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Title: Conjunctivitis caused by Neisseria gonorrhoeae with reduced 1
cephalosporin susceptibility and multidrug resistance 2
3
Running title: Neisseria gonorrhoeae Conjunctivitis 4
5
Takashi Suzuki, M.D., Ph.D. ,1,# Hiroshi Kitagawa, MSc, 1 Yosuke Maruyama,1 6
Satoshi Yamaguchi, MSc,1, * Yuri Sakane, M.D.,1 Hitoshi Miyamoto, 2 Yuichi 7
Ohashi, M.D., Ph.D. 1 8
1Department of Ophthalmology, Ehime University, Graduate School of Medicine, 9
Shitsukawa, Toon, Ehime 791-0295, Japan 10
2Department of Clinical Laboratory, Ehime University Hospital, Shitsukawa, 11
Toon, Ehime 791-0295, Japan 12
Footnote * Present address: ROHTO Pharmaceutical Co., Ltd, 6-5-4, 13
Kunimidai, Kizugawa, Kyoto 619-0216, Japan 14
15
#To whom correspondence should be addressed: Department of 16
Ophthalmology, Ehime University, Graduate School of Medicine, Shitsukawa, 17
Toon, Ehime 791-0295, Japan. 18
Phone: +81-89-960-5361, Fax: +81-89-960-5364, 19
E-mail: [email protected] 20
21
JCM Accepts, published online ahead of print on 11 September 2013J. Clin. Microbiol. doi:10.1128/JCM.01946-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 22
We report two cases of conjunctivitis caused by Neisseria gonorrhoeae with 23
reduced cephalosporin susceptibility. Patients showed no response to 24
cefmenoxime eye drops and intravenous ceftriaxone administration. The 25
patients’ condition improved after addition of oral minocycline. The isolates 26
contained the mosaic penA for reduction of く-lactam susceptibility. 27
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CASE REPORT 28
Case 1. A 31-year-old man visited a private clinic and presented with 29
conjunctival injection and discharge in his right eye. A diagnosis of acute 30
conjunctivitis was made and he was treated with a combination of 1.5% 31
levofloxacin (LVFX) and 0.1% betamethasone eye drops 4 times per day. 32
However, his symptoms had worsened 2 days later and he was referred to our 33
hospital. Slit-lamp biomicroscopy revealed severe purulent discharge, 34
conjunctival injection, and eyelid edema in the right eye (Fig. 1A). However he 35
did not have conjunctival papillae or follicles, and corneal epithelial damage. 36
Direct microscopy and bacterial culture of the discharge were performed. Direct 37
microscopy demonstrated the presence of gram-negative diplococci, and the 38
culture reports confirmed the presence of Neisseria gonorrhoeae. The patient 39
did not have other gonococcal infection, such as pharyngitis or urethritis. We 40
considered conjunctivitis caused by N. gonorrhoeae, and began treatment with 41
topical 0.5% cefmenoxime (CMX) every hour and intravenous administration of 42
ceftriaxone (CTRX) (1 g/day) for 3 days. Although the amount of discharge was 43
slightly decreased 1 week after initiating the therapy, conjunctival injection was 44
still active (Fig. 1B). We added oral minocycline (MINO; 200 mg/day) for 2 weeks 45
after obtaining drug susceptibility testing results, and the conjunctivitis resolved 46
within 7 days (Fig. 1C). 47
Case 2. A 28-year-old woman visited a local hospital and presented with 48
conjunctival injection and discharge in the right eye. She was treated with a 49
combination of 1.5% LVFX and 0.1% fluorometholone eye drops 4 times per day. 50
However, the amount of discharge increased and she was referred to our 51
hospital. Slit-lamp biomicroscopy revealed severe purulent discharge and eyelid 52
edema. Patient's condition was similar to the condition shown in Fig. 1A. 53
Microbiological tests revealed the presence of Neisseria gonorrhoeae. She did 54
not have other gonococcal infection. We started treatment with topical 0.5% 55
CMX every hour and intravenous administration of CTRX (1 g/day) for 3 days. 56
As conjunctival infection was still active 3 days later, oral MINO (200 mg/day) 57
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was administered for 1 week. Conjunctival inflammation subsided within 5 days 58
after addition of MINO. 59
60
Antimicrobial susceptibility testing 61
Three N. gonorrhoeae conjunctivitis isolates (EC358, EC359, and EC985) 62
were obtained between 2003 and 2013 at Ehime University Hospital. These 63
isolates, along with two other isolates (EC1025 from case 1 and EC1050 from 64
case 2) were used The ID-test HN-20 Rapid system confirmed identification of 65
N. gonorrhoeae. Antimicrobial susceptibility testing was performed using the E 66
test as described elsewhere (1). E test strips containing CTRX, cefuroxime 67
(CXM), cefalotin (CET), benzylpenicillin (PCG), and MINO, along with various 68
other antimicrobial agents, including routine ophthalmic agents such as LVFX, 69
gatifloxacin (GFLX), moxifloxacin (MFLX), chloramphenicol (CP), tobramycin 70
(TOB), erythromycin (EM), and azithromycin (AZM), were purchased from AB 71
bioMérieux. The minimum inhibitory concentration (MIC) was determined 72
according to the manufacturer’s instructions. After incubation, an ellipse 73
appeared on the MIC value scale (in たg/mL) where the concentration of the 74
antibiotic tested inhibited bacterial growth. All results were interpreted using 75
cut-off values for susceptibility and resistance according to the Clinical and 76
Laboratory Standards Institute (CLSI [M100-S22]) and The European 77
Committee on Antimicrobial Susceptibility Testing (EUCAST [www.eucast.org]). 78
No organization has established cut-off values for CET, MINO, CP, TOB, and 79
EM. MICs of the drugs tested are summarized in Table 1. The MICs of various 80
antimicrobials, except TOB and MINO, were higher for EC985, EC1025, and 81
EC1050, as compared to EC358 and EC359. EC985, EC1025, and EC1050 82
showed decreased susceptibility to cephalosporins, fluoroquinolones and 83
macrolides. The MICs of MINO against EC358, EC359, EC1025, and EC1050 84
were similar. 85
Genetic characterization 86
For molecular epidemiological examination, strains were genotyped by 87
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multilocus sequence typing (MLST) (2). PCR and sequencing of resistance 88
determinants, i.e., the penA, mtrR, ponA, porB1b (penB), gyrA, and parC genes, 89
were performed as described elsewhere (3 – 5). The genetic characteristics of 90
isolates are summarized in Table 2. The sequence types (STs) of EC985 and 91
EC1025 by MLST were ST 1901, which was the same as that of a 92
multidrug-resistant gonococcal clone that has spread worldwide (6 – 9). The ST 93
of EC1050 was ST 7363, which was the same as that of cefixime-resistant 94
isolates circulating in Japan (2). EC985, EC1025, and EC1050 were found to 95
have penA mosaic allele X, which has correlated with reduced susceptibility to 96
cephalosporins, including CTRX, in Japan (7 – 10). We investigated mutations in 97
the gyrA and parC genes, which confer fluoroquinolone resistance. EC358 and 98
EC 359 strains contained two amino acid substitutions, i.e., Ser91›Phe and 99
Asp95›Asn mutations within GyrA protein, while EC985 and EC1050 had 100
Ser87›Arg mutation and EC1025 had Ser87›Arg and Ser88›Pro mutations 101
within the ParC protein along with two mutations within GyrA protein. 102
Sequencing of other resistance determinants showed that all strains contained 103
the identical mtrR and penB alterations, i.e., an A deletion in the inverted repeat 104
of the mtrR promoter and two consecutive amino acid alterations in penB. In 105
addition, they also showed alteration of amino acid 421 in ponA. 106
107
Discussion 108
N. gonorrhoeae, which is the gram-negative coccus responsible for sexually 109
transmitted infection, rarely causes acute conjunctivitis. Transmission to the eye 110
could be by contact with infected urine or genital secretions. The incidence rates 111
of gonococcal conjunctivitis increase during spring and summer (11). This is a 112
potentially devastating ocular infection because N. gonorrhoeae can cause 113
severe ulcerative keratitis, which may rapidly progress to corneal perforation (11 114
– 14). Kawashima et al. reported that the durations between conjunctivitis and 115
corneal perforation were from 9 to 11 days in 5 cases of gonococcal 116
keratoconjunctivitis (12). Although it is critical to obtain an accurate diagnosis 117
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and start treatment as early as possible, it is difficult to diagnose in cases that do 118
not show systemic involvement, as in the present cases. As neither case 1 nor 2 119
had systemic gonococcal infection and the infection routes in these cases were 120
not known, diagnosis was delayed. Parenteral and topical antibiotics are 121
necessary to treat gonococcal conjunctivitis. A parenteral cephalosporin, such 122
as CTRX, is recommended as a first-line treatment for gonococcal infection. 123
Along with parenteral cephalosporins, topical antibiotics are generally added for 124
treatment of conjunctivitis. As the commercial cephalosporin available for 125
ophthalmic solution in Japan is CMX, which is a third-generation cephalosporin 126
antibiotic, CMX eye drops are generally chosen for gonococcal conjunctivitis. 127
Isolates from patients 1 and 2 contained the identical penA mosaic allele X and 128
showed reduced susceptibility to cephalosporin, including first-generation (CXM), 129
second-generation (CET), and third-generation agents (CTRX). The global 130
spread of gonococcal strains with decreased susceptibility to cephalosporins, 131
including CTRX, from Asia to Europe is now a major public health concern (6, 8, 132
9). Patients 1 and 2 did not respond promptly to parenteral CTRX and CMX eye 133
drops. Reduced in vitro susceptibility could be related to treatment failure. 134
CTRX-resistant N. gonorrhoeae, which had high MIC of CTRX (2 og/mL), was 135
isolated from female commercial sex workers in Japan (15). As STs of isolates 136
from patients 1 and 2 by MLST were the same as those of a 137
cephalosporin-resistant gonococcal clone that is spreading worldwide, the clonal 138
spread of resistant N. gonorrhoeae is a matter of concern. Due to the emergence 139
of CTRX-resistant strains in Japan, it is critical to consider several possibilities 140
for treatment of gonococcal infection. To treat gonococcal conjunctivitis, it is 141
necessary to use systemic antibiotics or antibiotic eye drops that can penetrate 142
into the ocular tissue. In our cases, inflammation of the conjunctival sac 143
decreased promptly after addition of oral MINO. The MICs of MINO against 144
isolates from patients 1 and 2 were 0.125 and 0.25 og/mL, respectively, which 145
were similar to those against isolates in 2003. Although the isolates tested had 146
penB mutations that reduce porin permeability of the outer membrane to 147
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hydrophilic antibiotics, such as tetracycline, susceptibility to MINO was not 148
markedly different among the isolates tested. MINO is known to penetrate into 149
ocular tissue very well, and has antiinflammatory, antimetalloproteinase, and 150
antiapoptotic properties (16, 17). Thus, MINO may be useful for treatment of 151
gonococcal conjunctivitis. Although fluoroquinolone eye drops are increasingly 152
used for treatment of bacterial conjunctivitis in general clinical practice, due to 153
their drug stability and broad spectra, the gonococcal resistance level to 154
fluoroquinolones is increasing in Japan (5, 18). Gonococcal isolates in 2003 had 155
mutations within the GyrA protein, but isolates from our cases also had 156
mutations within not only GyrA protein but also the ParC protein and showed 157
higher fluoroquinolone MICs. Consistent with the results of in vitro susceptibility 158
testing, the LVFX eye drops initially administered were ineffective for treatment 159
of conjunctivitis in our cases. However, the MICs of GFLX and MFLX (2 og/mL) 160
were lower than that of LVFX. It is likely that eye drops with high concentrations 161
of GFLX (3 mg/mL) and MFLX (5 mg/mL) would have efficacy for gonococcal 162
conjunctivitis rather than LVFX. The MICs of CP, EM, and AZM, which are 163
available for ophthalmic solutions, against isolates from patients 1 and 2 were 164
higher than those against isolates in 2003. However, AZM eye drops could be 165
effective because this drug can penetrate into conjunctival tissue and show a 166
sustained high concentration exceeding the MIC (0.125 – 0.25 og/mL) (19). For 167
effective treatment of conjunctivitis caused by multidrug-resistant gonococcus, 168
antibiotics should be chosen carefully with reference to the results of 169
confirmatory susceptibility testing. 170
In conclusion, we encountered two cases of conjunctivitis caused by N. 171
gonorrhoeae with reduced cephalosporin susceptibility. Ophthalmologists should 172
diagnose gonococcal conjunctivitis as early as possible and refer patients for 173
gonococcal antimicrobial susceptibility testing. Further epidemiological 174
surveillance of N. gonorrhoeae isolated from conjunctivitis is necessary. 175
176
177
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Acknowledgements 178
This study was supported in part by a Grant-in-Aid for scientific research from 179
the Japan Society for the Promotion of Science (KAKENHI: Grants-in-Aid for 180
Young Scientists B, 24791858). S.Y. is an employee of ROHTO Pharmaceutical 181
Co., Ltd. (Osaka, Japan). The other authors declare no conflict of interest. 182
183
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257
Figure legends 258
Figure 1. Case- 1 (A) Photograph of the eye before treatment with CTRX and 259
CMX, showing the massive conjunctival infection and severe purulent 260
discharge. (B) Photograph of the eye one week after treatment with CTRX 261
and CMX, showing conjunctival infection. (C) Photograph of the eye 7 262
days after addition of MINO, showing normal appearance of the 263
conjunctival sac. 264
265
266
267
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Table 1. Antibiotic susceptibilities of N. gonorrhoeae strains
strain Isolate
year
MIC(たg/mL)
CTRX LVFX GFLX MFLX CXM CET PCG CP TOB EM AZM MINO
E358 2003 0.008 2 0.25 0.5 0.032 1 0.25 1 8 0.032 0.032 0.125
E359 2003 0.008 2 0.25 0.5 0.064 2 0.25 1 8 0.032 0.032 0.125
E985 2009 0.125 8 4 2 4 16 4 4 8 1 0.25 0.5
E1025
(Patients1) 2011 0.125 8 2 2 4 16 2 4 4 1 0.25 0.125
E1050
(Patients2) 2012 0.125 8 2 2 2 8 0.5 4 8 0.5 0.125 0.25
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Table 2. Genetic characterization of N. gonorrhoeae strains
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EC358 UV9582" X"Ugt;3比Rjg" "
Cur;7比Cup"YVd" Ngw643比Rtq"
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EC359 UV9582" X"Ugt;3比Rjg" "
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In{323比N{u"
Cnc324比Cur"C/fgn"
EC985 UV3;23" Z"*oqucke+"Ugt;3比Rjg"
" Cur;7比Cup"Ugt:9比Cti" " Ngw643比Rtq" "
In{323比N{u"
Cnc324比Cur"C/fgn"
EC1025 (Patients1) UV3;23" Z"*oqucke+"Ugt;3比Rjg" "
Cur;8比Cup"
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In{323比N{u"
" Cnc324比Cur"C/fgn"
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EC1050 (Patients2) UV9585" Z"*oqucke+"Ugt;3比Rjg" "
Cur;9比Cup"Ugt:9比Cti" Ngw643比Rtq"
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