The Antibiotics Resistance Changes And Related Resistance Mechanism Of Clinical Stenotrophomonas Maltophilia Isolates

ObjectivesTo study the sensitivity of Stenotrophomonas maltophilia (S. maltophilia) clinical isolats from several hospitals in Anhui Province ranging from 2005 to 2010 against 6 antimicrobial agents, analyze the clinical infection characteristics, explore risk factors lead to nosocomial infection and investigate the epidemiology by genotyping among isolates, in order to provide a scientific basis for the control of nosocomial infection.To detect the various types of resistance gene and antimicrobial agents resistance phenotypes and analyze their relationships in clinical isolates from 2005-2008, in order to offer the theoretical basis and measures to prevent and control Stenotrophomonas maltophilia infection.Materials and MethodsIsolatesIn this study, during the month of September from 2005 to 2010, 188 clinical S. maltophilia isolates were collected from 34 different graded hospitals which were the members of Anhui Center for Surveillance of Bacterial Resistance. The isolates were identified by using the Microscan Walkaway-40 System.Methods According to a standard procedure described by the Clinical and Laboratory Standards Institute (CLSI), 2009, the susceptibility test to 188 clinical S. maltophilia isolates against 6 antimicrobial agents was performed by means of Agar Dilution Method. The epidemic spread of S. maltophilia isolates was detected by random primer PCR method for genotyping. The results of antimicrobial susceptibility testing and nosocomial infection characteristics were analysed by statistics.Total DNA of the 102 clinical isolates from 2005 to 2008 collection was extracted by suspending several overnight colonies in 0.5mL of double-distilled water and heating the mixture at 100 ?C for 10 min. The class 1, 2 and 3 integrons as well asβ-lactamase, quinolone resistance gene qnr, and sulfamethoxazole resistance gene sul and dfrA were detected by PCR, and their contribution to resistance of antimicrobial agents were analysed.Of the integrase-positive isolates, resistance gene cassettes embedded in integron were detected by PCR using specific primers. The positive PCR products of each fragment were purified after gel electrophoresis, and then cloned into T vector, expressed in E. coli JM109 competent cells, the plasmid DNA of E. coli JM109 was extracted and sequenced, the various resistance gene cassettes embedded in integron were carried by comparing the DNA sequence to the GenBank.In order to understand whether the sul2 gene were connected to integron resistance gene, insertion sequence (ISCR) and transposon, the unknown 3 'and 5' flanking nucleotide sequences of sul2 gene were studied by thermal asymmetric interlaced PCR and sequencing methods, so as to predict whether the sul2 genes were spread by rapid horizontal or vertical transmission initially. ResultsWe found S. maltophilia clinical infection rate among common non-fermentative bacteria increased year by year from 2005 to 2009, of which, the difference of growth rate changes from 2005 to 2008 years and 2005 to 2009 was statistically significant. 188 isolates were mainly from intensive care units (ICUs), respiratory care, departments of geriatrics, cadre ward in hospital, and 70% of which were isolated from patients above 70 years old, 153 (81%) of 188 were isolated from sputum specimens.According to the antimicrobial susceptibility results, minocycline showed good in vitro activity with susceptibility rate up to79%, followed by levofloxacin and trimethoprim/sulfamethoxazole with sensitivity rate higher than 60%, and the susceptibility rates to ticarcillin / clavulanic acid and chloramphenicol were lower than 50%. By analysing susceptibility changes of S. maltophilia isolates against the 6 antimicrobial agents from 2005 to 2010, we found the difference of resistance rates to ceftazidime was statistically significant, and the resistance rates to trimethoprim/ sulfamethoxazole from 2005 to 2010 had an increasing trend.By genotyping of the 188 isolates, we found all the isolates in 2005, 2007 and 2009 showed different geneotypes, but isolate 678 and 702 in 2006,isolate 824 and 826 in 2008,isolate 186 and 187 in 2010 shared the same geneotypes respectively, after tracking clinical data we found that they were from the same department in the same hospital respectively. Our results revealed that cross-colonization might be possible among the patients who are followed up at the same center.Among the 102 S. maltophilia isolates from 2005 to 2008 in this research, positive rates of L1 gene,L2 gene,and L1 gene together L2 gene were 76.47%,67.65%,56.86% respectively, and the positive rates of qnrX gene, qnrY gene and qnrZ gene were all 100%. With regard to the 102 isolates, 52 contained sul1 genes, of which 25 were SXT-resistant and 27 were SXT-susceptible. 13 isolates contained sul2 genes (all SXT-resistant), amongst which 7 also contained sul1 genes. Moreover, 16 of the 102 isolates carried dfrA genes (dfrA1, 1 isolate; dfrA12, 8 isolates; dfrA17, 7 isolates; dfrA5, 0 isolates).The gene for class 1 integrase (intI1) was detected in 66 (64.7%) of the 102 S. maltophilia isolates, no class 2 or 3 integrons were found. Of the 66 class 1 integrase-positive isolates, 72.7% (48 isolates) were shown to contain gene cassettes, the most common gene cassettes located in the class 1 integrons of S. maltophilia isolates in this region were dfrA17–aadA5, dfrA12–aadA2, aacA4–catB8–aadA1, aadB–aadA4, aacA4, aadA5, aadA1, aadB–aac(6′)-Ⅱ–blaCARB-8, arr-3–aacA4 and cmlA1. And the sequences of the gene cassettes embedded in class 1 integron have been deposited in GenBank and assigned the following accession numbers: GQ924479 (dfrA17–aadA5); GQ981416 (dfrA12–aadA2); GQ906532 (aacA4–catB8–aadA1); GQ866976 [aadB–aac(6′)-Ⅱ–blaCARB-8]; and GU137303 (arr-3–aacA4). Of the 31 SXT-resistant isolates, 29 carried class 1 integron (dfrA17–aadA5,dfrA12–aadA2,aadB–aadA4,aacA4–catB8–aadA1), the next two isolates were found to contain sul1 and sul2 genes. Other gene cassettes in the 19 SXT-susceptible and integrase-positive isolates included those for resistance to aminoglycosides,β-lactams, rifampicin and chloramphenicol (aacA4, aadA5, aadA1, aadB–aac(6′)-Ⅱ–blaCARB-8, arr-3–aacA4 and cmlA1).According to results of tail-PCR and senqence, 3 ' flanking nucleotide beside sul2 gene encoding a conserved hypothetical protein, 5' flanking nucleotide beside sul2 gene encoding conservative dihydropteroate synthase and transposase gene. ConclusionsIn this study, we found that as the growth of elderly population number, improvement of healthcare and the large irrational antibiotic use, the infections caused by S. maltophilia showed gradual increasing trend. Due to its intrinsical resistance and novel acquired resistance to the drugs as the empirical treatment for S. maltophilia infection, treatment of infections caused by S. maltophilia is problematic. The occurence of the same geneotype isolates in the same department suggested that the bacteria had the risk of causing nosocomial infection epidemic. Therefore, clinicians should be aware of the bacteria in control underlying debilitating diseases of patients, and take measures to reduce the infection of the bacteria.The chromosome-encoded quinolone resistance gene qnr was ubiquitous in all the isolates,and its contribution to fluoroquinolones is not found. The findings from this study indicated that the sul1 gene, in combination with dfrA17 and dfrA12 gene and sul2 genes lead to high resistance to SXT as well as multidrug resistance. The classⅠintegron was prevalent in S.maltophilia in our province, the resistance gene cassettes embedded in classⅠintegron confered resistance to sulfamethoxazole/trimethoprim aminoglycosides, chloramphenicol,β-lactams and resistance to rifampin. The transposon linked with sul2 gene and integron may accelerate the spread of resistant genes in S.maltophilia isolates and increase sulfamethoxazole/trimethoprim resistance rate.