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Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany Nicole Nari Horn a , Michael Kresken b,c , Barbara Körber-Irrgang b , Stephan Göttig a , Cornelia Wichelhaus d , Thomas A. Wichelhaus a,∗ , Working Party Antimicrobial Resistance of the Paul Ehrlich Society for Chemotherapy a
Institute of Medical Microbiology and Infection Control, Hospital of Goethe-University, Frankfurt am Main, Germany Antiinfectives Intelligence GmbH, Campus of the University of Applied Sciences, Rheinbach, Germany c Rheinische Fachhochschule Köln, Cologne, Germany d Institute of Applied Mathematics, University of Heidelberg, Germany b
a r t i c l e
i n f o
Article history: Received 2 January 2014 Received in revised form 4 March 2014 Accepted 10 April 2014 Keywords: Neisseria gonorrhoeae Antimicrobial resistance NG-MAST Gonorrhoea
a b s t r a c t Antimicrobial drug resistance in Neisseria gonorrhoeae has become an increasing public health problem. Hence, surveillance of resistance development is of crucial importance to implement adequate treatment guidelines. Data on the spread of antibiotic resistance among gonococcal isolates in Germany, however, is scarce. In a resistance surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send N. gonorrhoeae isolates to the study laboratory in Frankfurt am Main. Species verification was performed biochemically using ApiNH and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Etest method. For molecular epidemiological analysis, N. gonorrhoeae strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing. A total of 213 consecutive gonococcal isolates were analyzed in this nationwide study. Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (74%) and tetracycline (41%). Penicillin non-susceptibility was detected in 80% of isolates. The rate of azithromycin resistance was 6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone. Molecular typing of gonococcal isolates revealed a great heterogeneity of 99 different sequence types (ST), but ST1407 predominated (n = 39). This is the first comprehensive German multi-centre surveillance study on antibiotic susceptibility and molecular epidemiology of N. gonorrhoeae with implications for antibiotic choice for treatment of gonorrhoea. The World Health Organization supports the concept that an efficacious treatment of gonorrhoea results in at least 95% of infections being cured. Accordingly, as spectinomycin is not available on the German market, only the third generation cephalosporins cefixime and ceftriaxone are regarded as valuable drugs for empirical treatment of gonorrhoea in Germany. © 2014 Elsevier GmbH. All rights reserved.
Introduction Neisseria gonorrhoeae is one of the most frequent pathogens causing sexual transmitted diseases worldwide. According to the
∗ Corresponding author at: Institute of Medical Microbiology and Infection Control, Hospital of Goethe-University, Paul-Ehrlich-Straße 40, 60596 Frankfurt am Main, Germany. Tel.: +49 69 6301 6438; fax: +49 69 6301 5767. E-mail address: [email protected] (T.A. Wichelhaus).
latest WHO assessment, gonococcal infections represent 106 million of the estimated 499 million new cases of curable sexually transmitted infections (STI) that occur globally every year (WHO, 2008). Antimicrobial resistance in N. gonorrhoeae has progressively developed over the years and the emerging threat of untreatable gonococcal infection becomes apparent (Bolan et al., 2012; ECDC, 2012; Unemo and Nicholas, 2012a; Ison et al., 2013; WHO, 2012). The European surveillance report on antimicrobial resistance in N. gonorrhoeae reveals high rates of resistance to many previously recommended therapeutic agents in many European countries such
http://dx.doi.org/10.1016/j.ijmm.2014.04.001 1438-4221/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Horn, N.N., et al., Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.001
G Model IJMM-50809; No. of Pages 6
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as ciprofloxacin, tetracycline and penicillin (Cole et al., 2010). The report further concludes that continual surveillance of antimicrobial resistance is essential to monitor for increasing and emerging resistance to clinical relevant antibiotics and to adapt treatment guidelines accordingly (Cole et al., 2010). Hence, the present study was performed to provide first nationwide data on the epidemiology of antibiotic resistance among gonococcal isolates in Germany.
the age groups: 0–24 years, 25–34 years, 35–44 years and ≥45 years), were analyzed using the Fisher’s exact test with the exception for datasets where a cell equalled zero. In these cases, the Pearson, Mantel and Haenszel chi-squared test was applied using a continuity correction. Odds ratios (OR) and 95% confidence intervals (CI) are given. All tests were performed two-tailed, and a p-value < 0.05 was considered to be significant.
Materials and methods
Results
N. gonorrhoeae isolates
A total of 213 gonococcal isolates were analyzed in this nationwide study. Isolates were recovered from 179 males and 34 females. Mean age of the males and females was 31.9 years (median age: 31 years; range: 18–67 years) and 25.4 years (median age: 25 years; range: 18–55 years), respectively. The sites of isolation were urethra 69.0%, cervix 8.0%, rectum 3.8%, throat 0.5%, and 18.8% not reported.
In a prospective surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send consecutive N. gonorrhoeae isolates to the study laboratory at the Institute of Medical Microbiology and Infection Control at the Hospital of Goethe-University in Frankfurt am Main according to a study protocol. Data concerning participating laboratories with regard to location, type of laboratory and number of isolates provided are available as Supplementary Table 1. Informational data regarding gonococcal isolates, i.e. age and gender of patient, postal code, date of isolation, patient material, were provided anonymously. Species verification was performed biochemically using ApiNH (bioMérieux, Marcy l’Etoile, France) and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using VITEK-MS (bioMérieux). Supplementary Table 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.04.001. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed using the Etest method (bioMérieux) on commercially available Mueller Hinton chocolate agar, i.e. Mueller Hinton + 1% IsoVitaleX + 1% haemoglobin, (Becton Dickinson, Heidelberg, Germany), according to the manufacturer’s instructions. Clinical breakpoints set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, version 3.1) were used for interpretation of susceptibility results. N. gonorrhoeae strain ATCC 49226 was used for quality control. Isolates were tested for penicillinase production using the chromogenic reagent nitrocefin (Oxoid, Wesel, Germany). Consensus antimicrobial susceptibility data for Neisseria gonorrhoeae are calculated for multi-antigen sequence typing genogroups represented by ≥5 isolates. Therefore, the mode of the susceptibility results among isolates of a given genogroup was determined. Molecular typing For molecular epidemiological analysis, strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing (NGMAST) as described previously (Martin et al., 2004; Unemo and Dillon, 2011a). In brief, a two-digit allelic profile of a strain was generated by sequencing internal regions of the genes encoding two variable outer membrane proteins, the porin PorB and the subunit B of the transferrin binding protein TbpB. Sequence types (ST) were assigned using the NG-MAST website (www.ng-mast.net). In addition, NG-MAST genogroups based on sequence similarity at porB and tbpB alleles were defined as described previously (Chisholm et al., 2013).
Antimicrobial susceptibility Quality control testing with reference strain ATCC 49226 revealed that MICs of all antibiotics were within the given quality control ranges (data not shown). Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (73.7%) and tetracycline (41.3%). Penicillin nonsusceptibility was detected in 79.8% of isolates. The rate of azithromycin resistance was 5.6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone (Table 1). Beta-lactamase production was observed in 11.7% of the isolates as determined by the nitrocefin assay, all of them were classified as penicillin-resistant according to MIC determination. A consensus antimicrobial susceptibility result for genogroups represented by ≥5 isolates is shown in Table 2. Thereby, it becomes obvious that ciprofloxacin resistance is linked to distinct genogroups, i.e. G1407, G225, G1582, G304, G661, G6164. Interestingly, azithromycin resistance is solely associated with G1407. Moreover, isolates (n = 3) with the highest cefixime MICs, i.e. 0.094 mg/L, also belonged to G1407, more specifically ST1407. Molecular typing Molecular typing of the 213 gonococcal isolates revealed 99 different STs, representing 82 porB alleles and 32 tbpB alleles. 30 clusters, i.e. ≥2 isolates with the same ST, 69 STs represented by a single isolate and 46 new STs were identified. The most frequently observed ST was ST1407 (n = 39) as shown in Table 3. Based on sequence similarity of the porB and tbpB alleles nine major genogroups (represented by ≥5 isolates per genogroup) were defined, encompassing 149 (70%) of all isolates (Table 3). The proportion of various NG-MAST genogroups in Germany as well as within different parts of Germany is shown in Fig. 1. For definition on how the regions of Germany were defined see Supplementary Table 1. Genogroup 1407 was predominant in all regions, whereas G304 was exclusively found in Southern and Central Germany. Spatiotemporal analysis of gonococcal disease, i.e. an aggregation of cases caused by the same bacterial strain closely grouped in space (defined by the participating laboratory) and time (defined by a maximum time interval of two weeks between sample collection) was performed. A total of 15 spatiotemporal clusters were detected. On average, a cluster consisted of 2.3 patients. Statistical analysis
Statistical analysis Potential associations between genogroups, antimicrobial susceptibilities and patient characteristics, i.e. gender and age (with
Statistical analysis revealed that genogroup G1407 was significantly more common among males than females (OR: 3.6; CI: 1.21–10.68; p = 0.01). In contrast, genogroups G25 and G387 were
Please cite this article in press as: Horn, N.N., et al., Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.001
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Table 1 Antimicrobial susceptibility of 213 Neisseria gonorrhoeae isolates from Germany 2010/2011.
Penicillin Cefixime Ceftriaxone Ciprofloxacin Tetracyclin Azithromycin Spectinomycin
Susceptible (%)
Intermediate (%)
20.2 100 100 26.3 29.6 65.7 100
67.6 NA NA 0 29.1 28.6 NA
Resistant (%) 12.2 0 0 73.7 41.3 5.6 0
MIC range (mg/L) 0.004–16
ARTICLE IN PRESS
IJMM-50809; No. of Pages 6
International Journal of Medical Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm
Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany Nicole Nari Horn a , Michael Kresken b,c , Barbara Körber-Irrgang b , Stephan Göttig a , Cornelia Wichelhaus d , Thomas A. Wichelhaus a,∗ , Working Party Antimicrobial Resistance of the Paul Ehrlich Society for Chemotherapy a
Institute of Medical Microbiology and Infection Control, Hospital of Goethe-University, Frankfurt am Main, Germany Antiinfectives Intelligence GmbH, Campus of the University of Applied Sciences, Rheinbach, Germany c Rheinische Fachhochschule Köln, Cologne, Germany d Institute of Applied Mathematics, University of Heidelberg, Germany b
a r t i c l e
i n f o
Article history: Received 2 January 2014 Received in revised form 4 March 2014 Accepted 10 April 2014 Keywords: Neisseria gonorrhoeae Antimicrobial resistance NG-MAST Gonorrhoea
a b s t r a c t Antimicrobial drug resistance in Neisseria gonorrhoeae has become an increasing public health problem. Hence, surveillance of resistance development is of crucial importance to implement adequate treatment guidelines. Data on the spread of antibiotic resistance among gonococcal isolates in Germany, however, is scarce. In a resistance surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send N. gonorrhoeae isolates to the study laboratory in Frankfurt am Main. Species verification was performed biochemically using ApiNH and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Etest method. For molecular epidemiological analysis, N. gonorrhoeae strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing. A total of 213 consecutive gonococcal isolates were analyzed in this nationwide study. Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (74%) and tetracycline (41%). Penicillin non-susceptibility was detected in 80% of isolates. The rate of azithromycin resistance was 6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone. Molecular typing of gonococcal isolates revealed a great heterogeneity of 99 different sequence types (ST), but ST1407 predominated (n = 39). This is the first comprehensive German multi-centre surveillance study on antibiotic susceptibility and molecular epidemiology of N. gonorrhoeae with implications for antibiotic choice for treatment of gonorrhoea. The World Health Organization supports the concept that an efficacious treatment of gonorrhoea results in at least 95% of infections being cured. Accordingly, as spectinomycin is not available on the German market, only the third generation cephalosporins cefixime and ceftriaxone are regarded as valuable drugs for empirical treatment of gonorrhoea in Germany. © 2014 Elsevier GmbH. All rights reserved.
Introduction Neisseria gonorrhoeae is one of the most frequent pathogens causing sexual transmitted diseases worldwide. According to the
∗ Corresponding author at: Institute of Medical Microbiology and Infection Control, Hospital of Goethe-University, Paul-Ehrlich-Straße 40, 60596 Frankfurt am Main, Germany. Tel.: +49 69 6301 6438; fax: +49 69 6301 5767. E-mail address: [email protected] (T.A. Wichelhaus).
latest WHO assessment, gonococcal infections represent 106 million of the estimated 499 million new cases of curable sexually transmitted infections (STI) that occur globally every year (WHO, 2008). Antimicrobial resistance in N. gonorrhoeae has progressively developed over the years and the emerging threat of untreatable gonococcal infection becomes apparent (Bolan et al., 2012; ECDC, 2012; Unemo and Nicholas, 2012a; Ison et al., 2013; WHO, 2012). The European surveillance report on antimicrobial resistance in N. gonorrhoeae reveals high rates of resistance to many previously recommended therapeutic agents in many European countries such
http://dx.doi.org/10.1016/j.ijmm.2014.04.001 1438-4221/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Horn, N.N., et al., Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.001
G Model IJMM-50809; No. of Pages 6
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2
as ciprofloxacin, tetracycline and penicillin (Cole et al., 2010). The report further concludes that continual surveillance of antimicrobial resistance is essential to monitor for increasing and emerging resistance to clinical relevant antibiotics and to adapt treatment guidelines accordingly (Cole et al., 2010). Hence, the present study was performed to provide first nationwide data on the epidemiology of antibiotic resistance among gonococcal isolates in Germany.
the age groups: 0–24 years, 25–34 years, 35–44 years and ≥45 years), were analyzed using the Fisher’s exact test with the exception for datasets where a cell equalled zero. In these cases, the Pearson, Mantel and Haenszel chi-squared test was applied using a continuity correction. Odds ratios (OR) and 95% confidence intervals (CI) are given. All tests were performed two-tailed, and a p-value < 0.05 was considered to be significant.
Materials and methods
Results
N. gonorrhoeae isolates
A total of 213 gonococcal isolates were analyzed in this nationwide study. Isolates were recovered from 179 males and 34 females. Mean age of the males and females was 31.9 years (median age: 31 years; range: 18–67 years) and 25.4 years (median age: 25 years; range: 18–55 years), respectively. The sites of isolation were urethra 69.0%, cervix 8.0%, rectum 3.8%, throat 0.5%, and 18.8% not reported.
In a prospective surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send consecutive N. gonorrhoeae isolates to the study laboratory at the Institute of Medical Microbiology and Infection Control at the Hospital of Goethe-University in Frankfurt am Main according to a study protocol. Data concerning participating laboratories with regard to location, type of laboratory and number of isolates provided are available as Supplementary Table 1. Informational data regarding gonococcal isolates, i.e. age and gender of patient, postal code, date of isolation, patient material, were provided anonymously. Species verification was performed biochemically using ApiNH (bioMérieux, Marcy l’Etoile, France) and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using VITEK-MS (bioMérieux). Supplementary Table 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.04.001. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed using the Etest method (bioMérieux) on commercially available Mueller Hinton chocolate agar, i.e. Mueller Hinton + 1% IsoVitaleX + 1% haemoglobin, (Becton Dickinson, Heidelberg, Germany), according to the manufacturer’s instructions. Clinical breakpoints set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, version 3.1) were used for interpretation of susceptibility results. N. gonorrhoeae strain ATCC 49226 was used for quality control. Isolates were tested for penicillinase production using the chromogenic reagent nitrocefin (Oxoid, Wesel, Germany). Consensus antimicrobial susceptibility data for Neisseria gonorrhoeae are calculated for multi-antigen sequence typing genogroups represented by ≥5 isolates. Therefore, the mode of the susceptibility results among isolates of a given genogroup was determined. Molecular typing For molecular epidemiological analysis, strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing (NGMAST) as described previously (Martin et al., 2004; Unemo and Dillon, 2011a). In brief, a two-digit allelic profile of a strain was generated by sequencing internal regions of the genes encoding two variable outer membrane proteins, the porin PorB and the subunit B of the transferrin binding protein TbpB. Sequence types (ST) were assigned using the NG-MAST website (www.ng-mast.net). In addition, NG-MAST genogroups based on sequence similarity at porB and tbpB alleles were defined as described previously (Chisholm et al., 2013).
Antimicrobial susceptibility Quality control testing with reference strain ATCC 49226 revealed that MICs of all antibiotics were within the given quality control ranges (data not shown). Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (73.7%) and tetracycline (41.3%). Penicillin nonsusceptibility was detected in 79.8% of isolates. The rate of azithromycin resistance was 5.6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone (Table 1). Beta-lactamase production was observed in 11.7% of the isolates as determined by the nitrocefin assay, all of them were classified as penicillin-resistant according to MIC determination. A consensus antimicrobial susceptibility result for genogroups represented by ≥5 isolates is shown in Table 2. Thereby, it becomes obvious that ciprofloxacin resistance is linked to distinct genogroups, i.e. G1407, G225, G1582, G304, G661, G6164. Interestingly, azithromycin resistance is solely associated with G1407. Moreover, isolates (n = 3) with the highest cefixime MICs, i.e. 0.094 mg/L, also belonged to G1407, more specifically ST1407. Molecular typing Molecular typing of the 213 gonococcal isolates revealed 99 different STs, representing 82 porB alleles and 32 tbpB alleles. 30 clusters, i.e. ≥2 isolates with the same ST, 69 STs represented by a single isolate and 46 new STs were identified. The most frequently observed ST was ST1407 (n = 39) as shown in Table 3. Based on sequence similarity of the porB and tbpB alleles nine major genogroups (represented by ≥5 isolates per genogroup) were defined, encompassing 149 (70%) of all isolates (Table 3). The proportion of various NG-MAST genogroups in Germany as well as within different parts of Germany is shown in Fig. 1. For definition on how the regions of Germany were defined see Supplementary Table 1. Genogroup 1407 was predominant in all regions, whereas G304 was exclusively found in Southern and Central Germany. Spatiotemporal analysis of gonococcal disease, i.e. an aggregation of cases caused by the same bacterial strain closely grouped in space (defined by the participating laboratory) and time (defined by a maximum time interval of two weeks between sample collection) was performed. A total of 15 spatiotemporal clusters were detected. On average, a cluster consisted of 2.3 patients. Statistical analysis
Statistical analysis Potential associations between genogroups, antimicrobial susceptibilities and patient characteristics, i.e. gender and age (with
Statistical analysis revealed that genogroup G1407 was significantly more common among males than females (OR: 3.6; CI: 1.21–10.68; p = 0.01). In contrast, genogroups G25 and G387 were
Please cite this article in press as: Horn, N.N., et al., Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.001
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N.N. Horn et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
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Table 1 Antimicrobial susceptibility of 213 Neisseria gonorrhoeae isolates from Germany 2010/2011.
Penicillin Cefixime Ceftriaxone Ciprofloxacin Tetracyclin Azithromycin Spectinomycin
Susceptible (%)
Intermediate (%)
20.2 100 100 26.3 29.6 65.7 100
67.6 NA NA 0 29.1 28.6 NA
Resistant (%) 12.2 0 0 73.7 41.3 5.6 0
MIC range (mg/L) 0.004–16
