Antimicrobial Resistance and Type III Secretion System Virulotypes of Pseudomonas aeruginosa Isolates from Dogs and Cats in Primary Veterinary Hospitals in Japan: Identification of the International High-Risk Clone Sequence Type 235.

This study aimed to investigate the current trends in antimicrobial resistance among Pseudomonas aeruginosa clinical isolates of canine and feline origin and the prevalence of their sequence types (STs) and type III secretion system (T3SS) virulotypes, which remains unknown in Japan. A total of 240 nonduplicate clinical isolates of P. aeruginosa from dogs (n = 206) and cats (n = 34) collected from 152 primary care animal hospitals between August 2017 and October 2019 were examined. PCR detection of T3SS genes (exoU and exoS) and carbapenemase genes, multilocus sequence typing, and whole-genome sequencing of the representative carbapenem-resistant isolates were performed. Resistance rates to imipenem and meropenem were 6.67% and 2.08%, respectively. A high resistance rate (17.92%) was encountered with ciprofloxacin. The exoU-/exoS+ was the predominant T3SS virulotype (195 isolates, 81.3%), followed by exoU+/exoS- (35 isolates, 14.6%), exoU-/exoS- (7 isolates, 2.9%), and exoU+/exoS+ (3 isolates, 1.3%). A high frequency of the high-risk clones ST235 and clonal complex 235 (CC 235) (28.9%), followed by ST357 (21.1%), were noted among these 38 exoU+ isolates. Seventeen carbapenem-resistant isolates comprising 2 exoU+ isolates, including an ST235 isolate, and 15 exoU-/exoS+ isolates belonging to non-ST235/CC235 were detected, of which all were carbapenemase negative. Different combinations of mutations among oprD, efflux pump regulatory genes, and AmpC ß-lactamase regulatory genes were identified among representative isolates with high-level resistance to imipenem. This study emphasizes the occurrence of ST235 isolates among companion animals, which may represent a threat to public health because of the ability of this clone to acquire and spread resistance elements, including carbapenemase genes. IMPORTANCE Pseudomonas aeruginosa is an environmentally ubiquitous and important opportunistic human pathogen responsible for life-threatening health care-associated infections. Because of its extensive repertoire of virulence determinants and intrinsic and acquired resistance mechanisms, the organism could be one of the most clinically and epidemiologically important causes of morbidity and mortality. In recent years, worldwide spreading of multidrug-resistant high-risk clones, particularly sequence type 235 (ST235), has become a serious public health threat. Companion animals which share much of their living environment with humans could be important reservoirs and spreaders of antimicrobial-resistant bacteria and resistance genes of clinical importance in humans, such as extended-spectrum ß-lactamase-producing Enterobacterales and methicillin-resistant Staphylococcus aureus. However, antimicrobial resistance, virulence, and genotyping of P. aeruginosa in companion animals remain largely unknown. This work sheds light on the potential spread of high-risk clones in companion animals.

Presence of Colistin- and Tigecycline-Resistant Klebsiella pneumoniae ST29 in Municipal Wastewater Influents in Japan.

The aim of this study was to investigate the presence of colistin- and/or tigecycline-resistant Klebsiella spp. in influents from four wastewater treatment plants (WWTPs), which partly reflect the gut microbiome of human populations. Colistin- and tigecycline-resistant Klebsiella pneumoniae isolates (K30/ST29) were detected four times from the WWTP A during a period of 3 months. Disruptions of the mgrB and ramR genes by ISEc68 and ISKpn21, respectively, were identified in those four isolates. They also shared the IncL/M 86,197-bp plasmids carrying a blaCTX-M-3 and Tn1548-associated armA [IS26-IntI1-dfrA12-gucF-aadA2-qacEΔ1-sul1-ISCR1-ISEc28-armA-ISEc29-msr(E)-mph(E)-IS26]. Those isolates formed a distinct cluster within wgMLST clusters of ST29 K30 public reference strains of human origin and were unique due to harboring of Tn21-like mercury resistance operon transposons in addition to silver, copper, and arsenic resistance determinants. Five K. pneumoniae strains with different STs and 1 Klebsiella quasipneumoniae strain, exhibiting colistin resistance, were detected in WWTPs B, C, and D. For these isolates, disruptions of mgrB by ISEc68 (three isolates) or ISEcl1 (one isolate), insertion of IS2 in the mgrB promoter region (one isolate), and inactivation of MgrB by a nonsense mutation (one isolate) were identified. Close monitoring of these mcr-negative colistin- and/or tigecycline-resistant bacteria in wastewater influents is imperative to avoid further limiting of treatment options.

Genomic characterisation and epidemiology of nosocomial Serratia marcescens isolates resistant to ceftazidime and their plasmids mediating rare blaTEM-61.

OBJECTIVES: We determined the whole DNA sequences of plasmids carrying a rare extended-spectrum ß-lactamase gene (blaTEM-61) to precisely understand the spread of resistance among nosocomial Serratia marcescens populations. METHODS: Twenty non-duplicate ceftazidime-resistant S. marcescens nosocomial isolates (ceftazidime MICs, 32 to >128 mg/L) collected over 1 year were pulsotyped and nucleotide sequences of the blaTEM-61 gene and its promoter region were determined. Twelve representative isolates were analysed by whole-genome sequencing. RESULTS: The 20 isolates comprised two distinct pulsotypes: I (14 isolates) and II (6 isolates). They all contained the blaTEM-61 gene. A polymorphism in the repeat number of a 15-nucleotide sequence (5'-ATGTCATGATAATAA-3') was found in the promoter region of blaTEM-61; two, three and four repeat units were found in 6, 12 and 2 isolates, respectively. Single nucleotide polymorphism (SNP)-based phylogenetic analysis of 12 isolates revealed that 7 isolates of pulsotype I (12-44 SNP differences) and 5 isolates of pulsotype II (15-55 SNP differences) formed two distinct clusters of genotypes 1 and 2, respectively. All 12 isolates harboured a plasmid carrying the Tn1-blaTEM-61 element, although they were slightly different in size (78 883 bp, 78 898 bp and 78 913 bp) owing to differences in the number of 15-bp repetitive sequences. A 42 542-bp broad-host-range plasmid carrying the Tn1-blaTEM-61 element was also found in one of the isolates. CONCLUSIONS: We characterised a plasmid-encoded novel Tn1-blaTEM-61 element and transposon-dependent mechanisms underlying the propagation of antibiotic resistance, together with repeated new polymorphic 15-bp units in the promoter of blaTEM-61.