Apoptosis Inhibitor of Macrophages Contributes to the Chronicity of Mycobacterium avium Infection by Promoting Foamy Macrophage Formation.

In Mycobacterium avium infections, macrophages play a critical role in the host defense response. Apoptosis inhibitor of macrophage (AIM), also known as CD5L, may represent a novel supportive therapy against various diseases, including metabolic syndrome and infectious diseases. The mechanisms of AIM include modulating lipid metabolism in macrophages and other host cells. We investigated the role of AIM in M. avium infections in vitro and in vivo. In a mouse model of M. avium pneumonia, foamy macrophages were induced 6 wk after infection. The bacteria localized in these macrophages. Flow cytometric analysis also confirmed that the percentage of CD11chighMHCclassIIhigh interstitial and alveolar macrophages, a cell surface marker defined as foamy macrophages, increased significantly after infection. AIM in alveolar lavage fluid and serum gradually increased after infection. Administration of recombinant AIM significantly increased the number of bacteria in the lungs of mice, accompanied by the induction of inflammatory cytokine and iNOS expression. In mouse bone marrow-derived macrophages, the mRNA expression of AIM after M. avium infection and the amount of AIM in the supernatant increased prior to the increase in intracellular bacteria. Infected cells treated with anti-AIM Abs had fewer bacteria and a higher percentage of apoptosis-positive cells than infected cells treated with isotype control Abs. Finally, AIM in the sera of patients with M. avium-pulmonary disease was measured and was significantly higher than in healthy volunteers. This suggests that AIM production is enhanced in M. avium-infected macrophages, increasing macrophage resistance to apoptosis and providing a possible site for bacterial growth.

Biochemical characterization of the L1-like metallo-ß-lactamase from Stenotrophomonas lactitubi.

L1-like metallo-ß-lactamases (MBLs) exhibit diversity and are highly conserved. Although the presence of the blaL1-like gene is known, the biochemical characteristics are unclear. This study aimed to characterize an L1-like MBL from Stenotrophomonas lactitubi. It showed 70.9-99.7% similarity to 50 L1-like amino acid sequences. The characteristic kinetic parameter was its high hydrolyzing efficiency for ampicillin and nitrocefin. Furthermore, L1-like from S. lactitubi was distinctly more susceptible to inhibition by EDTA than that to inhibition by 2,6-pyridinedicarboxylic acid.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid ß-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-ß-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

External quality assessment survey for SARS-CoV-2 nucleic acid amplification tests in clinical laboratories in Tokyo, 2021.

INTRODUCTION: Nucleic acid amplification tests (NAATs) play a pivotal role in clinical laboratories for diagnosing COVID-19. This study aimed to elucidate the accuracy of these tests. METHODS: In 2021, an external quality assessment of NAATs for SARS-CoV-2 was conducted in 47 laboratories in Tokyo, Japan. In open testing, where the laboratories knew that the samples were intended for the survey, a simulated nasopharyngeal swab suspension sample was used, featuring a positive sample A with a viral concentration of 50 copies/µL, positive sample B with 5 copies/µL, and a negative sample. Laboratories employing real-time RT-PCR were required to report cycle threshold (Ct) values. In blind testing, where the samples were processed as normal test samples, a positive sample C with 50 copies/µL was prepared using a simulated saliva sample. RESULTS: Of the 47 laboratories, 41 were engaged in open testing. For sample A, all 41 laboratories yielded positive results, whereas for sample B, 36 laboratories reported positive results, 3 laboratories reported "test decision pending", 1 laboratory reported "suspected positive", and 1 laboratory did not respond. All 41 laboratories correctly identified the negative samples as negative. The mean Ct values were 32.2 for sample A and 35.2 for sample B. In the blind test, six laboratories received samples. Sample C was identified as positive by five laboratories and negative by one laboratory. CONCLUSIONS: The nature of the specimen, specifically the saliva, may have influenced the blind test outcomes. The identified issues must be meticulously investigated and rectified to ensure accurate results.

Assessing the discriminability of PCR-based open reading frame typing versus single-nucleotide polymorphism analysis via draft whole-genome sequencing of methicillin-resistant Staphylococcus aureus in nosocomial transmission analysis.

Phylogenetic analysis based on single-nucleotide polymorphism (SNP)-based through whole-genome sequencing is recognized as the standard method for probing nosocomial transmission. However, the application of WGS is constrained by the high cost of equipment and the need for diverse analysis tools, which limits its widespread use in clinical laboratory settings. In Japan, the prevalent use of PCR-based open reading frame typing (POT) for tracing methicillin-resistant Staphylococcus aureus (MRSA) transmission routes is attributed to its simplicity and ease of use. Although POT's discriminatory power is considered insufficient for nosocomial transmission analysis, conclusive data supporting this notion is lacking. This study assessed the discriminatory capabilities of SNP analysis and POT across 64 clinical MRSA strains. All 21 MRSA strains of ST5/SCCmec IIa, having more than 16 SNPs, demonstrated distinct clones. Conversely, two strains shared the same POT number and were identified as group A. Among the 12 MRSA strains of ST8/SCCmec IVl with over nine SNPs, five fell into POT group B, and five into POT group C. All four MRSA strains of ST8/SCCmec IVa were classified into POT group D, although they included strains with more than 30 SNPs. Among the 27 MRSA strains of ST1/SCCmec IVa, 14 were classified into POT group E. However, except for two clusters (each comprising two or three strains), all had SNP counts >10 (Fig. 1-D). SNP analysis of MRSA in CC1/SCCmec IV showed that several strains had the same number of SNPs in POT number (106-183-37), even among bacteria with >100 SNPs, indicating POT's limited use in detailed nosocomial transmission analysis.

Intrinsic clarithromycin heteroresistance in Mycobacterium avium.

BACKGROUND: Mycobacterium avium is associated with pulmonary disease in otherwise healthy adults. Several clarithromycin-refractory cases have been reported, including some cases caused by clarithromycin-susceptible strains. OBJECTIVES: To characterize the reason for the discrepancy between clinical response and antibiotic susceptibility results. METHODS: We conducted population analysis of clarithromycin-tolerant and heteroresistant subpopulations of M. avium cultured in vitro and in homogenates of infected lungs of mice. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for 28 M. avium and two M. kansasii strains. Mice were intranasally infected with M. avium and treated with or without clarithromycin (100 mg/kg) thrice weekly. They were sacrificed on day 35 and the bacteria in lung homogenates were tested for clarithromycin resistance. Population analysis assays were performed based on colony growth on plates containing two-fold dilutions of clarithromycin. RESULTS: The MBC/MIC ratios were ≥8 in all 28 strains of M. avium tested. In the population analysis assay, several colonies were observed on the plates containing clarithromycin concentrations above the MIC (2-64 mg/L). No growth of M. kansasii colonies was observed on the plates containing clarithromycin concentrations ≥2 mg/L. M. avium in the homogenates of infected lungs showed clearer clarithromycin-resistant subpopulations than in vitro, regardless of clarithromycin exposure. CONCLUSION: M. avium shows intrinsic heterogeneous resistance (heteroresistance) to clarithromycin. This may explain the observed discrepancies between clarithromycin susceptibility testing results and clinical response to clarithromycin treatment. Further studies are needed to confirm a link between heteroresistance and clinical outcomes.

Diversity of blaL1-like genes in Stenotrophomonas species: insights from genome analysis of publicly available genome sequences.

L1 metallo-ß-lactamases produced by Stenotrophomonas maltophilia exhibit high diversity. Here, we characterized the genomes of Stenotrophomonas species harboring blaL1-like genes using publicly available genome sequences. Our findings provide evidence that Stenotrophomonas species with blaL1-like genes constitute a complex comprising many species with high genetic diversity, and similarities between blaL1-like genes are lower than those of the genome. This suggests that the diversity of blaL1-like is attributable to species diversity in Stenotrophomonas species harboring blaL1-like and the rapid evolutionary changes in blaL1-like genes.

Pseudomonas otitidis bacteremia in an immunocompromised patient with cellulitis: case report and literature review.

BACKGROUND: Pseudomonas otitidis belongs to the genus Pseudomonas and causes various infections, including ear, skin, and soft tissue infections. P. otitidis has a unique susceptibility profile, being susceptible to penicillins and cephalosporins but resistant to carbapenems, due to the production of the metallo-ß-lactamase called POM-1. This revealed genetic similarities with Pseudomonas aeruginosa, which can sometimes lead to misidentification. CASE PRESENTATION: We report the case of a 70-year-old Japanese male who developed cellulitis and bacteremia during chemotherapy for multiple myeloma. He was initially treated with meropenem, but blood culture later revealed gram-negative bacilli identified as P. otitidis using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Carbapenem resistance was predicted from previous reports; therefore, we switched to dual therapy with levofloxacin and cefepime, and favorable treatment results were obtained. CONCLUSION: This is the first reported case of P. otitidis cellulitis and bacteremia in an immunocompromised patient. Carbapenems are typically used in immunocompromised patients and P. otitidis is often resistant to it. However, its biochemical properties are similar to those of Pseudomonas aeruginosa; therefore, its accurate identification is critical. In the present study, we rapidly identified P. otitidis using MALDI-TOF MS and switched from carbapenems to an appropriate antimicrobial therapy, resulting in a successful outcome.

Epidemiological analysis and antimicrobial susceptibility profile of Gram-negative bacilli that cause bacteremia in Japan.

We investigated the epidemiology and antimicrobial susceptibility profiles of seven major Gram-negative bacilli (Escherichia coli, Klebsiella spp., Enterobacter spp., Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia, and Bacteroides spp.) that caused bacteremia in Japan. We collected clinical information and isolates from patients aged 20 years or older who developed bacteremia during a year at three Japanese university hospitals and performed microbiological examination. In total, 628 cases were included, half of which were caused by E. coli (315 isolates). P. aeruginosa (56 isolates) was isolated most frequently among non-fermenting bacteria and 33 Bacteroides spp. were isolated. Mortality rates were the highest for P. aeruginosa (7-day, 16.1%; 30-day, 26.8%). The 7- and 30-day mortality rates ranged 3.8-9.0% and 8.3-17.6%, respectively, for Enterobacterales, and they were 15.2% each for Bacteroides spp. Regarding antimicrobial resistance, Enterobacterales and Acinetobacter spp. showed susceptibility to carbapenems and amikacin (98.0-100.0%). The susceptibility rates to ceftolozane/tazobactam ranged 82.4-99.0% for Enterobacterales and 92.9% for P. aeruginosa. More than 30.0% of E. coli isolates were resistant to fluoroquinolone. Extended-spectrum ß-lactamase (ESBL) producers were found in 21.0% of E. coli and approximately 80% of those were resistant to fluoroquinolones. The susceptibility of the 33 Bacteroides spp. to carbapenems, ampicillin/sulbactam, and piperacillin/tazobactam was 100.0%. Among the ESBL producers, blaCTX-M group 9 was the major subgroup in E. coli (77.3%), and blaCTX-M group 1 was detected in 18.2% of E. coli and 50.0% of Klebsiella spp. Continuous surveillance is needed to understand the epidemiology and consider appropriate therapeutic strategies.

Evaluation of the performance of GeneSoC®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures.

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/µL, respectively. The detection limit of the MRSA bacterial burden using this system was 104 and 103 CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles.

Biochemical Characterization of the Subclass B3 Metallo-ß-Lactamase PJM-1 from Pseudoxanthomonas japonensis.

Biochemical properties of the novel subclass B3 metallo-ß-lactamase (MBL) PJM-1 expressed in Pseudoxanthomonas japonensis, which is often isolated from the environment, were determined. The 906-bp blaPJM-1 gene in P. japonensis is a species-specific MBL gene, and PJM, with 301 predicted amino acids, has 81.8% amino acid identity with AIM-1. In this study, PJM-1 was recombinantly expressed and purified. PJM-1 showed a low catalytic activity against ceftazidime and cefepime, and it was strongly inhibited by EDTA.

Consensus on ß-Lactamase Nomenclature.

Assigning names to ß-lactamase variants has been inconsistent and has led to confusion in the published literature. The common availability of whole genome sequencing has resulted in an exponential growth in the number of new ß-lactamase genes. In November 2021 an international group of ß-lactamase experts met virtually to develop a consensus for the way naturally-occurring ß-lactamase genes should be named. This document formalizes the process for naming novel ß-lactamases, followed by their subsequent publication.

Full-length whole-genome sequencing analysis of emerged meropenem-resistant mutants during long-term in vitro exposure to meropenem for borderline meropenem-susceptible carbapenemase-producing and non-carbapenemase-producing Enterobacterales.

OBJECTIVES: Molecular analysis of meropenem-resistant mechanisms in mutants emerging from long-term in vitro meropenem exposure to borderline meropenem-susceptible carbapenemase-producing Enterobacterales (CPE) and non-CPE. METHODS: Escherichia coli TUM13867 harbouring both blaIMP-6- and blaCTX-M-2-carrying IncN plasmid and Citrobacter koseri TUM13189 with blaCTX-M-2-carrying chromosome were used. Meropenem MIC was 1 mg/L against both strains. Each strain was cultured in the hollow-fibre infection model (HFIM) to approximately 1 × 106 colony formation unit (cfu)/mL, and meropenem 1 g q8h treatment was initiated. Then, changes in total and meropenem-resistant populations were observed for 124 h. Meropenem resistance mechanisms were analysed using full-length whole-genome sequencing (WGS), reverse-transcription quantitative PCR and digital PCR. RESULTS: Meropenem reduced TUM13867 and TUM13189 to approximately 5 and 2 log10 cfu/mL, respectively, at 2 h after initiation, but regrowth was observed at 24 h. The meropenem-resistant mutant emergence frequency at 120 and 124 h was 4.4 × 10-4 for TUM13867 and 7.6 × 10-1 for TUM13189. Meropenem MIC of the mutants derived from TUM13867 (TUM20902) and TUM13189 (TUM20903) increased 4- and 16-fold, respectively. TUM20902, which harboured pMTY20902_IncN plasmid with a 27 505-bp deletion that included blaCTX-M-2, and blaIMP-6 showed 4.21-fold higher levels of transcription than the parental strain. TUM20903 had a 49 316-bp deletion that included ompC and a replicative increase of blaCTX-M-2 to three copies. CONCLUSIONS: Molecular analysis including full-length WGS revealed that the resistance mechanisms of meropenem-resistant mutants that emerged during long-term in vitro meropenem exposure were increased blaIMP-6 transcripts in CPE and increased blaCTX-M-2 transcripts due to gene triplication and OmpC loss resulting from ompC deletion in non-CPE.

Molecular and biochemical characterization of novel PAM-like MBL variants, PAM-2 and PAM-3, from clinical isolates of Pseudomonas tohonis.

BACKGROUND: There is no comprehensive study on PAM-like MBLs. OBJECTIVES: Our aim was to characterize novel B3 MBL variants, PAM-2 and PAM-3, from Pseudomonas tohonis clinical isolates. METHODS: We evaluated the antimicrobial susceptibility and the MBL gene composition of three novel P. tohonis clinical isolates identified at a Japanese hospital, using the broth microdilution method and WGS, respectively. We characterized the PAM-2 and PAM-3 proteins using recombinant protein expression and biochemical evaluations. RESULTS: Low carbapenem MICs (meropenem MIC = 0.125-1 mg/L) were observed for all three P. tohonis isolates; however, the isolates produced MBLs. We identified blaPAM-2 and blaPAM-3 as potential genes, belonging to a novel subclass of B3 MBLs. Their genomic sequence was similar to that of blaPAM-1 from Pseudomonas alcaligenes. PAM-2 and PAM-3 comprised 287 amino acids and exhibited 90% amino acid identity with PAM-1, 73% identity with POM-1 from Pseudomonas otitidis and 61% identity with L1 from Stenotrophomonas maltophilia. Biochemical evaluations of recombinant PAM-2 and PAM-3 revealed similar kcat/Km ratios and demonstrated catalytic activity against all the tested ß-lactams, except for aztreonam. In addition, the kcat/Km ratio for imipenem was 40-fold lower than that for meropenem. CONCLUSIONS: P. tohonis harbours a species-specific PAM-family MBL gene. This enzyme has higher hydrolytic activity against meropenem compared with that against imipenem.

Impact of Helicobacter pylori infection on fluid duodenal microbial community structure and microbial metabolic pathways.

BACKGROUND: The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16 S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16 S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. RESULTS: Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection changed the relative mean abundance of three phyla (Proteobacteria, Actinobacteria, and TM7) and ten genera (Neisseria, Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. CONCLUSIONS: H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

A case of Bacillus subtilis var. natto bacteremia caused by ingestion of natto during COVID-19 treatment in a maintenance hemodialysis patient with multiple myeloma.

A 70-year-old woman, who started on hemodialysis 7 months before for end-stage renal disease due to diabetic nephropathy and was diagnosed with symptomatic multiple myeloma 1 month before, was admitted to our hospital with critical coronavirus disease 2019 and treated with long-term immunosuppressive therapy such as steroids and tocilizumab. During treatment, Bacillus subtilis was detected in the blood cultures. We could not exclude the association of natto (fermented soybeans) with B. subtilis var. natto, which the patient had been eating every day from 8 days after admission. She was prohibited from eating natto and treated with vancomycin. Later, B. subtilis detected in the blood culture was identified as B. subtilis var. natto, which was identical with those contained in the natto that the patient consumed daily using a next-generation sequencer. Gut dysbiosis due to old age, malignant tumor, diabetes mellitus, end-stage renal disease, and intestinal inflammation caused by severe acute respiratory syndrome coronavirus 2 increased intestinal permeability and the risk of bacterial translocation, causing B. subtilis var. natto bacteremia. Therefore, careful consideration might be given to the intake of fermented foods containing live bacteria in patients with severe immunocompromised conditions.

A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020.

INTRODUCTION: The Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo. METHODS: We diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method. RESULTS: As a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative. CONCLUSION: The false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.

Metagenomic Shotgun Sequencing Analysis of Canalicular Concretions in Lacrimal Canaliculitis Cases.

Lacrimal canaliculitis is a rare infection of the lacrimal canaliculi with canalicular concretions formed by aggregation of organisms. Metagenomic shotgun sequencing analysis using next-generation sequencing has been used to detect pathogens directly from clinical samples. Using this technology, we report cases of successful pathogen detection of canalicular concretions in lacrimal canaliculitis cases. We investigated patients with primary lacrimal canaliculitis examined in the eye clinics of four hospitals from February 2015 to July 2017. Eighteen canalicular concretion specimens collected from 18 eyes of 17 patients were analyzed by shotgun metagenomics sequencing using the MiSeq platform (Illumina). Taxonomic classification was performed using the GenBank NT database. The canalicular concretion diversity was characterized using the Shannon diversity index. This study included 18 eyes (17 patients, 77.1 ± 6.1 years): 82.4% were women with lacrimal canaliculitis; canalicular concretions were obtained from 12 eyes using lacrimal endoscopy and six eyes using canaliculotomy with curettage. Sequencing analysis detected bacteria in all samples (Shannon diversity index, 0.05-1.47). The following genera of anaerobic bacteria (>1% abundance) were identified: Actinomyces spp. in 15 eyes, Propionibacterium spp., Parvimonas spp. in 11 eyes, Prevotella spp. in 9 eyes, Fusobacterium spp. in 6 eyes, Selenomonas spp. in 5 eyes, Aggregatibacter spp. in 3 eyes, facultative and aerobic bacteria such as Streptococcus spp. in 13 eyes, Campylobacter spp. in 6 eyes, and Haemophilus spp. in 3 eyes. The most common combinations were Actinomyces spp. and Streptococcus spp. and Parvinomonas spp. and Streptococcus spp., found in 10 cases. Pathogens were identified successfully using metagenomic shotgun sequencing analysis in patients with canalicular concretions. Canalicular concretions are polymicrobial with anaerobic and facultative, aerobic bacteria.

Structural and Biochemical Characterization of the Novel CTX-M-151 Extended-Spectrum ß-Lactamase and Its Inhibition by Avibactam.

The diazabicyclooctane (DBO) inhibitor avibactam (AVI) reversibly inactivates most serine ß-lactamases, including the CTX-M ß-lactamases. Currently, more than 230 unique CTX-M members distributed in five clusters with less than 5% amino acid sequence divergence within each group have been described. Recently, a variant named CTX-M-151 was isolated from a Salmonella enterica subsp. enterica serovar Choleraesuis strain in Japan. This variant possesses a low degree of amino acid identity with the other CTX-Ms (63.2% to 69.7% with respect to the mature proteins), and thus it may represent a new subgroup within the family. CTX-M-151 hydrolyzes ceftriaxone better than ceftazidime (kcat/Km values 6,000-fold higher), as observed with CTX-Ms. CTX-M-151 is well inhibited by mechanism-based inhibitors like clavulanic acid (inactivation rate [kinact]/inhibition constant [Ki ] = 0.15 µM-1 · s-1). For AVI, the apparent inhibition constant (Kiapp), 0.4 µM, was comparable to that of KPC-2; the acylation rate (k2/K) (37,000 M-1 · s-1) was lower than that for CTX-M-15, while the deacylation rate (koff) (0.0015 s-1) was 2- to 14-fold higher than those of other class A ß-lactamases. The structure of the CTX-M-151/AVI complex (1.32 Å) reveals that AVI adopts a chair conformation with hydrogen bonds between the AVI carbamate and Ser70 and Ser237 at the oxyanion hole. Upon acylation, the side chain of Lys73 points toward Ser130, which is associated with the protonation of Glu166, supporting the role of Lys73 in the proton relay pathway and Glu166 as the general base in deacylation. To our knowledge, this is the first chromosomally encoded CTX-M in Salmonella Choleraesuis that shows similar hydrolytic preference toward cefotaxime (CTX) and ceftriaxone (CRO) to that toward ceftazidime (CAZ).

Molecular characterization of Neisseria gonorrhoeae isolates collected through a national surveillance programme in Japan, 2013: evidence of the emergence of a ceftriaxone-resistant strain from a ceftriaxone-susceptible lineage.

OBJECTIVES: To investigate the spread of ceftriaxone-resistant Neisseria gonorrhoeae lineages similar to strains H041 (2009) and FC428 (2015), we characterized 55 strains collected in 2013 from hospitals across Japan. METHODS: Susceptibility testing and whole-genome sequencing. RESULTS: Susceptibility rates were 58% for cefixime and 98% for ceftriaxone. The 55 strains were whole-genome sequenced and classified into nine MLST-STs. MLST-ST1901 was the most prevalent (n = 19) followed by MLST-ST7363 (n = 12) and MLST-ST7359 (n = 11). The most prevalent penA [encoding penicillin binding protein 2 (PBP2)] mosaic types, based on the N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) scheme, were 10.001 (n = 20) followed by 34.001 (n = 13). The H041 and FC428 strains were not detected; however, a single ceftriaxone-resistant strain (TUM15748) with a MIC of 0.5 mg/L ceftriaxone was identified. The TUM15748 strain belonged to MLST-ST7359 and N. gonorrhoeae multiantigen sequence typing-ST6771, and had a novel PBP2 (PBP2TUM15748, penA type 169.001). The amino acid sequence of PBP2TUM15748 showed partial similarity to that of PBP2 from N. gonorrhoeae GU140106 and commensal Neisseria perflava and Neisseria cinerea. Natural transformation and recombination experiments using full-length TUM15748 penA showed that the ceftriaxone MICs of transformants increased 16-fold or more compared with the parental ceftriaxone-susceptible recipient strain (NG9807, belonging to MLST-ST7363). No ceftriaxone-resistant MLST-ST7359 strains have previously been reported. CONCLUSIONS: We showed here that a ceftriaxone-susceptible lineage acquired a mutant PBP2 mosaic type, integrating partial PBP2 sequences from commensal Neisseria species, resulting in the emergence of ceftriaxone-resistant strains.