New method for fully automated determination of the concentration of legionella in a water sample within a few hours

The hygien­ic neces­si­ty to con­trol the con­cen­tra­tion of legionel­la in tech­ni­cal water sys­tems from which aerosols can be dis­charged leads to the prob­lem that the cul­ti­va­tion method (ISO 11731–2017) used for this pur­pose only pro­vides reli­able results after a delay of 7–12 days. On this basis, nec­es­sary mea­sures can only be tak­en and con­trolled with a con­sid­er­able time delay. Rapid tests cur­rent­ly avail­able on the mar­ket either do not cor­re­late reli­ably with the accred­it­ed cul­ti­va­tion method or require (time-) con­sum­ing prepa­ra­tion steps. Some rapid tests pro­vide high­ly spe­cif­ic detec­tion for sin­gle Legionel­la species, but not for all Legionel­la species in a water sam­ple (Legionel­la spp. = species plu­ralis). The new­ly devel­oped mea­sur­ing device INWATROL L.nella+ from Inwa­tec is based on the method of mea­sur­ing the meta­bol­ic activ­i­ty of liv­ing cells and reli­ably deter­mines the para­me­ter Legionel­la spp. from a water sam­ple with­in a few hours. The mea­sur­ing device is direct­ly con­nect­ed to the tech­ni­cal water sys­tem with auto­mat­ic and self-dis­in­fect­ing sam­ple feed, includ­ing self-dis­in­fec­tion of the water con­tained in the mea­sur­ing cell after the mea­sure­ment is com­plet­ed. This enables the plant oper­a­tor to deter­mine the hygien­ic water qual­i­ty con­tin­u­ous­ly and safe­ly. In addi­tion to the direct con­trol of the suc­cess of the mea­sures car­ried out, it is also pos­si­ble to con­trol e.g. bio­cides accord­ing to requirements.

1. Intro­duc­tion
The hygien­ic rel­e­vance of the spread of path­o­gen­ic Legionel­la via aerosols from tech­ni­cal water sys­tems such as evap­o­ra­tive cool­ing sys­tems and cool­ing tow­ers has led to the cre­ation of tech­ni­cal hygiene guide­lines in many coun­tries. In Ger­many, VDI 2047 part 2 and 3, gen­er­al­ly accept­ed tech­ni­cal rules for ensur­ing the hygien­ic oper­a­tion of evap­o­ra­tive cool­ing sys­tems and cool­ing tow­ers came into force for the first time in 2015. In addi­tion, in many coun­tries the tol­er­a­ble con­cen­tra­tion of legionel­la in the cir­cu­la­tion water of the respec­tive plants is lim­it­ed by the leg­is­la­tor. In Ger­many, the forty-sec­ond ordi­nance for the imple­men­ta­tion of the Fed­er­al Immis­sion Con­trol Act (Ordi­nance on Evap­o­ra­tive Cool­ing Sys­tems, Cool­ing Tow­ers and Wet Sep­a­ra­tors — 42nd BIm­SchV) came into force on 19.08.2017, which also includes wet sep­a­ra­tors. So far, the basis for hygiene con­trol has always been the deter­mi­na­tion of the con­cen­tra­tion of legionel­la in the water by cul­ti­va­tion accord­ing to ISO 11731:2017 with sys­tem-depen­dent thresh­old val­ues. In this cul­ti­va­tion method, cell divi­sion pro­duces vis­i­ble and there­fore count­able colonies. In com­par­i­son to oth­er bac­te­r­i­al species, Legionel­la bac­te­ria divide rel­a­tive­ly slow­ly, so that the results of the mea­sure­ment are only avail­able after 7–12 days, where­by in some cas­es fur­ther inves­ti­ga­tions to con­firm sus­pi­cious colonies follow.

For the oper­a­tor of a plant with mon­i­tor­ing oblig­a­tion, this means a strong­ly delayed con­trol of the hygiene sta­tus. Fur­ther­more, the effi­cien­cy of any nec­es­sary mea­sures can only be deter­mined with a long delay. Addi­tion­al rapid tests for esti­mat­ing the con­t­a­m­i­na­tion of water with Legionel­la are avail­able, e.g. based on immuno­log­i­cal reac­tions (anti­body reac­tion), detec­tion of genet­ic mate­r­i­al (PCR) or by means of col­or flu­o­res­cence microscopy. The lim­i­ta­tions of these rapid tests are the live/dead quan­tifi­ca­tion, the com­pa­ra­bil­i­ty to the cul­ture method or the com­plex sam­ple preparation.
The new­ly devel­oped and patent­ed auto­mat­ic mea­sur­ing device INWATROL L.nella+ allows the reli­able and con­tin­u­ous deter­mi­na­tion of the para­me­ter Legionel­la species plu­ralis with high cor­re­la­tion to the cul­ti­va­tion method accord­ing to ISO 11731:2017 with­in a few hours with­out fur­ther prepa­ra­tion steps by the user.

2. Rapid test for the ful­ly auto­mat­ed deter­mi­na­tion of Legionel­la species pluralis 

2.1 Mea­sur­ing principle
The detec­tion of meta­bol­i­cal­ly active Legionel­la bac­te­ria is based on a non-spe­cif­ic enzy­mat­ic con­ver­sion of a non-polar flu­o­res­cein acid ester, which only pass­es through the cell mem­brane of liv­ing cells into the cell inte­ri­or where it is con­vert­ed into col­or-active flu­o­res­cein. The increase in flu­o­res­cence as a func­tion of time is direct­ly pro­por­tion­al to the num­ber of liv­ing cells and is con­vert­ed into colony form­ing units per 100 ml. Due to a com­bined heat and pH pre­treat­ment and the high mea­sur­ing tem­per­a­ture com­pared to the cul­ti­va­tion method, the accom­pa­ny­ing flo­ra is killed. The mea­sure­ment is per­formed undi­lut­ed in a sam­ple vol­ume of approx. 350 ml. In com­par­i­son to the cul­ti­va­tion method, the mea­sure­ment is not sig­nif­i­cant­ly influ­enced by accom­pa­ny­ing flo­ra and a high mea­sur­ing inac­cu­ra­cy due to a high dilution.

2.2 Con­tin­u­ous, auto­mat­ed mea­surement
For con­tin­u­ous mea­sure­ments, the mea­sur­ing device is direct­ly con­nect­ed to a water sys­tem. A ther­mal­ly self-dis­in­fect­ing sam­pling line ensures that no repro­duc­tion of legionel­la in the sup­ply line affects the mea­sure­ment result. Ide­al­ly, the sam­pling tap is in con­tin­u­ous oper­a­tion to exclude stag­na­tion of water between two mea­sure­ments. The mea­sur­ing cell in the device is rinsed sev­er­al times dur­ing fill­ing. After the rins­ing process is com­plet­ed, the com­bined heat and pH pre­treat­ment starts. When the pre-treat­ment is com­plet­ed, the mea­sur­ing cell cools down to the mea­sur­ing tem­per­a­ture and the mea­sure­ment begins. The mea­sur­ing cell is ther­mal­ly dis­in­fect­ed before the device is filled again for the fol­low-up exam­i­na­tion. The mea­sur­ing cell is ready for the next mea­sure­ment. Usu­al­ly, a sam­pling tap is installed direct­ly at the sam­pling point before the sam­pling line. This tap can be used to take micro­bi­o­log­i­cal sam­ples at the time of fill­ing the mea­sur­ing cell or at any oth­er times, e.g. for fur­ther val­i­da­tion measurements.

2.3 Auto­mat­ed mea­sure­ment of man­u­al­ly loaded samples
The con­tin­u­ous mea­sur­ing oper­a­tion can be inter­rupt­ed for man­u­al feed­ing of fur­ther water sam­ples via the fill­ing fun­nel. For clean­ing, rins­ing and fill­ing the mea­sur­ing cell, only the valve posi­tion on the device has to be changed. When the fill­ing is com­plet­ed, the valve posi­tion is returned to its orig­i­nal posi­tion and the mea­sur­ing device switch­es back to auto­mat­ic mode when the mea­sure­ment is com­plet­ed. The mea­sur­ing pro­ce­dure itself does not dif­fer from the auto­mat­ic mode.

2.4 Cul­ti­va­tion accord­ing to ISO 11731:2017/ UBA1 
The cul­ti­va­tion method uses sev­er­al approach­es with dif­fer­ent dilu­tion and pre­treat­ment stages (heat or acid). The aim is to obtain evalu­able results for both low and high lev­els of Legionel­la. For the result, the prepa­ra­tion with the high­est num­ber of con­firmed Legionel­la colonies is used (if the mea­sure­ment accuracy/number of colonies is suf­fi­cient­ly high). The lim­its of the accu­ra­cy of the cul­ti­va­tion method are main­ly due to the pos­si­ble influ­ence of the accom­pa­ny­ing flo­ra, i.e. oth­er microor­gan­isms which can sup­press the growth of the legionel­la or over­grow their colonies. Fur­ther­more, bac­te­ria are par­ti­cles in a water sam­ple and are not homo­ge­neous­ly dis­trib­uted. There­fore, when tak­ing small vol­umes from the sam­ple bot­tle, inac­cu­ra­cies may occur due to the some­times high dilu­tion fac­tors. Dur­ing cul­ti­va­tion, liv­ing but non-cul­tivable cells in the so-called VBNC2 sta­tus are not detect­ed. Many Legionel­la from a coher­ent agglom­er­ate, e.g. by prop­a­ga­tion with­in an amoe­ba, are only vis­i­ble and eval­u­at­ed as one colony dur­ing cul­ti­va­tion (see Lind­ner, Hahn: Micro­bi­o­log­i­cal analy­ses of the cool­ing water accord­ing to the 42nd BIm­SchV, p. 74, VGB Pow­erTech 9, 2018).

3. Exam­ples of appli­ca­tion and cor­re­la­tion to the cul­ture method

The INWATROL L.nella+ is being used in var­i­ous prac­ti­cal appli­ca­tions. Case stud­ies include the oper­a­tion in the fol­low­ing plants:

3.1 Mon­i­tor­ing of the cir­cu­la­tion water in the cool­ing tow­er of a coal-fired pow­er plant
Chal­lenges for the mea­sur­ing mode:
Chang­ing oper­at­ing con­di­tions due to load changes between full load, par­tial load and oper­a­tion with­out load at vary­ing flow rates (auto­mat­ic sam­pling direct­ly from the line behind the main cool­ing water pump) and cir­cuit water temperatures.
Increased influ­ence of VBNC cells espe­cial­ly at low cir­cuit water temperatures.
A sta­ble mea­sur­ing oper­a­tion has been achieved over sev­er­al months. The inter­im influ­ence of VBNC cells can be suc­cess­ful­ly sup­pressed in the instru­ment by chang­ing the auto­mat­ed pre­treat­ment adapt­ed to the main cool­ing water.

3.2 Mon­i­tor­ing of the cir­cu­lat­ing water in the evap­o­ra­tive cool­ing sys­tem of a starch factory
Chal­lenges for the mea­sur­ing operation:
Out­door loca­tion of the instru­ment (wall mount­ing) with strong­ly chang­ing ambi­ent temperatures
Par­tial­ly strong sol­id mat­ter input into the cir­cu­la­tion water with high organ­ic load
A sta­ble mea­sur­ing oper­a­tion over sev­er­al months was achieved. In par­tic­u­lar, the influ­ence of the bio­cide treat­ment on the con­cen­tra­tion of legionel­la could be proven direct­ly. When chang­ing from a non-oxi­diz­ing to an oxi­diz­ing bio­cide, a direct­ly mea­sur­able effect on both the con­cen­tra­tion of the legionel­la and the reac­tion speed could be observed.

3.3 Mon­i­tor­ing the cir­cu­la­tion water of a met­al cast house
Chal­lenge for the mea­sur­ing operation:
Heavy con­t­a­m­i­na­tion of the water with inor­gan­ic and organ­ic impu­ri­ties (cast­ing oil)
With strong fluc­tu­a­tions in the water qual­i­ty, reli­able mea­sure­ments have been achieved over a peri­od of sev­er­al months. Cast­ing plants are often equipped with a hot water stor­age tank. Depend­ing on the require­ments of the cast­ing plant(s), the tem­per­a­ture and hydraulic reten­tion time (stag­na­tion), as well as the load of organ­ic and inor­gan­ic con­t­a­m­i­na­tion fluc­tu­ates strong­ly with a sig­nif­i­cant influ­ence on the repro­duc­tion rate of legionella.

3.4 Mon­i­tor­ing the drink­ing water net­work of a bev­er­age manufacturer
Chal­lenge for the mea­sur­ing operation:
Reli­able detec­tion of low and increas­ing con­cen­tra­tion of legionel­la at chang­ing drink­ing water tem­per­a­ture in the pipeline network
Sup­pres­sion of the influ­ence of VBNC cells on mea­sure­ment results, espe­cial­ly at low water temperatures
With this char­ac­ter­is­ti­cal­ly low-nutri­ent and sol­id-free water, fluc­tu­at­ing Legionel­la con­t­a­m­i­na­tion could be reli­ably detect­ed over sev­er­al months, depend­ing on the con­sump­tion struc­ture and tem­per­a­tures in the pipeline network.

3.5 Hygien­ic mon­i­tor­ing of dif­fer­ent cool­ing sys­tems of a food pro­duc­ing com­pa­ny using a lab­o­ra­to­ry device
Chal­lenge for the mea­sur­ing operation:
Man­u­al sam­ple appli­ca­tion of cool­ing water sam­ples dif­fer­ent in quality
Dis­in­fec­tion of the feed fun­nel before sam­ple preparation
Guar­an­tee of low work effort for man­u­al sam­ples includ­ing result evaluation
A reli­able, auto­mat­ed adjust­ment of the para­me­ters for the pre-treat­ment in the device could be ensured over sev­er­al months even with dif­fer­ent­ly buffered and pre­loaded water sam­ples. Both drink­ing water sam­ples (mon­i­tor­ing of the make-up water for the cool­ing sys­tems) and the cool­ing water sam­ples showed a good cor­re­la­tion to the cul­ti­va­tion method accord­ing to UBA with clear­ly dif­fer­ent results.

3.6 Cor­re­la­tion of the rapid test INWATROL L.nella+ with the cul­ti­va­tion method
The cor­re­la­tion of the rapid test was car­ried out over a high num­ber of mea­sure­ments with the cul­ti­va­tion method accord­ing to ISO 11731:2017. Sam­pling, sam­ple trans­port as well as prepa­ra­tion and eval­u­a­tion of the mea­sure­ment results were car­ried out in accor­dance with the cur­rent rec­om­men­da­tion of the Fed­er­al Envi­ron­men­tal Agency for sam­pling and detec­tion of Legionel­la in evap­o­ra­tive cool­ing sys­tems, cool­ing tow­ers and wet sep­a­ra­tors (UBA). Val­i­da­tion mea­sure­ments were made with dif­fer­ent accred­it­ed lab­o­ra­to­ries. In order to obtain a reli­able qual­i­ta­tive com­par­i­son between the rapid test and the cul­ti­va­tion method, the fol­low­ing mea­sure­ments were car­ried out in only one accred­it­ed lab­o­ra­to­ry (IWW Rheinisch-West­fälis­ches Insti­tut für Wass­er Beratungs- und Entwick­lungs­ge­sellschaft mbH, D‑45476 Mül­heim an der Ruhr).

4. Dis­cus­sion
The cor­re­la­tion to the cul­tur­al prepa­ra­tions car­ried out in the lab­o­ra­to­ry can be rat­ed as very high over­all. Two devices showed sig­nif­i­cant short-term devi­a­tions from the lab­o­ra­to­ry results in the form of addi­tion­al find­ings. Here the influ­ence of VBNC cells on the mea­sure­ment result of the INWATROL L.nella+ rapid test was inves­ti­gat­ed. Meta­bol­ic activ­i­ty mea­sure­ments using flu­o­res­cein diac­etate are used in micro­bi­o­log­i­cal tests in addi­tion to oth­er meth­ods (mem­brane integri­ty, pro­tein syn­the­sis (FISH), intact polar mem­brane lipid analy­sis, cell exten­sion (“direct viable count”)) for the detec­tion of VBNC bac­te­ria. This can be an addi­tion­al ben­e­fit for the oper­a­tor, because recon­t­a­m­i­na­tion of water sys­tems with Legionel­la can also be a “revival” of VBNC organ­isms (see Hans-Curt Flem­ming, Jost Win­gen­der — IWW Zen­trum Wass­er, Biofilm Cen­tre, Uni­ver­si­ty Duis­burg-Essen). Often, how­ev­er, the aim of the oper­a­tor is to achieve the high­est pos­si­ble cor­re­la­tion to the legal­ly required exam­i­na­tion by means of cul­ti­va­tion in the lab­o­ra­to­ry. By adjust­ing the pre-treat­ment con­di­tions (main­ly by increas­ing the tem­per­a­ture and length­en­ing the pre-treat­ment time), the cor­re­la­tion to the cul­ti­va­tion method can be suc­cess­ful­ly restored in case of mul­ti­ple find­ings with VBNC cells.

Authors
Hol­ger Ohme, Jen­nifer Beck­er, Pas­cal Jahn, Dirk Heinecke