Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence.

Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiella-containing vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis.Abbreviations: ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: Coxiella-containing vacuole; Cvp: Coxiella vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.

Sample pooling methods for efficient pathogen screening: Practical implications.

Due to the large number of negative tests, individually screening large populations for rare pathogens can be wasteful and expensive. Sample pooling methods improve the efficiency of large-scale pathogen screening campaigns by reducing the number of tests and reagents required to accurately categorize positive and negative individuals. Such methods rely on group testing theory which mainly focuses on minimizing the total number of tests; however, many other practical concerns and tradeoffs must be considered when choosing an appropriate method for a given set of circumstances. Here we use computational simulations to determine how several theoretical approaches compare in terms of (a) the number of tests, to minimize costs and save reagents, (b) the number of sequential steps, to reduce the time it takes to complete the assay, (c) the number of samples per pool, to avoid the limits of detection, (d) simplicity, to reduce the risk of human error, and (e) robustness, to poor estimates of the number of positive samples. We found that established methods often perform very well in one area but very poorly in others. Therefore, we introduce and validate a new method which performs fairly well across each of the above criteria making it a good general use approach.

Microbial community structure reveals instability of nutritional symbiosis during the evolutionary radiation of Amblyomma ticks.

Mutualistic interactions with microbes have facilitated the adaptation of major eukaryotic lineages to restricted diet niches. Hence, ticks with their strictly blood-feeding lifestyle are associated with intracellular bacterial symbionts through an essential B vitamin supplementation. In this study, examination of bacterial diversity in 25 tick species of the genus Amblyomma showed that three intracellular bacteria, Coxiella-like endosymbionts (LE), Francisella-LE and Rickettsia, are remarkably common. No other bacterium is as uniformly present in Amblyomma ticks. Almost all Amblyomma species were found to harbour a nutritive obligate symbiont, Coxiella-LE or Francisella-LE, that is able to synthesize B vitamins. However, despite the co-evolved and obligate nature of these mutualistic interactions, the structure of microbiomes does not mirror the Amblyomma phylogeny, with a clear exclusion pattern between Coxiella-LE and Francisella-LE across tick species. Coxiella-LE, but not Francisella-LE, form evolutionarily stable associations with ticks, commonly leading to co-cladogenesis. We further found evidence for symbiont replacements during the radiation of Amblyomma, with recent, and probably ongoing, invasions by Francisella-LE and subsequent replacements of ancestral Coxiella-LE through transient co-infections. Nutritional symbiosis in Amblyomma ticks is thus not a stable evolutionary state, but instead arises from conflicting origins between unrelated but competing symbionts with similar metabolic capabilities.

Molecular survey of tick-borne pathogens in small mammals from Brazilian Amazonia / Levantamento molecular de patógenos transmitidos por carrapatos em pequenos mamíferos da Amazônia brasileira

Abstract Small non-volant mammals (marsupials and small rodents) were captured at three different timepoints from 23 forest fragments across three municipalities (Alta Floresta, Sinop and Cláudia) covering the Amazonian biome of the Mato Grosso State in Midwestern Brazil. The animal tissues (liver and spleen) and blood were screened using molecular tools for the detection of Babesia, Coxiella, Cytauxzoon, Hepatozoon, Theileria, and Anaplasmataceae agents. A total of 230 specimens (78 rodents and 152 marsupials) were trapped. Hepatozoon and Piroplasmorida agents were detected in the common opossums (Didelphis marsupialis). In turn, all samples (blood, liver, or spleen) collected from the small mammals were negative for the genus Coxiella and the family Anaplasmataceae, as detected by polymerase chain reaction (PCR). Phylogenetic analyses inferred from partial sequences of the 18S rRNA gene highlighted the occurrence of new Hepatozoon and Piroplasmorida haplotypes. Future studies determining the role of common opossum (D. marsupialis) in the epidemiological cycles of Hepatozoon and Babesia under natural conditions in the Amazonian biome are necessary.

Resumo Pequenos mamíferos não voadores (marsupiais e pequenos roedores) foram capturados em três diferentes períodos, ao longo de 23 fragmentos florestais de três municípios (Alta Floresta, Sinop e Cláudia), localizados no bioma amazônico do Estado de Mato Grosso, no centro-oeste do Brasil. Os tecidos dos animais (fígado e baço) e sangue foram selecionados e submetidos a ensaios moleculares para a detecção do DNA de Babesia, Coxiella, Cytauxzoon, Hepatozoon, Theileria e agentes Anaplasmataceae. Um total de 230 espécimes (78 roedores e 152 marsupiais) foram capturados. Hepatozoon e agentes Piroplasmorida foram detectados em gambás (Didelphis marsupialis). Ao contrário, todas as amostras (sangue, fígado ou baço) coletadas dos pequenos mamíferos foram negativas para o gênero Coxiella e a família Anaplasmataceae, conforme detectado pela reação em cadeia da polimerase (PCR). Análises filogenéticas inferidas pelas sequências parciais do gene 18S rRNA evidenciaram a ocorrência de novos haplótipos de Hepatozoon e Piroplasmorida. Futuros estudos determinando a importância do gambá-comun (D. marsupialis) nos ciclos epidemiológicos de Hepatozoon e Babesia em condições naturais, no bioma amazônico, são necessários.

Cytokine profiles in patients with Q fever fatigue syndrome.

BACKGROUND: Q fever fatigue syndrome (QFS) is a state of prolonged fatigue following around 20% of acute Q fever cases. It is thought that chronic inflammation plays a role in its etiology. To test this hypothesis we measured circulating cytokines and the ex-vivo cytokine production in patients with QFS and compared with various control groups. MATERIALS/METHODS: Peripheral blood mononuclear cells (PBMCs), whole blood, and serum were collected from 20 QFS patients, 19 chronic fatigue syndrome (CFS) patients, 19 Q fever seropositive controls, and 25 age- and sex-matched healthy controls. Coxiella-specific ex-vivo production of tumor necrosis factor (TNF)α, interleukin (IL)-1ß, IL-6, and interferon (IFN) was measured, together with a total of 92 circulating inflammatory proteins. RESULTS: PBMCs of QFS patients produced more IL-6 (P = 0.0001), TNFα (P = 0.0002), and IL-1ß (P = 0.0005) than the various control groups when stimulated with Coxiella antigen. QFS patients had distinct differences in circulating inflammatory markers compared to the other groups, including higher concentrations of circulating IL-6 and IFNγ. CONCLUSION: QFS patients showed signs of chronic inflammation compared to asymptomatic Q fever seropositive controls, CFS patients, and healthy controls, of which the monocyte-derived cytokines TNFα, IL-1ß, and especially IL-6, are likely crucial components.

Case Report: Scalp Eschar and Neck Lymphadenopathy Associated with Bacteremia due to Coxiella-Like Bacteria.

Coxiella-like bacteria have been recently proposed as human pathogens. Using molecular techniques, we detected Coxiella-like bacteria in the blood and serum samples of a patient with a scalp eschar, neck lymphadenopathy, severe urticaria, edema, fever, and arthralgia indicating that this organism can provide systemic complications.

Seroprevalence of Bartonella species, Coxiella burnetii and Toxoplasma gondii among patients with hematological malignancies: A pilot study in Romania.

Patients receiving immunosuppressive cancer treatments in settings where there is a high degree of human-animal interaction may be at increased risk for opportunistic zoonotic infections or reactivation of latent infections. We sought to determine the seroprevalence of selected zoonotic pathogens among patients diagnosed with haematologic malignancies and undergoing chemotherapeutic treatments in Romania, where much of the general population lives and/or works in contact with livestock. A convenience sample of 51 patients with haematologic cancer undergoing chemotherapy at a referral clinic in Cluj-Napoca, Romania, was surveyed regarding animal exposures. Blood samples were obtained and tested for evidence of infection with Bartonella species, Coxiella burnetii and Toxoplasma gondii, which are important opportunistic zoonotic agents in immunocompromised individuals. 58.8% of participants reported living or working on a farm, and living or working on a farm was associated with contact with livestock and other animals. 37.5% of participants were IgG seroreactive against one or more of five Bartonella antigens, and seroreactivity was statistically associated with living on farms. Farm dwellers were 3.6 times more likely to test IgG seroreactive to Bartonella antibodies than non-farm dwellers. 47.1% of the participants tested T. gondii IgG positive and 13.7% tested C. burnetii IgG positive, indicating past or latent infection. C. burnetii IgM antibodies were detected in four participants (7.8%), indicating possible recent infection. These results indicate that a large proportion of patients with haematologic cancer in Romania may be at risk for zoonotic infections or for reactivation of latent zoonotic infections, particularly with respect to Bartonella species. Special attention should be paid to cancer patients' exposure to livestock and companion animals in areas where much of the population lives in rural settings.

Antemortem Diagnosis of Coxiellosis in a Blue and Gold Macaw ( Ara ararauna).

A 15-year-old female blue and gold macaw ( Ara ararauna) was presented for evaluation after being found laterally recumbent, reluctant to move, and lethargic. Results of a complete blood count showed an increased number of immature heterophils with increased cytoplasmic basophilia and degranulation and the presence of a left shift. Radiographs and a computed tomography scan were performed and revealed a markedly enlarged spleen. An ultrasound-guided fine-needle aspirate of the spleen was submitted for cytologic examination and aerobic bacterial culture. While the culture revealed no growth, cytologic examination identified mononuclear phagocytes with cytoplasmic vacuoles containing structures consistent with bacteria. Pan-bacterial 16S rRNA polymerase chain reaction of the splenic sample followed by direct sequencing identified a Coxiella-like agent identical to one previously isolated in the liver of a golden-mantled rosella ( Platycercus eximius). Phylogenetic analysis shows that avian coxiellosis agents and Coxiella burnetii, the agent of Q fever, represent 2 independent events of development of vertebrate pathogenicity in this group of tick endosymbionts. This report suggests diagnostic and treatment directions for coxiellosis in avian patients and indicates where further study is needed.

Candidatus Coxiella massiliensis Infection.

Bacteria genetically related to Coxiella burnetii have been found in ticks. Using molecular techniques, we detected Coxiella-like bacteria, here named Candidatus Coxiella massiliensis, in skin biopsy samples and ticks removed from patients with an eschar. This organism may be a common agent of scalp eschar and neck lymphadenopathy after tick bite.

Seroprevalencia de Coxiella burnetii en trabajadores de fincas ganaderas del departamento de Antioquia / Seroprevalence of Coxiella Burnetii in cattle farm workers in the department of Antioquia

Introducción: Coxiella burnetii es el agente etiológico de la fiebre Q, zoonosis asociada principalmente al contacto con ganado bovino y caprino. El principal modo de transmisión es el contacto con productos del parto, sangre, leche, lana, además de la inhalación de las esporas de la bacteria, que permite que se produzca infección aun en sitios alejados del reservorio. Objetivo: describir la seroprevalencia de C. burnetii en una población de riesgo como los trabajadores de fincas ganaderas del departamento de Antioquia 2011-2012. Metodología: se determinaron los niveles de anticuerpos IgG, por inmunofluorescencia, determinantes del contacto previo con C. burnetti, en 102 trabajadores de fincas ganaderas localizadas en tres municipios del departamento de Antioquia. Resultados: se encontraron 15 (14.70%) muestras positivas para Ig G (fase I y II) contra Coxiella burnetii en un grupo de 92 hombres y 10 mujeres, sin que se estableciera relación entre el género y su seropositividad (p=0.167), edad (p: 0.889) y tiempo de exposición (p: 0.118). Conclusión: la infección por C. burnetti es una zoonosis de importancia en nuestro medio y debe pensarse en ella al momento de enfrentarse a cuadros febriles tanto agudos como crónicos, en poblaciones de riesgo. (Acta Med Colomb 2015; 40: 20-23).

Abstract Introduction: Coxiella burnetii is the etiologic agent of Q fever, zoonosis mainly associated to contact with cattle and goats. The main mode of transmission is contact with products of delivery, blood, milk, wool, in addition to inhalation of spores of the bacterium, that allows to occur the infection even in places far from the reservoir. Objective: to describe the seroprevalence of C. burnetii in a population at risk such as farm workers in Antioquia department 2011-2012. Methodology: IgG antibody levels were determined by immunofluorescence, determinants of previous contact with C. burnetii in 102 workers of farms localized in three municipalities of Antioquia. Results: 15 (14.70%) samples positive for IgG (phase I and II) were found against Coxiella burnetii in a group of 92 men and 10 women, with no relationship between gender and seropositivity (p = 0.167), age (p = 0.889) and exposure time (p = 0.118) established. Conclusion: C. burnetii infection is a zoonosis of importance in our environment and should be thought of when faced with febrile pictures both acute and chronic, in populations at risk. (Acta Med Colomb 2015; 40: 20-23).

Bartonella and Coxiella infective endocarditis in Brazil: molecular evidence from excised valves from a cardiac surgery referral center in Rio de Janeiro, Brazil, 1998 to 2009.

PCR was used to detect Coxiella burnetii and Bartonella spp in heart valves obtained during the period 1998-2009 from patients operated on for blood culture-negative endocarditis in a cardiac surgery hospital in Brazil. Of the 51 valves tested, 10 were PCR-positive; two were positive for Bartonella and one for C. burnetii.

Ten years of viral and non-bacterial serology in adults with cystic fibrosis.

Viral infections are associated with pulmonary exacerbations in children with cystic fibrosis (CF), but few studies have addressed the frequency in adults. This paper investigates the frequency and impact of viral infections in adults with CF receiving intravenous antibiotics. Pre- and post-treatment spirometry, inflammatory markers and antibody titres against influenza A, influenza B, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, Chlamydia psittaci, and Coxiella burnetti were analysed over a 10-year period. Non-bacterial infections were identified in 5.1% of 3156 courses of treatment. The annual incidence of admissions per patient associated with viral infection was 4.9%. The presence of viral infection in association with a pulmonary exacerbation did not adversely affect lung function or inflammatory markers in the short term. Adults with CF have a lower incidence of respiratory viral infections associated with pulmonary exacerbations requiring intravenous antibiotics compared to children and infants with CF.

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.

BACKGROUND: Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importance. RESULTS: The lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25 degrees C for LPS and 10 and 40 degrees C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40 degrees. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor alpha (TNFalpha) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay. CONCLUSIONS: Despite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident.

Survival of Mycobacterium avium and Mycobacterium tuberculosis in acidified vacuoles of murine macrophages.

Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth of M. avium was not impaired following fusion with the acidified vacuole. In contrast, the growth rate of M. tuberculosis was reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome.

IFN-gamma-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF.

The treatment of infectious diseases caused by intracellular bacteria, such as Q fever, may benefit from cytokines acting on macrophages. Monocytic THP-1 cells were infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with IFN-gamma. While C. burnetii multiplied in untreated monocytes, IFN-gamma reduced bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h. IFN-gamma also affected the viability of infected cells. Cell death resulted from apoptosis; occurring 24 h after the addition of IFN-gamma, it reached a maximum after 48 h and was followed by necrosis. Reactive oxygen intermediates were not required for C. burnetii killing, since monocytes from patients with chronic granulomatous disease were microbicidal in response to IFN-gamma. The role of cytokines was also investigated. IFN-gamma elicited a moderate release of IL-1beta in infected monocytes. Moreover, the IL-1 receptor antagonist did not affect C. burnetii survival, suggesting that IL-1beta was not involved in the bacterial killing induced by IFN-gamma. TNF was involved in IFN-gamma-induced killing of C. burnetii and cell death. IFN-gamma induced mRNA expression and sustained secretion of TNF. Neutralizing Abs to TNF as well as Abs directed against TNF receptors I and II, significantly prevented IFN-gamma-dependent killing of C. burnetii and cell death. These results suggest that IFN-gamma promotes the killing of C. burnetii in monocytes through an apoptotic mechanism mediated in part by TNF.

Analysis of the cbhE' plasmid gene from acute disease-causing isolates of Coxiella burnetii.

A gene termed cbhE' was cloned from the QpH1 plasmid of Coxiella burnetii. Expression of recombinants containing cbhE' in vitro and in Escherichia coli maxicells, produced an insert-encoded polypeptide of approx. 42 kDa. The CbhE protein was not cleaved when intact maxicells were treated with trypsin. Hybridizations of total DNA isolated from the six strains of C. burnetii indicate that this gene is unique to C. burnetii strains associated with acute disease, i.e., Hamilton[I], Vacca[II], and Rasche[III]. The cbhE' gene was not detected in strains associated with chronic disease (Biotzere[IV] and Corazon[V]) or the Dod[VI] strain. The cbhE' open reading frame (ORF) is 1022 bp in length and is preceded by a predicted promoter/Shine-Dalgarno (SD) region of TCAACT(-35)-N16-TAAAAT(-10)-N14-AGAAGGA (SD) located 10 nucleotides (nt) before the presumed AUG start codon. The ORF ends with a single UAA stop codon and has no apparent Rho-factor-independent terminator following it. The cbhE' gene codes for the CbhE protein of 341 amino acid (aa) residues with a deduced Mr of 39,442. CbhE is predominantly hydrophilic with a predicted pI of 4.43. The function of CbhE is unknown. No nt or aa sequences with homology to cbhE' or CbhE, respectively, were found in searches of a number of data bases.

Analysis of the cells involved in the lymphoproliferative response to Coxiella burnetii antigens.

Vaccination with an inactivated, whole cell, Q fever vaccine (Q-vax) induces lasting antibody conversion and a positive delayed-type hypersensitivity (DTH) skin reaction in about 60% of recipients but a long-lasting positive lymphoproliferative or mitogenic response to C. burnetii antigens with peripheral blood mononuclear cells (PBMC) in 85-95% of subjects. Analysis of the lymphoproliferative response to C. burnetii antigens has now been made by fractionation-reconstitution experiments with PBMC from vaccines, from past infections, and from healthy controls. The major contributor to the response in immune subjects proved to be the T lymphocyte. T cells were stimulated by both the phase I and phase II antigens of two prototype strains of C. burnetii and responses were greatly amplified by addition of IL-2. Similar T lymphocyte stimulation profiles were obtained with the 'Priscilla' strain of C. burnetii which represents a different biotype of Coxiella isolated from Q fever endocarditis; Q-vax is therefore likely to protect against endocarditis strains. Fractionation-reconstitution experiments with T and B cells from vaccines and subjects infected in the past, using various antigenic or haptenic fractions from C. burnetii indicate that protein, non-lipopolysaccharide components of the organism are responsible for the mitogenic response of immune T cells. However, the role of the lipopolysaccharide in the protective immunogen has still to be defined.

[An immunoenzyme method for demonstrating Coxiella burnetii antigens]. / Immunofermentnyi metod indikatsii antigenov Coxiella burnetii.

Materials on the development of an enzyme immunoassay (EIA) system for the detection of the antigens of C. burnetii, the causative agent of Q rickettsiosis, are presented. The system is highly specific and effective with respect to both corpuscular antigens of phases 1 and 2 and soluble antigen (lipopolysaccharide). The sensitivity of this method varies within the range 5-100 ng/ml. The effectiveness of EIA as a quantitative (semiquantitative) control test used in the process of the production of Coxiella preparations has been demonstrated.

Isolation of 16 strains of Coxiella burnetii from patients by using a sensitive centrifugation cell culture system and establishment of the strains in HEL cells.

Q fever, caused by Coxiella burnetii, may be acute or chronic. Only a few strains of C. burnetii have been isolated due to the difficulty and hazard of isolation. We report here the isolation using a centrifugation shell vial technique of 16 new strains from patients suffering chronic Q fever. Twenty-four samples were inoculated onto human embryonic lung (HEL) fibroblast cell monolayers growing in shell vials. C. burnetii was detected 6 days later by using immunofluorescence. Samples from valves (n = 10), arterial prostheses (n = 2), bone (n = 3), skin biopsy (n = 1), bone marrow (n = 1), and blood (n = 5) from 16 patients were successfully cultured. Two cerebrospinal fluid samples from two patients were negative. The strains were subcultured in HEL cells and are now established. The technique is sensitive and less hazardous than animal inoculation. We recommend the shell vial technique for isolation of C. burnetii.

Bactericidal effect of doxycycline associated with lysosomotropic agents on Coxiella burnetii in P388D1 cells.

There is no consistently reliable treatment for endocarditis resulting from chronic Coxiella burnetii infection, the causative agent of Q fever. Although certain antibiotics are recommended on the basis of their in vitro bactericidal activities, results of therapy with these antibiotics are often disappointing. To evaluate whether the currently recommended antibiotic susceptibility tests for C. burnetii give misleading results because of continued division of uninfected cells, thereby resulting in the dilution of infected cells and, hence, a false picture of antibiotic efficacy, we blocked cell division during antibiotic susceptibility testing with cycloheximide. Using this new method, we found that the currently recommended antibiotics for the treatment of Q fever, doxycycline, pefloxacin, and rifampin, did not reduce the ratio of infected to noninfected cells (either L929 or P388D1) by 9 days postinfection. To test the hypothesis that this lack of antibacterial activity is due to antibiotic inactivation by the low pH of the phagolysosomes in which C. burnetii is found, we used alkalinizing lysosomotropic agents (chloroquine or amantadine) concurrently with doxycycline. This resulted in the sterilization of C. burnetii infection in P388D1 cells. This finding seems to confirm our suspicion that the acidic conditions of the phagolysosomes in which C. burnetii is located inhibit antibiotic activity. This inhibition can be reversed in vitro when lysosomotropic alkalinizing agents are used.