Critical care ORIGINAL ARTICLE Thorax: first published as 10.1136/thoraxjnl-2015-208050 on 12 April 2016. Downloaded from Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia 1 2 3 2 4 5 Open Access T J Nolan, N J Gadsby, T P Hellyer, K E Templeton, R McMullan, J P McKenna, Scan to access more 1 1 1 1 1,6 3 free content J Rennie, C T Robb, T S Walsh, A G Rossi, A Conway Morris, A J Simpson ▸ Additional material is ABSTRACT published online only. To view Background Ventilator-acquired pneumonia (VAP) Key messages this file please visit the journal fi online (http://dx.doi.org/10. remains a signi cant problem within intensive care units 1136/thoraxjnl-2015-208050) (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the What is the key question? pathogenesis of VAP, but the mechanisms remain ▸ 1 fl What is the prevalence of Mycoplasmataceae MRC Centre for In ammation incompletely understood. We hypothesised that, because Research, University of among patients with ventilator-acquired Edinburgh, Edinburgh, UK of limitations in their routine detection, pneumonia (VAP), and does this have any 2Clinical Microbiology, NHS Mycoplasmataceae are more prevalent among patients pathophysiological relevance? Lothian, Edinburgh, UK with VAP than previously recognised, and that these 3 Institute of Cellular Medicine, organisms potentially impair immune cell function. What is the bottom line? Newcastle University, Methods and setting 159 patients were recruited from ▸ Patients with VAP have a high prevalence of Newcastle, UK Mycoplasmataceae compared with similar 4Centre for Infection and 12 UK ICUs. All patients had suspected VAP and underwent Immunity, Queen’s University, bronchoscopy and bronchoalveolar lavage (BAL). VAP was patients without VAP, and the most common Belfast, UK fi 4 species found can impair immune cell function. 5 de ned as growth of organisms at >10 colony forming Department of Microbiology, units per ml of BAL fluid on conventional culture. Samples Belfast Health & Social Care Why read on? Trust, Belfast, UK were tested for Mycoplasmataceae (Mycoplasma and ▸ These findings provide a novel insight into the 6 Division of Anaesthesia, Ureaplasma spp.) by PCR, and positive samples underwent biology of VAP and suggest new potential Department of Medicine, sequencing for speciation. 36 healthy donors underwent prevention strategies. University of Cambridge, BAL for comparison. Additionally, healthy donor monocytes Cambridge, UK and macrophages were exposed to Mycoplasma salivarium http://thorax.bmj.com/ Correspondence to and their ability to respond to lipopolysaccharide and Dr A Conway Morris, Division undertake phagocytosis was assessed. patients admitted to the intensive care unit (ICU).1 of Anaesthesia, University of Results Mycoplasmataceae were found in 49% (95% CI There is a growing recognition of failure of Cambridge, Box 93, 33% to 65%) of patients with VAP, compared with 14% Addenbrooke’s Hospital, Hills immune cell function (immunoparesis) in the lung2 (95% CI 9% to 25%) of patients without VAP. Patients Road, Cambridge CB2 2QQ, and peripherally34in the pathogenesis of VAP. The with sterile BAL fluid had a similar prevalence to healthy UK; [email protected] mediators of this immunoparesis remain incom- donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% pletely understood, although it is likely that it on September 27, 2021 by guest. Protected copyright. TJN and NJG contributed CI 2% to 22%)). The most common organism identified equally. arises from multifactorial insults involving both was M. salivarium. Blood monocytes from healthy ACM and AJS indicate joint host and microbial factors. It is now widely recog- volunteers incubated with M. salivarium displayed an senior authorship of this work. nised that the predominant route for infection is impaired TNF-α response to lipopolysaccharide (p=0.0003), via ‘microaspiration’ of organisms from the hypo- Received 10 November 2015 as did monocyte-derived macrophages (MDMs) (p=0.024). pharynx, allowing colonisation and subsequent Revised 12 January 2016 MDM exposed to M. salivarium demonstrated impaired Accepted 4 February 2016 infection of the lower airways.1 phagocytosis (p=0.005). Published Online First The organisms which cause VAP have tradition- Discussion and conclusions This study demonstrates 12 April 2016 ally been thought to be conventional bacterial a high prevalence of Mycoplasmataceae among patients species, such as Staphylococcus aureus and with VAP, with a markedly lower prevalence among patients Pseudomonas aeruginosa.5 However our under- with suspected VAP in whom subsequent cultures refuted standing of the microbiology of VAP is heavily the diagnosis. The most common organism found, M. influenced by the relative ease of microbial culture. salivarium, is able to alter the functions of key immune In recent years there has been a greater understand- cells. Mycoplasmataceae may contribute to VAP ing of the human microbiome and the influence of pathogenesis. bacteria that are not cultured.6 Recent reports have suggested that non-classical organisms, such as Mycoplasma spp., may be present in patients with VA P. 78The methods of detection used in these To cite: Nolan TJ, INTRODUCTION studies have been variable and there was little evi- Gadsby NJ, Hellyer TP, et al. Ventilator-acquired pneumonia (VAP) remains a sig- dence of the impact these species may have on host – Thorax 2016;71:594 600. nificant cause of morbidity and mortality in immunity. 594 Nolan TJ, et al. Thorax 2016;71:594–600. doi:10.1136/thoraxjnl-2015-208050 Critical care Mycoplasmataceae are members of the mollicutes class of bac- of consumables in one or other laboratory. Further confirmation Thorax: first published as 10.1136/thoraxjnl-2015-208050 on 12 April 2016. Downloaded from teria, which lack a cell wall and have the smallest genomes found came by running the PCR on saline passed through the same in self-replicating organisms.9 Their limited biosynthetic capabil- extraction columns but without contact with the patient or vol- ity makes culture and detection problematic, needing specialised unteer BAL fluid samples. growth media and prolonged incubation or reliance on indirect PCR for Mycoplasma spp. was based on the method of van methods such as serology.9 A range of Mycoplasmataceae such as Kuppeveld et al15 targeting the 16S rRNA gene using forward Mycoplasma salivarium and M. orale are generally considered to primer general prokaryotic oligonucleotides-1 (GPO-1) and be human commensals,10 while others such as M. pneumoniae reverse primer mycoplasma genus-specific oligonucleotides and Ureaplasma urealyticum are recognised pathogens of the (MGSO) and gel electrophoresis for detection. Positive extracts respiratory and genitourinary tract, respectively. However, even were sequenced on the reverse strand to determine Mycoplasma the Mycoplasma spp. commonly thought to be commensals have spp. using the QIAquick PCR purification kit (Qiagen) for puri- been shown to cause infections in immunocompromised hosts.11 fication, and ABI Prism BigDye Terminator and the ABI 3730 Additionally, co-infection with Mycoplasma spp. has been impli- instrument (Applied Biosystems, Life Technologies, Paisley, UK) cated in accelerated progression of HIV/AIDS.12 for Sanger sequencing. Ureaplasma spp. real-time PCR was We hypothesised that patients with confirmed VAP would based on an inhouse unpublished method targeting the urease have a higher prevalence of Mycoplasmataceae than patients gene using the following oligonucleotides; UreF: ACG WCG without VAP,and that the dominant organism found (M. salivar- TTT CGA TAT TCC AT, UreR: TTC CRT TAA CTA AGC CRT ium) would impair the ability of monocytes and macrophages to TT and UreP: 60FAM—TCG TTT TGA ACC AGG AGA YAA respond to further bacterial stimuli. Accordingly, this study had AA—BHQ1. Reactions comprised HotStarTaq (Qiagen) two aims; first, to quantify the prevalence of Mycoplasmataceae reagents, 0.5 μM primer Ure-F, 0.65 μM primer UreR, 0.2 μM in two cohorts of patients with suspected VAP and determine probe UreP and 10 μL nucleic acid extract to a final volume of which species were present. The second aim was to examine the 25 μL. Real-time PCR was carried out on the ABI 7500 instru- effect of a classically non-pathogenic Mycoplasma, M. salivar- ment (Applied Biosystems). Ureaplasma spp. positive extracts ium, on the ability of monocytes and macrophages to respond were speciated using the method of Kong et al16 targeting the 50 to pathogenic stimuli. end of the MBA gene with primers UMS-57c and UMA222 for U. parvum and UMS-170c and UMA263 for U. urealyticum, METHODS followed by gel-based detection. Patients and volunteers We constructed the subject cohorts for this study from clinical Human inflammatory cell/Mycoplasma interactions samples and data from two recent patient studies and a healthy Human mononuclear cells were obtained from whole blood volunteer cohort.13 14 The first patient cohort was recruited from healthy donors using previously described methods.17 18 from two general, teaching hospital ICUs from 2005 to 2009. Briefly, leucocytes were obtained by dextran sedimentation, with Patients were included if they fulfilled clinical criteria for sus- separation of mononuclear and granulocytic cells over Percoll pected VAP, that is, mechanical ventilation for >48 h, new infil- gradients. CD14+ve monocytes were extracted from the mono- trates on chest radiograph, and at least two of the following: nuclear fraction using a magnetic bead system (MACS, Miltenyi http://thorax.bmj.com/ temperature >38°C, leucocyte count >11×109 per litre of per- Biotec, Bisley, UK), and >95% purity confirmed by flow cyto- ipheral blood or purulent tracheal secretions. metry. Monocytes were either used fresh at 300 000 per well, The second patient cohort was recruited from 12 ICUs across or matured into monocyte-derived macrophages (MDMs). the UK from 2012 to 2013, and met similar enrolment criteria MDMs were produced by adhering 300 000 monocytes to to those in patient cohort 1.