Evidence of transmission of New Delhi metallo-ß-lactamase-producing Klebsiella pneumoniae through a gastrointestinal endoscope without an elevator channel.

OBJECTIVE: To investigate the source and transmission dynamics of an endoscope-associated New Delhi metallo-ß-lactamase-producing Klebsiella pneumonia (NDM-KP) outbreak. DESIGN: Epidemiological and genomic investigation. SETTING: Academic acute care hospital in New Jersey. PATIENTS: Five patients with active NDM-KP infection identified on clinical isolates, and four NDM-KP colonized patients identified via rectal swab screening. RESULTS: Over a twelve-month period, nine patients were identified with NDM-KP infection or colonization. Whole-genome sequencing (WGS) revealed that all of the identified cases were related by 25 mutational events or less. Seven of the cases were linked to gastrointestinal endoscopic procedures (four clinical cases and three positive screens among patients exposed to endoscopes suspected of transmission). Two cases demonstrated delayed transmission that occurred five months after the initial outbreak, likely through shared usage of a non-therapeutic gastroscope without an elevator channel. CONCLUSIONS: Although all endoscope cultures in our investigation were negative, the epidemiological link to gastrointestinal endoscopes, the high degree of relatedness via WGS, and the identification of asymptomatic NDM-KP colonization among patients exposed to shared endoscopes make the endoscopic mode of transmission most likely. This investigation highlights the probable transmission of NDM-KP via a gastroscope without an elevator channel, observed several months after an initial outbreak. We hypothesize that persistent mechanical defects may have contributed to the delayed device-related transmission of NDM-KP.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Genomic Analysis of Vancomycin-Resistant Staphylococcus aureus Isolates from the 3rd Case Identified in the United States Reveals Chromosomal Integration of the vanA Locus.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a human pathogen of significant public health concern. Although the genome sequences of individual VRSA isolates have been published over the years, very little is known about the genetic changes of VRSA within a patient over time. A total of 11 VRSA, 3 vancomycin-resistant enterococci (VRE), and 4 methicillin-resistant S. aureus (MRSA) isolates, collected over a period of 4.5 months in 2004 from a patient in a long-term-care facility in New York State, were sequenced. A combination of long- and short-read sequencing technologies was used to obtain closed assemblies for chromosomes and plasmids. Our results indicate that a VRSA isolate emerged as the result of the transfer of a multidrug resistance plasmid from a coinfecting VRE to an MRSA isolate. The plasmid then integrated into the chromosome via homologous recombination mediated between two regions derived from remnants of transposon Tn5405. Once integrated, the plasmid underwent further reorganization in one isolate, while two others lost the staphylococcal cassette chromosome mec element (SCCmec) determinant that confers methicillin-resistance. The results presented here explain how a few recombination events can lead to multiple pulsed-field gel electrophoresis (PFGE) patterns that could be mistaken for vastly different strains. A vanA gene cluster that is located on a multidrug resistance plasmid that is integrated into the chromosome could result in the continuous propagation of resistance, even in the absence of selective pressure from antibiotics. The genome comparison presented here sheds light on the emergence and evolution of VRSA within a single patient that will enhance our understanding VRSA genetics. IMPORTANCE High-level vancomycin-resistant Staphylococcus aureus (VRSA) began to emerge in the United States in 2002 and has since then been reported worldwide. Our study reports the closed genome sequences of multiple VRSA isolates obtained in 2004 from a single patient in New York State. Our results show that the vanA resistance locus is located on a mosaic plasmid that confers resistance to multiple antibiotics. In some isolates, this plasmid integrated into the chromosome via homologous recombination between two ant(6)-sat4-aph(3') antibiotic resistance loci. This is, to our knowledge, the first report of a chromosomal vanA locus in VRSA; the effect of this integration event on MIC values and plasmid stability in the absence of antibiotic selection remains poorly understood. These findings highlight the need for a better understanding of the genetics of the vanA locus and plasmid maintenance in S. aureus to address the increase of vancomycin resistance in the health care setting.

Comparative analysis of multiplexed PCR and short- and long-read whole genome sequencing to investigate a large Klebsiella pneumoniae outbreak in New York State.

In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel. Additionally, short-read whole genome sequencing (WGS) analysis was used to identify antimicrobial resistance (AMR) genes and assess isolate relatedness. Long-read Oxford Nanopore MinION WGS was used to characterize the plasmid content of a subset of isolates. All methods showed overall concordance, identifying four clusters, with a few discrepancies in the clustering of individual isolates. Though short- and long-read WGS results provided a more nuanced understanding of the molecular epidemiology of this outbreak, this study highlights the utility of the Acuitas® PCR-based approach, which can more easily be performed by health care facilities, for rapid clustering of patient isolates.

A Bioinformatic Pipeline for Improved Genome Analysis and Clustering of Isolates during Outbreaks of Legionnaires' Disease.

Legionnaires' disease, a severe lung infection caused by the bacterium Legionella pneumophila, occurs as single cases or in outbreaks that are actively tracked by public health departments. To determine the point source of an outbreak, clinical isolates need to be compared to environmental samples to find matching isolates. One confounding factor is the genome plasticity of L. pneumophila, making an exact sequence comparison by whole-genome sequencing (WGS) challenging. Here, we present a WGS analysis pipeline, LegioCluster, that is designed to circumvent this problem by automatically selecting the best matching reference genome prior to mapping and variant calling. This approach reduces the number of false-positive variant calls, maximizes the fraction of all genomes that are being compared, and naturally clusters the isolates according to their reference strain. Isolates that are too distant from any genome in the database are added to the list of candidate references, thereby creating a new cluster. Short insertions or deletions are considered in addition to single-nucleotide polymorphisms for increased discriminatory power. This manuscript describes the use of this automated and "locked down" bioinformatic pipeline deployed at the New York State Department of Health's Wadsworth Center for investigating relatedness between clinical and environmental isolates. A similar pipeline has not been widely available for use to support these critically important public health investigations.

Nanopore MinION Sequencing Reveals Possible Transfer of bla KPC-2 Plasmid Across Bacterial Species in Two Healthcare Facilities.

Carbapenemase-producing Enterobacteriaceae are a major threat to global public health. Klebsiella pneumoniae carbapenemase (KPC) is the most commonly identified carbapenemase in the United States and is frequently found on mobile genetic elements including plasmids, which can be horizontally transmitted between bacteria of the same or different species. Here we describe the results of an epidemiological investigation of KPC-producing bacteria at two healthcare facilities. Using a combination of short-read and long-read whole-genome sequencing, we identified an identical 44 kilobase plasmid carrying the bla KPC-2 gene in four bacterial isolates belonging to three different species (Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli). The isolates in this investigation were collected from patients who were epidemiologically linked in a region in which KPC was uncommon, suggesting that the antibiotic resistance plasmid was transmitted between these bacterial species. This investigation highlights the importance of long-read sequencing in investigating the relatedness of bacterial plasmids, and in elucidating potential plasmid-mediated outbreaks caused by antibiotic resistant bacteria.

Donor-derived transmission through lung transplantation of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 carbapenemase during an ongoing healthcare facility outbreak.

We describe a rare instance of donor-derived OXA-23-producing carbapenem-resistant Acinetobacter baumannii transmission during lung transplantation and the subsequent public health response. This investigation highlights how transplantation can introduce rare multidrug-resistant organisms into different healthcare facilities and regions.

Fungal Infections in Boston Keratoprosthesis Patients: Lessons Learned and Novel Developments on the Horizon.

The Boston Keratoprosthesis (B-KPro) is a widely accepted modality of corneal restoration in eyes where traditional penetrating keratoplasty has little chance of success. It is the most commonly used keratoprosthesis worldwide. While the introduction of broad-spectrum antibiotic prophylaxis has virtually eliminated cases of bacterial endophthalmitis, fungal colonization and infections are a growing concern. This review of the literature summarizes risk factors for fungal infections in KPro eyes, rates of fungal infection and colonization, clinical presentation, causative organisms, management, and outcomes. We also focus on current recommendations for antifungal prophylaxis, and highlight the role of translational research at the Massachusetts Eye and Ear Infirmary (MEEI, Boston, USA) with its aim of developing novel strategies for reducing rates of fungal infections in KPro patients.

Fungal Infections After Boston Type 1 Keratoprosthesis Implantation: Literature Review and In Vitro Antifungal Activity of Hypochlorous Acid.

PURPOSE: To review the current literature describing cases of fungal keratitis and endophthalmitis after Boston keratoprosthesis (KPro) implantation and to characterize the antifungal activity of 0.01% hypochlorous acid against medically relevant fungi. METHODS: A literature review of fungal keratitis or endophthalmitis in KPro patients from January 2001 to April 2015, and an in vitro time kill assay characterizing the fungicidal activity of 0.01% hypochlorous acid against fungi causing ocular infections. RESULTS: Fifteen publications, predominantly retrospective case series, were identified. Infection rates after KPro implantation ranged from 0.009 to 0.02 fungal infections per patient-year of follow-up. The largest single-surgeon series reported an incidence of 2.4% for fungal endophthalmitis during a 10-year period. Causative organisms included both yeasts and molds. Outcomes were favorable if infections were caught early and treated appropriately; less favorable outcomes were reported in developing countries where fungal species are endemic and resources are limited. 0.01% hypochlorous acid is rapidly fungicidal, reducing the number of viable yeast cells or mold conidia by at least 99.99% within 60 seconds. The antifungal activity extended to all molds (Acremonium kiliense, Aspergillus flavus, Aspergillus fumigatus, Fusarium solani, and Mucor indicus) and yeast species (Candida albicans and Candida parapsilosis) tested. CONCLUSIONS: Fungal infections remain a lifelong concern in patients after KPro implantation. There is a growing need for a standard antifungal prophylaxis regimen, especially in the developing world. The rapid broad-spectrum in vitro fungicidal activity of 0.01% hypochlorous acid against all fungi tested makes it an attractive candidate as an antifungal prophylaxis in KPro patients.

Biofilms in infections of the eye.

The ability to form biofilms in a variety of environments is a common trait of bacteria, and may represent one of the earliest defenses against predation. Biofilms are multicellular communities usually held together by a polymeric matrix, ranging from capsular material to cell lysate. In a structure that imposes diffusion limits, environmental microgradients arise to which individual bacteria adapt their physiologies, resulting in the gamut of physiological diversity. Additionally, the proximity of cells within the biofilm creates the opportunity for coordinated behaviors through cell-cell communication using diffusible signals, the most well documented being quorum sensing. Biofilms form on abiotic or biotic surfaces, and because of that are associated with a large proportion of human infections. Biofilm formation imposes a limitation on the uses and design of ocular devices, such as intraocular lenses, posterior contact lenses, scleral buckles, conjunctival plugs, lacrimal intubation devices and orbital implants. In the absence of abiotic materials, biofilms have been observed on the capsule, and in the corneal stroma. As the evidence for the involvement of microbial biofilms in many ocular infections has become compelling, developing new strategies to prevent their formation or to eradicate them at the site of infection, has become a priority.

Unencapsulated Streptococcus pneumoniae from conjunctivitis encode variant traits and belong to a distinct phylogenetic cluster.

Streptococcus pneumoniae, an inhabitant of the upper respiratory mucosa, causes respiratory and invasive infections as well as conjunctivitis. Strains that lack the capsule, a main virulence factor and the target of current vaccines, are often isolated from conjunctivitis cases. Here we perform a comparative genomic analysis of 271 strains of conjunctivitis-causing S. pneumoniae from 72 postal codes in the United States. We find that the vast majority of conjunctivitis strains are members of a distinct cluster of closely related unencapsulated strains. These strains possess divergent forms of pneumococcal virulence factors (such as CbpA and neuraminidases) that are not shared with other unencapsulated nasopharyngeal S. pneumoniae. They also possess putative adhesins that have not been described in encapsulated pneumococci. These findings suggest that the unencapsulated strains capable of causing conjunctivitis utilize a pathogenesis strategy substantially different from that described for S. pneumoniae at other infection sites.

Contribution of the R8 substituent to the in vitro antibacterial potency of besifloxacin and comparator ophthalmic fluoroquinolones.

INTRODUCTION: Previous work has shown that besifloxacin, an 8-chloro-fluoroquinolone, has more potent activity against gram-positive pathogens than moxifloxacin and gatifloxacin, which carry an 8-methoxy group. This study was conducted to determine the contribution of the R7 and R8 substituent to fluoroquinolone antibacterial activity. MATERIALS AND METHODS: Besifloxacin, moxifloxacin, gatifloxacin, their R8 structural analogs, and ciprofloxacin were tested against representative isolates of various gram-positive and gram-negative species and previously characterized fluoroquinolone-resistant mutants of Staphylococcus aureus. Minimum inhibitory and minimum bactericidal concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Reserpine was used to determine the effect of efflux pumps on antibacterial activity. RESULTS: In general, exchanging the R8 residue in besifloxacin slightly reduced the molecule's potency, while introducing an 8-chloro group in moxifloxacin increased its potency. A similar change in gatifloxacin had little to no effect. Substituting the R8 residues did not increase the susceptibility to the efflux pump inhibitor reserpine or result in a loss of bactericidal activity. In contrast, the positive control, ciprofloxacin, was shown to be a substrate for reserpine and lost bactericidal activity against some fluoroquinolone-resistant isolates of S. aureus. CONCLUSION: The data presented here show that, depending on the R7 substituent, replacing an 8-methoxy group with an 8-chloro substituent can improve potency or can have little-to-no effect. These findings highlight the importance of the interplay between the R7 and R8 substituents in determining antibacterial potency.

Microbiological etiology and susceptibility of bacterial conjunctivitis isolates from clinical trials with ophthalmic, twice-daily besifloxacin.

INTRODUCTION: Bacterial conjunctivitis is a contagious infection of the surface of the eye usually treated empirically with topical antibiotics. Since the etiologic agent is rarely identified, it is important to monitor which bacteria cause conjunctivitis and determine their antibacterial resistance profiles. METHODS: A total of 496 bacterial samples were isolated during a randomized, double-masked, vehicle-controlled, parallel-group study conducted in the United States with besifloxacin ophthalmic suspension 0.6% dosed twice daily. Species were determined by standard biochemical and/or molecular identification methods. Minimum inhibitory concentrations were determined according to Clinical and Laboratory Standards Institute standards. RESULTS: The most prevalent species was Haemophilus influenzae, followed by Staphylococcus epidermidis, Staphylococcus aureus, the Streptococcus mitis group, and Streptococcus pneumoniae. One species identified in this study, which was not previously noted as a common cause of bacterial conjunctivitis, was Dolosigranulum pigrum. Ampicillin resistance was common among H. influenzae isolates, while macrolide resistance was high among S. pneumoniae, S. epidermidis, and S. aureus. The latter two species also included a number of isolates resistant to methicillin and ciprofloxacin. CONCLUSION: Antibiotic resistance among isolates remains a concern and the appearance of an emerging ocular pathogen, D. pigrum, suggests the need for continued observation. The topical ophthalmic fluoroquinolones continue to provide a good balance of low to moderate (i.e., manageable) levels of resistance plus broad-spectrum coverage for empiric treatment of ocular infections.

Integrated analysis of three bacterial conjunctivitis trials of besifloxacin ophthalmic suspension, 0.6%: microbiological eradication outcomes.

PURPOSE: To assess clinical antimicrobial efficacy results obtained with besifloxacin ophthalmic suspension, 0.6%, administered three times a day (TID) for 5 days, integrated across three clinical trials of bacterial conjunctivitis and to investigate any microbiological eradication failures. METHODS: Clinical microbiological eradication data from three randomized, double-masked, parallel group studies of patients with bacterial conjunctivitis (two vehicle controlled; one active controlled with moxifloxacin ophthalmic solution, 0.5%) were integrated. All bacterial samples isolated at baseline above the species-specific threshold value were subjected to antimicrobial susceptibility testing. Samples isolated at subsequent visits were subjected to susceptibility testing and pulsed-field gel electrophoresis (PFGE) to investigate the cause of eradication failures and the potential for drug resistance development. RESULTS: Visit 2 (day 4 or 5) and visit 3 (day 8) overall microbiological eradication rates were 92.2% and 88.4% for besifloxacin ophthalmic suspension compared with 61.4% and 72.5% for vehicle and 91.6% and 85.7% for moxifloxacin ophthalmic solution. Visit 2 and visit 3 microbiological eradication rates for Gram-positive and Gram-negative isolates and for individual species were consistent with the overall eradication rates. The majority of observed eradication failures in any treatment group were due to the persistence of the pathogen isolated at baseline. Eradication failures in the besifloxacin treatment group were not associated with lower antimicrobial susceptibility at baseline. PFGE data showed that the majority of bacterial strains in eyes with eradication failures were identical to the strain isolated at baseline; these eradication failures were not associated with a lower antimicrobial susceptibility at the follow-up visit. CONCLUSION: Treatment with besifloxacin ophthalmic suspension, 0.6%, administered TID for 5 days resulted in microbiological eradication rates that were ≥ 90% across the three clinical studies for the common pathogens of bacterial conjunctivitis. The few eradication failures were not due to fluoroquinolone resistance at baseline and/or resistance development during treatment.

Integrated analysis of three bacterial conjunctivitis trials of besifloxacin ophthalmic suspension, 0.6%: etiology of bacterial conjunctivitis and antibacterial susceptibility profile.

BACKGROUND: The purpose of this paper is to report on the bacterial species isolated from patients with bacterial conjunctivitis participating in three clinical trials of besifloxacin ophthalmic suspension, 0.6%, and their in vitro antibacterial susceptibility profiles. METHODS: Microbial data from three clinical studies, conducted at multiple clinical sites in the US and Asia were integrated. Species were identified at a central laboratory, and minimum inhibitory concentrations were determined for various antibiotics, including ß-lactams, fluoroquinolones, and macrolides. RESULTS: A total of 1324 bacterial pathogens representing more than 70 species were isolated. The most common species were Haemophilus influenzae (26.0%), Streptococcus pneumoniae (22.8%), Staphylococcus aureus (14.4%), and Staphylococcus epidermidis (8.4%). H. influenzae was most frequently isolated among patients aged 1-18 years, while S. aureus was most prevalent among those >65 years. Drug resistance was prevalent: Of H. influenzae isolates, 25.3% were ß-lactamase positive and 27.2% of S. pneumoniae isolates were penicillin-intermediate/ resistant; of S. aureus isolates, 13.7% were methicillin-resistant (MRSA), and of these, 65.4% were ciprofloxacin-resistant, while 45.9% of S. epidermidis isolates were methicillin-resistant (MRSE), and, of these, 47.1% were ciprofloxacin-resistant. Besifloxacin was more potent than comparator fluoroquinolones overall, and particularly against Gram-positive bacteria. Against ciprofloxacin-resistant MRSA and MRSE, besifloxacin was four-fold to ≥ 128-fold more potent than other fluoroquinolones. CONCLUSIONS: While the pathogen distribution in bacterial conjunctivitis has not changed, drug resistance is increasing. Patient age and local antibiotic resistance trends should be considered in the treatment of this ocular infection. Besifloxacin showed broad-spectrum in vitro activity and was particularly potent against multidrug-resistant staphylococcal isolates.

High proportion of nontypeable Streptococcus pneumoniae isolates among sporadic, nonoutbreak cases of bacterial conjunctivitis.

PURPOSE: Outbreaks of bacterial conjunctivitis have been linked to nontypeable strains of Streptococcus pneumoniae that lack a capsule, a key virulence factor for invasive infections. In contrast, isolates from sporadic, nonoutbreak cases of conjunctivitis were thought to be similar to invasive or nasopharyngeal isolates with respect to their capsular serotype and antibiotic resistance profile. This hypothesis was tested for 302 strains isolated during three prospective, multicenter clinical studies of bacterial conjunctivitis. MATERIALS AND METHODS: S. pneumoniae capsular serotypes were determined by agglutination assay and confirmed by the Statens Serum Institute. The presence of the cpsAB capsule genes was determined by polymerase chain reaction (PCR). Minimum inhibitory concentrations were measured for 17 antibacterial drugs by the broth microdilution method. RESULTS: Only 25 (8.3%) isolates reacted with the capsule-specific antisera and only one (0.3%) of these serotypes was covered by the capsule-specific PCV7 vaccine. The remaining 277 (91.7%) isolates were nontypeable, suggesting that they did not produce a capsule. PCR analysis indicated the loss of the capsule operon in 24/25 randomly selected nontypeable strains. Resistance rates were highest for azithromycin, trimethoprim, and tetracycline, while no resistance was detected for the fluoroquinolones, linezolid, and vancomycin. Antibiotic resistance rates were generally lower than those reported for invasive isolates, although some highly resistant or multidrug-resistant isolates were identified. CONCLUSIONS: The prevalence of nontypeable strains of S. pneumoniae was higher than expected, while the number of isolates responsive to the PCV7 vaccine was surprisingly low. These results highlight the need for new vaccines that can target all S. pneumoniae strains regardless of the presence or nature of a capsule. In addition, resistance to azithromycin, erythromycin, tetracycline, and trimethoprim was greater than 10%, which may be relevant when selecting empiric treatments for ocular surface infections.

Topoisomerase mutations that are associated with high-level resistance to earlier fluoroquinolones in Staphylococcus aureus have less effect on the antibacterial activity of besifloxacin.

BACKGROUND: The impact of mutations in DNA gyrase and topoisomerase IV on minimum inhibitory concentrations (MICs) was investigated to better understand why besifloxacin has a higher potency against Staphylococcus aureus when compared to other fluoroquinolones, which was especially pronounced against ciprofloxacin-resistant isolates. METHODS: MICs were determined for 52 clinical isolates against besifloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, and levofloxacin. The genes encoding GyrA, GyrB, ParC, and ParE were sequenced and the potential impact of mutations assessed in light of recent structural data. RESULTS: For all fluoroquinolones tested, the MICs increased with the number of mutations in the quinolone resistance-determining regions. However, this increase was the smallest for besifloxacin and the largest for ciprofloxacin and levofloxacin. In addition to the commonly observed mutations in ParC and GyrA, more unusual mutations in ParE, such as Asp-432→His or Pro-585→Ser, were also detected. CONCLUSIONS: Compared to earlier fluoroquinolones, the higher potency of besifloxacin suggests that the drug's unique combination of a 7-azepinyl ring and an 8-chloro-substituent results in unique interactions with DNA gyrase and topoisomerase IV.

Monitoring antibiotic resistance in ocular microorganisms: results from the Antibiotic Resistance Monitoring in Ocular micRorganisms (ARMOR) 2009 surveillance study.

PURPOSE: To determine the antibacterial susceptibility profile of bacterial pathogens from ocular infections against relevant aminoglycoside, ß-lactam, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, lincosamide, and macrolide antibacterial agents. DESIGN: Laboratory investigation. METHODS: Isolates from patients with bacterial eye infections were collected prospectively by 34 institutions across the United States and were submitted to a central laboratory for inclusion in the Antibiotic Resistance Monitoring in Ocular micRorganisms (ARMOR) study. Minimum inhibitory concentrations were determined by microbroth dilution for 200 Staphylococcus aureus (S. aureus), 144 coagulase-negative staphylococci, 75 Streptococcus pneumoniae (S. pneumoniae), 73 Haemophilus influenzae (H. influenzae), and 100 Pseudomonas aeruginosa (P. aeruginosa) isolates. RESULTS: A large proportion of S. aureus and coagulase-negative staphylococci isolates were resistant to oxacillin/methicillin, azithromycin, or fluoroquinolones; 46.5% of S. aureus, 58.3% of coagulase-negative staphylococci, 9.0% of P. aeruginosa, and 9.3% of pneumococcal isolates were nonsusceptible to 2 or more antibacterial drug classes. Only 2.7% of H. influenzae isolates were nonsusceptible to 1 of the agents tested. Methicillin-resistant staphylococci were statistically more likely (all P < .0038) also to be resistant to fluoroquinolones, aminoglycosides, and macrolides. CONCLUSIONS: Resistance to 1 or more antibiotics is prevalent among ocular bacterial pathogens. Current resistance trends should be considered before initiating empiric treatment of common eye infections.

In vitro time-kill experiments with besifloxacin, moxifloxacin and gatifloxacin in the absence and presence of benzalkonium chloride.

OBJECTIVES: To compare the bactericidal activity of besifloxacin, moxifloxacin and gatifloxacin and determine the contribution of the preservative benzalkonium chloride (BAK) to bactericidal activity. METHODS: Time-kill experiments were performed against four species (n=12) with besifloxacin, moxifloxacin and gatifloxacin, in the presence or absence of BAK, at t=0, 5, 15, 30, 45, 60, 120 and 360 min, according to standard CLSI methods. RESULTS: In the presence of BAK, bactericidal activity was observed within 5 min, regardless of the fluoroquinolone tested. The bactericidal activity of BAK was unaffected by the concurrent presence of besifloxacin and rapid killing (within 5 to 15 min) was not observed at BAK concentrations below 50 mg/L. However, when tested without BAK, besifloxacin was bactericidal in as little as 45 min, while moxifloxacin and gatifloxacin required at least 120 min; besifloxacin kill rates against fluoroquinolone-susceptible and -resistant strains were at least 2- to 4-fold faster than those of gatifloxacin or moxifloxacin. CONCLUSIONS: Besifloxacin was the most rapidly bactericidal fluoroquinolone tested, followed by gatifloxacin and moxifloxacin, both of which had similar activity. Our studies demonstrate that the previously reported rapid in vitro killing by gatifloxacin formulations was probably due to the concurrent presence of 50 mg/L BAK, which is much higher than the 3.2 mg/L BAK observed in human tears 1 min after instillation of ophthalmic gatifloxacin solutions [Friedlaender MH, Breshears D, Amoozgar B et al. The dilution of benzalkonium chloride (BAK) in the tear film. Adv Ther 2006; 23: 835-41].

Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolated from the eye.

PURPOSE: Methicillin-resistant Staphylococcus aureus (MRSA) strains are commonly classified as hospital-acquired (HA) or community-acquired (CA). Typical HA-MRSA isolates are characterized by multidrug resistance and the SCCmec type II cassette, while CA-MRSA isolates are generally susceptible to more drug classes, are often of SCCmec type IV, and frequently carry the Panton-Valentine leukocidin (PVL) genes. This study determined the presence of traits characteristic for CA and HA strains in ocular MRSA isolates. MATERIALS AND METHODS: Fifty-six recent ocular isolates, consisting of 40 MRSA and 16 methicillin-susceptible Staphylococcus aureus (MSSA) comparator strains, were characterized. Minimum inhibitory concentration (MIC) testing was done according to current Clinical and Laboratory Standards Institute guidelines. Detection of the PVL encoding genes and determination of the SCCmec type was done by polymerase chain reaction (PCR), while spa typing and cluster analysis was performed following DNA sequencing. RESULTS: Of the 38 typeable MRSA isolates, 22 were of SCCmec type II and 16 were of SCCmec type IV. All SCCmec type II isolates were multidrug-resistant, lacked the PVL genes, and were of spa type t002 or closely related spa types. In contrast, the SCCmec type IV isolates were resistant to fewer classes of antimicrobial agents, often possessed the PVL genes (75.0%), and were of spa type t008 or closely related spa types. CONCLUSIONS: While the majority of ocular MRSA strains in this study fit the classical profile of HA- and CA-MRSA, some CA-MRSA isolates exhibited higher levels of antimicrobial resistance, which should be of particular concern to eye-care professionals. Furthermore, the apparent association of spa types and SCCmec types observed here warrants further investigation and suggests that spa typing may be useful in future HA- and CA-MRSA characterization studies.