Emergence of aztreonam/avibactam and tigecycline-resistant Pseudomonas putida group Co-producing blaIMP-1, blaAFM-4 and blaOXA-1041 with a novel sequence type ST268 in Southwestern China.

OBJECTIVES: The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM-AVI), with a focus on their microbial and genomic characteristics. METHODS: We assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of blaIMP-1 and blaAFM-4 was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining. RESULTS: Both strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced blaIMP-1, blaAFM-4, tmexCD-toprJ, and blaOXA-1041, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the blaIMP-1 and blaAFM-4 genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors. CONCLUSION: We initially identified two P. putida ST268 strains co-producing blaIMP-1, blaAFM-4, blaOXA-1041, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.

Role of efflux pumps, their inhibitors, and regulators in colistin resistance.

Colistin is highly promising against multidrug-resistant and extensively drug-resistant bacteria clinically. Bacteria are resistant to colistin mainly through mcr and chromosome-mediated lipopolysaccharide (LPS) synthesis-related locus variation. However, the current understanding cannot fully explain the resistance mechanism in mcr-negative colistin-resistant strains. Significantly, the contribution of efflux pumps to colistin resistance remains to be clarified. This review aims to discuss the contribution of efflux pumps and their related transcriptional regulators to colistin resistance in various bacteria and the reversal effect of efflux pump inhibitors on colistin resistance. Previous studies suggested a complex regulatory relationship between the efflux pumps and their transcriptional regulators and LPS synthesis, transport, and modification. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 1-(1-naphthylmethyl)-piperazine (NMP), and Phe-Arg-ß-naphthylamide (PAßN) all achieved the reversal of colistin resistance, highlighting the role of efflux pumps in colistin resistance and their potential for adjuvant development. The contribution of the efflux pumps to colistin resistance might also be related to specific genetic backgrounds. They can participate in colistin tolerance and heterogeneous resistance to affect the treatment efficacy of colistin. These findings help understand the development of resistance in mcr-negative colistin-resistant strains.

Identification and genomic analysis of a Vibrio cholerae strain isolated from a patient with bloodstream infection.

Vibrio cholerae is a bacterium ubiquitous in aquatic environments which can cause widespread infection worldwide. V. cholerae gradually became a rare species of bacteria in clinical microbiology laboratories with the control of the cholera epidemic. In this study, we isolated a V. cholerae strain, named VCHL017, from the blood of an elderly patient without gastrointestinal symptoms. The patient had a history of hookworm infection and multiple myeloma. Furthermore, she was immunocompromised, and received long-term chemotherapy and antimicrobial agents. VCHL017 was inoculated on blood agar and thiosulfate citrate bile salt sucrose plates (TCBS) to observe morphological characteristics. Then this isolate was identified by matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF MS). The minimum inhibitory concentrations (MICs) for cefazolin, ceftazidime, cefepime, meropenem, tetracycline, ciprofloxacin, chloramphenicol, and gentamicin of VCHL017 were determined by the microbroth dilution method. PCR and serum agglutination tests were used to determine whether the serogroups of the isolate belonged to the O1/O139 and cholera toxin encoding genes. Finally, the genomic features and phylogeny of VCHL017 were analyzed by whole genome sequencing (WGS). VCHL017 was a non-O1/O139 V cholerae strain that did not carry the ctxA gene. Antimicrobial susceptibility tests revealed that VCHL017 was susceptive to chloramphenicol and tetracycline. Although it did not carry the genes encoding the cholera toxin, WGS indicated that VCHL017 carried a variety of other virulence factors. By calculating the average nucleotide identity (ANI), we precisely identified the species of VCHL017 as V. cholerae. There are also A171S and A202S missense mutations in gyrA of VCHL017. The phylogenetic analysis indicated that VCHL017 was closely related to V. cholerae strains isolated from aquatic environments. Our results suggest that continuous monitoring is necessary for non-O1/O139 V cholerae strains isolated from outside the digestive tract, which could be pathogenic through multiple virulence factors.

Emergence of a Hypervirulent Tigecycline-Resistant Klebsiella pneumoniae Strain Co-producing bla NDM-1 and bla KPC-2 With an Uncommon Sequence Type ST464 in Southwestern China.

Emergence of bla NDM-1 and bla KPC-2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both bla NDM-1 and bla KPC-2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained bla NDM-1, bla KPC-2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying bla NDM-1 and bla KPC-2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying bla NDM-1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR, rcsA, lon, and csrD genes of AHSWKP25. Our results highlighted the potential of bla NDM-1 and bla KPC-2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence.

Phenotypic and Genotypic Characteristics of a Tigecycline-Resistant Acinetobacter pittii Isolate Carrying bla NDM-1 and the Novel bla OXA Allelic Variant bla OXA-1045.

A tigecycline-resistant Acinetobacter pittii clinical strain from pleural fluid carrying a bla NDM-1 gene and a novel bla OXA gene, bla OXA-1045, was isolated and characterized. The AP2044 strain acquired two copies of the bla NDM-1 gene and six antibiotic resistance genes (ARGs) from other pathogens. According to the whole-genome investigation, the GC ratios of ARGs (50-60%) were greater than those of the chromosomal backbone (39.46%), indicating that ARGs were horizontally transferred. OXA-1045 belonged to the OXA-213 subfamily and the amino acid sequence of OXA-1045 showed 89% similarity to the amino acid sequences of OXA-213. Then, bla OXA-1045 and bla OXA-213 were cloned and the minimum inhibitory concentrations (MICs) of ß-lactams in the transformants were determined using the broth microdilution method. OXA-1045 was able to confer a reduced susceptibility to piperacillin and piperacillin-tazobactam compared to OXA-213. AP2044 strain exhibited low pathogenicity in Galleria mellonella infection models. The observation of condensed biofilm using the crystal violet staining method and scanning electron microscopy (SEM) suggested that the AP2044 strain was a weak biofilm producer. Quantitative reverse transcription-PCR (qRT-PCR) was used to detect the expression of resistance-nodulation-cell division (RND) efflux pump-related genes. The transcription level of adeB and adeJ genes increased significantly and was correlated with tigecycline resistance. Therefore, our genomic and phenotypic investigations revealed that the AP2044 strain had significant genome plasticity and natural transformation potential, and the emergence of antibiotic resistance in these unusual bacteria should be a concern for future investigations.

Genomic and Phenotypic Characterization of a Colistin-Resistant Escherichia coli Isolate Co-Harboring bla NDM-5, bla OXA-1, and bla CTX-M-55 Isolated from Urine.

Background: Colistin is one of the few options for treating carbapenem-resistant Enterobacterales (CREs). There is little available information about the epidemic status of colistin-resistant CREs in Southern Sichuan, China. This study mainly investigated the genomic and phenotypic characteristics of an extensively drug resistant E. coli LZ00114 isolated from Luzhou, China. Materials and Methods: In 2020, LZ00114 was isolated from the urine of a patient with hydronephrosis and urinary tract infection in Luzhou, China. We assessed the resistance profile of LZ00114 in the presence and absence of the protonophore carbonyl cyano-m-chlorophenylhydrazine (CCCP) and 1-(1-naphthylmethyl)-piperazine (NMP) by antimicrobial susceptibility testing. The growth kinetics, motility, and pathogenicity of LZ00114 were determined to evaluate its microbial characteristics. In combination with whole genome sequencing (WGS) and real-time reverse transcription PCR (RT-PCR), we comprehensively analyzed the resistance mechanisms of LZ00114. Results: LZ00114 was resistant to various antimicrobial agents, including meropenem, tetracycline, ciprofloxacin, gentamicin, fosfomycin, and polymyxin B. Notably, CCCP reversed the resistance of LZ00114 to polymyxin B. LZ00114 displayed high pathogenicity in the infection model (P<0.01) compared with the Lab-WT strain, and its growth rate and motility were not significantly different from the Lab-WT strain. WGS and conjugation revealed that LZ00114 belonged to ST410 and carried a bla NDM-5-harboring self-transmissible IncX3 plasmid and a multi-replicon IncFII/FIA/FIB plasmid carrying bla OXA-1-bla CTX-M-55-tet(B)-aac(6')-Ib-cr-dfrA17-sul1-fosA3. Comparative genomics revealed genetic relatedness between LZ00114 and strains isolated from other regions. Furthermore, there were point mutations in pmrA (S29G, G144S), pmrB (D283G, Y358N), marR (G103S, Y137H), emrA (I219V), and emrD (G323D) of LZ00114. RT-PCR confirmed the overexpression of efflux pumps and PmrABC in LZ00114. Conclusion: This study provides valuable information for the surveillance of antimicrobial resistance and a theoretical basis for the prevention and control of colistin-resistant E. coli. There is still a need to be vigilant about the clone spread of the high-risk clone group E. coli ST410.

Pathogenic Characteristics and Risk Factors for ESKAPE Pathogens Infection in Burn Patients.

OBJECTIVE: This study aimed to determine the clinical manifestations, antimicrobial resistance, molecular characteristics, and risk factors for ESKAPE pathogens infection in burn patients. METHODS: A retrospective study of 187 burn patients infected with ESKAPE pathogens was conducted at the Department of Plastic and Burn Surgery of the Affiliated Hospital of Southwest Medical University (Luzhou, China) from October 2018 to June 2021. All strains were identified using a MicroScan WalkAway 96 Plus System, and antimicrobial susceptibilities were determined using the VITEK system or the disk diffusion method. The antimicrobial resistance genes of multi-drug resistant ESKAPE (MDR-ESKAPE) were detected by polymerase chain reaction (PCR). The multivariable logistic regression analysis was used to estimate the risk factors for ESKAPE infection and MDR-ESKAPE infection. RESULTS: A total of 255 strains were isolated in various types of clinical specimens from 187 burn patients, of which 47.5% were ESKAPE pathogens (121/255). Among these, MDR-ESKAPE pathogens accounted for 55% (67/121). Additionally, aph3'III, mecA, bla SHV, bla TEM, bla PDC, and bla SHV were the most prevalent genes detected in Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., respectively. The independent risk factors for ESKAPE infection were total body surface area (TBSA) >30-50% (odds ratio [OR] = 10.428; 95% confidence interval [CI], 2.047 to 53.108), TBSA >50% (OR = 15.534; 95% CI, 1.489 to 162.021), and parenteral nutrition (OR = 3.597; 95% CI, 1.098 to 11.787). No independent risk factors were found for MDR-ESKAPE infection. CONCLUSION: Clinical staff should be alert to the risk of nosocomial infection with ESKAPE pathogens in burn patients receiving parenteral nutrition and under TBSA >30%. Full attention should also be paid to the ESKAPE resistance, strict adherence to infection control protocols for the rational use of antimicrobial agents, and enhanced clinical standardization of antimicrobial agents management.

The oxidative degradation of Caffeine in UV/Fe(II)/persulfate system-Reaction kinetics and decay pathways.

In this study, the degradation of caffeine was investigated by UV/Fe2+ /persulfate (PS) process. Caffeine (CAF) degradation in sole-UV, UV/Fe2+ , UV/PS, and Fe2+ /PS systems was also conducted to examine the contribution of isolated processes to CAF degradation. The effects of pH levels, the concentration of Fe2+ and PS, inorganic anions, and initial concentration of CAF on the performance of UV/Fe2+ /PS process were evaluated. Radical competitive reactions indicated both hydroxyl radicals and sulfate radicals played important roles in CAF degradation in UV/Fe2+ /PS system. Nine intermediates, among which three were detected for the first time, were identified by ultra-performance liquid chromatography/electrospray-time-of-flight mass spectrometry (UPLC/ESI-TOF-MS) and SPME (solid-phase microextraction)/GC/MS. The possible degradation pathways of CAF were proposed, among which demethylation, hydroxylation, the oxidation of olefinic double bond, and the cleavage of pyrimidine ring and imidazole ring were involved in the degradation of CAF in UV/Fe2+ /PS system. PRACTITIONER POINTS: Caffeine degradation by UV/Fe2+ /PS process was investigated. Caffeine degradation did not follow a simple pseudo-first order kinetics Chloride ions promoted CAF degradation. The anions NO3 - , SO4 2- , and H2 PO4 - exerted a negative influence on caffeine degradation. Nine intermediates were detected, and decay pathways were proposed.

Epidemiological Characteristics and Formation Mechanisms of Multidrug-Resistant Hypervirulent Klebsiella pneumoniae.

Multi-drug resistance (MDR) and hypervirulence (hv) were exhibited by different well-separated Klebsiella pneumoniae lineages in the past, but their convergence clones-MDR-hypervirulent K. pneumoniae (HvKPs)-both highly pathogenic and resistant to most available antibiotics, have increasingly been reported. In light of the clonal lineages and molecular characteristics of the studied MDR-HvKP strains found in the literature since 2014, this review discusses the epidemiology of MDR-HvKPs, in particular summarizing the three general aspects of plasmids-associated mechanisms underlying the formation of MDR-HvKPs clones: MDR-classic K. pneumoniae (cKPs) acquiring hv plasmids, hvKPs obtaining MDR plasmids, and the acquisition of hybrid plasmids harboring virulence and resistance determinants. A deeper understanding of epidemiological characteristics and possible formation mechanisms of MDR-HvKPs is greatly needed for the proper surveillance and management of this potential threat.