Preparation and Performance Evaluation of Self-Cementing Nanoscale Polymeric Microspheres with Salt and Temperature Tolerance.

Polymer microspheres with temperature and salt resistance were synthesized using the anti-suspension polymerization method, incorporating the functional monomers AMPS, AM, and AA. To enhance their self-gelling properties, the microspheres were designed with a core-shell structure. The shell is composed of a polymeric surfactant, fatty alcohol polyoxyethylene ether methacrylate (AEOMA), which serves as a thermosensitive crosslinking agent, enabling self-crosslinking upon shell decomposition, addressing compatibility with reservoir pore throat dimensions. Comprehensive characterizations including infrared spectroscopy, scanning electron microscopy, optical microscopy, and laser particle size analysis were conducted. The microspheres exhibited successful synthesis, a nanoscale size, and regular spherical morphology. They demonstrated excellent temperature and salt resistance, making them suitable for high-temperature, high-salinity reservoir profile control. With a stable three-dimensional network structure, the microspheres displayed good expansion behavior due to hydrophilic groups along the polymer chains, resulting in favorable water affinity. Even after aging, the microspheres maintained their gelling state with a distinct and stable microscopic network skeleton. They exhibited superior plugging performance in low-permeability reservoirs, while effectively improving water absorption profiles in reservoirs with permeability contrasts of 10 to 80, thereby enhancing oil recovery.

Genome sequence of a sequence type 1 NDM-5-producing carbapenem -resistant Klebsiella pneumoniae in China.

OBJECTIVE: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents significant health challenges. Here, we present the structural genome sequence of an NDM-5-producing K. pneumoniae (HZKP2) in China. METHODS: Antimicrobial susceptibility tests were conducted via broth microdilution. Whole-genome sequencing (WGS) was performed for genomic analysis. Wzi and capsular polysaccharide (KL) were analysed using Kaptive. Resistance genes, virulence factors, and comparative genomics analyses were also conducted. Multilocus sequence typing (MLST), replicons type, and core genome multilocus sequence typing (cgMLST) analysis were further conducted using BacWGSTdb server. RESULTS: HZKP2 was resistant to cefepime, ceftazidime, ciprofloxacin, ciprofloxacin, meropenem, and ertapenem. It harbored fosA, blaSHV-187, oqxA, oqxB, sul1, dfrA1, tet(A), floR, aph(6)-Id, aph(3'')-Ib, sul2, blaCTX-M-55, and blaNDM-5. Based on the RAST results, 5563 genes that belonged to 398 subsystems were annotated. The complete genome sequence of HZKP2 was characterized as ST1, wzi 19, and KL19, with five contigs totaling 5,654,446 bp, including one chromosome and four plasmids. Further analysis found that blaNDM-5 was located in a 46,161 bp IncX3 plasmid (pHZKP2-3). The genetic structure of blaNDM-5 gene was ISKox3-IS26-bleMBL-blaNDM-5-IS5-ISAb125-IS3000. Further analysis revealed that insertion sequences mediated the dissemination of blaNDM-5 from other species of Enterobacterales. Phylogenetic analysis showed that the closest relative was from a human stool specimen in China, which differed by 53 cgMLST alleles. CONCLUSION: Our study provides the first structural perspective of the ST1 K. pneumoniae isolate producing NDM-5 in China. These results could provide valuable insights into the genetic characteristics, antimicrobial resistance mechanisms, and transmission dynamics of CRKP in clinical settings.

The dynamic evolution and IS26-mediated interspecies transfer of a blaNDM-1-bearing fusion plasmid leading to a hypervirulent carbapenem-resistant Klebsiella pneumoniae strain harbouring blaKPC-2 in a single patient.

OBJECTIVES: To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient. METHODS: Minimum inhibitory concentrations were measured using broth microdilution assays. Conjugation assays, string tests, and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants, insertion sequences, and virulence factors were identified using ABRicate/ResFinder database, ISFinder, and virulence factor database. Wzi and capsular polysaccharide (KL) were typed using Kleborate and Kaptive. Multi-locus sequence typing (MLST), replicon typing, and single nucleotide polymorphism analyses were conducted using the BacWGSTdb server. RESULTS: The carbapenem-resistant K. pneumoniae 2111KP was characterized as ST11, wzi64, and KL64, with a positive string test result and a relatively high virulence phenotype. Analysis of the 2111KP genome revealed that blaNDM-1 was located in a 268,400-bp IncFIB/IncHI1B/IncX3 conjugative plasmid (p2111KP-1), regulated by IS26, IS5, and ISKox3. p2111KP-1 was also a rmpA2-associated virulence plasmid with an iutA-iucABCD gene cluster and a IS26-mediated multidrug-resistant fusion plasmid, which contained 8-bp (AGCTGCAC or GGCCTTTG) target site duplications. Segments flanked by IS26 of p2111KP-1 were 99.99% identical to a 49,016-bp E. coli plasmid. CONCLUSIONS: This study provided direct evidence of plasmid fusion via IS26 between two different bacterial species within one patient and revealed the process by which genetic elements conferring carbapenem resistance and virulence were simultaneously transferred between these species. It highlights the need for strategic antibiotic use and rigorous monitoring to prevent the plasmid-mediated fusion and transmission of drug-resistance/virulence factors.

Increased Microbial Translocation is a Prognostic Biomarker of Different Immune Responses to ART in People Living with HIV.

Background: Microbial translocation (MT) is a characteristic of human immunodeficiency virus (HIV) infection. Whether MT is also a biomarker of different immune responses to antiretroviral therapy (ART) received by people living with HIV (PLWH) is not known. Methods: We examined the presence of MT in a cohort of 33 HIV-infected immunological responders (IRs) and 28 immunological non-responders (INRs) (≥500 and <200 cluster of differentiation (CD)4+ T-cell counts/µL after 2 years of HIV-1 suppression, respectively) with no comorbidities. Plasma samples were used to measure the circulating levels of MT markers. All enrolled study participants had received 2 years of viral-suppression therapy. Results: Levels of lipopolysaccharide (P = 0.0185), LPS-binding protein (P < 0.0001), soluble-CD14 (P < 0.0001), and endogenous endotoxin-core antibody (P < 0.0001) at baseline were significantly higher in INRs than in IRs and were associated with an increased risk of an immunological non-response, whereas the level of intestinal fatty acid-binding protein did not show this association. Analysis of receiver operating characteristic (ROC) curves demonstrated the utility of these individual microbial markers in discriminating INRs after ART in people living with HIV with high sensitivity, specificity, and area under the ROC curve. Conclusion: INRs in HIV infection are characterized by increased MT at baseline. These markers could be used as a rapid prognostic tool for predicting immune responses in people infected with the HIV.

Immune interference in effectiveness of influenza and COVID-19 vaccination.

Vaccines are known to function as the most effective interventional therapeutics for controlling infectious diseases, including polio, smallpox, rabies, tuberculosis, influenza and SARS-CoV-2. Smallpox has been eliminated completely and polio is almost extinct because of vaccines. Rabies vaccines and Bacille Calmette-Guérin (BCG) vaccines could effectively protect humans against respective infections. However, both influenza vaccines and COVID-19 vaccines are unable to eliminate these two infectious diseases of their highly variable antigenic sites in viral proteins. Vaccine effectiveness (VE) could be negatively influenced (i.e., interfered with) by immune imprinting of previous infections or vaccinations, and repeated vaccinations could interfere with VE against infections due to mismatch between vaccine strains and endemic viral strains. Moreover, VE could also be interfered with when more than one kind of vaccine is administrated concomitantly (i.e., co-administrated), suggesting that the VE could be modulated by the vaccine-induced immunity. In this review, we revisit the evidence that support the interfered VE result from immune imprinting or repeated vaccinations in influenza and COVID-19 vaccine, and the interference in co-administration of these two types of vaccines is also discussed. Regarding the development of next-generation COVID-19 vaccines, the researchers should focus on the induction of cross-reactive T-cell responses and naive B-cell responses to overcome negative effects from the immune system itself. The strategy of co-administrating influenza and COVID-19 vaccine needs to be considered more carefully and more clinical data is needed to verify this strategy to be safe and immunogenic.

Global, regional, and national HIV/AIDS disease burden levels and trends in 1990-2019: A systematic analysis for the global burden of disease 2019 study.

Background: Since the first HIV/AIDS case appeared in 1980s, HIV/AIDS has been the focus of international attention. As a major public health problem, there are epidemiological uncertainties about the future of HIV/AIDS. It is important to monitor the global statistics of HIV/AIDS prevalence, deaths, disability adjusted life years (DALYs), and risk factors for adequate prevention and control. Methods: The Global Burden of Disease Study 2019 database was used to analyze the burden of HIV/AIDS in 1990-2019. By extracting global, regional, and national data on HIV/AIDS prevalence, deaths, and DALYs, we described the distribution by age and sex, explored the risk factors, and analyzed the trends in HIV/AIDS. Results: In 2019, there were 36.85 million HIV/AIDS cases (95% UI: 35.15-38.86 million), 863.84 thousand deaths (95% UI: 78.61-99.60 thousand), and 47.63 million (95% UI: 42.63-55.65 million) DALYs. The global age-standardized HIV/AIDS prevalence, death, and DALY rates were 454.32 (95% UI: 433.76-478.59), 10.72 (95% UI: 9.70-12.39), and 601.49 (95% UI: 536.16-703.92) per 100,000 cases, respectively. In 2019, the global age-standardized HIV/AIDS prevalence, death, and DALY rates increased by 307.26 (95% UI: 304.45-312.63), 4.34 (95% UI: 3.78-4.90), and 221.91 (95% UI: 204.36-239.47) per 100,000 cases, respectively, compared to 1990. Age-standardized prevalence, death, and DALY rates decreased in high sociodemographic index (SDI) areas. High age-standardized rates were observed in low sociodemographic index areas, while low age-standardized rates were observed in high sociodemographic index areas. In 2019, the high age-standardized prevalence, death, and DALY rates were predominant in Southern Sub-Saharan Africa, and global DALYs peaked in 2004 and subsequently decreased. The highest global HIV/AIDS DALYs were in the 40-44 age group. The main risk factors affecting HIV/AIDS DALY rates included behavioral risks, drug use, partner violence, and unsafe sex. Conclusions: HIV/AIDS disease burden and risk factors vary by region, sex, and age. As access to health care increases across countries and treatment for HIV/AIDS infection improves, the HIV/AIDS disease burden is concentrated in areas with low SDIs, particularly in South Africa. Regional differences should be fully considered to target optimal prevention strategies and treatment options based on risk factors.

Activation of NRF2 blocks HIV replication and apoptosis in macrophages.

Abnormal oxidative stress caused by human immunodeficiency virus (HIV) infection affects viral replication and causes non-acquired immune deficiency syndrome-related complications in infected individuals. The transcription factor NFE2-related factor 2 (NRF2), a key regulator of oxidative stress, responds to abnormal oxidative stress by regulating the expression of NRF2-dependent cytoprotective genes. The present study aimed to determine whether inhibition of oxidative stress could control HIV replication and improve cell survival. In this study, the NRF2 activator, methyl bardoxolone, was used to treat cells for HIV infection. The effects on HIV replication and apoptosis pathways were confirmed by NRF2 activation or knockdown. The results showed that NRF2 activation could block HIV replication in macrophages before the integration phase and inhibited the expression of apoptotic pathways in virus-exposed macrophages. The study presents an unconventional anti-viral strategy of activation antioxidant response for HIV infection blocking.

Antibacterial and Anti-biofilm Efficacy of Chinese Dragon's Blood Against Staphylococcus aureus Isolated From Infected Wounds.

Chinese dragon's blood (CDB), a characteristic red resin, is an important traditional Chinese medicine (TCM), and empiric therapy of infected wounds with CDB is performed in clinical settings. For the first time, we herein report the antibacterial and anti-biofilm efficacy of CDB against Staphylococcus aureus (S. aureus). Antimicrobial susceptibility testing, growth curve assay, time-kill curve assay, crystal violet biofilm assay, scanning electron microscope (SEM) analysis, cell membrane tests, and quantitative real-time polymerase chain reaction (qRT-PCR) were used for this purpose. The results suggested that the minimum inhibitory concentration (MIC) values of CDB against S. aureus ranged from 32 to 128 µg/mL. Growth curves and time-kill curves confirmed that CDB could inhibit the growth of S. aureus. The biofilm formation ability and the expression levels of saeR, saeS, and hla of S. aureus in the presence and absence of CDB were statistically significant (P < 0.01). The results of SEM analysis and cell membrane tests revealed that exposure to CDB had some destructive effects on S. aureus cells. In conclusion, CDB exhibits positive antibacterial activity against S. aureus. Moreover, CDB could reduce the biofilm formation and the virulence factors of S. aureus by downregulating the expression levels of saeR, saeS, and hla genes. These findings indicated that CDB has immense potential to serve as a viable alternative for the treatment of infected wounds caused by S. aureus in clinical settings.

Resistance Profiles and Biological Characteristics of Rifampicin-Resistant Staphylococcus aureus Small-Colony Variants.

BACKGROUND: Staphylococcus aureus (S. aureus) is a major contributor to nosocomial and community-acquired infections. S. aureus small colony variants (SCVs) which changed in relevant phenotype have made more limited and difficult for therapeutic options against S. aureus infections increasingly. Rifampicin is considered as the "last-resort" antibiotic against S. aureus. Our study investigated resistance profiles and biological characteristics of rifampicin-resistant S. aureus SCVs. METHODS: We collected S. aureus SCVs that were selected from 41 rifampicin-resistant clinical isolates. Then, biological characteristics, resistance spectrum, and rifampicin resistance mechanisms of tested S. aureus SCVs and corresponding parental strains were investigated by classic microbiological methods, agar dilution method, polymerase chain reaction (PCR). Moreover, the fitness cost of S. aureus SCVs, including growth, biofilm formation ability, and virulence profile, was also determined by bacterial growth curve assay, biofilm formation assay, and Galleria mellonella infection model. RESULTS: There were three S. aureus SCVs (JP310 SCVs, JP1450 SCVs, JP1486 SCVs) that were selected from 41 rifampicin-resistant S. aureus. S. aureus SCVs colonies were tiny, with decreased pigmentation, and the hemolysis circle was not obvious compared with corresponding parental strains. And SCVs could not be restored to normal-colony phenotype after hemin, menaquinone, or thymidine supplementation. Different rpoB mutations occurred in JP1486 SCVs. Antimicrobial susceptibility testing revealed MICs of SCVs were higher than corresponding parental strains. Besides, the growth ability and virulence of SCVs were lower, and biofilm formation ability of which increased compared with parental strains. CONCLUSION: S. aureus SCVs share the rifampicin resistance mechanisms with parental strains, although there were some differences in the position of rpoB mutations. Moreover, we found that the biological characteristics of SCVs were significantly different from corresponding parental strains. In contrast, decreased susceptibility to other antibiotics of SCVs was observed during phenotype switch. Furthermore, SCVs incur the fitness cost.

Difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution.

OBJECT: To analyze the difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution and to further explore the function of mrkH for biofilm formation from the perspective of gene regulation. METHODS: 40 imipenem-resistant strains and 40 imipenem-sensitive strains were selected to conduct experiments. Carbapenem (imipenem) susceptibility test was performed by the agar-dilution method. blaKPC resistance gene, type 3 fimbriae-related coding genes (mrkA and mrkD) and regulation gene (mrkH) were screened by PCR. Biofilm formation assay was performed using crystal violet staining method in MHB. The relative expression of genes that critically involved in biofilm formation (mrkA, luxS, pgaA) and carbapenem resistance (ompk35, ompk36, acrB) were measured by quantitative real-time PCR (qRT-PCR). Furthermore, the mrkH cassette was cloned into pGEM-T Easy plasmid to yield pGEM:pmrkH and expressed in Escherichia coli DH5α and K. pneumoniae FK1911, and the biofilm formation assay after transformation was further tested. RESULTS: The MICs of imipenem were all more than 16 µg/mL in 40 imipenem-resistant strains and ranged from 0.125 µg/mL to 0.5 µg/mL in 40 imipenem-sensitive strains. Moreover, the blaKPC was identified in the 40 imipenem-resistant K. pneumoniae strains. All 80 K. pneumoniae strains were found to carry mrkA and mrkD genes. Interestingly, the mrkH gene was detected in 43 strains, of which 32 were carbapenem-sensitive strains. The biofilm formation capacity of strains carried mrkH cassette was significantly higher than other 37 strains in MHB media. The relative expression of mrkA in K. pneumoniae carrying mrkH gene was significantly up-regulated. Importantly, the biofilm formation ability of FK1911-pGEM:pmrkH strain was more higher than the strain of FK1911 in MHB medium. CONCLUSIONS: Our data demonstrated that MrkH played a crucial role in the regulation of biofilm formation by K. pneumoniae. In contrast to carbapenem-sensitive K. pneumoniae, carbapenem-resistant K. pneumoniae was less likely to have strong biofilm-forming capacity because it does not carry the mrkH gene.

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling.

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC ß-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism of resistance of K. pneumoniae during carbapenem treatment. RESULTS: Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 was found to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored blaSHV-187 and blaTEM-116. The blaSHV-187 and blaTEM-116 genes were not detected in FK-2723 and FK-2820. blaDHA-1, qnrB4, aac (6')-IIc, and blaSHV-12, EreA2, CatA2, SulI, and tetD, were identified in both FK-2723 and FK-2820. Moreover, the genes blaDHA-1, qnrB4, aac (6')-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate. CONCLUSION: This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.

Mechanisms of Heteroresistance and Resistance to Imipenem in Pseudomonas aeruginosa.

BACKGROUND: Heteroresistance is a phenomenon that occurs in all bacteria and can cause treatment failure. Yet, the exact mechanisms responsible for heteroresistance still remain unknown. The following study investigated the mechanisms of imipenem-heteroresistance and -resistance in Pseudomonas aeruginosa clinical isolates from Wenzhou, China. METHODS: Imipenem resistance was detected by the agar dilution method; heteroresistance was determined by population analysis profiles. Biofilm formation assay and modified carbapenem inactivation methods were also performed. Polymerase chain reaction (PCR) was conducted to detect oprD, and quantitative real-time PCR was used to determine expression levels of oprD, ampC and four efflux pump coding genes (mexB, mexD, mexE and mexY). RESULTS: Six imipenem-heteroresistant and -resistant P. aeruginosa isolates were selected respectively. Deficient oprD was detected in all resistant isolates and two heteroresistant isolates. No strains produced carbapenemases. Expression levels of oprD were down-regulated in heteroresistant isolates. Transcription levels of the mexE and mexY were significantly increased in all heterogeneous subpopulations compared with their respective native ones. Compared with the susceptible group, increased mean relative expression levels of mexE and mexY or the decreased mean relative expression levels of oprD were observed in the resistant group (P < 0.05), whereas transcription levels of the mexB and mexD remained unchanged. CONCLUSION: Down-regulation of oprD contributed to the resistance and heteroresistance of imipenem in our P. aeruginosa clinical isolates. In addition, the marginal up-regulation of efflux systems may indirectly affect imipenem resistance. Contrarily, defective oprD was less common in our experimental heteroresistant strains than resistant strains.

Mutations in the MepRAB efflux system contribute to the in vitro development of tigecycline resistance in Staphylococcus aureus.

OBJECTIVE: To characterize the evolutionary pathways of tigecycline (TGC) resistance and alterations in the biological characteristics of hospital-derived Staphylococcus aureus isolates under selective pressure. METHODS: Three clinical S. aureus strains and one standard S. aureus strain, ATCC 29213, were used for the in vitro selection of TGC-resistant S. aureus variants with gradient concentrations of TGC. Changes in drug resistance and genetic alterations in resistance-related genes (operon mepRAB and rpsJ) in mutant strains were determined. The efflux inhibitor assay for MepA and the fitness cost, determined by comparing the growth and virulence of parental and mutant strains, were also investigated. RESULTS: Mutants induced in vitro showed a 64- to 128-fold increase in the minimum inhibitory concentration (MIC) of TGC. Substitution mutations were detected in the transcriptional repressor mepR and the efflux pump gene mepA. A K57M amino acid substitution occurred in the ribosomal S10 protein-encoding gene rpsJ. The MICs of TGC in the final mutants were significantly decreased in the presence of efflux pump inhibitors. It was worth noting that growth was unaffected by TGC resistance selection in vitro, with the exception of one strain, and the MICs of other antibiotics and virulence were also unaffected. CONCLUSIONS: The evolution of TGC resistance in S. aureus in vitro is associated with a loss-of-function mutation in the efflux pump transcriptional repressor mepR and a missense mutation in the efflux pump-encoding gene mepA. Our work further validated the resistance mechanisms of S. aureus to TGC and reported previously undiscovered mutations.

Emergence of a multidrug-resistant ST 27 Escherichia coli co-harboring blaNDM-1, mcr-1, and fosA3 from a patient in China.

In this study, we report a clinical isolate of a carbapenem-, colistin-, and fosfomycin-resistant Escherichia coli isolate DC-3737 co-harboring blaNDM-1, mcr-1, and fosA3 from an inpatient in China. Antimicrobial susceptibility testing, polymerase chain reaction, multi-locus sequence typing, conjugation experiment, and Southern blot hybridization were performed on E. coli DC-3737 isolated from the wound. Plasmid analysis is presented and the locations of blaNDM-1, mcr-1, fosA3, and other resistance genes were identified as well. E. coli DC-3737 was resistant to ampicillin, ceftriaxone, ceftazidime, ciprofloxacin, levofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, imipenem, meropenem, ertapenem, fosfomycin and colistin, and with intermediate susceptibility to amikacin. It was typed as sequence type 27. The isolate possessed blaNDM-1, mcr-1, fosA3, blaCTX-M-9, blaTEM-1, aac (6')-Ib-cr and sul1 simultaneously. In addition, the mutations in quinolone resistance-determinant regions (QRDRs) such as Ser83Leu and Asp87Asn in gyrA, and Ser80Ile in parC were detected. Conjugation assays revealed that blaNDM-1, fosA3, sul1, mcr-1, and blaCTX-M-9 genes could successfully transfer their resistance phenotype to E. coli strain J53. Plasmid analysis and Southern hybridization showed that DC-3737 possessed Z-type self-transmissible plasmid bearing blaNDM-1, fosA3, and sul1. Moreover, mcr-1, blaCTX-M-9, and blaTEM-1 were located on a ~60-kb IncFIB type self-transmissible plasmid. This is the first report of blaNDM-1, mcr-1 and fosA3 co-harboring in E. coli in China. Moreover, it is also the first description of the co-harboring of blaNDM and fosA3 in a single Z plasmid in Enterobacteriaceae species. The identification of E. coli DC-3737 co-harboring blaNDM-1, mcr-1, and fosA3 in this study highlights the need to increase epidemiologic surveillance and the need for new classes of antibiotics to address multidrug-resistant bacteria.

Molecular Mechanisms and Epidemiology of Carbapenem-Resistant Escherichia coli Isolated from Chinese Patients During 2002-2017.

BACKGROUND: The emergence and spread of carbapenem-resistant Escherichia coli (E. coli) pose a serious threat to human health worldwide. This study aimed to investigate the molecular mechanisms underlying carbapenem resistance and their prevalence among E. coli in China. METHODS: A collection of 5796 E. coli clinical isolates were collected from the First Affiliated Hospital of Wenzhou Medical University from 2002 to 2017. Sensitivity to antibiotics was determined using the agar dilution method. The detection of carbapenemases production and the prevalence of resistance-associated genes were investigated through modified carbapenem inactivation method (mCIM), PCR and sequencing. The mutations in outer membrane porins genes (ompC and ompF) were also analyzed by PCR and sequencing assays. The effect of efflux pump mechanism on carbapenem resistance was also tested. E. coli were typed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: A total of 58 strains (1.0%) of carbapenem-resistant E. coli were identified. The strains carrying bla KPC-2 and bla NDM accounted for 22.4% (13/58) and 51.7% (30/58), respectively. Among bla NDM- positive strains, 27 bla NDM genes were assigned to bla NDM-5, while the remaining three strains were bla NDM-1, whereas bla VIM, bla IMP, bla OXA-48, and bla SHV were not found. The CTX-M-type ß-lactamase genes accounted for 96.6% (56/58). In addition, bla TEM-1 genes were identified in 58.6% of tested strains. In carbapenem-resistant isolates, mutations in OmpC (the majority of mutated sites were D192G and Q104_F141del, accounting for 54.5%) and OmpF (large deletions S75_V127del, W83_D135del and Q88_D135del) were detected. Of note, the antibiotic resistance was not associated with overexpression of efflux pump. Moreover, MLST categorized the 58 carbapenem-resistant isolates into 19 different sequence types. PFGE analysis revealed that homology among the carbapenem-resistant isolates was low and sporadic. CONCLUSION: The bla NDM was the principal resistance mechanism of carbapenem-resistant E. coli in the hospital. bla NDM-5 is becoming a new threat to public health and the alteration of outer membrane porins might help further increase the MIC of carbapenem.

Evaluation of resazurin-based assay for rapid detection of polymyxin-resistant gram-negative bacteria.

BACKGROUND: Colistin resistance is considered a serious problem due to a lack of alternative antibiotics. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test is a resazurin reduction-based technique that relies on the visual detection of bacterial growth in the presence of a defined concentration of colistin. The aim of this study was to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test in the detection of colistin susceptibility in common clinical Gram-negative bacteria. RESULTS: A total of 253 clinical isolates from a teaching hospital, including Acinetobacter baumanii (n = 58, 8 colistin-resistant), Pseudomonas aeruginosa (n = 61, 11 colistin-resistant), Klebsiella pneumoniae (n = 70, 20 colistin-resistant) and Escherichia coli (n = 64, 14 colistin-resistant) were tested in this study. The sensitivity and specificity of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test compared to Broth microdilution method was 100 and 99%, respectively. CONCLUSIONS: Our results suggest that Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test could be used as an accurate detection method for colistin resistance.

Evolution of resistance mechanisms and biological characteristics of rifampicin-resistant Staphylococcus aureus strains selected in vitro.

BACKGROUND: We aimed to determine the evolutionary pathways of rifampicin resistance in Staphylococcus aureus, and the impact of resistance mutations in the rpoB gene on fitness. METHODS: Three clinical strains and one reference strain were used to select for rifampicin-resistant S. aureus variants. The mutations responsible for rifampicin resistance in all of the selected isolates in vitro were investigated by polymerase chain reaction (PCR) and DNA sequencing. To compare the fitness cost of rpoB mutations against their corresponding original isolates, we performed bacterial growth curve assays, static biofilm assays, in vitro competition experiments and an infection model of Galleria mellonella larvae. RESULTS: We obtained four rifampicin-resistant S. aureus isolates that showed high levels of resistance to rifampicin with a minimal inhibitory concentration (MIC) of 128 mg/L, and all isolates had a mutation at position 481 (H481F/Y) in RpoB. A broth microdilution assay indicated that mutation of H481F/Y did not affect susceptibility to common antibacterial drugs but slightly increased the vancomycin MIC. To identify the pathways involved in the development of rifampicin resistance, 32 variants (eight mutants for each strain) and four original isolates were selected for gene sequencing. Different generations of isolates were found to harbor various mutations sites. Compared with the corresponding original isolates, an in vitro fitness assay of the variant isolates showed that growth and virulence were reduced, with a statistically significantly decreased fitness, whereas the capacity for biofilm formation was elevated. CONCLUSIONS: Our findings suggested that the acquisition of rifampicin resistance in S. aureus was dynamic and was associated with a significant fitness cost.