Antimicrobial Original Research Paper
Antimicrobial susceptibility of Listeria monocytogenes isolates from human cases in northern Italy, 2008–2010: MIC determination according, to EUCAST broth microdilution method* { Moira Madeo1, Rosario Musumeci1, Anna Maria Laura Careddu1, Ettore Amato2, Mirella Maria Pontello2, Clementina Elvezia Cocuzza1 1
Dipartimento di Chirurgia e Medicina Traslazionale, Universita` di Milano-Bicocca, Monza, MB, Italy, Dipartimento di Scienze della Salute, Universita` degli Studi di Milano, MI, Italy
2
Susceptibility of 96 Listeria monocytogenes human isolates collected in northern Italy between 2008 and 2010, to 15 antimicrobials, was investigated. Minimum inhibitory concentration (MIC) was evaluated by means of the standardized broth microdilution method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) international guidelines. All L. monocytogenes human isolates were susceptible to penicillin G (MIC90#0.06 mg/ml), meropenem (MIC90#0.06 mg/ml), and erythromycin (MIC9050.12 mg/ml). Susceptibility to the other tested antimicrobials could not be interpreted due to the lack of breakpoint values although two (2%) isolates were shown to have tetracycline MICs above EUCAST epidemiological cut-off values (ECOFF). Bactericidal activity for amoxicillin, gentamicin, and levofloxacin was generally observed at concentrations 2–4 times higher than MIC values. Though L. monocytogenes human strains, isolated in the north of Italy, appear to be susceptible to most antimicrobial agents used in human therapy, this study provides new data for epidemiological surveillance and clinical breakpoints definition. Keywords: Listeria monocytogenes, Antimicrobials susceptibility, Minimum inhibitory concentration, Bactericidal activity, EUCAST guidelines
Introduction Listeria monocytogenes is a facultative intracellular pathogen causing a broad spectrum of clinical manifestations: gastroenteritis in immunocompetent people, and septicaemia, meningitis, encephalitis, and/or brain abscesses in immunocompromised subjects;1,2 furthermore, infections during pregnancy can lead to miscarriage, stillbirth, premature delivery, or life-threatening infection of the newborn (pregnancyassociated listeriosis).3 Its case fatality rate ranges between 20 and 50% accounting for approximately 30% of all foodborne diseases associated deaths.4–6 The incidence of invasive listeriosis reported from different countries varies from 0.32 to 1 cases per 100 000 population and since 2000 an increase in the * The material is original and has not been submitted elsewhere. { The corresponding author, on behalf of all co-authors, has read and agreed to the Journal of Chemotherapy terms on data protection notice.
Correspondence to: Moira Madeo, Dipartimento di Chirurgia e Medicina Traslazionale, Universita` di Milano-Bicocca, Via Cadore 48, 20900 Monza, MB, Italy. Email: [email protected]
ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000187
incidence of human listeriosis has been reported in several European countries.7–9 Clonal diversity and virulence properties of isolates from human infection and from food and environment samples have been widely studied.10–12 Although few antibiotics have bactericidal activity against L. monocytogenes,13 clinical isolates are generally considered to be susceptible to a wide range of antimicrobial agents, such as penicillins, aminoglycosides, trimethoprim, tetracycline, macrolides, and vancomycin.14 Beta-lactams, such as penicillin G and ampicillin, alone or in combination with gentamicin, represent the first choice in the treatment of human listeriosis but trimethoprim–sulphamethoxazole (TMP–SMX), vancomycin, and erythromycin can also be used.2,14 Presently, the proportion of antibiotic-resistant strains of L. monocytogenes remains low worldwide; in the last decades however an overall increase in the incidence of resistance to tetracyclines, fluoroquinolones, streptomycin, chloramphenicol, macrolides,
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and trimethoprim has been reported.15,16 Moreover, isolates with innate reduced susceptibility or resistance to cephalosporins, fosfomycin, and first generation quinolones have been described.2,14,16 The emergence of resistant strains has been attributed to the acquisition by L. monocytogenes of known antibiotic resistance genes principally from enterococci and streptococci within the gastrointestinal tract.16–18 Comparison of L. monocytogenes antimicrobial susceptibility results, reported by previous studies, has however been hindered by the number of variables in the testing methods as well as to the different interpretive criteria used. Broth dilution tests, disc-diffusion tests, and automated systems are the most commonly reported methodologies for studying antimicrobial susceptibility.19 Among available tests, the broth dilution method for determining minimum inhibitory concentration (MIC) represents the gold standard for evaluating the antibacterial activity of antimicrobial agents, allowing to define epidemiological, pharmacological, and clinical parameters.20 Data correlating susceptibility profile of L. monocytogenes to clinical outcome, pharmacokinetic/ pharmacodynamic parameters are still lacking in order to establish clinical breakpoints;20,21 neither Clinical and Laboratory Standards Institute (CLSI) nor European Committee on Antimicrobial Susceptibility Testing (EUCAST) presently provide comprehensive interpretative criteria for Listeria spp. susceptibility testing. A consultation on L. monocytogenes breakpoints definition, based on epidemiological cut-off, was proposed by EUCAST in October 2011, and susceptibility breakpoints for ampicillin, penicillin G, erythromycin, meropenem, and TMP–SMX are now available. Overall to ensure effective measures in the treatment and infection control of human listeriosis, a constant surveillance of potentially emerging antimicrobial resistance in this pathogen represents an important public health issue. In this study, we investigated the antimicrobial susceptibility of 96 human isolates of L. monocytogenes collected in northern Italy between 2008 and 2010. The International Standards Organization (ISO 207761:2006) broth microdilution method, as recommended by EUCAST for L. monocytogenes, was employed.22
Materials and Methods Bacterial strains Ninety-six L. monocytogenes strains consecutively isolated between 2008 and 2010 from human cases of listeriosis in the north of Italy were kindly supplied by the laboratory-based network of Lombardia region (the major northern Italian region, accounting for 16% of the Italian population). All these strains were previously characterized by means of serotyping, pulsed field gel electrophoresis, and multilocus sequence typing.23
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Streptococcus pneumoniae ATCC 49619 was used as internal quality control in antimicrobial susceptibility testing as recommended by EUCAST. A reference strain of L. monocytogenes NCTC11994, kindly provided by Unichim (Milano, Italy), was also included for susceptibility testing. All bacterial strains were subcultured on brain– heart infusion (BHI) (Becton Dickinson, Milano, Italy) broth and stored at 280uC.
Antimicrobial susceptibility testing The following antimicrobials were included in the analysis: penicillin G, amoxicillin, cefotaxime, meropenem, vancomycin, gentamicin, azithromycin, erythromycin, linezolid, ciprofloxacin, levofloxacin, tetracycline, chloramphenicol, and clindamycin (all from SigmaAldrich, Milano, Italy). Trimethoprim–sulphamethoxazole was also included and used in fixed combination (1 : 5) (Roche, Milano, Italy). Antimicrobial agents’ stock solutions were stored at -20uC and appropriately diluted before use. Minimum inhibitory concentrations of the above mentioned antimicrobials against L. monocytogenes clinical strains were determined using the broth microdilution method according to EUCAST recommendations for fastidious bacteria.22 Inocula were prepared in 5 ml BHI broth (Becton Dickinson, Milano, Italy) by inoculating four to five colonies from a pure culture on BHI agar, to avoid selection of atypical variants. Tubes were incubated at 37uC to obtain broth turbidity equivalent to 0.5 McFarland standard in order to contain approximately 1.56108 cfu/ml. Inocula were further diluted in fresh cation-adjusted Mueller–Hinton broth (CAMHB) (Becton Dickinson, Milano, Italy), supplemented with 5% lysed horse blood (Oxoid, Milano, Italy) and 20 mg/ml beta-NAD (Serva, Euroclone, Milano, Italy) to obtain a final inoculum containing approximately 56105 cfu/ml. Viable counts were randomly performed, by plating an aliquot of the diluted inoculum suspension on BHI agar, in order to confirm the number of colony forming units in the final inocula for MIC determination. Fifty microliters of the final bacterial inoculum was added to U-bottom 96well microtitre plates containing 50 ml of the serially diluted antimicrobials, giving final concentrations ranging from 0.06 to 128 mg/ml. Streptococcus pneumoniae ATCC 49619 was used as control reference strain for antimicrobial susceptibility testing, as recommended by EUCAST. After incubation at 37uC for 16–20 hours the MIC endpoints were defined as the lowest antimicrobial concentration resulting in the absence of visible bacterial growth, as indicated by the lack of turbidity. Minimum inhibitory concentration values were interpreted using available EUCAST L. monocytogenes MIC clinical susceptibility breakpoints for penicillin G, erythromycin, and meropenem, equal
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or below 1.0, 0.25, and 1.0 mg/ml, respectively. The same document reports epidemiological cut-off values (ECOFF) based on MIC distribution collated data from multiple sources, geographical areas, and time periods. Values of ECOFF #4 mg/ml and #1 mg/ml are proposed for linezolid and tetracycline, respectively.24 MIC50 and MIC90 values were calculated, encompassing on the basis MIC values for § found to inhibit 50 and 90% of the isolates tested. To verify the bactericidal activity of the tested antibiotics towards L. monocytogenes strains, drops of 10 ml were collected from wells corresponding to concentration 16 MIC, 26 MIC, and 46 MIC wells (three replicates for each), plated on BHI agar without antimicrobials and further incubated for 30 hours. The antibiotics amoxicillin, gentamicin, and levofloxacin, known for their bactericidal activity, were investigated. Results were recorded as presence or absence of growth.
Antimicrobial susceptibility of L. monocytogenes
erythromycin, according to EUCAST breakpoints. Tested TMP–SMX (ratio 1 : 5) demonstrated MIC90#0.06 mg/ml; results however could not be interpreted with the available EUCAST breakpoints, as guidelines refer to the compounds at a fixed combination 1 : 19. For most of the other investigated antimicrobials, the lack of available clinical breakpoints did not allow to define susceptibility. However 2 out of the 96 strains tested showed MIC values for tetracycline above the ECOFF (#1 mg/ml). Amongst the other tested antimicrobials, cefotaxime demonstrated a wide distribution of MIC values, ranging from 0.06 to 32 mg/ml, with 85% of strains showing MIC values §2 mg/ml and MIC90516 mg/ml (Table 1). Similarly broader MICs distributions were observed for linezolid and chloramphenicol, with MIC9054 mg/ml. For the other antimicrobials agents MIC distributions appeared to be within a narrow range, with relatively low MIC50 and MIC90 values. The association between the previously described 12 molecular subtype clusters of the studied L. monocytogenes isolates23 and a reduced susceptibility to the tested antimicrobials (MICs.MIC90) was evaluated (Table 2). The bactericidal activity of amoxicillin, gentamicin, and levofloxacin against the 96 human strains of L. monocytogenes was evaluated following 18–24 hours incubation at 16 MIC, 26 MIC, and 46 MIC concentrations. Percentages of 22%, 49%, and 80% of the bacterial strains exposed to amoxicillin from wells corresponding to 16 MIC, 26 MIC, and 46 MIC,
Results Susceptibility of 96 L. monocytogenes human isolates against the 15 antimicrobial agents tested is shown in Table 1. The MIC distributions for each antimicrobial agent is illustrated as the number of isolates showing specific MIC values, as well as indicating MIC range, MIC50 and MIC90. Listeria monocytogenes NCTC 11994 reference strain was shown to be susceptible to penicillin G (MIC#0.06 mg/ml), meropenem (MIC50.12 mg/ml), and erythromycin (MIC#0.06 mg/ml). All 96 human L. monocytogenes isolates tested were found to be susceptible to penicillin G, meropenem, and
Table 1 Minimum inhibitory concentrations’ distribution, MIC range, MIC50 and MIC90 of Listeria monocytogenes human isolates, against a panel of 15 antimicrobials Number of isolates with MIC (mg/ml) of
Antibiotic
Lmo NCTC #0.06 0.12 0.25 0.5 11994a
Penicillin G Amoxicillin Cefotaxime Meropenem Vancomycin Gentamicin TMP–SMX (1 : 5) Azithromycin Erythromycin Linezolid Ciprofloxacin Levofloxacin Tetracycline Chloramphenicol Clindamycin
#0.06 0.12 4 0.12 1 0.12 #0.06 0.5 #0.06 2 0.5 0.5 0.25 4 0.25
84 61 6 81 0 41 87 35 82 12 2 47
9 25 3 12 2 34 6 16 8 7 11 8 13
34
18
3 7 2 3 18 10 2 17 3 15 30 16 24 1 24
2 1
1
2
4
b
8 16 32 64 128 MIC range MIC50 MIC90 S#
1 6 17 22 31 8
31 38 7 6 4 1 1 21 7 2 1 21 16 18 19 36 6 1 43 22 5 9 1 1 1 7 44 37 14 4 2
6
1
#0.06–0.25 #0.06–1 #0.06–32 #0.06–0.25 0.125–2 #0.06–2 #0.06–0.5 #0.06–1 #0.06–1 0.125–4 #0.06–2 #0.06–2 #0.06–4 0.25–16 #0.06–2
Sc (ECOFF) #
#0.06 #0.06 1.0 #0.06 0.12 NA 8 16 NA #0.06 #0.06 0.25 0.5 1 NA 0.12 0.50 NA #0.06 #0.06 0.064d #0.06 0.5 NA #0.06 0.12 1.0 1 4 NA 0.25 0.5 NA 0.5 1 NA #0.06 0.25 NA 4 4 NA 0.12 0.5 NA
1.0 NA NA 0.25 NA NA 0.064d NA 1.0 4.0 NA NA 1.0 NA NA
NA: not available. Bold numbers indicate isolates with minimum inhibitory concentration (MIC) values .MIC90. a MIC values of L. monocytogenes NCTC 11994 reference standard are reported. b Susceptibility according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints. c EUCAST epidemiological cut-off values (ECOFF) based on MIC distribution collated data from multiple sources, geographical areas, and time periods.24 d MIC breakpoint for concentrations of trimethoprim–sulphamethoxazole (TMP–SMX) ratio of 1 : 19.
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resistance determinants. Furthermore, the same study reported two strains cross-resistant to streptomycin, sulphamethoxazole, and kanamycin, one to streptomycin, sulphamethoxazole, kanamycin, and rifampin, and one to the latter four antibiotics as well as to erythromycin and chloramphenicol out of 98 food strains isolated in Italy in 1991; plasmid resistance determinants were demonstrated in three out of four of these resistant isolates.26 Aureli et al.27 described the susceptibility of 148 strains isolated from food in Italy, to antimicrobials used in veterinary and human therapy, by means of standard agar dilution and discdiffusion methods. In general, all strains were susceptible to the tested antibiotics with the exception of phosphomycin, lincomycin, and flumequine to which they were all resistant. More recently, Conter18 reported susceptibility data on 120 L. monocytogenes strains isolated in Italy from food and food-processing environments, performed by using the automated VITEK2 system, to 19 antibiotics currently used in veterinary and human therapy. Among the tested strains, 12% displayed resistance to at least one antibiotic. Resistance to clindamycin was found to be the most common, followed by that to linezolid and ciprofloxacin. In our study, the antimicrobial susceptibility of 96 human strains of this foodborne pathogen was evaluated by means of the standardized EUCAST protocol for MIC determinations. Minimum inhibitory concentration obtained in the present study were interpreted by the now available EUCAST breakpoints for L. monocytogenes24 as compared to previous studies in which Listeria susceptibility data were evaluated using Staphylococcus spp. and Enterococcus spp. breakpoints, as suggested by Aureli and coauthors.27 Using EUCAST criteria, no isolates were found to be resistant to penicillin G, meropenem, and erythromycin. For all other antimicrobial agents tested, although no breakpoints are
respectively, failed to grow even in the absence of antimicrobials. Similarly 19%, 55%, and 85% of the strains exposed to gentamicin and 40%, 86%, and 98% of those exposed to levofloxacin, from 16 MIC, 26 MIC, and 46 MIC wells failed to grow, respectively.
Discussion In northern Italy, the number of human listeriosis cases, reported by the local surveillance system, has shown a marked increased in incidence during the last decade thus calling for a continued surveillance to monitor the emergence of antimicrobial resistance. Antimicrobial resistance of human isolates of Listeria monocytogenes represents an important public health issue, particularly in view of the rise in antibiotic resistance for the treatment of severe systemic disease in immunocompromised patients, recently reported for this pathogen.2,14–16 Critical aspects in performing and comparing results of antimicrobial susceptibility tests are the standardization of methods and the availability of international guidelines for results’ interpretation. Susceptibility breakpoint setting is not an exact science, as widely reviewed,21 but determining MIC distributions using standardized methods represents the first step in defining susceptibility breakpoints and ECOFF.25 Getting together, susceptibility results for specific bacterial pathogens, from different laboratories that use standardized methods, is very important not only to define breakpoints but also to monitor the spread of antimicrobial resistance. To our knowledge, few studies have reported MIC distributions of L. monocytogenes strains circulating in Italy, particularly for human isolates. A study conducted in Italy from 1987 to 1991, using disc and agar dilution assays, revealed that 2 out of 54 human isolates were resistant to streptomycin (MIC564 mg/ ml), as a result of the acquisition of plasmid-encoded
Table 2 Molecular subtype clusters of Listeria monocytogenes of isolates with MIC values .MIC90 Cluster1
Antimicrobial agents (MIC90 values, mg/ml)
Number of isolates Penicillin G (.0.06) Amoxicillin (0.12) Cefotaxime (16) Meropenem (.0.06) Vancomycin (1) Gentamicin (0. 5) TMP–SMX(1 : 5) (0.06) Azithromycin (0.5) Erythromycin (0.12) Ciprofloxacin (0.5) Levofloxacin (1) Tetracycline (0.25) Chloramphenicol (4) Clindamycin (0.5)
1
1
2
3
4
5
6
7
8
9
10
11
12
Other2
3 1 1 – – 1 – – – – 2 1 – – –
10 – 1 – 2 1 – 1 1 – – – – – 1
6 1 – – 3 1 – 1 1 1 – – – – –
7 – 1 1 2 1 1 – – – 2 – – – –
7 2 2 1 1 – – – 1 – – – – – 1
1 – – – – – – 1 – – – – – – –
3 – 1 – – – – – 1 1 – – – – –
3 – – – – – – – 1 – – – 1 1 –
5 1 – 1 – – – – 1 – – 1 2 1 –
3 – – – – – – – – – – – – – –
28 3 1 3 2 – 2 3 1 2 – 2 3 1 1
4 – 1 – 1 1 – 1 – – – – 1 – –
19 4 2 2 4 2 2 2 – 2 3 1 5 4 3
For the naming of clusters see previous publication (23). ,2 isolates belonging to same pulsotype. No strains were found to have MIC values . MIC90 for Linezolid. 2
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available, the relatively low MIC values obtained against the tested L. monocytogenes strains suggest susceptibility to the tested agents. A correlation between some L. monocytogenes serotypes and antimicrobial resistance has been previously reported although no explanation for these observations has been proposed.28 For instance, Barbosa found that resistance to erythromycin was correlated with the serogroup IVb, while no correlation was observed between ciprofloxacin resistance and L. monocytogenes serotypes.29 On the contrary, Kovacevic16 found that serotype 1/2a isolates were more frequently resistant to ciprofloxacin compared to serotype 4b. In this study, no correlation was found between L. monocytogenes clonal clusters and a reduced susceptibility to the tested antimicrobials (MICs.MIC90), based on previously published data on the molecular characterization of the tested isolates.23 Listeria monocytogenes isolates, showing higher MIC values, were found to be heterogeneously distributed amongst the commonly identified clonal clusters with several isolates belonging to cluster 11, which however represents the most common molecular subtype circulating in our geographical area.23 In general, the overall MIC distribution range obtained in this study was comparable to L. monocytogenes susceptibility data available from EUCAST,22 which includes multiple sources, geographical areas, time periods, as well as wider antimicrobial concentration’s intervals than those selected for this study (0.002–512 mg/ml). Our findings suggest that there is no significant difference in the antimicrobial susceptibility of L. monocytogenes strains collected from human cases in north Italy and that previously described in isolates from other geographical areas in Europe. The temporal evolution of resistance to erythromycin, chloramphenicol, ciprofloxacin, and tetracycline was analyzed in a large study performed in France on 4816 clinical L. monocytogenes strains isolated between 1926 and 2007. The prevalence of resistant strains was low, estimated at 1.27% among human isolates, although resistance to tetracyclines and fluoroquinolones was found to be emerging.15 Recently, Barbosa reported susceptibility data obtained from human (n595) and food (n5353) strains isolated in Portugal between 2003 and 2007, by means of agar microdilution method. In this study, resistance to at least one of the tested antibiotics: nitrofurantoin, ciprofloxacin, erythromycin, tetracycline, gentamicin, and rifampicin was found in both food (21.0%) and clinical (28.3%) isolates. Resistance to two or more antimicrobials was demonstrated only for food isolates (1.8%).28 Kovacevic,16 in a study conducted in Canada on 54 L. monocytogenes strains, isolated from food and
Antimicrobial susceptibility of L. monocytogenes
food-processing environments, reported resistance to cefoxitin (98%), ciprofloxacin (7%), clindamycin (33%), and tetracycline (6%). Furthermore, reduced susceptibility was observed in linezolid (6%), rifampicin (2%), streptomycin (6%), ciprofloxacin (67%), and clindamycin (65%) according to CLSI guidelines.16 Although, no resistant strains were demonstrated in our study, the wider MICs distribution of L. monocytogenes against cephalosporin (cefotaxime), is in keeping with data from a previous study,28 performed on strains isolated from patients undergoing cancer treatment at a Cancer Centre in New York from 1991 to 1997. Listeria monocytogenes strains isolated from these patients were susceptible to a wide variety of antimicrobial agents, although resistance to broad-spectrum cephalosporin antibiotics was consistently observed.29 The bactericidal activity of amoxicillin, gentamicin, and levofloxacin evaluated in this study suggests that concentrations higher than MIC values were generally necessary to obtain bacterial killing. This preliminary finding supports the high capacity of L. monocytogenes to generally tolerate and survive the action of bactericidal antimicrobial agents. This intracellular bacteria pathogen is able to survive cellular oxidative stress, probably as a consequence of its interrupted tricarboxylic acid (TCA) cycle, resulting in its reduced sensitivity to bactericidal antibiotics.13 Further investigations might be useful to better elucidate the mechanisms implicated in such intrinsic resistance. Acquisition of self-transferable, mobile, and conjugative genetic elements has also been associated with the emergence of antimicrobials resistance in this pathogen. These genes have been reported to have spread to L. monocytogenes through mobile genetic elements, such as transposons and plasmids, within the gastrointestinal tract.16–18 In spite of the low prevalence of antimicrobial resistance presently observed in human strains of L. monocytogenes isolated in our geographical area, the emergence of resistant strains worldwide underlines the need for a continuous surveillance for this pathogen. Moreover, new susceptibility data for human strains of L. monocytogenes can provide useful information for epidemiological and public health purposes.
Disclaimer Statements Contributors MM carried out the antimicrobial susceptibility experiments, analyzed the data, interpreted the results, and wrote the manuscript. RM contributed to the study coordination. AMLC contributed to perform antimicrobial susceptibility testing. EA contributed to the laboratory analysis of clinical isolates and managed the specific database. MMP conceived and coordinated the study, provided
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constructive comments, revised the first draft of the paper critically, and approved the final version of the paper. CEC conceived and coordinated the study, coworked on associated data interpretation, and coauthored the manuscript. All authors discussed the results, interpretation, and commented on the manuscript at all stages. Funding PROGRAMMA CCM 2001 Ministero della Salute - Project title ‘‘Invasive human Listeriosis: set up of an integrated surveilance network’’ coordinated by Prof. Mirella Maria Pontello. Conflicts of interest The authors declare that there are no conflicts of interest. Ethics approval Ethical approval was not required from the Ethics Committee.
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12 Lomonaco S, Verghese B, Gerner-Smidt P, Tarr C, Gladney L, Joseph L, et al. Novel epidemic clones of Listeria monocytogenes, United States, 2011. Emerging Infect Dis. 2013;19:147–50. 13 Feld L, Knudsen GM, Gram L. Bactericidal antibiotics do not appear to cause oxidative stress in Listeria monocytogenes. Appl Environ Microbiol. 2012;78(12):4353–7. 14 Swaminathan B, Gerner-Smidt P. The epidemiology of human listeriosis. Microbes Infect. 2007;9:1236–43. 15 Morvan A, Moubareck C, Leclercq A, Herve´-Bazin M, Bremont S, Lecuit M, et al. Antimicrobial resistance of Listeria monocytogenes strains isolated from humans in France. Antimicrob Agents Chemother. 2010;54(6):2728–31. 16 Kovacevic J, Sagert J, Wozniak A, Gilmour MW, Allen KJ. Antimicrobial resistance and co-selection phenomenon in Listeria spp. recovered from food and food production environments. Food Microbiol. 2013;34:319–27. 17 Charpentier E, Courvalin P. Antibiotic resistance in Listeria spp. Antimicrob Agents Chemother. 1999;43(9):2103–8. 18 Conter M, Paludi D, Zanardi E, Ghidini S, Vergara A, Ianieri A. Characterization of antimicrobial resistance of foodborne Listeria monocytogenes. Int J Food Microbiol. 2009;128:497– 500. 19 Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis. 2009;49:1749–55. 20 Cornaglia G, Hryniewicz W, Jarlier V, Kahlmeter G, Mittermayer H, Stratchounski L, et al. European recommendations for antimicrobial resistance surveillance. Clin Microbiol Infect. 2004;10:349–83. 21 Turnidge J, Paterson DL. Setting and revising antibacterial susceptibility breakpoints. Clin Microbiol Rev. 2007;20(3):391–408. 22 European Committee on Antimicrobial Susceptibility Testing. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth microdilution. EUCAST Discussion Document E.Def 5.1. 2003. 23 Mammina C, Parisi A, Guaita A, Aleo A, Bonura C, Nastasi A, et al. Enhanced surveillance of invasive listeriosis in the Lombardy region, Italy, in the years 2006–2010 reveals major clones and an increase in serotype 1/2a. BMC Infect Dis. 2013;13:152. 24 European Committee on Antimicrobial Susceptibility Testing. Antimicrobial wild type distributions of microorganisms. Available from: http://mic.eucast.org/Eucast2/SearchController/ search.jsp?action5performSearch&BeginIndex50&Micdif5mic &NumberIndex550&Antib5-1&Specium5220, May 2007. 25 Leclercq R, Canton R, Brown DF, Giske CG, Heisig P, MacGowan AP, et al. EUCAST expert rules in antimicrobial susceptibility testing. Clin Microbiol Infect. 2013;19(2):141–60. 26 Facinelli B, Giovanetti E, Varaldo PE, Casolari P, Fabio U. Antibiotic resistance in foodborne listeria. Lancet. 1991;338:1272. 27 Aureli P, Ferrini AM, Mannoni V, Hodzic S, WedellWeergaard C, Oliva B. Susceptibility of Listeria monocytogenes isolated from food in Italy to antibiotics. Int J Food Microbiol. 2003;83(3):325–30. 28 Barbosa J, Magalha˜es R, Santos I, Ferreira V, Branda˜o TR, Silva J, et al. Evaluation of antibiotic resistance patterns of food and clinical Listeria monocytogenes isolates in Portugal. Foodborne Pathog Dis. 2013;10(10):1–6. 29 Safdar A, Armstrong D. Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a comprehensive cancer center (1955–1997). J Clin Microbiol. 2003;41(1):483–5.
Antimicrobial susceptibility of Listeria monocytogenes isolates from human cases in northern Italy, 2008–2010: MIC determination according, to EUCAST broth microdilution method* { Moira Madeo1, Rosario Musumeci1, Anna Maria Laura Careddu1, Ettore Amato2, Mirella Maria Pontello2, Clementina Elvezia Cocuzza1 1
Dipartimento di Chirurgia e Medicina Traslazionale, Universita` di Milano-Bicocca, Monza, MB, Italy, Dipartimento di Scienze della Salute, Universita` degli Studi di Milano, MI, Italy
2
Susceptibility of 96 Listeria monocytogenes human isolates collected in northern Italy between 2008 and 2010, to 15 antimicrobials, was investigated. Minimum inhibitory concentration (MIC) was evaluated by means of the standardized broth microdilution method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) international guidelines. All L. monocytogenes human isolates were susceptible to penicillin G (MIC90#0.06 mg/ml), meropenem (MIC90#0.06 mg/ml), and erythromycin (MIC9050.12 mg/ml). Susceptibility to the other tested antimicrobials could not be interpreted due to the lack of breakpoint values although two (2%) isolates were shown to have tetracycline MICs above EUCAST epidemiological cut-off values (ECOFF). Bactericidal activity for amoxicillin, gentamicin, and levofloxacin was generally observed at concentrations 2–4 times higher than MIC values. Though L. monocytogenes human strains, isolated in the north of Italy, appear to be susceptible to most antimicrobial agents used in human therapy, this study provides new data for epidemiological surveillance and clinical breakpoints definition. Keywords: Listeria monocytogenes, Antimicrobials susceptibility, Minimum inhibitory concentration, Bactericidal activity, EUCAST guidelines
Introduction Listeria monocytogenes is a facultative intracellular pathogen causing a broad spectrum of clinical manifestations: gastroenteritis in immunocompetent people, and septicaemia, meningitis, encephalitis, and/or brain abscesses in immunocompromised subjects;1,2 furthermore, infections during pregnancy can lead to miscarriage, stillbirth, premature delivery, or life-threatening infection of the newborn (pregnancyassociated listeriosis).3 Its case fatality rate ranges between 20 and 50% accounting for approximately 30% of all foodborne diseases associated deaths.4–6 The incidence of invasive listeriosis reported from different countries varies from 0.32 to 1 cases per 100 000 population and since 2000 an increase in the * The material is original and has not been submitted elsewhere. { The corresponding author, on behalf of all co-authors, has read and agreed to the Journal of Chemotherapy terms on data protection notice.
Correspondence to: Moira Madeo, Dipartimento di Chirurgia e Medicina Traslazionale, Universita` di Milano-Bicocca, Via Cadore 48, 20900 Monza, MB, Italy. Email: [email protected]
ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000187
incidence of human listeriosis has been reported in several European countries.7–9 Clonal diversity and virulence properties of isolates from human infection and from food and environment samples have been widely studied.10–12 Although few antibiotics have bactericidal activity against L. monocytogenes,13 clinical isolates are generally considered to be susceptible to a wide range of antimicrobial agents, such as penicillins, aminoglycosides, trimethoprim, tetracycline, macrolides, and vancomycin.14 Beta-lactams, such as penicillin G and ampicillin, alone or in combination with gentamicin, represent the first choice in the treatment of human listeriosis but trimethoprim–sulphamethoxazole (TMP–SMX), vancomycin, and erythromycin can also be used.2,14 Presently, the proportion of antibiotic-resistant strains of L. monocytogenes remains low worldwide; in the last decades however an overall increase in the incidence of resistance to tetracyclines, fluoroquinolones, streptomycin, chloramphenicol, macrolides,
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and trimethoprim has been reported.15,16 Moreover, isolates with innate reduced susceptibility or resistance to cephalosporins, fosfomycin, and first generation quinolones have been described.2,14,16 The emergence of resistant strains has been attributed to the acquisition by L. monocytogenes of known antibiotic resistance genes principally from enterococci and streptococci within the gastrointestinal tract.16–18 Comparison of L. monocytogenes antimicrobial susceptibility results, reported by previous studies, has however been hindered by the number of variables in the testing methods as well as to the different interpretive criteria used. Broth dilution tests, disc-diffusion tests, and automated systems are the most commonly reported methodologies for studying antimicrobial susceptibility.19 Among available tests, the broth dilution method for determining minimum inhibitory concentration (MIC) represents the gold standard for evaluating the antibacterial activity of antimicrobial agents, allowing to define epidemiological, pharmacological, and clinical parameters.20 Data correlating susceptibility profile of L. monocytogenes to clinical outcome, pharmacokinetic/ pharmacodynamic parameters are still lacking in order to establish clinical breakpoints;20,21 neither Clinical and Laboratory Standards Institute (CLSI) nor European Committee on Antimicrobial Susceptibility Testing (EUCAST) presently provide comprehensive interpretative criteria for Listeria spp. susceptibility testing. A consultation on L. monocytogenes breakpoints definition, based on epidemiological cut-off, was proposed by EUCAST in October 2011, and susceptibility breakpoints for ampicillin, penicillin G, erythromycin, meropenem, and TMP–SMX are now available. Overall to ensure effective measures in the treatment and infection control of human listeriosis, a constant surveillance of potentially emerging antimicrobial resistance in this pathogen represents an important public health issue. In this study, we investigated the antimicrobial susceptibility of 96 human isolates of L. monocytogenes collected in northern Italy between 2008 and 2010. The International Standards Organization (ISO 207761:2006) broth microdilution method, as recommended by EUCAST for L. monocytogenes, was employed.22
Materials and Methods Bacterial strains Ninety-six L. monocytogenes strains consecutively isolated between 2008 and 2010 from human cases of listeriosis in the north of Italy were kindly supplied by the laboratory-based network of Lombardia region (the major northern Italian region, accounting for 16% of the Italian population). All these strains were previously characterized by means of serotyping, pulsed field gel electrophoresis, and multilocus sequence typing.23
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Streptococcus pneumoniae ATCC 49619 was used as internal quality control in antimicrobial susceptibility testing as recommended by EUCAST. A reference strain of L. monocytogenes NCTC11994, kindly provided by Unichim (Milano, Italy), was also included for susceptibility testing. All bacterial strains were subcultured on brain– heart infusion (BHI) (Becton Dickinson, Milano, Italy) broth and stored at 280uC.
Antimicrobial susceptibility testing The following antimicrobials were included in the analysis: penicillin G, amoxicillin, cefotaxime, meropenem, vancomycin, gentamicin, azithromycin, erythromycin, linezolid, ciprofloxacin, levofloxacin, tetracycline, chloramphenicol, and clindamycin (all from SigmaAldrich, Milano, Italy). Trimethoprim–sulphamethoxazole was also included and used in fixed combination (1 : 5) (Roche, Milano, Italy). Antimicrobial agents’ stock solutions were stored at -20uC and appropriately diluted before use. Minimum inhibitory concentrations of the above mentioned antimicrobials against L. monocytogenes clinical strains were determined using the broth microdilution method according to EUCAST recommendations for fastidious bacteria.22 Inocula were prepared in 5 ml BHI broth (Becton Dickinson, Milano, Italy) by inoculating four to five colonies from a pure culture on BHI agar, to avoid selection of atypical variants. Tubes were incubated at 37uC to obtain broth turbidity equivalent to 0.5 McFarland standard in order to contain approximately 1.56108 cfu/ml. Inocula were further diluted in fresh cation-adjusted Mueller–Hinton broth (CAMHB) (Becton Dickinson, Milano, Italy), supplemented with 5% lysed horse blood (Oxoid, Milano, Italy) and 20 mg/ml beta-NAD (Serva, Euroclone, Milano, Italy) to obtain a final inoculum containing approximately 56105 cfu/ml. Viable counts were randomly performed, by plating an aliquot of the diluted inoculum suspension on BHI agar, in order to confirm the number of colony forming units in the final inocula for MIC determination. Fifty microliters of the final bacterial inoculum was added to U-bottom 96well microtitre plates containing 50 ml of the serially diluted antimicrobials, giving final concentrations ranging from 0.06 to 128 mg/ml. Streptococcus pneumoniae ATCC 49619 was used as control reference strain for antimicrobial susceptibility testing, as recommended by EUCAST. After incubation at 37uC for 16–20 hours the MIC endpoints were defined as the lowest antimicrobial concentration resulting in the absence of visible bacterial growth, as indicated by the lack of turbidity. Minimum inhibitory concentration values were interpreted using available EUCAST L. monocytogenes MIC clinical susceptibility breakpoints for penicillin G, erythromycin, and meropenem, equal
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or below 1.0, 0.25, and 1.0 mg/ml, respectively. The same document reports epidemiological cut-off values (ECOFF) based on MIC distribution collated data from multiple sources, geographical areas, and time periods. Values of ECOFF #4 mg/ml and #1 mg/ml are proposed for linezolid and tetracycline, respectively.24 MIC50 and MIC90 values were calculated, encompassing on the basis MIC values for § found to inhibit 50 and 90% of the isolates tested. To verify the bactericidal activity of the tested antibiotics towards L. monocytogenes strains, drops of 10 ml were collected from wells corresponding to concentration 16 MIC, 26 MIC, and 46 MIC wells (three replicates for each), plated on BHI agar without antimicrobials and further incubated for 30 hours. The antibiotics amoxicillin, gentamicin, and levofloxacin, known for their bactericidal activity, were investigated. Results were recorded as presence or absence of growth.
Antimicrobial susceptibility of L. monocytogenes
erythromycin, according to EUCAST breakpoints. Tested TMP–SMX (ratio 1 : 5) demonstrated MIC90#0.06 mg/ml; results however could not be interpreted with the available EUCAST breakpoints, as guidelines refer to the compounds at a fixed combination 1 : 19. For most of the other investigated antimicrobials, the lack of available clinical breakpoints did not allow to define susceptibility. However 2 out of the 96 strains tested showed MIC values for tetracycline above the ECOFF (#1 mg/ml). Amongst the other tested antimicrobials, cefotaxime demonstrated a wide distribution of MIC values, ranging from 0.06 to 32 mg/ml, with 85% of strains showing MIC values §2 mg/ml and MIC90516 mg/ml (Table 1). Similarly broader MICs distributions were observed for linezolid and chloramphenicol, with MIC9054 mg/ml. For the other antimicrobials agents MIC distributions appeared to be within a narrow range, with relatively low MIC50 and MIC90 values. The association between the previously described 12 molecular subtype clusters of the studied L. monocytogenes isolates23 and a reduced susceptibility to the tested antimicrobials (MICs.MIC90) was evaluated (Table 2). The bactericidal activity of amoxicillin, gentamicin, and levofloxacin against the 96 human strains of L. monocytogenes was evaluated following 18–24 hours incubation at 16 MIC, 26 MIC, and 46 MIC concentrations. Percentages of 22%, 49%, and 80% of the bacterial strains exposed to amoxicillin from wells corresponding to 16 MIC, 26 MIC, and 46 MIC,
Results Susceptibility of 96 L. monocytogenes human isolates against the 15 antimicrobial agents tested is shown in Table 1. The MIC distributions for each antimicrobial agent is illustrated as the number of isolates showing specific MIC values, as well as indicating MIC range, MIC50 and MIC90. Listeria monocytogenes NCTC 11994 reference strain was shown to be susceptible to penicillin G (MIC#0.06 mg/ml), meropenem (MIC50.12 mg/ml), and erythromycin (MIC#0.06 mg/ml). All 96 human L. monocytogenes isolates tested were found to be susceptible to penicillin G, meropenem, and
Table 1 Minimum inhibitory concentrations’ distribution, MIC range, MIC50 and MIC90 of Listeria monocytogenes human isolates, against a panel of 15 antimicrobials Number of isolates with MIC (mg/ml) of
Antibiotic
Lmo NCTC #0.06 0.12 0.25 0.5 11994a
Penicillin G Amoxicillin Cefotaxime Meropenem Vancomycin Gentamicin TMP–SMX (1 : 5) Azithromycin Erythromycin Linezolid Ciprofloxacin Levofloxacin Tetracycline Chloramphenicol Clindamycin
#0.06 0.12 4 0.12 1 0.12 #0.06 0.5 #0.06 2 0.5 0.5 0.25 4 0.25
84 61 6 81 0 41 87 35 82 12 2 47
9 25 3 12 2 34 6 16 8 7 11 8 13
34
18
3 7 2 3 18 10 2 17 3 15 30 16 24 1 24
2 1
1
2
4
b
8 16 32 64 128 MIC range MIC50 MIC90 S#
1 6 17 22 31 8
31 38 7 6 4 1 1 21 7 2 1 21 16 18 19 36 6 1 43 22 5 9 1 1 1 7 44 37 14 4 2
6
1
#0.06–0.25 #0.06–1 #0.06–32 #0.06–0.25 0.125–2 #0.06–2 #0.06–0.5 #0.06–1 #0.06–1 0.125–4 #0.06–2 #0.06–2 #0.06–4 0.25–16 #0.06–2
Sc (ECOFF) #
#0.06 #0.06 1.0 #0.06 0.12 NA 8 16 NA #0.06 #0.06 0.25 0.5 1 NA 0.12 0.50 NA #0.06 #0.06 0.064d #0.06 0.5 NA #0.06 0.12 1.0 1 4 NA 0.25 0.5 NA 0.5 1 NA #0.06 0.25 NA 4 4 NA 0.12 0.5 NA
1.0 NA NA 0.25 NA NA 0.064d NA 1.0 4.0 NA NA 1.0 NA NA
NA: not available. Bold numbers indicate isolates with minimum inhibitory concentration (MIC) values .MIC90. a MIC values of L. monocytogenes NCTC 11994 reference standard are reported. b Susceptibility according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints. c EUCAST epidemiological cut-off values (ECOFF) based on MIC distribution collated data from multiple sources, geographical areas, and time periods.24 d MIC breakpoint for concentrations of trimethoprim–sulphamethoxazole (TMP–SMX) ratio of 1 : 19.
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resistance determinants. Furthermore, the same study reported two strains cross-resistant to streptomycin, sulphamethoxazole, and kanamycin, one to streptomycin, sulphamethoxazole, kanamycin, and rifampin, and one to the latter four antibiotics as well as to erythromycin and chloramphenicol out of 98 food strains isolated in Italy in 1991; plasmid resistance determinants were demonstrated in three out of four of these resistant isolates.26 Aureli et al.27 described the susceptibility of 148 strains isolated from food in Italy, to antimicrobials used in veterinary and human therapy, by means of standard agar dilution and discdiffusion methods. In general, all strains were susceptible to the tested antibiotics with the exception of phosphomycin, lincomycin, and flumequine to which they were all resistant. More recently, Conter18 reported susceptibility data on 120 L. monocytogenes strains isolated in Italy from food and food-processing environments, performed by using the automated VITEK2 system, to 19 antibiotics currently used in veterinary and human therapy. Among the tested strains, 12% displayed resistance to at least one antibiotic. Resistance to clindamycin was found to be the most common, followed by that to linezolid and ciprofloxacin. In our study, the antimicrobial susceptibility of 96 human strains of this foodborne pathogen was evaluated by means of the standardized EUCAST protocol for MIC determinations. Minimum inhibitory concentration obtained in the present study were interpreted by the now available EUCAST breakpoints for L. monocytogenes24 as compared to previous studies in which Listeria susceptibility data were evaluated using Staphylococcus spp. and Enterococcus spp. breakpoints, as suggested by Aureli and coauthors.27 Using EUCAST criteria, no isolates were found to be resistant to penicillin G, meropenem, and erythromycin. For all other antimicrobial agents tested, although no breakpoints are
respectively, failed to grow even in the absence of antimicrobials. Similarly 19%, 55%, and 85% of the strains exposed to gentamicin and 40%, 86%, and 98% of those exposed to levofloxacin, from 16 MIC, 26 MIC, and 46 MIC wells failed to grow, respectively.
Discussion In northern Italy, the number of human listeriosis cases, reported by the local surveillance system, has shown a marked increased in incidence during the last decade thus calling for a continued surveillance to monitor the emergence of antimicrobial resistance. Antimicrobial resistance of human isolates of Listeria monocytogenes represents an important public health issue, particularly in view of the rise in antibiotic resistance for the treatment of severe systemic disease in immunocompromised patients, recently reported for this pathogen.2,14–16 Critical aspects in performing and comparing results of antimicrobial susceptibility tests are the standardization of methods and the availability of international guidelines for results’ interpretation. Susceptibility breakpoint setting is not an exact science, as widely reviewed,21 but determining MIC distributions using standardized methods represents the first step in defining susceptibility breakpoints and ECOFF.25 Getting together, susceptibility results for specific bacterial pathogens, from different laboratories that use standardized methods, is very important not only to define breakpoints but also to monitor the spread of antimicrobial resistance. To our knowledge, few studies have reported MIC distributions of L. monocytogenes strains circulating in Italy, particularly for human isolates. A study conducted in Italy from 1987 to 1991, using disc and agar dilution assays, revealed that 2 out of 54 human isolates were resistant to streptomycin (MIC564 mg/ ml), as a result of the acquisition of plasmid-encoded
Table 2 Molecular subtype clusters of Listeria monocytogenes of isolates with MIC values .MIC90 Cluster1
Antimicrobial agents (MIC90 values, mg/ml)
Number of isolates Penicillin G (.0.06) Amoxicillin (0.12) Cefotaxime (16) Meropenem (.0.06) Vancomycin (1) Gentamicin (0. 5) TMP–SMX(1 : 5) (0.06) Azithromycin (0.5) Erythromycin (0.12) Ciprofloxacin (0.5) Levofloxacin (1) Tetracycline (0.25) Chloramphenicol (4) Clindamycin (0.5)
1
1
2
3
4
5
6
7
8
9
10
11
12
Other2
3 1 1 – – 1 – – – – 2 1 – – –
10 – 1 – 2 1 – 1 1 – – – – – 1
6 1 – – 3 1 – 1 1 1 – – – – –
7 – 1 1 2 1 1 – – – 2 – – – –
7 2 2 1 1 – – – 1 – – – – – 1
1 – – – – – – 1 – – – – – – –
3 – 1 – – – – – 1 1 – – – – –
3 – – – – – – – 1 – – – 1 1 –
5 1 – 1 – – – – 1 – – 1 2 1 –
3 – – – – – – – – – – – – – –
28 3 1 3 2 – 2 3 1 2 – 2 3 1 1
4 – 1 – 1 1 – 1 – – – – 1 – –
19 4 2 2 4 2 2 2 – 2 3 1 5 4 3
For the naming of clusters see previous publication (23). ,2 isolates belonging to same pulsotype. No strains were found to have MIC values . MIC90 for Linezolid. 2
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available, the relatively low MIC values obtained against the tested L. monocytogenes strains suggest susceptibility to the tested agents. A correlation between some L. monocytogenes serotypes and antimicrobial resistance has been previously reported although no explanation for these observations has been proposed.28 For instance, Barbosa found that resistance to erythromycin was correlated with the serogroup IVb, while no correlation was observed between ciprofloxacin resistance and L. monocytogenes serotypes.29 On the contrary, Kovacevic16 found that serotype 1/2a isolates were more frequently resistant to ciprofloxacin compared to serotype 4b. In this study, no correlation was found between L. monocytogenes clonal clusters and a reduced susceptibility to the tested antimicrobials (MICs.MIC90), based on previously published data on the molecular characterization of the tested isolates.23 Listeria monocytogenes isolates, showing higher MIC values, were found to be heterogeneously distributed amongst the commonly identified clonal clusters with several isolates belonging to cluster 11, which however represents the most common molecular subtype circulating in our geographical area.23 In general, the overall MIC distribution range obtained in this study was comparable to L. monocytogenes susceptibility data available from EUCAST,22 which includes multiple sources, geographical areas, time periods, as well as wider antimicrobial concentration’s intervals than those selected for this study (0.002–512 mg/ml). Our findings suggest that there is no significant difference in the antimicrobial susceptibility of L. monocytogenes strains collected from human cases in north Italy and that previously described in isolates from other geographical areas in Europe. The temporal evolution of resistance to erythromycin, chloramphenicol, ciprofloxacin, and tetracycline was analyzed in a large study performed in France on 4816 clinical L. monocytogenes strains isolated between 1926 and 2007. The prevalence of resistant strains was low, estimated at 1.27% among human isolates, although resistance to tetracyclines and fluoroquinolones was found to be emerging.15 Recently, Barbosa reported susceptibility data obtained from human (n595) and food (n5353) strains isolated in Portugal between 2003 and 2007, by means of agar microdilution method. In this study, resistance to at least one of the tested antibiotics: nitrofurantoin, ciprofloxacin, erythromycin, tetracycline, gentamicin, and rifampicin was found in both food (21.0%) and clinical (28.3%) isolates. Resistance to two or more antimicrobials was demonstrated only for food isolates (1.8%).28 Kovacevic,16 in a study conducted in Canada on 54 L. monocytogenes strains, isolated from food and
Antimicrobial susceptibility of L. monocytogenes
food-processing environments, reported resistance to cefoxitin (98%), ciprofloxacin (7%), clindamycin (33%), and tetracycline (6%). Furthermore, reduced susceptibility was observed in linezolid (6%), rifampicin (2%), streptomycin (6%), ciprofloxacin (67%), and clindamycin (65%) according to CLSI guidelines.16 Although, no resistant strains were demonstrated in our study, the wider MICs distribution of L. monocytogenes against cephalosporin (cefotaxime), is in keeping with data from a previous study,28 performed on strains isolated from patients undergoing cancer treatment at a Cancer Centre in New York from 1991 to 1997. Listeria monocytogenes strains isolated from these patients were susceptible to a wide variety of antimicrobial agents, although resistance to broad-spectrum cephalosporin antibiotics was consistently observed.29 The bactericidal activity of amoxicillin, gentamicin, and levofloxacin evaluated in this study suggests that concentrations higher than MIC values were generally necessary to obtain bacterial killing. This preliminary finding supports the high capacity of L. monocytogenes to generally tolerate and survive the action of bactericidal antimicrobial agents. This intracellular bacteria pathogen is able to survive cellular oxidative stress, probably as a consequence of its interrupted tricarboxylic acid (TCA) cycle, resulting in its reduced sensitivity to bactericidal antibiotics.13 Further investigations might be useful to better elucidate the mechanisms implicated in such intrinsic resistance. Acquisition of self-transferable, mobile, and conjugative genetic elements has also been associated with the emergence of antimicrobials resistance in this pathogen. These genes have been reported to have spread to L. monocytogenes through mobile genetic elements, such as transposons and plasmids, within the gastrointestinal tract.16–18 In spite of the low prevalence of antimicrobial resistance presently observed in human strains of L. monocytogenes isolated in our geographical area, the emergence of resistant strains worldwide underlines the need for a continuous surveillance for this pathogen. Moreover, new susceptibility data for human strains of L. monocytogenes can provide useful information for epidemiological and public health purposes.
Disclaimer Statements Contributors MM carried out the antimicrobial susceptibility experiments, analyzed the data, interpreted the results, and wrote the manuscript. RM contributed to the study coordination. AMLC contributed to perform antimicrobial susceptibility testing. EA contributed to the laboratory analysis of clinical isolates and managed the specific database. MMP conceived and coordinated the study, provided
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constructive comments, revised the first draft of the paper critically, and approved the final version of the paper. CEC conceived and coordinated the study, coworked on associated data interpretation, and coauthored the manuscript. All authors discussed the results, interpretation, and commented on the manuscript at all stages. Funding PROGRAMMA CCM 2001 Ministero della Salute - Project title ‘‘Invasive human Listeriosis: set up of an integrated surveilance network’’ coordinated by Prof. Mirella Maria Pontello. Conflicts of interest The authors declare that there are no conflicts of interest. Ethics approval Ethical approval was not required from the Ethics Committee.
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