Carlos Florindo

Rui Pereira

Márcia Boura

Baltazar Nunes

Albertina Paulino

João P Gomes

Maria J Borrego

Genotypes and

HAL is

INTRODUCTION

Neisseria gonorrhoeae (N. gonorrhoeae), the etiologic agent of gonorrhoea, remains one of the most common bacterial sexually transmitted infections (STIs), as more than 60 millions of new cases are reported annually worldwide. [START_REF] Gerbase | Global epidemiology of sexually transmitted diseases[END_REF][START_REF]Gonorrhoeaincidence rate (per 100,000 population[END_REF] Recent trends in gonorrhoea have shown a progressive increase in several countries of Western Europe (e.g. Sweden and Switzerland), resulting in the resurgence of an old problem for the public health. [START_REF]Gonorrhoeaincidence rate (per 100,000 population[END_REF][START_REF] Fenton | Recent trends in the epidemiology of sexually transmitted infections in the European Union[END_REF] These alarming data may indicate a return to high risk sexual behaviours, contributing to onward transmission of STIs, as the HIV, which highlights the need for routine surveillance, prevention and control measures. [START_REF] Lowndes | European Surveillance of STI's Network. Surveillance systems for STIs in the European Union: facing a changing epidemiology[END_REF] Over the past decades, N. gonorrhoeae has repeatedly developed resistance to traditional antimicrobial agents, conditioning the choice of the first-line treatment. [START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF] Thus, third-generation cephalosporins are now recommended for European countries, despite emergence and spread of isolates displaying reduced susceptibility to cefixime and ceftriaxone has been recently reported. [START_REF] Bignell | 2009 European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Monfort | First Neisseria gonorrhoeae genotyping analysis in France: identification of a strain cluster with reduced susceptibility to ceftriaxone[END_REF][START_REF] Tanaka | Analysis of mutations within multiple genes associated with resistance in a clinical isolate of Neisseria gonorrhoeae with reduced ceftriaxone susceptibility that shows a multidrug-resistant phenotype[END_REF] In Portugal, data resulting from the mandatory notification reveal that gonorrhoea incidence remained low and stable during the last decade (0.27 -0.7 cases per 100 000 inhabitants). [START_REF]Gonorrhoeaincidence rate (per 100,000 population[END_REF] However, current data on antimicrobial susceptibility profiles of Portuguese N. gonorrhoeae circulating isolates are not available, as well as information about the molecular epidemiology of pathogen. The only evaluation performed so far in Portugal regarded the determination of tetracycline resistance (which is no longer used for treatment) back in 1997. [START_REF] Ferreira | High-level tetracycline resistant Neisseria gonorrhoeae isolated in Portugal[END_REF] Typing strategies of N. gonorrhoeae isolates are valuable tools to define the sexual networks, to distinguish between relapse and re-infection episodes, and to monitor the emergence and spread of antibiotic resistant gonococci clones. [START_REF] Choudhury | Identification of individuals with gonorrhoea within sexual networks: a population-based study[END_REF][START_REF] Tapsall | Two cases of failed ceftriaxone treatment in pharyngeal gonorrhoea verified by molecular microbiological methods[END_REF][START_REF] Palmer | Prediction of antibiotic resistance using Neisseria gonorrhoeae multi-antigen sequence typing[END_REF] N. gonorrhoeae multiantigen sequence typing (NG-MAST) is a high-discriminatory tool to assess the genetic diversity of N. gonorrhoeae, through the evaluation of allelic combination between two highly polymorphic genes, por and tbpB. This can be especially important for short-term epidemiology enabling the identification of recent chains of transmission. [START_REF] Martin | Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area[END_REF] In the present study we aimed to determine the antibiotic phenotype and MASTgenotype distribution of N. gonorrhoeae population in Portugal between 2004 and 2009, which constitutes the first molecular epidemiological study held in this country. Specific associations within genotypes and sexual orientation, age, and antibiotic resistance were also evaluated.

METHODS

N. gonorrhoeae isolates

Between 2006 and 2009, more than one hundred laboratories dispersed through the whole country were asked to send N. gonorrhoeae isolates to the national bacterial sexually transmitted infections laboratory located in the Portuguese NIH. The national collection was sent yearly to the "European Surveillance of Sexually Transmitted Infections" network. Twenty-five laboratories (including the NIH reference laboratory) from the Lisbon area (n=15), Porto area (n=6), Leiria (n=2) and Algarve (n=2) participated, leading to the collection of a total of 274 N. gonorrhoeae isolates.

Participating laboratories provided the following data: date of isolation, specimen anatomic site, gender and age of the infected individual, as well as sexual orientation.

All data were anonymous in order to avoid any ethical conflict.

A total of 236 from the whole 274 N. gonorrhoeae isolates collection were fully typed through NG-MAST. The infected individuals were aged from under one year to 58 years, median age 28. From these, 26 were isolated from women and 210 from men Considering the sexual orientation, 74 were heterosexual (18 women and 56 men), 70 were MSM and for 92 infected individuals no information was provided.

Antimicrobial Susceptibility testing

The isolates were sub-cultured once in chocolate agar (BioMérieux, France) before susceptibility testing was performed. Suspensions of cultures aged 24 hours were prepared equivalent to McFarland's standard 0.5. The ceftriaxone, ciprofloxacin, penicillin, and spectinomycin MIC of isolates was determined in GC agar (Oxoid, Basingstoke, UK) with 1% Isovitalex (Becton, Dickinson and Company, Sparks, USA) using Etests (AB bioMérieux, Solna, Sweden) according to the manufacturer's instructions. Plates were allowed to incubate for 24 hours at 36ºC in 5% CO 2 .

Isolates were categorised according to the following definitions: Ciprofloxacin resistant (MICs≥1 mg/L), spectinomycin resistant (MICs≥128 mg/L) ceftriaxone resistant (MIC≥0,5 mg/L), decreased-susceptibility to ceftriaxone (0.125≥MICs≤0,25 mg/L), and penicillin resistant (MIC≥2 mg/L). All penicillin intermediate or resistant isolates were tested for penicillinase production using the chromogenic reagent, Nitrocefin (Oxoid, Basingstoke, UK), according to manufacturer's instructions.

NG-MAST

The molecular genotyping of all N. gonorrhoeae isolates was performed using the NG-MAST method, as previously described. [START_REF] Martin | Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area[END_REF] Briefly, N. gonorrhoeae DNA was extracted with QIAamp DNA mini kit (Qiagen, Valencia, CA), according to manufacturer's instructions. Two highly polymorphic loci, por and tbpB were subjected to PCR amplification and both strands of internal fragments were sequenced (490 and 390 bp, respectively) by using Big Dye v.1.1 chemistry on an ABI3700 capillary sequencer (Applied BioSystems, Foster City, USA). Sequences were aligned by using Lasergene software (DNASTAR, Madison, USA). Alleles and sequence types (STs) were assigned in the international database of the NG-MAST website (www.ngmast.net).

Genetic analysis

In order to evaluate the polymorphism degree and the phylogenetic relatedness among NG-MAST STs, concatenated sequences of por and tbpb alleles were created in a head-to-tail fashion, as previously described for other pathogens. [START_REF] Nunes | Chlamydia trachomatis diversity viewed as a tissue-specific coevolutionary arms race[END_REF] Subsequently, a Neighboor-Joining phylogenetic tree of all concatenated sequences was generated with MEGA4 software using Kimura two-parameter and p-distance models. [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF][START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF][START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF] We have also generated matrices of pairwise comparisons to estimate the total number of variable sites, the overall mean genetic distance among all STs, and the genetic relatedness between putative ST clusters. The pairwise-deletion option was chosen to remove all sites containing missing data or alignment gaps for all genetic distance estimations. No analyses were done at the protein level because the tbpb allele becomes out-of-frame within the concatenated sequence.

Statistical analysis

Chi-square, Fisher Exact and Linear-by-Linear tests were used to evaluate the associations between ST types and antibiotic resistance, year of isolation and population characteristics (sexual orientation, age, and gender). Observed differences were considered statistically significant for p<0.05. All statistical results were obtained with the package of statistical programs SPSS 15.0.

RESULTS

Antimicrobial susceptibility phenotypes

From the 236 N. gonorrhoeae isolates, 187 were subjected to antimicrobial susceptibility testing. Antibiotic resistance to spectinomycin and ceftriaxone was not observed in the current studied population. Furthermore, only four isolates presented reduced susceptibility to ceftriaxone, which occurred in 2007. Contrarily, 148 (79.1%) and 70 (37.4%) isolates displayed resistance to penicillin and ciprofloxacin, respectively, where 15.5% of the former were penicillinase producers. Multiresistance was detected in 35.8% of isolates considering that 95.7% of ciprofloxacin resistant isolates were also resistant to penicillin.

Genetic diversity of N. gonorrhoeae isolates

According to the NG-MAST method, 236 N. gonorrhoeae isolates were distributed into 104 different STs, 60 (57.7%) of which had not been reported before to the international database. The majority of STs (66.3%, 69 of 104) were represented by a single isolate, whereas the remaining STs included two to 19 isolates. This high genetic diversity arose from the allelic combination of 86 por and 39 tbpB alleles, which is supported by the fully-branched phylogenetic tree based on concatenated sequences of these alleles (fig 1). The overall mean genetic distance was 97. The most frequent STs were ST210 (n=19, 8.1%) and ST225 (n=18, 7.6%), which, together with other eight STs (table 1) represent 42% (100/236) of all isolates.

There was a statistically significant non-homogeneous distribution over the five years for ST225 (p<0.001), ST210 (p=0.011), and ST2 (p=0.007), with a decreasing tendency for the first two (p=0.016 and p=0.002, respectively) and an increasing tendency for the latter (p=0.005).

The evaluation of STs from nine pairs of isolates recovered from known sexual partners has shown 88.9% of concordance. These are represented by a diverse set of STs and are dispersed throughout the study period. The single pair of isolates that presented non-identical STs, involved two novel STs (ST3628 and ST3630), which share the por 2171 allele.

STs versus socio-demographic data

There were substantial differences in ST distribution by age and sexual orientation. Indeed, ST2 and ST783 were predominantly found in patients under 25 years of age (p=0.02 and p=0.009, respectively) (table 1). On the other hand, one of the most common STs, ST225, was identified predominantly in patients above 25 years (p=0.028). Regarding sexual orientation, there was a statistically significant association between ST225 and the MSM (p=0.001) whereas ST783 and ST1318 were associated with heterosexual individuals (p=0.014). No statistical association regarding geographic origin of isolates and ST was found in the present study.

STs versus antibiotic resistance

There was a statistical significant association between resistance to ciprofloxacin and isolates with ST1479 (100%, p<0.001), ST3615 (100%, p=0.007), ST225 (94.4%, p<0.001) and ST1407 (88.9%, p=0.002). For all the other antibiotics no association was found.

DISCUSSION

In Portugal it is assumed that only a small fraction of the N. gonorrhoeae infections are notified to health authorities, which may be in the basis of the apparently stable incidence rates, contradicting reports from other countries that evidence increasing rates. Globally, the results from the antibiotic susceptibility profiles matched the ones reported for other countries. [START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Monfort | First Neisseria gonorrhoeae genotyping analysis in France: identification of a strain cluster with reduced susceptibility to ceftriaxone[END_REF][START_REF] Olsen | The Neisseria gonorrhoeae population in Sweden during 2005-phenotypes, genotypes and antibiotic resistance[END_REF][START_REF] Starnino | Phenotypic and genotypic characterization of Neisseria gonorrhoeae in parts of Italy: detection of a multiresistant cluster circulating in a heterosexual network[END_REF][START_REF] Wong | Molecular epidemiological identification of Neisseria gonorrhoeae clonal clusters with distinct susceptibility profiles associated with specific groups at high risk of contracting human immunodeficiency virus and syphilis[END_REF] In fact, no isolates displayed resistance to spectinomycin and ceftriaxone whereas 79.1% and 37.4% were resistant to penicillin and ciprofloxacin, respectively, which supports the current recommend therapy strategy. [START_REF] Bignell | 2009 European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults[END_REF] However, contrarily to what has been described in some countries, we did not observed an increase tendency for reduced susceptibility to ceftriaxone. [START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Monfort | First Neisseria gonorrhoeae genotyping analysis in France: identification of a strain cluster with reduced susceptibility to ceftriaxone[END_REF][START_REF] Tanaka | Analysis of mutations within multiple genes associated with resistance in a clinical isolate of Neisseria gonorrhoeae with reduced ceftriaxone susceptibility that shows a multidrug-resistant phenotype[END_REF] Nevertheless, the continuous monitorization of susceptibility profiles to this antibiotic is advisable.

Considering the association between antibiotic susceptibility phenotypes and STs, our results also supported previous reports. Indeed, ST225, which was the second most frequent ST in our study, and the only disseminated worldwide, was mostly found in ciprofloxacin resistant N. gonorrhoeae isolates (p<0.001). [START_REF] Monfort | First Neisseria gonorrhoeae genotyping analysis in France: identification of a strain cluster with reduced susceptibility to ceftriaxone[END_REF][START_REF] Palmer | Prediction of antibiotic resistance using Neisseria gonorrhoeae multi-antigen sequence typing[END_REF][START_REF] Wong | Molecular epidemiological identification of Neisseria gonorrhoeae clonal clusters with distinct susceptibility profiles associated with specific groups at high risk of contracting human immunodeficiency virus and syphilis[END_REF][START_REF] Todd | Using epidemiological and molecular methods to investigate an outbreak of gonorrhoea associated with heterosexual contact in Newcastle, NSW, Australia[END_REF] We also found a similar association for ST1459 (p<0.001), so far only described in France, and that had comprised solely isolates resistant to this antibiotic. Curiously, the rate of ciprofloxacin resistance within a subgroup of isolates that includes ST225 was 96.4%.

Indeed, the probability of ciprofloxacin resistance among these isolates was 73.5-fold higher than for the remaining isolates with other STs. Interestingly, the genetically and phylogenetically closest subgroup (subgroup A1 in fig 1), which includes the most prevalent ST210, presented only 8.0% resistance to ciprofloxacin. Thus, based on our results and on the literature we can speculate that ciprofloxacin resistance is associated with ST225 and genetically highly-related STs. [START_REF] Monfort | First Neisseria gonorrhoeae genotyping analysis in France: identification of a strain cluster with reduced susceptibility to ceftriaxone[END_REF] However, the opposite cannot be stated as isolates resistant to ciprofloxacin can be found phylogenetic distant from ST225, as for ST1479 (within group B in fig 1), which reveals 148 [SE 11] nucleotide differences to ST225. Further studies comprehending more isolates and STs are essential to understand this phenomenon.

The phylogenetic tree evidenced a huge strain diversity which would be expected due to the highly polymorphism of the two alleles involved in the NG-MAST method, and grouped STs in two major groups (fig 1) that present a genetic distance of 159.8 [SE 8.9] nucleotide differences. However, there is a high number of STs represented by a single isolate, and no predominant ST exists, which may be due to the local emergence of new STs, recent introduction of foreign STs or incomplete/insufficient epidemiological surveillance. In Portugal, although our data do not represent the entire country, this heterogeneous STs scenario may represent the "tip of the iceberg", reflecting a high number of undiagnosed and not reported gonorrhoea cases. Thus, such diversity of STs within the studied population did not allow the identification of sexual networks that spread this infection, contrarily to what has been successfully achieved in the UK. [START_REF] Risley | Geographical and demographic clustering of gonorrhoea in London[END_REF] However, eight of the nine sexual partners included in this study were infected by isolates with the same ST, which reinforces the interest of NG-MAST as a tool for the construction of sexual networks. Nonetheless, the high rate of polymorphism of the two genes could lead to slight differences in the resulting combined genotype. In fact, the discordant couple (ST3628 and ST3630) evidenced the same por allele but a tbpB allele diverging by a single nucleotide mutation. Overall, the couple concordance rate (88.9%) was similar to the described in the literature. [START_REF] Bilek | Concordance between Neisseria gonorrhoeae genotypes recovered from known sexual contacts[END_REF][START_REF] Liao | Comparison of Neisseria gonorrhoeae multiantigen sequence typing and porB sequence analysis for identification of clusters of N. gonorrhoeae isolates[END_REF] Considering the inexistence of sexual networks in the present study and the dynamic fluctuation on the predominance of N. gonorrhoeae types per year, these apparently prevailing types likely represent ecological succeeded STs during a short period of time, although N. gonorrhoeae is typically considered as 'nonclonal'

where alleles are at linkage equilibrium. [START_REF] Smith | How clonal are bacteria?[END_REF][START_REF] O'rourke | Further evidence for the non-clonal population structure of Neisseria gonorrhoeae: extensive genetic diversity within isolates of the same electrophoretic type[END_REF] This can explain the short-term emergence of STs in all the NG-MAST studies performed to date. As an example, ST210 and ST225 frequency has been declining in Portugal since 2004 and are eventually being replaced by other STs.

In the future, we expect that the laboratory-based national surveillance of N.

gonorrhoeae infections will provide a broader spectrum of isolates that will allow the establishment of the scenario of the Portuguese sexual networks. Furthermore, it would be desirable that molecular epidemiology studies could be extended to a large number of countries, in order to evaluate the accuracy of the associations reported so far, involving STs, age, sexual orientation, and antibiotic resistance, and to perform further research to investigate such associations.

  during the years 2004 (n=17), 2006 (n=29), 2007 (n=75), 2008 (n=50) and 2009 (n=65).

  3 [SE 5.1] nucleotide differences, where a maximum distance of 205 [SE 12.5] nucleotides was observed between ST4324 and ST4334. Interestingly, the 104 STs formed two major groups (fig 1) separated by 159.8 [SE 8.9] nucleotide differences, yielding several subgroups of genetically close STs, as the one that includes the worldwide prevalent ST225 (subgroup A2 in fig 1), that shows a genetic distance within the same-group strains solely of 5.3 [SE 1.1].

[ 2 ]

 2 It is expected that the implementation of laboratory and clinical notification (according to ECDC guidelines) will provide more accurate epidemiological data on this STI. Considering the lack of data in Portugal and the recent development of the NG-MAST technique, we aimed to determine the antibiotic phenotype and MAST-genotype distribution of N. gonorrhoeae population in Portugal since 2004.

Figure 1 - 8 [

 18 Figure 1 -Phylogenetic reconstruction of concatenated sequences of por and tbpB

Table 1 -

 1 Characterisation of the most frequent STS

										Antibiotic susceptibility a :
			Sexual Orientation		Age
									Penicillin	Ciprofloxacin
	ST	n
			MSM	HS b		<25	≥25		S	I/R	S	I/R
			n (%) c	n (%) c	p	n (%)	n (% )	p	n (%) c	n (%) c	n (%) c	n (%) c	p
	210	19 5 (35.7)	9 (64.3)		5 (26.3) 14 (73.7)		6 (40.0) 9 (60.0)	14 (93.3) 1 (6.7)	0.02
	225	18 9 (100)	0 (0.0)	0.002 2 (11.1) 16 (88.9) 0.028 0 (0.0)	18 (100) 0 (0.0)	18 (100) <0.001
	783	9	0 (0.0)	7 (100)	0.014 7 (77.8) 2 (22.2)	0.009 1 (11.1) 8 (88.9)	9 (100)	0 (0.0)	0.02
	1318	9	0 (0.0)	7 (100)	0.014 5 (55.6) 4 (44.4)		2 (22.2) 7 (77.8)	9 (100)	0 (0.0)	0.02
	1407	9	5 (71.4)	2 (28.6)		3 (33.3) 6 (66.7)		0 (0.0)	9 (100)	1 (11.1)	8 (88.9)	0.002
	1479	9	1 (25.0)	3 (75.0)		4 (44.4) 5 (55.6)		2 (22.2) 7 (77.8)	0 (0.0)	9 (100)	<0.001
	1780	9	5 (71.4)	2 (28.6)		3 (33.3) 6 (66.7)		5 (55.6) 4 (44.4)	9 (100)	0 (0.0)	0.02
	2	6	0 (0.0)	4 (100)		5 (83.3) 1 (16.7)	0.02 4 (66.7) 2 (33.3)	6 (100)	0 (0.0)
	1034	6	1 (33.3)	2 (66.7)		3 (50.0) 3 (50.0)		0 (0.0)	5 (100)	5 (100)	0 (0.0)

a S, Susceptible; I, Intermediate; R, Resistant b HS, Heterosexual c Percentage according to known information

Competing interests: None.


Dr Craig Tipple
email: c.tipple@imperial.ac.uk

Dr Alan Smith

Elzbieta Bakowska

Melanie C Corbett

Corneal perforation requiring corneal grafting -A rare complication of Gonococcal Eye Infection

Keywords: Neisseria gonorrhoeae, Corneal Perforation, HIV, Grafting, Corneal

published or not. The documents may come   L'archive ouverte pluridisciplinaire

1. Early recognition and treatment of Neisseria gonorrhoea eye infection, wherever it may present, is paramount to prevent sight-threatening complications.

2. A sexual history is important in the management of patients with purulent eye discharge as patients do not readily associate genital and eye symptoms.

3. The optimal antimicrobial management of this condition is unknown and there is presently no UK guideline for its management.

Introduction

The adult conjunctiva is susceptible to infection by Neisseria gonorrhoea (GC) by both direct and indirect contact. Once infection is established the bacteria can quickly spread to the cornea leading to keratitis, ulceration, descemetocoele formation and ultimately perforation. The rapid identification and treatment of the infection is paramount as these complications can occur within 24 hours of infection [START_REF] Day | Don't forget Gonococcus! Eye[END_REF].

Case Report

A 25 year old man initially presented to the ophthalmologists via the emergency department. He reported three days of irritation and discharge from the right eye attributed to a chemical injury. Swabs were taken and he was treated with dexamethasone, chloramphenicol, ascorbic acid and cyclopentolate eye drops. 7 days later the eye had become increasingly painful with intense conjunctival injection and copious purulent discharge (Image 1A). A corneal abscess had developed under the upper lid together with corneal melt and associated perforation (Image 1B). Unaided visual acuity was right eye 6/36 and left 6/4.

Culture results from the initial visit were found to be positive for GC, which was resistant to ciprofloxacin and nalidixic acid. He was admitted by the ophthalmologists for intensive topical antibiotics.

The patient was referred to the genito-urinary (GU) service a full GU sexual history and screening. The male was a sexually active homosexual. He reported a urethral discharge 4 weeks prior to his eye symptoms, which had resolved spontaneously. He was offered and accepted a full GU screen. This initially revealed urethral GC and a positive HIV point-of-care test.

Subsequent results confirmed HIV-1 infection, urethral GC and Chlamydia.

The patient received initial treatment with ceftriaxone 250mg IM and azithromycin 1g PO. IV ceftriaxone was continued for 7 days together with topical chloramphenicol and gentamicin hourly day and night; dexamethasone and cyclopentolate eye drops. On day 7 of admission, once the eye was quieter, he underwent tectonic corneal grafting of the perforated area of cornea (Image 1C). A kidney-shaped lamellar graft was overlaid on the perforation and area of melt just inside the superior limbus.

During close outpatient follow-up his vision in the affected eye improved to 6/18 unaided (6/5 pinhole) (Image 1D). He was found to have a CD4 count of 180 (22%).

Discussion

The presentation of gonococcal eye infection can have a similar initial presentation to the common infective forms of conjunctivitis. However conjunctival swabs are not routinely taken, so the diagnosis of GC can initially be missed. In this patient a swab was taken because the purulent discharge was inconsistent with the history of chemical injury. Sexual history taking is particularly important to identify patients most at risk of gonococcal eye infection.

GC infection of the eye has been reported both with and without concomitant anogenital infection [START_REF] Harry | Unilateral gonococcal ophthalmia without genital infection: an unusual presentation in an adult[END_REF][START_REF] Gambrelle | Gonococcal conjunctivitis complicated by perforating corneal abscess in an adult[END_REF]. Patients may not readily associate their eye and genital symptoms. In this case in particular there was a 4-week delay between the onset of genital and eye symptoms. The importance of considering the diagnosis is therefore paramount.

The British Association of Sexual Health and HIV (BASHH) do not have a recommendation for the treatment of GC eye in their current guidelines(4).

For genital infection, World Health Organisation (WHO) 2003 guidelines recommend ceftriaxone 125mg IM stat(5) whilst the American Centers for Disease Control (CDC) advise a 1g dosage [START_REF] Workowski | Sexually transmitted diseases treatment guidelines, 2006[END_REF]. The GC isolated in this case had confirmed sensitivity to third generation cephalosporins, namely cefixime and ceftriaxone. For eye infections, topical antibiotics achieve much higher concentrations in the infected tissues than systemic treatments. Four times a day treatments are usually sufficient for conjunctivitis, but for corneal involvement, dual topical therapy hourly throughout the day and night is required [START_REF] Day | Don't forget Gonococcus! Eye[END_REF]. In this case, in view of the breach of the cornea, systemic treatment more akin to that for disseminated GC was employed.

Case reports of GC eye infection with associated corneal perforation have been previously described in the ophthalmology literature but not that of GU medicine. This case highlights both the potential severe complications of this infection, which should be treated immediately and followed up closely by ophthalmologists, and the shortcomings of current guidelines for it's management.

Legend for

Figure

  Anterior segment photographs of a patient with gonoccocal keratitis: (A) on admission, 10 days after the onset of symptoms, there was copious pus and a superior corneal perforation; (B) after 2 days of systemic and intensive topical antibiotics, the infection began to settle, but necrotic stroma remained around the perforation. Tectonic corneal grafting was performed at 1 week. (C) One week postoperatively the perforation was sealed. (D) Ten months later the eye was white and quiet, sutures were removed and he achieved an unaided visual acuity of 6/18. Authors Roles All authors contributed to the preparation of the manuscript with CT as the main contributor and corresponding author. MC drafted the detailed ophthalmic management and figure legend. CT = Craig Tipple MC = Melanie Corbett


Daniel Golparian

Bengt Hellmark

Hans Fredlund

Magnus Unemo
email: magnus.unemo@orebroll.se

Emergence, spread and characteristics of Neisseria gonorrhoeae isolates with in vitro decreased susceptibility and resistance to extended-spectrum cephalosporins in Sweden

Keywords: Neisseria gonorrhoeae, antimicrobial resistance (AMR), ceftriaxone, cefixime, penA mosaic allele

Emergence, spread and characteristics of Neisseria gonorrhoeae isolates with in vitro decreased susceptibility and resistance to extended-spectrum cephalosporins in Sweden

Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which remains a global public health problem. Most worrying, N. gonorrhoeae has developed high-level resistance to all antimicrobials used in the traditional treatment of gonorrhoea, e.g. penicillins, tetracyclines and fluoroquinolones. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF] Furthermore, recent years gonococcal strains with resistance and/or decreased susceptibility to azithromycin, including high-level resistance, and the currently ideal treatment options extended-spectrum cephalosporins (ESCs) have emerged. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Chisholm | Emergence of high-level azithromycin resistance in Neisseria gonorrhoeae in England and Wales[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Whiley | Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Australia, that are less susceptible to Ceftriaxone[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF][START_REF] Lee | Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone[END_REF][START_REF] Osaka | Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone[END_REF][START_REF] Muratani | Outbreak of Cefozopran (penicillin, oral cephems, and aztreonam)-resistant Neisseria gonorrhoeae in Japan[END_REF][START_REF] Ameyama | Mosaic-like structure of penicillinbinding protein 2 gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime[END_REF][START_REF] Ito | Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in central Japan[END_REF] Treatment failures using oral ESCs including cefixime are occurring, especially in the WHO Western Pacific Region (WPR), [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF][START_REF] Yokoi | Threat to cefixime treatment of gonorrhea[END_REF][START_REF] Lo | Ceftibuten resistance and treatment failure of Neisseria gonorrhoeae infection[END_REF][START_REF] Tapsall | Implications of current recommendations for third-generation cephalosporin use in the WHO Western Pacific region following the emergence of multiresistant gonococci[END_REF] and the susceptibility to all ESCs (oral and injectable) is decreasing globally. Verified treatment failures of urogenital gonorrhoea using ceftriaxone (injectable) are still lacking. However, the emergence and spread of decreased susceptibility and resistance to ESCs are serious concerns globally and gonorrhoea may become untreatable in certain circumstances.

The first molecular mechanism for decreased in vitro susceptibility and resistance to ESCs is alteration of the penA gene, i.e. acquisition of a penA mosaic allele that encodes an altered penicillin binding-protein 2 (PBP2). [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Whiley | Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Australia, that are less susceptible to Ceftriaxone[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF][START_REF] Lee | Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone[END_REF][START_REF] Osaka | Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone[END_REF][START_REF] Muratani | Outbreak of Cefozopran (penicillin, oral cephems, and aztreonam)-resistant Neisseria gonorrhoeae in Japan[END_REF][START_REF] Ameyama | Mosaic-like structure of penicillinbinding protein 2 gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime[END_REF][START_REF] Ito | Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in central Japan[END_REF][START_REF] Tapsall | Implications of current recommendations for third-generation cephalosporin use in the WHO Western Pacific region following the emergence of multiresistant gonococci[END_REF][START_REF] Ohnishi | Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF] However, also alterations of a single amino acid, i.e. A501, of PBP2 in non-mosaic penA alleles may decrease the ESC susceptibility. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Whiley | Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Australia, that are less susceptible to Ceftriaxone[END_REF][START_REF] Lee | Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone[END_REF][START_REF] Powell | Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism of antibiotic resistance[END_REF] Furthermore, mutations in the promoter and/or coding sequence of the repressor gene mtrR, which cause an over-expression of the MtrCDE efflux pump system further decrease the ESC susceptibility. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF][START_REF] Folster | MtrR modulates rpoH expression and levels of antimicrobial resistance in Neisseria gonorrhoeae[END_REF] In addition, specific porB1b gene mutations (the penB resistance determinant) that alter amino acid G101 and A102 in the outer membrane PorB1b porin result in a decreased permeability and accordingly further decreased susceptibility to ESCs. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF][START_REF] Olesky | Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations[END_REF][START_REF] Olesky | Identification and analysis of amino acid mutations in Porin IB that mediate intermediate-level resistance to penicillin and tetracycline in Neisseria gonorrhoeae[END_REF] Finally, at least one unknown resistance determinant exists, [START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF] and accordingly all mechanisms that underlay the development of ESC decreased susceptibility and resistance are far from known.

Most, if not all, resistance in N. gonorrhoeae to different antimicrobials has historically emerged in the WHO Western Pacific Region (WPR). This resistance has subsequently and rapidly spread globally, by sex tourists and other travellers. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF] The emergence and spread of ESC decreased susceptibility and resistance globally will most probably follow the same pattern. Accordingly, already in 2001 two cases of treatment failure with oral cephalosporins were reported in Japan, [START_REF] Akasaka | Emergence of cephem-and aztreonamhigh-resistant Neisseria gonorrhoeae that does not produce beta-lactamase[END_REF] and in 2006 all oral ESCs were excluded in the treatment guidelines for gonorrhoea in Japan, due to the high level of resistance. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF] In Sweden, the first gonococcal isolate with decreased susceptibility to ESCs, which also contained the characteristic penA mosaic allele X, [START_REF] Ito | Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in central Japan[END_REF] and was indistinguishable to the Japanese isolates causing treatment failures, was found in 2002. [START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF] The aims of this study were to examine the emergence, spread, and phenotypic as well as genotypic characteristics of N. gonorrhoeae isolates with decreased susceptibility and resistance to extended-spectrum cephalosporins (ESCs) in Sweden.

METHODS

Neisseria gonorrhoeae isolates

All examined isolates were obtained at the National Reference Laboratory for Pathogenic Neisseria, Örebro University Hospital, Sweden from 1998 through 2009. All N. gonorrhoeae clinical isolates displaying a minimum inhibitory concentration [MIC] of ≥0.032 to cefixime and/or ceftriaxone were included (n=331). These isolates were from 331 patients (females 18% and males 82%). The mean age of the males were 34 years (median age: 31 years; and range: 16 to 68 years) and the mean age of the females were 34 years (median age: 32 years; and range: 15 to 59 years). The gender distribution and age distribution were relatively similar during the years examined. Unfortunately, complete data regarding sexual orientation of the patients and country of exposure to infection were not available. However, data available showed that the patients belonged to both heterosexual and men who have sex with men (MSM) networks. Furthermore, especially the heterosexuals were exposed to the infection in many countries worldwide, e.g. European countries (n=14, spread all over Europe), African countries (n=3), Asian countries (n=6), Australia, and USA. The isolates were species confirmed with sugar utilization test and/or Phadebact GC Monoclonal Test (Boule Diagnostics AB, Huddinge, Sweden), and preserved as previously described. [START_REF] Unemo | Molecular epidemiology of Neisseria gonorrhoeae: sequence analysis of the porB gene confirms presence of two circulating strains[END_REF] The WHO 2008 N. gonorrhoeae reference strains were included as control strains. [START_REF] Unemo | Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes[END_REF]

Antimicrobial susceptibility testing

The susceptibility (mg/L) to cefixime, ceftriaxone, ampicillin, azithromycin, ciprofloxacin, and spectinomycin was determined using the Etest method (AB Biodisk, Solna, Sweden), as previously described. [START_REF] Berglund | One year of Neisseria gonorrhoeae isolates in Sweden: the prevalence study of antibiotic susceptibility shows relation to the geographic area of exposure[END_REF] The breakpoints used for susceptibility (S) and resistance (R) were as follows: cefixime (S≤0.125 mg/L, R>0.125 mg/L; only tentative in vitro breakpoint due to the lack of appropriate correlates between in vitro findings and clinical outcome), ceftriaxone (S≤0.125 mg/L, R>0.125 mg/L; only tentative in vitro breakpoint due to the lack of appropriate correlates between in vitro findings and clinical outcome), ampicillin (S≤0.125 mg/L, R>2 mg/L), azithromycin (S≤0.25 mg/L, R>0.5 mg/L), ciprofloxacin (S≤0.032 mg/L, R>0.064 mg/L) and spectinomycin (S≤64 mg/L, R>64 mg/L), according to The European committee on antimicrobial susceptibility testing (EUCAST [www.eucast.org]). β-lactamase production was detected using nitrocefin discs.

Isolation of genomic DNA

Bacterial DNA was isolated in the robotized NorDiag Bullet instrument (NorDiag ASA Company, Oslo, Norway) using the BUGS'n BEADS™ STI-fast kit (NorDiag ASA Company), according to the instructions from the manufacturer.

Detection of penA mosaic alleles and alteration of amino acid A501 in PBP2

All gonococcal isolates (n=331) were screened for the presence of a penA mosaic allele using a previously described penA mosaic allele conventional PCR, [START_REF] Whiley | Use of a novel screening PCR indicates presence of Neisseria gonorrhoeae isolates with a mosaic penA gene sequence in Australia[END_REF] as well as an earlier depicted method based on real-time PCR and pyrosequencing. [START_REF] Unemo | Real-time PCR and subsequent pyrosequencing for screening of penA mosaic alleles and prediction of reduced susceptibility to expanded-spectrum cephalosporins in Neisseria gonorrhoeae[END_REF] Both these methods were used in order to identify most/all types of penA mosaic alleles, and the pyrosequencing method also identifies alterations of A501 in PBP2. DNA of the WHO 2008 N. gonorrhoeae WHO reference strain K, [START_REF] Unemo | Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes[END_REF] which contains the characteristic penA mosaic allele X, [START_REF] Ito | Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in central Japan[END_REF] and sterile water were included in each run as positive control and negative control, respectively.

mtrR and porB1b sequencing

The promoter and coding sequence of mtrR, and the full-length porB1b gene were sequenced in all isolates possessing a penA mosaic allele or any alteration of A501 in PBP2 (n=115), as previously described. [START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Unemo | Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes[END_REF] DNA of the WHO 2008 N. gonorrhoeae reference strain M and sterile water were included in all runs as positive control and negative control, respectively. [START_REF] Unemo | Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes[END_REF] Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) NG-MAST, was performed on all isolates possessing a penA mosaic allele or any alteration of A501 in PBP2 (n=115), as previously described. [START_REF] Unemo | Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain[END_REF] NG-MAST allele numbers of porB and tbpB, and sequence types (STs) were assigned using the NG-MAST (www.ng-mast.net) database.

Sequence alignments and phylogenetic analysis

Multiple-sequence alignments of nucleotide sequences and the deduced corresponding amino acid sequences were performed in the software BioEdit Sequence Alignment Editor version 7.0.9.0 with manual adjustment.

Phylogenetic analysis of the full-length porB1b gene encoding the mature PBP2 was performed with the software TREECON (version 1.3b) by using the Jin and Nei substitution model, the Kimura evolutionary model, an α-value of 0.5 and the neighbor-joining method, as previously described. [START_REF] Unemo | Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain[END_REF]

RESULTS

Longitudinal trends in the MICs of cefixime and ceftriaxone

The distributions of MICs of cefixime (2003, which was the first year all isolates were examined, and 2009) and ceftriaxone (1998 and 2009) displayed distinct shifts towards higher MICs over the years for both antimicrobials. An increased proportion of isolates with MICs above the tentative in vitro resistance breakpoints, cefixime 5.2% (n=12) in 2009 and ceftriaxone 0.4% (n=1) in 2009, were identified (Fig. 1).

Antimicrobial susceptibility of all selected N. gonorrhoeae isolates from 1998 to 2009 (n=331)

Based on the EUCAST breakpoints, 30 (9.1%) of the strains displayed in vitro resistance to cefixime. However, 330 (99.7%) of the isolates were susceptible to ceftriaxone, and accordingly only one isolate showed resistance (MIC=0.250 mg/L). Among all the isolates, 8% were resistant to ampicillin, 17% to azithromycin, and 91% to ciprofloxacin, however, no isolates resistant to spectinomycin were found. Beta-lactamase production was identified in 41 (12%) of the isolates.

The year of collection, number of isolates, presence of ESC resistance determinants, and susceptibility to ESCs of all isolates are summarized in Table 1.

penA mosaic allele and alteration of A501 in PBP2

Seventy-eight (24%) isolates contained a penA mosaic allele, identified using the conventional PCR (n=77) and the pyrosequencing method (n=78), and 37 (11%) isolates comprised an alteration of A501 (A501V [n=33] and A501T [n=4]) in PBP2 (Table 1 and2). mean MIC: 0.094 mg/L), respectively (Table 1 and2). The majority of isolates, 62 (80%) out of 79, that displayed an increased MIC (>0.032) to both cefixime and ceftriaxone, also possessed either a penA mosaic allele or an alteration of A501 in PBP2.

Specific mutations in mtrR and porB1b (penB alteration)

Mutations in the promoter region or coding sequence of the mtrR gene that evidently cause an over-expression of the MtrCDE efflux pump were observed in 110 (96%) of the isolates examined (n=115). Ninety-three (81%) had the characteristic single nucleotide (A) deletion in the inverted repeat of the promoter region and eight isolates (7%) displayed alterations of amino acid G45 in MtrR (Table 1). Nine (8%) of the isolates contained both these alterations. penB alterations in amino acid 101 and/or 102 were detected in 113 (98%) of the isolates. One of these isolates lacked the alteration in amino acid position 101 and two isolates did not possess the alteration in 102. In only five (4%) of the isolates a mutation in mtrR occurred in the absence of the penB alteration. All the 31 isolates displaying in vitro resistance to cefixime (n=30) and ceftriaxone (n=1) according to EUCAST breakpoints contained penA alterations (mosaic allele [n=30] and A501 [n=1]) and penB alteration, and 30 of these isolates also comprised the single nucleotide (A) deletion in the promoter region of mtrR.

N. gonorrhoeae multiantigen sequence typing and phylogenetic analysis

Among the isolates containing a penA mosaic allele (n=78), 22 different NG-MAST STs were identified, of which 11 had not been previously described (Table 2). However, the phylogenetic analysis of the full-length porB gene sequences (25 different sequences) showed that 60 (77%; 90% [60/67] of all found after 2003) of these penA mosaic allele isolates belonged to a cluster comprising all ST1407 (n=29) and very closely related genotypes (n=31), i.e. differing by a maximum of 14 nucleotides in the full-length porB gene (Fig. 2).

The isolates comprising this cluster were from patients infected in Sweden, but also many other European Union (EU) as well as East-European countries, USA, South-East Asia (Thailand), and WHO WPR (Japan, The Philippines, China, Korea, Australia). Among the isolates comprising an alteration of A501 in PBP2 (n=37), a total of 16 different STs were observed, of which 4 had not been previously described. These isolates were phylogenetically more heterogeneous (data not shown), however, nine isolates belonged to the identical ST (ST1103) (Table 2).

DISCUSSION

In the present study, it was clearly depicted that the susceptibility to extended-spectrum cephalosporins (both oral and injectable), the last remaining options for treatment of gonorrhoea, is rapidly decreasing and, according to the tentative EUCAST breakpoints [www.eucast.org], in vitro resistance to both cefixime and ceftriaxone has already been identified in Sweden. Furthermore, it was clearly shown that the decreased susceptibility and resistance to extended-spectrum cephalosporins such as cefixime and ceftriaxone is associated with both penA mosaic allele and alteration of A501 in PBP2, in synergy with the mtrR and penB resistance determinants, which has been previously reported. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF] However, the effects of the different resistance determinants on the fold-increase of the MICs differ for cefixime and ceftriaxone. [START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF] Interestingly, no clinical N. gonorrhoeae isolate with a penA mosaic allele that also contains mutations causing an alteration of A501 in PBP2 has ever been reported. The alteration of A501 in PBP2 still seems to be a gonococcal specific alteration, perhaps due to ESC selection pressure instead of transformation, which may reflect the lower extent of clonal spread observed in the present study. It cannot be excluded that an isolate with a penA mosaic allele that develops mutations resulting in an alteration of A501 comprises a lower biological fitness. However, probably this is not the case and instead it is only a matter of time before these type of strains will emerge, which may even be resistant to both cefixime and ceftriaxone.

The selection of isolates with MIC-values of ≥0.032 for cefixime and/or ceftriaxone in the present study made it possible to detect diverse combinations of the resistance determinants mentioned above. Accordingly, in order to detect isolates containing the initial ESC resistance determinants, i.e. in isolates that cannot be considered as fully susceptible to ESCs, the breakpoints stated by Clinical and Laboratory Standards Institute (CLSI [www.clsi.org]) and EUCAST [www.eucast.org] are too high. Nevertheless, detection of the initial resistance determinants (penA mosaic allele or alteration of A501 in PBP2, mtrR and penB alterations) is currently only for surveillance purposes, i.e. to monitor the emergence and spread of isolates with decreased ESC susceptibility, and not for identification of isolates with clinical resistance causing treatment failures. However, especially for emergence and spread of decreased susceptibility and resistance to ESCs, especially ceftriaxone, this type of surveillance (using lower MIC breakpoints), if possible, supplemented by genetic screening for detection of penA mosaic alleles as well as alterations of A501 in PBP2, in addition to the mtrR and penB determinants, and monitoring of longitudinal trends in ESC MICs over time would be exceedingly valuable. Alterations in additional genes have been previously suggested to contribute to the decreased susceptibility and resistance to ESCs such as mutations in pilQ (previously known as penC), [START_REF] Zhao | The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer stability[END_REF] and mutations in ponA encoding the PBP1. [START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF] However, recent studies have shown that, in contrast to the penicillins, pilQ and ponA do not significantly affect the susceptibility to ESCs in N. gonorrhoeae, [START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF][START_REF] Whiley | The Neisseria gonorrhoeae pilQ gene is an unlikely contributor to reduced susceptibility to extended spectrum cephalosporins in clinical strains[END_REF] therefore these two genes were not examined in the present study. Most important, in many cases it remains difficult to accurately correlate the known resistance determinants (alterations in penA, mtrR, and penB) to the ESC MICs of the isolates. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF] Partly this is due to the fact that there are different types of penA mosaic and PBP2 A501 altered alleles, which continue to evolve, and their effects on the ESC MICs of the corresponding gonococcal isolates highly vary, e.g. some A501 altered alleles do not significantly increase the ESC MICs. In addition, the presence or absence of the mtrR and penB resistance determinants further affects the MICs of the ESCs. However, it is also clear that at least one unknown resistance determinant exists and accordingly the mechanisms and their interplay that underlay the development of ESC decreased susceptibility and resistance are far from known. [START_REF] Tapsall | Meeting the public health challenge of multidrug-and extensively drug-resistant Neisseria gonorrhoeae[END_REF][START_REF] Lindberg | Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: Association with genetic polymorphisms in penA, mtrR, porB1b, and ponA[END_REF][START_REF] Zhao | Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae[END_REF] Accordingly, an enhanced understanding and better correlates of the genetic resistance determinants, the in vitro ESC MICs of the corresponding isolates, treatment outcome, and spread of isolates with decreased susceptibility and resistance to ESC are essential.

Finally, the present study revealed, using NG-MAST and full-length porB gene sequencing, that several different gonococcal strains with penA mosaic allele or alterations of A501 in PBP2 are circulating in Sweden and other countries.[www.ng-mast.net; present study; 2,3,4,6,29,30] However, specific gonococcal strains such as ST1407, and its evolving genetically highly similar subtypes, containing a penA mosaic allele mainly identical to the penA mosaic allele X associated with treatment failures using oral ESCs in e.g. Japan, [START_REF] Ito | Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in central Japan[END_REF] are now spreading in increasing prevalence in the WHO WPR (Japan, The Philippines, China, Korea, Australia), South-East Asia (Thailand), many EU as well as East-European countries, USA, etc.[www.ng-mast.net; present study; 2,4,29,30] Accordingly, a more or less global spread, after initial emergence in the WHO WPR, of some gonococcal penA mosaic allele strains with decreased susceptibility and resistance to ESCs can already be seen.

In conclusions, the proportions of N. gonorrhoeae isolates with decreased susceptibility and resistance to cefixime and ceftriaxone have substantially increased over the years in Sweden. Several different gonococcal strains containing penA mosaic allele or alterations of A501 in PBP2 with decreased susceptibility to ESCs are circulating in Sweden and other countries. However, specific gonococcal strains such as ST1407, and its evolving genetically highly similar subtypes, account for the majority of the isolates spreading in increasing prevalence in Sweden and many other countries worldwide. Nevertheless, the magnitude of this spread as well as the presence of treatment failures are far from known, and it is essential to more effectively monitor and also consider this in a global context. Finally, an enhanced surveillance, understanding and better correlates of the genetic resistance determinants, the in vitro ESC MICs of the corresponding isolates, treatment outcome, and spread of isolates with decreased susceptibility and resistance to ESC are essential.

Key messages

• Antimicrobial resistance (AMR) in gonococci is a major public health problem worldwide, and gonorrhoea may become untreatable in certain circumstances.

• The gonococcal susceptibility to extended-spectrum cephalosporins (ESCs), the last remaining treatment options in many settings globally, is rapidly decreasing in Sweden as observed worldwide.

• penA mosaic alleles and A501 alterations, together with mtrR and penB, are important for decreased susceptibility/resistance to ESCs, however, unknown resistance determinant(s) also exist(s).

• At least one gonococcal penA mosaic strain (ST1407), including its evolving genetically related subtypes, with decreased susceptibility/resistance to ESCs seems to be circulating worldwide.

  MIC, minimum inhibitory concentration a The selection of isolates with increased MICs of cefixime and ceftriaxone was performed from a collection of 348 isolates (1998), 85 (1999), 131 (2000), 141 (2001), 120 (2002), 130 (2003), 149 (2004), 486 (2005), 431 (2006), 487 (2007), 523 (2008) and 434 (2009). b Number of isolates that contained the characteristic single nucleotide (A) deletion in the inverted repeat of the promoter region and alteration of G45 in MtrR, respectively. c MIC-values for isolates that possessed a penA mosaic allele or an alteration of A501 in PBP2. d The alterations of A501 in PBP2 were A501V (n=33) and A501T (n=4).

  However, no isolates contained a penA mosaic allele including any mutations causing an alteration of A501 in PBP2.The proportion of isolates with penA mosaic allele substantially increased from the year 2006 (0.5%) to 2009 (8.1%), while there were no isolates with penA mosaic alleles during 1998-2001, in 2004 or in 2005. There were in total 11 isolates with penA mosaic allele already in 2002 (n=6) and 2003 (n=5), however, eight of those were received from USA (n=5) and UK (n=3) for a specific study regarding ESC decreased susceptibility. The prevalence of isolates with PBP2 A501 alteration ranged from 0.5% to 2.3% between 2003, when these were firstly detected, and 2009. The MICs of cefixime and ceftriaxone for the penA mosaic allele isolates ranged from 0.023-0.500 mg/L (mean MIC: 0.064 mg/L) and 0.008-0.250 mg/L (mean MIC: 0.047), respectively. The MICs of the isolates with PBP2 A501 alteration were 0.016-0.19 mg/L (cefixime, mean MIC: 0.064 mg/L) and 0.023-0.125 mg/L (ceftriaxone,

  Figures: 2

Figure 1 Figure 2

 12 Figure 1 The distribution of Neisseria gonorrhoeae MICs of A. ceftriaxone (1998 and 2009)

Table 1 .

 1 Neisseria gonorrhoeae displaying increased MICs of cefixime and/or ceftriaxone (≥0.032 mg/L) isolated during 1998-2009 in Sweden (n=331) Eight of the 11 isolates with decreased susceptibility and also penA mosaic allele in 2002 (n=6) and 2003 (n=5) were received from the USA (n=5) and UK (n=3) for a specific study regarding ESC decreased susceptibility.

	Yr of isolation	penA alteration (% of isolates)	mtrR alteration b	PorB1b (penB alteration)	MIC (mg/L) range c
	(No. of isolates; % of isolates) a	Mosaic allele Alteration of A501 in PBP2 d	Promoter region	Coding region	G101	A102	Cefixime	Ceftriaxone
	1998 (0; 0%), 1999 (2; 2%), 2000 (0;	None	•	•	•	•	•	•
	0%), 2001 (2; 1%), 2005 (32; 6%)	None	•	•	•	•	•	•
	2002 (21; 17%) e	6 (5.0) e None	6 •	6 •	6 •	6 •	0.25-0.5 •	0.064-0.125 •
	2003 (27; 21%) e	5 (3.8) e	5	None	5	5	0.25-0.5	0.125
		3 (2.3)	None	3	3	3	0.064-0.094	0.064-0.125
	2004 (17; 11%)	None 3 (2.0)	• 1	• 2	• 3	• 3	• 0.032-0.064	• 0.064-0.125
	2006 (49; 11%)	2 (0.5)	2	None	2	2	0.047-0.094	0.064-0.125
		3 (0.7)	3	None	2	3	0.032-0.047	0.064
	2007 (43; 9%)	10 (2.1) 9 (1.8)	10 5	None 6	10 9	10 9	0.032-0.25 0.016-0.047	0.032-0.125 0.032-0.064
	2008 (81; 15%)	20 (3.8) 12 (2.3)	20 11	None None	19 12	19 10	0.023-0.25 0.023-0.19	0.023-0.25 0.023-0.125
	2009 (57; 13%)	35 (8.1) 7 (1.6)	33 6	None None	34 7	34 7	0.032-0.38 0.023-0.064	0.008-0.125 0.023-0.094

e

Table 2 .

 2 The NG-MAST STs, year of isolation, and MICs of cefixime and ceftriaxone for all Neisseria gonorrhoeae with a Unique STs represented by single isolates; ST3128, ST3499, ST3709, ST3787, ST4265 a , ST4266 a , ST4267 a , ST4273 a , ST4276 a and ST4642 a .

	penA mosaic allele (n=78) or an alteration of A501 in PBP2 (n=37) isolated during 2002-2009 in Sweden
						No. of isolates with MIC (mg/L)
	NG-MAST ST (No. of isolates)	Year		≤0.032	0.047	0.064	0.094	0.125 0.19 0.25 0.38	0.5
	penA mosaic allele isolates (n=78)
	1407 (29)	2007-2009	IX TX	2 14	4 5	7 4	3 1	7 4	1	4 1	1
	3378 (8)	2008-2009	IX TX	5	1	2	2 1	1 1	1	1	1
	326 (6)	2002	IX TX			1		5		5		1
	925 (5)	2003	IX TX					5		2		3
	4272 (3) a	2007-2008	IX TX		1			2		3
	4275 (3) a	2008	IX TX	1		3		2
	4269 (3) a	2008-2009	IX TX				1 1	2		1	1
	3431 (3)	2009	IX TX	3	1	2
	4274 (2) a	2008-2009	IX TX	2	1	1
	2212 (2)	2009	IX TX	2	2
	3158 (2)	2009	IX TX	2		2
	4268 (2) a	2009	IX TX	1				1 1		1
	Unique STs (10) b	2006-2009	IX TX	1 3	1 2	2 1	2 1	1 3	2	1
	A501 alteration in PBP2 (n=37)
	1103 (9)	2008-2009	IX TX	2	2 1	3 3	1 2	1 3
	1619 (6)	2003, 2007	IX TX	1	2	2 4	1 1	1
	437 (3)	2006, 2008	IX TX		3	3
	2943 (3)	2007	IX TX	3 1	1	1
	1288 (3)	2009	IX TX	3 3
	1723 (2)	2004	IX TX			2 1		1
	1405 (2)	2007	IX TX	2 2
	Unique STs (9) c	2003-2009	IX TX	7 4	1	1 1	3		1
	NG-MAST, Neisseria gonorrhoeae multiantigen sequence typing; ST, sequence type; MIC, minimum inhibitory
	concentration; IX, cefixime; TX, ceftriaxone.
	c Unique STs represented by single isolates; ST1493, ST1582, ST1724, ST1766, ST3755, ST4271 a , ST4277 a , ST4278 a , and
	ST4279 a .

a New sequence types found in the present study.

b

Funding

This study was supported by grants from the Örebro County Council Research committee and the Foundation for Medical Research at Örebro University Hospital, Sweden.

Contributors

MU in collaboration with DG, BH and HF initiated and designed the study. DG performed all the laboratory work. DG and MU analysed all the data and wrote the manuscript, in collaboration with BH and HF.

Competing Interest: None declared.

EXCLUSIVE LICENCE

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in STI and any other BMJPGL products and sub-licences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms).

Conflict of interest


B Hellmark
email: bengt.hellmark@orebroll.se

M Unemo

Å Nilsdotter-Augustinsson

Söderquist

B Söderquist

In vitro antimicrobial synergy testing of coagulase-negative staphylococci isolated from prosthetic joint infections using Etest and with a focus on rifampicin and linezolid

In vitro antimicrobial synergy testing of coagulase-negative staphylococci isolated from prosthetic joint infections using Etest and with a focus on rifampicin and linezolid B. Hellmark, M. Unemo, Å. Nilsdotter-Augustinsson, B.

Introduction

Prosthetic joint infections (PJIs) are uncommon complications of prosthetic device surgery procedures, but represent harmful conditions with high morbidity and substantial costs. Coagulase-negative staphylococci (CoNS), and especially Staphylococcus epidermidis, is the most important pathogen involved in delayed or late onset PJIs [START_REF] Zimmerli | Prosthetic-joint infections[END_REF]. There are no standards for the antimicrobial treatment of PJIs [START_REF] Esposito | Prosthetic joint infections: microbiology, diagnosis, management and prevention[END_REF] and, during the recent decades, the treatment consisted predominately of cell wall antimicrobials such as isoxazolyl penicillin or glycopeptides [START_REF] Zavasky | Reconsideration of rifampin: a unique drug for a unique infection[END_REF]. However, without prosthetic joint exchange surgery, the long-term outcome has been disappointing, with a high rate of failures following finite treatment regimens [START_REF] Collins | Infections at the site of a hip implant. Successful and unsuccessful management[END_REF][START_REF] Marculescu | Outcome of prosthetic joint infections treated with debridement and retention of components[END_REF][START_REF] Zimmerli | Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial. Foreign-Body Infection (FBI) Study Group[END_REF]. S. epidermidis involved in a foreign body infection transforms into a stationary growth phase and produces biofilm. Therefore, cell wall antimicrobials may not be effective, since they do not efficiently penetrate the biofilm and have only a modest effect on bacteria in a non-growing phase [START_REF] Trampuz | Molecular and antibiofilm approaches to prosthetic joint infection[END_REF]. Accordingly, additional antimicrobial agents and/or combinations of antimicrobials for the treatment of foreign body infections have been assessed. Predominantly antimicrobial combinations including rifampicin, which penetrates the biofilm and is effective also against stationary phase bacteria, have been investigated in experimental studies [START_REF] Schwank | Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics[END_REF][START_REF] Svensson | Pharmacodynamic effects of antibiotics and antibiotic combinations on growing and nongrowing Staphylococcus epidermidis cells[END_REF][START_REF] Widmer | Correlation between in vivo and in vitro efficacy of antimicrobial agents against foreign body infections[END_REF] as well as in clinical trials [START_REF] Zimmerli | Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial. Foreign-Body Infection (FBI) Study Group[END_REF][START_REF] Berdal | Use of rifampicin and ciprofloxacin combination therapy after surgical debridement in the treatment of early manifestation prosthetic joint infections[END_REF][START_REF] Soriano | Treatment of acute postsurgical infection of joint arthroplasty[END_REF]. Resistance to rifampicin may rapidly emerge and, therefore, monotherapy should be avoided. To reduce the emergence of resistance to rifampicin, it is, accordingly, recommended to combine it with additional antimicrobials and several antimicrobials have been proposed, including fluoroquinolones [START_REF] Zimmerli | Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial. Foreign-Body Infection (FBI) Study Group[END_REF], linezolid [START_REF] Soriano | Efficacy and tolerability of prolonged linezolid therapy in the treatment of orthopedic implant infections[END_REF] and clindamycin [START_REF] Soriano | Treatment of acute postsurgical infection of joint arthroplasty[END_REF]. In addition, linezolid may be a treatment option for multidrug-resistant (MDR) S. epidermidis, as monotherapy [START_REF] Soriano | Efficacy and tolerability of prolonged linezolid therapy in the treatment of orthopedic implant infections[END_REF] or in combination with rifampicin [START_REF] Senneville | Effectiveness and tolerability of prolonged linezolid treatment for chronic osteomyelitis: a retrospective study[END_REF]. However, concerns have been raised due to supposed in vitro antagonism between rifampicin and other antimicrobial agents [START_REF] Zavasky | Reconsideration of rifampin: a unique drug for a unique infection[END_REF]. This strengthens the need for synergy testing of antimicrobial combinations including rifampicin that are considered for treatment, which may also be needed for all antimicrobial combinations.

The aim of this study was to investigate antimicrobial combinations against S. epidermidis that may be of clinical interest for the long-term treatment of PJIs, focusing on rifampicin and linezolid. In vitro synergy testing was performed using Etest.

Materials and methods

Thirty-seven clinical CoNS isolates obtained during revision surgery for PJIs with extraction or exchange of the prosthetic devices were analysed. Multiple tissue biopsy samples, usually five or more, were taken perioperatively. The isolates were collected from patients with infected hip (n=27), knee (n=9) and elbow joint (n=1) prostheses. The isolates comprised S. epidermidis (n=33), S. capitis (n=1), S. haemolyticus (n=1), S. simulans (n=1) and S. lugdunensis (n=1). The minimum inhibitory concentrations (MICs, µg/mL) of all antimicrobials were determined using the Etest method (AB Biodisk, Solna, Sweden). Rifampicin and linezolid were tested in combination with each other and with clindamycin, fusidic acid, ciprofloxacin and trimethoprim-sulfamethoxazole (SXT), respectively. First, the MIC for each antimicrobial was determined according to the guidelines from the Swedish Reference Group for Antibiotics (SRGA; http://www.srga.org). For the combination (synergy) testing, two methods, fixed ratio and MIC/ MIC ratio, were performed [START_REF] Manno | Use of the E test to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis[END_REF][START_REF] Pankey | In vitro synergy of ciprofloxacin and gatifloxacin against ciprofloxacin-resistant Pseudomonas aeruginosa[END_REF][START_REF] Tsuji | Etest synergy testing of clinical isolates of Staphylococcus aureus demonstrating heterogeneous resistance to vancomycin[END_REF]. Briefly, in the fixed ratio method, an Etest strip of the first antimicrobial was placed on an agar plate, its exact location marked on the back of the plate, and incubated for 1 h at room temperature. Then, the Etest strip was removed and an Etest strip of the second antimicrobial was placed at the same location on the agar plate as the first strip. In the MIC/ MIC ratio method, an Etest strip of the first antimicrobial was placed on an agar plate, with the location and the MIC value from the single testing marked on the back of the plate, and incubated for 1 h at room temperature. Then, the Etest strip was removed and an Etest strip of the second antimicrobial was superimposed so that the respective MIC values from the single testing were aligned. All tests were performed on IsoSensitest agar (Oxoid, Hampshire, England) and at 36°C aerobic atmosphere for 18-24 h.

In order to determine whether each specific combination of antimicrobials resulted in a synergistic, additive, indifferent or antagonistic effect, the fractional inhibitory concentration (FIC) index was calculated as previously described [START_REF] Manno | Use of the E test to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis[END_REF]. The following cut-off values and interpretative criteria in regards of the achieved effect were used; FIC index ≤0.5 indicated a synergistic effect, FIC index >0.5 and ≤1.0 an additive effect, FIC index >1.0 and ≤4.0 indifferent effect, and FIC index >4.0 an antagonistic effect [START_REF] Manno | Use of the E test to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis[END_REF]. Random isolates were repeatedly analysed in order to assess the reproducibility.

Results and discussion

The MIC values for all isolates and all antimicrobial agents in single use are shown in Table 1.

Altogether, 888 MIC determinations were performed using the fixed ratio method and the MIC/MIC ratio method. The results of the MIC/MIC ratio method were more reproducible and easier to interpret, especially when evaluating two antimicrobials with substantial differences in their MIC values. Accordingly, the results of the antimicrobial synergy testing, using Etest and the MIC/ MIC ratio method, of all isolates and antimicrobial combinations are summarised in Table 2.

Overall, 16 (43%) of the isolates displayed a synergistic (n=5), additive (n=14) or antagonistic (n=11) effect when combining two antimicrobials (Table 2).

The combination of rifampicin and ciprofloxacin displayed no synergistic effect in vitro. Instead, there were two (5%) Including one strain not possible to distinguish between indifferent and additive effect c Including two strains not possible to distinguish between additive and synergistic effect d Including two strains not possible to distinguish between indifferent and additive effect e Including one strain not possible to distinguish between indifferent and additive effect and one strain not possible to completely distinguish between additive and synergistic effect

isolates showing an antagonistic effect with an increase of the ciprofloxacin MIC from 6 µg/ml and 8 µg/ml, respectively, to >32 µg/ml. For rifampicin and fusidic acid, two isolates (5%) displayed a synergistic effect, with a decrease of the rifampicin MIC from >32 µg/ml to 8 µg/ml and 0.125 µg/ml, respectively, as well as of fusidic acid from 6 µg/ml and 12 µg/ml to 1.5 µg/ml and 0.064 µg/ml, respectively. For rifampicin and clindamycin, four isolates (11%) displayed an additive effect, with a decrease of the clindamycin MIC from >256 µg/ml (n=3) to 0.94 µg/ml (n=2) and 128 µg/ml, respectively, and one additional isolate displayed a decrease from 0.19 µg/ml to 0.064 µg/ml. Linezolid comprises no known cross-resistance to other antimicrobials and is highly effective against Gram-positive bacteria. However, the treatment is expensive and long-term use may cause adverse effects [START_REF] Soriano | Efficacy and tolerability of prolonged linezolid therapy in the treatment of orthopedic implant infections[END_REF][START_REF] Rao | Similar hematologic effects of long-term linezolid and vancomycin therapy in a prospective observational study of patients with orthopedic infections[END_REF]. For the combination rifampicin and linezolid, no synergistic or additive effects were found in the present in vitro test, but in a previous study by Jacqueline et al. [START_REF] Jacqueline | In vitro activity of linezolid alone and in combination with gentamicin, vancomycin or rifampicin against methicillin-resistant Staphylococcus aureus by time-kill curve methods[END_REF], using the time-kill curve method, an additive effect could be seen. This has also been indicated in clinical studies [START_REF] Soriano | Efficacy and tolerability of prolonged linezolid therapy in the treatment of orthopedic implant infections[END_REF][START_REF] Senneville | Effectiveness and tolerability of prolonged linezolid treatment for chronic osteomyelitis: a retrospective study[END_REF]. Accordingly, this combination treatment may have a synergistic effect in vivo. Regarding linezolid and fusidic acid, almost 20% of the isolates displayed a positive effect (synergistic or additive). For linezolid and clindamycin, all three effects were found, i.e. two isolates (5%) displayed synergistic effect, two (5%) additive effects and three (8%) antagonistic effects. Clindamycin might be of clinical interest as the empirical treatment of PJIs, since it has a good effect on aerobic as well as anaerobic bacteria. Linezolid in combination with trimethoprim-sulfamethoxazole displayed the highest percentage of antagonistic effect (four isolates; 11%), with an increase of linezolid MIC by 2 to 3 whole MIC steps, while rifampicin and trimethoprim-sulfamethoxazole displayed no antagonistic effect and one isolate (3%) displayed an additive effect.

The reproducibility of the antimicrobial synergy testing using Etest was high (data not shown). Ideally, the Etest method for synergy testing of staphylococci should be further validated to a "gold standard," such as checkerboard titration or any in vitro kinetic model for synergy testing. However, there have been studies comparing Etest, the time-kill curve method and/or checkerboard methods for organisms such as Acinetobacter baumannii [START_REF] Bonapace | Evaluation of antibiotic synergy against Acinetobacter baumannii: a comparison with Etest, time-kill, and checkerboard methods[END_REF], Candida species [START_REF] Lewis | Comparison of Etest, chequerboard dilution and timekill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species[END_REF] and Burkholderia cepacia [START_REF] Manno | Use of the E test to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis[END_REF]. All of these studies concluded that Etest is simple to use, time-efficient, reproducible and has good agreement to other methods for synergy testing. However, additional investigations are needed. A limitation of this study was that only bacteria in the growth phase, and not in the stationary phase protected by biofilm, were examined. Accordingly, the results may not completely reflect the situation in vivo during a PJI, but examine interesting antimicrobial combinations and their interactions in vitro.

In conclusion, Etest is an objective and easily performed in vitro method for the antimicrobial synergy testing of CoNS. However, the effects of different combinations of antimicrobials were strain-dependent and, accordingly, each isolate needs to be tested for the specific antimicrobial combination considered for treatment.

Table 1

 1

	Minimum inhibitory concentrations (MICs; µg/ml), using Etest, of six antimicrobials in coagulase-negative staphylococcal isolates
	(n=37) from prosthetic joint infections
		Rifampicin	Linezolid	Ciprofloxacin	Fusidic acid	Clindamycin	Trimethoprim-sulfamethoxazole
	S. epidermidis	>32	0.19	8	0.047	0.094	>640
	S. epidermidis	>32	0.047	>32	6	>256	>640
	S. epidermidis	0.002	0.19	16	12	>256	>640
	S. epidermidis	>32	0.19	>32	6	0.023	>640
	S. epidermidis	>32	0.19	>32	0.047	>256	>640
	S. epidermidis	<0.002	0.125	>32	6	0.047	>640
	S. epidermidis	>32	0.19	>32	12	>256	>640
	S. epidermidis	0.25	0.125	>32	12	>256	>640
	S. epidermidis	0.003	0.19	>32	16	>256	>640
	S. epidermidis	>32	0.19	>32	0.047	>256	>640
	S. epidermidis	0.003	0.19	>32	0.032	>256	2.5
	S. epidermidis	0.003	0.25	>32	12	0.064	>640
	S. epidermidis	>32	0.25	>32	0.047	>256	>640
	S. epidermidis	6	0.125	3	0.032	>256	>640
	S. epidermidis	0.003	0.19	>32	0.032	>256	>640
	S. epidermidis	0.003	0.25	>32	0.023	>256	320
	S. epidermidis	>32	0.125	>32	6	>256	>640
	S. epidermidis	>32	0.25	>32	0.047	>256	>640
	S. epidermidis	0.003	0.25	>32	0.032	>256	>640
	S. epidermidis	0.002	0.25	>32	6	>256	>640
	S. epidermidis	>32	0.19	0.19	0.023	>256	>640
	S. epidermidis	>32	0.19	6	0.023	>256	>640
	S. epidermidis	>32	0.19	0.047	0.047	>256	>640
	S. epidermidis	0.002	0.38	16	6	0.094	480
	S. epidermidis	0.003	0.25	0.19	0.047	0.125	5
	S. epidermidis	0.003	0.38	16	0.064	0.064	>640
	S. epidermidis	0.003	0.25	>32	4	>256	>640
	S. epidermidis	0.003	0.25	0.19	0.047	0.094	0.64
	S. epidermidis	0.003	0.25	>32	0.016	0.064	480
	S. epidermidis	0.003	0.19	>32	8	0.094	>640
	S. epidermidis	0.003	0.25	0.25	0.125	>256	0.94
	S. epidermidis	0.004	0.19	0.19	0.032	>256	3.8
	S. epidermidis	0.003	0.38	0.38	0.032	0.125	0.94
	S. capitis	0.012	0.38	0.125	0.064	0.19	1.88
	S. haemolyticus	0.006	0.38	>32	12	>256	240
	S. simulans	0.006	0.5	4	0.047	0.19	15
	S. lugdunensis	<0.002	0.094	0.25	0.047	0.064	1.88

Table 2

 2 Antimicrobial synergy testing, using Etest, of coagulase-negative staphylococcal isolates (n=37) from prosthetic joint infections

	Antimicrobial combination a	Number (%) of isolates
		Synergism	Addition	Indifference	Antagonism
	RI + LZ	0	0	36 (97%)	1 (3%)
	RI + CI	0	0	35 (95%) b	2 (5%)
	RI + FU	2 (5%)	0	35 (95%)	0
	RI + CM	0	4 (11%) c	33 (89%)	0
	RI + SXT	0	1 (3%)	36 (97%) d	0
	LZ + CI	0	1 (3%)	35 (95%)	1 (3%)
	LZ + FU	1 (3%)	6 (16%)	30 (81%) e	0
	LZ + CM	2 (5%)	2 (5%)	30 (81%)	3 (8%)
	LZ + SXT	0	0	33 (89%)	4 (11%)
	a RI, rifampicin; LZ, linezolid; CI, ciprofloxacin; FU, fusidic acid; CM, clindamycin; SXT, trimethoprim-sulfamethoxazole
	b