In Vitro Antibacterial Activity of Ceftobiprole and Comparator Compounds against Nation-Wide Bloodstream Isolates and Different Sequence Types of MRSA.

Bloodstream infections by bacteria, especially multidrug-resistant bacteria, remain a worldwide public health concern. We evaluated the antibacterial activity of ceftobiprole and comparable drugs against different bloodstream isolates and different sequence types of methicillin-resistant Staphylococcus aureus (MRSA) in China. We found that MRSA, methicillin-susceptible Staphylococcus aureus (MSSA), and methicillin-susceptible coagulase-negative Staphylococcus (MSCNS) displayed ceftobiprole sensitivity rates of >95%, which are similar to the rates for linezolid, daptomycin, and vancomycin. Of the tested MRCNS strains, 90.4% were sensitive to ceftobiprole. The sensitivities of ST59, ST398, and ST22 MRSA to ceftobiprole were higher than that of ST239. Ceftobiprole's MIC50/90 value against Enterococcus faecalis was 0.25/2 mg/L, whereas Enterococcus faecium was completely resistant to this drug. Ceftobiprole exhibited no activity against ESBL-positive Enterobacterales, with resistance rates between 78.6% and 100%. For ESBL-negative Enterobacterales, excluding Klebsiella oxytoca, the sensitivity to ceftobiprole was comparable to that of ceftazidime, ceftriaxone, and cefepime. The MIC50/90 value of ceftobiprole against Pseudomonas aeruginosa was 2/16 mg/L, and for Acinetobacter baumannii, it was 32/>32 mg/L. Thus, ceftobiprole shows excellent antimicrobial activity against ESBL-negative Enterobacterales and Pseudomonas aeruginosa (comparable to that of ceftazidime, ceftriaxone, and cefepime); however, it is not effective against ESBL-positive Enterobacterales and Acinetobacter baumannii. These results provide important information to clinicians.

Epidemiology and clinical features of Skin and Soft Tissue Infections Caused by PVL-Positive and PVL-Negative Methicillin-Resistant Staphylococcus aureus Isolates in inpatients in China: a single-center retrospective 7-year study.

Previous studies have mainly focused on outpatient cases of skin and soft tissue infections (SSTIs), with limited attention to inpatient occurrences. Thus, we aimed to compare the clinical parameters of inpatients with SSTIs, performed genomic characterization, and determined the subtypes of Panton-Valentine leucocidin (PVL) bacteriophages of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from these patients. We found that PVL-positive patients had shorter hospital stays (mean, 9 vs. 24 days; p < 0.001) and abscess resolution durations (mean, 8 vs. 13 days; p < 0.01). PVL-positive MRSA-induced SSTIs were more frequently associated with abscesses [36/55 (65.5%) vs. 15/124 (12.1%), p < 0.001], with 52.7% undergoing incision and drainage; over 80% of PVL-negative patients received incision, drainage, and antibiotics. In PVL-positive patients receiving empirical antibiotics, anti-staphylococcal agents such as vancomycin and linezolid were administered less frequently (32.7%, 18/55) than in PVL-negative patients (74.2%, 92/124), indicating that patients with PVL-positive SSTIs are more likely to require surgical drainage rather than antimicrobial treatment. We also found that the ST59 lineage was predominant, regardless of PVL status (41.3%, 74/179). Additionally, we investigated the linear structure of the lukSF-PV gene, revealing that major clusters were associated with specific STs, suggesting independent acquisition of PVL by different strain types and indicating that significant diversity was observed even within PVL-positive strains detected in the same facility. Overall, our study provides comprehensive insights into the clinical, genetic, and phage-related aspects of MRSA-induced SSTIs in hospitalized patients and contributes to a more profound understanding of the epidemiology and evolution of these pathogens in the Chinese population.

Clinical relevance, mechanisms, and evolution of polymyxin B heteroresistance carbapenem-resistant Klebsiella pneumoniae: A genomic, retrospective cohort study.

OBJECTIVES: To study the clinical relevance, mechanisms, and evolution of polymyxin B (POLB) heteroresistance (PHR) in carbapenem-resistant Klebsiella pneumoniae (CRKP), potentially leading to a significant rise in POLB full resistant (FR) CRKP. METHODS: Total of 544 CRKP isolates from 154 patients treated with POLB were categorized into PHR and POLB non-heteroresistance (NHR) groups. We performed statistical analysis to compare clinical implications and treatment responses. We employed whole-genome sequencing, bioinformatics, and PCR to study the molecular epidemiology, mechanisms behind PHR, and its evolution into FR. RESULTS: We observed a considerable proportion (118 of 154, 76.62%) of clinically undetected PHR strains before POLB exposure, with a significant subset of them (33 of 118, 27.97%) evolving into FR after POLB treatment. We investigated the clinical implications, epidemiological characteristics, mechanisms, and evolutionary patterns of PHR strains in the context of POLB treatment. About 92.86% (39 of 42) of patients had PHR isolates before FR, highlighting the clinical importance of PHR. the ST15 exhibited a notably lower PHR rate (1 of 8, 12.5% vs. 117 of 144, 81.25%; p < 0.01). The ST11 PHR strains showing significantly higher rate of mgrB mutations by endogenous insertion sequences in their resistant subpopulation (RS) compared with other STs (78 of 106, 73.58% vs. 4 of 12, 33.33%; p < 0.01). The mgrB insertional inactivation rate was lower in FR isolates than in the RS of PHR isolates (15 of 42, 35.71% vs. 84 of 112, 75%; p < 0.01), whereas the pmrAB mutation rate was higher in FR isolates than in the RS of PHR isolates (8 of 42, 19.05% vs. 2 of 112, 1.79%; p < 0.01). The evolution from PHR to FR was influenced by subpopulation dynamics and genetic adaptability because of hypermutability. DISCUSSION: We highlight significant genetic changes as the primary driver of PHR to FR in CRKP, underscoring polymyxin complexity.

Comparative analysis of genomic characteristics, virulence and fitness of community-associated Staphylococcus aureus ST121 clone causing fatal diseases in China and other CA-MRSA clones.

The increasing rate of community-associated Staphylococcus aureus (CA-SA) worldwide has aroused global public concern for decades. Although ST121 clone is one of the prevalent CA-SA in China, there is still limited knowledge about it. In this study, we conducted a genomic analysis of 28 CA-SA ST121 isolates from severe bloodstream infection cases and 175 ST121 isolates from the public database. Phylogenetic analysis revealed the consistency and the complexity of global ST121 lineages, and suggested potential cross-country even cross-continental transmission of ST121 isolates. By investigating the virulence and fitness between ST121-CA-methicillin-resistant SA (CA-MRSA) and other CA-MRSA clones, we found that ST121-MRSA exhibits virulence comparable to the highly virulent USA300 clone, exceeding that of the predominant CA-MRSA lineage ST59 in China and the other American CA-MRSA clone MW2. Notably, based on analyses of virulence genes, eta, etb, edin-C and egc were only found in ST121, suggesting that the high virulence of ST121 may be attributed to the combination of these virulence factors encoded by mobile genetic elements. However, results of experiments in mice nasal and human alveolar epithelial cells showed that the colonization capacity of ST121 is much lower than that of other clones. Moreover, ST121-MRSA displayed much lower acid tolerance, suggesting that ST121-MRSA may not have such capacity to achieve the epidemiological success of other CA-MRSA clones and become the dominant lineage. Our findings expand current understanding of the epidemiology and pathogenicity of the hypervirulent ST121 clone, and highlight the importance of colonization capacity and environmental adaption in MRSA epidemiological success.

Novel SCCmec variants in clonal complex 398 and lineage-specific pseudo-SCCmec identified in ST88 MRSA from invasive bloodstream infections in China.

BACKGROUND: Methicillin resistance in Staphylococcus aureus is primarily due to the mecA gene found in highly diverse staphylococcal cassette chromosome mec (SCCmec) elements, with an increasing number of variants being continually discovered. OBJECTIVES: To characterize two novel SCCmec variants identified in clonal complex (CC) 398 strains and lineage-specific pseudo-SCCmec elements in the ST88 clone. METHODS: WGS and comparative genomic analysis were used to elucidate the SCCmec element diversity of representative isolates. RESULTS: The non-typeable 47 kb SCCmec found in the CC398 strain SKLX55795 represents a novel subtype of XIV, showing significant differences in structural organization and genetic content within the joining regions compared with the XIV element from the prototype strain SC792. This unique subtype comprised remnants from various mobile genetic elements that encode antimicrobial resistance genes, ultimately forming a large MDR region. Genome analysis of CC398 strain SKLX61416 revealed the presence of a novel 50 kb composite SCCmec with two distinct domains, carrying the ccr gene complexes 5/8 and containing genes for the detoxification of arsenic and sulphide. Further sequence analysis disclosed that 44.23% (23/52) of ST88 strains in our collection carried a lineage-specific pseudo-SCCmec, termed ΨSCCmecST88. This ΨSCCmecST88 harboured the mec gene complex C2, along with a series of genes associated with heavy metal resistance, but lacked an approximately 28 kb region encompassing the ccr gene complex. CONCLUSIONS: Our findings provide evidence for the ongoing evolution of SCCmec elements within the CC398 and ST88 clones, underscoring the need for further surveillance to understand the biological significance of these elements.

Recombination Drives Evolution of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 KL47 to KL64 in China.

Carbapenem-resistant Enterobacteriaceae, especially carbapenemase-producing Klebsiella pneumoniae, is an urgent problem in health care facilities worldwide. K. pneumoniae isolates classified as sequence type 11 (ST11) are largely responsible for the spread of carbapenem-resistant K. pneumoniae (CRKP) in China. Our previous phylogenetic reconstruction suggested that CRKP ST11 capsular locus 64 (KL64) was derived from an ST11-KL47 ancestor through recombination. However, the molecular origin of KL64 remains largely unknown, and our understanding of the recombination is incomplete. Here, we screened a global sample of 22,600 K. pneumoniae genomes and searched for KL64-harboring STs, which were found to be ST1764, ST3685, ST1764-1LV, ST30, ST505, ST147, and ST11, wherein ST1764, ST3685, ST1764-1LV, and ST30 belonged to a clonal complex, CC1764. We compared the genetic structures of representative strains from ST11-KL47, ST11-KL64, CC1764-KL64, ST505-KL64, and ST147-KL64 and further performed phylogenetic analysis and single-nucleotide polymorphism analysis among 248 isolates from all these STs. The results suggested a recombination event has occurred in a homologous ~154-kb region covering KL and the lipopolysaccharide biosynthesis locus (OL) between a recipient ST11-KL47-OL101 and a donor CC1764 (except ST30), giving rise to ST11-KL64-O2v1 strains (recombination I). Furthermore, we also found an infrequent ST11-KL64-O2v1 subclone which was not produced by recombination I but was hybridized from ST11-KL47-OL101 and ST147-KL64-O2v1 strains through recombination of a homologous ~485-kb region covering KL and OL (recombination II). Our findings provide important insights into the role of recombination in the evolution of clinical strains and the diversity of capsule and lipopolysaccharide of widely distributed KPC-associated ST11 K. pneumoniae in China. IMPORTANCE Chromosomal recombination events are considered to contribute to the genetic diversification and ultimate success of many bacterial pathogens. A previous study unravelled the molecular evolution history of ST258 strains, which have been largely responsible for the spread of KPC in the United States. Here, we used comparative genomic analyses to discover two recombination events in ST11 CRKP strains, which is a predominant KPC-associated CRKP clone in China. Two new ST11-CRKP subclones with altered capsule and lipopolysaccharide, which are two primary determinants of antigenicity and antigenic diversity among K. pneumoniae, were produced through these two recombination events, respectively. Horizontal transfer of the KL and OL appears to be a crucial element driving the molecular evolution of K. pneumoniae strains. These findings not only extend our understanding of the molecular evolutionary history of ST11 but also are an important step toward the development of preventive, diagnostic, and therapeutic strategies for CRKP.

Comparison of Genetic Features and Evolution of Global and Chinese Strains of Community-Associated Methicillin-Resistant Staphylococcus aureus ST22.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 22, especially the epidemic MRSA-15 (EMRSA-15), has been one of the most important disease-causing clones transmitting rapidly within and between hospitals globally. However, the genetic features and evolution of Chinese MRSA ST22 remain to be determined. Herein, we performed comparative genomics analysis of 12 ST22 community-associated (CA) MRSA isolates from China with 9 Chinese ST22 CA-MSSA isolates and 284 ST22 genomes from global sources, to clarify the genotypic features and potential transmission of MRSA ST22 strains isolated in China. Phylogenetic reconstruction and time estimation suggested that the Chinese subclade emerged around 2006, and the ST22-SCCmec V clone may have evolved from the native ST22-MSSA clone rather than spread from other regions, indicating that the Chinese ST22-MRSA-V clone is independent of the EMRSA-15 and Gaza clone, with differences in lukSF-PV and tsst-1 carriage. Virulence assays suggested that the ST22-MRSA clone was highly virulent, displaying higher or similar virulence potential as MSSA ST22 predecessors and the epidemic USA300 and ST22-MSSA. However, two nonsense mutations caused by a frameshift in agrC were identified in two ST22-MSSA isolates, resulting in a significant attenuation of virulence. RT-qPCR also demonstrated that the high virulence potential of these ST22 strains may be attributed to elevated expression of agr. This study provides insight into the epidemiology of the novel and highly virulent CA-MRSA ST22 clones. IMPORTANCE Staphylococcus aureus sequence type 22 (ST22) is the main HA-MRSA clone spreading in Europe. It has strong capacity to supplant and replace other formerly epidemic MRSA clones. Previous work has described genotypic characteristics of ST22 belonging to EMRSA-15 and Gaza clone; however, the genetic feature and virulence potential of Chinese spread of ST22 strains are still limited. We conducted a detailed analysis of genomic evolution of global ST22 strains, to clarify the genotypic features and potential transmission of MRSA ST22 strains isolated from China. Our results suggested that the Chinese subclade is highly virulent, and emerged around 2006. We also demonstrated that the ST22-SCCmec V may have evolved from the native ST22-MSSA clone rather than spread from other regions, and the high virulence potential of these ST22 strains may be attributed to the high expression of agr based on the results of virulence assays of Chinese ST22 clones. Our findings are of great importance for providing insights into the epidemiology and pathogenicity of global and Chinese ST22 clones.

Genomic epidemiology and characterisation of penicillin-sensitive Staphylococcus aureus isolates from invasive bloodstream infections in China: an increasing prevalence and higher diversity in genetic typing be revealed.

Many countries have reported increasing rates of penicillin-susceptible methicillin-sensitive Staphylococcus aureus (MSSA-PENS). To date, there is relatively little known about the current situation and molecular characteristics of MSSA-PENS in China. In this study, we carried out a laboratory-based multi-region retrospective study to investigate the genomic epidemiology and characterisation of MSSA-PENS isolated from invasive bloodstream infections (BSIs) across 17 provinces. The prevalence of MSSA-PENS isolates increased significantly over the 6-year period, with the proportion increasing from 3.51% in 2014-8.80% in 2019, an average relative increase of 22.14% per year (95% confidence interval 9.67%-34.61%, P for trend <0.001), suggesting that China is experiencing a resurgence of MSSA-PENS. Phylogenetic analysis showed a higher strain diversity occurred; the most frequent clonal complexes (CCs) identified were CC188 (17.14%), CC398 (15.71%) and CC5 (15.71%). Over half of MSSA-PENS strains were pan-susceptible, with erythromycin the most frequent resistance observed. Moreover, 25 isolates were identified as immune evasion cluster negative, including CC15, CC188 and CC1, and 6 strains encoded the Panton-Valentine leucocidin gene. Importantly, virulence assays showed that MSSA-PENS exhibited a level of virulence comparable to that of penicillin-resistant MSSA (MSSA-PENR), indicating that more-sensitive strains should not be mistaken for lacking aggressiveness in vivo. Furthermore, 11 of these isolates were confirmed as blaZ positive but phenotype sensitive, with different amino acid changes in blaZ. Our data support the recommendation to clinicians regarding the usage of penicillin in invasive BSIs caused by MSSA-PENS, which might create a novel opportunity for better antimicrobial stewardship in the future.

Comparative genomic and transmission analysis of Clostridioides difficile between environmental, animal, and clinical sources in China.

Clostridioides difficile is the most common pathogen causing antibiotic-associated diarrhea. Previous studies showed that diverse sources, aside from C. difficile infection (CDI) patients, played a major role in C. difficile hospital transmission. This study aimed to investigate relationships and transmission potential of C. difficile strains from different sources. A prospective study was conducted both in the intensive care unit (ICU) and six livestock farms in China in 2018-2019. Ninety-eight strains from CDI patients (10 isolates), asymptomatic hospitalized carriers (55), the ICU environment (12), animals (14), soil (4), and farmers (3) were collected. Sequence type (ST) 3/ribotype (RT) 001, ST35/RT046, and ST48/RT596 were dominant types, distributed widely in multiple sources. Core-genome single-nucleotide polymorphism (cgSNP) analysis showed that hospital and farm strains shared several common clonal groups (CGs, strains separated by ≤ 2 cgSNPs) (CG4/ST3/RT001, CG7/ST35/RT046, CG11/ST48/RT596). CDI patients, asymptomatic carriers, and the ICU environment strains also shared several common CGs. The number of virulence genes was not statistically different between strains from different sources. Multi-source strains in the same CG carried identical virulence gene sequences, including pathogenicity genes at the pathogenicity locus and adhesion-related genes at S-layer cassette. Resistance genes (ermB, tetM, etc.) were widespread in multiple sources, and multi-source strains in the same CG had similar resistance phenotypes and carried consistent transposons and plasmid types. The study indicated that interspecies and cross-regional transmission of C. difficile occurs between animals, the environment, and humans. Community-associated strains from both farms and asymptomatic hospitalized carriers were important reservoirs of CDI in hospitals.

Genomic Epidemiology and Characterization of Methicillin-Resistant Staphylococcus aureus from Bloodstream Infections in China.

Since 2010, methicillin-resistant Staphylococcus aureus (MRSA) ST59 began to increase in prevalence in China, gradually replacing ST239 and has become the dominant clone in most hospitals in China. Here, we investigated the changing epidemiology, phylogenetic reconstruction, and genomic characterization of MRSA clones in China to identify the genomic driving factors in the prevalence of ST59. Most MRSA isolates were identified as ST59 (36.98%; 277/749), which increased from 25.09% in 2014 to 35.53% in 2019. The phylogenetic analysis of the 749 MRSA isolates showed a high level of diversity and the copresence of hospital-associated, community-associated, livestock-associated, and hypervirulent clones. Furthermore, minimum spanning trees revealed that ST59 MRSA clones from different hospitals and regions were integrated, suggesting that frequent exchanges had occurred between regions and hospitals. ST59 clones displayed higher susceptibility to antimicrobials than did ST239 and ST5 MRSA clones, indicating that resistance to non-ß-lactam and fluoroquinolone antibiotics may be not critical for the epidemic success of ST59 clones. Virulence factors detection showed that sak and chp genes enriched in MRSA ST59 may be associated with the enhanced spreading success of ST59, whereas qacA may have contributed to the predominance of ST5 in East China. Our refined analysis of different clones among ST239, ST5, ST59, and ST398 demonstrated the existence of potential driving factors for the evolution of nosocomial MRSA populations and diversity of the evolutionary events surrounding clonal replacement. IMPORTANCE As a developing country, China has an unbalanced health care system due to regional differences in economic development. However, China is also a country worthy of study with regard to the population dynamics of MRSA within the more resource-rich health care systems. In this study, we carried out genomic analysis to investigate the genomic epidemiology and characterization of MRSA isolated from bloodstream infections over a timespan of 6 years. Our refined analysis of different MRSA clones among ST59, ST5, ST239, and ST398 demonstrated the existence of driving factors for the evolution of nosocomial MRSA populations and diversity of the evolutionary events surrounding clonal replacement.

Comparative Genomic Analysis Provides Insights into the Evolution and Genetic Diversity of Community-Genotype Sequence Type 72 Staphylococcus aureus Isolates.

Staphylococcus aureus sequence type (ST) 72, the predominant community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) lineage in South Korea, has emerged as a major cause of bloodstream infection in hospital settings. However, relatively little information is available regarding the genomic characteristics and dissemination of ST72. Here, we characterized the whole-genome sequence of 24 ST72 isolates from China, along with 83 ST72 genomes from global sources. Of these 107 ST72 isolates, 63 were MRSA and 44 were methicillin-susceptible S. aureus (MSSA). Phylogenetic analysis revealed four distinct clades (A, B, C, and D), of which clade D contained only MSSA isolates. By characterizing the evolutionary dynamics of the ST72 lineage, we found that the MRSA from China might not have developed from the MSSA in China. Furthermore, we observed both international transmission of ST72 isolates and interregional transmission within China. The distributions of the SCCmec and spa types of isolates differed among clades. Additionally, in silico analyses revealed that the distributions of resistance genes, virulence genes, and mobile genetic elements (MGEs) also differed among isolates of the four clades. This was especially true for clade D isolates, which had the lowest level of antimicrobial resistance and had obtained specific virulence genes such as tsst-1 by acquisition of specific MGEs. Notably, ST72 MRSA isolates were more antibiotic resistant than ST72 MSSA isolates, but comparably virulent. Our findings provide insight into the potential transmission and genotypic features of ST72 clones across the globe. IMPORTANCE Understanding the evolution and dissemination of community-genotype ST72 Staphylococcus aureus isolates is important, as isolates of this lineage have rapidly spread into hospital settings and caused serious health issues. In this study, we first carried out genome-wide analysis of 107 global ST72 isolates to characterize the evolution and genetic diversity of the ST72 lineage. We found that the MSSA lineage in China might have evolved independently from the MRSA isolates from China, and that ST72 isolates have the potential to undergo both international transmission and interregional transmission within China. The diversity of isolates correlated with distinct acquisitions of SCCmec elements, antibiotic resistance genes, virulence genes, and mobile genetic elements. The comprehensive information on the ST72 lineage emerging from this study will enable improved therapeutic approaches and rapid molecular diagnosis.

Genomic Analysis of Delftia tsuruhatensis Strain TR1180 Isolated From A Patient From China With In4-Like Integron-Associated Antimicrobial Resistance.

Delftia tsuruhatensis has become an emerging pathogen in humans. There is scant information on the genomic characteristics of this microorganism. In this study, we determined the complete genome sequence of a clinical D. tsuruhatensis strain, TR1180, isolated from a sputum specimen of a female patient in China in 2019. Phylogenetic and average nucleotide identity analysis demonstrated that TR1180 is a member of D. tsuruhatensis. TR1180 exhibited resistance to ß-lactam, aminoglycoside, tetracycline and sulphonamide antibiotics, but was susceptible to phenicols, fluoroquinolones and macrolides. Its genome is a single, circular chromosome measuring 6,711,018 bp in size. Whole-genome analysis identified 17 antibiotic resistance-related genes, which match the antimicrobial susceptibility profile of this strain, as well as 24 potential virulence factors and a number of metal resistance genes. Our data showed that Delftia possessed an open pan-genome and the genes in the core genome contributed to the pathogenicity and resistance of Delftia strains. Comparative genomics analysis of TR1180 with other publicly available genomes of Delftia showed diverse genomic features among these strains. D. tsuruhatensis TR1180 harbored a unique 38-kb genomic island flanked by a pair of 29-bp direct repeats with the insertion of a novel In4-like integron containing most of the specific antibiotic resistance genes within the genome. This study reports the findings of a fully sequenced genome from clinical D. tsuruhatensis, which provide researchers and clinicians with valuable insights into this uncommon species.

Identification of floR Variants Associated With a Novel Tn4371-Like Integrative and Conjugative Element in Clinical Pseudomonas aeruginosa Isolates.

Florfenicol is widely used to control respiratory diseases and intestinal infections in food animals. However, there are increasing reports about florfenicol resistance of various clinical pathogens. floR is a key resistance gene that mediates resistance to florfenicol and could spread among different bacteria. Here, we investigated the prevalence of floR in 430 Pseudomonas aeruginosa isolates from human clinical samples and identified three types of floR genes (designated floR, floR-T1 and floR-T2) in these isolates, with floR-T1 the most prevalent (5.3%, 23/430). FloR-T2 was a novel floR variant identified in this study, and exhibited less identity with other FloR proteins than FloRv. Moreover, floR-T1 and floR-T2 identified in P. aeruginosa strain TL1285 were functionally active and located on multi-drug resistance region of a novel incomplete Tn4371-like integrative and conjugative elements (ICE) in the chromosome. The expression of the two floR variants could be induced by florfenicol or chloramphenicol. These results indicated that the two floR variants played an essential role in the host's resistance to amphenicol and the spreading of these floR variants might be related with the Tn4371 family ICE.

High-Level Aminoglycoside Resistance in Human Clinical Klebsiella pneumoniae Complex Isolates and Characteristics of armA-Carrying IncHI5 Plasmids.

Aminoglycosides are important options for treating life-threatening infections. However, high levels of aminoglycoside resistance (HLAR) among Klebsiella pneumoniae isolates have been observed to be increasing frequently. In this study, a total of 292 isolates of the K. pneumoniae complex from a teaching hospital in China were analyzed. Among these isolates, the percentage of HLAR strains was 13.7% (40/292), and 15 aminoglycoside resistance genes were identified among the HLAR strains, with rmtB being the most dominant resistance gene (70%, 28/40). We also described an armA-carrying Klebsiella variicola strain KP2757 that exhibited a high-level resistance to all aminoglycosides tested. Whole-genome sequencing of KP2757 demonstrated that the strain contained one chromosome and three plasmids, with all the aminoglycoside resistance genes (including two copies of armA and six AME genes) being located on a conjugative plasmid, p2757-346, belonging to type IncHI5. Comparative genomic analysis of eight IncHI5 plasmids showed that six of them carried two copies of the intact armA gene in the complete or truncated Tn1548 transposon. To the best of our knowledge, for the first time, we observed that two copies of armA together with six AME genes coexisted on the same plasmid in a strain of K. variicola with HLAR. Comparative genomic analysis of eight armA-carrying IncHI5 plasmids isolated from humans and sediment was performed, suggesting the potential for dissemination of these plasmids among bacteria from different sources. These results demonstrated the necessity of monitoring the prevalence of IncHI5 plasmids to restrict their worldwide dissemination.

The genetic feature and virulence determinant of highly virulent community-associated MRSA ST338-SCCmec Vb in China.

ST59 is the predominant pathotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in China. As a variant of ST59, there is relatively little known about the detailed information of ST338. To address this issue, here, we described thirteen ST338 CA-MRSA strains isolated from severe bloodstream infection cases, and focused on their epidemiology, genetic features and virulence potential. Phylogenetic analysis showed the earliest isolated strain of this study is likely a predecessor of recent ST338 lineage (after year of 2014). Furthermore, the phylogenetic reconstruction and time estimation suggested that ST338 evolved from ST59 in 1991. Notably, the carrying patten of virulence factors of all ST338 strains were similar, and the genomic islands νSaα, νSaγ and SaPI and the core virulence factors like hla and psm were detected in ST338 isolates. However, all ST338 isolates lacked some adhesion factors such as clfA, clfB, eap, cna and icaD. Additionally, among these ST338 strains, one PVL-negative ST338 isolate was detected. Experiment on mice nose and human alveolar epithelial cell showed that the nasal colonization ability of ST338 was weaker than that of CA-MRSA MW2. In a mouse bloodstream infection model and skin infection model, PVL+ and PVL- strains had the similar virulence, which was dependent on upregulation of toxin genes rather than the presence of mobile genetic elements such as ΦSa2 carrying PVL. Our findings provide important insight into the epidemiology and pathogenicity of the novel and highly virulent ST338-SCCmec Vb clone.

CXCL10 is a Tumor Microenvironment and Immune Infiltration Related Prognostic Biomarker in Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is the 10th most common cancer worldwide and the outcomes for patients with the disease remain extremely poor. Precision biomarkers are urgently needed to increase the efficiency of early diagnosis and to improve the prognosis of patients. The tumor microenvironment (TME) and tumor immune infiltration are thought to impact the occurrence, progression, and prognosis of PAAD. Novel biomarkers excavated originating from the TME and immune infiltration may be effective in predicting the prognosis of PAAD patients. In the current study, the ESTIMATE and CIBERSORT algorithms were applied to estimate the division of immune and stromal components and the proportion of tumor-infiltrating immune cells in 182 PAAD cases downloaded from The Cancer Genome Atlas database. Intersection analyses of the Protein-Protein Interaction networks and Cox regression analysis identified the chemokine (CXC-motif) ligand 10 (CXCL10) as a predictive biomarker. We verified that CXCL10 in the TME negatively correlates with prognosis in PAAD and positively correlates with tumor cell differentiation. GSE62452 from the GEO database and cumulative survival analysis were performed to validate CXCL10 expression as an independent prognostic indicator. We also found that memory B cells, regulatory T cells, and macrophages M0 and M1 were correlated with the expression of CXCL10 indicating that expression of CXCL10 influenced the immune activity of the TME. Our data suggest that CXCL10 is beneficial as a prognostic indicator in PAAD patients and highlights the potential for immune targeted therapy in the treatment of PAAD.

Rifaximin Modulates the Gut Microbiota to Prevent Hepatic Encephalopathy in Liver Cirrhosis Without Impacting the Resistome.

The gut microbiota has an important role in the pathogenesis of hepatic encephalopathy(HE). Rifaximin, an intestinal non-absorbable antibacterial agent, is effective in the treatment of HE. However, whether long-term prophylactic use induces antibacterial resistance and its mechanism for treating HE remains unclear. This prospective study assessed the impact of 12 weeks rifaximin administration on the gut microbiota and resistome in cirrhotic patients. Fecal sampling was conducted 1 day before the first rifaximin administration and at Weeks 1, 2, 4, 6, 8, 10, 12 of the study. Thirty cirrhotic patients who were in remission from recurrent HE was enrolled to receive rifaximin (400mg TID for 12 weeks). Rifaximin improved hyperammonemia and cognitive function in the 21 patients who completed rifaximin treatment. The dynamic observations showed the gut microbiota diversity, composition and the number of resistance genes, plasmids, insertion sequences did not change significantly during the period(P>0.05). Metabolic pathways such as aromatic amino acids, tryptophan synthesis, urea cycle, and LPS synthesis reduced. No new antimicrobial resistance genes was emergenced. However, the number of aminoglycosides, rifamycin and phenolic resistance genes increased, whereas tetracycline, fosfomycin and cephamycin decreased (P<0.05). Changes in the abundance of E. coli, K. pneumoniae, and B. longum strains correlated with changes of resistance genes. Prophylactic use of rifaximin for 12 weeks improved hyperammonemia and neurophysiological function, maintained gut microbiota diversity, composition and did not change the overall resistome. Rifaximin altered expression of HE-related metabolic pathways. All of these effects could play a key role in preventing HE. Clinical Trial Registration: ChiCTR1900022234 (registered at the Chinese Clinical Trial Registry).

Characterization of an IncR Plasmid with Two Copies of ISCR-Linked qnrB6 from ST968 Klebsiella pneumoniae.

To characterize the molecular structure of IncR plasmid-related sequences, comparative genomic analysis was conducted using 261 IncR plasmid backbone-related sequences. Among the sequences, 257 were IncR plasmids including the multidrug-resistance IncR plasmid pR50-74 from Klebsiella pneumoniae strain R50 of this work, and the other four were from bacterial chromosomes. The IncR plasmids were derived from different bacterial genera or species, mainly Klebsiella pneumoniae (70.82%, 182/257), Escherichia coli (11.28%, 29/257), Enterobacter cloacae (7.00%, 18/257), and Citrobacter freundii (3.50%, 9/257). The bacterial chromosomes carrying IncR plasmid backbone sequences were derived from Proteus mirabilis AOUC-001 and Klebsiella pneumoniae KPN1344, among others. The IncR backbone sequence of P. mirabilis AOUC-001 chromosome shows the highest identity with that of pR50-74. Complex class 1 integrons carrying various copies of ISCR1-sdr-qnrB6-△qacE/sul1 (ISCR1-linked qnrB6 unit) were identified in IncR plasmids. In addition to two consecutive copies of qnrB6-qacE-sul1, the other resistance genes encoded on pR50-74 are all related to mobile genetic elements, such as IS1006, IS26, and the class 1 integron. This study provides a clear understanding of the mobility and plasticity of the IncR plasmid backbone sequence and emphasizes the important role of ISCR in the recruitment of multicopy resistance genes.

Characterization of a Novel Chromosomal Class C ß-Lactamase, YOC-1, and Comparative Genomics Analysis of a Multidrug Resistance Plasmid in Yokenella regensburgei W13.

Yokenella regensburgei, a member of the family Enterobacteriaceae, is usually isolated from environmental samples and generally resistant to early generations of cephalosporins. To characterize the resistance mechanism of Y. regensburgei strain W13 isolated from the sewage of an animal farm, whole genome sequencing, comparative genomics analysis and molecular cloning were performed. The results showed that a novel chromosomally encoded class C ß-lactamase gene with the ability to confer resistance to ß-lactam antibiotics, designated bla YOC - 1, was identified in the genome of Y. regensburgei W13. Kinetic analysis revealed that the ß-lactamase YOC-1 has a broad spectrum of substrates, including penicillins, cefazolin, cefoxitin and cefotaxime. The two functionally characterized ß-lactamases with the highest amino acid identities to YOC-1 were CDA-1 (71.69%) and CMY-2 (70.65%). The genetic context of the bla YOC - 1 -ampR-encoding region was unique compared with the sequences in the NCBI nucleotide database. The plasmid pRYW13-125 of Y. regensburgei W13 harbored 11 resistance genes (bla OXA - 10, bla LAP - 2, dfrA14, tetA, tetR, cmlA5, floR, sul2, ant(3″)-IIa, arr-2 and qnrS1) within an ∼34 kb multidrug resistance region; these genes were all related to mobile genetic elements. The multidrug resistance region of pYRW13-125 shared the highest identities with those of two plasmids from clinical Klebsiella pneumoniae isolates, indicating the possibility of horizontal transfer of these resistance genes between bacteria of various origins.

Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates.

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5-82.1%), and MIC levels (MIC90 > 256 µg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1-3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria's resistance to macrolides.