Oxford Scheme Research Articles

611. Fosfomycin Resistance of Multidrug-Resistant Escherichia coli and Mechanisms of Fosfomycin Resistance

BackgroundFosfomycin is one of the antibiotics that may be a candidate for the next-generation antimicrobial agents againt multidrug-resistant bacteria. To date, it is known that the resistance rate is not high for Escherichia coli. However, it is necessary to update the fosfomycin resistance rates in E. coli according to the studies that extended spectrum β-lactamase (ESBL) producing E. coli strains are highly resistance to fosfomycin. We evaluated the resistance rate of fosfomycin, the resistant mechanism of fosfomycin in E. coli, and the activity of fosfomycin against susceptible and resistant strains of E. coli.MethodsA total of 283 clinical isolates was collected from patients with Escherichia coli species during the period of January 2018 to June 2018, in three tertiary hospitals of Republic of Korea. In vitro antimicrobial susceptibility tests were performed in all E. coli isolates using the broth microdilution method according to the Clinical and Laboratory Standard Institute (CLSI). Multilocus sequence typing (MLST) of the Oxford scheme was conducted to determine the genotypes of E. coli isolated. Fosfomycin genes were investigated for all fosfomycin-resistant E. coli strains.ResultsThe overall resistance rate to fosfomycin was 10.2%, compared with 53.4%, 46.3%, 41.3%, 31.1%, 10.6%, 2.5%, and 2.1% for ciprofloxacin, cefixime, cefepime, piperacillin/tazobactam, colistin, ertapenem, and amikacin, respectively. The 29 fosfomycin-resistant isolates did not show a clonal pattern on the phylogenetic tree. MurA and glp genes were identified in all strains. FosA3 were identified in two strains and uhp gene were identified in 4 strains. In time-kill curve studies, fosfomycin was more bactericidal than cefixime against all sensitive E. coli strain. Morever, fosfomycin was more bactericidal than piperacillin/tazobactam against ESBL-producing E. coli strain.ConclusionThe resistant rate of fosfomycin to E. coli is still low. Fosfomycin was active against E. coli including ESBL producing strains.Disclosures All authors: No reported disclosures.

Emerging challenges of whole-genome-sequencing–powered epidemiological surveillance of globally distributed clonal groups of bacterial infections, giving Acinetobacter baumannii ST195 as an example

Whole-genome sequencing (WGS) has revolutionized the genotyping of bacterial pathogens and is expected to become the new gold standard for tracing the transmissions of bacterial infectious diseases for public health purposes. However, it is still unexpectedly demanding to employ WGS for global epidemiological surveillance because of the high degree of similarity between the genomes of intercontinental isolates. The aim of this study was to utilize genomically derived bioinformatics analysis to identify globally distributed A. baumannii ST195 lineage and differentiation outbreaks to address this issue. The genomic sequences and their related epidemiological metadata of 2850 A. baumannii isolates were recruited from NCBI Genbank database. Assignment into sequence type (Oxford scheme) and lineage (global clone 2/CC92) were performed. A total of 91 ST195 A. baumannii isolates were subsequently classified to perform the bacterial source tracking analysis by implementing both core genome MLST (cgMLST) and core genome SNP (cgSNP) strategy that were integrated in our recently updated BacWGSTdb 2.0 server. Antibiotic resistance genes were identified using the ResFinder database. The ST195 A. baumannii isolates distributed widely in eight countries and harboured multiple antimicrobial resistance genes simultaneously. In most cases, the bacterial isolates recovered from geographically distant sources may present less genomic sequence similarity, i.e., the phylogenetic relationship between these ST195 isolates worldwide was roughly congruent with their country of isolation. However, a few isolates collected from distant geographic regions were revealed to possess smaller genetic distances (less than 8 loci or 20 SNPs) than the threshold without an observable epidemiological link. Our study highlights the emerging challenges entailed in the WGS-powered epidemiological surveillance of globally distributed clonal groups. Standardization is urgently required before WGS can be routinely applied to infectious diseases outbreak investigations.

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates reveals the emergence of blaOXA-23 and blaNDM-1 encoding international clones in India

Acinetobacter baumannii is a nosocomial pathogen increasingly affecting the critically ill patients and represents a major public health challenge. Carbapenem-resistant A. baumannii (CRAB) is found to be associated with International Clones (ICs) and different classes of carbapenemases. The objective of the present study was to investigate the prevalence of carbapenem resistance genes, clonal relationship and genetic structure of clinical isolates of A. baumannii. In the present study, multi-locus sequence typing (MLSTOX) and analysis were carried out using Oxford scheme for 86 clinical isolates of CRAB along with 11 carbapenem sensitive A. baumannii (CSAB) collected over a period of two years (2014–2016) from two tertiary care hospitals of North India. We observed a high prevalence of the blaOXA-23-like (97.7%) among the CRAB followed by blaNDM-1 (29.1%) and blaOXA58-like (3.5%). Forty-seven Sequence Types (STs) were represented by all 97 isolates, out of which, 28 (59.6%) were novel STs that were assigned to 41 isolates. STs 451 (13%), 447 (7%), 195 (6%) and 848 (5%) were the most common STs. The majority of CRAB isolates (44.3%) belonged to the CC92, followed by the CC447 (15.1%), CC109 (9.3%) and CC110 (3.4%), which corresponds to the IC2, 8, 1 and 7 respectively. Phylogenetic and recombination analysis suggested two major and one minor lineage in the population. Further linkage disequilibrium analysis suggested clonal nature of the population as recombination was noticed at a low frequency, which was not enough to split the clonal relationship. The knowledge of genetic structure of CRAB from this study will be invaluable to illustrate epidemiology, surveillance and understanding its global diversity.

Comparative Analysis of the Two Acinetobacter baumannii Multilocus Sequence Typing (MLST) Schemes.

Acinetobacter species assigned to the Acinetobacter calcoaceticus-baumannii (Acb) complex, are Gram-negative bacteria responsible for a large number of human infections. The population structure of Acb has been studied using two 7-gene MLST schemes, introduced by Bartual and coworkers (Oxford scheme) and by Diancourt and coworkers (Pasteur scheme). The schemes have three genes in common but underlie two coexisting nomenclatures of sequence types and clonal complexes, which complicates communication on A. baumannii genotypes. The aim of this study was to compare the characteristics of the two schemes to make a recommendation about their usage. Using genome sequences of 730 strains of the Acb complex, we evaluated the phylogenetic congruence of MLST schemes, the correspondence between sequence types, their discriminative power and genotyping reliability from genomic sequences. In silico ST re-assignments highlighted the presence of a second copy of the Oxford gdhB locus, present in 553/730 genomes that has led to the creation of artefactual profiles and STs. The reliability of the two MLST schemes was tested statistically comparing MLST-based phylogenies to two reference phylogenies (core-genome genes and genome-wide SNPs) using topology-based and likelihood-based tests. Additionally, each MLST gene fragment was evaluated by correlating the pairwise nucleotide distances between each pair of genomes calculated on the core-genome and on each single gene fragment. The Pasteur scheme appears to be less discriminant among closely related isolates, but less affected by homologous recombination and more appropriate for precise strain classification in clonal groups, which within this scheme are more often correctly monophyletic. Statistical tests evaluate the tree deriving from the Oxford scheme as more similar to the reference genome trees. Our results, together with previous work, indicate that the Oxford scheme has important issues: gdhB paralogy, recombination, primers sequences, position of the genes on the genome. While there is no complete agreement in all analyses, when considered as a whole the above results indicate that the Pasteur scheme is more appropriate for population biology and epidemiological studies of A. baumannii and related species and we propose that it should be the scheme of choice during the transition toward, and in parallel with, core genome MLST.

Dry eye in patients with allergic conjunctivitis

Aim: Ocular allergy is an ocular surface disease deteriorates the patient’s quality of life. This situation continues and repeats inflammation via allergen contacts. Inflammation disrupted the tear film instability and may lead to dry eye. The aim of our study is to investigate concurrently appearing signs and symptoms of dry eye in a group of patients with symptomatic allergic conjunctivitis. Material and Methods: To be evaluated 141 patients with ocular allergy and 138 patient without ocular allergy in terms of dry eye symptoms and signs by Oxford scheme, OSDI scores, Break up time (BUT) and Schirmer test. Results: There was a statistically significant difference was detected between the dry eye scores of the two groups. Conclusion: It may be related that ocular allergy and dry eye. Because of this relation, patients have ocular allergy evaluated in terms of dry eye simultaneously.

Conjunctival Impression Cytology and Tear-Film Changes in Patients With Inflammatory Bowel Disease.

To evaluate the ocular surface changes and tear-film functions in patients with inflammatory bowel disease (IBD). The IBD group included 57 patients, and the control group included 29 healthy individuals. The Schirmer I test, tear breakup time (TBUT), corneal and conjunctiva fluorescent staining, and conjunctival impression cytology tests were performed to both groups. The rate of dry eye was 56.1% and 10.3% in the IBD and control groups, respectively, as assessed by the Schirmer I test (P<0.001). The rate of dry eye was 52.6% and 20.8% in the IBD and control groups, respectively through TBUT (P=0.004). The Ocular Surface Disease Index scores of the IBD group (15.25±7.31) were significantly higher than the control group (11.75±7.33) (P=0.039). According to the Oxford scheme, mild-to-moderate staining was 54.4% in the IBD group and 6.9% in the control group (P<0.001). Nelson Staging System showed that 69% of the IBD group had stage 2 or 3 impression cytology, whereas none of the control group had stage 2 or 3 impression cytology (P<0.001). Using the Schirmer I test, the rate of dry eye was 55.8% of the patients with IBD receiving 5-aminosalicylic acid (5-ASA) and 61.5% of the patients with IBD receiving both 5-ASA and azathioprine (P=0.485). Our study results showed that dry eye was 3 times higher in the IBD group than the control group. But, the duration of disease seems not to have effect on dry eye.

Lateral tarsal strip versus lateral tarsal strip with three-snip punctoplasty for managing epiphora in involutional ectropion

To compare the efficacy of two surgical techniques-lateral tarsal strip (canthoplasty) alone, and lateral tarsal strip with three-snip punctoplasty-in reducing epiphora arising from involutional ectropion with partial punctal stenosis. Fourty patients with involutional ectropion and partial stenosis of the lacrimal punctum were randomly allocated to two treatment groups. Group 1 patients received lateral tarsal strip alone with only non-invasive stenting of the punctum, and group 2 patients received tarsal strip plus three-snip punctoplasty. Subjective assessment of epiphora was achieved via completion of a quality of life (QoL) questionnaire preoperatively and at postoperative month 3. Eyelid position, adverse outcomes and corneal dryness (via Oxford grading scheme) were also assessed postoperatively. Only patients with unilateral problems were included in the study. Forty eyes of 40 patients were included: 20 in each group. The mean ages of group 1 and group 2 patients were 79±11 and 80±9 years, respectively. All patients reported significantly reduced eye watering after surgery, with no significant intergroup difference in subjective outcomes, except that computer usage and night driving (P<0.05), improved in a more significant way in group 2. Eyelid malposition was corrected in all cases, there were no cases of postoperative punctal eversion, and no significant adverse events or complications occurred. Finally, the mean improvements in the dryness/keratitis score (using the Oxford scheme) were comparable between the 2 groups (P=0.34). The study findings indicate that treatment of involutional ectropion with partial punctal stenosis by lateral tarsal strip with three snip punctoplasty does not provide greater reduction in discomfort secondary to epiphora than conventional lateral tarsal strip alone, except for specific situations such as night driving or computer use.

Complete Genome Sequencing of Acinetobacter baumannii Strain K50 Discloses the Large Conjugative Plasmid pK50a Encoding Carbapenemase OXA-23 and Extended-Spectrum β-Lactamase GES-11.

Multidrug-resistant (MDR) Acinetobacter baumannii strains appeared as serious emerging nosocomial pathogens in clinical environments and especially in intensive care units (ICUs). A. baumannii strain K50, recovered from a hospitalized patient in Kuwait, exhibited resistance to carbapenems and additionally to ciprofloxacin, chloramphenicol, sulfonamides, amikacin, and gentamicin. Genome sequencing revealed that the strain possesses two plasmids, pK50a (79.6 kb) and pK50b (9.5 kb), and a 3.75-Mb chromosome. A. baumannii K50 exhibits an average nucleotide identity (ANI) of 99.98% to the previously reported Iraqi clinical isolate AA-014, even though the latter strain lacked plasmid pK50a. Strain K50 belongs to sequence type 158 (ST158) (Pasteur scheme) and ST499 (Oxford scheme). Plasmid pK50a is a member of the Aci6 (replication group 6 [RG6]) group of Acinetobacter plasmids and carries a conjugative transfer module and two antibiotic resistance gene regions. The transposon Tn2008 carries the carbapenemase gene blaOXA-23, whereas a class 1 integron harbors the resistance genes blaGES-11, aacA4, dfrA7, qacEΔ1, and sul1, conferring resistance to all β-lactams and reduced susceptibility to carbapenems and resistance to aminoglycosides, trimethoprim, quaternary ammonium compounds, and sulfamethoxazole, respectively. The class 1 integron is flanked by MITEs (miniature inverted-repeat transposable elements) delimiting the element at its insertion site.

Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii.

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.

Effect of diquafosol three per cent ophthalmic solution on tear film and corneal aberrations after cataract surgery

BackgroundThe aim was to evaluate the effect of diquafosol on tear film parameters and corneal aberrations after cataract surgery.MethodsThis retrospective, comparative, observational case series included patients who underwent cataract surgery with or without peri‐operative use of diquafosol three per cent ophthalmic solution. Patients in group I (31 eyes) were treated with diquafosol three per cent ophthalmic solution six times a day from one week before surgery to three months after surgery. Patients in group II (33 eyes) underwent cataract surgery without being administered diquafosol. Tear film break‐up time (BUT), the ocular surface disease index (OSDI), the Oxford Scheme score, Schirmer's test I and corneal aberrations were determined before and at one and three months post‐surgery.ResultsIn group I, the BUT, OSDI and Oxford Scheme score showed significant improvement at one and three months post‐surgery (p = 0.002 for BUT at one month and three months, p = 0.023 and p = 0.049 for OSDI at one month and three months and p = 0.001 and p = 0.026 for Oxford Scheme at one month and three months), compared to baseline. In group II, the BUT, OSDI and Oxford Scheme score did not show any significant improvement during the follow‐up period. In both groups, Schirmer's test I significantly decreased at three months post‐surgery (p = 0.011 for group I and p = 0.034 for group II), compared to baseline. There were significant differences in the BUT and OSDI between the groups at three months post‐surgery (p = 0.037 for and p = 0.011 for ). Regarding the corneal aberration, there were no significant differences between the groups at three months post‐surgery.ConclusionPeri‐operative application of diquafosol three per cent ophthalmic solution may prevent dry eye‐related ocular surface changes accompanied by improvement of ocular symptom. No definite changes in corneal aberrations were noted.

Emergence of the Uncommon Clone ST944/ST78 Carrying blaOXA-40-like and blaCTX-M-like Genes Among Carbapenem-Nonsusceptible Acinetobacter baumannii in Moscow, Russia.

Carbapenem-nonsusceptible (Carba-NS) Acinetobacter baumannii has emerged as an important cause of nosocomial infections. In the present study, we characterized 91 Carba-NS A. baumannii isolates collected from patients of surgical departments and intensive care units at three hospitals in Moscow in 2012-2015. Multilocus sequence typing (MLST) using the Oxford (Oxf) scheme identified 16 sequence types (STs) of three clonal complexes (CCs), including CC92Oxf (67%), CC109Oxf (1%), CC944Oxf (29%), and the singleton ST1100Oxf (3%). CC944Oxf was composed of ST944Oxf (n = 16) and two of its newly described single locus variants ST1103Oxf (n = 3) and ST1104Oxf (n = 7); all the three STs were identical to the Pasteur (Pas) MLST scheme ST78. All CC944Oxf/ST78Pas isolates were blaOXA-40-like positive and all but one isolate harbored a blaCTX-M-like gene. ST944Oxf was the only ST found in each of the three study hospitals. Biofilm growth capacity was similar among Carba-NS and nonclonal carbapenem-susceptible isolates. Our data demonstrate the predominance of two clonal lineages among Carba-NS A. baumannii. One of these, the uncommon blaOXA-40-like/blaCTX-M-like-positive clone of CC944Oxf/ST78Pas, seems to be endemic in Russia.

OXA-23 and ISAba1–OXA-66 class D β-lactamases in Acinetobacter baumannii isolates from companion animals

Acinetobacter baumannii is recognised as a major pathogen of nosocomial infections that frequently show resistance to last-resort antimicrobials. To investigate whether A. baumannii from companion animals harbour carbapenem resistance mechanisms, 223 clinical isolates obtained from veterinary clinics between 2000 and 2013 in Germany were screened for carbapenem-non-susceptibility employing meropenem-containing Mueller–Hinton agar plates. Minimum inhibitory concentration (MIC) data were obtained using the VITEK®2 system. Assignment to international clones (ICs) was done by multiplex PCR or repetitive sequence-based PCR employing the DiversiLab system. Clonality was studied using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Genes encoding carbapenemases and aminoglycoside-modifying enzymes were detected by PCR. In three samples from dogs, carbapenem-resistant A. baumannii carrying the blaOXA-23 gene on plasmids and located on transposon Tn2008 were identified. The isolates belonged to sequence type ST1P (clonal complex CC1/IC1/pulsotype II) and ST10P (CC10/IC8/pulsotype IV) according to the Pasteur MLST scheme, and to ST231Ox (CC109) and ST585Ox (CC447) following the Oxford scheme. Insertion sequence ISAba1 was identified upstream of blaOXA-66 in 58 A. baumannii isolates. MLST referred them to ST2P (CC2/IC2/pulsotypes I and III), ST208Ox, ST350Ox and ST556Ox (all CC118), respectively. PFGE suggested nosocomial spread of these highly related strains, which frequently demonstrated a multidrug-resistant phenotype, in one veterinary clinic. These data show that A. baumannii from companion animals reveal resistance determinants and clonal lineages of strains globally emerging in humans. This suggests an interspecies transmission and warrants molecular surveillance of A. baumannii in veterinary clinics to mitigate its further spread.

Glaucoma therapy and dry eye

Patients with glaucoma have increased prevalence of dry eye (DE) compared to age-matched population without glaucoma. Clinical presentation of DE varies among individuals and may significantly reduce quality of life. The onset and deterioration of DE is caused by the toxic-inflammatory effects of preservatives in eye drops, active substance itself, and added, pharmacologically inactive substances or excipients. Ocular surface changes most frequently include superficial punctate keratitis, tear film instability, and allergic reactions. Despite the lack of symptoms, clinical signs may indicate ocular surface damage. Discordance between signs and symptoms is partly caused by decreased corneal sensitivity induced by neurotoxicity of the preservative benzalkonium chloride (BAK). Therefore, it is important to evaluate ocular surface before initiating glaucoma therapy and during follow-up also in asymptomatic patients. Preservative-free and/or BAK-free therapy is indicated in patients with severe DE and allergy to preservatives, and recommended in patients with DE of moderate severity, blepharitis, in symptomatic patients, before filtering surgery to reduce preoperative inflammation, in those with moderate fluorescein staining of grade 2 on Oxford scheme, and reduced tear film break-up time.

Zdravljenje glavkoma in suho oko

Patients with glaucoma have increased prevalence of dry eye (DE) compared to age-matched population without glaucoma. Clinical presentation of DE varies among individuals and may significantly reduce quality of life. The onset and deterioration of DE is caused by the toxic-inflammatory effects of preservatives in eye drops, active substance itself, and added, pharmacologically inactive substances or excipients. Ocular surface changes most frequently include superficial punctate keratitis, tear film instability, and allergic reactions. Despite the lack of symptoms, clinical signs may indicate ocular surface damage. Discordance between signs and symptoms is partly caused by decreased corneal sensitivity induced by neurotoxicity of the preservative benzalkonium chloride (BAK). Therefore, it is important to evaluate ocular surface before initiating glaucoma therapy and during follow-up also in asymptomatic patients. Preservative-free and/or BAK-free therapy is indicated in patients with severe DE and allergy to preservatives, and recommended in patients with DE of moderate severity, blepharitis, in symptomatic patients, before filtering surgery to reduce preoperative inflammation, in those with moderate fluorescein staining of grade 2 on Oxford scheme, and reduced tear film break-up time.

Size of the Lesions of Superficial Punctate Keratitis in Dry Eye Syndrome Observed With a Slit Lamp.

To evaluate the size distribution of epithelial lesions of superficial punctate keratitis (SPK) in dry eye after staining of the ocular surface by sodium fluorescein. Fluorescein was instilled in 10 patients with dry eye graded using the Oxford Scheme. Pictures were taken using a standard Topcon slit lamp with cobalt blue light, without barrier filter. Two magnifications (×10 and ×16) were used and calibrated using a certified standard reference grating, allowing the diameter of the observed objects to be determined with ImageJ software. The most visible and isolated SPK lesions (green dots) were selected. The size of 254 SPK lesions was measured by tracing the irradiance profile and manually measuring the full width at half maximum. For all patients, with the 2 magnifications combined, the median diameter was 20.9 μm (15.2-26.6 μm, 10-90 percentile). There was a significant difference between the size of SPK lesions measured with ×10 and ×16 magnifications, respectively, 24.3 μm (18.2-29.8) versus 19.0 μm (15.2-26.6) (P < 0.001). Lesions seem to be smaller than normal superficial epithelial cells (which are approximately 25 × 50 μm) and might correspond to the staining of dying shrunken cells, according to recent investigations. These new quantitative data will help in developing automated recognition algorithms to obtain reliable objective classification of corneal staining.

Loss and gain of aminoglycoside resistance in global clone 2 Acinetobacter baumannii in Australia via modification of genomic resistance islands and acquisition of plasmids.

The objective of this study was to examine the evolution of carbapenem-resistant global clone 2 (GC2) Acinetobacter baumannii in Australia focusing on the complement of aminoglycoside resistance genes and their location in resistance islands and plasmids. Sixty-two carbapenem-resistant GC2 A. baumannii isolates with various aminoglycoside resistance profiles and resistance gene content that were recovered over the period 1999-2010 from hospitals on the east coast of Australia were examined. PCR was used to link relevant contigs retrieved from whole genomes sequenced using Illumina HiSeq and assembled de novo using Velvet. Resistance phenotypes were extended to include additional antibiotics using a disc diffusion assay. Sixty-one isolates were ST208 (formerly ST92; Oxford scheme) and one was ST425. All isolates included the oxa23 carbapenem resistance gene in Tn2006 located in the same position in AbGRI1-2, along with the ISAba1-sul2-CR2Δ-tetA(B)-tetA(R)-CR2-strB-strA configuration. All isolates harboured either AbGRI2-1 carrying the aacC1 (gentamicin resistance) cassette or a variant derived from it via loss of some of the island content. When aacC1 was lost, aminoglycoside resistance was sometimes regained via acquisition of aadB (gentamicin, kanamycin and tobramycin resistance) in pRAY*-v1 or TnaphA6 (amikacin, kanamycin and neomycin resistance) in a repAci6 plasmid. A small cryptic plasmid or a deletion variant of this plasmid was always present and a large cryptic plasmid was also variably present. The extensively antibiotic-resistant GC2 isolates from Sydney, Brisbane and Canberra appear to have arisen from a single import that was introduced into Australia in, or prior to, 1999 that then evolved and spread.

Prediction of Putative Resistance Islands in a Carbapenem-Resistant Acinetobacter baumannii Global Clone 2 Clinical Isolate.

BackgroundWe investigated the whole genome sequence (WGS) of a carbapenem-resistant Acinetobacter baumannii isolate belonging to the global clone 2 (GC2) and predicted resistance islands using a software tool.MethodsA. baumannii strain YU-R612 was isolated from the sputum of a 61-yr-old man with sepsis. The WGS of the YU-R612 strain was obtained by using the PacBio RS II Sequencing System (Pacific Biosciences Inc., USA). Antimicrobial resistance genes and resistance islands were analyzed by using ResFinder and Genomic Island Prediction software (GIPSy), respectively.ResultsThe YU-R612 genome consisted of a circular chromosome (ca. 4,075 kb) and two plasmids (ca. 74 kb and 5 kb). Its sequence type (ST) under the Oxford scheme was ST191, consistent with assignment to GC2. ResFinder analysis showed that YU-R612 possessed the following resistance genes: four β-lactamase genes blaADC-30, blaOXA-66, blaOXA-23, and blaTEM-1; armA, aadA1, and aacA4 as aminoglycoside resistance-encoding genes; aac(6')Ib-cr for fluoroquinolone resistance; msr(E) for macrolide, lincosamide, and streptogramin B resistance; catB8 for phenicol resistance; and sul1 for sulfonamide resistance. By GIPSy analysis, six putative resistant islands (PRIs) were determined on the YU-R612 chromosome. Among them, PRI1 possessed two copies of Tn2009 carrying blaOXA-23, and PRI5 carried two copies of a class I integron carrying sul1 and armA genes.ConclusionsBy prediction of resistance islands in the carbapenem-resistant A. baumannii YU-R612 GC2 strain isolated in Korea, PRIs were detected on the chromosome that possessed Tn2009 and class I integrons. The prediction of resistance islands using software tools was useful for analysis of the WGS.

Clinical evaluation of the effect of diquafosol ophthalmic solution in glaucoma patients with dry eye syndrome

To investigate the effects of diquafosol on intraocular pressure (IOP) and dry eye symptoms in glaucoma patients with dry eye syndrome (DES). This study evaluated a total of 138 glaucoma patients with DES who were treated with diquafosol ophthalmic solution (DIQUAS(®)). Before treatment and 1, 4, 12, 36, and 52weeks after treatment, IOP, ocular surface disease index (OSDI), tear film break-up time (BUT), Schirmer I test scores, fluorescein staining, conjunctival impression cytology, and adverse drug reactions were evaluated. Throughout the treatment period, the mean IOP for all the patients remained stable after treatment with diquafosol (15.4±2.8mmHg at baseline and 16.0±2.8mmHg at 52weeks). The mean OSDI score improved significantly at 4, 12, and 52weeks after diquafosol treatment. The BUT and Schirmer I test scores were significantly increased after diquafosol treatment. The Oxford scheme score was significantly decreased at 1, 4, 12, 36, and 52weeks after diquafosol treatment. A significant improvement in goblet cell density was observed after 4weeks of treatment with diquafosol. Adverse drug reactions were reported in 22 (15.9%) patients. There were no serious adverse drug reactions. Diquafosol was effective in improving objective and subjective symptoms and maintained a stable IOP in glaucoma patients with DES. Therefore, the addition of diquafosol treatment in glaucoma patients with DES or ocular surface side effects using anti-glaucoma medication may be beneficial.

Treatment with sodium hyaluronate eye drops in a patient who had early-onset bleb leakage after trabeculectomy with mitomycin C

We present the case of a 47-year-old man who had bilateral proliferative diabetic retinopathy and neovascular glaucoma. Schirmer I test revealed tear secretions of 5 mm and 3 mm in the right and left eyes, respectively. Tear breakup times in the right and left eyes were 7 and 8 seconds, respectively. The ocular surface staining in both eyes was scored as Grade 1 as per the Oxford scheme. Retinal photocoagulation was performed for correction of the proliferative diabetic retinopathy and rubeosis iridis, which resolved with treatment. However, the intraocular pressure in the left eye could not be adequately controlled. Therefore, trabeculectomy with mitomycin C using limbal-based conjunctival flap was performed. Three hours after the surgery, the patient developed a large and diffuse filtering bleb, but no leakage occurred from the conjunctival scar. However, on the first postoperative day, leakage was noted and the conjunctiva was at the leakage point. The leakage resolved transiently, but recurred the next day. Severe keratoconjunctival epithelial failure was detected, and the patient was administrated 0.1% sodium hyaluronate eye drops six times daily. The epithelial failure improved, and many microcysts were detected on the bleb surface where the epithelial failure improved. The leakage resolved 2 days after initiation of the sodium hyaluronate eye drops. The microcysts disappeared and the bleb surface became smooth 1 month later.

Assessment of the size spectrum of epithelial lesions of punctuate superficial keratitis during dry eye

PurposeQuantification of staining of the ocular surface by fluorescein and lissamine green is important for diagnosis and follow up of dry eye syndromes, especially in clinical trials. Analysis of digital pictures may help improving the reliability of this quantification but require objective data on the basic lesions constitutive of punctuate superficial keratitis (PSK) to develop adequate algorithms. Aim: to evaluate the size spectrum of epithelial lesions of PSK during dry eye to improve the existing diagnosis tools.MethodsFluorescein was instilled to six patients suffering from dry eye graded according to the Oxford scheme. Pictures (3008x1960px) were taken using a Topcon slit‐lamp using the Cobalt blue light without barrier filter. Two magnifications (x10 and x16) were used and calibrated using a certified standard reference grating. It allowed determining the size of observed objects with the software ImageJ. The most visible and isolated PSK lesions (green dots) were selected. The size of 180 different PSK lesions (30/patient, 15/magnification) were measured by tracing the light intensity profile and manually measuring the width at half maximum.ResultsAll patients combined, the mean was 21.6 ± 4.8 μm (15.2‐30 μm, 10°‐90° percentile). Lesion diameters ranged between 11.4 and 30.4 μm.ConclusionsLesions seem to be smaller than epithelial cells (approximatively 25 μm×50 μm for superficial cells) and might correspond to the beginning of cell detachment with fluorescein retained between cells. These new quantitative data will help developing automatic recognition algorithm to obtain reliable objective classification of the corneal staining.