Antimicrobial Susceptibility Patterns of Legionella spp. Strains Isolated from Water Systems in Morocco

Abstract

Objective:

Legionella is a waterborne pathogen that causes a severe form of pneumonia called Legionnaires' diseases, which is normally acquired by inhalation of aerosols containing Legionella originating from natural and man-made water systems. The aim of this study was to describe the level of antimicrobial susceptibility of environmental Legionella spp. strains to preferred and recommended therapeutic agents to treat Legionella disease.

Methods:

The minimum inhibitory concentrations (MICs) of 60 environmental Legionella spp. strains were tested using the broth dilution method. Susceptibility testing was performed for 12 antimicrobial agents: macrolides (erythromycin, azithromycin [AZI], and clarithromycin [CLA]), fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin, and gemifloxacin), a ketolide (telithromycin), cefotaxime (CEF), tigecycline (TIG), doxycycline (DOX), and rifampicin (RIF).

Results:

All tested strains of Legionella spp. were inhibited by low concentrations of fluoroquinolones and macrolides. Regarding the macrolides, CLA was the most active antibiotic, and AZI was the least active. RIF was the most effective antibiotic against the isolates in vitro. All isolates were inhibited by the following antibiotics (in decreasing order of their MICs): DOX>CEF>TIG.

Conclusions:

No resistance against these drugs was detected, and all isolates were inhibited by low concentrations of the tested antibiotics. Susceptibility testing of environmental Legionella spp. isolates must be monitored often to detect and evaluate the possible development of antibiotic resistance.

Introduction

Legionella are gram-negative bacteria and one of the emerging waterborne pathogens responsible for two types of disease (legionellosis): Legionnaire's disease (LD), a severe form of pneumonia, and Pontiac fever, which includes a flu-like and self-limiting illness; resulting from the inhalation of aerosols containing these bacteria.^1,2 Because of their ubiquity, Legionella are present in natural and man-made environments such as premise plumbing materials, cooling towers, spas, faucets, and shower heads.³ Various parameters favor the proliferation of Legionella in water systems, such as hardness, temperature (between 25°C and 45°C), corrosion, scale, stagnation, flow regimes,^4,5 and biofilms.⁶ Biofilm offers protection against disinfection and can harbor amoebas, a growth vector for Legionella.¹ Moreover, bacteria exposed to disinfection and stressed environmental conditions can enter a viable but nonculturable state (VBNC). Although undetected by standard culture methods,⁷ the cultivability of VBNC cells can recover under more favorable conditions (lower water temperature, loss of disinfectant, and presence of biofilm).⁸

Some outbreaks of legionellosis have been linked to aerosol-producing devices, including water systems, cooling towers, and fountains.^9–11 Legionellosis affects especially elderly, immunocompromised and immunosuppressed patients.¹² Person-to-person transmission has only been reported once.¹³

Today, the family Legionellaceae consists of a single genus, Legionella, but contains ∼60 different Legionella spp., and >73 serogroups (sgs) have been identified^14,15; among the species, Legionella pneumophila causes the most diseases and is responsible for 91.5% of human infections, especially sg 1, which is the most pathogenic, causing the majority of LDs (84.2%).^16,17

L. pneumophila is recognized as an intracellular pathogen that can not only replicate in eukaryotic phagocytic cells in the environment but also multiply and survive in human macrophages and cause potentially lethal pneumonia.¹⁸ Thus, antibiotics with intracellular activity are needed for successful therapy.

Historically, erythromycin (ERY) has been the first line antibiotic.¹⁹ In fact, macrolides, fluoroquinolones, and rifampicin (RIF) are the most commonly used antibiotics in the treatment of LD, based on their greater in vitro activity and intracellular penetration.²⁰ Furthermore, in severe cases of LD, RIF is used most often in combination with other drugs. However, some studies have reported therapy failure in patients with LD receiving treatment with these antibiotics.^21,22

In Morocco, there are not enough data on clinical and environmental isolates to evaluate the in vitro activity of antimicrobial agents. The aim of our study was to describe the antimicrobial susceptibility of environmental strains of Legionella spp. isolated from hot water systems of residential facilities in Morocco.

Materials and Methods

Bacterial strain selection

A total of 60 environmental Legionella spp. strains were used in this study; 20 of them were Legionella pneumophila sg 1 (33.33%), 38 Legionella pneumophila sg 2–15 (63.33%), and 2 Legionella anisa (3.33%). The strains were isolated from hot water systems of residential facilities in different Moroccan cities during the period from 2016 to 2018 according to the T90-431/A1 (April 2006) standard “Detection and enumeration of Legionella spp. and Legionella pneumophila—Method by direct inoculation and after concentration by membrane filtration or centrifugation.” All Legionella isolates were first serologically identified by the latex agglutination test using a commercial kit (SLIDEX^® Legionella-Kit; bioMérieux, Marcy l'Etoile, France) and then stored at −70°C in glycerol stocks before analysis.

Antimicrobial agents

Twelve antimicrobials were tested with different concentrations ranging from 0.016 to 8 mg/L: macrolides (ERY, azithromycin [AZI], and clarithromycin [CLA]), fluoroquinolones (ciprofloxacin [CIP], levofloxacin [LEV], moxifloxacin [MOX], and gemifloxacin [GEM]), a ketolide (telithromycin [TELI]), cefotaxime (CEF), tigecycline (TIG), doxycycline (DOX), and RIF. The antimicrobials were purchased from commercial sources (Sigma-Aldrich, Germany).

Broth dilution susceptibility testing

Antimicrobial susceptibility was assessed by microdilution in buffered yeast extract (BYE) broth supplemented with 0.04% L-cysteine in 96-well microtiter plates, following the National Committee for Clinical Laboratory Standards guidelines. Legionella isolates were subcultured on Buffered Charcoal Yeast Extract Alpha-Ketoglutarate (BCYE-α) (bioMérieux) and incubated for 72 hours at 37°C ± 2.5% carbon dioxide (CO₂). A suspension of each strain was prepared in BYE broth, and the turbidity was adjusted to an optical density equivalent to 0.5 McFarland standards. Antibiotic serial dilutions were prepared in BYE broth (50 μL) and added to an equal volume of inoculum in each well. After incubation for 72 hours at 37°C ± 2.5% CO₂, the minimum inhibitory concentration (MIC), the concentration in the first well with no visible growth, was determined. All tests were performed in duplicate, and the results were expressed as the mean of the two values.

To determine the effect of charcoal (BCYE-α) on the MICs, Legionella pneumophila ATCC 33152 and Staphylococcus aureus ATCC 29213 were used as control strains against the same antibiotics using BYE, Mueller–Hinton (MH), and BCYE-α (bioMérieux) as previously described by the European Committee on Antimicrobials Susceptibility Testing.²³

Results

The cumulative percentages, MIC₅₀ and MIC_90, of the 60 environmental Legionella isolates inhibited by different concentrations of the 12 antimicrobials tested are shown in Table 1.

Table 1.

Cumulative Distribution of the Minimum Inhibitory Concentrations of 60 Environmental Legionella Strains (n = 60)

Antibiotics	Cumulative % of strains inhibited at indicated concentrations (mg/L)																		Range (mg/L) of susceptibility	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)
Antibiotics	0.016	0.032	0.047	0.064	0.094	0.125	0.19	0.25	0.38	0.5	0.75	1	1.5	2	3	4	6	8	Range (mg/L) of susceptibility	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)
ERY				3.33	18.3		46.8	50		71.7	91.7	96.7		100					0.064–2	0.25	0.75
AZI			13.3	21.7		41.7	48.3	56.7	76.7	90	98.3		100						0.047–1.5	0.25	0.5
CLA		3.33		40	50			78.3	83.3	93.3	98.3	100							0.032–1	0.094	0.5
CIP			6.7	16.7		46.7	56.7			81.7	96.7		100						0.047–1.5	0.19	0.75
LEV			3.33	58.3	68.3		76.7	85	90		93.3	100							0.047–1	0.064	0.38
MOX				46.7	18.3		36.7	50		93.3	95		98.3		100				0.064–3	0.25	0.5
GEM			15	28.3		45		70	81.7	98.3			100						0.047–1.5	0.25	0.5
DOX							5	16.7		28.3	40		48.3	60	80	91.7	95	100	0.19–8	2	4
CEF								20		31.7	41.7			75	90	98.3	100		0.25–6	2	3
RIF	5	51.7		95	96.7		98.3	100											0.016–0.25	0.032	0.094
TELI							25	38.3		51.7	75	76.7		85	96.7	100			0.19–4	0.5	3
TIG						1.7		6.7	23.3	38.3	48.3		63.3	83.3	95		100		0.125–6	1.5	3

AZI, azithromycin; CEF, cefotaxime; CIP, ciprofloxacin; CLA, clarithromycin; DOX, doxycycline; ERY, erythromycin; GEM, gemifloxacin; LEV, levofloxacin; MIC, minimum inhibitory concentration; MOX, moxifloxacin; RIF, rifampicin; TELI, telithromycin; TIG, tigecycline.

The MIC₅₀ and MIC₉₀ values for the fluoroquinolones (CIP, LEV, GEM, and MOX) ranged from 0.047 to 3. Specifically, they were 0.19 and 0.75 mg/L for CIP, 0.25 and 0.5 mg/L for MOX, 0.25 and 0.5 mg/L for GEM, and 0.064 and 0.38 mg/L for LEV, respectively.

The MIC₅₀ and MIC₉₀ values for the macrolides (AZI, CLA, and ERY) ranged from 0.032 to 2. In particular, they were 0.25 and 0.75 mg/L for ERY, 0.25 and 0.5 mg/L for AZI, and 0.094 and 0.5 mg/L for CLA, respectively (Table 2).

Table 2.

Minimum Inhibitory Concentration (MIC)₅₀, MIC₉₀ and MIC Range (mg/L) of the 12 Antibiotics Tested Against Legionella spp. Isolates

Antibiotics	Legionella pneumophila sg 1 (n = 20)			Legionella pneumophila sg 2–15 (n = 38)			Legionella anisa (n = 2)
Antibiotics	MIC₅₀	MIC₉₀	Range	MIC₅₀	MIC₉₀	Range	MIC₅₀	MIC₉₀	Range
ERY	0.25	0.75	0.19–2	0.19	0.75	0.064–0.75	0.094	0.25	0.094–0.25
AZI	0.125	0.38	0.064–1.5	0.19	0.5	0.047–0.75	0.064	0.25	0.064–0.25
CLA	0.094	0.25	0.032–1	0.064	0.38	0.032–0.75	0.064	0.094	0.064–0.094
CIP	0.125	0.75	0.064–1.5	0.19	0.5	0.047–0.75	0.19	0.5	0.19–0.5
LEV	0.064	0.25	0.064–1	0.064	0.38	0.047–1	0.094	0.19	0.094–0.19
MOX	0.25	0.5	0.19–3	0.25	0.5	0.064–1.5	0.19	0.75	0.19–0.75
GEM	0.25	0.5	0.047–1.5	0.125	0.38	0.047–0.5	0.094	0.19	0.094–0.19
DOX	2	4	0.19–8	1.5	4	0.19–8	2	2	2–2
CEF	2	3	0.5–6	0.5	3	0.25–4	0.5	1	0.5–1
RIF	0.032	0.094	0.016–0.25	0.032	0.064	0.016–0.19	0.032	0.064	0.032–0.064
TELI	0.5	2	0.19–4	0.25	2	0.19–3	0.25	0.5	0.25–0.5
TIG	3	6	0.38–6	0.75	3	0.125–3	1	2	1–2

The MIC₅₀ and MIC₉₀ values for CEF were 2 and 3 mg/L, 1.5 and 3 mg/L for TIG, 0.5 and 3 mg/L for TELI, and 2 and 4 mg/L for DOX, respectively.

RIF was the most active drug, with MIC₅₀ = 0.032 mg/L and MIC₉₀ = 0.064 mg/L (range 0.016–0.25); DOX was the least active drug tested in our study.

The activity of the tested antibiotics against Legionella pneumophila sg 1, Legionella pneumophila sg 2–15, and L. anisa is given in Table 2. When comparing the MIC values of Legionella pneumophila sg 1 and Legionella pneumophila sg 2–15, no differences were observed for CLA, DOX, LEV, MOX, and RIF. However, the MIC values of CEF, ERY, GEM, TELI, and TIG were higher for Legionella pneumophila sg 1 than for Legionella pneumophila sg 2–15. Furthermore, the MIC₅₀ and MIC₉₀ values of the tested antibiotics were lower for L. anisa than for L. pneumophila.

It is worth mentioning that the MIC values of RIF were the lowest, while DOX and CEF presented high MICs against the tested strains (Table 2).

Table 3 shows the MIC (mg/L) value of the control strains in the corresponding media. Legionella pneumophila ATCC 33152 generally showed MICs similar to the MIC values of the environmental strains. The MICs of Staphylococcus aureus ATCC 29213 on MH agar fell within the range of published values (Table 3). Comparing the results obtained from control strains on two different media, BCYE-α agar provided higher MICs in 75% of the performed tests. Nine of the 12 antimicrobials showed an elevated MIC compared with the control strains tested on BCYE-α medium. Macrolides (ERY and AZI) and fluoroquinolones (CIP, LEV, MOX, and GEM) were affected by charcoal (BCYE-α medium). A similar effect was observed with RIF, TELI, TIG, and CEF. The MICs of RIF, MOX, and GEM were the most influenced by charcoal.

Table 3.

Minimum Inhibitory Concentrations (mg/L) Value of the Reference Strains in Corresponding Media

Antibiotics	Legionella pneumophila ATCC 33152		Staphylococcus aureus ATCC 29213
Antibiotics	BYE	BCYE-α	MH	BCYE-α
ERY	0.094	0.19	0.25	0.75
AZI	0.032	0.094	0.5	1.5
CLA	0.064	0.064	0.19	0.19
CIP	0.25	0.5	0.25	0.75
LEV	0.094	0.125	0.25	0.5
MOX	0.19	1	0.064	1
GEM	0.125	0.75	0.094	2
DOX	2	2	0.5	0.5
CEF	2	3	0.75	1
RIF	0.032	0.064	0.016	0.125
TELI	0.125	0.125	0.25	0.5
TIG	1.5	3	0.38	2

BCYE-α, Buffered Charcoal Yeast Extract Alpha-Ketoglutarate; BYE, buffered yeast extract; MH, Mueller–Hinton.

Discussion

Environmental surveillance of Legionella spp. is essential to prevent and minimize Legionella infection and to identify any changes in antimicrobial susceptibility. Although Legionella pneumophila sg 1 actually causes the majority of LD cases,¹⁶ other sgs and species such as Legionella pneumophila sg 2–15²⁴ and L. anisa²⁵ have been implicated in either pneumonia or Pontiac fever. In the present study, we tested for the first time in Morocco the susceptibility of environmental Legionella strains to 12 antibiotics often used in LD therapy.

All 60 strains were fully susceptible and inhibited by low concentrations of the tested antibiotics. Among these antibiotics, and in accordance with previous reports, RIF (MIC range: 0.016–0.25 mg/L) was the most effective drug against the L. pneumophila and L. anisa isolates.^22,24,26 Despite its high activity, the use of RIF as a monotherapy for Legionella increases the possibility of bacterial resistance development; hence, RIF combination therapy is recommended for patients with refractory or severe LD.^22,27

Macrolides (especially AZI) and fluoroquinolones (especially LEV) are the first-choice drugs for LD therapy. Among the macrolides, AZI and CLA showed the greatest activity, which was even more than the activity of ERY.²⁸ According to a previous study, CLA was more active against all isolates than AZI and ERY.²⁸ The MIC₅₀ and MIC₉₀ values of AZI and ERY (Table 2) were slightly higher than previously reported for environmental isolates.^24,29,30 The most likely explanation of this difference seems to be the origin of the strains and their geographical environment.^26,29,31 The MIC₅₀ and MIC₉₀ values of macrolides were lower for the L. anisa and Legionella pneumophila sg 2–15 isolates than for the Legionella pneumophila sg 1 isolates.

Currently, fluoroquinolones have become the most widely used antibiotics for lower respiratory tract infections therapy because of their broad-spectrum coverage, ease of administration, and comparatively few adverse effects.²²

In line with other reports,^26,28 the MIC₉₀ values in our study indicate that the L. pneumophila isolates were most susceptible to LEV (Tables 1 and 2). Indeed, we found that LEV was the most active antibiotic among all the fluoroquinolones and the second active drug amid the tested antimicrobial agents. In accordance with previous studies, fluroquinolones have a greater activity toward Legionella compared to macrolides.^24,28,32 In contrast, other studies reported that the activities of fluoroquinolones toward L. pneumophila were similar to those of macrolides based on MIC₉₀ values²⁶; these differences may be attributed to geographical distribution and the susceptibility testing methods used.

The MIC₅₀ and MIC₉₀ values of LEV against Legionella (Table 1) were 0.064 and 0.38 mg/L, respectively. Our LEV MIC₉₀ values were two to four times higher than the MIC₉₀ values reported by several authors.^22,28,32

A previous study that included 50 Legionella spp. isolated from water systems of hotels reported that the MIC₉₀ values for RIF, LEV, AZI, and CLA using the broth dilution method were 0.015, 0.125, 0.125, and 0.125, respectively.²² These values were lower than the MIC₉₀ values for RIF, LEV, AZI, and CLA in our assessment (Table 1).

In our study, the MIC values of AZI, DOX, and TIG were lower than those reported by De Giglio et al.²⁶; this dissimilarity could be explained by the use of different susceptibility testing methods. It is known that the E-test method yields higher MIC values compared with other methods.¹⁹ In fact, no systematic difference in MICs values was observed between Legionella pneumophila sg 1 and sg 2–15 for ERY and TELI (Table 2). In contrast, the MIC values for other drugs were lower for the Legionella pneumophila sg 2–15 and sg 1 strains. The MIC₉₀ values of the majority of antibiotics were lower for L. anisa than for L. pneumophila (Table 2). According to several studies, the antimicrobial susceptibility of Legionella pneumophila sg 2–15 was slightly higher than that of Legionella pneumophila sg 1; furthermore, Legionella pneumophila sg 1 remained less susceptible to the majority of drugs, even less than the Legionella anisa and Legionella pneumophila sg 2–15 isolates.^24,33

In our study, TIG was the least active drug followed by DOX. The MICs of TIG, DOX, and CEF were higher than those of the other tested antibiotics. In other words, TIG (MIC range: 0.125–6 mg/L) and DOX (MIC range: 0.19–8 mg/L) were clearly the least effective drugs against L. pneumophila and L. anisa (Tables 1 and 2). CEF was more active than TIG and DOX, and the activities were in the following order: CEF>TIG>DOX. In accordance with different studies, DOX was reported as the least active drug among the antimicrobial agents commonly used in LD therapy; in fact, attention should be paid when prescribing such antibiotics.^26,29

Our study confirms that RIF provided the lowest MICs and tetracyclines (DOX and TIG) provided the highest.^26,30,34

With regard to RIF, it should be noted that the MIC of this antimicrobial toward Legionella pneumophila ATCC 33152 was below the tentative highest MIC for wild-type and the MIC reported by March et al.³⁰ and Bruin et al.³⁴ In contrast, it was above the MIC reported by De Giglio et al.²⁶ These disparities in MIC values can be due to differences in the amount of charcoal used in the media, the geographical origins of strains, and the antibiotic susceptibility testing (AST) methodology.

Given that we used a medium without charcoal, it should be interesting to mention that our MICs for Legionella pneumophila ATCC 33152 were lower than the MICs published by several authors using BCYE-α medium^30,34,35; however, they were in accordance with those published by De Giglio et al.,²⁶ for the majority of the tested antimicrobials.

Various methods have been used to determine the antibiotic susceptibility of Legionella, such as broth and agar dilution, E-tests and disk diffusion.^{24,26,28,29,34} None of these methods offers a gold standard, and some studies have reported that using a different methodology leads to variability in the range of MIC values.^26,36 Several studies have indicated that charcoal or other components of BCYE agar can inhibit various antibiotics, including fluoroquinolones and macrolides.^{22,24,33,36,37} To prevent any inactivation of antimicrobials by charcoal, we used the broth dilution method in BYE. Some authors have indicated that the use of BYE (without charcoal) instead of BCYE may be a good option for conducting susceptibility testing for Legionella spp. to antimicrobials.^22,33 Overall, the results from this study are comparable with previous works where the same standard dilution testing in broth was used.^22,28

In general, the results of antibiotic susceptibility obtained with either method must be interpreted with caution. It should be considered that in vitro testing of antibiotic agents sometimes poorly correlates with clinical efficacy, as the intracellular location of the microorganisms may protect them from an otherwise effective antimicrobial therapy.^26,37

Legionella in environmental water systems can be exposed to antimicrobial agents from medical or veterinary practice or to those secreted by other microorganisms.³⁸ Consequently, the presence of these antibiotics in the environment can promote the development of bacterial resistance,³⁹ which can increase the risk of failed antibiotic treatment in patients with LD. In fact, antibiotic susceptibility surveillance of environmental Legionella strains is essential to evaluate the possible emergence of resistance.

Conclusions

All isolates were inhibited by low concentrations of the tested antibiotics, and no resistance against these drugs was detected. The microdilution method may be the right choice to carry out susceptibility testing of Legionella to antimicrobials due to the potential inhibitory effect of charcoal using the AST method on BCYE-α. Overall, susceptibility testing of environmental Legionella spp. isolates should be monitored often to detect and evaluate the possible development of antibiotic resistance.

Footnotes

Acknowledgments

A. ASSAIDI thanks the Centre National pour la Recherche Scientifique et Technique of Morocco for the Scholarships 3USMS2015.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the Centre National pour la Recherche Scientifique et Technique of Morocco in the framework of a priority research project (Grant No. PPR2015/16).

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