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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields.

Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.

There are few diagnostic methods that readily distinguish among community-acquired methicillin (meticillin)-resistant Staphylococcus aureus strains, now frequently transmitted within hospitals. We describe a rapid and high-throughput method for bacterial profiling of staphylococcal isolates. The method couples PCR to electrospray ionization-mass spectrometry (ESI-MS) and is performed on a platform suitable for use in a diagnostic laboratory. This profiling technology produces a high-resolution genetic signature indicative of the presence of specific genetic elements that represent distinctive phenotypic features. The PCR/ESI-MS signature accurately identified genotypic determinants consistent with phenotypic traits in well-characterized reference and clinical isolates of S. aureus . Molecular identification of the antibiotic resistance genes correlated strongly with phenotypic in vitro resistance. The identification of toxin genes correlated with independent PCR analyses for the toxin genes. Finally, isolates were correctly classified into genotypic groups that correlated with genetic clonal complexes, repetitive-element-based PCR patterns, or pulsed-field gel electrophoresis types. The high-throughput PCR/ESI-MS assay should improve clinical management of staphylococcal infections.

In the treatment of serious bacterial infections, the rapid institution of appropriate antimicrobial chemotherapy may be lifesaving. Choosing the correct antibiotic or combination of antibiotics is becoming very important, as multidrug resistance is found in many pathogens. Using a collection of 75 well-characterized multidrug-resistant (MDR) Acinetobacter sp. isolates, we show that PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and base composition analysis of PCR amplification products can quickly and accurately identify quinolone resistance mediated by mutations in the quinolone resistance-determining regions of gyrA and parC , two essential housekeeping genes. Single point mutations detected by PCR/ESI-MS in parC (found in 55/75 of the isolates) and in gyrA (found in 66/75 of the isolates) correlated with susceptibility testing and sequencing. By targeting resistance determinants that are encoded by genes with highly conserved DNA sequences (e.g., gyrA and parC ), we demonstrate that PCR/ESI-MS can provide critical information for resistance determinant identification and can inform therapeutic decision making in the treatment of Acinetobacter sp. infections.

The prevalence of Streptococcus pneumoniae serotype 6C, a recently recognized serotype that cross-reacts serologically with serotype 6A, was investigated. Isolates of serotype 6A in various collections were recovered, and serotype 6C was differentiated from 6A by multiplex PCR of DNA extracts by using appropriate primers. Antimicrobial susceptibility was performed by Clinical and Laboratory Standards Institute broth microdilution, and selected isolates were typed by pulsed-field gel electrophoresis, repetitive sequence-based PCR typing, and rapid multilocus sequence typing (MLST) by electrospray ionization mass spectrometry of PCR products. A total of 60 serotype 6C isolates were found: 30 of 122 Cleveland isolates collected from 1979 to 2007, 19 of 39 pediatric isolates collected nationwide in 2005 and 2006, and 11 pediatric isolates from Massachusetts collected in 2006 and 2007. Only four isolates were recovered prior to introduction of the conjugate pneumococcal vaccine in 2000; the earliest isolate was recovered in 1989. The sources of the isolates included blood ( n = 5), the lower respiratory tract ( n = 27), the sinus ( n = 5), the ear ( n = 2), and the nasopharynx ( n = 18); isolates were recovered from 49 children and 11 adults. Pediatric isolates were found in all six major U.S. geographic regions. Antimicrobial susceptibility showed that 22 isolates were nonsusceptible to penicillin, macrolides, and trimethoprim-sulfamethoxazole, 8 had other resistance patterns, and 30 were fully susceptible. The three typing methods used showed similar clusters of up to eight isolates per cluster. MLST showed five clusters related to serotype 6A, two clusters related to serotype 6B, one cluster related to serotype 3, and one cluster related to serotype 34. This study documents the occurrence, nationwide distribution, diversity, likely origins, and increasing incidence after 2001 of this recently recognized serotype. Serotype 6C warrants consideration for addition to future conjugate pneumococcal vaccines.

We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to “weigh” the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.

In a previous bioinformatics-based search for novel small-RNA genes encoded by theEscherichia coligenome, we identified a region, IS063, located between theompNandydbKgenes, that encodes an ∼100-nucleotide small-RNA transcript. Here we show that the expression of this small RNA is increased at a low temperature and in minimal medium. Twenty-two nucleotides at the 5′ end of this transcript have the potential to form base pairs with the leader sequence of the mRNA encoding the outer membrane protein OmpC. The deletion of IS063 increased the expression of anompC-luctranslational fusion 1.5- to 2-fold, and a 10-fold overexpression of the small RNA led to a 2- to 3-fold repression of the fusion. Deletion and overexpression of the IS063 RNA also resulted in increases and decreases, respectively, in OmpC protein levels. Taken together, these results suggest that IS063is a regulator of OmpC expression; thus, the small RNA has been renamed MicC. The antisense regulation was further demonstrated by the finding thatmicCmutations were suppressed by compensatory mutations in theompCmRNA. MicC was also shown to inhibit ribosome binding to theompCmRNA leader in vitro and to require the Hfq RNA chaperone for its function. We suggest that the MicF and MicC RNAs act in conjunction with the EnvZ-OmpR two-component system to control the OmpF/OmpC protein ratio in response to a variety of environmental stimuli.

We used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating in regions of high and low tuberculosis (TB) endemicity in China. The dominant principal genetic group (PGG) circulating in China was PGG1, and drug-resistant gene mutations were more diversified in the region of low rather than high TB endemicity.

Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.

We previously showed, using a single-copy papBAp-lac fusion (previously designated papBA-lac), that pyelonephritis-associated pili (pap) pilin gene transcription is subject to both phase variation and thermoregulatory control mechanisms (L. B. Blyn, B. A. Braaten, C. A. White-Ziegler, D. H. Rolfson, and D. A. Low, EMBO J. 8:613-620, 1989). At 37 degrees C, Escherichia coli strains carrying the papBAp-lac fusion displayed both Lac+ and Lac- colony phenotypes. In contrast, at 23 degrees C, colonies displayed a uniform Lac- phenotype, suggesting that pilin was not transcribed at this temperature. In this study, a strain carrying the papBAp-lac fusion was subjected to mini-Tn10 (mTn10) mutagenesis to isolate mutants that could initiate transcription of pilin at the nonpermissive temperature. Two classes of thermoregulatory mutants were identified in which the mTn10 mutation was linked to the mutant phenotype. Class I mutants displayed a phase variation phenotype at both 37 degrees C and 23 degrees C, whereas class II mutants displayed a uniform Lac+ colony phenotype at both temperatures. Preliminary analysis of these mutants showed that the mTn10 insertions in the class I mutants were chromosomally located, whereas the mTn10 insertions in the class II mutants were located within the papBAp-lac fusion phage. Southern blot analysis of the class I mutants demonstrated that mTn10 was present in the same 5.9-kilobase SalI DNA fragment in each mutant. Two of the class I mTn10 mutations were mapped to approximately 23.4 min on the E. coli K-12 chromosome. The locus defined by the class I mTn10 mutations was designated tcp, for thermoregulatory control of pap. Analysis of phase transition rates of the class I mutants showed that the phase-off (Lac-)----phase-on (Lac+) transition rates were higher than those observed with the nonmutant E. coli strain.

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.