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.

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.

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.

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.

Whole-Genome Sequencing of the Novel Pseudomonas Strain TUM22785 Harboring blaPAM-1, Isolated from a Patient with Urinary Tract Infection in Japan.

Pseudomonas species are Gram-negative aerobic bacteria that cause opportunistic infections. Here, we report the whole-genome sequence of the Pseudomonas sp. strain TUM22785, isolated from an outpatient with a urinary tract infection at a medical institution in Japan. This strain harbors a metallo-ß-lactamase (MBL) blaPAM-1 gene.

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.

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.

Pseudomonas tohonis sp. nov., isolated from the skin of a patient with burn wounds in Japan.

Strain TUM18999T was isolated from the skin of a patient with burn wounds in Japan. The strain was successfully cultured at 20-42 °C (optimum, 30-35 °C) in 1.0-4.0% NaCl (w/v) and at pH 5.5-9.5, optimum pH 5.5-8.5. The phylogenetic tree reconstructed using 16S rRNA, gyrB, rpoB and rpoD gene sequences indicated that strain TUM18999T is closely related to Pseudomonas otitidis MCC10330T. Although the partial 16S rRNA gene sequence (1412 bp) of TUM18999T exhibits high similarity to those of Pseudomonas alcaligenes NBRC 14159T (99.08 %) and Pseudomonas otitidis MCC10330T (98.51 %), multi-locus sequence analysis using 16S rRNA, gyrB, rpoB and rpoD genes reveals a clear distinction between TUM18999T and other Pseudomonas species. In addition, an average nucleotide identity >90 % was not observed in the P. aeruginosa group. Moreover, TUM18999T and P. otitidis can be distinguished based on the minimum inhibitory concentration for carbapenem. Meanwhile, the cellular fatty acids are enriched with C18 : 1 ω7c/C18 : 1 ω6c (34.35 %), C16 : 1 ω7c/C16 : 1 ω6c (24.22 %), C16 : 0 (19.79 %) and C12 : 0 (8.25 %). Based on this evidence, strain TUM18999T can be defined as representing a novel Pseudomonas species, with the proposed name Pseudomonas tohonis sp. nov. The type strain is TUM18999T (GTC 22698T=NCTC 14580T).

Evaluation of the simplified carbapenem inactivation method as a phenotypic detection method for carbapenemase-producing Enterobacterales.

Carbapenemase-producing Enterobacterales (CPE) have become a global health concern. Current molecular detection methods require special equipment and reagents. Thus, there is an urgent need for a highly sensitive, specific, and simple method for phenotypic detection of CPE in clinical microbiology laboratories. A simplified carbapenem inactivation method (sCIM) was recently reported. However, its utility for CPE detection has not been sufficiently evaluated to date. We evaluated the sCIM and compared it with the modified CIM (mCIM), using 133 CPE strains (producing IMP, 92; NDM, 11; NDM and OXA-48-like, 1; KPC, 13; OXA-48-like, 12; GES-24, 3; Nmc-A, 1) and 82 non-CPE strains (extended spectrum ß-lactamase, 61; AmpC, 21). The sCIM was conducted by loading bacteria onto imipenem and meropenem disks. When imipenem disks with a 1+ bacterial load were used, the sensitivity and specificity of the sCIM were 97.0% and 100%, and those of the mCIM were 97.0% and 96.3%, respectively. The specificity of the sCIM decreased to 57.3% when the bacterial load on imipenem disks was increased to 2+. In contrast, when meropenem disks with a 1+ bacterial load were used, the sCIM had a lower sensitivity (78.2%) and an equivalent specificity (100%). When meropenem disks with a bacterial load of 2+ were used, the sensitivity and specificity of the sCIM increased to 96.2% and 93.9%, respectively. The diameter of the inhibition zone on meropenem disks was larger than that on imipenem disks, and the sCIM was less sensitive when meropenem disks were used. In addition, sCIM detection rates when using meropenem disks were particularly low for OXA-48-like producers (bacterial load 1+, 0/12; bacterial load 2+, 10/12). Our results indicate that the sensitivity and specificity of the sCIM was dependent on the bacterial load and that large bacterial loads led to false positives for AmpC and extended spectrum ß-lactamase producers. Thus, the sCIM has high sensitivity and specificity for appropriate bacterial loads when imipenem disks are used.

Immunochromatography and chemiluminescent enzyme immunoassay for COVID-19 diagnosis.

INTRODUCTION: The rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required to prevent the spread of COVID-19. This study evaluated the utility of two SARS-CoV-2 antigen detection methods. METHODS: We evaluated two types of antigen detection methods using immunochromatography (Espline) and quantitative chemiluminescent enzyme immunoassay (Lumipulse). RT-PCR was performed as a standard procedure for COVID-19 diagnosis. Lumipulse and RT-PCR were performed for all 486 nasopharyngeal swabs and 136 saliva samples, and the Espline test was performed for 271 nasopharyngeal swabs and 93 saliva samples. RESULTS: The sensitivity and specificity of the Espline test were 10/11 and 260/260 (100%), respectively for the nasopharyngeal swabs and 3/9 and 84/84 (100%), respectively for the saliva samples. High sensitivities for both saliva (8/9) and nasopharyngeal swabs (22/24) were observed in the Lumipulse test. The specificities of the Lumipulse test for nasopharyngeal swabs and saliva samples were 460/462 (99.6%) and 123/127 (96.9%), respectively. CONCLUSION: The Espline test is not effective for saliva samples but is useful for simple and rapid COVID-19 tests using nasopharyngeal swabs because it does not require special devices. The Lumipulse test is a powerful high-throughput tool for COVID-19 diagnosis because it has high detection performance for nasopharyngeal swabs and saliva samples.

Complete whole-genome sequence of the novel Pseudomonas species strain TUM18999, isolated from a patient with a burn wound in Japan.

OBJECTIVES: Pseudomonas is a Gram-negative bacterial genus with numerous member species. In this study, using whole-genome sequencing, we characterized a novel Pseudomonas sp. strain TUM18999, isolated as a pathogen from a human patient. METHODS: The TUM18999 strain was isolated from a patient's burn wound. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. The whole-genome sequence was obtained using Miseq and MinION, and we conducted phylogenetic analysis based on single nucleotide polymorphisms of the core genome. RESULTS: Antimicrobial susceptibility testing revealed a high ceftazidime MIC (32 mg/L). Moreover, carbapenemase production was confirmed using the modified carbapenem inactivation method. We found that the complete genome of TUM18999 was 6,826,062 bp long, with 6175 coding sequences (CDS) and a DNA G+C content (non-plasmid) of 66.4 mol%. Consistent with the high similarities with the 16S rRNA sequences of P. otitidis MCC10330 (98.6%) and P. alcaligenes NBRC 14159 (99.2%), similarities (<90%) were also observed with the gyrB genes of both strains. The average nucleotide identities for P. alcaligenes NBRC 14159 and P. otitidis MCC10330 were also <90%. The core-genome single nucleotide polymorphism phylogenetic tree indicated that the TUM18999 strain was most closely related to P. otitidis MCC10330. In addition, the TUM18999 strain carried the novel gene, species-specific subclass B3 metallo-ß-lactamase (MBL), and its similarities with P. alcaligenes metallo-ß-lactamase-1 (PAM-1) and P. otitidis metallo-ß-lactamase-1 (POM-1) were 90.24% and 73.14%, respectively. CONCLUSION: We characterized the complete whole genome sequence of the novel Pseudomonas sp. TUM18999 carrying the novel gene species-specific subclass B3 MBL.

Carbapenem inactivation method using bacterial lysate and MOPS (LCIM): a very sensitive method for detecting carbapenemase-producing Acinetobacter species.

OBJECTIVES: Detection of carbapenem-hydrolysing class D ß-lactamase (CHDL)-producing Acinetobacter spp. is critical for understanding antibiotic resistance. In this study, we compared the available detection techniques derived from the carbapenem inactivation method (CIM), using CHDL-producing Acinetobacter spp., and developed a modified method that uses bacterial lysate (lysate CIM; LCIM). METHODS: A total of 159 Acinetobacter spp. (102 carbapenemase producers and 57 non-producers) and 14 Pseudomonas spp. (7 carbapenemase producers and 7 non-producers) were tested. Modified CIM, simplified CIM, CIMTris, Triton-CIM and LCIM were compared using these strains. Distinct from the CIM, LCIM includes a longer incubation period (4 h) with 2.0% Triton X-100 (v/v) in 20 mM MOPS buffer instead of water. RESULTS: The sensitivity/specificity of the modified CIM, simplified CIM, CIMTris, Triton-CIM and LCIM were 71.6%/100%, 66.1%/89.1%, 88.1%/95.3%, 80.7%/100% and 97.2%/100%, respectively. LCIM was the most sensitive and specific. CONCLUSIONS: Use of bacterial lysate and MOPS increased the sensitivity of the CIM in detecting CHDL-producing Acinetobacter spp.

Bacterial keratoconjunctivitis caused by Staphylococcus argenteus belonging to sequence type 1223 isolated in Japan.

Staphylococcus argenteus, characterized by the formation of non-pigmented (white) colonies, was recently identified as a new lineage separated from Staphylococcus aureus. However, correct identification of this lineage is difficult because of the similar characteristics to S. aureus. Here, we describe the first known case of keratoconjunctivitis due to S. argenteus in a 64-year-old man with diabetes. The symptoms of the patient were not improved by antibiotic therapy using levofloxacin eye drops (15 mg/mL). The conjunctival scraping was cultured, and coagulase-positive staphylococci forming white colonies were detected. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed the species as S. argenteus with a spectral score of 1.97. After the antibiotic was changed to vancomycin eye drops (10 mg/mL), the patient's symptom clearly improved. Multi-locus sequence typing showed that this isolate belonged to sequence type 1223, which has been predominantly isolated worldwide. Furthermore, this isolate harbored various virulence genes associated with S. aureus, such as staphylococcal enterotoxins and leukocidin. Since only limited information is available for this organism, further studies are needed to establish the epidemiology of S. argenteus.

Evaluation of inhibitor-combination mCIM for detecting MBL-producing Enterobacterales using three MBL inhibitors.

The increase in carbapenemase-producing Enterobacterales (CPE), including metallo-ß-lactamase (MBL) producers, is a severe global health concern. Thus, highly sensitive and specific methods for detecting MBL producers are needed. In this study, we tested the detectability of MBL-producing Enterobacterales against three types of MBL inhibitors (sodium mercaptoacetate, SMA; ethylenediaminetetraacetic acid, EDTA; and dipicolinic acid, DPA) used in combination with a modified carbapenem inactivation method (mCIM). These inhibitor-combination mCIMs were tested against 129 CPE (IMP, 93; NDM, 11; KPC, 13; NMC, 1; OXA-48, 11) and 75 non-CPE. For evaluation of MBL inhibitors, we used two concentrations for each of the three inhibitors: DPA (200 and 300 mg l- 1), EDTA (5 and 10 mM), and SMA (1500 and 3000 mg l- 1). The overall sensitivities of SMA, EDTA and DPA were 97.1-99.0 %, 81.7-99.0 % and 88.5-96.2 %, respectively. Moreover, each method showed high specificity (99.0-100 %). Although inhibitor-combination mCIMs were highly sensitive and specific for the detection of MBL producers, we found that sensitivity was dependent on the concentration of inhibitors.

Long-term observation of antimicrobial susceptibility and molecular characterisation of Campylobacter jejuni isolated in a Japanese general hospital 2000-2017.

OBJECTIVES: Campylobacter jejuni (C. jejuni) is one of the most common pathogens that causes gastroenteritis. Because there is currently insufficient epidemiological information about the antimicrobial susceptibility and molecular characterisation of clinical isolates of C. jejuni in Japan, this study carried out antimicrobial susceptibility testing and multilocus sequence typing (MLST) of clinical C. jejuni isolates in Tokyo between 2000-2017. METHODS: Antimicrobial susceptibility to erythromycin and ciprofloxacin was tested using the broth microdilution method in 430 C. jejuni clinical isolates collected over 18 years, between 2000-2017, at a Tokyo general hospital. To observe the sequence type (ST) evolution, 82 isolates were chosen from three non-consecutive years (16 isolates from 2000, 25 isolates from 2008, and 41 isolates from 2017) and analysed by MLST as a molecular characterisation test. Mutations in the quinolone resistance-determining region of the gyrA and gyrB genes were identified. RESULTS: The rate of resistance to erythromycin was low, but that of ciprofloxacin resistance was 34.9% in 2000-2008 and 41.9% in 2009-2017. The most common clonal complex (CC) identified during the entire period was CC21; ST4526 with ciprofloxacin resistance was highly prevalent in 2017 (6 of 11; 54.5%). CONCLUSION: The results indicate that the rate of resistance to quinolone has gradually increased. Since ST4526 was not isolated in 2000 and 2008, it is likely that ST4526 is rapidly increasing in Japan.

Novel mechanism responsible for high-level macrolide resistance in Moraxella catarrhalis.

BACKGROUND: High-level macrolide-resistant Moraxella catarrhalis strains have been isolated; however, the underlying mechanism has not been well elucidated. We investigated the role of mutations in the 23S rRNA gene and the L4 and L22 ribosomal proteins using spontaneous erythromycin-resistant mutants and transformants. MATERIALS AND METHODS: The erythromycin-susceptible M. catarrhalis ATCC25238 and clinical isolate Mc19 were used as parental strains. To obtain spontaneous erythromycin-resistant mutants, in vitro stepwise selection was performed using brain-heart infusion agar plates containing various concentrations of erythromycin. The role of the mutations identified in the spontaneous mutants was validated using transformation experiments. RESULTS: We obtained two spontaneous mutants with high-level resistance to erythromycin, S25-32-af10 and S19-256-af10, from ATCC25238 and Mc19, respectively. S25-32-af10 exhibited mutations of Q61R in L4 and Insertion98SRADRIS in L22. S19-256-af10 exhibited three C2611T-mutated alleles in the 23S rRNA gene and G65A in L4. Transformants with single mutations identified in S25-32-af10 or S19-256-af10 showed higher erythromycin and azithromycin minimum inhibitory concentrations (MICs) than those of each parental strain. However, transformants with multiple mutations identified in S25-32-af10 or S19-256-af10 showed macrolide MICs similar to those of each parental strain. CONCLUSION: Our results provide the first evidence suggesting that Q61R in L4 and Insertion98SRADRIS in L22 are involved in the synergistic acquisition of high-level resistance to both 14- and 15-member macrolides, and that C2611T in the 23S rRNA gene and G65A in L4 also synergistically contribute toward conferring high-level 14-member macrolide resistance to M. catarrhalis.

Molecular Characterization of Fluoroquinolone-Resistant Moraxella catarrhalis Variants Generated In Vitro by Stepwise Selection.

Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis resistance to fluoroquinolones were revealed.

Pantoea calida bacteremia in an adult with end-stage stomach cancer under inpatient care.

Pantoea calida is a gram-negative bacillus that was first identified in 2010. Here, we describe the first known case of P. calida bacteremia in a 77-year-old woman with end-stage stomach cancer under inpatient care. The patient was admitted to our hospital for pain after receiving anti-cancer therapy at outpatient facility. Thirteen days after admission, her temperature rose to 39.6 °C. A blood culture was ordered for suspected bacterial infection, and the patient was treated empirically with ampicillin/sulbactam. Cultures showed white pitting colonies later identified as a Pantoea sp. by biochemical analysis. The isolate's 16S rRNA sequence was identical to that of P. calida (100%), and showed 99.1% similarity with that of Pantoea gaviniae. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed the species as P. calida with an average spectral score >2.0. The primary isolate was ampicillin-resistant, but susceptible to other antibiotics and the bacteremia was cleared after three days of antibiotic therapy. Since P. calida infection is relatively rare, limited information exists on the pathogen's portal of entry and bacterial characteristics; thus, further studies are necessary to establish the pathophysiological mechanisms P. calida infection.

Antimicrobial susceptibility to ß-lactam antibiotics and production of BRO ß-lactamase in clinical isolates of Moraxella catarrhalis from a Japanese hospital.

We investigated BRO-ß-lactamase production of Moraxella catarrhalis isolates and its antimicrobial susceptibility to ß-lactams. Of the 233 isolates, 232 were BRO producers and 224 were BRO-1 producers. Four isolates exhibited elevated ceftriaxone minimum inhibitory concentration (2 µg/mL) and different pulsed-field gel electrophoresis patterns and we expect this number to increase in the near future.

Evaluation of the modified carbapenem inactivation method and sodium mercaptoacetate-combination method for the detection of metallo-ß-lactamase production by carbapenemase-producing Enterobacteriaceae.

We evaluated the effectiveness of carbapenem inactivation method (CIM) and modified CIM (mCIM). Our results indicated that mCIM with 4h incubation improved sensitivity and specificity for detecting carbapenemase-producing Enterobacteriaceae compared to CIM. Additionally, we developed a sodium mercaptoacetate-combination method (SMA-mCIM) to detect metallo-ß-lactamase (MBL) with high sensitivity and specificity.