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Introduction

These include air conditioning cooling towers or evaporative condensers^1-4. It has also been isolated from surface water from a stream, from mud from a stream bottom⁵, and from lake waters not associated with epidemics. In its natural environment, the bacterium resides within freshwater amoebas such as Acanthamoeba and Hartmenella, but the organism can also infect humans upon the inhalation of contaminated aerosols. Invasion and replication in alveolar macrophages can lead to pneumonia or "Legionnaires' disease" in individuals who are immunocompromised or have some underlying conditions that reduce the efficiency of their lung defense mechanisms⁶.

These data suggest thatLegionellasecretes type IV effectors into the host cell cytosol that function to target specific host trafficking pathways in order to remodel the nascent vacuole^7、8.L. pneumophilahas been detected only sporadically by use of the cultivation method in untreated groundwater (GW) and in treated water at temperatures below 20°C^9-11. Primary macrophages derived from bone marrow of the permissive A/J mouse, the interaction with the lysosomal pathway is only delayed, as the RV eventually matures into an acidified vacuole in a process that resembles autophagy¹².

In addition toL. pneumophila, the agent of Legionnaires disease, there are now four other proposed species ofLegionella¹³:Legionellamicdadei (pittsburgensis),Legionelladumoffii,Legionellabozemanii, andLegionellagormanii. All of these species but L. gormanii have been isolated as causative agents for illnesses involving pneumonia^14,15.

However, it is not clear if these or other organisms represent potential pathogens, because representatives ofL. pneumophilaserogroups and genotypes also show distinct differences in infectivity. The semiselective buffered charcoal yeast extract (BCYE) agar medium supplemented with antibiotics was used to detect culturableLegionella^{16, 17}.

Strains ofL. micdadeiwere isolated from blood from patients.L. micdadei从病人和证明也被孤立be the causative agent of pneumonia^{18, 19}. The organism has also been isolated from environmental sourcesspecifically, nebulizers from respiratory therapy equipment²⁰.L. pneumophilais responsible for more than 90% of the reported cases of legionellosis²¹. Currently, about 50Legionellaspecies have been defined, nearly half of which have been associated with cases of disease (e.g.,L. pneumophila,L. micdadei, L. bozemanii, L. longbeachae,andL. dumoffii)²².

The ability ofLegionellaspp. to multiply in biofilms in low-pH environments has recently been demonstrated, and those authors also concluded that water temperature affected species composition²³.L. gormaniiwas isolated from soil collected from a creek bank during a Legionnaires disease outbreak investigation. The relationship of this organism to Legionnaires disease or other human diseases is not known at this time, although there is evidence that it can cause human as well as animal infections²⁴. These isolates, referred to asLegionella-like organisms (LLO).

The procedure currently used to isolateL. pneumophilaand otherLegionellaspp. Theorganism can be isolated either from the guineapig tissues or the egg yolk sacs on charcoal-yeastextract (CYE) agar plating medium²⁵. In addition tobeing tedious, this method is costly in terms oflaboratory animals, eggs, and time. The proceduremay take several weeks and does not allow for rapidprocessing of specimens, which might be of criticalimportance during an epidemic investigation.

This report describes a selective isolation procedure that has been used to isolateLegionelladirectly from environmental samples without animal or egg inoculation. This procedure utilizes a selective medium consisting of CYE agar with four antibiotics added: cephalothin, colistin, vancomycin, and cycloheximide.L. pneumophilais relatively resistant to low pH for a short period of time²⁶. Our results show that the rate of recovery ofLegionellafrom environmental water samples is improved if samples are treated with acid and then plated on the selective agar medium.

Materials and Methods

Bacterial strains

A stock culture ofL. pneumophilathat had been passed on artificial media several times was used for plate counts and antibiotic minimum inhibitory concentration determinations. This strain is referred to as the "stock strain" in this report. A 48-h CYE agar culture of the stock strain was suspended in yeast extract broth with 40% glycerol, divided into small aliquots, and frozen at -70°C. A vial was thawed and streaked on CYE agar for use each week.

Another culture ofL. pneumophila, referred to in this report as the "tissue strain," had never been passed on artificial media. The tissue strain was stored as macerated spleen tissue from a guinea pig infected with the disease. The infected spleen, after having been macerated, was divided into small aliquots and frozen at -70°C. For each experiment, a vial was thawed and diluted 1:5,000 in phosphate buffered saline before being plated on the media to be tested.

Environmental water samples

The environmental samples used in thisstudy were from various parts of the Uttar Pradeshand were specimens from whichLegionellaor LLOhad already been isolated with the guinea pig/egginoculation method. Some of the specimens werecollected during epidemic investigations ofLegionnaires disease outbreaks but may not havebeen related to the outbreaks. The samples werestored at 4°C for 2 to 18 months before being usedin the study.

Media

The CYE agar used in this study contained1.0% yeast extract (Hi-Media ), 1.7% agar (Biogene), 0.15% activated charcoal, 0.04% Lcysteine hydrochloride, and 0.025% ferric pyrophosphate (soluble). The pH was adjusted to 6.9 to 7.0 after autoclaving and the addition of Lcysteine and ferric pyrophosphate. Selective media were prepared by adding solutions of antibiotics to autoclaved CYE agar held in a water bath at 50°C until immediately before the plates were poured.

酵母提取物汤由酵母extr 1.0%act (Hi-Media) solution to which 0.04% Lcysteine hydrochloride and 0.025% ferric pyrophosphate were added after autoclaving. The pH of the broth was adjusted to 6.9 to 7.0. FG agar (Mueller-Hinton, with 0.04% L-cysteine hydrochloride and 0.025% ferric pyrophosphate, pH 6.9 to 7.0 was used to detect the production of brown pigment by isolates presumed to beLegionella. Another medium, yeast extract-tyrosine agar, consisting of 1.0% yeast extract (Hi Media), 1.7% agar (Biogene), and 0.04% L-tyrosine, was also used for detection of brown pigment production since many organisms that were difficult to grow on FG agar grew satisfactorily on yeast extracttyrosine agar.

Direct plating of environmental samples

Two 10-fold dilutions of each environmentalspecimen were prepared in phosphate-buffered saline (pH 7.2 to 7.3). A 0.1-ml quantity of eachdilution and of the undiluted specimen was spreadonto the surface of CYE and selective agar plates一式两份。板块在35°C孵化2.5% C02. The plates were read after 5, 6, 7, and10 days of incubation for colonies with morphologysimilar to that ofLegionella(white, glistening,convex, circular, entire, and from 1 to 2 mm indiameter with a "ground glass" appearance undermagnification). At the time of each reading, allcolonies thought to beLegionellawere subculturedto fresh CYE agar. A total plate count of all colonieswas recorded at 10 days.

Acid buffer treatment of environmental samples

The HCl-KCl buffer used for pretreatingwater samples was prepared by mixing 3.9 ml of0.2 M HCl with 25 ml of 0.2 M KCl to yield a buffersolution with a pH of approximately 2.2. Twoprocedures designated "A" and "B" for acid治疗使用。过程中,10毫升water sample was centrifuged at 4,000 rpm for 10 min in aerosol-free centrifuge containers. The supernatant was poured off, and the sediment was resuspended in 1 ml of the original water sample. This suspension was diluted 1:10 with the HCl-KCl buffer. At intervals ranging from 5 to 60 min, 0.1-ml quantities were removed from the acid suspension and were plated on duplicate agar plates of CYE and selective media. After each such interval, a sample was diluted 1:10 with phosphate-buffered saline and then plated in 0.1-ml quantities on duplicate CYE and selective media plates.

Procedure B did not involve centrifugation but involved adding 0.5 ml of the water sample to 4.5 ml of HCl-KCl buffer. At intervals of 5 to 60 min, 0.1-ml quantities of undiluted and diluted (1:10 in phosphate buffered saline) acid-treated water sample were plated onto CYE and selective media. This procedure resulted in plating a 10 and a 10-2 dilution of the water sample, which was 10-fold more dilute when plated than samples prepared according to procedure A.

The plates inoculated with the acid-treated suspensions were incubated, read, and counted as described above for direct plating of water samples.

Screening suspectLegionellaisolates

Each colony thought to beLegionellawasstreaked for isolation to a CYE agar plate (containingno antibiotics) and incubated for 2 to 5 days. Fromeach such plate, the isolate was streaked onto yeastextract-tyrosine agar (or FG) and 5% sheep bloodagar. If growth did not occur on blood agar after 5天的孵化(Legionellacharacteristically failsto grow on blood agar) or if the isolate producedbrowning of the yeast extract-tyrosine agar or FGagar, the isolate was examined by the fluorescentantibody(FA) staining method with conjugatedantisera to each of the five species ofLegionella. Isolates that were positive by FA in the polyvalentL. pneumophilaconjugate were then furtherexamined by FA with type-specific conjugates toL.pneumophilaserogroups 1 to 6. If the FA resultsindicated aLegionellaspecies, the isolate wasexamined for the cellular fatty acids characteristicof theLegionellaspecies to confirm theidentification, If the fatty acid profile demonstrateda pattern ofa high percentage of branched-chain cellular fatty acids, and if the isolate did not grow on blood agar or produce browning on FG or yeast extract tyrosine agar, was gram negative, and was otherwise similar toLegionella, the isolate was considered to be an LLO^{27, 28}.

Selective media experiments

To determine their suitability for inclusionin a selective medium, colistin and cephalothin wereadded separately to CYE agar in several differentlevels. Plate counts of theL. pneumophilastock这些selec应变进行tive media andon CYE agar with no antibiotics. In addition, 10environmental samples were direct plated on thesemedia as described above. Subsequently,cephalothin at a level of4μg/ml was combined inCYE agar with different levels of colistin (2, 4, 8,16, and 32μg/ml) to determine the optimumconcentration. TheL. pneumophilastock strain andtwo environmental specimens were plated on thesefive selective media with cephalothin and and 16μgof colistin per ml was selected as optimal, and tothis medium were added two concentrations ofvancomycin (3 and 0.5μg/ml). On these twoselective media, and also on CYE medium, platecounts of the stock strain ofL. pneumophilaweredone. Using cephalothin-colistinvancomycin (CCV)agar and CYE agar, five environmental watersamples were plated by using the methoddescribed.

Edelstein-Finegold (E-F) medium (CYE agar with 40 U of polymyxin B and 0.5μg of vancomycin per ml) was compared with CCV medium (CYE agar with 4μg of cephalothin, 16μg of colistin, and 0.5μg of vancomycin per ml) for isolation ofLegionellaand suppression of non-Legionellaorganisms. Ten environmental water samples from whichLegionellahad been isolated by guinea pig/egg inoculation were used. Samples were diluted and plated as described above on three media: E-F medium, CCV medium, and CYE medium without antibiotics. These plates were read and picked forLegionellacolonies, and total plate counts were determined to assess the inhibition of total flora.

In a final attempt to improve the selectivity of CCV medium, we added cycloheximide (an antifungal agent) because the prolonged incubation time (10 days) necessary to recoverLegionellacan sometimes result in overgrowth by fungi. A comparison of plate counts of the stock and tissue strains ofL. pneumophilaon CCV medium containing various concentrations of cycloheximide resulted in the selection of 80μg of cycloheximide per ml as the optimum level. Thirteen environmental water samples that had already been shown to bepositive forLegionellaand LLO were plated on CYE agar with, per ml, 4μg of cephalothin, 16μg of colistin, 0.5μg of vancomycin, and 80μg of cycloheximide (CCVC medium) and on CYE agar without antibiotics.

Acid treatment experiments

To evaluate the effects of low pH onsurvival ofLegionellaover a period of time, weexposed the stock strain ofL. pneumophilato theHCl-KCl buffer (pH 2.0) for periods of 5, 15, 30,and 60 min. Plate counts of the acid suspension ofthe stock strain were performed after each timeinterval.

Five water samples already shown to be positive forL. pneumophilaby the guinea pig/egg inoculation method were subjected to the HCl-KCl buffer treatment for 30 min according to procedure A, and attempts were made to recoverLegionella. Two naturally contaminated environmental water samples, LS- 4 and MI-56, were retested using 0-, 5-, 15-, and 30- min exposure times to HCl-KCl buffer to determine the most appropriate exposure interval for acid buffer treatment.In the last experiment the acid buffer treatment (procedures A and B), with an exposure time of 5 min and with samples directly plated onto CYE medium and CCVC selective medium, were compared for efficacy in isolatingLegionellafrom 11 environmental water samples already shown to be positive forLegionellaby the guinea pig/eg inoculation method.

Results

The growth of theL. pneumophilastockstrain was inhibited on CYE medium containing 8or 16μg of cephalothin per ml, although the minimuminhibitory concentration of cephalothin forL.pneumophilahas been reported to be 16μg/ml21, 22. Although the minimum inhibitory concentrationof colistin was reported to be 3.6μg/ml, colistin didnot inhibit growth ofL. pneumophila(stock andtissue strain) even at a level of 7.2μg/ml in our study.If an antibiotic is to be an effective agent in selectivemedia it must inhibit bacteria other thanLegionella,however, colistin at 7.2μg/ml did not sufficientlyreduce the total plate counts of 10 environmentalspecimens as compared to the total plate countson CYE without colistin. The average total platecount with no antibiotic was 7.7 X 105, and thatwith 7.2μg of colistin per ml was 2.5 X 105.

Using cephalothin-colistin medium (CYE agar with 4μg of cephalothin and 16μg of colistin per ml), we isolatedL. pneumophilagroup 1 from 1 to 2 environmental samples, but the average amount of suppression of competing non-Leginellaflora was less than 1 log. CCV medium (CYE agar with, per ml, 4μg of cephalothin, 16μg of colistin, and 0.5μg of vancomycin) was more inhibitory for grampositive spreading colonies than cephalothin-colistin medium and showed little suppression of theL.pneumophilastock strain. Three of five water samples cultured on CCV agar yieldedL.pneumophila, one sample yielded two serogroups ofL. pneumophila.

Table 1: Comparison of total plate counts of all organisms (Legionellaeand nonLegionellae) from 10 environmental specimens Click here to view table

Table 1 lists the isolates ofLegionellaand LLO and gives the results of the total plate counts of all organisms for the comparison of inhibitory characteristics of CCV, E-F, and CYE media. CCV medium was superior to E-F and CYE media in inhibiting competing flora in the environmental specimens. In addition,Legionellaand LLO were isolated from 4 of the 10 samples on CCV medium, whereas no isolations were made from E-F or CYE media.

When CCV plus 80μg of cycloheximide per ml (CCVC medium) was plated with 13 environmental water samples that had already been shown to be positive forLegionellaand LLO, 7 were positive on CCVC medium, and only 3 were positive on CYE without antibiotics. However, two of the three isolates on CYEwere recovered from chlorinated water samples relatively free of competing flora.

In the experiments done to develop the acid buffer method, theL. pneumophilastock strain was shown to be resistant to the acid for up to 30 min. The plate count of the stock strain was reduced from 6.3 X 108 colony-forming units per ml at 0 min of acid exposure to 6.1 X 108 at 30 min and to 6.8 X 107 at 60 min. When five environmental samples were exposed to the acid buffer from 30 min by procedure A (see Materials and Methods),L.pneumophilagroup 1 was recovered from two of the five samples. One of the three samples that did not yieldL. pneumophilayielded an LLO isolate. However, two samples tested later yielded isolates ofL. pneumophilaat 5 min but not at 30 min of exposure to the acid buffer. The total microbial flora for both samples in this instance was reduced by about 2 logs after 5 min of exposure to HCl-KCl buffer, indicating that 5 min is an effective exposure interval for acid buffer treatment.

Table 2: Comparison of three methods for efficacy in isolatingLegionellafrom water samples known to be positive forLegionella Click here to view table

Table 3: Comparison of types of Legionella isolates by the animal inoculation/egg method and direct media isolation method Click here to view table

Table 2 lists the results of the comparison of acid buffer procedures A and B with direct plating onto CYE medium and CCV selective medium. Direct plating yielded isolates ofL. pneumophilagroup 5 from one water sample (MI-56) on both CYE and CCVC. Five of the water samples yielded LLO after direct plating. When procedure B was used, 2 of 11 samples yieldedLegionella, and 3 yielded only LLO. When procedure A was used, five water samples yieldedLegionellaand five yielded LLO.

Analysis of the isolates from 15 environmental samples, tested by both the guinea pig/egg inoculation method and the acid pretreatment/ plating media method discussed above, revealed that multiplespecies and serogroups ofLegionellawere isolated by both methods (Table 3). The most obvious difference in the groups or species was that LLO was isolatedfrom six samples cultured directlyon plating media, but only one LLO isolate was obtained with the guinea pig/egg inoculation method.

Discussion

Procedure A was superior to both directplating on CYE and CCVC and to procedure B forisolatingLegionella. After the acid buffer treatment,CCVC selective medium used for plating gaveslightly better results than CYE medium. However,oneL. pneumophilaisolate was recovered on CYEthat was not recovered on CCVC medium. This mayhave been coincidence rather than the result ofinhibition ofL. pneumophilaby the selective medium,but because it occurred we recommend that bothCYE and CCVC media be used when samples arepretreated with acid buffer^{29, 30, 31}.

At this time no isolation method forLegionellacan completely replace the guinea pig/ egg inoculation method. Since our environmental specimens contained numerous and diverse flora, no single combination of antibiotics tested was effective in suppressing competing flora in all specimens. Even the acid buffer procedure used in combination with CCVC selective medium and CYE medium led to the recovery ofLegionellafrom only 45% of water samples known to containLegionella. However, it had been as long as a year in some cases between the timesLegionellahad been isolated from guinea pigs or eggs and the time we did this study.Legionellamay not have been still viable in some of the water samples, and the acid pretreatment/ media method may be more effective than our results indicate^{32, 33, 34, 35}.

Direct plating without acid buffer treatment on the selective medium, CCVC agar, led to a recovery rate of up to 36% forLegionellafrom environmental samples used in our study. It does appear to be more sensitive than animal inoculation for isolation of LLO, possibly indicating that this group of organisms is less pathogenic for guinea pigs (Table 3). These organisms are being considered as possible newLegionellaspecies since they have many characteristics similar to otherLegionella.

The utility of CCVC medium for clinical isolation ofL. pneumophilaand otherLegionellaspecies remains to be evaluated. E-F medium, which in our study was not as effective as CCV medium for environmental isolation ofLegionella, has been demonstrated to be effective for isolation ofL. pneumophilafrom clinical specimens, although its efficacy for isolation of other species ofLegionellais unknown.

Acknowledgements

Authors are thankful to J.C. Bose Institute of Life Science, Department of Biotechnology, Bundelkhand University, Jhansi, 284128 (U.P.), India, for providing financial support and lab facilities for this study.

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