The Brucella Cell Envelope.

The cell envelope is a multilayered structure that insulates the interior of bacterial cells from an often chaotic outside world. Common features define the envelope across the bacterial kingdom, but the molecular mechanisms by which cells build and regulate this critical barrier are diverse and reflect the evolutionary histories of bacterial lineages. Intracellular pathogens of the genus Brucella exhibit marked differences in cell envelope structure, regulation, and biogenesis when compared to more commonly studied gram-negative bacteria and therefore provide an excellent comparative model for study of the gram-negative envelope. We review distinct features of the Brucella envelope, highlighting a conserved regulatory system that links cell cycle progression to envelope biogenesis and cell division. We further discuss recently discovered structural features of the Brucella envelope that ensure envelope integrity and that facilitate cell survival in the face of host immune stressors.

Intracellular growth of Brucella is mediated by Dps-dependent activation of ferritinophagy.

Bacteria of the genus Brucella cause brucellosis, one of the world's most common zoonotic diseases. A major contributor to Brucella's virulence is the ability to circumvent host immune defense mechanisms. Here, we find that the DNA-binding protein Dps from Brucella is secreted within the macrophage cytosol, modulating host iron homeostasis and mediating intracellular growth of Brucella. In addition to dampening iron-dependent production of reactive oxygen species (ROS), a key immune effector required for immediate bacterial clearance, cytosolic Dps mediates ferritinophagy activation to elevate intracellular free-iron levels, thereby promoting Brucella growth and inducing host cell necrosis. Inactivation of the ferritinophagy pathway by Ncoa4 gene knockout significantly inhibits intracellular growth of Brucella and host cell death. Our study uncovers an unconventional role of bacterial Dps, identifying a crucial virulence mechanism used by Brucella to adapt to the harsh environment inside macrophages.

Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins.

Toll-like receptors (TLRs) are essential components of innate immunity that serves as the first line of defense against the invaded microorganisms. However, successful infectious pathogens subvert TLR signaling to suppress the activation of innate and adaptive responses. Brucella species are infectious intracellular bacterial pathogens causing the worldwide zoonotic disease, brucellosis, that impacts economic growth of many countries. Brucella species are considered as stealthy bacterial pathogens as they efficiently evade or suppress host innate and adaptive immune responses for their chronic persistence. However, the bacterial effectors and their host targets for modulating the immune responses remain obscure. Brucella encodes various outer membrane proteins (Omps) that facilitate their invasion, intracellular replication, and immunomodulation. Outer membrane protein 25 (Omp25) of Brucella plays an important role in the immune modulation through suppression of proinflammatory cytokines. However, the mechanism and the signaling pathways that are targeted by Omp25 to attenuate the production of proinflammatory cytokines remain obscure. Here, we report that Omp25 and its variants, viz. Omp25b, Omp25c, and Omp25d, suppress production of proinflammatory cytokines that are mediated by various TLRs. Furthermore, we demonstrate that Omp25 and its variants promote enhanced ubiquitination and degradation of TLRs and their adaptor proteins to attenuate the expression of proinflammatory cytokines. Targeting multiple TLRs and adaptor proteins enables Omp25 to effectively suppress the expression of proinflammatory cytokines that are induced by diverse pathogen-associated molecular patterns. This can contribute to the defective adaptive immune response and the chronic persistence of Brucella in the host.

Brucella targets the host ubiquitin-specific protease, Usp8, through the effector protein, TcpB, for facilitating infection of macrophages.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host ubiquitin-specific protease, USP8, negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Upon silencing or chemical inhibition of USP8, the membrane localization of the CXCR4 receptor was enriched, which augmented the invasion of Brucella into macrophages. Activation of USP8 through chemical inhibition of 14-3-3 protein affected the invasion of Brucella into macrophages. Brucella suppressed the expression of Usp8 at its early stage of infection in the infected macrophages. Furthermore, we found that only live Brucella could negatively regulate the expression of Usp8, suggesting the role of secreted effector protein of Brucella in modulating the gene expression. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB, plays a significant role in downregulating the expression of Usp8 by targeting the cyclic-AMP response element-binding protein pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonists showed a diminished bacterial load. Our experimental data demonstrate a novel role of Usp8 in the host defense against microbial intrusion. The present study provides insights into the microbial subversion of host defenses, and this information may ultimately help to develop novel therapeutic interventions for infectious diseases.

Genomic Diversity and Zoonotic Potential of Brucella neotomae.

After reports in 2017 of Brucella neotomae infections among humans in Costa Rica, we sequenced 12 strains isolated from rodents during 1955-1964 from Utah, USA. We observed an exact strain match between the human isolates and 1 Utah isolate. Independent confirmation is required to clarify B. neotomae zoonotic potential.

Expression of cytokine and Apoptosis-Associated genes in mice bone Marrow-Derived Macrophages stimulated with Brucella recombinant type IV secretion effectors.

BACKGROUND: Brucellosis is an economically important infectious caused by most commonly by Brucella. Detection of infected animals at the early stage is important for controlling the disease. The diagnostic antigens, usually protein antigens, have attracted much interest. However, the accurate mechanism of immune response is still unknown. The secretory effectors (BPE005, BPE275, and BPE123) of the type IV secretion system (T4SS) were involved in the intracellular circulation process of Brucella and the immune responses of the host. METHODS: Genes encoding three B. abortus effector proteins (BPE005, BPE275, and BPE123) of T4SS were cloned and the recombinant proteins were expressed and purified. The purified recombinant proteins were named rBPE005, rBPE275 and rBPE123. Then, the expressions of Th1- and Th2-related cytokine genes were analyzed in mice bone marrow-derived macrophages (BMDMs) after stimulation with rBPE005, rBPE275, and rBPE123. Furthermore, four apoptosis-associated genes (Caspase-3, Caspase-8, Bax, and Bcl-2) were also detected to explore the damage of the proteins to the cells. RESULTS: Expressions of all Th1- and Th2-related cytokine genes were induced with three proteins, and different cytokine expression patterns induced by each protein depend on the stimulation time and dose of protein. However, expressions of apoptosis-related genes did not change. CONCLUSION: These results showed that the secreted antigens of Brucella induced an immune reaction via the production of Th1- and Th2-type cytokines in BMDMs without exerting any damage on the cells.

Efficacy of antibiotic prophylaxis to preventing brucellosis in accidental exposure: A systematic review.

BACKGROUND: Antibiotic prophylaxis to prevent brucellosis after accidental exposure to Brucella is an important topic in public health. This study aimed to systematically review the efficacy of antibiotic prophylaxis following accidental exposure to Brucella in preventing human brucellosis disease. METHODS: The study protocol was registered in PROSPERO (CRD42023456812). The outcomes included the incidence of brucellosis disease, adverse events rate, and antibiotic prophylaxis adherence. A comprehensive literature search, conducted until 20 November, 2023, involved Medline, Embase, Cochrane Library, and LILACS databases. Descriptive analysis and meta-analysis using R software were performed, risk of bias was assessed using JBI Critical appraisal tools, and certainty of evidence was assessed using the GRADE tool. RESULTS: Among 3102 initially identified records, eight studies involving 97 individuals accidentally exposed, all focused on high-risk accidental exposure to Brucella in laboratory settings, were included in the review. All studies reported the prophylactic treatment comprising doxycycline at a dosage of 100 mg twice daily, combined with rifampicin at 600 mg, both administered over 21 days. Prophylaxis adherence was reported in 86% of cases, and incidence of brucellosis post-treatment was 0.01. Adverse events, mainly gastrointestinal, occurred in 26% of cases. Critical appraisal revealed limitations in reporting demographics and clinical information. The certainty of evidence was rated as 'very low,' emphasising the need for caution in interpreting the observed outcomes due to study design constraints and the absence of comparative groups. CONCLUSIONS: PEP is an alternative practice reported in the literature, used in accidents with high-risk exposure to Brucella. The currently available evidence of the efficacy of antibiotic prophylaxis is insufficient to support a recommendation for or against the widespread use of antibiotic prophylaxis, so caution is needed in interpreting results due to the very low certainty of evidence, primarily stemming from case series and lack of comparative groups.

Pleurisy due to brucellosis.

Any system or organ involvement can be seen in brucellosis, which is still a significant public health problem in developing countries. The rate of respiratory system involvement is lower than that of other systems and which is also difficult to document. Brucellosis-associated pleurisy is a rare complication even in endemic regions. In this case report, a 78-year-old male patient who was assessed for pleural effusion etiology is presented. Brucella spp. were isolated on the 14th day of the pleural fluid incubation in the blood culture set and the patienthas been treated successfully for brucellosis. Based on our experience we think that it is important to use blood culture media for sterile body fluids, particularly for microorganisms that are difficult to isolate such as Brucella spp.

Motor polyradiculoneuropathy as an unusual presentation of neurobrucellosis: a case report and literature review.

Brucellosis, a zoonotic disease caused by Brucella species, poses a significant global health concern. Among its diverse clinical manifestations, neurobrucellosis remains an infrequent yet debilitating complication. Here, we present a rare case of neurobrucellosis with unusual presentations in a 45-year-old woman. The patient's clinical course included progressive lower extremity weakness, muscle wasting, and double vision, prompting a comprehensive diagnostic evaluation. Notable findings included polyneuropathy, elevated brucella agglutination titers in both cerebrospinal fluid and blood, abnormal EMG-NCV tests, and resolving symptoms with antibiotic therapy. The clinical presentation, diagnostic challenges, and differentiation from other neurological conditions are discussed. This case underscores the importance of considering neurobrucellosis in regions where brucellosis is prevalent and highlights this rare neurological complication's distinctive clinical and radiological features. Early recognition and appropriate treatment are crucial to mitigate the significant morbidity associated with neurobrucellosis.

Neurobrucellosis: laboratory features, clinical characteristics, antibiotic treatment, and clinical outcomes of 21 patients.

BACKGROUND: Neurobrucellosis (NB) is a rare and serious complication of brucellosis. Its clinical manifestations vary, with no obvious specificity. At present, there is no clear clinical diagnosis or treatment for reference. In this study, we retrospectively analyzed the clinical data for 21 patients with NB to provide reference data for its further study. METHODS: We analyzed the epidemiological and clinical manifestations, laboratory tests, imaging examinations, cerebrospinal fluid, and treatment plans of 21 patients diagnosed with NB in the Department of Neurology, Xuanwu Hospital, Capital Medical University Beijing, China. RESULTS: The ages of the patients ranged from 15 to 60 years old (mean age 40.1 ± 13.33 years), the male: female ratio was 4.25:1. Thirteen patients had a history of animal (sheep, cattle) contact, three had no history of animal contact, and the contact status of four was unknown. Brucella can invade various systems of the body and show multi-system symptoms, the main general manifestations were fever (66.67%), fatigue (57.14%) and functional urination or defecation disturbance (42.86%). The main nervous system manifestations were limb weakness (52.38%) and hearing loss (47.62%).The main positive signs of the nervous system included positive pathological signs (71.43%), sensory abnormalities (52.38%), limb paralysis (42.86%). Nervous system lesions mainly included spinal cord damage (66.67%), cranial nerve involvement (61.90%), central demyelination (28.57%) and meningitis (28.57%). In patients with cranial nerve involvement, 69.23% of auditory nerve, 15.38% of optic nerve and 15.38% of oculomotor nerve were involved. The blood of eight patients was cultured for Brucella, and three (37.5%) cultures were positive and five (63.5%) negative. The cerebrospinal fluid (CSF) of eight patients was cultured for Brucella, and two (25.00%) cultures were positive and six (75.00%) negative. Nineteen of the patients underwent a serum agglutination test (SAT), 18 (94.74%) of whom were positive and one (5.26%) of whom were negative. A biochemical analysis of the CSF was performed in 21 patients, and the results were all abnormal. Nineteen patients underwent magnetic resonance imaging (MRI). Twenty-one patients were treated with doxycycline and/or rifampicin, combined with ceftriaxone, quinolone, aminoglycoside, or minocycline. After hospitalization, 15 patients improved (71.43%), two patients did not recover, and the status of four patients was unknown. CONCLUSIONS: The clinical manifestations, CSF parameters, and neurological imaging data for patients with NB show no significant specificity or correlations. When patients with unexplained neurological symptoms accompanied by fever, fatigue, and other systemic manifestations in a brucellosis epidemic area or with a history of contact with cattle, sheep, animals, or raw food are encountered in clinical practice, the possibility of NB should be considered. Treatment is based on the principles of an early, combined, and long course of treatment, and the general prognosis is good.

A study on the epidemiology of brucellosis in bovine population of peri-urban and rural areas of district Multan, southern Punjab, Pakistan.

BACKGROUND: Brucellosis is a zoonotic disease caused by a bacterial pathogen belonging to the genus Brucella. It is one of the most frequent bacterial zoonoses globally but unfortunately, it is still considered as a neglected disease in the developing world. Keeping in view, this study was conducted to determine the prevalence and risk determinants of brucellosis in large ruminants of peri-urban and rural areas of district Multan-Pakistan. For this purpose, blood samples (n = 490) were collected from the cattle (n = 245) and buffalo (n = 245) population of the study area and subjected to preliminary screening of brucellosis using local and imported RBPT reagents. All the samples were further analyzed using commercially available multi-specie indirect ELISA kit followed by their confirmation by PCR using genus and species-specific primers. Data obtained from lab analysis and questionnaires were subjected to statistical analysis for Pearson Chi-square, Odds Ratio and Confidence intervals (95%). RESULTS: The results showed that the maximum seropositivity was recorded with local RBPT reagent (VRI, Pakistan; 12.45%; 95%CI = 9.72-15.65%) followed by RBPT-IDEXX (12.24%; 95%CI = 9.52-15.45%) and RBPT-ID.vet (11.84%; 95%CI = 9.18-14.95%) however statistical difference was non-significant (P = 0.956). The ELISA results showed an overall seroprevalence rate of 11.22% (95%CI = 8.59-14.33%) with comparatively higher rate in cattle (12.65%; 95%CI = 8.82-17.44%) as compared to buffaloes (9.80%; 95%CI = 6.49-14.15%). The PCR analysis confirmed the presence of genus Brucella in all seropositive samples whereas frequency of B. abortus and B. melitensis in seropositive samples was 80% and 20%, respectively. The co-existence of both species was also observed in 5.45% samples. The statistical analysis showed a significant association of bovine brucellosis with herd size, breed, reproductive disorders, mode of insemination, educational status and farmers' awareness about brucellosis (P < 0.05). Conversely, locality, age, weight, gender, pregnancy status, parity and puberty status had no associations with brucellosis (P > 0.05). CONCLUSION: In conclusion, brucellosis is prevalent in large ruminants of district Multan, Pakistan. It is suggested to devise and implement stringent policies for the effective control and prevention of brucellosis in the region. Further, the current situation also warrants the need to strengthen interdisciplinary coordination among veterinarians and physicians in one health perspective to ensure and strengthen the human and animal health care systems in the region.

Resolution of N-acetyl-DL-methionine methyl ester by the lipase from Brucella thiophenivorans.

In this study, lipase-catalyzed resolution of N-acetyl-DL-methionine methyl ester (N-Ac-DL-MetOMe) was evaluated. A lipase from Brucella thiophenivorans was prone to exhibit high activity and excellent enantioselectivity toward N-Ac-DL-MetOMe to produce the key chiral intermediate N-acetyl-L-methionine methyl ester (N-Ac-L-MetOMe). The results showed that the enzymatic reaction was carried out in 100 g/L racemic substrate for 2 h, the conversion reached 51.3%, the enantiomeric excess value N-Ac-L-MetOMe exceeded 99%, and the enantiomeric ratio value >200. Therefore, the lipase from B. thiophenivorans has potential prospects for the resolution of N-Ac-DL-MetOMe to produce the important intermediate N-Ac-L-MetOMe.

A subunit vaccine based on Brucella rBP26 induces Th1 immune responses and M1 macrophage activation.

Brucellosis is a global zoonotic infection caused by Brucella bacteria, which poses a significant burden on society. While transmission prevention is currently the most effective method, the absence of a licenced vaccine for humans necessitates the urgent development of a safe and effective vaccine. Recombinant protein-based subunit vaccines are considered promising options, and in this study, the Brucella BP26 protein is expressed using prokaryotic expression systems. The immune responses are evaluated using the well-established adjuvant CpG-ODN. The results demonstrate that rBP26 supplemented with a CpG adjuvant induces M1 macrophage polarization and stimulates cellular immune responses mediated by Th1 cells and CD8 + T cells. Additionally, it generates high levels of rBP26-specific antibodies in immunized mice. Furthermore, rBP26 immunization activates, proliferates, and produces cytokines in T lymphocytes while also maintaining immune memory for an extended period of time. These findings shed light on the potential biological function of rBP26, which is crucial for understanding brucellosis pathogenesis. Moreover, rBP26 holds promise as an effective subunit vaccine candidate for use in endemic areas.

Is it safe to use ovarian follicular fluid from cows seropositive for brucellosis in the production of in vitro embryos?

Animal reproduction biotechniques are important tools for the technological advancement of livestock, as they allow the selection of the reproductive potential of superior quality females and males; however, infectious diseases that have a predilection for the reproductive system can be a hindrance for the use of these technologies. Therefore, the present study aimed to detect Brucella spp. in the ovarian follicular fluid of brucellosis-positive bovine cows. A total of 47 bovine ovarian follicular fluid aspirates from cows, positive in tests for brucellosis and from Brucella-positive herd, were submitted to PCR. The primers used in the PCR were specific to the genus Brucella (bcsp31 gene). All 47 bovine aspirates were negative for Brucella spp. 0.00% (95% CI: 0.00-4.00%). Our results demonstrated that Brucella spp. was absent in the ovarian follicular fluid from seropositive cows, which indicates that Brucella spp.-infected cows could be used for reproductive biotechnologies carried out with follicular aspirates. Future studies are needed to more precisely evaluate the feasibility and safety of using these oocytes from brucellosis-seropositive cows to transfer embryos to heifers/cows not infected by Brucella, aiming to produce calves free of the infection.

An Unusual Manifestation of Brucella-Associated Uveitis in a Young Male Patient.

This is a unique case of retinoschisis as an ocular manifestation of brucellosis. A 38-year-old male presented with recurrent episodes of bilateral eye redness, predominately in his left eye. His visual acuity was not affected, and he did not report any other symptoms. On slit lamp examination, binocular Koeppe nodules of the iris and cells in the left anterior chamber were observed. Fundoscopy followed by meticulous multimodal imaging confirmed left inferior retinoschisis. The patient was diagnosed with panuveitis, and a series of laboratory examinations revealed positive anti-IgM Brucella antibodies. Ocular brucellosis can cause variable, atypical, and serious presentations, hence, early diagnosis is paramount to avoid complications.

Can Hematological Inflammatory Indices Be Used to Differentiate Modic Type 1 Changes from Brucella Spondylodiscitis?

Background and Objectives: Differentiation between brucella spondylodiscitis and Modic type I changes (MC1) includes difficulties. Hematological inflammatory indices (HII) such as neutrophil to lymphocyte ratio (NLR) and aggregate index of systemic inflammation (AISI) are suggested as indicators of inflammation and infection and have diagnostic, prognostic, and predictive roles in various diseases. This study aimed to evaluate differences between brucella spondylodiscitis and MC1 in terms of HII. Materials and Methods: Thirty-five patients with brucella spondylodiscitis and thirty-seven with MC1 were enrolled in the study. Brucella spondylodiscitis and MC1 were diagnosed by microbiological, serological, and radiological diagnostic tools. HII (NLR, MLR, PLR, NLPR, SII, SIRI, AISI) were derived from baseline complete blood count. Results: The two groups were similar for age (p = 0.579) and gender (p = 0.092), leukocyte (p = 0.127), neutrophil (p = 0.366), lymphocyte (p = 0.090), and monocyte (p = 0.756) scores. The Brucella spondylodiscitis group had significantly lower pain duration (p < 0.001), higher CRP and ESR levels (p < 0.001), and lower platelet count (p = 0.047) than the MC1 group. The two groups had similarity in terms of HII: NLR (p = 0.553), MLR (p = 0.294), PLR (p = 0.772), NLPR (p = 0.115), SII (p = 0.798), SIRI (p = 0.447), and AISI (p = 0.248). Conclusions: Increased HII can be used to differentiate infectious and non-infectious conditions, but this may be invalid in brucellosis. However, pain duration, CRP and ESR levels, and platelet count may be useful to distinguish brucella spondylodiscitis from MC1.

Cell and Tissue Tropism of Brucella spp.

Brucella spp. are facultatively intracellular bacteria that can infect, survive, and multiply in various host cell types in vivo and/or in vitro. The genus Brucella has markedly expanded in recent years with the identification of novel species and hosts, which has revealed additional information about the cell and tissue tropism of these pathogens. Classically, Brucella spp. are considered to have tropism for organs that contain large populations of phagocytes such as lymph nodes, spleen, and liver, as well as for organs of the genital system, including the uterus, epididymis, testis, and placenta. However, experimental infections of several different cultured cell types indicate that Brucella may actually have a broader cell tropism than previously thought. Indeed, recent studies indicate that certain Brucella species in particular hosts may display a pantropic distribution in vivo. This review discusses the available knowledge on cell and tissue tropism of Brucella spp. in natural infections of various host species, as well as in experimental animal models and cultured cells.

Novel Species of Brucella Causing Human Brucellosis, French Guiana.

Human brucellosis is a zoonoses caused by bacteria of the genus Brucella. Infection results in subacute or chronic debilitating disease with nonspecific clinical manifestations and is often associated with consuming unpasteurized dairy products. We report 2 cases of brucellosis in male patients who were hospitalized in distinct towns of French Guiana, an overseas territory of France located on the northeastern shore of South America. Both men were citizens of Brazil working as clandestine goldminers in the deep Amazonian rainforest. Characterization of the 2 bacterial isolates revealed that they represent a potential new species of Brucella. Medical practitioners working in contact with wildlife in this region of the world should be aware of the existence of these pathogens and the potential for human infection.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

An Update on Novel Taxa and Revised Taxonomic Status of Bacteria Isolated from Domestic Animals Described in 2018 to 2021.

Novel bacterial taxonomy and nomenclature revisions can have significant impacts on clinical practice, disease epidemiology, and veterinary microbiology laboratory operations. Expansion of research on the microbiota of humans, animals, and insects has significant potential impacts on the taxonomy of organisms of clinical interest. Implications of taxonomic changes may be especially important when considering zoonotic diseases. Here, we address novel taxonomy and nomenclature revisions of veterinary significance. Noteworthy discussion centers around descriptions of novel mastitis pathogens in Streptococcaceae, Staphylococcaceae, and Actinomycetaceae; bovine reproductive tract pathogens in Corynebacteriaceae; novel members of Mannheimia spp., Leptospira spp., and Mycobacterium spp.; the transfer of Ochrobactrum spp. to Brucella spp.; and revisions to the genus Mycoplasma.