A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic species 13TU

A new PCR-based method that exploits differences in gyrB gene sequences was developed to distinguish between Acinetobacter baumannii and Acinetobacter genomic sp. 13TU. Among 118 clinical and reference Acinetobacter strains, 102 of which were previously speciated by amplified rDNA restriction analysis as belonging to the Acinetobacter calcoaceticus-A. baumannii complex, the method correctly identified 31 A. baumannii and 54 Acinetobacter genomic sp. 13TU isolates to the species level. The method was rapid, specific and easy to interpret.     

Serological cross-reactions between Acinetobacter calcoaceticus and chlamydiae.

A cross-reaction between Acinetobacter calcoaceticus and chlamydiae is described. A water-soluble, heat stable, non-dialyzable antigen was extracted from Acinetobacter species by boiling. This antigen fixed complement in the presence of homologous hyperimmune sera from rabbits or guinea pigs and in the presence of heterologous human or hyperimmunized animal sera containing chlamydial antibodies. Hyperimmune antisera to the extracted antigen, or to suspensions of live acinetobacters, also reacted in complement fixation with a group-specific antigen.     

Nosocomial infections due to Acinetobacter calcoaceticus

Fifty four isolates of Acinetobacter calcoaceticus were studied in a period of 6 months. Maximum isolates were from burns cases and environmental sampling from burns ward also grew the same organism, indicating their role as nosocomial pathogen. Acinetobacter may initially be mistaken for Neisseria species. As the organisms show multidrug resistance to commonly used antibiotics their correct identification is important.     

22种抗生素对醋酸钙不动杆菌抑菌浓度的研究

用ＡＭＳ法测定了２２种抗生素对１０２株自临床标本分离的醋酸钙不动杆菌的抑菌浓度，结果显示ＣＩＰ的抑菌浓度为０．７６ｍｇ／Ｌ，ＴＯＢ、ＧＥＮ、ＩＭＩ、ＴＥＴ、ＡＭＩ和ＣＦＴ的抑菌浓度为１．７７～９．５４ｍｇ／Ｌ，ＣＦＺ、ＡＭＰ、ＣＦＳ、ＣＦＵ、ＴＩＡ和ＡＺＴ的抑菌浓度为１０．５０～１９．６２ｍｇ／Ｌ，ＰＩＰ、ＣＦＸ、ＴＩＣ、ＣＦＡ、ＣＥＰ、ＭＥＺ、ＣＦＰ和ＣＡＲ的抑菌浓度为２１．４４～３７．９３ｍｇ／Ｌ，ＮＩＴ的抑菌浓度为１８７．２８ｍｇ／Ｌ。在痰液、伤口及其它标本分离的醋酸钙不动杆菌的抑菌浓度测定结果中，多种抗生素的几何均数有显著性差异（Ｐ＜０．０５～０．００１）。     

Immune response to Acinetobacter calcoaceticus infection in man

After growth in an iron-depleted chemically-defined medium Acinetobacter calcoaceticus expressed four high mol. wt outer-membrane proteins (OMPs) which were repressed under iron supplementation or in a complex laboratory medium. Immunoblotting with serum from a septicaemic patient infected with A. calcoaceticus revealed antibody binding to these iron-repressible OMPs, indicating that they were expressed in vivo, and also to the 42- and 18-Kda OMPs. Although the antibody response to the OMPs did not vary significantly during convalescence, the response to the O-polysaccharide component of lipopolysaccharide decreased significantly. However, antibodies in serum from patients with A. calcoaceticus wound infections reacted with the iron repressible OMPs and a 54-Kda antigen suggesting a difference in immune recognition between local and systemic infection.     

Cryptococcus species identification by multiplex PCR.

Members of the Cryptococcus species complex are encapsulated basidiomycetous yeasts, which can affect the central nervous system (CNS) and if untreated may cause meningitis. Cryptococcus neoformans is an opportunistic pathogen causing infections mainly in immunocompromised individuals. Cryptococcus gattii is a primary pathogen responsible for a high incidence of cryptococcomas in the lung and brain and shows a delayed response to antifungal therapy. The differentiation between the two species is primarily based on their growth on and color change of canavanine - glycine-bromothymol blue agar (CGB). Since this test is not always reliable, a multiplex PCR to identify both Cryptococcus species using more than 130 samples was standardized and the results obtained compared to those with the CGB test, using the Crypto Check serotyping kit as the standard. The multiplex PCR was shown to be more specific than the CGB test, in that results obtained with it were in agreement with those from serotyping all the samples, while the data from the CGB test disagreed with 6 out of 131 samples.     

Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU)

Acinetobacter genomic species (gen. sp.) 3 and gen. sp. 13TU are increasingly recognized as clinically important taxa within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. To define the taxonomic position of these genomic species, we investigated 80 strains representing the known diversity of the ACB complex. All strains were characterized by AFLP analysis, amplified rDNA restriction analysis and nutritional or physiological testing, while selected strains were studied by 16S rRNA and rpoB gene sequence analysis, multilocus sequence analysis and whole-genome comparison. Results supported the genomic distinctness and monophyly of the individual species of the ACB complex. Despite the high phenotypic similarity among these species, some degree of differentiation between them could be made on the basis of growth at different temperatures and of assimilation of malonate, l-tartrate levulinate or citraconate. Considering the medical relevance of gen. sp. 3 and gen. sp. 13TU, we propose the formal names Acinetobacter pittii sp. nov. and Acinetobacter nosocomialis sp. nov. for these taxa, respectively. The type strain of A. pittii sp. nov. is LMG 1035^T (=CIP 70.29^T) and that of A. nosocomialis sp. nov. is LMG 10619^T (=CCM 7791^T).     

Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii

Representatives (n = 31) of outbreak strains of Acinetobacter baumannii from five countries fell into three clear groups, designated Groups 1-3, based on their ompA (outer-membrane protein A), csuE (part of a pilus assembly system required for biofilm formation) and bla(OXA-51-like) (the intrinsic carbapenemase gene in A. baumannii) gene sequences. With the exception of the closely related alleles within the Group 1 clonal complex, alleles at each locus were highly distinct from each other, with a minimum of 14 nucleotide differences between any two alleles. Isolates within a group shared the same combination of alleles at the three loci, providing compelling evidence that the outbreak strains investigated belonged to three clonal lineages. These corresponded to the previously identified European clones I-III. Sequence differences among the alleles were used to design multiplex PCRs to rapidly assign isolates belonging to particular genotypes to sequence groups. In the UK, genotypes belonging to the Group 1 clonal complex have been particularly successful, accounting for the vast majority of isolates referred from hospitals experiencing problems with Acinetobacter.     

Periplasmic aminopeptidases in Acinetobacter calcoaceticus and Pseudomonas aeruginosa

The greater part of the intracellular aminopeptidases in Pseudomonas aeruginosa and Acinetobacter calcoaceticus is soluble. The localization of aminopeptidases in the cells was examined using the osmotic shock method with some modifications. When the cells of A. calcoaceticus and P. aeruginosa of the logarithmic phase were subjected to an osmotic shock, all aminopeptidases investigated were mainly localized in the sucrose supernatants and in the periplasm. Acid phosphatase as marker enzyme for periplasm showed a similar distribution between the fractions as the aminopeptidases. The periplasmic aminopeptidases of both microorganisms were separated by FPLC on Superose 12 and their molecular masses were determined. The results obtained show that at least four different aminopeptidases occur in the periplasm, a leucyl aminopeptidase (LAP, cleaving Leu-NH-NH₂. 400 kDa), a glutamyl aminopeptidase (GAP, 200 kDa), an alanyl aminopeptidase (AAP, 80 kDa) and a prolyl aminopeptidase (PAP, 65 kDa). The results are in agreement for both species. Our results show clearly that aminopeptidases of these typical members of Gram-negative bacteria are mainly periplasmic like degrading enzymes (alkaline and acid phosphatases, 5′-nucleotidase, cyclic phosphodiesterase), detoxifying enzymes and binding proteins for amino acids and sugars.     

Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry

Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.     

Delineation of new proteolytic genomic species in the genus Acinetobacter

Twenty-seven proteolytic Acinetobacter strains differing phenotypically from the 12 previously described Acinetobacter species were studied by DNA/DNA hybridization using the S1 nuclease method to assess their relatedness. Five DNA groups (genomic species 13 to 17) containing 20 strains were delineated. Seven strains remained ungrouped. Within species, the level of DNA relatedness to the reference strains ranged from 64 to 99%, with delta Tm values below 3.5 degrees C. DNA group 13 was 31 to 42% related to group 14. DNA group 15 was 59 to 69% related to group 16, with delta Tm values between 4.5 and 6 degrees C. DNA group 17 was 51 to 61% related to DNA groups 15 and 16 with delta Tm values between 5.5 and 7.5 degrees C. The seven ungrouped strains were 28 to 60% related to the five newly delineated genomic species with delta Tm between 6.5 and 13.5 degrees C. Reference strains of the five genomic species were 5 to 22% related to the type or reference strains of the 12 Acinetobacter genomic species previously described. Biochemically, DNA groups 13 to 17 and ungrouped strains could not be separated unambiguously and therefore are not named.     

Distinct antimicrobial resistance patterns and antimicrobial resistance-harboring genes according to genomic species of Acinetobacter isolates

Using 58 isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005, we performed genomic identification by amplified rRNA gene restriction analysis (ARDRA) and investigated the existence of metallo-beta-lactamase (MBL) producers and extended-spectrum beta-lactamase (ESBL) producers. Genomic species identification of Acinetobacter strains using ARDRA showed that 40 strains were genomic species 2 (Acinetobacter baumannii), 9 were 13 sensu Tjernberg and Ursing (13TU), 5 were Acinetobacter phenon 6/ct 13TU, and 4 were Acinetobacter genospecies 3. Among 58 strains, 13 isolates were MBL producers carrying bla(IMP-1) or bla(VIM-2) and 13 isolates were ESBL producers carrying bla(PER-1). Notably, the MBL producers were mostly 13TU, Acinetobacter phenon 6/ct 13TU, and Acinetobacter genospecies 3, which showed susceptibility to ciprofloxacin and ampicillin-sulbactam. However, 12 of 13 strains carrying bla(PER-1) were A. baumannii, showing multidrug resistance. The data revealed that the antimicrobial resistance patterns and resistance-harboring genes of Acinetobacter species are remarkably distinct according to the genomic species of Acinetobacter isolates.     

Adherence of Acinetobacter calcoaceticus RAG-1 to human epithelial cells and to hexadecane.

The ability of Acinetobacter calcoaceticus RAG-1 to adhere to human epithelial cells was investigated and compared with its ability to adhere to a test hydrocarbon (hexadecane). RAG-1, a microorganism originally isolated for growth on hydrocarbon, adhered to epithelial cells when grown under conditions which promote its adherence to hexadecane; similarly, RAG-1 cells adhered poorly to epithelial cells when grown under conditions which cause the cells to possess low affinity towards hexadecane. A mutant derived from RAG-1, MR-481, deficient in its ability to adhere to hydrocarbon, was similarly unable to adhere to epithelial cells. RAG-1 adherence to epithelial cells was not blocked by a number of sugars tested. Streptococcus pyogenes, whose adherence to epithelial cells has been previously attributed to hydrophobic interactions, was also able to adhere to hexadecane. Results suggest that hydrophobic interactions mediate adherence of the strains studied to both epithelial cells and hydrocarbon.     

Differential detection of five mouse-infecting helicobacter species by multiplex PCR

Several species of helicobacter have been isolated from laboratory mice, including H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, which appear to be the most common. The most widely used published method for molecular detection of these agents is PCR amplification of a conserved region of 16S rRNA, but differential speciation requires restriction enzyme digestion of the amplicons. This study was undertaken to determine PCR conditions that would simultaneously and specifically identify each of the five common species without restriction enzyme analyses. First, we designed novel and specific PCR primers for H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, using sequences from the heterologous regions of 16S rRNA. Because of comigration of amplified products, we next identified P17, an H. bilis-specific protein; P25, an H. hepaticus-specific protein; and P30, an H. muridarum-specific protein by screening genomic DNA expression libraries of each species. Primers were designed from these three genes, plus newly designed, species-specific 16S rRNA primers for H. rodentium and H. typhlonius that could be utilized for a five-plex PCR. The sizes of the amplicons from H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius were 435, 705, 807, 191, and 122 bp, respectively, allowing simultaneous detection and effective discrimination among species.     

Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR.

In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310- and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA+) staphylococci and to differentiate S. aureus (femA+) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA+, and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.     

Direct genomic multiplex PCR for BRCA1 and application to mutation detection by single-strand conformation and heteroduplex analysis

Most mutation detection methods are based on analysis of PCR amplified segments and the application of multiplex PCR is one central approach to improving screening efficiency. Genes like the breast-ovarian cancer susceptibility gene BRCA1 pose a difficult challenge to efficient mutation screening because of large coding regions, numerous exons, and complex mutational spectra. The application to BRCA1 of a general approach to effective multiplex PCR is described here. Fifteen triplex PCRs and a single PCR reaction condition were used for amplification of all BRCA1 coding regions and the BRCA1-specific segments from the duplicated promoter region. SSCP/HDX gel analysis of the multiplex products detected mobility distinctions for 34/34 sets of allelic BRCA1 fragments. A novel polymorphism was found, CTTCT(4)CT(10)CT(12) >CT(4)CT(11), a compound deletion in a region beginning at the +33 position of IVS7 and resulting in a net deletion of 15 bp. This change was shown to be one of the common polymorphisms that define the two major haplotypes of the BRCA1-RNU2 region in a large proportion of the world population. A triplex PCR for SSCP detection of this deletion and two other distantly located common polymorphisms may be used to screen haplotype content and facilitate comparison of samples with similar haplotypes in subsequent mutation screening. The approach for robust multiplex amplification is generally applicable and allows rapid development of efficient testing for a wide variety of mutations in any gene(s) encompassing a large coding region or numerous exons and including as many as 50 different genomic PCR products.