Genomic epidemiology and ceftazidime-avibactam high-level resistance mechanisms of Pseudomonas aeruginosa in China from 2010 to 2022.

Ceftazidime-avibactam (CZA) resistance is a huge threat in the clinic; however, the underlying mechanism responsible for high-level CZA resistance in Pseudomonas aeruginosa (PA) isolates remains unknown. In this study, a total of 5,763 P. aeruginosa isolates were collected from 2010 to 2022 to investigate the ceftazidime-avibactam (CZA) high-level resistance mechanisms of Pseudomonas aeruginosa (PA) isolates in China. Fifty-six PER-producing isolates were identified, including 50 isolates carrying blaPER-1 in PA, and 6 isolates carrying blaPER-4. Of these, 82.1% (46/56) were classified as DTR-PA isolates, and 76.79% (43/56) were resistant to CZA. Importantly, blaPER-1 and blaPER-4 overexpression led to 16-fold and >1024-fold increases in the MICs of CZA, respectively. WGS revealed that the blaPER-1 gene was located in two different transferable IncP-2-type plasmids and chromosomes, whereas blaPER-4 was found only on chromosomes and was carried by a class 1 integron embedded in a Tn6485-like transposon. Overexpression of efflux pumps may be associated with high-level CZA resistance in blaPER-1-positive strains. Kinetic parameter analysis revealed that PER-4 exhibited a similar kcat/Km with ceftazidime and a high (∼3359-fold) IC50 value with avibactam compared to PER-1. Our study found that overexpression of PER-1 combined with enhanced efflux pump expression and the low affinity of PER-4 for avibactam contributes to high-level resistance to CZA. Additionally, the Tn6485-like transposon plays a significant role in disseminating blaPER. Urgent active surveillance is required to prevent the further spread of high-level CZA resistance in DTR-PA isolates.

Overexpression of blaGES-1 due to a strong promoter in the class 1 integron contributes to decreased ceftazidime-avibactam susceptibility in carbapenem-resistant Pseudomonas aeruginosa ST235.

Sequence type 235 (ST235) Pseudomonas aeruginosa, harboring so-called international, high-risk, or widespread clones, is associated with relatively high morbidity and mortality, partly due to multiantibiotic and high-level antibiotic resistance. Treatment of infections caused by such strains with ceftazidime-avibactam (CZA) is often successful. However, CZA resistance in carbapenem-resistant P. aeruginosa (CRPA) strains has been consistently reported with the increasing use of this drug. Likewise, we identified thirty-seven CZA-resistant ST235 P. aeruginosa strains from among 872 CRPA isolates. A total of 10.8% of the ST235 CRPA strains were resistant to CZA. Site-directed mutagenesis, cloning, expression, and whole-genome sequencing analysis revealed that overexpression of blaGES-1, which was carried in a class 1 integron of the complex transposon Tn6584, occurred due to a strong promoter, contributing to CZA resistance. Moreover, such overexpression of blaGES-1 combined with an efflux pump resulted in high-level resistance to CZA, considerably reducing the therapeutic options available for treating infections caused by ST235 CRPA. Considering the widespread presence of ST235 P. aeruginosa strains, clinicians should be aware of the risk of CZA resistance development in high-risk ST235 P. aeruginosa. Surveillance initiatives for preventing further dissemination of high-risk ST235 CRPA isolates with CZA resistance are essential.

Feeling good and doing more: How does the customer respond to pro-customer deviance in the context of hospitality.

Guided by the service-dominant logic, hospitality employees have to occasionally engage in pro-customer deviance to offer customized service. While pro-customer deviance has been linked with several customer attitudinal outcomes, the different customers' emotional and behavioral responses have not yet been clarified. This study explored customers' responses toward customer-contact employees and enterprises. In addition, to investigate the emotional and cognitive mechanisms underlying those response processes, this study introduced gratitude toward employee and customer-company identification as mediators in the relationship between pro-customer deviance and a series of customer extra-role behaviors. A multisource field study was conducted to test a two-stage structural equation model. The results showed that pro-customer deviance is positively related to customers' positive feedback and service friendship toward employees via gratitude. Also, the customer-company identification is found to play a mediation role between pro-customer deviance and customers' advocacy and prohibitive voice toward an organization. Theoretical and managerial contributions are also discussed at the end.

Transmission of ST45 and ST2407 extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in neonatal intensive care units, associated with contaminated environments.

OBJECTIVES: Given the increasing frequency of infections due to extended-spectrum ß-lactamase (EBSL)-producing Klebsiella pneumoniae in humans over recent decades, infection control against this pathogen is of high importance. METHODS: In this study, the transmission mode of ESBL-producing K. pneumoniae in neonatal intensive care units (NICU) was investigated. We collected K. pneumoniae isolates from patients admitted to the NICU and performed environmental screening of the NICU and nearby obstetrics department. All isolates were analysed using antimicrobial susceptibility testing, whole-genome sequencing, molecular typing, and antimicrobial and virulence determinant screening. The phylogenetic relationships of all the isolates were analysed using core-genome multi-locus sequence type and single-nucleotide polymorphism-based analysis, and their plasmids harbouring antimicrobial resistance genes in ST2407 were compared. RESULTS: Eighteen K. pneumoniae isolates were collected, of which 10 isolates from patients belonged to ST45 and ST2407, and eight isolates from the environment belonged to various other clones. Although 80% and 100% of isolates from patients were ESBL-positive (blaCTX-M-14 and blaCTX-M-55) and possessed siderophores, respectively; fewer environmental isolates harboured antimicrobial resistance and virulence genes. For both ST45 and ST2407 isolates, the phylogenetic assessment revealed a close relationship between clinical and environmental isolates, indicating that bloodstream infections were associated with the contaminated environments. CONCLUSIONS: Based on these results, the environmental prevalence of K. pneumoniae should be considered given its pathogenicity in humans. Early and active infection control measures could decrease the spread of multidrug-resistant K. pneumoniae.

Alcaligenes faecalis metallo-ß-lactamase in extensively drug-resistant Pseudomonas aeruginosa isolates.

OBJECTIVES: To characterize Alcaligenes faecalis metallo-ß-lactamase (MBL) AFM-2 and AFM-3 from clinical Pseudomonas aeruginosa isolates NDTH10366, NDTH9845 and WTJH17. METHODS: Clinical isolates were whole-genome sequenced using the Illumina and Oxford Nanopore platforms. MICs of clinical isolates and transformants containing MBL genes were determined using broth microdilution methods. Kinetic parameters of purified AFM and NDM-1 were measured using a spectrophotometer. The AFM structure was modelled with SWISS-MODEL. RESULTS: NDTH10366 and NDTH9845 were extensively drug-resistant (XDR) isolates carrying blaAFM-2 and multiple copies of blaKPC-2, whereas WTJH17 was an XDR isolate carrying blaAFM-3. The plasmid-borne blaAFM-2 and blaAFM-3 genes are associated with a novel ISCR element, ISCR29. AFM-2 and AFM-3, differing from AFM-1 by one amino acid substitution each, shared 86.2% and 86.6% amino acid sequence identity with NDM-1, respectively. Phylogenetic analysis confirmed the close relationship between AFM and NDM. Expression of AFM and NDM-1 under their native promoters in DH5α and PAO1 led to elevated MICs for all tested ß-lactams except aztreonam. Comparable catalytic abilities were observed for AFM and NDM-1 when hydrolysing nitrocefin, cefepime, imipenem and biapenem, whereas for other tested ß-lactams AFM displayed weaker enzymatic activities. Modelling AFM structure revealed a characteristic αß/ßα fold with two zinc-binding active sites. CONCLUSIONS: AFM from clinical P. aeruginosa isolates demonstrated ß-lactamase activity comparable to NDM-1. Co-carriage of blaAFM and blaKPC renders clinical P. aeruginosa isolates non-susceptible to all antipseudomonal ß-lactams. The association of blaAFM genes with translocatable genetic elements and plasmids highlights their concerning potential for dissemination.

Emergence of Ceftazidime- and Avibactam-Resistant Klebsiella pneumoniae Carbapenemase-Producing Pseudomonas aeruginosa in China.

Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa (KPC-PA) has been reported sporadically. However, epidemiological and antimicrobial susceptibility data specific for KPC-PA are lacking. We collected 374 carbapenem-resistant P. aeruginosa (CRPA) isolates from seven hospitals in China from June 2016 to February 2019 and identified the blaKPC-2 gene in 40.4% (n = 151/374) of the isolates. Approximately one-half of all KPC-PA isolates (n = 76/151; 50.3%) were resistant to ceftazidime-avibactam (CAZ-AVI). Combining Kraken2 taxonomy identification and Nanopore sequencing, we identified eight plasmid types, five of which carried blaKPC-2, and 13 combination patterns of these plasmid types. In addition, we identified IS26-ΔTn6296 and Tn1403-like-ΔTn6296 as the two mobile genetic elements that mediated blaKPC-2 transmission. blaKPC-2 plasmid curing in 28 strains restored CAZ-AVI susceptibility, suggesting that blaKPC-2 was the mediator of CAZ-AVI resistance. Furthermore, the blaKPC-2 copy number was found to correlate with KPC expression and, therefore, CAZ-AVI resistance. Taken together, our results suggest that KPC-PA is becoming a clinical threat and that using CAZ-AVI to treat this specific pathogen should be done with caution. IMPORTANCE Previous research has reported several cases of KPC-PA strains and three KPC-encoding P. aeruginosa plasmid types in China. However, the prevalence and clinical significance of KPC-PA are not available. In addition, the susceptibility of the strains to CAZ-AVI remains unknown. Samples in this study were collected from seven tertiary hospitals prior to CAZ-AVI clinical approval in China. Therefore, our results represent a retrospective study establishing the baseline efficacy of the novel ß-lactam/ß-lactamase combination agent for treating KPC-PA infections. The observed correlation between the blaKPC copy number and CAZ-AVI resistance suggests that close monitoring of the susceptibility of the strain during treatment is required. It would also be beneficial to screen for the blaKPC gene in CRPA strains for antimicrobial surveillance purposes.

In vitro Effect of the Combination of Aztreonam and Amoxicillin/Clavulanic Acid Against Carbapenem-Resistant Gram-Negative Organisms Producing Metallo-ß-Lactamase.

INTRODUCTION: Antibiotics for treating infectious diseases caused by carbapenem-resistant Gram-negative pathogens (CR-GNOs) are very limited in clinical practice. We aim to provide supportive evidence by revealing the combined effect of aztreonam (ATM) and amoxicillin/clavulanic acid (AMC) against GNOs with carbapenem resistance mediated by metallo-ß-lactamase (MBL). METHODS: All isolates were identified by the VITEK system and EDTA inhibitory assays. PCR followed by sequencing was conducted to confirm the genotypes of MBL and extended spectrum ß-lactamase (ESBL). Time kill assay was performed to clarify the bactericidal effect of drug combination. RESULTS: A total of 59 MBL-producing CR-GNOs (33 Enterobacteriaceae spp. isolates and 26 Pseudomonadales isolates) were identified and there found three MBL genes, namely, bla IMP, bla NDM and bla VIM, with ratios of 76.2%, 11.8% and 11.8%, respectively. The Enterobacteriaceae spp. isolates were commonly positive for the ESBL genes, including bla TEM (18 isolates), bla SHV (20 isolates) and bla CTX-M-1 (8 isolates), while the P. aeruginosa isolates were positive for bla OXA-10 (11 isolates). The checkerboard microdilution assay was used to detect combination effect of ATM and AMC, which showed synergy (97.0%) and partial synergy (3.0%) in Enterobacteriaceae spp. isolates, and partial synergy (42.3%) and indifference (34.6%) in the Pseudomonadales isolates. Four Enterobacteriaceae spp. isolates were selected for a time-kill assay, and rapid bactericidal effects were observed in the combination groups compared to the control and mono-ATM groups; these effects began in the first hour and continued to the sixth hour, yielding a 5- to 7-fold reduction in Log10 CFU/mL. DISCUSSION: The combination of ATM and AMC would be an available option to control infections caused by MBL-producing CR-GNOs, especially Enterobacteriaceae spp. isolates that coproduce ESBLs, and exhibit significant synergic effects in vitro.

Detection and characterization of a clinical Escherichia coli ST3204 strain coproducing NDM-16 and MCR-1.

OBJECTIVES: A plasmid-mediated colistin resistance gene, mcr-1, has been reported worldwide and has caused concern regarding a major therapeutic challenge. Alarmingly, mcr-1 has spread into clinical carbapenem-resistant Enterobacteriaceae isolates, resulting in extensively drug-resistant and even pan drug-resistant isolates that can cause untreatable infections. In this study, we report isolation of an extensively drug-resistant Escherichia coli strain EC1188 that coproduces NDM-16 and MCR-1 from a urine sample taken from a patient with craniocerebral injury. MATERIALS AND METHODS: E. coli strain EC1188 was identified and subjected to genotyping, susceptibility testing and conjugation experiments. The genetic locations of blaNDM-16 and mcr-1 were established with southern blot hybridization. The complete genome sequence of this strain was obtained and the genetic characteristics of the mcr-1- and blaNDM-16-harboring plasmids were analyzed. In addition, comparative genetic analyses of mcr-1 and blaNDM-16 with closely related plasmids were also carried out. RESULTS: Whole-genome sequencing revealed that strain EC1188 possess various resistance genes and virulence genes. S1-pulsed-field gel electrophoresis and southern blot suggested that the blaNDM-16 and mcr-1 genes were located on an ~65 kb plasmid and an ~80 kb plasmid, respectively. Moreover, the two genes could successfully transfer their resistance phenotype to E. coli strain C600. Sequence analysis showed that these two plasmids possessed high sequence similarity to previously reported blaNDM-5-harboring and mcr-1-harboring plasmids in China. CONCLUSION: To the best of our knowledge, this is the first report to isolate an E. coli strain that coproduces NDM-16 and MCR-1. In addition, we characterized the blaNDM-16-harboring plasmid for the first time. Our study further emphasizes that the co-occurrence of the two prevalent transferrable resistance plasmids in a single isolate is highly significant because infections caused by MCR-1-producing carbapenem-resistant Enterobacteriaceae isolates are increasing each year. It is imperative to perform active surveillance to prevent further dissemination of MCR-1-producing CRE isolates.

Fosfomycin resistance among vancomycin-resistant enterococci owing to transfer of a plasmid harbouring the fosB gene.

The presence and characterisation of plasmid-mediated fosfomycin resistance determinants were investigated among 45 clinical vancomycin-resistant enterococci (VRE) isolated in Zhejiang Province, China. In total, 19 VRE were resistant to fosfomycin, of which 18 isolates had conjugative fosfomycin resistance and were positive for fosB. No reported fos genes were detected in the remaining isolate. Among the 18 fosB-carrying isolates, the fosB gene was always flanked by tnpA, suggesting the same novel fosB transposon. In 10 of the 18 fosB-carrying isolates, the fosB and tnpA genes were found reversely inserted in the vanA transposon Tn1546. In the remaining eight isolates the fosB and vanA genes were located on different plasmids. These findings indicate that acquisition of the conjugative plasmid harbouring the novel fosB transposon (ISL3-like transposon) and the Tn1546-like transposon (containing vanA and fosB) may explain, at least in part, the recent increase in fosfomycin-resistant Enterococcus faecium in China.

Multilocus sequence typing reveals genetic diversity of carbapenem- or ceftazidime-nonsusceptible Pseudomonas aeruginosa in China.

It is unclear whether the genetic background of drug-resistant Pseudomonas aeruginosa was disseminated from a certain clone. Thus, we performed MLST (multilocus sequence typing) of 896 P. aeruginosa isolates that were nonsusceptible to imipenem, meropenem, or ceftazidime. This revealed 254 sequence types (STs), including 104 new STs and 34 STs with novel alleles. Thirty-three clonal complexes and 404 singletons were found. In conclusion, drug-resistant P. aeruginosa clones can be developed from diverse genetic backgrounds.

Epidemiological characteristics and genetic structure of blaNDM-1 in non-baumannii Acinetobacter spp. in China.

OBJECTIVES: The goal of this study was to investigate the epidemiological characteristics and the surrounding genetic structure of bla(NDM-1) in non-baumannii Acinetobacter spp. in China. METHODS: Non-baumannii Acinetobacter spp. were collected from 28 provinces in China and were screened for the presence of bla(NDM-1) using PCR. The following four methods were used to classify the Acinetobacter isolates: the Vitek 2 system, 16S-23S rRNA gene intergenic spacer sequencing, amplified rDNA restriction analysis and partial rpoB sequence analysis. An S1-PFGE assay and Southern blot hybridization were performed to determine the plasmid location of bla(NDM-1). The transferability of bla(NDM-1)-harbouring plasmids was confirmed by conjugation experiments and electrotransformation. The surrounding genetic structure of the bla(NDM-1) gene was analysed using a restriction endonuclease-based cloning approach and primer walking. RESULTS: Among 726 non-baumannii Acinetobacter spp., nine isolates collected from six different provinces and assigned to seven different Acinetobacter spp. contained the bla(NDM-1) gene. None of these isolates was directly infectious to the patients or demonstrated an epidemiological importation from abroad. These bla(NDM-1) genes were located on plasmids that could be transferred to Escherichia coli J53 by conjugation and Acinetobacter baylyi ADP1 by electrotransformation. Seven of the nine strains shared a common genetic structure in which bla(NDM-1) was flanked by two copies of ISAba125. CONCLUSIONS: The clinical challenge posed by bla(NDM-1) is currently minimal in China; however, more attention should be devoted to monitoring the dissemination of this gene due to its potential transferability via the ISAba125-associated transposon.