Temporal dynamics of extended-spectrum ß-lactamase-producing Escherichia coli and carbapenemase-producing Gram-negative bacteria in hospital wastewater.

Hospital wastewater is a reservoir for the environmental spread of clinically relevant antimicrobial-resistant bacteria and resistance genes. The aim of this study was to quantify total Escherichia coli, extended-spectrum ß-lactamase (ESBL)-producing E. coli, and carbapenemase-producing organisms (CPOs) and perform whole-genome sequencing-based characterization of these bacterial isolates in hospital wastewater samples collected bimonthly in Japan from January to November 2021. Total E. coli counts were 8.1 × 103-8.8 × 104 colony-forming units (CFU)/mL. ESBL-producing E. coli were detected in the sampling months of January, March, May, and July, with the ratio of ESBL-producing E. coli to total E. coli being remarkably highest (95 %) in July. In contrast, DHA-1 Ambler class C ß-lactamase (AmpC)-producing E. coli was detected in September and November, accounting for 28 % and 3 % of total E. coli counts, respectively. All 140 ESBL-producing E. coli isolates harbored the blaCTX-M genes, with blaCTX-M-14 being the most common genotype (94.3 %), the vast majority of which were associated with the human virulent B2-O25b: H4-ST131-fimH30R/non-Rx. In September, E. coli clade I-O8:H33-ST3910-fimH1074 was primarily associated with blaDHA-1. Among 26 representative CPO isolates, Aeromonas caviae (34.6 %) and A. hydrophila subsp. hydrophila (30.8 %) were dominant. The most frequently detected carbapenemase gene was blaIMP-1 (57.7 %), followed by blaGES-24 (34.6 %) and blaGES-4 (7.7 %). Estimated bacterial counts of CPOs ranged from 4.0 × 10-1 to 4.7 × 103 CFU/mL over the six sampling months. blaIMP-1-positive A. hydrophila subsp. hydrophila ST860, which was repeatedly detected over the five sampling months, accounted for the highest total number of this bacterial clone (79 %). Overall, this study provides insights into the overwhelming presence and persistence of E. coli B2-O25b:H4-ST131-H30R/non-Rx with blaCTX-M-14 and Aeromonas spp. with blaIMP-1 in hospital wastewater, and the change in the dynamics of resistance gene prevalence from blaCTX-M-positive E. coli to blaDHA-1-positive E. coli.

Municipal wastewater monitoring revealed the predominance of bla GES genes with diverse variants among carbapenemase-producing organisms: high occurrence and persistence of Aeromonas caviae harboring the new bla GES variant bla GES-48.

IMPORTANCE: The emergence and spread of carbapenemase-producing organisms (CPOs) represent a global health threat because they are associated with limited treatment options and poor clinical outcomes. Wastewater is considered a hotspot for the evolution and dissemination of antimicrobial resistance. Thus, analyses of municipal wastewater are critical for understanding the circulation of these CPOs and carbapenemase genes in local communities, which remains scarcely known in Japan. This study resulted in several key observations: (i) the vast majority of bla GES genes, including six new bla GES variants, and less frequent bla IMP genes were carbapenemase genes encountered exclusively in wastewater influent; (ii) the most dominant CPO species were Aeromonas spp., in which a remarkable diversity of new sequence types was observed; and (iii) CPOs were detected from combined sewer wastewater, but not from separate sewer wastewater, suggesting that the load of CPOs from unrecognized environmental sources could greatly contribute to their detection in influent wastewater.

Zoonotic potential and antimicrobial resistance of Escherichia spp. in urban crows in Japan-first detection of E. marmotae and E. ruysiae.

Little is known about the prevalence of antimicrobial-resistant bacteria and pathogenic Escherichia coli in crows (carrion and jungle crows). We studied the phylogeny, virulence and antimicrobial resistance gene profiles of crow E. coli isolates to investigate their zoonotic potential and molecular epidemiology. During the winter of 2021-2022, 34 putative E. coli isolates were recovered from 27 of the 65 fresh fecal samples collected in urban areas. Three strains of the B1-O88:H8-ST446-fimH54 lineage, classified as extraintestinal pathogenic E. coli (ExPEC) and necrotoxigenic E. coli type 2, were colistin-resistant and harbored mcr-1.1-carrying IncI2 plasmids. The blaCTX-M-55 was identified in a multidrug-resistant B1-O non-typeable:H23-ST224-fimH39 strain. In phylogroup B2, two lineages of O6:H1-ST73-fimH30 and O6:H5-ST83-fimH21 were classified as ExPEC, uropathogenic E. coli, and necrotoxigenic E. coli type 1 (O6:H5-ST83-fimH21), and contained several virulence genes associated with avian pathogenic E. coli. Noteworthy is that three isolates, identified as E. coli by MALDI-TOF MS, were confirmed to be two Escherichia marmotae (cryptic clade V) and one Escherichia ruysiae (cryptic clade III) based on ANI and dDDH analyses. Our results provide the first evidence of these new species in crows. E. marmotae and E. ruysiae isolates in this study were classified as ExPEC and contained the enteroaggregative E. coli heat-stable toxin 1 gene. In addition, these two E. marmotae isolates displayed a close genetic relationship with human isolates associated with septicemia. This study provides the first insights into the prevalence and zoonotic significance of Escherichia spp. in urban crows in Japan, posing a significant risk for their transmission to humans.

Genomic Traits Associated with Virulence and Antimicrobial Resistance of Invasive Group B Streptococcus Isolates with Reduced Penicillin Susceptibility from Elderly Adults.

This study aimed to investigate genomic traits underlying the antimicrobial resistance and virulence of multidrug-resistant (MDR) group B streptococci with reduced penicillin susceptibility (PRGBS) recovered from elderly patients with bloodstream infections, which remain poorly characterized. The pangenome was found to be open, with the predicted pan- and core genome sizes being 3,531 and 1,694 genes, respectively. Accessory and unique genes were enriched for the Clusters of Orthologous Groups (COG) categories L, Replication, recombination, and repair, and K, Transcription. All MDR PRGBS isolates retained a core virulence gene repertoire (bibA, fbsA/-B/-C, cspA, cfb, hylB, scpB, lmb, and the cyl operon), supporting an invasive ability similar to that of the other invasive GBS, penicillin-susceptible GBS (PSGBS), and noninvasive PRGBS isolates. The putative sequence type 1 (ST1)-specific AlpST-1 virulence gene was also retained among the serotype Ia/ST1 PRGBS isolates. In addition to tet(M) and erm(B), mef(A)-msr(D) elements or the high-level gentamicin resistance gene aac(6')-aph(2″), which are both rare in PSGBS, were detected among those MDR PRGBS isolates. In the core single-nucleotide polymorphism (SNP) phylogenetic tree, all invasive ST1 PRGBS isolates with serotypes Ia and III were placed together in a clade with a recombination rate of 3.97, which was 36 times higher than the value found for a clade formed by serotype V/ST1 PSGBS isolates derived mostly from human blood. ST1 has been the predominant sequence type among the PRGBS isolates in Japan, and serotypes Ia and III have been very rare among the ST1 PSGBS isolates. Thus, these lineages that mostly consisted of serotypes Ia/ST1 and III/ST1 PRGBS could possibly emerge through recombination within the ST1 populations. IMPORTANCE Streptococcus agalactiae, or group B Streptococcus (GBS), is recognized as the leading cause of neonatal invasive infections. However, an increasing incidence of invasive GBS infections among nonpregnant adults, particularly the elderly and those with underlying diseases, has been observed. There is a trend toward the increasing occurrence of penicillin nonsusceptibility among GBS clinical isolates, from 4.8% in 2008 to 5.8% in 2020 in Japan. Also, in the United States, the frequency of adult invasive GBS isolates suggestive of ß-lactam nonsusceptibility increased from 0.7% in 2015 to 1.0% in 2016. In adults, mortality has been significantly higher among patients with bacteremia than among those without bacteremia. Our study revealed that invasive GBS with reduced penicillin susceptibility (PRGBS) isolates harbor major virulence and resistance genes known among GBS, highlighting the need for large population-based genomic surveillance studies to better understand the clinical relevance of invasive PRGBS isolates.

Genomic landscape of blaGES-5- and blaGES-24-harboring Gram-negative bacteria from hospital wastewater: emergence of class 3 integron-associated blaGES-24 genes.

OBJECTIVES: This study aimed to characterize Gram negative bacteria carrying blaGES carbapenemase genes detected in wastewater from a hospital with no history of detection of clinical isolates producing GES carbapenemases. METHODS: Six hospital effluent samples were screened for carbapenemase-producing organisms (CPO) using CHROMagar mSuperCARBA and MacConkey agar with 1 µg/mL imipenem. Polymerase chain reaction (PCR) amplification and sequencing of carbapenemase genes, multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. RESULTS: Among 21 CPO isolates, 11 Klebsiella spp. and 5 Enterobacter kobei isolates carried blaGES-24, and 4 E. roggenkampii and 1 Pseudomonas aeruginosa isolates carried blaGES-5. Genomic analysis of 8 representative isolates comprising 6 blaGES-24-positive and 2 blaGES-5-positive revealed that class 3 integrons with complete or defective Tn402-like transposition modules were predominantly associated with two tandem copies of blaGES-24. Furthermore, a total of 5 new class 3 integrons, In3-18 to In3-22, were identified among 5 blaGES-24 and 1 blaGES-5 plasmids. One strain each of K. pneumoniae subsp. pneumoniae and K. quasipneumoniae subsp. similipneumoniae harboring blaGES-24 plasmids also carried a rare blaVEB-1-positive class 1 integron on a non-typeable plasmid, where these blaVEB-1 plasmids had high sequence similarity. Virulence gene profiles differed between Klebsiella spp. and Enterobacter spp.; the former harbored type III fimbriae cluster, salmochelin, and T6SS type i2 gene clusters, while the latter had curli pili operon, aerobactin, T2SS gene clusters, and T6SS type i3 gene clusters. CONCLUSION: Our findings confirmed the linkage of blaGES-24 with rare Tn402-like class 3 integrons and the structural diversity of their gene cassette arrays.

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.

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.