Enhanced mycobacterial diagnostics in liquid medium by microaerobicbubble flow in Portable Microbe Enrichment Unit
Elias Hakalehto ∗Institute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
Received 3 June 2012; received in revised form 23 June 2013; accepted 24 June 2013
bstract
Portable Microbe Enrichment Unit (PMEU) method with microaerobic bubbling speeded up the growth of otherwise slowly starting andropagating Mycobacterium sp. Mycobacterium fortuitum growth was detected after 10–11 h and Mycobacterium marinum produced clearrowth in 4 days. A mycobacterial environmental isolate was verified in 2 days in the PMEU Spectrion® equipped with infrared sensors. Inarallel static (without gas bubbling) cultures hardly any growth occurred. In conclusion, PMEU technology provided thus a rapid detection of
nvironmental and clinical mycobacterial isolates. It would also help in the field diagnosis of antibiotic resistant Mycobacterium tuberculosis.
2013 Published by Elsevier Ireland Ltd.
eywords: Mycobacteria; Infection; Early diagnosis; Tuberculosis; PMEU; Rapid microbiology; MDR; XDR; Antibiotic resistance; Field microbiology
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. Introduction
Mycobacteria grow slowly in laboratory and also in vivo.ractically all of them are either pathogens or opportunis-
ic pathogens. In global perspective, about 1.4 M lives areost annually due to Mycobacterium tuberculosis infec-ions [1]. In a multi-national study in Estonia, Latvia,eru, Philippines, Russia, South Africa, South Korea, andhailand, on average of 6.7% of patients had XDR (exten-ively drug-resistant) tuberculosis [2]. The range betweentudy sites was 0.8–15.2% and 43.7% of the patients had
strain resistant to at least one of the second-line antibi-tics. Clinical confirmation of tuberculosis with antibioticesistance analysis usually takes several weeks and delayshe treatment as well precaution actions [1]. Among newiseases, 4% is caused by a multi-resistant strain (MDR).
M. tuberculosis infections are often associated with lungs,ut different mycobacteria cause diseases in different tis-
Please cite this article in press as: E. Hakalehto, Enhanced mycobacterPortable Microbe Enrichment Unit, Pathophysiology (2013), http://dx.d
ues. Both Mycobacterium fortuitum and M. tuberculosis, forxample, can also turn into intracellular pathogens [3].
PMEU technologies with gas bubble flow comprise ofifferent techniques to monitor hygiene in water and otheramples. Enhanced microbiological cultivation and detectionethods have been developed for clinical, industrial and envi-
onmental samples [4,5]. The essential point is the optiono boost the microbes with gas bubble flow [6,7]. Varioustandard media can be used in cultivations. The optimationf gas bubbling in PMEU has improved the recovery ratef the enterobacterial isolates about 2.5 times when com-ared to the standard cultivation without PMEU acceleration8].
Regarding the field monitoring of tuberculosis, Kim-rough et al. [9] stated: “Findings suggest the need for earlystablishment of the tuberculosis services, especially in dis-laced populations from high-burden areas and for continuednnovation and prioritisation of tuberculosis control in crisisetting”.
The aim of this study was to test if the PMEU methodnd especially its gas flow could enhance the cultivation ofycobacterium sp. As the device is portable these results
ould also be exploited in field conditions in early detection
ial diagnostics in liquid medium by microaerobic bubble flow inoi.org/10.1016/j.pathophys.2013.08.005
f M. tuberculosis infection and epidemics, and also otherathogenic or opportunistically pathogenic mycobacteria.heir cultivation is at present still time consuming and forms
Fig. 1. Growth curves on M. fortuitum from PMEU Spectrion® analysis.Tbl
UStms6
sPMEU Spectrion® equipment has been validated in the
Finnish State Research Centre (VTT) for the analysis ofhygiene indicating coliforms [12]. An automated sampling
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E. Hakalehto / Pathop
he bottle-neck in overall diagnosis of these diseases andampers the control of the epidemics.
. Materials and methods
.1. Bacterial strains and their cultivation
M. tuberculosis is a hazardous study organism. Therefore,. fortuitum (ATCC 6841) and Mycobacterium marinum
ATCC 927) were used in this study. They were obtainedrom the American Type Culture Collection. Our environ-ental isolate E40 (from river water) was derived from theinnish National Institute of Health and Welfare.
The cultivation took place at 33–35 ◦C in PMEUpectrion® with diminished oxygen concentration (5%) [10]sing M7H9 broth with Middlebrook ADC Enrichment sup-lement (Beckton Dickinson, USA) and used according tohe instructions of the manufacturer. Parts of the PMEU cul-ivation syringes were bubbled with gas flow, while othersemained stagnant.
Plate cultures were made on M7H9 broth medium supple-ented with 1.25% agar and ChromAgarTM.
. Results and discussion
Enrichment cultivation is crucial in mycobacterial diag-osis. It was remarkably speeded up in case of M. fortuitumsing PMEU Spectrion®, which is an easily portable culti-ation and detection unit. When a heavy inoculum of about05–106 cells was used for a broth culture, the onset of growthas recorded within 10–11 h (Fig. 1).As gas bubbling has been previously shown to promote
he onset of growth in the PMEU [6,7]. The gas mixture wasbtained from the pressurized bottles and contained 5% O2,0% CO2, and 85% N2, corresponding to the atmospheren campylobacterial cultivation in the PMEU [10]. In static
ycobacterial reference cultures, hardly any growth occurredn five days. For the colony counts from the static and PMEUultures, see Table 1.
Strain of Mycobacterium sp., isolated from river water ininland by the National Institute of Health and Welfare, waslso tested in the PMEU Spectrion®. The growth started inbout 2 days (Fig. 2).
Purity of mycobacterial cultures was tested on Petri dishesFig. 3A and B) and by microscopical monitoring (Fig. 4).inal cell concentrations of Mycobacterium sp. and M. mar-
num after 3 and 5 days, respectively, are presented in Table 2.he PMEU technique turned out to be a potential approach ineveloping fast methods to monitor clinical and environmen-al mycobacteria. It can also be developed for the diagnostics
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f M. tuberculosis in the laboratory and in the field.Antibiotic resistance monitoring with PMEU Spectrion®
as been carried out e.g. with Enterobacter cloaceae hospitaltrain No. 165/08 obtained from the Neonatal Intensive Care
FS2
he culture with gas bubble flow above (A), with static reference cultureelow (B). In the former one A case, the onset of growth was recorded iness than 12 h.
nit of Kuopio University Hospital (a gift from Drs. Ullaankilampi and Jouni Pesola) [11]. Corresponding to the cul-
ivation of different mycobacteria, the bacterial growth wasonitored by the IR sensors also in this case. This strain was
hown to be resistant to penicillin G and cefuroxime in 5 and h, respectively.
Versions of the PMEU could be applied widely for theensitive detection of pathogenic bacteria [5].
ial diagnostics in liquid medium by microaerobic bubble flow inoi.org/10.1016/j.pathophys.2013.08.005
ig. 2. Development of a Mycobacterium sp. culture during 6 days of PMEUpectrion® cultivation. The early signs of increasing density were seen after4 h, and clear onset of growth occurred in 48 h.
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ARTICLE IN PRESSPATPHY-780; No. of Pages 4
E. Hakalehto / Pathophysiology xxx (2013) xxx–xxx 3
Fig. 3. (A and B) Close images of the mycobacterial colonies. On the left growth of Mycobacterium sp. E40 isolated from river water on CromAgarTM medium.On the right a colony of M. marinum strain ATCC 927 grown on M7H9 agar (1.25%).
Table 1Bacterial densities in the PMEU. The colony growth was followed in fivegas bubbling PMEU syringe cultures (No. 1–5) and four static cultures (No.6–9) of M. fortuitum. The final cell yields were measured in colonies on theM7H9 agar medium. Dilutions −5 (1:100,000) and −7 (1:10,000,000) of theparallel PMEU cultures were used in inoculations. Resulting average con-centration for M. fortuitum in the PMEU gas flow was calculated as 740 M,and for the static cultures 24 M, after the period of five days of cultivation.Bacterial concentrations indicated by Colony Forming Units (CFU’s).
Culture no −5 M7H9/ChromAgar −7 M7H9
1 882 613 274 1175 766 2107 1988 3839 171
Fig. 4. Dark field image of M. marinum ATCC 927 cells photographed byNikon Eclipse E3 microscope (1000×). Capsular structures were visiblearound some cells.
Table 2Growth yields of two mycobacterial strains in microaerobic PMEU cultureswith and without bubble flow. CFU = Colony Forming Units. The bacterialconcentrations are calculated from CFU’s (Colony Forming Units).
Bacterial concentrationafter 3 days cultivation inthe PMEU
ystem has been designed e.g. for water departments [13].ll PMEU versions can be connected to the internet, or used
lso by mobile phone systems to follow up the growth. Furthertudies regarding the applications are warranted.
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