Characterization of Shiga toxin-producing Escherichia coli isolated from cattle around Ulaanbaatar city, Mongolia.

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0 to 40 percent. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from 8 farms. This is the first report on the characterization of STEC in cattle in Mongolia and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Genomic analysis of Salmonella isolated from canal water in Bangkok, Thailand.

Antimicrobial resistance (AMR) poses an escalating global public health threat. Canals are essential in Thailand, including the capital city, Bangkok, as agricultural and daily water sources. However, the characteristic and antimicrobial-resistance properties of the bacteria in the urban canals have never been elucidated. This study employed whole genome sequencing to characterize 30 genomes of a causal pathogenic bacteria, Salmonella enterica, isolated from Bangkok canal water between 2016 and 2020. The dominant serotype was Salmonella Agona. In total, 35 AMR genes and 30 chromosomal-mediated gene mutations were identified, in which 21 strains carried both acquired genes and mutations associated with fluoroquinolone resistance. Virulence factors associated with invasion, adhesion, and survival during infection were detected in all study strains. 75.9% of the study stains were multidrug-resistant and all the strains harbored the necessary virulence factors associated with salmonellosis. One strain carried 20 resistance genes, including mcr-3.1, mutations in GyrA, ParC, and ParE, and typhoid toxin-associated genes. Fifteen plasmid replicon types were detected, with Col(pHAD28) being the most common type. Comparative analysis of nine S. Agona from Bangkok and 167 from public databases revealed that specific clonal lineages of S. Agona might have been circulating between canal water and food sources in Thailand and globally. These findings provide insight into potential pathogens in the aquatic ecosystem and support the inclusion of environmental samples into comprehensive AMR surveillance initiatives as part of a One Health approach. This approach aids in comprehending the rise and dissemination of AMR and devising sustainable intervention strategies.IMPORTANCEBangkok is the capital city of Thailand and home to a large canal network that serves the city in various ways. The presence of pathogenic and antimicrobial-resistant Salmonella is alarming and poses a significant public health risk. The present study is the first characterization of the genomic of Salmonella strains from Bangkok canal water. Twenty-two of 29 strains (75.9%) were multidrug-resistant Salmonella and all the strains carried essential virulence factors for pathogenesis. Various plasmid types were identified in these strains, potentially facilitating the horizontal transfer of AMR genes. Additional investigations indicated a potential circulation of S. Agona between canal water and food sources in Thailand. The current study underscores the role of environmental water in an urban city as a reservoir of pathogens and these data obtained can serve as a basis for public health risk assessment and help shape intervention strategies to combat AMR challenges in Thailand.

Affinity of ß-Lactam Antibiotics for Neisseria gonorrhoeae Penicillin-Binding Protein 2 Having Wild, Cefixime-Reduced-Susceptible, and Cephalosporin (Ceftriaxone)-Resistant penA Alleles.

Multidrug-resistant Neisseria gonorrhoeae is a serious concern worldwide. Resistance to ß-lactam antibiotics occurs through mutations in penicillin-binding proteins (PBPs), acquisition of ß-lactamases, and alteration of antibiotic penetration. Mosaic structures of penA, which encodes PBP2, play a major role in resistance to ß-lactams, especially cephalosporins. Ceftriaxone (CRO) is recognized as the only satisfiable antibiotic for the treatment of gonococcal infections; however, CRO-resistant isolates have emerged in the community. Here, we examined the affinity of ß-lactam antibiotics for recombinant PBP2 in a competition assay using fluorescence-labeled penicillin. We found no or little difference in the affinities of penicillins and meropenem (MEM) for PBP2 from cefixime (CFM)-reduced-susceptible strain and cephalosporin-resistant strain. However, the affinity of cephalosporins, including CRO, for PBP2 from the cephalosporin-resistant strain was markedly lower than that for PBP2 from the CFM-reduced-susceptible-resistant strain. Notably, piperacillin (PIP) showed almost the same affinity for PBP2 from penicillin-susceptible, CFM-reduced-susceptible, and cephalosporin (including CRO)-resistant strains. Thus, PIP/tazobactam and MEM are candidate antibiotics for the treatment of CRO-resistant/multidrug-resistant N. gonorrhoeae.

Human pathogenic bacteria on high-touch dry surfaces can be controlled by warming to human-skin temperature under moderate humidity.

Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p<0.05). Experimentally, warming to human-skin temperature (37°C with 90% humidity) for 18~72h significantly suppressed the survival of pathogenic bacteria on dry surfaces, such as plastic surfaces [p<0.05 vs. 15°C (Escherichia coli DH5α, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and blaNDM-5 E. coli)] or handrails [p<0.05 vs. 15~25°C (E. coli DH5α, S. aureus, P. aeruginosa, A. baumannii)], under moderate 55% humidity. Furthermore, intermittent heating to human-skin temperature reduced the survival of spore-forming bacteria (Bacillus subtilis) (p<0.01 vs. continuous heating to human-skin temperature). NhaA, an Na+/H+ antiporter, was found to regulate the survival of bacteria on dry surfaces, and the inhibitor 2-aminoperimidine enhanced the effect of warming at human-skin temperature on the survival of pathogenic bacteria (E. coli DH5α, S. aureus, A. baumannii) on dry surfaces. Thus, warming to human-skin temperature under moderate humidity is a useful method for impairing live pathogenic bacteria on high-touch surfaces, thereby helping to prevent the spread of healthcare-associated infections.

Serotype replacement and an increase in non-encapsulated isolates among community-acquired infections of Streptococcus pneumoniae during post-vaccine era in Japan.

Objectives: It is feared that the serotype replacement of Streptococcus pneumoniae occurred by the introduction of pneumococcal vaccines as periodical inoculation leads to reduced efficacy of the approved vaccines and altered antimicrobial susceptibility. Methods: We determined serotypes of 351 S. pneumoniae isolates collected at a commercial clinical laboratory in Hokkaido prefecture, Japan, from December 2018 to February 2019 by using the polymerase chain reaction procedure of the US Centers for Disease Control and Prevention. Antimicrobial susceptibility and resistance gene profiles were also examined. Results: Vaccine coverage rates were 7.9% for 13-valent conjugate vaccine, and 32.5% for 23-valent polysaccharide vaccine, respectively. Non-typable strains were 19.7%. cpsA-positive isolates (group I), and null capsule clade (NCC)1, NCC2 and NCC3 (group II) comprised 31.3%, 28.4%, 32.8%, and 7.5% of the 69 non-typable strains, respectively. No penicillin-resistant/intermediate isolates were found; however, serotypes 35B and 15A/F showed low susceptibility to ß-lactams. Only five strains (1.4%) were levofloxacin-resistant, and all were from the older persons, and three strains were serotype 35B. Conclusion: The progression of serotype replacement in non-invasive pneumococcal infections has occurred during the post-vaccine era in Japan, and non-encapsulated isolates, such as NCC, have increased. Antimicrobial susceptibility is not worsened.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.

BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

Pseudo-outbreak of Mycobacterium lentiflavum at a general hospital in Japan.

BACKGROUND: Mycobacterium lentiflavum is a slow-growing nontuberculous mycobacterium that is widely distributed in soil and water systems, but it is sometimes pathogenic to humans. Although cases of M. lentiflavum infections are rare, 22 isolates of M. lentiflavum were identified at a single hospital in Japan. We suspected a nosocomial outbreak; thus, we conducted transmission pattern and genotype analyses. METHODS: Cases of M. lentiflavum isolated at Kushiro City General Hospital in Japan between May 2020 and April 2021 were analyzed. The patient samples and environmental culture specimens underwent whole-genome sequencing (WGS). Additionally, we retrospectively collected clinical data from patient medical records. RESULTS: Altogether, 22 isolates of M. lentiflavum were identified from sputum and bronchoalveolar lavage samples. Clinically, the instances with M. lentiflavum isolates were considered contaminants. In the WGS analysis, 19 specimens, including 18 patient samples and 1 environmental culture from the hospital's faucet, showed genetic similarity. The frequency of M. lentiflavum isolation decreased after we prohibited the use of taps where M. lentiflavum was isolated. CONCLUSIONS: WGS analysis identified that the cause of M. lentiflavum pseudo-outbreak was the water used for patient examinations, including bronchoscopy.

Discovery of Biologically Optimized Polymyxin Derivatives Facilitated by Peptide Scanning and In Situ Screening Chemistry.

Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically identified by peptide scanning methodologies, there is no practical method for optimization at the "scanned" position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the "scanning and direct derivatization" strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure-activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.

Synthesis of macrocyclic nucleoside antibacterials and their interactions with MraY.

The development of new antibacterial drugs with different mechanisms of action is urgently needed to address antimicrobial resistance. MraY is an essential membrane enzyme required for bacterial cell wall synthesis. Sphaerimicins are naturally occurring macrocyclic nucleoside inhibitors of MraY and are considered a promising target in antibacterial discovery. However, developing sphaerimicins as antibacterials has been challenging due to their complex macrocyclic structures. In this study, we construct their characteristic macrocyclic skeleton via two key reactions. Having then determined the structure of a sphaerimicin analogue bound to MraY, we use a structure-guided approach to design simplified sphaerimicin analogues. These analogues retain potency against MraY and exhibit potent antibacterial activity against Gram-positive bacteria, including clinically isolated drug resistant strains of S. aureus and E. faecium. Our study combines synthetic chemistry, structural biology, and microbiology to provide a platform for the development of MraY inhibitors as antibacterials against drug-resistant bacteria.

Chromosomal coharboring of blaIMP-60 and mcr-9 in Enterobacter asburiae isolated from a Japanese woman with empyema: a case report.

BACKGROUND: Polymyxin E (colistin) is a last-resort antibiotic to treat infections caused by carbapenemase-producing Enterobacteriaceae (CPE). However, reports of CPEs resistant to colistin have been increasing, and the mcr genes are emerging as resistance mechanisms. Among them, plasmid-mediate mcr-9 is known to be associated with colistin resistance, whereas reports on chromosomal mcr-9 and its association with colistin resistance in humans are few. CASE PRESENTATION: We identified Enterobacter asburiae harboring mcr-9 and blaIMP-60 in the pleural fluid of a patient with empyema. The long-read sequencing technique revealed that these genes were located on its chromosome. Despite the lack of exposure to colistin, the organism showed microcolonies in the inhibition circle in the E-test and disk diffusion test. Antibiotic susceptibility testing by broth microdilution confirmed its resistance to colistin. CONCLUSION: Our case report showed that mcr-9 can be present not only on plasmids but also on the chromosome in E. asburiae, and that the presence of mcr-9 on its chromosome may influence its susceptibility to colistin.

Colistin Susceptibility in Companion Animal-Derived Escherichia coli, Klebsiella spp., and Enterobacter spp. in Japan: Frequent Isolation of Colistin-Resistant Enterobacter cloacae Complex.

Transmission of colistin-resistant Enterobacterales from companion animals to humans poses a clinical risk as colistin is a last-line antimicrobial agent for treatment of multidrug-resistant Gram-negative bacteria including Enterobacterales. In this study, we investigated the colistin susceptibility of 285 Enterobacterales (including 140 Escherichia coli, 86 Klebsiella spp., and 59 Enterobacter spp.) isolated from companion animals in Japan. We further characterized colistin-resistant isolates by multilocus sequence typing (MLST), phylogenetic analysis of hsp60 sequences, and population analysis profiling, to evaluate the potential clinical risk of companion animal-derived colistin-resistant Enterobacterales to humans in line with the One Health approach. All E. coli isolates were susceptible to colistin, and only one Klebsiella spp. isolate (1.2%, 1/86 isolates) was colistin resistant. Enterobacter spp. isolates were frequently colistin resistant (20.3%, 12/59 isolates). In colistin-resistant Enterobacter spp., all except one isolate exhibited colistin heteroresistance by population analysis profiling. These colistin-heteroresistant isolates belonged to clusters I, II, IV, VIII, and XII based on hsp60 phylogeny. MLST analysis revealed that 12 colistin-resistant Enterobacter spp. belonged to the Enterobacter cloacae complex; five Enterobacter kobei (four ST591 and one ST1577), three Enterobacter asburiae (one ST562 and two ST1578), two Enterobacter roggenkampii (ST606 and ST1576), and Enterobacter hormaechei (ST1579) and E. cloacae (ST765) (each one strain). Forty-two percent of the colistin-resistant E. cloacae complex isolates (predominantly ST562 and ST591) belonged to lineages with human clinical isolates. Four E. kobei ST591 isolates were resistant to third-generation cephalosporines, aminoglycosides, and fluroquinolones but remained susceptible to carbapenems. In conclusion, our study is the first to our knowledge to report the frequent isolation of the colistin-resistant E. cloacae complex from companion animals. Furthermore, a subset of isolates belonged to human-associated lineages with resistance to multiple classes of antibiotics. These data warrant monitoring carriage of the colistin-resistant E. cloacae complex in companion animals as part of a domestic infection control procedure in line with the One Health approach.

Solid-Phase Total Synthesis of Plusbacin A3.

The total synthesis of the depsipeptide natural product plusbacin A3 (1) utilizing solid-phase peptide synthesis (SPPS) was disclosed. A 3-hydroxy-proline derivative compatible with Fmoc SPPS was prepared by a diastereoselective Joullié-Ugi three-component reaction (JU-3CR)/hydrolysis sequence. After peptide elongation on the solid support, cleavage of the peptide from the resin, followed by macrolactamization and global deprotection, gave plusbacin A3 (1).

A hydroxypropyl methylcellulose plaque assay for human respiratory syncytial virus.

Quantifying proliferative virus particles is one of the most important experimental procedures in virology. Compared with classical overlay materials, newly developed cellulose derivatives enable a plaque-forming assay to produce countable clear plaques easily. HEp-2 cells are widely used in plaque assays for human respiratory syncytial virus (RSV). It is crucial to use an overlay material to keep HEp-2 cell proliferation and prevent RSV particles from spreading over the fluid. Among four cellulose derivatives, carboxymethyl cellulose sodium salt (CMC), hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), and hydroxyethyl cellulose (HEC), we found that HPMC was the optimal overlay material because HPMC maintained HEp-2 cell proliferation and RSV infectivity. Although MCC was unsuitable for RSV, it assisted the plaque-forming by human metapneumovirus in TMPRSS2-expressing cells. Therefore, depending on the cells and viruses, it is necessary to use different overlay materials at varying concentrations.

Establishment of a reference panel of Helicobacter pylori strains for antimicrobial susceptibility testing.

BACKGROUND: Eradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing. METHOD: A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined. RESULTS: Three H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella-based horse-serum-containing agar medium were as follows: JSHR3 (CLR 16 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), JSHR6 (CLR 0.016 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), and JSHR31 (CLR 16 µg/ml, AMX 1 µg/ml and MNZ 64 µg/ml). CONCLUSIONS: A reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic-susceptible, JSHR3, which was CLR-resistant, and JSHR31, which was multi-resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.

Reduction of susceptibility to azoles and 5-fluorocytosine and growth acceleration in Candida albicans in glucosuria.

Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia and glucosuria, and is a risk factor for Candida infections. To reveal the potential effects of glucosuria on Candida spp., we investigated their growth and antifungal susceptibilities in normal human urine to which glucose was added. The viable cell numbers of Candida spp. were more than 10 fold higher in the urine added 3000 mg/dL glucose than in plain urine. In antifungal susceptibility, more than 80% of Candida albicans clinical isolates increased minimum inhibitory concentrations of azoles and 5-fluorocytosine with the addition of glucose, and exceeded their breakpoints. In most of the C. albicans clinical isolates, the mRNA expression of the azole resistance genes ERG11, CDR1, CDR2, and MDR1 in the presence of glucose in urine. These observations provide valuable information about the clinical course and therapeutic effects of azoles against C. albicans infections in patients with diabetes mellitus and hyperglucosuria.

Isolation of Human Lineage, Fluoroquinolone-Resistant and Extended-ß-Lactamase-Producing Escherichia coli Isolates from Companion Animals in Japan.

An increase in human and veterinary fluoroquinolone-resistant Escherichia coli is a global concern. In this study, we isolated fluoroquinolone-resistant E. coli isolates from companion animals and characterized them using molecular epidemiological analysis, multiplex polymerase chain reaction to detect E. coli ST131 and CTX-M type extended-spectrum ß-lactamases (ESBL), and multi-locus sequence typing analysis. Using plain-CHROMagar ECC, 101 E. coli isolates were isolated from 34 rectal swabs of dogs and cats. The prevalence of resistance to fluoroquinolone and cefotaxime was 27.7% and 24.8%, respectively. The prevalence of fluoroquinolone-resistant isolates (89.3%) was higher when CHROMagar ECC with CHROMagar ESBL supplement was used for E. coli isolation. The prevalence of cefotaxime resistance was also higher (76.1%) when 1 mg/L of ciprofloxacin-containing CHROMagar ECC was used for isolation. The cefotaxime-resistant isolates possessed CTX-M type ß-lactamase genes (CTX-M-14, CTX-M-15, or CTX-M-27). Seventy-five percent of fluoroquinolone-resistant isolates were sequence types ST131, ST10, ST1193, ST38, or ST648, which are associated with extensive spread in human clinical settings. In addition, we isolated three common fluoroquinolone-resistant E. coli lineages (ST131 clade C1-M-27, C1-nM27 and ST2380) from dogs and their respective owners. These observations suggest that companion animals can harbor fluoroquinolone-resistant and/or ESBL-producing E. coli, in their rectums, and that transmission of these isolates to their owners can occur.

[Bacteriological analysis of therapeutic important antimicrobial resistance].

Antimicrobial resistance in bacterial infections is a major concern for clinical settings. In recent years, the number of Extended-spectrum ß-lactamase producing (ESBL)- and fluoroquinolones (FQ)-resistant Escherichia coli has been increasing in Japan, especially against third-generation cephalosporins and FQs, which are frequently used in medical practice. On the other hand, antimicrobial agents such as tazobactam-piperacillin, colistin, and tigecycline, which are not general-purpose agents but last-line drugs for multidrug-resistant bacteria, are also important. Enterobacteriaceae that are resistant to these antimicrobials have been reported, although the isolation rate of resistant bacteria is lower than that of frequent used antimicrobial resistance. The author has been studying antimicrobial drug resistance and multidrug resistance of bacteria isolated from clinical settings. In particular, bacteriological analysis of antimicrobial resistance, which is important for treatment, has been conducted mainly on E. coli isolated from clinical specimens at medical facilities in Sapporo City. In this article, the author describes the findings obtained so far.

Clonal/subclonal changes and accumulation of CTX-M-type ß-lactamase genes in fluoroquinolone-resistant Escherichia coli ST131 and ST1193 strains isolated during the past 12 years, Japan.

OBJECTIVES: Fluoroquinolone (FQ)- and third-generation cephalosporin-resistant Escherichia coli are increasing in Japan. In the early 2000s, the FQ-resistant E. coli clone ST131 increased in clinical settings worldwide. It frequently produces extended-spectrum ß-lactamases (ESBLs) such as CTX-M. This study aimed to explore the characteristics of FQ-resistant E. coli isolated in Japan during 2008-2009 and 2020. METHODS: We compared FQ-resistant E. coli clinical isolates from urine samples collected in 2020 (151 isolates) with a FQ-resistant E. coli collection isolated in 2008-2009 (42 isolates). Identification of E. coli ST131 clades and blaCTX-M were determined by multiplex PCR. Sequence types of non-ST131 isolates were determined by whole-genome sequencing. RESULTS: Although the prevalence of ST131 was comparable in 2020 (74.2%) and 2008-2009 (78.6%), the subclades differed during the two time periods (C1-nM27: 40.2% in 2008-2009 vs. 78.8% in 2020; C1-M27: 32.1% in 2008-2009 vs. 9.1% in 2020). The incidence of blaCTX-M among ST131 isolates increased from 27.3% in 2008-2009 to 64.3% in 2020. blaCTX-M was found in 80.6% and 93.8% of C1-M27 and C2 in 2020, respectively, and blaCTX-M possession in C1-nM27 increased from 19.2% in 2008-2009 to 40% in 2020. FQ-resistant ST1193 was detected only in 2020 (17.9% of 151 isolates, of which 14.8% possessed blaCTX-M). CONCLUSION: Increased resistance of E. coli to FQs and third-generation cephalosporins in Japan can be attributed to the accumulation of blaCTX-M in C1-nM27 and the increase of C1-M27 and C2 clades with high blaCTX-M possession, alongside the spread of ST1193.