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There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei . We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella . Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

This paper studied automatic identification of malaria infected cells using deep learning methods. We used whole slide images of thin blood stains to compile an dataset of malaria-infected red blood cells and non-infected cells, as labeled by a group of four pathologists. We evaluated three types of well-known convolutional neural networks, including the LeNet, AlexNet and GoogLeNet. Simulation results showed that all these deep convolution neural networks achieved classification accuracies of over 95%, higher than the accuracy of about 92% attainable by using the support vector machine method. Moreover, the deep learning methods have the advantage of being able to automatically learn the features from the input data, thereby requiring minimal inputs from human experts for automated malaria diagnosis.

Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface.

Bacteriuria, or the presence of bacteria in urine, is associated with both asymptomatic and symptomatic urinary tract infection and underpins much of the dynamic of microbial colonization of the urinary tract. The prevalence of bacteriuria in dissimilar patient groups such as healthy adults, institutionalized elderly, pregnant women, and immune-compromised patients varies widely. In addition, assessing the importance of 'significant bacteriuria' in infected individuals represents a diagnostic challenge, partly due to various causal microorganisms, and requires careful consideration of the distinct etiologies of bacteriuria in different populations and circumstances. Recent molecular discoveries have revealed how some bacterial traits can enable organisms to grow in human urine, which, as a fitness adaptation, is likely to influence the progression of bacteriuria in some individuals. In this review, we comprehensively analyze currently available data on the prevalence of causal organisms with a focus on asymptomatic bacteriuria in dissimilar populations. We evaluate recent advances in the molecular detection of bacteriuria from a diagnostic viewpoint and briefly discuss the potential benefits and some of the challenges of these approaches. Overall, this review provides an update on the comparative prevalence and etiology of bacteriuria from both microbiological and clinical perspectives.

Pneumococcal surface protein A (PspA) is the only pneumococcal surface protein known to strongly bind lactoferrin on the bacterial surface. In the absence of PspA Streptococcus pneumoniae becomes more susceptible to killing by human apolactoferrin (apo-hLf), the iron-free form of lactoferrin. In the present study we examined diverse strains of S. pneumoniae that differed by 2 logs in their susceptibility to apo-hLf. Among these strains, the amount of apo-hLf that bound to cell surface PspA correlated directly with the resistance of the strain to killing by apo-hLf. Moreover examination of different pspA alleles on shared genetic backgrounds revealed that those PspAs that bound more lactoferrin conferred greater resistance to killing by apo-hLf. The effects of capsule on killing of pneumococci by apo-hLf were generally small, but on one genetic background, however, the lack of capsule was associated with 4-times as much apo-hLf binding and 30-times more resistance to killing by apo-hLf. Overall these finding strongly support the hypothesis that most of the variation in the ability of apo-hLf is dependent on the variation in the binding of apo-hLf to surface PspA and this binding is dependent on variation in PspA as well as variation in capsule which may enhance killing by reducing the binding of apo-hLf to PspA.

Streptococcus pneumoniae expresses more than 90 capsule types, and currently available pneumococcal vaccines are designed to provide serotype-specific protection. Consequently, serotyping of pneumococcal isolates is important for determining the serotypes to be included in pneumococcal vaccines and to monitor their efficacy. Yet serotyping of pneumococcal isolates has remained a significant technical challenge. By multiplexing many assays, we have now developed a simple yet comprehensive serotyping assay system that can not only identify all known pneumococcal serotypes but also subdivide nontypeable (NT) isolates into those with or without the conventional capsule locus. We have developed this assay system to require only six key reagents: two are used in one multiplex inhibition-type immunoassay, and four are required in two multiplex PCR-based assays. The assay system is largely automated by a seamless combination of monoclonal antibody-based and PCR-based multiplex assays using the flow cytometric bead array technology from Luminex. The assay system has been validated with a panel of pneumococci expressing all known pneumococcal serotypes and was found to be easily transferable to another laboratory.

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae . Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA , lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin ( P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

Antiretroviral therapy (ART) quickly suppresses HIV-1 replication in patients, to nondetectable levels. However, even after years of effective ART, cessation of therapy results in the immediate rebound of viremia. This is attributed to a long-lived reservoir of latently HIV-1-infected memory CD4+ T cells (2-4). As a result of the long life span of memory T cells that serve as cellular hosts to latent HIV-1 infection, the latent HIV-1 reservoir is extremely stable. Natural eradication in the absence of any replenishment of the reservoir by de novo infection events is predicted to take about 70 years (26). Since natural depletion of the latent reservoir is unlikely to be achievable, HIV-1 latency is believed to represent the principal obstacle to curative AIDS therapy (2-9). Strategies to purge this viral reservoir will have to be included in any HIV-1 therapy with curative intent. Based on the molecular understanding of HIV-1 latency, several attempts to therapeutically deplete the latent HIV-1 reservoir by activating the integrated but transcriptionally silent viral promoter have previously been made. This was initially attempted using strategies that would increase NF-κB activity levels in latently infected T cells. With NF-κB being the major activating transcription factor for HIV-1, this was thought to promote HIV-1 reactivation (6, 18, 21). Patients on ART were thus treated with interleukin-2 (IL-2) or the anti-CD3 monoclonal antibody (MAb) OKT3, two known inducers of NF-κB activity in T cells (2-9). Later, histone deacetylase (HDAC) inhibitors (valproic acid) were applied therapeutically, as it had been proposed that similar to the case for inactive but inducible cellular genes, a restrictive histone code would govern HIV-1 latency. HDAC inhibitors were thought to trigger changes in the histone composition at the viral promoter to favor viral transcription in the absence of cellular activation. To date, all clinical HIV-1 reactivation protocols have failed to reduce the latent reservoir, or the clinical significance of reported reductions has been disputed (27, 28). This situation is further complicated by a recent report that latent HIV-1 infection events in vivo are found integrated predominantly into actively expressed host genes (12). While some have voiced concerns that the detected integration events were likely to be functionally inactive, we recently confirmed this idea in an unbiased cellular model of HIV-1 latency establishment (10). In this system, we found that all of the analyzed latently infected cell clones carried the HIV-1 genome in actively expressed host genes. However, actively expressed genes are an unlikely environment for the establishment of a restrictive histone code. In this setting, HIV-1 latency could be controlled by transcriptional interference of the host gene promoter by the integrated viral long terminal repeat (LTR) (10, 13, 22). These findings favor the development of reactivation strategies that would aim to directly transcriptionally activate the latent HIV-1 promoter by stimulating the host cell. However, if HIV-1 reactivation should be achieved by direct LTR activation, then in order to prevent the induction of a cytokine storm, therapeutics would have to be developed that, unlike anti-CD3 MAbs or IL-2, dissociate cellular gene activation from HIV-1 reactivation. Here we present proof-of-concept data showing that this can be achieved. In this study, we report that a protein activity secreted by the nonpathogenic bacterium Massilia timonae, termed HIV-1-reactivating factor (HRF), efficiently reactivated latent HIV-1 infection without inducing cellular gene expression. HRF produced a strong but short burst of NF-κB activity. This type of hit-and-run activation was sufficient to trigger initial HIV-1 Tat production, which then induced self-perpetuation of HIV-1 expression. Since cellular gene expression is not regulated by trans-acting positive feedback mechanisms, no sustainable gene expression was established, despite the strong initial trigger. These findings suggest that the control of both the amplitude and the duration of NF-κB activity can be used to selectively trigger latent HIV-1 expression without induction of cellular genes.

Group B Streptococcus (GBS) causes urinary tract infections, but the pathogenic mechanisms underlying GBS urinary tract infections are unknown. We investigated whether uropathogenic GBS can bind to bladder uroepithelium to initiate urinary tract infection. Uropathogenic GBS isolated from a patient with acute cystitis bound to human T24 bladder uroepithelial cells in close association with F-actin in statistically significantly higher numbers compared with nonuropathogenic GBS. In vivo modeling using transurethrally infected mice revealed superior fitness of uropathogenic GBS for bladder colonization and potent uropathogenic GBS-specific up-regulation of interleukin 1alpha during infection. Thus, binding of uropathogenic GBS to uroepithelium and vigorous induction of interleukin 1alpha represents the initial stages of GBS urinary tract infection.

Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection, PIR-B-deficient (PIR-B−/−) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica Typhimurium (WB335). PIR-B−/− mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns of inflammatory lesions were found in their livers at 7–14 days postinfection: diffuse spreading along the sinusoids in PIR-B−/− mice vs nodular restricted localization in WT mice. PIR-B−/− mice have more inflammatory cells in the liver but fewer B cells and CD8+ T cells in the spleen than WT mice at 14 days postinfection. PIR-B−/− bone marrow-derived macrophages (BMMφ) failed to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared with WT BMMφ. PIR-B−/− BMMφ also produced more nitrite and TNF-α upon exposure to Salmonella than WT BMMφ did. These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial infection.

This review demonstrates that funeral service professionals (FSPs) have a risk of exposure to bacterial and viral pathogens as well as to prion-mediated diseases. It reveals a lack of published studies focusing on the implementation and effectiveness of infection control policies for this occupational group as well as the difficulty involved in determining actual infection rates related to workplace exposure events. Possible reasons for this lack of data include the categorization of these workers by the Bureau of Labor Statistics Standard Occupational Classification System as service providers rather than health care professionals.

Asplenic individuals have increased susceptibility to septicemia caused by encapsulated bacteria. Streptococcus pneumoniae, a pathogen carried in the nasal passages of many humans without complication, is responsible for a large proportion of infections seen in asplenic individuals. Our studies have evaluated the efficacy of antibodies to pneumococcal surface protein A (PspA) in protection of asplenic mice. In passive immunity studies, pneumococci were more completely cleared from the blood of splenectomized mice receiving passive antiserum to PspA than those receiving normal rabbit serum. From active mucosal (intranasal) and systemic (subcutaneous) immunizations with rPspA, we determined that the levels of PspA antibodies produced in splenectomized mice were not significantly different from levels seen in mock-splenectomized animals. This active immunity to PspA was able to protect splenectomized mice against death following infection with live pneumococci. Our results suggest that PspA immunization may also protect asplenic humans from pneumococcal infections.

Fluoroquinolone resistance in Streptococcus pneumoniae is primarily mediated by point mutations in the quinolone resistance–determining regions of gyrA and parC. Antimicrobial resistance mutations in housekeeping genes often decrease fitness of microorganisms. To investigate the fitness of quinolone-resistant S. pneumoniae (QRSP), the relative growth efficiencies of 2 isogenic QRSP double mutants were compared with that of their fluoroquinolone-susceptible parent, EF3030, by using murine nasopharyngeal colonization and pneumonia models. Strains containing the GyrA: Ser81Phe, ParC: Ser79Phe double mutations, which are frequently seen in clinical QRSP, competed poorly with EF3030 in competitive colonization or competitive lung infections. However, they efficiently produced lung infection even in the absence of EF3030. The strain containing the GyrA: Ser81Phe, ParC: Ser79Tyr double mutations, which is seen more frequently in laboratory-derived QRSP than in clinical QRSP, demonstrated reduced nasal colonization in competitive or noncompetitive lung infections. However, the strain was equally able to cause competitive or noncompetitive lung infections as well as EF3030.

With a tuberculosis case detection rate of 5.9 per 100,000 population in 2001, Alabama ranked twelfth highest in the United States. However, cases among foreign-born and human immunodeficiency virus-infected individuals remain low in Alabama. To understand the endemic statewide disease pattern, tuberculosis strains were studied for clustering in a long-term population-based study from January 1994 to May 2000. IS6 110 restriction fragment length polymorphism analysis was performed for 1,834 strains. Spoligotyping was used as a secondary typing method for the 37% of isolates displaying a restriction fragment length polymorphism pattern with 6110 copies. A total of 721 (41%) patients provided isolates that composed 114 clusters, each containing isolates from 2 to 136 patients, suggesting that recent transmission accounted for 35% of tuberculosis cases. Demographic, behavioral, and clinical characteristics of patients with clustered versus nonclustered isolates stratified by low-copy-number strains (6110 copies) versus high-copy-number strains (≥6 IS 6110 copies) were evaluated. Younger age, black race, a history of alcohol abuse, and homelessness were predictors of clustering of low-copy-number, strains and younger age, urban residency, alcohol abuse, homelessness, noninjection drug use, and a history of incarceration and/or cavitary disease were predictors of clustering of high-copy-number strains. By identifying local characteristics of tuberculosis clustering through molecular fingerprinting, control programs can distribute their limited resources to impact the transmission of tuberculosis in high-risk populations and evaluate strain distribution across geographical areas.

Intestinal macrophages, which are thought to orchestrate mucosal inflammatory responses, have received little investigative attention compared with macrophages from other tissues. Here we show that human intestinal macrophages do not express innate response receptors, including the receptors for LPS (CD14), Fcalpha (CD89), Fcgamma (CD64, CD32, CD16), CR3 (CD11b/CD18), and CR4 (CD11c/CD18); the growth factor receptors IL-2 (CD25) and IL-3 (CD123); and the integrin LFA-1 (CD11a/CD18). Moreover, resident intestinal macrophages also do not produce proinflammatory cytokines, including IL-1, IL-6, IL-10, IL-12, RANTES, TGF-beta, and TNF-alpha, in response to an array of inflammatory stimuli but retain avid phagocytic and bacteriocidal activity. Thus, intestinal macrophages are markedly distinct in phenotype and function from blood monocytes, although intestinal macrophages are derived from blood monocytes. To explain this paradox, we show that intestinal stromal cell-derived products downregulate both monocyte receptor expression and, via TGF-beta, cytokine production but not phagocytic or bacteriocidal activity, eliciting the phenotype and functional profile of intestinal macrophages. These findings indicate a mechanism in which blood monocytes recruited to the intestinal mucosa retain avid scavenger and host defense functions but acquire profound "inflammatory anergy," thereby promoting the absence of inflammation characteristic of normal intestinal mucosa despite the close proximity of immunostimulatory bacteria.

Prior infection of mice with a field strain of mouse hepatitis virus (MHV) increased the early resistance of euthymic mice to virulent Salmonella typhimurium strain SR-11 infections (as defined by significantly fewer salmonella colony-forming units (cfu) present in spleens and livers 4 days after salmonella infection). This increase in salmonella resistance was observed when the interval between MHV and salmonella infections was 6 days, but not at 3, 10, or 14 day intervals. The mouse lty locus, which controls the number of intracellular salmonella, had a significant effect on the ability of MHV to induce resistance to salmonella. MHV caused an increase in resistance to salmonella in ltys (salmonella susceptible) mice at all doses of salmonella tested (100 to 10000 cfu). In the ltyr (salmonella resistant) mice tested the beneficial effect of MHV on salmonella resistance was small and when observed, was only present at salmonella doses of 10000 cfu or greater. Neither the Lpsd nor Xid mutations affected the ability of MHV to increase resistance to salmonella infection. In contrast to euthymic mice, MHV infection greatly decreased the resistance of athymic (nude) mice to salmonella infection. Since the Nu locus does not affect the resistance of mice to salmonella (at 4 days post salmonella infection), these results indicate that MHV infection and the nude phenotype interact to increase susceptibility to salmonella. These findings re-emphasize the importance of keeping laboratory mice used in research free of MHV and other immunomodulatory pathogens.

Macrolide resistance was detected in 64 of 77 (83.1%) Streptococcus pneumoniae isolates from South Korea. Seven (10.9%) isolates contained only mef (A), 32 (50%) contained only erm (B), and 25 (39.1%) contained mef (A) and erm (B). Nineteen isolates containing mef (A) and erm (B) belonged to serotype 19F, and seven isolates were identical to the Taiwan 19F -14 clone.

Intranasal infection of mice with certain strains of capsular group 19 Streptococcus pneumoniae can result in focal pneumonia in the absence of bacteremia. Using this model of murine pneumonia, we demonstrated that immunization with recombinant forms of either pneumococcal surface protein A (PspA) or PdB (a genetically detoxified derivative of pneumolysin) elicited significant protection against focal pulmonary infection. This may be the first demonstration that a proposed vaccine antigen can protect against pneumococcal pneumonia. The best protection was obtained by immunizing mice with a mixture of PspA and PdB, indicating that the protection elicited by these antigens can complement each other. This result is in agreement with previous studies that used pneumococcal sepsis and nasal colonization models and demonstrate that the best protein vaccines for prevention of infection may be those that include more than one protection-eliciting pneumococcal protein.

Streptococcus pneumoniae is a major human pathogen causing pneumonia, sepsis, meningitis, and otitis media ([12][1]). It causes infections most often in young children ([12][1]) and elderly adults ([1][2]) because their immune systems are either unprepared or unable to respond effectively to

We characterized 32 levofloxacin-nonsusceptible Streptococcus pneumoniae (LNSP) isolates obtained from a broad geographic region of North America over a 5-year period by using capsular serotypes, antimicrobial susceptibility profiles, BOX-PCR, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sixteen international clones identified by the Pneumococcal Molecular Epidemiology Network also were included for comparison. Fifteen serotypes were represented, with serogroups 6, 9, 14, 19, and 23 accounting for 63% of isolates. Among isolates whose quinolone resistance-determining regions were sequenced, all contained gyrA and parC point mutations. Sixty-three percent were penicillin susceptible, and 84% were erythromycin susceptible. BOX-PCR analysis identified 39 different band patterns among 32 LNSP and 16 international clones and grouped 16 isolates, including 2 international clones, into seven unrelated groups of 2 to 4 isolates each. PFGE analysis identified 35 different band patterns among 32 LNSP and 16 international clones and grouped 21 isolates, including 3 international clones, into eight unrelated groups of 2 to 6 isolates each. MLST performed on 10 isolates identified five allelic profiles and separated 9 isolates into four groups of 2 to 3 isolates each. Overall, each typing method indicated that the LNSP were heterogeneous and that resistance to fluoroquinolones was not closely associated with a particular serotype or with coresistance to other antimicrobial classes and suggests that LNSP have likely arisen through independent mutational events as a result of selective pressure. However, seven LNSP were found to be related to three international clones by PFGE.

Although tuberculosis (TB) remains a major public health threat worldwide (1), its declining incidence in the United States has led health policy makers to develop plans for disease elimination (less than one patient per million) by 2010 (2). Although targeted screening and treatment of latent TB infection has been recommended for groups at high risk (3), learning more about recent TB transmission will help identify specific program interventions that may prevent infection and disease. Molecular epidemiology has been used to identify groups most at risk for recent TB transmission in high-incidence urban and rural areas of the United States (4–9), but little data have been available from sites with a low-to-moderate disease incidence. Maryland’s varied culture and geography provide a microcosm for the study of TB transmission in the United States. The population of 5.1 million resides in distinct areas: urban (city of Baltimore), suburban (5 counties), and rural coastal and mountainous areas (18 counties). Baltimore reports high rates of homelessness, HIV infection, and illegal drug use. Foreign immigration to suburban and some rural areas of the state has increased by 53% in the past decade, causing Maryland to rank third in the nation in rate of foreign population growth (10). In spite of the presence of these groups at high risk of acquiring TB, excellent treatment regimens utilizing directly observed therapy (87% vs. 47% nationally) and four-drug initial therapy (89% vs. 77% nationally) resulted in a 15-year decline in disease incidence (11). Since 1989, the state’s TB incidence has remained lower than the national average (4.9/100,000 vs. 5.6/100,000 population, respectively, in 2001) (11), and Baltimore ranks 26th among 31 major U.S. cities for TB incidence (Centers for Disease Control and Prevention [CDC], unpub. data, 2000). As part of the CDC-supported National Tuberculosis Genotyping and Surveillance Network, we used DNA fingerprinting of Mycobacterium tuberculosis isolates and patient information to conduct a statewide epidemiologic study of culture-positive Maryland TB patients. We quantified the problem of recent TB transmission in the state, characterized circumstances and settings for transmission, and used our findings to review programmatic interventions.

The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis . Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli . Upon induction of the TrcR protein, there was a >500-fold increase in β-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

C-reactive protein (CRP) is an acute-phase protein with a well-known association with infection and other inflammatory conditions. We have shown that expression of human CRP by CRP transgenic (CRPtg) mice is protective against lethal infection byStreptococcus pneumoniae, an effect likely mediated by CRP's ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection withSalmonella entericaserovar Typhimurium, a gram-negative pathogen that causes the murine equivalent of typhoid fever. CRPtg mice experimentally infected with a virulent Typhimurium strain lived longer and had significantly lower mortality than their non-tg littermates. The greater resistance of CRPtg mice could be attributed to significantly increased early (0 to 4 h) blood clearance of salmonellae and significantly decreased numbers of bacteria in the liver and spleen on day 7 postinfection. In addition, 14 days after infection with an avirulentSalmonellastrain, the serum titer of anti-Salmonellaimmunoglobulin G antibodies was higher in CRPtg than non-tg mice. This study provides unequivocal evidence that CRP plays an important role in vivo in host defense against salmonellae during the early stages of infection. In addition, as the beneficial effect of CRP includes enhancement of the host's humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.

We studied 198 macrolide-resistant S. pneumoniae isolates obtained from adults and children to evaluate whether 2-μg clindamycin disks can distinguish between isolates manifesting ermB - versus mefE -mediated resistance to clarithromycin and to determine the relative frequency with which each resistance mechanism occurred in these populations. The mefE gene was predominant among 109 isolates from children, occurring in 73.4% versus 50.6% of 89 isolates from adults. Three isolates (1.5%) did not amplify either gene. Among 125 mefE + isolates, the MIC of clarithromycin at which 90% of the isolates tested were inhibited, determined by Etest, was 32 μg/ml versus >256 μg/ml in 70 ermB + isolates. All ermB + isolates were highly resistant to clindamycin (MICs >256 μg/ml), whereas all mefE + isolates were susceptible to clindamycin using the 2-μg disk. Testing S. pneumoniae from the respiratory tract for susceptibility to clindamycin by agar disk diffusion is an easy and inexpensive method to estimate the frequency of resistance mediated by ermB in specific patient populations. Macrolide resistance mediated by ermB is usually of greater magnitude than that due to mefE . Clinical studies are needed to determine the significance of high- versus low-level macrolide resistance in S. pneumoniae .

The MB/BacT system (MB/BacT) with a revised antibiotic supplement kit was compared with the BACTEC 460 system (BACTEC 460) in a test of 488 specimens submitted for mycobacterial culture from 302 patients. Twenty-four Mycobacterium tuberculosis isolates were detected by the BACTEC 460 versus 23 isolates by the MB/BacT. Mean time until detection of M. tuberculosis isolates identified by both systems was 11.9 days for the BACTEC 460 versus 13.7 days for the MB/BacT ( P = 0.046). M. avium complex was detected in 12 specimens by the MB/BacT versus 10 specimens by the BACTEC 460. Only 8 of 14 (57%) M. avium isolates were detected by both systems, with a mean time until detection of 10.1 days for the BACTEC 460 and 14.2 days for the MB/BacT ( P = 0.009). The BACTEC 460 and the MB/BacT detected M. gordonae in four specimens, but only a single specimen was positive by both systems. One M. fortuitum isolate and one of five M. kansasii isolates were recovered only by the BACTEC 460. The bacterial overgrowth rate was 7.0% for the MB/BacT versus 4.1% for the BACTEC 460. We found the MB/BacT to be comparable to the BACTEC 460 for mycobacterial detection. Even though time until detection with the MB/BacT was slightly longer (1.8 days longer for M. tuberculosis and 4.1 days for M. avium [mean values]) and the bacterial overgrowth rate was somewhat higher, the decreased labor, the availability of a computerized data management system, and the noninvasive, nonradiometric aspects of the MB/BacT offset these relative disadvantages and make it an acceptable alternative for use in the diagnostic laboratory.

The focus of this study was the phenotypic characterization of Salmonella typhimurium mutants lacking the function of the response regulator mviA. The inactivation of mviA+ (mviA::kan) is shown to induce a significant change in the growth of most virulent strains, as reflected in the size of the colonies formed on agar plates. The colony phenotype observed in these strains has been designated as the small colony morphology (Scm+) phenotype. Mutants exhibiting the Scm+ phenotype are shown to be significantly attenuated for virulence in susceptible (ItyB) mice. The Scm+ phenotype therefore provides an in vitro phenotypic marker for mviA+ activity. Further examination of Scm+ mutants has revealed that they lack expression of a 55 kDa periplasmic protein which is detected in isogenic mviA+ strains. This protein has been designated mviA+ related protein A (MrpA) and was expressed in direct correlation with virulence in all S. typhimurium strains examined.

Serotypes of group B streptococcus (GBS) that cause urinary tract infection (UTI) are poorly characterized. We conducted a prospective study of GBS UTI in adults to define the clinical and microbiological characteristics of these infections, including which serotypes cause disease. Patients who had GBS cultured from urine over a 1-year period were grouped according to symptoms, bacteriuria, and urinalysis. Demographic data were obtained by reviewing medical records. Isolates were serotyped by latex agglutination and multiplex PCR-reverse line blotting (mPCR/RLB). Antibiotic susceptibilities were determined by disc diffusion. GBS was cultured from 387/34,367 consecutive urine samples (1.1%): 62 patients had bacteriuria of >10 7 CFU/liter and at least one UTI symptom; of these patients, 31 had urinary leukocyte esterase and pyuria (others not tested), 50 (81%) had symptoms consistent with cystitis, and 12 (19%) had symptoms of pyelonephritis. Compared with controls (who had GBS isolated without symptoms), a prior history of UTI was an independent risk factor for disease. Increased age was also significantly associated with acute infection. Serotyping results were consistent between latex agglutination and mPCR/RLB for 331/387 (85.5%) isolates; 22 (5.7%) and 7 (1.8%) isolates were nontypeable with antisera and by mPCR/RLB, respectively; and 45/56 (80.4%) isolates with discrepant results were typed by mPCR/RLB as belonging to serotype V. Serotypes V, Ia, and III caused the most UTIs; serotypes II, Ib, and IV were less common. Nontypeable GBS was not associated with UTI. Erythromycin (39.5%) and clindamycin (26.4%) resistance was common. We conclude that a more diverse spectrum of GBS serotypes causes UTI than previously recognized, with the exception of nontypeable GBS.

Monoclonal antibodies (MAbs) were raised against the outer membrane (OM) antigens ofSalmonella typhimurium.Enzyme-linked immunosorbent assays and Western immunoblots indicated that 10 MAbs in the panel were specific for surface epitopes, and 10 recognized buried epitopes of OmpC or OmpD porins; three MAbs reacted with smooth lipopolysaccharide (LPS), two bound rough LPS, and the remaining three MAbs apparently reacted with a porin–LPS complex. We screened these MAbs and immune polyclonal sera in CAF1(Ityr) mice for their relative immunoprotective potential against a challenge with 10 to 500 LD50of the virulentS. typhimuriumLT-2 strain WB600, or against two LD50of purified OM from this organism. Polyclonal sera that contained high titers of antibodies to porin monomers and trimers, and LPS, provided significant protection (33 to 100% survivors). Antiporin MAbs, when administered individually, did not protect or prolong the survival of mice. A mixture of MAbs with specificity for the surface, but not buried epitopes of porins, prolonged the survival of mice against endotoxemia, but none provided significant protection against mouse typhoid. MAbs specific for smooth (but not rough) LPS on the other hand, conferred significant protection against endotoxemia and mouse typhoid. Finally, MAbs that presumably recognized epitopes present in porin–LPS complexes, were also protective against endotoxemia and mouse typhoid. These results support the role of antibodies to LPS O-chains, porin–LPS complexes, and to a lesser degree, native porins in acquired resistance to infection byS. typhimurium.

Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments. The organism can survive potentially lethal acid conditions (pH

To reliably transport blood samples for cryoglobulin analysis, we have created a sample transport device containing a mixture of two waxes that solidifies at 38°C and maintains sample temperature at 38°C. Samples arriving at the laboratory at 37 to 38°C increased to 95% from 34% with the use of the device.

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli ; however, Gram-positive organisms, including Streptococcus agalactiae , or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

We conducted a program of population-based molecular typing of all Mycobacterium tuberculosis isolates obtained in Alabama since 1994. Of 2452 isolates, 1013 (41%) had fewer than 6 bands of IS6110; 348 (14%) had a single two-band pattern (JH2). With conventional epidemiologic methods, we identified three groups of related patients with JH2 isolates. Spoligotyping and pattern of variable number of tandem repeats identified 10 molecular groups; two found by conventional methods were subdivided.

From 1996 to 2000, 23 Maryland and Washington, D.C., tuberculosis cases were identified in one six-band DNA cluster. Cases were clustered on the basis of their Mycobacterium tuberculosis isolates. Medical record reviews and interviews were conducted to identify epidemiologic linkages. Eighteen (78%) of the 23 case-patients with identical restriction fragment length polymorphism patterns were linked to another member; half the patients were associated with a Washington, D.C., homeless shelter. Molecular epidemiology defined the extent of this large, cross-jurisdictional outbreak.

The virulent Salmonella typhimurium strain WB600 carries the mviA allele of the gene mouse virulence A. As shown here, the virulent phenotype of WB600 is the result of a nonfunctional mviA gene. As compared to the functional allele mviA+, mviA increases virulence in Itys mice, but not in Ityr mice. A specific BglII site, mviA4185, between osmZ and galU, located at approximately 35 min on the salmonella chromosome, was within mviA. Insertion of an antibiotic cassette in the mviA4185 site of mviA+ or the homologous mviA4093 site of mviA DNA resulted in virulence when either cassette was recombined into the chromosome. When mviA and mviA+ were both expressed in the same strain with one carried in the chromosome and the other on a plasmid, avirulence was dominant. Replacement of the mviA allele of strain WB600 using P22 transductions of linked antibiotic cassettes cloned into the chromosome of virulent S. typhimurium strains (SR-11, TML, SL1344, C5, ATCC14028, W118-2, and WB600) showed that all but WB600 contained the avirulent mviA+ allele. Southern hybridizations provided no evidence for a second mviA allele anywhere in the genome of the six non-WB600 strains.

Two commercial swab transport systems, Copan Amies gel agar with and without charcoal (Copan Diagnostics, Corona, Calif.), were compared to direct inoculation onto modified Thayer-Martin medium for detection of Neisseria gonorrhoeae in 1,490 endocervical specimens obtained from women attending a sexually transmitted disease clinic. Copan swabs were held in the transport system for 24 h at room temperature prior to inoculation onto modified Thayer-Martin medium. All cultures were incubated at 35°C in 5% CO 2 , and bacteria were identified on the basis of Gram stain, oxidase, and biochemical reactions. Copan Amies gel agar transport system without charcoal detected 77 of 81 (95%) direct inoculation culture-positive specimens, and Copan Amies gel agar transport system with charcoal detected 53 of 56 (95%) directly inoculated culture-positive specimens. Copan Amies gel agar without charcoal inoculated after 6 h supported growth of 56 (98%) positive cultures out of only 55 directly inoculated culture-positive specimens. This study demonstrates that Copan swabs represent a reasonable alternative, providing convenience, low cost, and ease of use while still maintaining a satisfactory recovery rate of N. gonorrhoeae from clinical specimens, if specimens can be inoculated onto selective media within a relatively short time period not involving overnight shipment.

Maternal-fetal ABO incompatibility is a common hematological problem affecting the newborn. In general, hemolysis is minimal and the clinical course is relatively benign, rarely causing the escalating levels of hyperbilirubinemia and significant anemia commonly associated with Rh hemolytic disease of the newborn (HDN). The incidence of HDN ranges from one in 150 births to 1:3000 births, depending on the degree of anemia and level of serum bilirubin. The etiology of ABO hemolytic disease of the newborn (ABO-HDN) is complex because anti-A and anti-B antibodies are composed mainly of IgM. Since only IgG antibodies cross the placenta, those pregnant women with high levels of IgG anti-A,B, anti-A, or anti-B with an ABO incompatible fetus will be the ones to give birth to an infant with ABO-HDN. We describe a case of a B/Rh positive term newborn born to an O/Rh negative African-American mother demonstrating aggressive hemolysis and a robust response of the bone marrow. This case was successfully managed with phototherapy and simple RBC transfusion without the need for exchange transfusion.

The Salmonella typhimurium virulence gene mviA+ has a predicted amino acid sequence with homology to the N-terminal 112-amino-acid sequence of response regulator proteins. A previously described mutant allele (mviA), which restores virulence to avirulent LT2 strains, was shown to contain a point mutation which would be predicted to cause a single amino acid change, V-102-->G (W. H. Benjamin, Jr., J. Yother, P. Hall, and D. E. Briles, J. Exp. Med. 1,74:1073-1083, 1991). A comparison of the nucleotide sequence of mviA+ with that of the Escherichia coli and Salmonella typhi genes revealed a high degree of conservation.

Recipients of organ transplants are at increased risk for infection both because of immunosuppression and because of the transfer of microbes through the donor organs. We report two cases of M. tuberculosis disease in recipients of single lung transplants who shared a common donor. Both recipients developed pulmonary tuberculosis, one having fever and pulmonary infiltrates and the other having subclinical disease with M. tuberculosis organisms being recovered from bronchoalveolar lavage. Restriction fragment length polymorphism analysis on both isolates of M. tuberculosis revealed a common source. The donor of both lungs had a normal chest radiograph and no known prior history of M. tuberculosis infection of disease. These cases are the first report of two single lung recipients developing pulmonary tuberculosis from a common donor.

PsaA ofStreptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+transporter. Mutations inpsaAcause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. ApsaBCdeletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather,psaAmutations indirectly affect this process through the disruption of Mn2+transport. The deficiency in Mn2+transport also causes hypersensitivity to oxidative stress from H2O2and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+and Mn2+cations after a lag of about 15 h. Addition of Mn2+alone or together with Fe2+allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+alone extended the 15-h lag phase to 25 h. Thus, while Fe2+adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.

Vancomycin-resistant enterococci (VRE) have become important causes of nosocomial infections. This study evaluated the association between a variety of intravenous antimicrobial exposures and the isolation of VRE using two control groups: (1) a vancomycin-susceptible enterococci (VSE) group, to assess factors associated with development of VRE, and (2) a nonenterococci control group, to assess factors associated with positive cultures for enterococci without regard to vancomycin resistance. After adjusting for the effect of other antimicrobials, time at risk, and patient morbidity, compared to vancomycin-susceptible enterococci controls, exposures to imipenem (OR = 4.9, 95% CI = 1.6-14.1) and ceftazidime (OR = 2.6, 95% CI = 1.1-6.1) were significant predictors of VRE. When compared to nonenterococci controls, exposures to ampicillin (OR = 20.1, 95% CI = 1.5-263.1) and imipenem (OR = 5.1, 95% CI = 1.5-17.1) were significantly associated with VRE. Neither piperacillin nor vancomycin was associated with VRE compared to either control group. This study offers further evidence that the replacement of broad-spectrum cephalosporins by extended-spectrum penicillins, specifically piperacillin, may be effective in reducing VRE.

We review the history of vancomycin-resistant enterococci (VRE) and propose a causal model illustrating the roles of exposure to VRE reservoirs, patient characteristics, antimicrobial exposure, and prevalence of VRE in the progression from potential VRE reservoirs to active disease in hospitalized patients. Differences in VRE colonization and VRE infection are discussed with respect to hospital surveillance methodology and implications for interventions. We further document clonal transmission of VRE in a large, urban, teaching hospital and demonstrate VRE susceptibility to a wide array of antimicrobial agents. This model can guide the identification of mutable factors that are focal points for intervention.

We conducted a population-based study to assess demographic and risk-factor correlates for the most frequently occurring Mycobacterium tuberculosis genotypes from tuberculosis (TB) patients. The study included all incident, culture-positive TB patients from seven sentinel surveillance sites in the United States from 1996 to 2000. M. tuberculosis isolates were genotyped by IS6110-based restriction fragment length polymorphism and spoligotyping. Genotyping was available for 90% of 11,923 TB patients. Overall, 48% of cases had isolates that matched those from another patient, including 64% of U.S.-born and 35% of foreign-born patients. By logistic regression analysis, risk factors for clustering of genotypes were being male, U.S.-born, black, homeless, and infected with HIV; having pulmonary disease with cavitations on chest radiograph and a sputum smear with acid-fast bacilli; and excessive drug or alcohol use. Molecular characterization of TB isolates permitted risk correlates for clusters and specific genotypes to be described and provided information regarding cluster dynamics over time.

Objective This study demonstrates the value of Mycobacterium tuberculosis fingerprinting used in conjunction with traditional epidemiologic methods to identify smoldering outbreaks of tuberculosis in endemic areas where background rates of tuberculosis are high. Methods IS 6110 DNA fingerprinting was performed on isolates of M tuberculosis from verified cases of tuberculosis in Alabama from 1994 to 1998. A statewide database groups isolates into“ clusters” and tracks them cumulatively over time. A large cluster was identified and was secondarily investigated using traditional epidemiologic methods. Results Twenty-five isolates were found to be identical by fingerprinting analysis. Patients were living within 10 counties across the state, and 12 cases were localized to a single county. This represented an ongoing, statewide tuberculosis outbreak previously unrecognized by local and state health officials. Secondary investigation of the cases revealed the primary sites of transmission to be a correctional facility and two homeless shelters. Conclusions Population surveillance using M tuberculosis fingerprinting was successfully utilized to detect a significant and smoldering tuberculosis outbreak. Measures are currently in place to identify and prevent further transmission in the involved locations.