Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR

The antibiotic susceptibility of Acinetobacter calcoaceticus-Acinetobacter baumannii complex strains recovered from the intensive care unit (ICU) of West China Hospital, Sichuan, PR China, from 2006 to 2009 was investigated. The identification of A. baumannii and analysis of carbapenemase-encoding genes and their relationship with ISAba1 were performed by PCR. Furthermore, a DiversiLab repetitive extragenic palindromic sequence-based PCR (rep-PCR) microbial typing system and a multilocus sequence typing (MLST) scheme were applied to assess the genetic relationship of the isolates. The results showed that the antibiotic susceptibility of the A. calcoaceticus-A. baumannii complex isolates changed and imipenem resistance increased rapidly between 2006 and 2009. The bla OXA-51-like and ISAba1-associated bla OXA-23 genes were prevalent in the imipenem-resistant A. baumannii isolates. However, the bla OXA-58-like gene was found in only one isolate and no metallo-b-lactamase genes were detected. The representative multidrug-resistant A. baumannii isolates were identified as one cluster by rep-PCR fingerprinting and belonged to the clonal complex 92 (CC92) according to MLST. These findings indicate a situation of increasing resistance and wide distribution of class D b-lactamase genes, especially the acquired ISAba1-associated bla OXA-23 gene, in A. baumannii isolates in the ICU of West China Hospital, probably caused by expansion of the CC92 clone.

Section: Methodsmentioning

confidence: 99%

Increasing imipenem resistance and dissemination of the ISAba1-associated bla OXA-23 gene among Acinetobacter baumannii isolates in an intensive care unit

Xie

Zhang

et al. 2011

“…DiversiLab, which is one of those methods, is a commercial repetitive-sequence-based PCR (rep-PCR) typing system that amplifies strain-specific noncoding repetitive sequences. The system contains qualitycontrolled reagents in a kit format, automated detection and analysis using microfluidics with the corresponding information digitized in a software package that allows data archiving, retrieval and reporting (Healy et al, 2005). Rep-PCR libraries can therefore be easily assembled to enable, for example, the comparison of strains over time and probably across laboratories, with a view to charting the epidemiology of isolates.…”

Section: Introductionmentioning

confidence: 99%

Interlaboratory reproducibility of DiversiLab rep-PCR typing and clustering of Acinetobacter baumannii isolates

Higgins

Hujer

et al. 2012

We have investigated the reproducibility of DiversiLab rep-PCR fingerprints between two laboratories with the aim of determining if the fingerprints and clustering are laboratory-specific or portable. One-hundred non-duplicate Acinetobacter baumannii isolates were used in this study. DNA isolation and rep-PCR were each performed separately in two laboratories and rep-PCR patterns generated in laboratory A were compared with those from laboratory B. Twelve A. baumannii isolates processed in laboratory A showed ¢98 % pattern similarity with the corresponding 12 isolates tested in laboratory B and were considered identical. Sixty-four isolates showed 95-97.9 % similarity with their corresponding isolates. Twenty-three isolates showed 90-94 % similarity with the corresponding isolates, while one isolate showed only 87.4 % similarity. However, intra-laboratory clustering was conserved: isolates that clustered in laboratory A also clustered in laboratory B. While clustering was conserved and reproducible at two different laboratories, demonstrating the robustness of rep-PCR, interlaboratory comparison of individual isolate fingerprints showed more variability. This comparison allows conclusions regarding clonality to be reached independent of the laboratory where the analysis is performed.

“…b-Lactamase activity was detected in 2/26 (7.7 %) Hif strains, R3788 and R3907, by an ampicillin hydrolysis assay. Consistent with the detected b-lactamase activity, strain R3907, a blood isolate, and R3788, a sinus isolate, were the only isolates determined to carry a large conjugative plasmid associated with blactamase activity, as they produced an~450 bp amplicon by PCR with primers topoF and topoR (Erwin et al, 2005).Hif isolate typing by repPCR fingerprinting DNA fingerprinting for the confirmed Hif isolates was performed using repPCR with primers detecting ERIC sequences (Healy et al, 2005a), and amplicons were resolved using bioMérieux Diversilab Bacterial Barcodes software to generate a similarity index. Isolates with similarity indices .85 % were considered identical in this study (Healy et al, 2005a, b).…”

mentioning

confidence: 99%

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates

Watson

Nelson

Nguyen

et al. 2013

The incidence of invasive infections due to Haemophilus influenzae has decreased significantly in developed countries with high rates of vaccination against H. influenzae serotype b (Hib). This vaccine provides no protection against H. influenzae serotype f (Hif), typically associated with invasive infections in adults with chronic disease and/or immunodeficiency, and rarely in otherwise healthy adults and children. The specific properties of Hif associated with virulence remain largely uncharacterized. A panel of 26 Hif strains consisting of both invasive disease-associated and mucosal surface non-invasive disease-associated isolates was surveyed by DNA fingerprinting, biotyping and PCR detection of hmw1, hmw2, hsf, the hif fimbrial locus and the lipooligosaccharide (LOS) biosynthetic island, and assessment of b-lactamase expression and determination of resistance to the bactericidal activity of normal adult human serum. Repetitive sequence-based PCR fingerprinting differentiated the 26 strains into three clusters, with the majority of isolates (22/26, 84.6 %) clustered into a single indistinguishable group. Most isolates (24/26, 92.3 %) were of biotype I and two isolates produced b-lactamase with detection of a conjugative plasmid, and the isolates displayed a range of resistances to the bactericidal activity of human serum. All 26 isolates carried the adhesin hsf, 21 carried a partial hif fimbrial operon and 4 had the adhesin genes hmw1/2. A LOS biosynthetic island was detected in 20 isolates consisting of the genes lic2BC. It was concluded that Hif has many recognized virulence properties and comprises a relatively homogeneous group independent of the anatomical source from which it was isolated. INTRODUCTIONHaemophilus influenzae, a human-restricted Gram-negative coccobacillus, is a commensal of the upper respiratory mucosa and a pathogen commonly causing airway mucosal disease and occasional invasive disease. Since Margaret Pittman's original description of capsular serotypes among H. influenzae isolates in 1931 (Pittman, 1931), H. influenzae serotype b (Hib) had been the most clinically significant strain causing invasive disease (e.g. meningitis, epiglottitis, septicaemia and osteomyelitis) in previously well infants and children (Aubrey & Tang, 2003). Since the early 1990s, routine administration of the Hib conjugate vaccines (which induce protective levels of anti-capsular antibody) has virtually eliminated Hib disease among infants and young children in developed countries (Agrawal & Murphy, 2011;Ladhani, 2012). However, the vaccine provides no protection from infection due to nonb serotypes, including H. influenzae serotype f (Hif), which remains a rare but significant cause of invasive infection. Given the reduction in Hib carriage among Hib conjugate vaccine recipients, concern exists as to whether invasive disease due to Hif or other non-b encapsulated strains will become more prevalent due to serotype replacement Abbreviations: BLNAR, b-lactamase-negative ampicillin-resistant; CSF, cerebrospinal ...