Treatment of refractory localized pulmonary nocardiosis caused by Nocardia mexicana with a combination of medication and surgery.

Pulmonary nocardiosis is a rare disease that is often difficult to cure because of its tendency to recur. Here, we report a case of refractory localized pulmonary nocardiosis caused by Nocardia mexicana. A 60-year-old Japanese woman had recurring pulmonary nocardiosis four times previously and each time she was treated with antibiotics for a sufficient duration; nevertheless, the disease continued to recur, probably because of resistance to antibiotics. As a fifth treatment, we performed middle lobe resection and pre- and post-operative antimicrobial therapy for 6 months. The combination of medication and surgery was useful for treating refractory localized pulmonary nocardiosis.

Evaluation of the VITEK2 AST-XN17 card for the detection of carbapenemase-producing Enterobacterales in isolates primarily producing metallo ß-lactamase.

Carbapenemase-producing Enterobacterales (CPE) are not always resistant to carbapenem antimicrobial susceptibility testing (AST) and can be difficult to detect. With the newly created VITEK2 AST-XN17 card, the types of antibiotics measured in AST can be increased. In this study, we evaluated the detectability of CPE using the results of AST with multiple antimicrobial agents with additional measurements of the AST-XN17 card. In addition, we evaluated the CPE detectability of comments on CPE using the VITEK2 Advance Expert System (AES). In total, 169 Enterobacterales samples, including 76 non-CPE and 93 CPE, collected from multiple medical institutions in the Kinki region of Japan, were used in this investigation. AST with VITEK2 was performed by adding the AST-XN17 card in addition to the AST-N268 or AST-N404 card. Measurement results were identified using cutoff values, primarily Clinical and Laboratory Standards Institute breakpoints, and the CPE detection capability of each antibiotic was evaluated in several terms, including sensitivity and specificity. The drugs highly sensitive to CPE detection were faropenem (FRPM) > 2 µg/mL at 100% and meropenem > 0.25 µg/mL at 98.9%; the highest specificity to CPE detection was for avibactam/ceftazidime (AVI/CAZ) > 8 µg/mL at 100%. The sensitivity and specificity of each card in the AES output were 86.2% and 94.7% for AST-N404 and AST-XN17 and 91.5% and 90.8% for AST-N268 and AST-XN17, respectively. AST using the VITEK2 AST-XN17 card is a useful test method of screening for CPE.

Genomic Characterization of ESBL- and Carbapenemase-Positive Enterobacteriaceae Co-harboring mcr-9 in Japan.

Worldwide spread of Enterobacteriaceae resistant to colistin, a polypeptide antibacterial drug for last-resort treatment of carbapenemase-producing Enterobacteriaceae (CPE) infections, is concerning. This study aimed to elucidate colistin MICs and molecular characteristics of mcr-1 to mcr-9 of ESBL-producing Escherichia coli (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring mcr genes (especially mcr-9). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting mcr-1 to mcr-9 were performed for all strains with S1-nuclease pulsed-field gel electrophoresis, Southern blot hybridization, and whole-genome sequencing (WGS) with hybrid assembly performed for mcr gene-carrying strains. Two CPE strains showed a MIC ≥ 4 µg/ml in colistin susceptibility testing, with no known resistance mechanism detected. However, PCR conducted on all target strains detected three mcr-9-carrying strains showing colistin susceptibility. The bla CTX-M-62 -positive E. coli THUN648 strain simultaneously carried bla CTX-M-62 and mcr-9 on a 275-kbp plasmid. Besides, bla IMP-6 + bla CTX-M-2 -positive Klebsiella pneumoniae THUN262 and bla GES-24 -positive Enterobacter kobei THUN627 had mcr-9 encoded on the chromosome. Only THUN627 encoded qseB/C, which is suggested to be a regulatory gene for mcr-9, downstream of mcr-9. However, this strain showed no increased expression of these genes in mRNA quantitative analysis under colistin exposure. Colistin MICs of ESBL-Ec and CPE in Japan were all below 2 µg/ml, which is below the epidemiological cutoff (ECOFF) value (https://eucast.org/) or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither "microbiological" nor "clinical" resistance. Several colistin-susceptible Enterobacteriaceae carrying silent mcr-9 encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by mcr-9 remains unclear.

Comparison of the performance of three carbapenem inactivation methods for the detection of carbapenemase-producing gram-negative bacilli.

INTRODUCTION: The carbapenem inactivation method test (CIM) was developed as a method for detecting carbapenemase-producing Gram-negative bacilli, and the modified CIM (mCIM) was recommended by the CLSI for as an improved method in M100-S27. However, few studies have evaluated the influence of bacterial species and genotype on its sensitivity and specificity. In this study, we evaluate the performance of these improved modified CIM methods with mCIM. METHODS: As strains, clinical isolates from Naga Municipal Hospital and stored strains from the Study of Bacterial Resistance in the Kinki Region of Japan were used. The mCIM, CIM-Tris, and simple CIM (sCIM) test methods were applied to 120 Enterobacterales, 40 Pseudomonas aeruginosa, and 37 Acinetobacter spp. The procedure and criteria for each method were based on the original papers and the CLSI M - 100 S27 documents. RESULTS: The sensitivity of the test methods in the detection of carbapenemase in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. was as follows: mCIM, 98.9%, 90.0%, and 76.5%, respectively; CIM-Tris, 94.4%, 100%, 100%; and sCIM 98.9%, 85.0%, 76.5%. All methods showed 100% specificity in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. Each method performed well in the detection of metallo ß-lactamase-producing strains, however, the sensitivity tended to be low in the detection of the organisms producing serine-type carbapenemase, such as GES, OXA-23, and OXA-51. CONCLUSIONS: Care must be taken when selecting test methods because the sensitivity of the detection differs depending on the bacterial species and genotype.

The microcolony detection method (MCD), a simple and rapid screening test for antimicrobial resistance bacteria on positive blood cultures.

Rapid detection of antibiotic-resistant bacteria in blood cultures is critical for the timely treatment of patients with sepsis. The aim of this study was to develop a simple method for the rapid detection of drug-resistant bacteria from blood cultures and to evaluate its performance. We developed an optical microscopy-based microcolony detection method (MCD) for the rapid detection of antibiotic-resistant bacterial colonies in media. This method was tested using staphylococci resistant to methicillin and gram-negative bacilli resistant to third-generation cephalosporins and carbapenem. The results of the investigations of clinical samples using this method were compared with the drug susceptibility testing results for each of the 457 isolates, which included 134 staphylococci and 323 g-negative bacilli. The MCD was successful in detecting antibiotic-resistant bacterial growth from culture-positive blood samples in approximately 3 h. The sensitivity/specificity for methicillin-resistant staphylococci was 100%/97.2%. In the case of gram-negative bacilli, the sensitivity/specificity values for bacteria resistant to ceftriaxone, ceftazidime, and carbapenem were 100%/98.7%, 100%/89.3%, and 100%/90.9%, respectively. Therefore, MCD is a clinically useful screening method for the efficient and rapid detection of antibiotic-resistant bacteria and can be easily implemented in laboratories.

Evaluation of presumptive identification of Enterobacterales using CHROMagar Orientation medium and rapid biochemical tests.

BACKGROUND: The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is gradually spreading among large-scale laboratories; however, this method is impractical for small-scale laboratories. In laboratories without access to these rapid identification methods, problems related to them remain unsolved. In this study, we aimed to develop a rapid and inexpensive method to presumptively identify Enterobacterales using CHROMagar Orientation medium. METHODS: The algorithm for presumptive identification of Enterobacteriaceae using CHROMagar Orientation medium was based on our previous studies. Modified property tests for indole, lysine decarboxylase, ornithine decarboxylase, and hydrogen sulfide were performed to evaluate the differentiation of the bacterial species. RESULTS: Using the type strains and clinical isolates, it was possible to conduct the property tests at a low cost, within 4 hours. The spot indole test was performed without any nonspecific reactions for the bacteria forming colored colonies. The presumptive identification of bacteria was thereby possible within 24 hours after specimen submission. CONCLUSION: All these results suggest that the rapid presumptive identification of Enterobacterales is possible with this new identification method using CHROMagar Orientation medium. This is therefore a prompt and economical method that can be used in routine laboratory work.

Antimicrobial susceptibility surveillance of obligate anaerobic bacteria in the Kinki area.

Obligate anaerobes exist as resident flora in various sites in humans, but they are also emphasized as endogenous causative microorganism of infections. We performed surveillance to understand the trend of drug susceptibility in obligate anaerobic bacteria in the Kinki area of Japan. In the experiment, we used 156 obligate anaerobe isolates collected from 13 institutions that participated in the Study of Bacterial Resistance Kinki Region of Japan. MALDI Biotyper was used to identify the collected strains, and among the 156 test strains, those that could be identified with an accuracy of Score Value 2.0 or more included 6 genera, 30 species, and 144 strains (Bacteroides spp. 77 strains, Parabacteroides sp. 2 strains, Prevotella spp. 29 strains, Fusobacterium spp. 14 strains, Porphyromonas spp. 2 strains, and Clostridioides difficile 20 strains), and they were assigned as subject strains for drug susceptibility testing. The drug susceptibility test was carried out by broth microdilution method using Kyokuto Opt Panel MP ANA (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) and judged according to CLSI criteria. As a result, Bacteroides and Parabacteroides species showed good sensitivities to tazobactam-piperacillin, imipenem, metronidazole and chloramphenicol, and low sensitivities to ampicillin, cefoperazone and vancomycin. Prevotella species showed good sensitivities to sulbactam-ampicillin, tazobactam-piperacillin, cefmetazole, imipenem, doripenem and metronidazole. Susceptibility rates to other drugs were slightly different depending on the bacterial species. Both Fusobacterium spp. and Porphyromonas spp. showed high sensitivities to many drugs. C. difficile was highly sensitive to vancomycin and metronidazole, having MIC90s of 0.5 µg/mL and ≤2 µg/mL, respectively.

First case report of sepsis caused by Rhizobium pusense in Japan.

INTRODUCTION: Species of the genus Rhizobium are opportunistic, usually saprophytic, glucose-non-fermenting, Gram-negative bacilli found in agricultural soil. Rhizobium pusense infections are the least common Rhizobium infections and have low incidence. CASE PRESENTATION: Herein, we report the first case of sepsis with R. pusense in Japan in a 67-year-old Japanese woman with a history of hyperlipidaemia, hypertension, diabetes, hypothyroidism and osteoporosis. She had undergone cerebrovascular treatment because she was diagnosed with a subarachnoid haemorrhage. The results of postoperative blood culture showed oxidase-positive, urease-positive, non-lactose-fermenting Gram-stain-negative rods. Using the Vitek2 system, the isolate was distinctly identified as Rhizobium radiobacter. However, 16S rRNA gene sequencing showed 99.93 % similarity with the type strain of R. pusense and 99.06 % similarity with the type strain of R. radiobacter. Additional gene sequencing analysis using recA (97.2 %) and atpD (96.2 %) also showed that the isolated strain is most closely related to R. pusense. The patient was cured by treatment using intravenous meropenem (3 g/d) for 4 weeks and was discharged safely. CONCLUSION: The definite source of sepsis was unknown. However, the possibility of having been infected through the catheter during the cerebrovascular operation was speculated.

Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae.

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory.

[The annual changes in antimicrobial susceptibility test results of Pseudomonas aeruginosa isolates from the Kinki district].

A study was conducted of the 1,225 Pseudomonas aeruginosa strains that were isolated at 20 medical institutions in the Kinki district between 2011 and 2013 to determine their antimicrobial susceptibility and to characterize the strains of multidrug-resistant Pseudomonas aeruginosa (MDRP) and the metallo-ß-lactamase (MBL) -producing strains. The MIC50/MIC90 values (µg/mL) of the various antimicrobial agents were as follows: imipenem, 2/>8; meropenem, 1/>8; doripenem, 0.5/8; biapenem, 1/>8; tazobactam/piperacillin, 8/>64; piperacillin, 8/>64; sulbactam/cefoperazone, 8/64; cefepime, 4/16; cefozopran, 2/>16; aztreonam, 8/>16; amikacin, 4/16; levofloxacin, 1/>4; and ciprofloxacin, 0.25/>2. From the viewpoint of the annual changes in the susceptibility rates (according to the CLSI guidelines [M100-S22]), the susceptibility to tazobactam/piperacillin, piperacillin, cefepime, cefozopran and aztreonam decreased in 2013. On the other hand, two antimicrobial agents showed high susceptibility rates each year; amikacin (94.0-95.6%) showed the highest rate, followed by doripenem (80.3-82.6%). With the exception of amikacin, there were substantial inter-institutional differences in antimicrobial susceptibility. In comparison to the previous CLSI guidelines (M100-S21), the new CLSI guidelines (M100-S22) on the use of carbapenems and penicillins show that the MIC80 has been affected. The MDRP detection rates in 2011, 2012 and 2013 were 1.8% (8 strains), 1.8% (8 strains), and 2.8% (10 strains), respectively. The MBL detection rates were as follows: bla(VIM-2), 0.2% (1 strain) in 2011; bla(IMP-1), 0.9% (4 strains) in 2012, and 1.7% (6 strains, including bla(IMP-1) [3 strains], bla(IMP-2) [2 strains] and bla(VIM-2) [1 strain]) in 2013.

First case report of pulmonary nocardiosis caused by Nocardia mexicana.

INTRODUCTION: Nocardia species usually cause opportunistic infections, and the frequency of these infections is increasing owing to the growing population of immunocompromised hosts. However, Nocardia species may sometimes cause an infection disease in immunocompetent hosts. Nocardia mexicana infections are the least common and are very rare. CASE PRESENTATION: Herein, we report the first case of a pulmonary infection with N. mexicana in a 61-year-old Japanese woman with a history of hyperlipidaemia and bronchiectasis and a 6-month history of non-productive hacking cough. A sample of bronchial lavage fluid obtained by bronchofiberscopy showed filamentous branching gram-positive rods and acid-fast filamentous branching rods, and a colony of suspected Nocardia was cultured. Based on 16S rRNA, gyrB,rpoB, secA1 and hsp65 gene sequence analyses and biochemical and physiological properties, the strain was identified as N. mexicana. The strain was resistant to the antimicrobial agents amoxicillin-clavulanic acid, clarithromycin, minocycline, gentamycin, tobramycin, ciprofloxacin and trimethoprim-sulfamethoxazole. The patient was treated with biapenem followed by intravenous amikacin and oral linezolid. CONCLUSION: Despite its rarity, the species require attention owing to the existence of multidrug-resistant strains.