Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis.

Whole-genome sequencing and antimicrobial resistance in Brucella melitensis from a Norwegian perspective.

Brucellosis is a rarely encountered infection in Norway. The aim of this study was to explore all Brucella melitensis isolates collected in Norway from 1999 to 2016 in relation to origin of infection and antimicrobial resistance patterns. A total of 23 isolates were analysed by whole-genome sequencing and compared with selected sequences of B. melitensis available from NCBI. Additionally, SNP analysis in antibiotic resistance determining genes was performed. The majority belonged to the East Mediterranean clade (genotype II), while the remaining isolates belonged to the African clade (genotype III). These results indicate that human brucellosis in Norway is related to travels or migration from the Middle East, Asia or Africa, in accordance with results from Germany, Denmark and Sweden. Antibiotic susceptibility patterns were determined by broth microdilution method and/or gradient strip method. All isolates were susceptible for all tested antibiotics, except for rifampicin where phenotypical results indicated resistance or intermediate resistance in all isolates based on broth microdilution method, and in four isolates based on gradient strip testing. In contrast, screening of the rpoB gene did not reveal any mutations in the previously described rpoB "hot spot" regions related to rifampicin resistance, indicating overestimation of resistance based on phenotypical results.

Novel insights into transmission routes of Mycobacterium avium in pigs and possible implications for human health.

Mycobacterium avium infection is a severe condition in humans, whereas pigs are often subclinically infected. Pig carcasses represent a possible source of human infection. Faecal excretion of M. avium was recently demonstrated in experimentally infected pigs, along with detection of M. avium in apparently normal lymph nodes. The present study investigates faecal excretion in naturally infected herds and the presence of live mycobacteria in lymph nodes. Two pig herds (A and B), with a history of sporadically suspected M. avium infection were sampled. Herd B used peat, as opposed to Herd A. Samples from peat, sawdust, drinking water, faeces and lymph nodes were collected. Identification of mycobacteria was performed by 16S rDNA sequencing and PCR. Mycobacterium avium isolates were analysed by Multi-Locus Variable Number of Tandem repeat Analysis (MLVA). Mycobacterium avium subsp. hominissuis was detected in samples of faeces, peat and lymph nodes from Herd B, often with identical MLVA profiles. Additionally, other non-tuberculous mycobacteria (NTM) were found in the same material. The absence of macroscopic lymph node lesions in the presence of M. avium subsp. hominissuis was frequently demonstrated. In Herd A, only one NTM isolate, which proved not to be M. avium, was found. Faeces might facilitate transmission of M. avium subsp. hominissuis between pigs and maintain the infection pressure in herds. The low incidence of macroscopic lesions together with the massive presence of M. avium subsp. hominissuis in lymph nodes from pigs kept on peat raises questions related to animal husbandry, food safety and human health.

Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection.

BACKGROUND: Mycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah) are environmental mycobacteria and significant opportunistic pathogens. Mycobacterium avium infections in humans and pigs are mainly due to Mah. It is not known whether this is caused by a difference in virulence or difference in exposure to the two subspecies. The aim of the present study was to investigate the ability of the M. avium subspecies to replicate intracellularly and to characterise the gene expression program triggered by infection of human primary macrophages. RESULTS: All isolates were able to invade and persist within human macrophages. However, intracellular replication was only evident in cells infected with the two Maa isolates. Transcriptional responses to the isolates were characterized by upregulation of genes involved in apoptosis, immune- and inflammatory response, signal transduction and NF-kB signaling, cell proliferation and T-cell activation. Although similar pathways and networks were perturbed by the different isolates, the response to the Maa subspecies was exaggerated, and there was evidence of increased activation of type I and II interferon signaling pathways. CONCLUSION: Mycobacterium avium isolates of different genetic characteristics invaded monocytes and induced different degree of macrophage activation. Isolates of Maa were able to replicate intracellularly suggesting that differences in exposure, uptake or induction of adaptive immunity are more likely explanations for the difference in prevalence between M. avium subspecies.

Molecular characterization of Mycobacterium avium subspecies hominissuis isolated from humans, cattle and pigs in the Uganda cattle corridor using VNTR analysis.

BACKGROUND: Members of the Mycobacterium avium complex (MAC) cause disease in both human and animals. Their ubiquitous nature makes them both successful microbes and difficult to source track. The precise characterization of MAC species is a fundamental step in epidemiological studies and evaluating of possible reservoirs. This study aimed at identifying and characterizing Mycobacterium avium subsp. hominissuis isolated from human, slaughter cattle and pigs in various parts of the Uganda cattle corridor (UCC) at two temporal points using variable number of tandem repeat (VNTR) analysis. METHODS: A total of 46 M. avium isolates; 31 from 997 pigs, 12 from 43 humans biopsies and three from 61 cattle lesions were identified to subspecies level using IS1245 and IS901 PCR, thereafter characterized using VNTR. Twelve loci from two previously described VNTR methods were used and molecular results were analyzed and interpreted using Bionumerics 6.1. PRINCIPAL FINDINGS: 37 of the isolates were identified as M. avium subsp. hominissuis and four as M. avium subsp. avium, while five could not be differentiated, possibly due to mixed infection. There was distinct clustering that coincides with the temporal and spatial differences of the isolates. The isolates from humans and cattle in the North Eastern parts of the UCC shared identical VNTR genotypes. The panel of loci gave an overall discriminatory power of 0.88. Some loci were absent in several isolates, probably reflecting differences in isolates from Uganda/Africa compared to isolates previously analyzed by these methods in Europe and Asia. CONCLUSIONS: The findings indicate a molecular difference between M. avium subsp. hominissuis isolates from pigs in Mubende and cattle and human in the rest of the UCC. Although human and cattle shared VNTR genotypes in the North Eastern parts of the UCC, it is most likely a reflection of a shared environmental source.

Molecular epidemiology, drug susceptibility and economic aspects of tuberculosis in Mubende district, Uganda.

BACKGROUND: Tuberculosis (TB) remains a global public health problem whose effects have major impact in developing countries like Uganda. This study aimed at investigating genotypic characteristics and drug resistance profiles of Mycobacterium tuberculosis isolated from suspected TB patients. Furthermore, risk factors and economic burdens that could affect the current control strategies were studied. METHODS: TB suspected patients were examined in a cross-sectional study at the Mubende regional referral hospital between February and July 2011. A questionnaire was administered to each patient to obtain information associated with TB prevalence. Isolates of M. tuberculosis recovered during sampling were examined for drug resistance to first line anti-TB drugs using the BACTEC-MGIT960(TM) system. All isolates were further characterized using deletion analysis, spoligotyping and MIRU-VNTR analysis. Data were analyzed using different software; MIRU-VNTR plus, SITVITWEB, BioNumerics and multivariable regression models. RESULTS: M. tuberculosis was isolated from 74 out of 344 patients, 48 of these were co-infected with HIV. Results from the questionnaire showed that previously treated TB, co-infection with HIV, cigarette smoking, and overcrowding were risk factors associated with TB, while high medical related transport bills were identified as an economic burden. Out of the 67 isolates that gave interpretable results, 23 different spoligopatterns were detected, nine of which were novel patterns. T2 with the sub types Uganda-I and Uganda-II was the most predominant lineage detected. Antibiotic resistance was detected in 19% and multidrug resistance was detected in 3% of the isolates. CONCLUSION: The study detected M. tuberculosis from 21% of examined TB patients, 62% of whom were also HIV positive. There is a heterogeneous pool of genotypes that circulate in this area, with the T2 lineage being the most predominant. High medical related transport bills and drug resistance could undermine the usefulness of the current TB strategic interventions.

Molecular investigation of multiple strain infections in patients with tuberculosis in Mubende district, Uganda.

Multiple strain tuberculosis (TB) infections are now an acceptable facet of tuberculosis epidemiology. Identification of patients infected with more than one strain gives an insight in disease dynamics at individual and population level. This study therefore aimed at identifying multiple strain infections among TB infected patients. Furthermore, to determine factors associated with multiple strain infections in Mubende district of Uganda. A total of 72 Mycobacterium tuberculosis isolates from patients at Mubende regional referral hospital were characterized using 15 loci MIRU-VNTR, Spoligotyping and deletion analysis. Genotypic and epidemiological data were analyzed using MIRU-VNTR plus, Bionumerics software version 6.1 and an exact logistic regression model respectively. Eight (11.1%) of the 72 patients had mixed TB infections. Five were exclusively pulmonary mixed infections while three had both pulmonary and extra-pulmonary infections (Compartmentalized TB infections). Unlike previous studies that have linked this phenomenon to Beijing strains, multiple strains in this study belonged to T2-Uganda, X2 and T1 lineages. Two of the pulmonary mixed infections were resistant to rifampicin or isoniazid. All except one were HIV positive, newly diagnosed cases and urban residents of Mubende district. The study revealed that one in nine urban dwelling, HIV/TB co-infected patient were infected with more than one M. tuberculosis strains. The molecular findings give indications of a vital component of the disease dynamics that is most likely under looked at clinical level.

Mycobacterium bovis infections in slaughter pigs in Mubende district, Uganda: a public health concern.

BACKGROUND: Bovine tuberculosis (TB) caused by Mycobacterium bovis is primarily a disease of ruminants, particularly cattle (Bos primigenius) and buffalo (Syncerus caffer), and is endemic in most developing countries. To date, studies done in Uganda have documented the prevalence of M. bovis in cattle, humans and wild life, in addition to non-tuberculous mycobacteria in pigs. Pigs are increasingly becoming an important component of the livestock sector and share the human ecosystem in rural Uganda. It is therefore of public health interest that they are not a source of human infections. As a follow up to previously published findings on mycobacteria in pigs, this study was aimed at investigating the occurrence and molecular characteristics of M. bovis detected in slaughter pigs in Mubende district, Uganda. One hundred fifty mesenteric lymph nodes with lesions suggestive of mycobacterial infections were collected from approximately one thousand slaughtered pigs in Mubende district over a period of five months. The isolation and identification of M. bovis was done using conventional mycobacteriological methods. Mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC) were identified to species level using deletion analysis. Molecular typing was done using Spoligotyping and MIRU-VNTR analysis. Molecular data were analysed and interpreted using MIRU-VNTR plus, SpolDB4.0 and the Mycobacterium bovis spoligo database. RESULTS: Of the examined animals, one boar and two sows from Madudu Sub County were infected with M. bovis which presented as lesions of a deep yellow colour and a grit-like texture in the mesenteric lymph nodes. This represents 2% (3/150) of the lymph nodes where lesions suggestive of mycobacterial infections were detected. Molecular analysis revealed that the isolates from the infected pigs showed identical MIRU-VNTR profile and spoligotype (SB1469). CONCLUSIONS: This is the first study documenting the occurrence of M. bovis in slaughter pigs in Uganda, revealing that one in fifty slaughter pigs with suspected lesions in mesenteric lymph nodes were infected. Molecular analysis revealed that the isolates were identical, showing a spoligotype previously reported from humans and cattle in the north eastern part of the Uganda cattle corridor. This finding is of public health importance, therefore there is a need for close cooperation between medical and veterinary professionals in designing and implementing control and prevention measures that safeguard the public from this potential source of zoonotic TB in Uganda.

Non-tuberculous mycobacteria isolated from slaughter pigs in Mubende district, Uganda.

BACKGROUND: The importance of infections caused by non-tuberculous mycobacteria (NTM) in animals and humans has gained considerable recognition during the past few years. In the developed world, where pig production is extensively practiced, studies on mycobacterial infections and related control strategies have received increasing attention. The infections are reported to be caused by a wide spectrum of NTM. Unfortunately, these infections have been less recognized in sub-Saharan Africa owing to lack of awareness and systematic studies. In this study we aimed at isolating and identifying species of mycobacteria involved in causing infections in slaughter pigs in Mubende district of Uganda. Furthermore we wanted to identify factors associated with infection prevalence in the study area. METHODS: A total of 363 lymph nodes were collected and cultured for the presence of mycobacteria. Isolates were identified by 16S rDNA gene sequencing. A questionnaire survey was administered to identify production related factors associated with infection prevalence. Data were assembled and analysed using descriptive statistics and mixed effects logistic regression analysis. RESULTS: Mycobacteria were detected in 39 % (143/363) of the examined lymph nodes, 63 % (59/93) of lymph nodes with gross lesions typical of mycobacteriosis and 31% (84/270) of lymph nodes with no visible lesions. Nineteen per cent of the isolated mycobacteria were identified as Mycobacterium (M) avium, of these 78% and 22% were M. avium sub sp. Hominissuis and avium respectively. Other mycobacterial species included M. senuense (16%), M. terrae (7%) and M. asiaticum (6%). This study found free range systems (OR = 3.0; P = 0.034) and use of water from valley dams (OR = 2.0; P = 0.049) as factors associated with high prevalence of mycobacteria in slaughter pigs. CONCLUSIONS: This study demonstrated a high prevalence of NTM infections among slaughter pigs in Mubende district of Uganda. M. avium was the most prevalent of all NTM isolated and identified. Free range system of pig management and valley dam water were the most significant factors associated with NTM prevalence in Mubende district. These findings could be of a major public health concern given that it is in a predominantly pork consuming population with 18% HIV/AIDS prevalence. Therefore, stringent post-mortem inspection at the slaughter houses is of paramount importance to reduce human exposure.

A comparative study of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis in experimentally infected pigs.

BACKGROUND: Mycobacterium avium subsp. avium (Maa) and Mycobacterium avium subsp. hominissuis (Mah) are opportunistic pathogens that may infect several species, including humans and pigs. Mah is however more frequently isolated from pigs than Maa, and it is unclear if this is due to difference in virulence or in exposure to the two organisms. Clinical isolates of each subspecies were administered perorally to ten domestic pigs, respectively. The animals were sacrificed at six and 12 weeks after inoculation. At necropsy, macroscopic lesions were recorded, and tissue samples were collected for mycobacterial culture, IS1245 real time PCR and histopathological examination. Culturing was also performed on faecal samples collected at necropsy. RESULTS: Macroscopic and histopathological lesions were detected in pigs infected with each subspecies, and bacterial growth and histopathological changes were demonstrated, also in samples from organs without gross pathological lesions. Six weeks after inoculation, live Mah was detected in faeces, as opposed to Maa. The presence of live mycobacteria was also more pronounced in Mah infected tonsils. In comparison, the Maa isolate appeared to have a higher ability of intracellular replication in porcine macrophages compared to the Mah isolate. CONCLUSIONS: The study shows that both subspecies were able to infect pigs. Additionally, the more extensive shedding of Mah might cause pig-to-pig transmission and contribute to the higher incidence of infection caused by this subspecies.

Investigation of an outbreak of mycobacteriosis in pigs.

BACKGROUND: A high proportion of pigs imported to Serbia from a Lithuanian breeding herd reacted positively against avian and/or bovine tuberculin. The pigs were euthanized and lesions characteristic for mycobacterial infection were detected. An investigation of potential mycobacteriosis in the pigs imported to Serbia and the possible source of infection in the Lithuanian herd were therefore initialised. RESULTS: Formalin fixed, paraffin embedded lymph nodes from tuberculin positive animals were examined by real-time PCR for IS1245 and IS6110. IS1245 was detected in 55% and IS6110 in 11% of the samples. Seven of the ten IS6110 positive samples were positive for IS1245. Eleven lymph nodes from 10 pigs and 15 environmental samples were collected from the Lithuanian breeding herd and cultured for mycobacteria. M. avium subsp. hominissuis was detected in all lymph nodes and from eight samples of peat and sawdust. Isolates with identical and related IS1245- and IS1311 RFLP profiles were detected from swine and peat. CONCLUSIONS: This study demonstrated cross reactions between avian and bovine tuberculin in pigs. Real-time PCR indicated infection with M. avium in the Serbian pigs. However, as a small proportion of the lymph nodes were positive for IS6110, infection with bacteria in the M. tuberculosis complex could not be ruled out. Analyses confirmed the presence of M. avium subsp. hominissuis in porcine and environmental samples from the Lithuanian breeding herd. The results indicate peat as a source of M. avium subsp. hominissuis infection in these pigs, and that the pigs imported to Serbia were infected with M. avium subsp. hominissuis.

Isolation of non-tuberculous mycobacteria from pastoral ecosystems of Uganda: public health significance.

BACKGROUND: The importance of non-tuberculous mycobacteria (NTM) infections in humans and animals in sub-Saharan Africa at the human-environment-livestock-wildlife interface has recently received increased attention. NTM are environmental opportunistic pathogens of humans and animals. Recent studies in pastoral ecosystems of Uganda detected NTM in humans with cervical lymphadenitis and cattle with lesions compatible with bovine tuberculosis. However, little is known about the source of these mycobacteria in Uganda. The aim of this study was to isolate and identify NTM in the environment of pastoral communities in Uganda, as well as assess the potential risk factors and the public health significance of NTM in these ecosystems. METHOD: A total of 310 samples (soil, water and faecal from cattle and pigs) were examined for mycobacteria. Isolates were identified by the INNO-Lipa test and by 16S rDNA sequencing. Additionally, a questionnaire survey involving 231 pastoralists was conducted during sample collection. Data were analysed using descriptive statistics followed by a multivariable logistic regression analysis. RESULTS: Forty-eight isolates of NTM were detected; 25.3% of soil samples, 11.8% of water and 9.1% from animal faecal samples contained mycobacteria. Soils around water sources were the most contaminated with NTM (29.8%). Of these samples, M. fortuitum-peregrinum complex, M. avium complex, M. gordonae, and M. nonchromogenicum were the most frequently detected mycobacteria. Drinking untreated compared to treated water (OR = 33), use of valley dam versus stream water for drinking and other domestic use (OR = 20), sharing of water sources with wild primates compared to antelopes (OR = 4.6), sharing of water sources with domestic animals (OR = 5.3), and close contact with cattle or other domestic animals (OR = 13.8) were the most plausible risk factors for humans to come in contact with NTM in the environment. CONCLUSIONS: The study detected a wide range of potentially pathogenic NTM from the environment around the pastoral communities in Uganda. Drinking untreated water and living in close contact with cattle or other domestic animals may be risk factors associated with the possibility of humans and animals acquiring NTM infections from these ecosystems.