Polymerase Chain Reaction Mapping Research Articles

Molecular Epidemiological Characteristics of Carbapenem Resistant Aeromonas from Hospital Wastewater.

Hospital wastewater (HWW) promotes the spread of carbapenem resistance genes (CRGs). Aeromonas carry a large number of CRGs in HWW, they may play a role as a suitable reservoir for CRGs, while resistomes in HWW are still poorly characterized regarding carbapenem resistant Aeromonas. Thus, the aim of the study was to evaluate the molecular epidemiological characteristics of carbapenem resistant Aeromonas in HWW. A total of 33 carbapenem resistant Aeromonas were isolated from HWW. Antimicrobial susceptibility testing and polymerase chain reaction (PCR) were used to assess the antimicrobial resistance profiles. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and multilocus sequence typing (MLST). The horizontal transmission mode of bla KPC was explored through conjugation and transformation experiments. The stability of bla KPC-IncP-6 plasmids was assessed through plasmid stability and in vitro competition test. The PCR mapping method was used to investigate the structural diversity of bla KPC. The detection rates of bla KPC and cphA in Aeromonas were 97.0% and 39.4% respectively. Aeromonas caviae were grouped into 13 clusters by ERIC-PCR and 12 STs by MLST. Aeromonas veronii were grouped into 11 clusters by ERIC-PCR and 4 STs by MLST. 56.3% bla KPC were located on mobilizable IncP-6 plasmids. bla KPC-IncP-6 plasmid showed high stability and low cost fitness. Carbapenem resistant Aeromonas from HWW mainly carried bla KPC, which exhibited great structural diversity. Aeromonas might serve as reservoirs for bla KPC and bla KPC might spread mainly through transformation in HWW.

Molecular Characteristics of Escherichia coli Causing Bloodstream Infections During 2010-2015 in a Tertiary Hospital, Shanghai, China.

BackgroundThe bloodstream infections (BSI) caused by Escherichia coli pose a serious threat to human health. To explore molecular characteristics of E. coli causing BSI, we collected E. coli isolates causing BSI in Huashan Hospital, Shanghai, China during 2010–2015.MethodsIn all E. coli isolates causing BSI collected from this study, polymerase chain reaction (PCR) was used to detect ESBLs and carbapenemase genes, and minimum inhibitory concentrations (MICs) were determined with agar dilution method. Outer membrane proteins were examined by SDS-PAGE in carbapenem-resistant strains. The genetic background of blaKPC gene was investigated by combining next-generation sequencing with a PCR mapping approach. Conjugation and transformation experiments were performed to verify the mobilization of blaKPC. The transcription levels of the blaKPC gene were measured by RT-PCR.ResultsDuring 2010–2015, a total of 207 E. coli BSI strains were isolated. The positive rates of β-lactamase resistant genes were 0.48% (blaKPC), 57% (blaTEM), 23.67% (blaCTX-M-1), 18.84% (blaCTX-M-9), and 1.93% (blaSHV). High rates of blaTEM, blaCTX-M-1, and blaCTX-M-9 were consistent with the poor activity of third-generation cephalosporins and aztreonam in vitro, except for carbapenem and β-lactamase inhibitor combinations. Low susceptibility rates were observed for piperacillin (25.1%) in contrast to the increased susceptibility when combined with β-lactamase inhibitors, namely piperacillin-tazobactam (90.8%). Only one KPC-producing E. coli strain was detected. Despite the combination of OmpC loss, the low expression level of KPC may be responsible for its lower resistance to carbapenems compared to E. coli DH5α (pKP12-100).ConclusionE. coli strains isolated from BSI were still highly susceptible to carbapenems and β-lactamase inhibitor combinations, and blaCTX-M was the dominant genotype of ESBLs. The low expression of blaKPC may be the reason for the low resistance to carbapenems.

Changing epidemiology of KPC-producing Klebsiella pneumoniae in Argentina: Emergence of hypermucoviscous ST25 and high-risk clone ST307.

To assess the epidemiological features of 76 Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) isolates recovered from three hospitals in Buenos Aires, Argentina, during 2015-2017. Antimicrobial susceptibilities were determined according to CLSI Clinical and Laboratoy Standards guidelines. Molecular typing of KPC-Kp was performed by pulsed-field gel electrophoresis (PFGE)-Xbal and multilocus sequence typing. Plasmid encoded genes involved in carbapenem, fosfomycin and colistin resistance were detected by polymerase chain reaction (PCR) and sequencing. Also, mgrB inactivation was investigated in those colistin-resistant isolates. Genetic platforms involved in horizontal spread of blaKPC were investigated by PCR mapping. Besides β-lactams, high resistance rates were observed for gentamycin, quinolones and trimethoprim-sulfamethoxazole. KPC-Kp sequence type (ST)258 corresponded to 26% of the isolates, while 42% corresponded to ST25. The other isolates were distributed in a diversity of lineages such as ST11 (10.5%), ST392 (10.5%), ST307, ST13, ST101, ST15 and ST551. blaKPC-2 was detected in 75 of 76 isolates, and one ST307 isolate harboured blaKPC-3. Tn4401 was identified as the genetic platform for blaKPC in epidemic lineages such as ST258 and ST307. However, in ST25 and ST392, which are usually not related to blaKPC, a blaKPC-bearing non-Tn4401 element was identified. Alterations in mgrB were detected in seven of 11 colistin-resistant isolates. Despite previous reports in Argentina, ST258 is no longer the absolute clone among KPC-Kp isolates. In the present study, dissemination of more virulent lineages such as the hypermucoviscous ST25 was detected. The emergence of the high-risk clone ST307 and occurrence of blaKPC-3 was noticed for the first time in this region.

1200. Molecular Epidemiology of Cephalosporinases and Extended Spectrum β-Lactamases (ESBLs) in Proteus mirabilis Isolates From Croatia: Following the Spread of Resistance Determinants Between Long-Term Care Facilities and the Community

BackgroundPrevious studies on P. mirabilis strains isolated from Croatian healthcare institutions revealed the predominance of TEM-52 extended spectrum β-lactamase (ESBL), as well as the emergence of plasmid AmpC β-lactamases. Our aim was to molecularly characterize cefalosporinases in P. mirabilis isolates from long-term care facilities (LTCFs) and to compare their resistance profile and dynamics with community isolates.MethodsFrom a total of 3,321 P. mirabilis isolates collected from two LCTFs and from outpatients between 2015 and 2017, 1.23% of them were resistant to third generation of cephalosporins. Antimicrobial sensitivity was tested by broth microdilution method. ESBLs and plasmid-mediated AmpC β-lactamases were detected with phenotypic inhibitor-based tests and polymerase chain reaction (PCR). Antibiotic resistance dissemination and genetic context of bla genes were interrogated by conjugal mating and PCR mapping, respectively. Plasmids were characterized by conjugation and transformation experiments, as well as PCR-based replicon typing.ResultsHigh-level of resistance to amoxicillin, co-amoxiclav, first, second and third generation of cephalosporins was found in all isolates. Three isolates tested positive in inhibitor-based test with clavulanic acid, and 38 both in Hodge test and combined disk test with phenylboronic acid, indicating the production of ESBLs and plasmid-mediated AmpC β-lactamases, respectively. Two ESBL-positive organisms yielded amplicons with primers for CTX-M β-lactamase of group 1 and one for TEM. All AmpC-positive organisms were identified by PCR as CMY (with an additional TEM). Insert sequence ISEcp-1 was found upstream of blaCMYi blaCTX-M genes. CTX-M positive strains harbored IncK plasmid, whereas AmpC-positive strains were negative for known plasmid types. This is also a first description of P. mirabilis harboring CTX-M-15 β-lactamase in Croatia.ConclusionOur study showed the persistence of CMY β-lactamases in one LTCF, but also the dissemination of characteristic resistance determinants to another LTCF and the community. Similar to some other studies, there was a clear trend of cephalosporinase dynamic switch from TEM variants to CMY and CTX-M, with impending consequences for treatment decisions.Disclosures All authors: No reported disclosures.

Mapping of genomic EGFRvIII deletions in glioblastoma: insight into rearrangement mechanisms and biomarker development.

Epidermal growth factor receptor (EGFR) variant III (vIII) is the most common oncogenic rearrangement in glioblastoma (GBM), generated by deletion of exons 2 to 7 of EGFR. The proximal breakpoints occur in variable positions within the 123-kb intron 1, presenting significant challenges in terms of polymerase chain reaction (PCR)-based mapping. Molecular mechanisms underlying these deletions remain unclear. We determined the presence of EGFRvIII and its breakpoints for 29 GBM samples using quantitative PCR, arrayed PCR mapping, Sanger sequencing, and whole genome sequencing (WGS). Patient-specific breakpoint PCR was performed on tumors, plasma, and cerebrospinal fluid (CSF) samples. The breakpoint sequences and single nucleotide polymorphisms (SNPs) were analyzed to elucidate the underlying biogenic mechanism. PCR mapping and WGS independently unveiled 8 EGFRvIII breakpoints in 6 tumors. Patient-specific primers yielded EGFRvIII PCR amplicons in matched tumors and in cell-free DNA (cfDNA) from a CSF sample, but not in cfDNA or extracellular-vesicle DNA from plasma. The breakpoint analysis revealed nucleotide insertions in 4 samples, an insertion of a region outside of the EGFR locus in 1, microhomologies in 3, as well as a duplication or an inversion accompanied by microhomologies in 2, suggestive of distinct DNA repair mechanisms. In the GBM samples that harbored distinct breakpoints, the SNP compositions of EGFRvIII and amplified non-vIII EGFR were identical, suggesting that these rearrangements arose from amplified non-vIII EGFR. Our approach efficiently "fingerprints" each sample's EGFRvIII breakpoints. Breakpoint sequence analyses suggest that independent breakpoints arose from precursor amplified non-vIII EGFR through different DNA repair mechanisms.

An Outbreak of NDM-1-Producing Klebsiella pneumoniae, Associated with OmpK35 and OmpK36 Porin Loss in Tunisia.

To describe clinical and molecular characteristics of an outbreak due to metallo-β-lactamases (MBLs) producing Klebsiella pneumoniae collected at Charles Nicolle Hospital of Tunis and to analyze the impact of outer membrane porin (OMP) loss on carbapenem resistance levels. Between 2010 and 2015, 178 carbapenem-resistant Enterobacteriaceae were isolated. Screening for MBL production was performed using combined disk diffusion method, with imipenem and ethylene diamine tetraacetic acid (EDTA) as inhibitors. Resistance genes and virulence factors were identified by polymerase chain reaction (PCR) and sequencing. Genotyping was performed by pulsed-field gel electrophoresis and multilocus sequence typing. Genetic environment of carbapenemase genes was determined by PCR mapping. Conjugation assays were performed, and plasmids were assigned to incompatibility groups by PCR-based replicon typing. OMPs were profiled by sodium dodecyl sulfate-polyacrilamide gel electrophoresis, and porin genes were sequenced. Nineteen K. pneumoniae (10.6%) showing MBL activity were isolated from patients hospitalized on four different wards. NDM-1 was the only MBL identified, in association with blaOXA-48. All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. blaNDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of blaNDM-1 was due to two clones, one major clone belonging to sequence type (ST) 147 (n = 16) and the other clone belonging to ST307 (n = 3). This study describes an outbreak of NDM-1-producing K. pneumoniae strains, isolated from a Tunisian hospital, caused by two clones belonging to ST147 and ST307; and highlights the role of OMPs loss, in combination with β-lactamase expression, in conferring high carbapenem resistance.

Characterization of Isolates Associated with Emergence of OXA-48-Producing Klebsiella pneumoniae in Croatia.

Here, we report a retrospective study conducted to elucidate emergence, epidemiology, and molecular mechanisms of resistance underlying the early spread of OXA-48 carbapenemase-producing Enterobacteriaceae in Croatia. Retrospective screening for OXA-48 producers was performed on a collection of 296 nonrepetitive, carbapenem-nonsusceptible enterobacterial isolates collected from January 2011 to December 2012 from 40 participating centers in Croatia. Antimicrobial susceptibility profiles and production of carbapenemases were assessed phenotypically. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used for epidemiological analysis. Resistance genes were characterized by polymerase chain reaction (PCR) and sequencing. Plasmid localization of blaOXA-48 in isolates and transconjugants was investigated by S1-PFGE and Southern hybridization. PCR mapping was used for identification of genetic platform surrounding blaOXA-48. Out of 296 carbapenem-nonsusceptible isolates, blaOXA-48 gene was detected in 12 Klebsiella pneumoniae isolates. All OXA-48-producing isolates showed varying resistance to carbapenems and 11 were multidrug resistant. All coproduced additional beta-lactamases, including CTX-M-15, which was detected in eight isolates. Isolates were delineated in five clonal types by PFGE corresponding to five sequence types (STs) assigned ST15, ST16, ST37, ST528, and ST1418. All OXA-48 isolates conjugated successfully and other resistance determinants were not cotransferred. blaOXA-48 was carried on a ∼60 kb IncL/M plasmid and was detected within Tn1999.2 composite transposon. OXA-48, a class D carbapenemase, is emerging as a potentially significant contributor among carbapenem-resistant Enterobacteriaceae in Croatia, alongside class A and B carbapenemases. Polyclonal genetic background of K. pneumoniae isolates carrying ∼60 kb incL/M plasmid indicates that dissemination of the blaOXA48 gene is not driven exclusively by the spread of a single clone.

Spread of imipenem-resistant Acinetobacter baumannii of European clone II in Western China

The aim of this study was to investigate the distribution of resistance genes and the clonal relationship amongst imipenem-resistant Acinetobacter baumannii isolates from ten hospitals in Western China as well as to compare the molecular epidemiological data with those of isolates from two hospitals in Hangzhou and Beijing. Genes encoding OXA carbapenemases, metallo-β-lactamases, AmpC cephalosporinase and carbapenem resistance-associated outer membrane protein (CarO) were screened using polymerase chain reaction (PCR) and sequencing. PCR mapping was performed to determine whether insertion sequence IS Aba1 elements preceded OXA carbapenemases and AmpC cephalosporinase. International clonal lineages were identified by sequence type multiplex PCR. Multilocus sequence typing (MLST) was performed to determine the sequence types (STs), and then eBURST algorithm was applied to assign clonal complexes (CCs). In this study, dissemination of acquired IS Aba1 preceding the bla OXA-23-like gene was the predominant enzymatic resistance mechanism amongst 272 imipenem-resistant isolates. Five isolates harboured the carO gene disrupted by insertion of IS Aba1 and three isolates lacked carO. All of the 36 representative isolates belonged to European clone II. Ten STs, including three novel types, were identified. These STs were clustered into CC92 and two distinct singletons. These observations suggest that imipenem-resistant A. baumannii of European clone II, which carries acquired IS Aba1 preceding the bla OXA-23-like gene and belongs to CC92, has spread within Western China.

Characterisation of the CTX-M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration

The localisation and genetic organisation of bla CTX-M-15 were studied in 37 CTX-M-15-producing Klebsiella pneumoniae isolates collected from 2005 to 2008 within the Barcelona metropolitan area. Polymerase chain reaction (PCR)-based replicon typing and Southern hybridisations were used to identify the bla CTX-M-15 location. The genetic environment was analysed by PCR mapping and sequencing, and transferability of bla CTX-M-15 was evaluated by conjugation and transformation assays. The majority of the 37 isolates carried bla CTX-M-15 in a plasmid location, frequently associated with the aac(6′)-Ib-cr gene. Plasmids encoding bla CTX-M-15 carried three distinct replicons, i.e. IncFII, IncR and IncFIIk, the latter two not having been described previously in association with bla CTX-M-15. Several of these plasmids were not self-transferable. Furthermore, in all isolates belonging to sequence type ST-1, bla CTX-M-15 was found integrated into the K. pneumoniae chromosome. In all the studied isolates, the mobile element IS Ecp1 was found upstream of bla CTX-M-15, whereas IS 26 was found inserted within IS Ecp1 in several isolates, in previously unreported positions. In conclusion, these findings indicate that among K. pneumoniae strains isolated in the Barcelona metropolitan area, bla CTX-M-15 is associated with diverse genetic elements, including the IncR and IncFIIk replicons, as reported for the first time here, and the chromosome.

Molecular Epidemiology and Genetic Environment of AcquiredblaACC-1inSalmonella entericaSerotype Livingstone Causing a Large Nosocomial Outbreak in Tunisia

Eighty-four isolates of Salmonella enterica serovar Livingstone were collected from patients hospitalized in a pediatric ward in Sfax Hospital (South Tunisia). These isolates were responsible for two nosocomial outbreaks in 2000 and 2002. Twenty-eight clinical isolates of S. enterica serovar Livingstone were also obtained in two other Tunisian hospitals in Monastir (Central Tunisia) and Tunis (North Tunisia), respectively, in 2002 and 2003. Pulsed-field gel electrophoresis yielded that these isolates were closely related. Antimicrobial susceptibility testing showed a particular beta-lactam resistance phenotype, suggestive of the presence of an AmpC-type enzyme in 111 of the 112 clinical isolates. bla(ACC-1) was characterized by polymerase chain reaction (PCR) and sequence analysis in the 111 isolates. TEM-1 was characterized in all strains and SHV-2a in only two strains. The genetic organization of bla(ACC-1) was determined by PCR mapping and sequencing. The plasmid-borne bla(ACC-1) gene mapped immediately downstream from ISEcp1. This ISEcp1 insertion sequence was itself disrupted by IS26 insertion sequences. A supplementary deletion of 13 bp was observed in ISEcp1 upstream IS26, in all isolates from Tunis, except one. PCR analysis and sequencing also revealed the presence of tnpR, bla(SCO-1), gdha, IS1353, and TniB Delta 1.

Conjugative Transferability of the A/C Plasmids fromSalmonella entericaIsolates That Possess or LackblaCMYin the A/C Plasmid Backbone

The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)-based replicon typing was used to type plasmid replicons. Conjugation experiments were preformed in triplicate at 30 degrees C and 37 degrees C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3-12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a bla(CMY) gene. A/C was the predominant replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugants, and most of these donors carried multiple replicons. A/C cotransferred with B/O, N, and I1 at both 30 degrees C and 37 degrees C and with HI2 at 30 degrees C. Seven Salmonella Newport isolates that produced transconjugants possessed only the single A/C replicon and lacked bla(CMY). PCR mapping of the A/C transfer gene operon in ten Salmonella Newport isolates that carried bla(CMY) revealed a bla(CMY) inverted repeat element integrated between the traA and traC genes. These results suggest that A/C may have been a conjugative plasmid before the integration of bla(CMY) into the transfer gene operon. Additionally, transfer deficient A/C replicons may be mobilized in the presence of certain compatible conjugative plasmids.

Characterization of the glutathione S-transferase GSTT1 deletion: discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype-phenotype correlation.

Glutathione S-transferase theta enzyme activity involved in the metabolism of toxic compounds is absent in approximately 20% of Caucasians due to a homozygous deletion of GSTT1 (*0/0). Because the exact manner of the GSTT1 deletion was unknown, current genotyping of GSTT1 was limited to detect the presence versus complete absence of the gene by a GSTT1-specific polymerase chain reaction (PCR). Thus, heterozygous (*A/0) and homozygous (*A/A) samples could not be discriminated. We have characterized the boundaries of the deletion of the human glutathione S-transferase theta (GSTT1) gene: PCR mapping and sequencing revealed a 54251 bp fragment including GSTT1 to be deleted from chromosome 22, most likely by a homologous recombination event between two highly homologous sequence stretches that flank GSTT1. Based on the knowledge of the GSTT1*0 region, a PCR assay was devised for unambiguous discrimination of homozygously deleted (*0/0), heterozygously (*A/0) and homozygously GSTT1 carrying (*A/A) individuals. Genotyping of 180 samples of a Caucasian population revealed that the deletion consists of one defined allele, whose distribution in the population fits the Hardy-Weinberg equilibrium with observed 20% *0/0, 46% *A/0 and 34% *A/A individuals. The number of GSTT1*A alleles detected by this procedure correlated highly significant with the enzyme activity in erythrocytes. Genotype-phenotype comparisons demonstrated a codominant type of inheritance by a gene-dose effect: samples with two active alleles expressed a statistically significant higher enzymatic activity compared to those with one null allele (P < 0.0001, ANOVA).

Optical PCR: genomic analysis by long-range PCR and optical mapping.

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

Multiple initiations in the c-myc replication origin independent of chromosomal location.

At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.

Mapping of the Gene Encoding the Regulatory Subunit RIIα of cAMP-Dependent Protein Kinase (Locus PRKAR2A) to Human Chromosome Region 3p21.3–p21.2

We have determined the chromosomal localization of the gene for the regulatory subunit RIIα of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome 3 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. Furthermore, PCR analysis of a chromosome 3 mapping panel revealed the presence of a human RIIα-specific amplification product only in cell lines containing the region 3p21.3–p21.2. The localization of PRKAR2A was confirmed by PCR mapping using the Stanford G3 Radiation Hybrid Panel as template. The results from this analysis demonstrated that PRKAR2A is most closely linked to D3S3334 (lod score 12.5) and flanked by D3S1322E and D3S1581.

Molecular epidemiology of aminoglycoside resistance in Acinetobacter spp.

Most aminoglycoside resistance in Acinetobacter spp. involves production of aminoglycoside-modifying enzymes. Previous studies have shown that the genes encoding these enzymes can be present on plasmids, transposons or within integron-type structures. To determine whether particular mechanisms of aminoglycoside resistance have developed in strains from specific geographical locations (with subsequent clonal spread), or whether common mechanisms have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. throughout the world, a genotypically heterogeneous collection of 24 multiresistant clinical isolates of Acinetobacter spp. from 15 hospitals in 11 countries worldwide was studied. All were resistant to two or more aminoglycoside antibiotics. The full aminoglycoside resistance profile was determined for each isolate, allowing a putative enzyme content to be inferred, with subsequent confirmation of enzyme content and genetic location by polymerase chain reaction (PCR) and hybridisation techniques. All produced at least one aminoglycoside-modifying enzyme, most commonly AAC(3)-I and ANT(3'')-I in various combinations. Other enzymes found were AAC(3)-II, AAC(6')-I, ANT(2''), APH(3')-I and APH(3')-VI. None was confined to strains from a particular geographical area. Nine isolates transferred resistance mediated by AAC(3)-I, ANT(2'')-I, APH(3')-I or APH(3)'-VI by conjugation to a sensitive strain of A. baumannii, but most resistance was non-transferable. PCR mapping revealed an integron location in six isolates for the aac(3)-Ia gene and in three isolates for the ant(3'')-Ia gene. Overall, the study demonstrated that similar aminoglycoside-modifying enzymes are found in unrelated isolates of Acinetobacter spp., and that particular genes are not restricted to specific areas of the world. The demonstration of certain genes on plasmids and integrons emphasises the probable importance of these structures in the dissemination of certain types of aminoglycoside resistance in Acinetobacter spp.

Molecular HLA typing--the brave new world.

Molecular HLA typing--the brave new world.

A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7.

Alterations in the amino acid sequence of the estrogen receptor (ER) have been shown to have dramatic effects on its function. Recently, mutant ERs have been isolated from both clinical samples and established breast cancer cell lines, primarily through the use of the polymerase chain reaction (PCR). All previously reported mutations have given rise to either alterations or truncations of the ER protein. We determined the structure of a novel 80 kDa ER which is expressed in an estrogen independent subclone of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was initially detected by Western blot analysis using a variety of ER specific antibodies. PCR mapping and partial PCR mediated subcloning of the ER cDNA were used to demonstrate that this protein was an ER containing an in-frame duplication of exons 6 and 7. This type of duplication has not been previously described for any members of the steroid receptor superfamily. Karyotype analysis coupled with fluorescence in situ hybridization (FISH) demonstrated that MCF-7:2A cells contained 4-5 copies of the ER gene in contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH analyses in both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is the source of the ER in normal human cells. The relative expression level of 2:1 is consistent with DNA gene dosage analysis. Genomic PCR was then used to demonstrate that the 80 kDa ER mRNA was not derived from the trans-splicing of two ER mRNAs but was the result of a genomic rearrangement in which exons 6 and 7 were duplicated in an in-frame fashion. This variant ER may prove to be useful in elucidating the mechanism of estrogen action in breast cancer cells.

A strategy for the identification of candidate genes for alcohol-related phenotypes and other human disorders using rapid polymerase chain reaction mapping of gene-based sequence-tagged sites.

We describe a method for the rapid identification and mapping of human genes, including those possibly contributing to disease and alcohol-related phenotypes. New human genes are identified from cDNA libraries through single-pass sequencing into the 3' untranslated (3'UT) regions of human brain cDNAs. Primers derived from the 3'UT region sequences [representing gene-based, sequence-tagged sites (STSs)] are used for polymerase chain reaction (PCR) analyses of the CEPH megabase insert yeast artificial chromosome (YAC) DNA pools. With this approach, approximately 18,000 megabase YACs can be screened and a single YAC identified using only 52 PCR reactions. The YAC localization in conjunction with other mapping techniques, such as PCR mapping to human chromosomes using somatic cell hybrids, allows identification of chromosomal band locations. In this manner, each gene can be associated with its own STS, which in turn specifies both a corresponding genomic clone and specific location in the genome. These locations can be compared with the purported locations of disease genes. The locations of the STSs can also be compared with those of Quantitative Trait Loci implicated for quantitative traits (e.g., alcohol-related phenotypes) on the basis of synteny between the mouse and human genes. Using this strategy, we found candidates for 78 human disease/syndrome genes among the first 220 genes mapped.

Localization of the gene for a novel human adenylyl cyclase (ADCY7) to chromosome 16.

A novel form of human adenylyl cyclase (ADCY7) has been discovered in the human erythroleukemia cell line (HEL). This cell line has been widely used as a model for studies of the characteristics of human platelets. Data from HEL cells suggests that ADCY7 may be the major AC form in human platelets. In the current study polymerase chain reaction (PCR) techniques coupled with use of human/rodent somatic hybrid panels and a yeast artificial chromosome (YAC) library were used to determine the chromosomal localization of the gene (adcy7) for ADCY7 enzyme. A 251-bp product from the 3' untranslated region of human adcy7 was amplified for PCR mapping and the results localize the adcy7 gene to region 16q12-16q13 of the human genome. The AC enzyme family is characterized by the presence of 12 membrane-spanning domains in its sequences, and this chromosomal region is known to contain other genes coding for proteins characterized by 12 membrane-spanning domains.