Preclinical safety and efficacy characterization of an LpxC inhibitor against Gram-negative pathogens.

The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC is an essential enzyme in the biosynthesis of lipid A, the outer membrane anchor of lipopolysaccharide and lipooligosaccharide in Gram-negative bacteria. The development of LpxC-targeting antibiotics toward clinical therapeutics has been hindered by the limited antibiotic profile of reported non-hydroxamate inhibitors and unexpected cardiovascular toxicity observed in certain hydroxamate and non-hydroxamate-based inhibitors. Here, we report the preclinical characterization of a slow, tight-binding LpxC inhibitor, LPC-233, with low picomolar affinity. The compound is a rapid bactericidal antibiotic, unaffected by established resistance mechanisms to commercial antibiotics, and displays outstanding activity against a wide range of Gram-negative clinical isolates in vitro. It is orally bioavailable and efficiently eliminates infections caused by susceptible and multidrug-resistant Gram-negative bacterial pathogens in murine soft tissue, sepsis, and urinary tract infection models. It displays exceptional in vitro and in vivo safety profiles, with no detectable adverse cardiovascular toxicity in dogs at 100 milligrams per kilogram. These results establish the feasibility of developing oral LpxC-targeting antibiotics for clinical applications.

Nocardia neocaledoniensis as Rare Cause of Spondylodiscitis.

Nocardia neocaledoniensis is a rare species of Nocardia bacteria, identified in 2004 in hypermagnesian ultramafic soil of New Caledonia. Culture of this opportunistic pathogen from spinal biopsy samples confirmed N. neocaledoniensis spondylodiscitis in an immunocompromised man. Isolation of this unusual species from spinal biopsy samples illustrates its underappreciated ability to cause invasive infection.

Clinical Features and Outcome of Multidrug-Resistant Osteoarticular Tuberculosis: A 12-Year Case Series from France.

The optimal treatment for osteoarticular infection due to multidrug-resistant tuberculosis strains (MDR-OATB) remains unclear. This study aims to evaluate the diagnosis, management and outcome of MDR-OATB in France. We present a case series of MDR-OATB patients reviewed at the French National Reference Center for Mycobacteria between 2007 and 2018. Medical history and clinical, microbiological, treatment and outcome data were collected. Twenty-three MDR-OATB cases were reported, representing 3% of all concurrent MDR-TB cases in France. Overall, 17 were male, and the median age was 32 years. Six patients were previously treated for TB, including four with first-line drugs. The most frequently affected site was the spine (n = 16). Bone and joint surgery were required in 12 patients. Twenty-one patients (91%) successfully completed the treatment with a regimen containing a mean of four drugs (range, 2-6) for a mean duration of 20 months (range, 13-27). Overall, high rates of treatment success were achieved following WHO MDR-TB treatment guidelines and individualized patient management recommendations by the French National TB Consilium. However, the optimal combination of drugs, duration of treatment and role of surgery in the management of MDR-OATB remains to be determined.

Molecular detection of isoniazid monoresistance improves tuberculosis treatment: A retrospective cohort in France.

OBJECTIVES: Isoniazid-monoresistant tuberculosis (HR-TB) requires early diagnosis and adapted treatment to achieve optimal outcomes. The primary aim of the study was to assess the impact of the implementation of rapid diagnostic tests on HR-TB treatment in France. METHODS: We designed a retrospective multicentre study including consecutive HR-TB patients diagnosed in 2016 and 2017. Implementation of a molecular assay detecting isoniazid resistance directly on a clinical sample was recorded. The association between early implementation of such assays and adequate treatment was assessed by a multivariable Cox proportional hazards model. RESULTS: Overall, 99 HR-TB patients were included from 20 University Hospitals. Among all smear-positive HR-TB patients, only 26% beneficiated from early molecular HR detection. This detection was independently associated with shorter time to adequate treatment (HR = 2.0 [1.1-3.8], p = 0.03). CONCLUSION: In our study, molecular detection of HR on an initial sample was independently associated with earlier treatment adaptation.

Retrospective Multicentric Study on Campylobacter spp. Bacteremia in France: The Campylobacteremia Study.

BACKGROUND: Campylobacter spp. bacteremia is a severe infection. A nationwide 5-year retrospective study was conducted to characterize its clinical features and prognostic factors. METHODS: The study included patients with Campylobacter spp. bacteremia diagnosed in 37 French hospitals participating in the surveillance network of the National Reference Center for Campylobacters and Helicobacters, from 1 January 2015 to 31 December 2019. The goal was to analyze the effects of a delay of appropriate antibiotic therapy and other risk factors on 30-day mortality rates, antibiotic resistance, patient characteristics, and prognosis according to the Campylobacter species. RESULTS: Among the 592 patients, Campylobacter jejuni and Campylobacter fetus were the most commonly identified species (in 42.9% and 42.6%, respectively). The patients were elderly (median age 68 years), and most had underlying conditions, mainly immunodepression (43.4%), hematologic cancers (25.9%), solid neoplasms (23%), and diabetes (22.3%). C. jejuni and Campylobacter coli were associated with gastrointestinal signs, and C. fetus was associated with secondary localizations. Among the 80 patients (13.5%) with secondary localizations, 12 had endocarditis, 38 vascular, 24 osteoarticular, and 9 ascitic fluid infections. The 30-day mortality rate was 11.7%, and an appropriate antibiotic treatment was independently associated with 30-day survival (odds ratio, 0.47 [95% confidence interval, .24-.93]; P = .03). The median efficient therapy initiation delay was quite short (2 days [interquartile range, 0-4 days]) but it had no significant impact on the 30-day mortality rate (P = .78). CONCLUSIONS: Campylobacter spp. bacteremia mainly occurred in elderly immunocompromised individuals with variable clinical presentations according to the species involved. Appropriate antimicrobial therapy was associated with improved 30-day survival.

Antibiotic Therapy of Plague: A Review.

Plague-a deadly disease caused by the bacterium Yersinia pestis-is still an international public health concern. There are three main clinical forms: bubonic plague, septicemic plague, and pulmonary plague. In all three forms, the symptoms appear suddenly and progress very rapidly. Early antibiotic therapy is essential for countering the disease. Several classes of antibiotics (e.g., tetracyclines, fluoroquinolones, aminoglycosides, sulfonamides, chloramphenicol, rifamycin, and ß-lactams) are active in vitro against the majority of Y. pestis strains and have demonstrated efficacy in various animal models. However, some discrepancies have been reported. Hence, health authorities have approved and recommended several drugs for prophylactic or curative use. Only monotherapy is currently recommended; combination therapy has not shown any benefits in preclinical studies or case reports. Concerns about the emergence of multidrug-resistant strains of Y. pestis have led to the development of new classes of antibiotics and other therapeutics (e.g., LpxC inhibitors, cationic peptides, antivirulence drugs, predatory bacteria, phages, immunotherapy, host-directed therapy, and nutritional immunity). It is difficult to know which of the currently available treatments or therapeutics in development will be most effective for a given form of plague. This is due to the lack of standardization in preclinical studies, conflicting data from case reports, and the small number of clinical trials performed to date.

Isoniazid-monoresistant tuberculosis in France: Risk factors, treatment outcomes and adverse events.

OBJECTIVES: Isoniazid-monoresistant tuberculosis (HR-TB) is the most prevalent form of drug-resistant TB worldwide and in France and is associated with poorer treatment outcomes compared with drug-susceptible TB (DS-TB). The objective of this study was to determine the characteristics of HR-TB patients in France and to compare outcomes and safety of treatment for HR-TB and DS-TB. METHODS: We performed a case-control multicenter study to identify risk factors associated with HR-TB and compare treatment outcomes and safety between HR-TB patients and DS-TB patients. RESULTS: Characteristics of 99 HR-TB patients diagnosed and treated in the university hospitals of Paris, Lille, Caen and Strasbourg were compared with 99 DS-TB patients. Female sex (OR = 2.2; 1.0-4.7), birth in the West-Pacific World Health Organization region (OR = 4.6; 1.1-18.7) and resistance to streptomycin (OR = 77.5; 10.1-594.4) were found to be independently associated with HR-TB. Rates of treatment success did not differ significantly between HR-TB and DS-TB. CONCLUSIONS: Factors associated with HR-TB are not significant enough to efficiently screen TB patients at risk of HR-TB. The systematic implementation of rapid molecular testing on clinical samples remains the only effective way to make the early diagnosis of HR-TB and adapt treatment.

Prospective evaluation of the Amplidiag® CarbaR+VRE assay for direct screening of carbapenemase producing gram-negative bacilli from rectal swabs.

This prospective study evaluated the ability of the qPCR Amplidiag® CarbaR+VRE assay to detect Carbapenemase-producing Gram-negative bacilli (CP-GNB) directly on 1830 rectal swabs extracted using the fully automated platform Amplidiag® Easy instrument. The Amplidiag® CarbaR+VRE assay gave a positive signal for 94 rectal swabs, whereas only 70 grew with CP-GNB on chromogenic media including 4 VIM-producing P. aeruginosa, 8 OXA-23-producing A. baumannii and 58 carbapenemase-producing Enterobacteriaceae. All the CP-GNB culture positive were detected by the Amplidiag® CarbaR+VRE assay. Twenty-four qPCR-positive and culture-negative samples were further investigated using targeted PCRs and subsequent DNA sequencing. Seventeen and 7 of these were positive and negative with PCR/DNA sequencing, respectively. Taken together, the Amplidiag® CarbaR+VRE could detect carbapenemases directly from rectal swabs in 3h 30 using a fully automated platform and showed high biological performances (sensitivity, specificity, and negative and positive predictive values were 100%, 98.6%, 100%, and 74.5%, respectively).

Polymorphism in the Yersinia LcrV Antigen Enables Immune Escape From the Protection Conferred by an LcrV-Secreting Lactococcus Lactis in a Pseudotuberculosis Mouse Model.

Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

Is a chlorine dioxide wiping procedure suitable for the high-level disinfection of nasendoscopes?

BACKGROUND: Nasendoscopes are widely used in the outpatient ENT setting. Their reprocessing requires high-level disinfection (HLD). Recently, a wiping procedure using chlorine dioxide (ClO2) has been proposed as an alternative to HLD traditional procedures. OBJECTIVE: To assess the effectiveness of the HLD wiping procedure versus soaking procedure on a contaminated nasendoscope. METHOD: A nasendoscope was contaminated with four strains of bacteria and Bacillus subtilis spores. After HLD either with the wiping procedure or with the soaking procedure (PA), the reduction of the initial contamination was determined. FINDINGS: The wiping procedure with ClO2 displayed more than 5 log reduction for vegetative bacteria after 30 s contact time (CT) and 4 log reduction on B. subtilis spores after 2 min CT. The soaking procedure with PA displayed similar results on planktonic bacteria after 10 min CT but the log reduction of B. subtilis remained below 4. CONCLUSION: The ClO2 wiping procedure showed bactericidal and sporicidal efficacy on a contaminated nasendoscope in a shorter time compared to the PA soaking procedure. Thus, ClO2 wiping procedure might be considered as an alternative to the traditional HLD procedure for nasendoscopes.

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

Characterization of the protective immune response to Yersinia pseudotuberculosis infection in mice vaccinated with an LcrV-secreting strain of Lactococcus lactis.

BACKGROUND: Pseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudotuberculosis and is considered to be a significant problem in veterinary medicine. We previously found that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseudotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited protective response and at determining the duration of vaccine-induced immunity. METHODS: Splenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4+, CD8+ or CD25+ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the number of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intravenous injection of Y. pseudotuberculosis was followed up to 9-months. RESULTS: We found that T and B lymphocytes but not non-conventional lymphoid cells were affected by Ll-LcrV immunization. We also observed that depletion of CD4+ and CD25+ but not CD8+ cells in immunized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that activated CD4+ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge at 9months post-vaccination. CONCLUSION: Mucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with ß-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

Drug design from the cryptic inhibitor envelope.

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study.

BACKGROUND: Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. METHODS: We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. FINDINGS: Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. INTERPRETATION: We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. FUNDING: National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.

Yersinia pestis requires the 2-component regulatory system OmpR-EnvZ to resist innate immunity during the early and late stages of plague.

Plague is transmitted by fleas or contaminated aerosols. To successfully produce disease, the causal agent (Yersinia pestis) must rapidly sense and respond to rapid variations in its environment. Here, we investigated the role of 2-component regulatory systems (2CSs) in plague because the latter are known to be key players in bacterial adaptation to environmental change. Along with the previously studied PhoP-PhoQ system, OmpR-EnvZ was the only one of Y. pestis' 23 other 2CSs required for production of bubonic, septicemic, and pneumonic plague. In vitro, OmpR-EnvZ was needed to counter serum complement and leukocytes but was not required for the secretion of antiphagocyte exotoxins. In vivo, Y. pestis lacking OmpR-EnvZ did not induce an early immune response in the skin and was fully virulent in neutropenic mice. We conclude that, throughout the course of Y. pestis infection, OmpR-EnvZ is required to counter toxic effectors secreted by polymorphonuclear leukocytes in the tissues.

First evaluation of drug-resistant Mycobacterium tuberculosis clinical isolates from Congo revealed misdetection of fluoroquinolone resistance by line probe assay due to a double substitution T80A-A90G in GyrA.

BACKGROUND: Tuberculosis (TB) is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC) isolates collected in the two main cities of Congo. METHODS: Over a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST). Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl) and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping. RESULTS: Of the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46%) were resistant to one or more first-line antiTB drugs. Of these, 15 (71%) were multidrug resistant (MDR). The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60%) in rpoB and S315T (87%) in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA. CONCLUSIONS: Taken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.

New insights into how Yersinia pestis adapts to its mammalian host during bubonic plague.

Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of Y. pestis genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of Y. pestis mutants, each lacking one or more of the genes previously identified as being up-regulated in vivo. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, "per-pool" screening method that we have developed. Our data showed that in addition to genes involved in physiological adaptation and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (ympt1.66c, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of ympt1.66c reduced Y. pestis' ability to spread to the lymph nodes draining the dermal inoculation site--probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.