Timothy Neal

Emergence of high-level azithromycin resistance in Neisseria gonorrhoeae in England and Wales

OBJECTIVES This study aimed to investigate the origin of high-level azithromycin resistance that emerged in isolates of Neisseria gonorrhoeae in England and Wales in 2007, and to establish methods for identifying high-level azithromycin resistance. METHODS The Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) data from 2001-07 were examined for emerging trends in azithromycin susceptibility. Further to the identification of six high-level azithromycin-resistant isolates in GRASP 2007, an additional 102 isolates were selected on the basis of azithromycin susceptibility and geographic origin from the GRASP 2006 and 2007 collections. Susceptibility testing by Etest and disc diffusion was performed on all 108 isolates and 75 of these were typed by N. gonorrhoeae multiantigen sequence typing. RESULTS A slight drift towards higher MICs of azithromycin was observed in the gonococcal population since 2001. Of greater concern was the first example of a shift to high-level resistance observed in six isolates in 2007. All six isolates were sequence type 649, which was not observed in any of the lower-level azithromycin-resistant isolates from 2007 or in any isolates tested from the same geographical locations. Contact tracing data for one patient suggested a link with Scotland. Disc diffusion testing of all 108 isolates showed that azithromycin, but not erythromycin, discs can differentiate between low-level and high-level resistance. CONCLUSIONS High-level azithromycin resistance has emerged in England and Wales. Contact tracing and typing data suggest this may have originated from Scotland. Surveillance of azithromycin resistance will be key in controlling its further dissemination.

Minimum Inhibitory Concentrations of Standard and Novel Antimicrobials for Isolates from Bacterial Keratitis

PURPOSE To determine the minimum inhibitory concentrations (MICs) of 12 antimicrobials in current ophthalmic use and 4 potentially new alternatives against isolates from bacterial keratitis. METHODS Bacteria were collected from cases of bacterial keratitis in six centers in the United Kingdom between 2003 and 2006. MICs were measured by using susceptibility strips containing a concentration gradient of the antimicrobials penicillin, cefuroxime, ceftazidime, chloramphenicol, gentamicin, amikacin, vancomycin, teicoplanin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, meropenem, linezolid, tigecycline, and daptomycin. RESULTS Isolates (n = 772) were collected including coagulase negative Staphylococcus (CNS) (30%), Pseudomonas aeruginosa (23%), Staphylococcus aureus (14%), Enterobacteriaceae (14%), and streptococci (13%). Meropenem had low MICs for most isolates. All isolates except P. aeruginosa were susceptible to tigecycline. Linezolid was active against the majority of Gram-positive pathogens. Ten percent of S. aureus and 20% of CNS isolates were methicillin resistant. When systemic breakpoints were used, 84% of S. aureus isolates were susceptible to ciprofloxacin and 98% to moxifloxacin. Of the P. aeruginosa isolates, 99% were susceptible to ceftazidime, 96% to gentamicin, 99% to ciprofloxacin and 100% to moxifloxacin. More than 97% of Enterobacteriaceae isolates were susceptible to ceftazidime, gentamicin, ciprofloxacin, and moxifloxacin. CONCLUSIONS Based on systemic breakpoint data, resistance to commonly used antimicrobials was apparent. Meropenem is a potentially effective agent for ophthalmic use, with low MICs throughout all the bacterial subgroups. Tigecycline and linezolid showed good activity against particular groups and may be useful for treating bacterial keratitis resistant to current antimicrobials. Of the fluoroquinolones, moxifloxacin showed the lowest MICs and resistance for both Gram-positive and -negative bacteria.

Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections.

ABSTRACT Pseudomonas aeruginosa is a common opportunistic bacterial pathogen that causes a variety of infections in humans. Populations of P. aeruginosa are dominated by common clones that can be isolated from diverse clinical and environmental sources. To determine whether specific clones are associated with corneal infection, we used a portable genotyping microarray system to analyze a set of 63 P. aeruginosa isolates from patients with corneal ulcers (keratitis). We then used population analysis to compare the keratitis isolates to a wider collection of P. aeruginosa from various nonocular sources. We identified various markers in a subpopulation of P. aeruginosa associated with keratitis that were in strong disequilibrium with the wider P. aeruginosa population, including oriC, exoU, katN, unmodified flagellin, and the carriage of common genomic islands. The genome sequencing of a keratitis isolate (39016; representing the dominant serotype O11), which was associated with a prolonged clinical healing time, revealed several genomic islands and prophages within the accessory genome. The PCR amplification screening of all 63 keratitis isolates, however, provided little evidence for the shared carriage of specific prophages or genomic islands between serotypes. P. aeruginosa twitching motility, due to type IV pili, is implicated in corneal virulence. We demonstrated that 46% of the O11 keratitis isolates, including 39016, carry a distinctive pilA, encoding the pilin of type IV pili. Thus, the keratitis isolates were associated with specific characteristics, indicating that a subpopulation of P. aeruginosa is adapted to cause corneal infection.

Bacterial susceptibility to topical antimicrobials and clinical outcome in bacterial keratitis.

PURPOSE To investigate the relationship between the susceptibility of bacteria to topical antimicrobials and clinical outcome in microbial keratitis. METHODS Clinical outcome data were collected from patients with microbial keratitis from whom a bacterium had been isolated during the period 2003 to 2006. The minimum inhibitory concentration (MIC) was determined for the isolates against 10 antimicrobials. The determinants of the primary clinical outcome, the ratio of healing time (closure of epithelial defect) to ulcer size (HT/UA), was analyzed in a general linear model. RESULTS Complete clinical outcome and MIC data were available for 421 patients. Sixteen (4%) patients required enucleation and 23 (5%) surgical treatment; in 382 (91%) the ulcer healed with intensive topical antimicrobial therapy. There were significant correlations between HT/UA and organism type (P = 0.001), nearest distance of the ulcer to the limbus (0.02), and MIC of the first antimicrobial used or lowest MIC of combined therapy (P = 0.006). In a model including patients who received monotherapy with a fluoroquinolone who had no subsequent change in their treatment and whose ulcers healed without surgical intervention, there were significant linear associations between clinical outcome and MIC for Pseudomonas spp. (P = 0.047), Staphylococcus aureus (P = 0.04), and Enterobacteriaceae (P = 0.045), but not for Streptococcus spp. (P = 0.85) and coagulase-negative staphylococci (CNS) (P = 0.88). CONCLUSION With fluoroquinolone monotherapy, there was significant association between the MIC of the antimicrobial prescribed and the clinical outcome with all bacteria except CNS and Streptococcus spp. The approach used in this study, if used prospectively, could allow topical breakpoint susceptibility concentrations to be determined for individual antimicrobial and bacterial combinations.

Emergence of a Neisseria gonorrhoeae clone showing decreased susceptibility to cefixime in England and Wales

OBJECTIVES The third-generation cephalosporins recommended in national guidelines are amongst the last remaining effective agents for treatment of gonorrhoea. This study characterizes gonococcal isolates with decreased cefixime susceptibility from England and Wales. METHODS A total of 96 isolates of Neisseria gonorrhoeae exhibiting cefixime MICs of ≥0.125 mg/L, either collected as part of the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) between 2005 and 2008 (54 from a total of 4649 isolates) or referred to the national reference laboratory in 2008 and 2009 (42 isolates), were tested for susceptibility to a range of antimicrobial agents and were typed using N. gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS All 96 isolates were also resistant to tetracycline (MIC ≥2 mg/L) and ciprofloxacin (MIC ≥16 mg/L) and 56% showed low-level chromosomal resistance to penicillin. Where data were available, the mean patient age was 31 years, and 88% (83/94) of patients were men. Isolates referred through GRASP were predominantly from men who have sex with men (MSM; 29/44, 66%) and from patients of white British ethnicity (25/43, 58%). The majority of isolates belonged either to sequence type (ST) 1407 (71/96, 74%) or to a highly related ST that shares the tpbB allele (allele 110), but with a different por allele (20/96, 21%). ST1407 was found in both MSM (22/29, 76%) and heterosexual patients (12/15, 80%) and among all eight isolates from patients reporting sex abroad. CONCLUSIONS The emergence of a clonal group of gonococci showing decreased susceptibility to cefixime in England and Wales highlights the need for continued surveillance.

An In Vitro Investigation of Synergy or Antagonism between Antimicrobial Combinations against Isolates from Bacterial Keratitis

PURPOSE To investigate antimicrobial combinations for synergy or antagonism against isolates of Staphylococcus aureus and Pseudomonas aeruginosa. METHODS Isolates were collected from cases of microbial keratitis from six centers in the United Kingdom. Minimum inhibitory concentrations (MICs) were determined by using E-test strips for 16 antimicrobials, including both current and potentially available agents. E-test strips were used to test selected antimicrobials in combination against a representative set of 10 S. aureus and 10 P. aeruginosa isolates. E-tests of the two antimicrobials were placed on sensitivity agar at right angles intersecting at their respective MICs. Antimicrobial combinations were classified as synergistic, additive, indifferent, or antagonistic, according to their fractional inhibitory concentration (FIC). RESULTS The combinations meropenem and ciprofloxacin, meropenem and teicoplanin, moxifloxacin and teicoplanin, and ciprofloxacin and teicoplanin, gave the lowest mean FICs for S. aureus, with synergy or additivity being seen in 60% to 80% of isolates. The meropenem/ciprofloxacin combination gave the lowest mean FIC for P. aeruginosa isolates, with 90% showing an additive or synergistic effect. The other combinations elicited a predominantly indifferent response. No consistent antagonistic effect was observed with the combinations used. CONCLUSIONS The combination of meropenem and ciprofloxacin was predominantly additive or synergistic for both S. aureus and P. aeruginosa. Teicoplanin combined with meropenem, ciprofloxacin, or moxifloxacin was also predominantly additive or synergistic against S. aureus.

Pharmacokinetics of vancomycin following intracameral bolus injection in patients undergoing phacoemulsification cataract surgery

Aim: To determine the elimination kinetics of intracameral vancomycin administered as a bolus injection at the end of phacoemulsification cataract surgery. Methods: Vancomycin 1 mg/0.1 ml saline solution was administered to 19 patients by intracameral bolus injection at the end of routine cataract surgery. The aqueous concentration of vancomycin was determined in nine patients 1 minute after administration and in 10 patients 18–24 hours postoperatively. Aqueous samples were obtained by inserting a Rycroft cannula into the anterior chamber via the side port incision. Fluorescence polarisation immunoassay was used to calculate the aqueous vancomycin concentration. Results: The median (interquartile range) vancomycin concentration was 5458 (4756–6389) mg/l at 1 minute and 40.6 (25.9–47.1) mg/l 18 to 24 hours (median 19 hours) postoperatively. The vancomycin concentration exceeded the minimum inhibitory concentration (MIC) of endophthalmitis-causing gram-positive bacteria by a factor of 4 for up to 26 hours postoperatively. No adverse event or reaction was noted. Conclusion: Following bolus intracameral injection at the end of cataract surgery the concentration of vancomycin in the anterior chamber vastly exceeds its MIC for at least 24 hours but is predicted to fall below the MIC after 33 hours.

Staphylococcus aureus with reduced glycopeptide susceptibility in Liverpool, UK

OBJECTIVES To investigate if colonization with heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) is associated with hGISA bacteraemia. METHODS Isolates of methicillin-resistant S. aureus (MRSA) from blood cultures and from swabs to detect MRSA colonization were screened for reduced susceptibility to glycopeptides by an agar incorporation method. Isolates detected by this screen were tested for glycopeptide resistance by MacroEtest, standard MIC Etest methods and population analysis profile-AUC (PAP-AUC) analysis. S. aureus isolates with and without reduced glycopeptide susceptibility were characterized by PFGE and spa typing. RESULTS MRSA isolates with reduced susceptibility to glycopeptides, as identified by the MacroEtest method, were detected in the colonization screens of 86 of 2550 MRSA-positive patients. The isolates were confirmed by Etest MIC and PAP-AUC analysis as hGISA. A total of 82/86 of the hGISA colonizing isolates were EMRSA-16 by PFGE; the remainder were EMRSA-15. Bacteraemia with hGISA was identified in five patients during the study period; two isolates were EMRSA-16 and three were EMRSA-15. hGISA colonization could not be linked to hGISA bacteraemia and hGISA bacteraemia could not be linked to hGISA colonization. Four of the five hGISA bacteraemias developed following teicoplanin therapy for a central venous catheter-associated MRSA bacteraemia. CONCLUSIONS Laboratory strategies to reduce morbidity associated with hGISA should focus on testing for hGISA in bacteraemic (rather than colonizing) MRSA isolates in patients with recurrent S. aureus bacteraemia following glycopeptide exposure.

In vivo development of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA), GISA and daptomycin resistance in a patient with meticillin-resistant S. aureus endocarditis

We report a patient who developed a meticillin-resistant Staphylococcus aureus (MRSA) central venous catheter infection complicated by infective endocarditis. The patient was initially treated with glycopeptides, which led to the development of heterogeneous glycopeptide resistance, the detection of which required the use of a macro Etest screening test. Subsequently, the causative strain, confirmed by PFGE as a UK epidemic MRSA-15, was treated with daptomycin, and again resistance developed in vivo. The development in vivo of resistance to both these agents suggests that the resistance mechanisms may be associated. We suggest that the clinician managing MRSA infection should anticipate daptomycin resistance when reduced glycopeptide susceptibility is detected.

Endophthalmitis After Penetrating Keratoplasty

PURPOSE To determine the incidence of endophthalmitis after penetrating keratoplasty (PK) and patient and donor risk factors. DESIGN Retrospective cohort study using national transplant registry data. PARTICIPANTS All corneal transplant recipients (n = 11 320) registered on the United Kingdom Transplant Registry undergoing their first PK between April 1999 and December 2006. METHODS Patients who developed endophthalmitis were identified on the transplant registry. In addition, cases where the fellow cornea from the same donor had been transplanted were included. Clinical information regarding donor and recipient characteristics, surgical details, and postoperative outcomes were collected and analyzed. In cases where endophthalmitis was reported, the diagnosis was verified by a follow-up supplementary questionnaire to the surgeon. Logistic regression was used to investigate differences in the factors associated with the development of endophthalmitis. MAIN OUTCOME MEASURES Incidence of endophthalmitis and graft survival. RESULTS The overall incidence of endophthalmitis occurring after primary PK in the UK was 0.67%. The incidence of endophthalmitis occurring within 6 weeks of surgery was 0.16%. Graft survival after endophthalmitis was 27% (95% confidence interval, 16-38) at 5 years, with a mean best-corrected visual acuity of 1.13 (logarithm of the minimum angle of resolution) for surviving grafts. Factors associated with endophthalmitis were donor cause of death (infection), high-risk cases, and indication for corneal transplantation. CONCLUSION Endophthalmitis remains a serious issue, with those affected having reduced graft survival and poor visual outcomes. Management of the identified recipient and donor risk factors are important to reduce endophthalmitis risk. In particular, the increased incidence of endophthalmitis when the donor dies of infection requires further explanation and review of current donor eye retrieval and eye bank practices. The delayed presentation of endophthalmitis cases also raises questions regarding possible sequestration of microbes within the corneal tissue and the effect of antimicrobials in storage media.