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

IntroductionThe 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.MethodsAs 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.ResultsThe 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 β-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.ConclusionsCare must be taken when selecting test methods because the sensitivity of the detection differs depending on the bacterial species and genotype.     

PCR-based detection of resistance genes in anaerobic bacteria isolated from intra-abdominal infections

Little information is available on the distribution of antimicrobial resistance genes in anaerobes in Japan. To understand the background of antimicrobial resistance in anaerobes involved in intra-abdominal infections, we investigated the distribution of eight antimicrobial resistance genes (cepA, cfiA, cfxA, ermF, ermB, mefA, tetQ, and nim) and a mutation in the gyrA gene in a total of 152 organisms (Bacteroides spp., Prevotella spp., Fusobacterium spp., Porphyromonas spp., Bilophila wadsworthia, Desulfovibrio desulfuricans, Veillonella spp., gram-positive cocci, and non-spore-forming gram-positive bacilli) isolated between 2003 and 2004 in Japan. The cepA gene was distributed primarily in Bacteroides fragilis. Gene cfxA was detected in about 9 % of the Bacteroides isolates and 75 % of the Prevotella spp. isolates and did not appear to contribute to cephamycin resistance. Two strains of B. fragilis contained the metallo-β-lactamase gene cfiA, but they did not produce the protein product. Gene tetQ was detected in about 81, 44, and 63 % of B. fragilis isolates, other Bacteroides spp., and Prevotella spp. isolates, respectively. The ermF gene was detected in 25, 13, 56, 64, and 16 % of Bacteroides spp., Prevotella spp., Fusobacterium spp., B. wadsworthia, and anaerobic cocci, respectively. Gene mefA was found in only 10 % of the B. fragilis strains and 3 % of the non-B. fragilis strains. Genes nim and ermB were not detected in any isolate. Substitution at position 82 (Ser to Phe) in gyrA was detected in B. fragilis isolates that were less susceptible or resistant to moxifloxacin. This study is the first report on the distribution of resistance genes in anaerobes isolated from intra-abdominal infections in Japan. We expect that the results might help in understanding the resistance mechanisms of specific anaerobes.     

Microbiological analysis of patients with first and recurrent episodes of acute cholangitis in a middle-sized hospital: A single-center retrospective study in rural North Kyoto,Japan

BackgroundKnowledge of the bacterial spectrum involved in acute cholangitis is essential for adequate empiric antibiotic treatment. There is a lack of published data comparative data between patients with first and recurrent episodes of acute cholangitis. This study aimed to analyze the microbial spectrum in patients with first and second episodes of acute cholangitis.MethodsWe retrospectively assessed 251 patients with first episodes of acute cholangitis between January 2014 to September 2020.ResultsAt the first episode of acute cholangitis, the predominant strains belonged to Escherichia coli (17.9%), followed by Klebsiella spp. (15.5%), Enterobacter spp. (6.4%), and Enterococcus spp. (5.6%). During follow-up, acute cholangitis recurred in 109 patients; at the second episode, the predominant strains belonged to Enterococcus spp. (35.8%), followed by Klebsiella spp. (27.5%), Enterobacter spp. (22.9%), and Escherichia coli (15.6%). Enterococcus spp. were the most common pathogen in patients with second episode of acute cholangitis, regardless of whether the cholangitis was caused by a malignant tumor or a benign disease.ConclusionsUnlike in patients with a first episode of acute cholangitis, clinicians should consider empirical treatment with anti-enterococcal antibiotics in patients with recurrent episodes of acute cholangitis.     

Characterization of Aspergillus spp. isolated from patients with coronavirus disease 2019

IntroductionInvasive pulmonary aspergillosis (IPA) is an important complication of coronavirus disease 2019 (COVID-19), and while there are case reports and epidemiological studies, few studies have isolated Aspergillus strains from patients. Therefore, we analyzed the strains, sensitivities, and genetic homology of Aspergillus spp. Isolated from patients with COVID-19.MethodsWe investigated the Aspergillus strains detected from patients with COVID-19 hospitalized in Osaka Metropolitan University Hospital from December 2020 to June 2021. A molecular epidemiological analysis of Aspergillus spp. was performed using drug susceptibility tests and TRESPERG typing, and data on patient characteristics were collected from electronic medical records.ResultsTwelve strains of Aspergillus were detected in 11 of the 122 patients (9%) with COVID-19. A. fumigatus was the most common species detected, followed by one strain each of Aspergillus aureolus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus. A. aureolus was resistant to voriconazole, and no resistance was found in other strains. All A. fumigatus strains were genetically distinct strains. Six of the 11 patients that harbored Aspergillus received antifungal drug treatment and tested positive for β-D-glucan and/or Aspergillus galactomannan antigen. The results indicated that Aspergillus infections were acquired from outside the hospital and not from nosocomial infections.ConclusionStrict surveillance of Aspergillus spp. is beneficial in patients at high-risk for IPA. When Aspergillus is detected, it is important to monitor the onset of IPA carefully and identify the strain, perform drug sensitivity tests, and facilitate early administration of therapeutic agents to patients with IPA.     

Genetic characteristics of piliated Streptococcus pneumoniae serotype 35B,increased after introduction of pneumococcal vaccines in Japan

IntroductionStreptococcus pneumoniae is a commensal bacterium of the human nasopharynx and a major causative pathogen of bacterial diseases worldwide. Pilus of S. pneumoniae is one of the virulence factors which enhance the adhesion to the host epitherial cells in the upper respiratory tract.MethodsWe analyzed the serotype distribution and presence of pilus genes, rrgC and sipA, among 785 S. pneumoniae isolates from specimens of patients with invasive or non-invasive disease in a regional Japanese hospital between October 2014 and August 2018. We next performed multilocus sequence typing and penicillin-resistant genotyping for 86 isolates of serotype 35B.ResultsSerotype 35B was the most frequent serotype which accounted for 11.0% of total isolates and had pilus genes at high rate (80.2%). Clonal complex (CC) 558 isolates accounted for 77.9% of serotype 35B and were highly positive for rrgC and gPRSP (98.5%). In contrast, all CC2755 isolates (19.8%) were rrgC-negative and gPISP.ConclusionsOur results suggest that CC558 may assist the prevalence of serotype 35B after the introduction of vaccines, as that clone has pili as adhesins in addition to non-susceptibility against penicillin. These results may be useful information for development of optimal preventive strategies. Continuous studies on serotype distribution and virulence factors of S. pneumoniae are necessary.     

Bacteriological analysis of Neisseria lactamica isolated from the respiratory tract in Japanese children

IntroductionNeisseria lactamica is a commensal bacterium of the upper respiratory tract in humans and is closely related to Neisseria meningitidis. N. lactamica colonization may contribute to preventing N. meningitidis colonization and invasive meningococcal disease. However, the transference of antimicrobial resistance genes from N. lactamica to N. meningitidis has been reported.MethodsIn this study, we aimed to identify N. lactamica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and performed multilocus sequence typing of seven N. lactamica strains isolated from Japanese children. We also analyzed the antimicrobial susceptibility of these strains and the mutations in their antimicrobial resistance genes (penA, gyrA, and parC).ResultsAll the N. lactamica strains could be identified using MALDI-TOF MS. All strains were of different sequence types (STs), including five new STs. Five strains had intermediate susceptibility, two were resistant to ampicillin, and all had five out of the five known PBP2 mutations. Six strains were resistant to levofloxacin. Among the quinolone-resistant strains, three had GyrA mutations, and three had both ParC and GyrA mutations.ConclusionsN. lactamica STs may vary in Japanese children, and penicillin- and quinolone-resistant strains may be prevalent. We should pay attention not only to the drug resistance of N. meningitidis but also to the drug susceptibility of N. lactamica whose drug-resistance genes may transfer to N. meningitidis.     

Epidemiology and molecular characterization of fecal carriage of third-generation cephalosporin-resistant Enterobacterales among elderly residents in Japan

IntroductionThe spread of third-generation cephalosporin-resistant Gram-negative bacteria is a serious concern in acute and post-acute care settings. This study aimed to understand the epidemiology and molecular background of fecal colonization of resistant Enterobacterales in elderly people.MethodsIn December 2015–December 2017, stool or rectal swab samples were collected from 101 elderly patients receiving home care, using long-term care facilities (LTCF), and living in nursing homes repeatedly at 3?9-month intervals. Patient clinical background data were collected from medical records. After phenotypic screening for extended-spectrum β-lactamase (ESBL), AmpC-type β-lactamase or carbapenemase production, drug resistance genes of isolates were analyzed using polymerase chain reaction (PCR). ESBL-producing Escherichia coli isolates obtained from the same patients in repetitive screenings were analyzed using PCR-based ORF typing. Risk factors for persistent carriage of resistant Enterobacterales were analyzed using multivariate analysis.ResultsResistant Enterobacterales isolates were detected in 37 of 101 (36.6%) and 29 of 80 (36.3%) residents in first and second screenings, respectively. ESBL-producing E. coli accounted for 80% isolates, the most common being CTX-M-9-group β-lactamase producers. Molecular epidemiological analysis revealed probable transmissions of ESBL-producing E. coli; 58% of ESBL-producing E. coli colonizers were persistent colonizers at least after 3 -month intervals. Age > 87 years and LTCF residence were independent risk factors for persistent carriage of ESBL-producing E. coli.ConclusionsWe showed, for the first time, high persistent colonization rate of ESBL-producing E. coli among elderly people in post-acute care settings with probable horizontal transmission. We also identified significant risk factors for persistent colonization.     

A mixed infection involving Bacteroides denticanum,Lactobacillus salivarius,and Streptococcus anginosus as causative agents of abscess around a pharyngo-esophageal anastomosis and acute vertebral osteomyelitis: Identification by ribosomal RNA sequencing of bacterial isolates

“Bacteroides denticanum” is an anaerobic, non-spore-forming, gram-negative bacterium with a rod morphology typical of canine, ovine, and macropod oral flora. There is only one report of bloodstream infection caused by “B. denticanum” from a dog bite in human. Here, we report a case with no history of animal contact who developed an abscess caused by “B. denticanum” around a pharyngo-esophageal anastomosis after undergoing balloon dilatation procedure for stenosis following laryngectomy. The patient was a 73-year-old man with laryngeal cancer, esophageal cancer, hyperuricemia, dyslipidemia, and hypertension with a 4-week history of cervical pain, sore throat, and fever. Computed tomography showed fluid collection on the posterior pharyngeal wall. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) identified Bacteroides pyogenes, Lactobacillus salivarius, and Streptococcus anginosus from abscess aspiration. 16S ribosomal RNA sequencing re-identified the Bacteroides species as “B. denticanum”. T2-weighted magnetic resonance images showed a high signal intensity adjacent to the anterior vertebral body of C3–C7. The diagnosis was peripharyngeal esophageal anastomotic abscess and acute vertebral osteomyelitis caused by “B. denticanum”, L. salivarius, and S. anginosus. The patient was treated with sulbactam ampicillin intravenously for 14 days and then switched to oral amoxicillin with clavulanic acid for 6 weeks. To our knowledge, this is the first report of a human infection caused by “B. denticanum” without a history of animal contact. Despite remarkable advancements facilitated by MALDI-TOF MS in microbiological diagnosis, the accurate identification of novel, emerging, or uncommon microorganisms and comprehending their pathogenicity, suitable therapy, and follow up necessitate sophisticated molecular approaches.     

Emergence of Haemophilus influenzae with low susceptibility to quinolones isolated from pediatric patients in Japan

IntroductionIn 2010, oral fluoroquinolone tosufloxacin (TFX) granules were released as the first oral respiratory quinolone for children in Japan.MethodsTo investigate the recent trend of H. influenzae strains with low susceptibility to quinolones in children, we analyzed the gene sequences of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE of 23 clinical isolates from 15 patients aged <15 years with an MIC of ≥0.5 μg/mL for TFX from 2010 to 2018.ResultsAmino acid substitutions were observed in both GyrA and ParC in 13 strains (81%, 13/16), except two strains with a TFX MIC of 0.5 μg/mL with amino acid substitution in only GyrA and one strain with a TFX MIC of 1 μg/mL with no amino acid substitution. Four ST422 strains were observed in 2018, the detection age range was wide (0–7 years), and the residential city was varied. A total of 3/15 patients had a clear history of TFX treatment.ConclusionsEven for the strain with an MIC of 0.5 μg/mL for TFX, it is highly possible that it harbors a mutation in gyrA, which is the first step toward quinolone resistance, and it may also harbor mutations in both gyrA and parC. Furthermore, several specific sequence type quinolone-resistant H. influenzae strains, particularly ST422, may be widespread among children in Japan. It is necessary to investigate changes in resistance both at the MIC and gene levels. The continuous monitoring of strains and the use of antimicrobial drugs in treatment should be carefully observed.     

In vitro and in vivo antibacterial activity of nitrofurantoin against clinical isolates of E. coli in Japan and evaluation of biological cost of nitrofurantoin resistant strains using a mouse urinary tract infection model

IntroductionNitrofurantoin is a well-established antibiotic, and is an important first-line oral treatment for uncomplicated urinary tract infections. However, little information is available with respect to its antibacterial activity in Japan, in vivo efficacy, or the in vivo biological cost of resistant strains.MethodsWe compared the susceptibility of six representative antibacterial agents—nitrofurantoin, sulfamethoxazole/trimethoprim, fosfomycin, mecillinam, ciprofloxacin, and cefdinir—against E. coli clinically isolated in Japan during 2017. We evaluated the in vivo efficacy of nitrofurantoin using a model of mouse urinary tract infection caused by ciprofloxacin resistant E. coli. We obtained nitrofurantoin resistant isolates through tests generating spontaneous mutations, and assessed the in vivo fitness of nitrofurantoin resistant isolates.ResultsThe MIC₉₀ of nitrofurantoin was 16 μg/mL, and was the lowest among the drugs tested. It was found that, in the mouse urinary tract infection model, 30 mg/kg and 100 mg/kg of nitrofurantoin reduced the count of viable bacterial cells in the kidney, while 100 mg/kg of ciprofloxacin did not. All spontaneous bacterial mutants resistant to nitrofurantoin had deletions in the nfsA gene, and we found that the resistant strain had lower growth in the mouse urinary tract infection model than in the parent strain.ConclusionsWe demonstrated promising in vitro and in vivo activity of nitrofurantoin against E. coli clinical isolates in Japan, and lower in vivo fitness of the resistant strain of nitrofurantoin.     

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae

PurposeIn Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains.Materials and methodsPneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to β-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling.ResultsIn PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations.ConclusionsNVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.     

Prospective survey of Aspergillus species isolated from clinical specimens and their antifungal susceptibility: A five-year single-center study in Japan

Aspergillus fumigatus is the most prevalent species that causes aspergillosis. A. fumigatus strains with tandem repeats in the cyp51A promoter have emerged in the environment. Aspergillus species other than A. fumigatus have also been recognized as causative agents of aspergillosis; however, they show lower susceptibility to antifungals compared with A. fumigatus. Therefore, it is important to precisely identify Aspergillus species and determine their antifungal susceptibility. Herein, we collected 119 mold strains isolated from clinical specimens collected at a hospital between November 2013 and December 2018. The collected strains were identified by sequencing several regions, including internal transcribed spacers, and determined their susceptibility to the antifungals itraconazole, voriconazole, and amphotericin B. Of 119 strains, 107 were Aspergillus species, which were identified as A. fumigatus (67), Aspergillus section Nigri (21), A. flavus (7), A. terreus (6), and A. nidulans (6). In Aspergillus section Nigri, the number of A. niger was less than the number of A. welwitschiae and A. tubingensis. Two azole-resistant A. fumigatus samples were included among the isolates. Four of the eight A. tubingensis isolates showed less susceptibility to voriconazole; however, all isolates of A. niger and A. welwitschiae were susceptible to itraconazole and voriconazole. Because of lack of susceptibility data for non-fumigatus Aspergillus and an increasing frequency of antifungal resistance among A. fumigatus, our data along with further surveillance may contribute to determining the frequency and susceptibility of Aspergillus spp. clinical isolates in Japan.     

Characteristics and antimicrobial choice of pediatric bacterial enteritis in the Kanto region of Japan: A multicenter retrospective observational study

IntroductionThere are few reports on the causative microorganisms of bacterial enteritis in children in Japan in recent years. The distribution of causative microorganisms is important for estimating pathogens and making decisions regarding the treatment plan, as antimicrobial agents are not required for mild bacterial enteritis cases but are used for severe cases or immunocompromised patients.MethodsWe retrospectively surveyed pediatric patients who underwent stool culture at eight hospitals in the Kanto region of Japan from 2014 to 2019 for patient characteristics, causative microorganisms, and prescribed antimicrobial agents.ResultsA total of 4,475 stool cultures were submitted, and the positivity rate for bacterial enteritis was 11%. The causative microorganisms were Campylobacter spp. in 338 cases (67.3%), Salmonella spp. in 85 cases (16.9%), enterohemorrhagic Escherichia coli O157 in 23 cases (4.6%), and Yersinia spp. in 45 cases (9.0%). Hospitals with pediatric infectious disease physicians had a lower rate of antimicrobial therapy for Campylobacter enteritis than hospitals without pediatric infectious disease physicians.ConclusionsCampylobacter spp. are the most common causative agent for bacterial enteritis in this study, and the presence of pediatric infectious disease physicians may promote the appropriate use of antimicrobial agents.     

Recent acute reduction in macrolide-resistant Mycoplasma pneumoniae infections among Japanese children

IntroductionMycoplasma pneumoniae contributes to numerous pneumonia cases among children and young adults. Therefore, this study aimed to investigate the prevalence of M. pneumoniae infections among Japanese children, occurring since 2008.MethodsNasopharyngeal swab specimens were obtained from all cases, following which real-time PCR was performed to identify M. pneumoniae. Further, the p1 genotypes of isolates were determined using the PCR restriction fragment length polymorphism typing method.ResultsThe annual rate of macrolide-resistant M. pneumoniae (MRMP) infections peaked at 81.8% in 2012 and decreased annually until 2015. Although the infection rate increased to 65.3% in 2016, it decreased again to 14.3% in 2018. Although >90% of isolates harbored the type 1 genotype until 2012, this rate decreased, and approximately 80% harbored p1 genotypes other than type 1 in 2018. Furthermore, the occurrence rate of MRMP among the type 1 isolates was very high (82.4%), whereas that among p1 genotypes other than type 1 was very low (6.5%).ConclusionsMRMP occurrence potentially decreased owing to changes in not only antibiotic usage but also in the distribution of p1 genotype among isolates.     

Risk factors for acute kidney injury in vancomycin and piperacillin/tazobactam combination therapy: A retrospective study

IntroductionCombined use of vancomycin (VCM) and piperacillin/tazobactam (PIPC/TAZ) has been reported to increase the incidence of acute kidney injury (AKI). However, the risk factors associated with AKI after VCM and PIPC/TAZ (VPT) administration have not yet been identified. Therefore, we retrospectively assessed patients treated with VPT to investigate the risk factors for AKI development.MethodsThe study involved patients who were treated with VPT from January 1, 2016 to March 31, 2020. The patients were divided into the AKI or non-AKI group. The clinical characteristics of patients and antimicrobial therapy were compared between the groups. Their association with AKI risk was evaluated using multivariate logistic regression analysis.ResultsIn total, 182 patients were included, with 118 in the non-AKI group and 64 in the AKI group. Therefore, the incidence of AKI was 35.2 %. The initiation of VPT combination therapy on the same day and concomitant use of vasopressors were associated with an increased risk of AKI (odds ratio [OR] 2.55, 95 % confidential interval [CI] 1.20–5.44 and OR 3.22, 95 % CI 1.31–7.89, respectively).ConclusionOur findings suggest that the concomitant use of vasopressors and initiating VPT combination therapy on the same day are likely risk factors for AKI development.     

Risk factors and pathogen characteristics associated with unfavorable outcomes among adults with pneumococcal meningitis in Japan, 2006 to 2016

PurposeIn this study, we aimed to clarify the risk factors associated with unfavorable outcomes in adults with pneumococcal meningitis (PnM).MethodsSurveillance was conducted between 2006 and 2016. Adults with PnM (n = 268) were followed up for outcomes within 28 days after admission using the Glasgow Outcome Scale (GOS). After classifying the patients into the unfavorable (GOS1–4) and favorable (GOS5) outcome groups, i) the underlying diseases, ii) biomarkers at admission, and iii) serotype, genotype, and antimicrobial susceptibility for all isolates were compared between both groups.ResultsOverall, 58.6% of patients with PnM survived,15.3% died, and 26.1% had sequelae. The number of living days in the GOS1 group was highly heterogeneous. Motor dysfunction, disturbance of consciousness, and hearing loss were the commonest sequelae. Of the underlying diseases identified in 68.9% of the PnM patients, liver and kidney diseases were significantly associated with unfavorable outcomes. Of the biomarkers, creatinine and blood urea nitrogen, followed by platelet and C-reactive protein had the most significant associations with unfavorable outcomes. There was a significant difference in the high protein concentrations in the cerebrospinal fluid between the groups. Serotypes 23F, 6C, 4, 23A, 22F, 10A, and 12F were associated with unfavorable outcomes. These serotypes were not penicillin-resistant isolates possessing three abnormal pbp genes (pbp1a, 2x, and 2b), except for 23F. The expected coverage rate of the pneumococcal conjugate vaccine (PCV) was 50.7% for PCV15 and 72.4% for PCV20.ConclusionsIn the introduction of PCV for adults, the risk factors for underlying diseases should be prioritized over age, and serotypes with unfavorable outcomes should be considered.