
we found a significant association of the haplotype gcg of tlr-4 with a. baumanii - infection ( p = 0.027 ) .
no significant differences were detected in the distribution of tlr-2 and tlr-5 haplotypes between a. baumanii - infected patients and controls . compared with the common haplotype atg of tlr-4 , the haplotye gcg contained two mutations both in the promoter region ( table 1 ) .
haplotype distribution of tlr-2 , tlr-4 and tlr-5 polymorphisms in ab infected patients and controls haplotype : tlr-2 [ rs76112010 , rs3804099 , rs7656411 ] ; tlr-4 [ rs1927914 , rs10759932 , rs11536889 ] ; tlr-5 [ rs1861172 , rs5744174 , rs2241097 , rs17163737 ] .
frequency : the haplotypefrequency in the whole population including ab - infected patients and controls . or : odd ratio ; ci : confidence interval .
the major risk factors of a. baumanii infection include the severity of the underlying disease , surgical operations , a large area of trauma ( especially burns ) , premature birth , ever living in icu , prolonged hospitalization , contact with contaminated ward or equipment , invasive procedures ( such as mechanical ventilation , in - dwelling tubes , etc . ) , and ever using antibiotics .
although these factors are statistically associated with a. baumanii infection , they can not accurately predict the risk of exposure , it is difficult to explain why some patients exposed to these factors are infected , and some are not .
tlrs are the most important pattern recognition receptors , through which the natural immune system recognizes foreign pathogens .
many studies indicate that gene polymorphisms in tlrs are closely related to the susceptibility to diseases in humans .
although tlr9 detect bacterial dna and recognize dsdna from viruses and the genomes of protozoa ( 20 ) , and the innate immune system appears to use tlr9 for detecting unmethylated cpg dinucleotides , which are relatively common in bacterial and viral genomes but are highly methylated and rare in vertebrate genomes ( 21 , 22 ) , but in this study , the snp of tlr-9 , rs187084 , was significantly related to a. baumanii infection especially under the recessive model . however , this snp was not in hwe in control patients .
therefore , more random and more extensive sampling may be required to confirm further this association .
interestingly , the haplotye gcg contained two mutations both in the promoter region , compared with the common haplotype atg .
we speculate that these two snps in the tlr-4 promoter , rs1927914 and rs10759932 , influence its transcriptional regulation .
one snp of tlr-9 ( rs187084 ) and the haplotype gcg of tlr-4 are significantly associated with a. baumanii infection .
our studies provide insights to the mechanism of a. baumanii infection from the perspective of the host .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
 background : during recent years , infection of acinetobacter baumanii showed a rapid growth in hospitals and community . 
 toll - like receptors ( tlrs ) are the most important pattern recognition receptors , which play a critical role during recognizing invading pathogens by the natural immune system . 
 our objective was to determine the associations of tlrs polymorphisms with the susceptibility to a. baumanii infection in a chinese population.methods:we carried out a case - control study , genotyping 13 polymorphisms of tlr-2 , tlr-4 , tlr-5 and tlr-9 genes on 423 a. baumanii - infected patients and 385 exposed controls . 
 thirteen snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes were analyzed . 
 snp genotyping was performed using an improved multiplex ligation detection reaction ( imldr ) technique.results:the snp of tlr-9 , rs187084 , was related to a. baumanii - infection significantly under recessive model ( g / g , to a / a + g / a , p = 0.0064 , or = 0.59 , 95% ci = 0.400.86 ) after adjustment with age . besides , the haplotype gcg of tlr-4 was significantly associated with a. baumanii infection ( p = 0.027).conclusion : tlr-4 and tlr-9 may be related to the susceptibility to a. baumanii infection in a chinese population . 
Introduction
Materials and Methods
Study subjects and DNA isolation
SNP selection and genotyping
Statistical analysis
Results
Discussion
Conclusion
Ethical considerations