Effect of Ultraviolet-c (UVc) light and ozone on the survival of Mycobacterium abscessus complex organisms associated with cystic fibrosis.

Background: Members of the Mycobacterium abscessus complex have now emerged as clinically significant respiratory pathogens in people with cystic fibrosis (CF), potentially leading to increased disease severity, antibiotic treatment, and persistence dilemmas. Many of these species are resistant to disinfectants and biocides commonly used to clean and disinfect the hospital environment, thus necessitating the need to examine innovative ways to eliminate these organisms from such environments. It was, therefore, the aim of this study to examine the individual effect of ultraviolet-c (UVc) light (λ = 254 nm) and ozone (O3) on the growth of the M. abscessus complex organisms, as well as on seven other clinically significant CF pathogens, including Achromobacter spp., Burkholderia gladioli, Burkholderia cenocepacia, Burkholderia multivorans, Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia. Methods: Bacterial isolates (n = 46), including M. abscessus complex (n = 6) (M. abscessus subsp abscessus [n = 2], M. abscessus subsp. bolletii [n = 2], M. abscessus subsp. massiliense [n = 2]), and other CF pathogens (n = 40) including Achromobacter spp., B. gladioli, B. cenocepacia, B. multivorans, P. aeruginosa, S. aureus, and S. maltophilia, were exposed for 1 h to UVc light (254 nm), as well as to ozone (O3; 26 ppm). Results: UVc light inactivated all M. abscessus complex organisms (n = 6), as well as the 40 isolates from the other genera and species. No bacterial species tested was able to survive the UVc treatment. O3 was unable to inactivate all isolates of M. abscessus subsp. abscessus (n = 2), M. abscessus subsp. bolletii (n = 2), and one isolate of M. abscessus subsp. massiliense, but killed one strain of M. abscessus subsp. massiliense. Overall, O3 inactivated only 20% of total isolates, allowing the posttreatment growth of the remaining 80% of isolates. There was no difference in the growth dynamic of P. aeruginosa from the environmental waters which had received O3 treatment and the control (untreated with O3). Bacterial growth, while occurring post-O3 treatment, was not as prolific in all remaining organisms, as in the untreated controls, demonstrating some but limited antibacterial effect. Conclusions: From the data presented by this study, UVc light at 254 nm was effective at eliminating all organisms examined, including members of the M. abscessus complex. Given the refractory nature of these organisms against conventional wet chemical disinfection, UVc potentially offers a physical method to control and eliminate the survival of these organisms on health-care surfaces and fomites. For many CF species examined in this study, these data represent the first reports of the organisms susceptibility to UVc light. Further work is now required to establish time/distance parameters incorporated into newly designed innovative devices, to allow disinfection protocols to be optimized, and delivered to exploit this vulnerability with these nontuberculous mycobacterial organisms, as well as with the other bacterial species examined.

Helping map the taxonomical position of the Nontuberculous Mycobacteria (NTM) in cystic fibrosis.

Background: Nontuberculous mycobacteria (NTMs) have now emerged as important opportunistic bacterial pathogens, particularly among patients with cystic fibrosis (CF). The development of improved molecular technologies and bioinformatics and the adoption of whole-genome sequencing to more isolates have allowed for a reanalysis of the existing taxa within the genus Mycobacterium, resulting in the renaming of some existing NTM Mycobacterium species to three novel genera, viz., Mycolicibacterium gen. nov., Mycolicibacter gen. nov. and Mycobacteroides gen. nov. This has resulted in controversy, particularly within the clinical community, accompanied by a reluctance to adopt and employ these new bacterial names. Therefore, the aims of this study were (i) to identify NTM organisms associated with CF lung disease that have been reported previously in the published literature, (ii) to examine the realignment of NTM organisms previously described in CF within the revised new mycobacterial taxonomy and renaming, and (iii) to identify and explore online taxonomical tools to help educate clinical medicine about recent changes in NTM taxonomy. Methods: Three tasks were performed, namely (i) to identify NTM organisms previously associated with people with CF, (ii) to examine the extent and scope of the reclassification of CF-related NTM species affected by changes in recent taxonomy and nomenclature, and (iii) to identify and examine the educational utility of online taxonomical educational tools/software (LifeMap [http://lifemap.univ-lyon1.fr/]; National Center for Biotechnology Information [NCBI] Taxonomy browser [https://www.ncbi.nlm.nih. gov/guide/taxonomy/]; and List of Prokaryotic names with Standing in Nomenclature [LPSN] [https://lpsn.dsmz.de/]). Mycobacterium (Mycobacteroides) abscessus was selected as the species to evaluate the application of these tools. Results: Twenty-one NTM species have been reported that have been associated with CF lung disease. Of these, two have been reclassified into the Mycobacteroides genus, two into the Mycolicibacter genus, and seven into the Mycolicibacterium genus. LifeMap, NCBI Taxonomy browser, and LPSN offered interactive visual support to better understand the taxonomy and nomenclature of NTM organisms. Conclusion: We, therefore, advocate that clinical and scientific parties employ these online tools to gain a better insight into the familiarization and understanding of such evolving NTM classification, thereby aiding a better lexicon and communication among all stakeholders.

Description of wylie-stanley agar for the recovery of mycobacterium abscessus.

Background: The microbiology of cystic fibrosis (CF) is complicated by the presence of several species, including Mycobacterium abscessus, which are highly resistant to antibiotics. Conventional selective bacteriological methods employ antibiotics which favor the growth of one bacterial component over others in a mixed population. For in vitro studies examining multiple species, for example, in dual biofilm models, it is difficult to successfully separate M. abscessus from nontuberculous mycobacterial (NTM) species. Therefore, it was the aim of this study to develop a selective agar medium that was able to isolate M. abscessus from a pool of other highly-resistant Gram-negative organisms, which would be useful to microbiologists performing co-culture experiments and which require re-isolation of the NTM organism. Methods: Wylie-Stanley agar (WSA) was developed consisting of glucose, 16 g/l; yeast extract, 30 g/l; peptone, 6.8 g/l; and agar, 20 g/l along with selective supplements including chloramphenicol, 50 mg/l; ceftazidime, 32 mg/l; colistin, 24 mg/l; trimethoprim, 21.3 mg/l; sulfamethoxazole, 106.7 mg/l; and novobiocin, 50 mg/l. This medium was (i) challenged with 10 non-NTM species (27 isolates) of common Gram-negative and Gram-positive organisms associated with CF and (ii) compared to Columbia Blood Agar and Middlebrook 7H10 Agar for the isolation of M. abscessus organisms from mixed cultures of NTM organisms and Pseudomonas aeruginosa and Stenotrophomonas maltophilia. Results: This medium was highly specific for the growth of M. abscessus organisms and grew all NTM organisms. WSA medium did not allow the growth of any of the non-NTM species. When mixed cultures of M. abscessus species and P. aeruginosa and S. maltophilia were inoculated onto WSA medium, only the NTM organism could be grown successfully, highlighting the specificity of this medium. In contrast, both Columbia Blood Agar and Middlebrook 7H10 Agar allowed the growth of both NTM and non-NTM organisms. Conclusion: While the specificity was high, the sensitivity of WSA was low, and therefore, we do not advocate employment of WSA medium for the primary isolation of M. abscessus organisms from CF sputum, rather for the purposes of separating M. abscessus populations of organisms from other highly-resistant organisms, including P. aeruginosa and S. maltophilia, which would be useful to microbiologists performing co-culture experiments and which require re-isolation of the pure M. abscessus organism.

Hospital ice, ice machines, and water as sources of nontuberculous mycobacteria: Description of qualitative risk assessment models to determine host-Nontuberculous mycobacteria interplay.

Over the last 30 years, there have been at least 17 published reports of nontuberculous mycobacteria (NTMs) being isolated from hospital ice or ice-making machines. Of these, 12 were reports of pseudo-outbreaks, i.e., the nosocomial transmission of organism from hospital ice/ice machines to patients, resulting in patient colonization, but with no disease manifestations. In addition, there were five outbreaks that resulted in clinical disease/pathology associated with NTM organism. Eleven different species of NTMs have been associated with these reports, where over half (59%) of the species identified were Mycobacterium fortuitum (18%), Mycobacterium gordonae (14%), Mycobacterium mucogenicum (14%), and Mycobacterium porcinum (14%). Several of these reports clearly documented that ice machines had been properly maintained, cleaned, and serviced in accordance with the CDC guidelines yet became contaminated with NTM organisms. These reports frequently detail that after extensive cleaning/disinfection following the discovery of NTM organisms, ice machines remained contaminated with NTM organisms, highlighting the difficulty in eradicating these from ice machines, once contaminated. Several reports identified that the only remedy to the contamination problem was to replace the ice machine with a new machine. Two qualitative risk assessment models are presented for (i) patients exposed to contaminated ice machine but before NTM colonization/infection and (ii) patients already colonized with NTMs from ice machines. Therefore, to protect immunocompromised/immunosuppressed patients' safety, especially during surgical or respiratory procedures, ice should not be sourced from the ice machine but should be made from sterile water and stored safely and separately away from the ice machine.

Comparison of four agar media for the enumeration of the Mycobacterium abscessus complex.

Background: Traditional culture of nontuberculous mycobacteria (NTMs) has involved egg-based formulations (Lowenstein-Jensen medium) or defined media (Middlebrook formulations), which have disadvantages of composition complexity, availability, and cost. This study quantitatively compared three non-selective, non-blood based basal agars with Columbia blood agar (CBA), to enumerate Mycobacterium abscessus complex organisms in pure culture. Methods: M. abscessus subsp. massiliense, M. abscessus subsp. bolletii, and M. abscessus subsp. abscessus were employed. Inocula of each of these were counted on three basal agar media, including (i) standard plate count agar (SPCA), (ii) tryptone soya agar (TSA), and (iii) Mueller-Hinton agar (MHA) and compared to counts on CBA. Results: All NTM isolates of all subspecies grew successfully on all four media examined. The growth was most profuse on SPCA, with a mean colony diameter of 3 mm, whereas the mean colony diameter on all other media was 1 mm. Statistically, there was no significant difference in counts when comparing CBA with SPCA or MHA (P > 0.05), whereas there was a statistically significant difference between CBA and TSA (P = 0.01). There was no statistically significant difference between SPCA and MHA (P = 0.53). Conclusion: This study indicates that SPCA and MHA are equally effective as CBA, when enumerating of M. abscessus complex organisms. Employment of TSA gave significantly lower counts than CBA (P = 0.01) and therefore should not be employed when enumerating these organisms. SPCA yielded the most profuse growth of all media examined. In addition to these advantages, given that SPCA does (i) not require blood as a medium constituent, (ii) is simple to reconstitute, (iii) is relatively cheap, and (iv) is widely available commercially, this study endorses employment of SPCA for the nonselective culture of M. abscessus complex organisms, including enumeration.

Susceptibility of the Mycobacterium abscessus complex to drying: Implications for nebulizer hygiene in patients with cystic fibrosis.

Background: Nebulizer hygiene and care is important in cystic fibrosis (CF) to minimize device contamination from bacteria, including nontuberculous mycobacteria (NTMs). Most nebulizer manufacturers recommend nebulizer drying, however there is little evidence to understand how nebulizer drying affects NTM survival. Methods: Mycobacterium abscessus subsp. massiliense (n = 2), M. abscessus subsp. bolletii (n = 2), and M. abscessus subsp. abscessus (n = 2) were evaluated for their ability to survive simulated drying conditions associated with routine nebulizer care. Bacterial inocula (circa. 107 colony-forming units) were added to plastic and allowed to dry to completeness for 24 h, employing passive and active drying. Results: NTM isolates of all subspecies could be recovered from all passive and active drying experiments, both in diluent and in sterile sputum, following drying (24 h). There was no combination of drying or physiology that supported NTM cell death, and there was no difference in observed survival with the three species of M. abscessus examined. Conclusion: This study indicates that drying, either passively or actively, for 24 h at room temperature, is unable to eradicate all M. abscessus organisms from dry plastic surfaces, even in the presence of residual sputum contamination. Whilst drying may be advantageous for nebulizer performance, it should not be regarded as an absolute control for the elimination of NTM organisms. With nebulizer hygiene, NTM organisms would be able to survive on a nebulizer following drying for 24 h, which has not undergone any formal disinfection protocol. Therefore, for NTM eradication from washed nebulizers, CF patients should therefore seek an effective alternative control to drying for NTM eradication, i.e., heat disinfection in baby bottle disinfectors. CF patients and health-care professionals should not rely solely on nebulizer drying to achieve NTM eradication.

The Role of 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in the Diagnosis of Left-sided Endocarditis: Native vs Prosthetic Valves Endocarditis.

BACKGROUND: 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) has emerged as a useful diagnostic tool for suspected infective endocarditis (IE) in patients with prosthetic valves or implantable devices. However, there is limited evidence regarding use of 18F-FDG-PET/CT for the diagnosis of native valve endocarditis (NVE). METHODS: Between 2014 and 2017, 303 episodes of left-sided suspected IE (188 prosthetic valves/ascending aortic prosthesis and 115 native valves) were studied. 18F-FDG-PET/CT accuracy was determined in the subgroups of patients with NVE and prosthetic valve endocarditis (PVE)/ascending aortic prosthesis infection (AAPI). Associations between inflammatory infiltrate patterns and 18F-FDG-PET/CT uptake were investigated in an exploratory ad hoc histological analysis. RESULTS: Among 188 patients with PVE/AAPI, the sensitivity, specificity, and positive and negative predictive values of 18F-FDG-PET/CT focal uptake were 93%, 90%, 89%, and 94%, respectively, while among 115 patients with NVE, the corresponding values were 22%, 100%, 100%, and 66%. The inclusion of abnormal 18F-FDG cardiac uptake as a major criterion at admission enabled a recategorization of 76% (47/62) of PVE/AAPI cases initially classified as "possible" to "definite" IE. In the histopathological analysis, a predominance of polymorphonuclear cell inflammatory infiltrate and a reduced extent of fibrosis were observed in the PVE group only. CONCLUSIONS: Use of 18F-FDG-PET/CT at the initial presentation of patients with suspected PVE increases the diagnostic capability of the modified Duke criteria. In patients who present with suspected NVE, the use of 18F-FDG-PET/CT is less accurate and could only be considered a complementary diagnostic tool for a specific population of patients with NVE.

Antimycobacterial strategies to evade antimicrobial resistance in the nontuberculous mycobacteria.

The nontuberculous mycobacteria (NTM) have recently emerged as important bacterial pathogens of both animals and humans. Of particular, concern is the high level of antimicrobial resistance (AMR) displayed by these organisms, which complicates treatment and potential successful outcomes. This review, therefore, wishes to examine novel compounds and approaches to combatting AMR in the NTMs, specifically examining antimycobacterial (NTM) compounds from plants and venoms, as well as examining synergistic and combination effects with other antimicrobials. Novel and modified drugs including new inhaled drugs are examined, as well as the repurposing of existing drugs for antimycobacterial activity. Many of these novel interventions are at various stages of development, from initial concept through to licensed intervention. The challenge remains to translate these interventions from in vitro laboratory models to effective in vivo interactions. When these are realized, then we will have the opportunity of overcoming NTM AMR, to the benefit of medicine, society, and humanity.

PET/Computed Tomography Evaluation of Infection of the Heart.

The 2015 European Society of Cardiology guidelines for the management of infective endocarditis included 18F-fluorodeoxyglucose (18F-FDG) PET/computed tomography (CT) in the diagnostic work-up of prosthetic valve endocarditis. This article examines the literature from the last 3 years to highlight the additional role 18F-FDG-PET/CT can contribute to an accurate diagnosis of cardiac infections and associated infectious complications. The challenges and pitfalls associated with 18F-FDG-PET/CT in such clinical settings must be recognized and these are discussed along with the suggested protocols that may be incorporated in an attempt to address these issues.

Molecular diagnostics of medically important bacterial infections.

Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world's biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. In developed countries, the emergence of new, rare or already-forgotten infectious diseases, such as HIV/AIDS, Lyme disease and tuberculosis, has stimulated public interest and inspired commitments to surveillance and control. Recently, it is reported that infectious diseases are responsible for more than 17 million deaths worldwide each year, most of which are associated with bacterial infections. Hence, the control of infectious diseases control is still an important task in the world. The ability to control such bacterial infections is largely dependent on the ability to detect these aetiological agents in the clinical microbiology laboratory. Diagnostic medical bacteriology consists of two main components namely identification and typing. Molecular biology has the potential to revolutionise the way in which diagnostic tests are delivered in order to optimise care of the infected patient, whether they occur in hospital or in the community. Since the discovery of PCR in the late 1980s, there has been an enormous amount of research performed which has enabled the introduction of molecular tests to several areas of routine clinical microbiology. Molecular biology techniques continue to evolve rapidly, so it has been problematic for many laboratories to decide upon which test to introduce before that technology becomes outdated. However the vast majority of diagnostic clinical bacteriology laboratories do not currently employ any form of molecular diagnostics but the use such technology is becoming more widespread in both specialized regional laboratories as well as in national reference laboratories. Presently molecular biology offers a wide repertoire of techniques and permutations of these analytical tools, hence this article wishes to explore the application of these in the diagnostic laboratory setting.