Richard C. Huard

PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions

ABSTRACT The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and “Mycobacterium tuberculosis subsp. canettii.” Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561′, Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or “M. canettii” or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use.

Genetic Organization of Transposase Regions Surrounding blaKPC Carbapenemase Genes on Plasmids from Klebsiella Strains Isolated in a New York City Hospital

ABSTRACT Carbapenem-resistant Klebsiella strains carrying Klebsiella pneumoniae carbapenemases (KPC) are endemic to New York City and are spreading across the United States and internationally. Recent studies have indicated that the KPC structural gene is located on a 10-kb plasmid-borne element designated Tn4401. Fourteen Klebsiella pneumoniae strains and one Klebsiella oxytoca strain isolated at a New York City hospital in 2005 carrying either blaKPC-2 or blaKPC-3 were examined for isoforms of Tn4401. Ten of the Klebsiella strains contained a 100-bp deletion in Tn4401, corresponding to the Tn4401a isoform. The presence of this deletion adjacent to the upstream promoter region of blaKPC in Tn4401a resulted in a different −35 promoter sequence of TGGAGA than that of CTGATT present in isoform Tn4401b. Complete sequencing of one plasmid carrying blaKPC from each of three nonclonal isolates indicated the presence of genes encoding other types of antibiotic resistance determinants. The 70.6-kb plasmid from K. pneumoniae strain S9 carrying blaKPC-2 revealed two identical copies of Tn4401b inserted in an inverse fashion, but in this case, one of the elements disrupted a group II self-splicing intron. In K. pneumoniae strain S15, the Tn4401a element carrying blaKPC-2 was found on both a large 120-kb plasmid and a smaller 24-kb plasmid. Pulsed-field gel electrophoresis results indicate that the isolates studied represent a heterogeneous group composed of unrelated as well as closely related Klebsiella strains. Our results suggest that endemic KPC-positive Klebsiella strains constitute a generally nonclonal population comprised of various alleles of blaKPC on several distinct plasmid genetic backgrounds. This study increases our understanding of the genetic composition of the evolving and expanding role of KPC-producing, healthcare-associated, gram-negative pathogens.

Down-Modulation of Lung Immune Responses by Interleukin-10 and Transforming Growth Factor β (TGF-β) and Analysis of TGF-β Receptors I and II in Active Tuberculosis

ABSTRACT Immune factors influencing progression to active tuberculosis (TB) remain poorly defined. In this study, we investigated the expression of immunoregulatory cytokines and receptors by using lung bronchoalveolar lavage cells obtained from patients with pulmonary TB, patients with other lung diseases (OLD patients), and healthy volunteers (VOL) by using reverse transcriptase PCR, a transforming growth factor β (TGF-β) bioactivity assay, and an enzyme immunoassay. TB patients were significantly more likely than OLD patients to coexpress TGF-β receptor I (RI) and RII mRNA, as well as interleukin-10 (IL-10) mRNA (thereby indicating the state of active gene transcription in the alveolar cells at harvest). In contrast, gamma interferon (IFN-γ) and IL-2 mRNA was seen in both TB and OLD patients. Likewise, significantly elevated pulmonary steady-state protein levels of IL-10, IFN-γ, and bioactive TGF-β were found in TB patients versus those in OLD patients and VOL. These data suggest that the combined production of the immunosuppressants IL-10 and TGF-β, as well as coexpression of TGF-β RI and RII (required for cellular response to TGF-β), may act to down-modulate host anti-Mycobacterium tuberculosis immunity and thereby allow uncontrolled bacterial replication and overt disease. Delineating the underlying mechanisms of M. tuberculosis-triggered expression of these immune elements may provide a molecular-level understanding of TB immunopathogenesis.

Tuberculosis Is Associated with a Down-Modulatory Lung Immune Response That Impairs Th1-Type Immunity

Immune mediators associated with human tuberculosis (TB) remain poorly defined. This study quantified levels of lung immune mediator gene expression at the time of diagnosis and during anti-TB treatment using cells obtained by induced sputum. Upon comparison to patients with other infectious lung diseases and volunteers, active pulmonary TB cases expressed significantly higher levels of mediators that counteract Th1-type and innate immunity. Despite the concomitant heightened levels of Th1-type mediators, immune activation may be rendered ineffectual by high levels of intracellular (SOCS and IRAK-M) and extracellular (IL-10 and TGF-βRII, IL-1Rn, and IDO) immune suppressive mediators. These modulators are a direct response to Mycobacterium tuberculosis as, by day 30 of anti-TB treatment, many suppressive factors declined to that of controls whereas most Th1-type and innate immune mediators rose above pretreatment levels. Challenge of human immune cells with M. tuberculosis in vitro up-regulated these immune modulators as well. The observed low levels of NO synthase-2 produced by alveolar macrophages at TB diagnosis, along with the heightened amounts of suppressive mediators, support the conclusion that M. tuberculosis actively promotes down-modulatory mediators to counteract Th1-type and innate immunity as an immunopathological strategy. Our data highlight the potential application of immune mediators as surrogate markers for TB diagnosis or treatment response.

Prevalence of methicillin-sensitive and methicillin-resistant Staphylococcus aureus in pregnant women.

OBJECTIVE: To estimate the extent of Staphylococcus aureus vaginal-rectal colonization among pregnant women as severe S aureus infections have emerged in pregnant and postpartum women and infants. METHODS: We conducted a prospective surveillance study for methicillin-sensitive S aureus and methicillin-resistant S aureus on all routine de-identified vaginal-rectal prenatal group B streptococcus (GBS) screening cultures submitted to the microbiology laboratory of a tertiary-care facility from January to July 2005. Standard microbiologic techniques and molecular analyses were used to detect community-associated methicillin-resistant S aureus strains. As opposed to health care–associated methicillin-resistant S aureus isolates, community-associated methicillin-resistant S aureus isolates were defined as those possessing the type IV or type V staphylococcal chromosomal cassette mec element and usually lacking a multidrug-resistant phenotype. RESULTS: A total of 2,963 GBS screening cultures were analyzed, from which 743 (25.1%, 95% confidence interval [CI] 23.5–26.7%) GBS isolates and 507 (17.1%, 95% CI 15.7–18.5%) S aureus isolates were identified. Group B streptococcus colonization was significantly associated with S aureus colonization (prevalence odds ratio 2.1, 95% CI 1.7–2.5, P<.001). Of the S aureus isolates, 14 (2.8%, 95% CI 1.4–4.2%) were methicillin-resistant, and 13 of these were determined to be community-associated methicillin-resistant S aureus. CONCLUSION: The prevalence of S aureus colonization identified in GBS screening cultures from pregnant women was substantial and associated with GBS co-colonization. Although we do not advocate routine screening of pregnant women for methicillin-sensitive S aureus and methicillin-resistant S aureus colonization, we recommend continued monitoring of both methicillin-sensitive S aureus and methicillin-resistant S aureus infections in this population and their infants. LEVEL OF EVIDENCE: II-3

Discovery of a Novel Mycobacterium tuberculosis Lineage That Is a Major Cause of Tuberculosis in Rio de Janeiro, Brazil

ABSTRACT The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in ∼30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RDRio. Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RDRio locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RDRio strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RDRio lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RDRio strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RDRio strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RDRioM. tuberculosis.

Nocardia cyriacigeorgica, an Emerging Pathogen in the United States

ABSTRACT Nocardia cyriacigeorgica is recognized as an emerging pathogen in many parts of the world. We present the first case description of invasive N. cyriacigeorgica pulmonary infection in the United States identified to the species level by 16S rRNA and hsp65 sequence analysis. A subsequent retrospective molecular screening of recent Nocardia clinical isolates at our New York City medical center yielded an additional six N. cyriacigeorgica isolates. Because routine laboratory algorithms for the phenotypic identification of Nocardia species are limited in practice, the true prevalence of N. cyriacigeorgica infections may be greater than currently appreciated. Indeed, we present evidence confirming that N. cyriacigeorgica is coincident with the unofficial species designation Nocardia asteroides complex antimicrobial susceptibility pattern type VI and distinct from the N. asteroides sensu stricto strain ATCC 19247T. As nocardial species identity can predict antimicrobial susceptibility and guide clinical management, we offer simplified phenotypic and molecular protocols to assist the identification of N. cyriacigeorgica.

Application of Sensitive and Specific Molecular Methods To Uncover Global Dissemination of the Major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis Spoligotype Family

ABSTRACT The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RDRio. Using the Brazilian strains, we pinpoint an Ag85C103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RDRio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RDRio strains, were then used to analyze an international collection of M. tuberculosis strains. RDRioM. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RDRio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RDRio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RDRio sublineage.

Changes in the Molecular Epidemiological Characteristics of Methicillin‐Resistant Staphylococcus aureus in a Neonatal Intensive Care Unit

OBJECTIVE To determine whether the molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA) had changed in a level III neonatal intensive care unit (NICU). DESIGN Retrospective review of medical records. SETTING Level III NICU of a university-affiliated childrens hospital in New York, New York. PATIENTS Case patients were neonates hospitalized in the NICU who were colonized or infected with MRSA. METHODS Rates of colonization and infection with MRSA during the period from 2000 through 2008 were assessed. Staphylococcal chromosomal cassette (SCC) mecA analysis and genotyping for S. aureus encoding protein A (spa) were performed on representative MRSA isolates from each clonal pulsed-field gel electrophoresis pattern. RESULTS Endemic MRSA infection and colonization occurred throughout the study period, which was punctuated by 4 epidemiologic investigations during outbreak periods. During the study period, 93 neonates were infected and 167 were colonized with MRSA. Surveillance cultures were performed for 1,336 neonates during outbreak investigations, and 115 (8.6%) neonates had MRSA-positive culture results. During 2001-2004, healthcare-associated MRSA clones, carrying SCC mec type II, predominated. From 2005 on, most MRSA clones were community-associated MRSA with SCC mec type IV, and in 2007, USA300 emerged as the principal clone. CONCLUSIONS Molecular analysis demonstrated a shift from healthcare-associated MRSA (2001-2004) to community-associated MRSA (2005-2008).

The Mycobacterium tuberculosis Complex-Restricted Gene cfp32 Encodes an Expressed Protein That Is Detectable in Tuberculosis Patients and Is Positively Correlated with Pulmonary Interleukin-10

ABSTRACT Human tuberculosis (TB) is caused by the bacillus Mycobacteriumtuberculosis, a subspecies of the M. tuberculosis complex (MTC) of mycobacteria. Postgenomic dissection of the M. tuberculosis proteome is ongoing and critical to furthering our understanding of factors mediating M. tuberculosis pathobiology. Towards this end, a 32-kDa putative glyoxalase in the culture filtrate (CF) of growing M. tuberculosis (originally annotated as Rv0577 and hereafter designated CFP32) was identified, cloned, and characterized. The cfp32 gene is MTC restricted, and the gene product is expressed ex vivo as determined by the respective Southern and Western blot testing of an assortment of mycobacteria. Moreover, the cfp32 gene sequence is conserved within the MTC, as no polymorphisms were found in the tested cfp32 PCR products upon sequence analysis. Western blotting of M. tuberculosis subcellular fractions localized CFP32 predominantly to the CF and cytosolic compartments. Data to support the in vivo expression of CFP32 were provided by the serum recognition of recombinant CFP32 in 32% of TB patients by enzyme-linked immunosorbent assay (ELISA) as well as the direct detection of CFP32 by ELISA in the induced sputum samples from 56% of pulmonary TB patients. Of greatest interest was the observation that, per sample, sputum CFP32 levels (a potential indicator of increasing bacterial burden) correlated with levels of expression in sputum of interleukin-10 (an immunosuppressive cytokine and a putative contributing factor to disease progression) but not levels of gamma interferon (a key cytokine in the protective immune response in TB), as measured by ELISA. Combined, these data suggest that CFP32 serves a necessary biological function(s) in tubercle bacilli and may contribute to the M. tuberculosis pathogenic mechanism. Overall, CFP32 is an attractive target for drug and vaccine design as well as new diagnostic strategies.