Marc Ouellette

Rapid detection of group B streptococci in pregnant women at delivery

BACKGROUND Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates. METHODS We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay. RESULTS Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture. CONCLUSIONS Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.

Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages

Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR‐knockout mutants by gene disruption in L.donovani and L.major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L.donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR‐containing region to a larger chromosome. Mutants of L.donovani and L.major possessing only one wild‐type TR allele express less TR mRNA and have lower TR activity compared with wild‐type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates.

Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels

ABSTRACT We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting thetuf gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of severaltuf genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Québec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticusdemonstrated sufficient interspecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of Staphylococcus-specific and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticus, S. hominis, or S. saprophyticus) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.

The amplified H circle of methotrexate-resistant leishmania tarentolae contains a novel P-glycoprotein gene.

Acquired resistance to methotrexate in Leishmania species is often associated with the amplification of H circles, 68 kb duplex DNA circles containing a 30 kb inverted repeat. We report here that the H circle of Leishmania tarentolae contains an open reading frame, ltpgpA, that has the attributes of P‐glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). Although amplification of H circles is associated with proportionally increased levels of a 5.5 kb transcript of the ltpgpA gene, such methotrexate resistant mutants are not cross‐resistant to any of the drugs extruded by mammalian multi‐drug resistant cells. In Leishmania, ltpgpA is part of a gene family containing at least two other members. Sequences homologous to one of the nucleotide binding sites of ltpgpA are conserved in other kinetoplastida.

A novel antifolate resistance gene on the amplified H circle of Leishmania

In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short‐chain dehydrogenases, enzymes that are involved in several oxido‐reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.

Co-amplification of the gamma-glutamylcysteine synthetase gene gsh1 and of the ABC transporter gene pgpA in arsenite-resistant Leishmania tarentolae.

Resistance to the oxyanion arsenite in the parasite Leishmania is multifactorial. We have described previously the frequent amplification of the ABC transporter gene pgpA, the presence of a non‐PgpA thiol–metal efflux pump and increased levels of glutathione and trypanothione in resistant cells. Other loci are also amplified, although their role in resistance is unknown. By gene transfection, we have characterized one of these novel genes. It corresponds to gsh1, which encodes γ‐glutamylcysteine synthetase, an enzyme involved in the rate‐limiting step of glutathione biosynthesis. Transfection of gsh1 in wild‐type cells increased the levels of glutathione and trypanothione to levels found in resistant mutants. These transfectants were not resistant to metals. However, when gsh1 was transfected in partial revertants, it conferred resistance. As pgpA is frequently co‐amplified with gsh1, we co‐transfected the two genes into both wild‐type and partial revertants. Arsenite resistance levels in wild‐type cells could be accounted for by the contribution of PgpA alone. In the partial revertant, the gsh1 and pgpA gene product acted synergistically. These results support our previous suggestion that PgpA recognizes metals conjugated to thiols. Furthermore, amplification of gsh1 overcomes the rate‐limiting step in the synthesis of trypanothione, contributing to resistance. In addition, the results suggest that at least one more factor acts synergistically with the gsh1 gene product.

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P‐glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.

Resistance to Antimony and Treatment Failure in Human Leishmania (Viannia) Infection

BACKGROUND Failure of antimonial therapy has been increasingly reported in anthroponotic visceral leishmaniasis and in cutaneous disease. The role of drug resistance in treatment failure has been difficult to ascertain because therapeutic response is multifactorial, and the efficacy of antimonial drugs depends on an effective immune response. In this study, we sought to determine whether standard treatment selects for resistant organisms and whether drug resistance contributes to treatment failure. METHODS We evaluated the susceptibility to antimony of 19 strains isolated before treatment with meglumine antimoniate and 21 strains isolated at treatment failure from 20 patients. The 50% effective dose (ED50) of antimony in the form of additive-free meglumine antimoniate was determined for intracellular amastigotes in human promonocytic U-937 cells. RESULTS Before treatment, 16% of strains (3/19) showed primary resistance (ED50 of >128 microg Sb/mL), whereas 84% (16/19) were susceptible (ED50 of <20 microg Sb/mL). However, 88% of susceptible strains (14/16) had ED90 values of >128 microg Sb/mL. At treatment failure, 40% of strains (8/20) were resistant. Secondary resistance was documented in 4 patients. CONCLUSIONS Primary and secondary resistance to antimony can contribute to treatment failure in American cutaneous leishmaniasis. Selection for resistance to antimony occurs during standard treatment with antimonial drugs, and primary resistance to antimony supports the plausibility of anthroponotic transmission.

A Proteomics Screen Implicates HSP83 and a Small Kinetoplastid Calpain-related Protein in Drug Resistance in Leishmania donovani Clinical Field Isolates by Modulating Drug-induced Programmed Cell Death

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials (Sb(V)), but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory isolates, but our understanding of resistance in field isolates is lagging behind. We describe here a comparative analysis of a genetically related pair of Sb(V)-sensitive and -resistant Leishmania donovani strains isolated from kala-azar patients. The resistant isolate exhibited cross-resistance to other unrelated Leishmania drugs including miltefosine and amphotericin B. A comparative proteomics screen has highlighted a number of proteins differentially expressed suggesting that programmed cell death (PCD) is modified in the resistant parasite. Indeed drug-induced PCD progression was altered in the Sb(V)-resistant strain as determined using early and late markers of apoptosis. Two proteins, the heat shock protein HSP83 and the small kinetoplastid calpain-related protein (SKCRP14.1) were shown to be intimately implicated in the drug-induced PCD phenotype. HSP83 increased drug resistance and reduced drug-mediated PCD activation by interfering with the mitochondrial membrane potential, whereas SKCRP14.1 promoted antimonial-induced PCD but protected against miltefosine-induced PCD. This study highlights the important role of PCD in drug susceptibility/resistance in the protozoan parasite Leishmania.