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In recent years, polymyxin has been used as a last-resort therapy for carbapenem-resistant bacterial infections. The emergence of heteroresistance (HR) to polymyxin hampers the efficacy of polymyxin treatment by amplifying resistant subpopulation. However, the mechanisms behind polymyxin HR remain unclear. Small noncoding RNAs (sRNAs) play an important role in regulating drug resistance. The purpose of this study was to investigate the effects and mechanisms of sRNA on polymyxin B (PB)-HR in carbapenem-resistant Klebsiella pneumoniae. In this study, a novel sRNA PhaS was identified by transcriptome sequencing. PhaS expression was elevated in the PB heteroresistant subpopulation. Overexpression and deletion of PhaS were constructed in three carbapenem-resistant K. pneumoniae strains. Population analysis profiling, growth curve, and time-killing curve analysis showed that PhaS enhanced PB-HR. In addition, we verified that PhaS directly targeted phoP through the green fluorescent protein reporter system. PhaS promoted the expression of phoP, thereby encouraging the expression of downstream genes pmrD and arnT. This upregulation of arnT promoted the 4-amino-4-deoxyL-arabinosaccharide (L-Ara4N) modification of lipid A in PhaS overexpressing strains, thus enhancing PB-HR. Further, within the promoter region of PhaS, specific PhoP recognition sites were identified. ONPG assays and RT-qPCR analysis confirmed that PhaS expression was positively modulated by PhoP and thus up-regulated by PB stimulation. To sum up, a novel sRNA enhancing PB-HR was identified and a positive feedback regulatory pathway of sRNA-PhoP/Q was demonstrated in the study. This helps to provide a more comprehensive and clear understanding of the underlying mechanisms behind polymyxin HR in carbapenem-resistant K. pneumoniae.

Background: Surveillance systems revealed that the prevalence of vancomycin-resistant Enterococcus faecium (VREfm) has increased. We aim to investigate the epidemiological and genomic characteristics of VREfm in China. Methods: We collected 20,747 non-redundant E. faecium isolates from inpatients across 19 hospitals in six provinces between January 2018 and June 2023. VREfm was confirmed by antimicrobial susceptibility testing. The prevalence was analyzed using changepoint package in R. Genomic characteristics were explored by whole-genome sequencing. Results: 5.59% (1159/20,747) of E. faecium isolates were resistant to vancomycin. The prevalence of VREfm increased in Guangdong province from 5% before 2021 to 20−50% in 2023 (p

ABSTRACTThis study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 μg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 μg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

Plasmid-mediated conjugation is a common mechanism for most bacteria to transfer antibiotic resistance genes (ARGs). The conjugative transfer of ARGs is emerging as a major threat to human beings. Although several transfer-related factors are known to regulate this process, small RNAs (sRNAs)-based regulatory roles remain to be clarified. Here, the Hfq-binding sRNA GadY in donor strain Escherichia coli (E. coli) SM10λπ was identified as a new regulator for bacterial conjugation. Two conjugation models established in our previous studies were used, which SM10λπ carrying a chromosomally integrated IncP-1α plasmid RP4 and a mobilizable plasmid pUCP24T served as donor cells, and P. aeruginosa PAO1 or E. coli EC600 as the recipients. GadY was found to promote SM10λπ-PAO1 conjugation by base-pairing with its target mRNA SdiA, an orphan LuxR-type receptor that responds to exogenous N-acylated homoserine lactones (AHLs). However, SM10λπ-EC600 conjugation was not affected due to EC600 lacking AHLs synthase. It indicates that the effects of GadY on conjugation depended on AHLs-SdiA signalling. Further study found GadY bound SdiA to negatively regulate the global RP4 repressors KorA and KorB. When under ciprofloxacin or levofloxacin treatment, GadY expression in donor strain was enhanced, and it positively regulated quinolone-induced SM10λπ-PAO1 conjugation. Thus, our study provides a novel role for sRNA GadY in regulating plasmid-mediated conjugation, which helps us better understand bacterial conjugation to counter antibiotic resistance.

Abstract Background and purpose Whether symptomatic unruptured intracranial aneurysms (UIAs) lead to change in circulating inflammation remains unclear. This study aims to evaluate the role of hematological inflammatory indicators in predicting symptomatic UIA. Methods Adult patients diagnosed with saccular intracranial aneurysm from March 2019 to September 2023 were recruited retrospectively. Clinical and laboratory data, including the white blood cells (WBC), neutral counts (NEUT), lymphocyte counts (LYM), and monocyte counts (MONO) of each patient, were collected. The neutrophil-to-lymphocyte ratio (NLR) and lymphocyte-to-monocyte ratio (LMR) were calculated as NLR = NEUT/LYM, LMR = LYM/MONO, SII = PLT*NEUT/LYM. The hematological inflammatory indicators were compared in symptomatic saccular and asymptomatic UIA patients. Multivariable logistic regression analyses were performed to explore the factors predicting symptomatic UIA. Results One hundred and fifty UIA patients with a mean age of 58.5 ± 12.4 were included, of which 68% were females. The NLR and LMR were significantly associated with symptomatic UIA, and the association remained in small UIAs (

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. Small regulatory RNAs (sRNAs) regulate multiple bacterial adaptations to environmental changes, especially virulence. Our previous study showed that sRNA PrrH negatively regulates the expression of a number of virulence factors, such as pyocyanin, rhamnolipid, biofilm, and elastase in the P. aeruginosa strain PAO1. However, previous studies have shown that the prrH-deficient mutant attenuates virulence in an acute murine lung infection model. All ΔprrH-infected mice survived the entire 28-day course of the experiment, whereas all mice inoculated with the wild-type or the complemented mutant succumbed to lung infection within 4 days of injection, but the specific mechanism is unclear. Herein, we explored how PrrH mediates severe lung injury by regulating the expression of virulence factors. In vivo mouse and in vitro cellular assays demonstrated that PrrH enhanced the pathogenicity of PAO1, causing severe lung injury. Mechanistically, PrrH binds to the coding sequence region of the mRNA of exsA, which encodes the type III secretion system master regulatory protein. We further demonstrated that PrrH mediates a severe inflammatory response and exacerbates the apoptosis of A549 cells. Overall, our results revealed that PrrH positively regulates ExsA, enhances the pathogenicity of P. aeruginosa, and causes severe lung injury.IMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium and the leading cause of nosocomial pneumonia. The pathogenicity of P. aeruginosa is due to the secretion of many virulence factors. Small regulatory RNAs (sRNAs) regulate various bacterial adaptations, especially virulence. Therefore, understanding the mechanism by which sRNAs regulate virulence is necessary for understanding the pathogenicity of P. aeruginosa and the treatment of the related disease. In this study, we demonstrated that PrrH enhances the pathogenicity of P. aeruginosa by binding to the coding sequence regions of the ExsA, the master regulatory protein of type III secretion system, causing severe lung injury and exacerbating the inflammatory response and apoptosis. These findings revealed that PrrH is a crucial molecule that positively regulates ExsA. Type III-positive strains are often associated with a high mortality rate in P. aeruginosa infections in clinical practice. Therefore, this discovery may provide a new target for treating P. aeruginosa infections, especially type III-positive strains.

As an important virulence phenotype of Escherichia coli, the regulation mechanism of biofilm by non-coding RNA and quorum sensing system has not been clarified. Here, by transcriptome sequencing and RT-PCR analysis, we found CsrB, a non-coding RNA of the carbon storage regulation system, was positively regulated by the LuxR protein SdiA. Furthermore, β-galactosidase reporter assays showed that SdiA enhanced promoter transcriptional activity of csrB. The consistent dynamic expression levels of SdiA and CsrB during Escherichia coli growth were also detected. Moreover, curli assays and biofilm assays showed sdiA deficiency in Escherichia coli SM10λπ or BW25113 led to a decreased formation of biofilm, and was significantly restored by over-expression of CsrB. Interestingly, the regulations of SdiA on CsrB in biofilm formation were enhanced by quorum sensing signal molecules AHLs. In conclusion, SdiA plays a crucial role in Escherichia coli biofilm formation by regulating the expression of non-coding RNA CsrB. Our study provides new insights into SdiA-non-coding RNA regulatory network involved in Escherichia coli biofilm formation.

ABSTRACT Pseudomonas aeruginosa (P. aeruginosa) is one of the leading causes of chronic infections, including reinfection, relapse, and persistent infection, especially in cystic fibrosis patients. Relapse P. aeruginosa infections are more harmful because of repeated hospitalization and undertreatment of antimicrobials. However, relapse P. aeruginosa infection in China remains largely unknown. Herein, we performed a 3-year retrospective study from 2019 to 2022 in a tertiary hospital, which included 442 P. aeruginosa isolates from 196 patients. Relapse infection was identified by screening clinical records and whole-genome sequencing (WGS). We found that 31.6% (62/196) of patients had relapsed infections. The relapse incidence of carbapenem-resistant P. aeruginosa infection (51.4%) is significantly higher than that of carbapenem-susceptible P. aeruginosa infection (20.2%, P < 0.0001). These isolates were assigned to 50 distinct sequence types and sporadically distributed in phylogeny, indicating that relapsed infections were not caused by certain lineages. Fast adaptation and evolution of P. aeruginosa isolates were reflected by dynamic changes of antimicrobial resistance, gene loss and acquisition, and single-nucleotide polymorphisms during relapse episodes. Remarkably, a convergent non-synonymous mutation that occurs in a pyochelin-associated virulence gene fptA (T1056C, M252T) could be a considerable target for the diagnosis and treatment of relapse P. aeruginosa infection. These findings suggest that integrated utilization of WGS and medical records provides opportunities for improved diagnostics of relapsed infections. Continued surveillance of the genomic dynamics of relapse P. aeruginosa infection will generate further knowledge for optimizing treatment and prevention in the future. IMPORTANCE Pseudomonas aeruginosa is a predominant pathogen that causes various chronic infections. Relapse infections promote the adaptation and evolution of antimicrobial resistance and virulence of P. aeruginosa, which obscure evolutionary trends and complicate infection management. We observed a high incidence of relapse P. aeruginosa infection in this study. Whole-genome sequencing (WGS) revealed that relapse infections were not caused by certain lineages of P. aeruginosa isolates. Genomic dynamics of relapse P. aeruginosa among early and later stages reflected a plasticity scattered through the entire genome and fast adaptation and genomic evolution in different ways. Remarkably, a convergent evolution was found in a significant virulence gene fptA, which could be a considerable target for diagnosis and treatment. Taken together, our findings highlight the importance of longitudinal surveillance of relapse P. aeruginosa infection in China since cystic fibrosis is rare in Chinese. Integrated utilization of WGS and medical records provides opportunities for improved diagnostics of relapse infections.

A sharp increase in multidrug-resistant tuberculosis (MDR-TB) threatens human health. Spontaneous mutation in essential gene confers an ability of Mycobacterium tuberculosis resistance to anti-TB drugs. However, conventional laboratory strategies for identification and prediction of the mutations in this slowly growing species remain challenging. Here, by combining XCas9 nickase and the error-prone DNA polymerase A from M. tuberculosis, we constructed a CRISPR-guided DNA polymerase system, CAMPER, for effective site-directed mutagenesis of drug-target genes in mycobacteria. CAMPER was able to generate mutagenesis of all nucleotides at user-defined loci, and its bidirectional mutagenesis at nick sites allowed editing windows with lengths up to 80 nucleotides. Mutagenesis of drug-targeted genes in Mycobacterium smegmatis and M. tuberculosis with this system significantly increased the fraction of the antibiotic-resistant bacterial population to a level approximately 60- to 120-fold higher than that in unedited cells. Moreover, this strategy could facilitate the discovery of the mutation conferring antibiotic resistance and enable a rapid verification of the growth phenotype-mutation genotype association. Our data demonstrate that CAMPER facilitates targeted mutagenesis of genomic loci and thus may be useful for broad functions such as resistance prediction and development of novel TB therapies.

ABSTRACT Haemophilus seminalis is a newly proposed species that is phylogenetically related to Haemophilus haemolyticus. The distribution of H. seminalis in the human population, its genomic diversity, and its pathogenic potential are still unclear. This study reports the finding of our comparative genomic analyses of four newly isolated Haemophilus strains (SZY H8, SZY H35, SZY H36, and SZY H68) from human sputum specimens (Guangzhou, China) along with the publicly available genomes of other phylogenetically related Haemophilus species. Based on pairwise comparisons of the 16S rRNA gene sequences, the four isolates showed 95% ANI values) with 17 strains that were previously identified as either “Haemophilus intermedius” or hemin (X-factor)-independent H. haemolyticus and therefore required a more detailed classification study. Phylogenetically, these isolates, along with the two previously described H. seminalis isolates (a total of 23 isolates), shared a highly homologous lineage that is distinct from the clades of the main H. haemolyticus and Haemophilus influenzae strains. These isolates present an open pangenome with multiple virulence genes. Notably, all 23 isolates have a functional heme biosynthesis pathway that is similar to that of Haemophilus parainfluenzae. The phenotype of hemin (X-factor) independence and the analysis of the ispD, pepG, and moeA genes can be used to distinguish these isolates from H. haemolyticus and H. influenzae. Based on the above findings, we propose a reclassification for all “H. intermedius” and two H. haemolyticus isolates belonging to H. seminalis with an emended description of H. seminalis. This study provides a more accurate identification of Haemophilus isolates for use in the clinical laboratory and a better understanding of the clinical significance and genetic diversity in human environments. IMPORTANCE As a versatile opportunistic pathogen, the accurate identification of Haemophilus species is a challenge in clinical practice. In this study, we characterized the phenotypic and genotypic features of four H. seminalis strains that were isolated from human sputum specimens and propose the “H. intermedius” and hemin (X-factor)-independent H. haemolyticus isolates as belonging to H. seminalis. The prediction of virulence-related genes indicates that H. seminalis isolates carry several virulence genes that are likely to play an important role in its pathogenicity. In addition, we depict that the genes ispD, pepG, and moeA can be used as biomarkers for distinguishing H. seminalis from H. haemolyticus and H. influenzae. Our findings provide some insights into the identification, epidemiology, genetic diversity, pathogenic potential, and antimicrobial resistance of the newly proposed H. seminalis.

The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied in routine clinical microbiology laboratories as an efficient and reliable technique for diagnostic purpose. In this work, we evaluated the performance of the newly developed Zybio EXS3000 (Zybio Inc., China) in microbial identification and compared it with VITEK MS (bioMérieux, France). For this study, a total of 1340 isolates from various clinical specimens were collected. These isolates were analyzed simultaneously on both EXS3000 and VITEK MS. The inconsistent or unidentifiable data were further identified using the help of either 16S rRNA gene or ITS region sequencing. During the study, we observed that EXS3000 and VITEK MS provided positive confirmatory diagnostics for 95.0% and 96.5% of the isolates, respectively, which were consistent with the sequencing results. However, it is worth noting that the EXS3000 system needs to improve the identification performance of Candida albicans in the follow-up. There are no significant differences between the two devices in terms of microbial identification performance. The advantage of EXS3000 over VITEK MS is in its ability to perform in significantly lesser time period. In conclusion, the results of this investigation showed that EXS3000 can be used to identify microorganisms in clinical microbiology laboratories.

The prevalence and genetic mechanism of antibiotic heteroresistance (HR) have attracted significant research attention recently. However, non-genetic mechanism of HR has not been adequately explored. The present study aimed to evaluate the role of quorum sensing (QS), an important mechanism of behavioral coordination in different subpopulations and consequent heteroresistance. First, the prevalence of HR to 7 antibiotics was investigated in 170 clinical isolates of P. aeruginosa using population analysis profiles. The results showed that P. aeruginosa was significantly heteroresistant to meropenem (MEM), amikacin (AMK), ciprofloxacin (CIP), and ceftazidime (CAZ). The observed HR was correlated with down-regulation of QS associated genes lasI and rhlI. Further, loss-of-function analysis results showed that reduced expression of lasI and rhlI enhanced HR of P. aeruginosa to MEM, AMK, CIP, and CAZ. Conversely, overexpression of these genes or treatment with 3-oxo-C12-HSL/C4-HSL lowered HR of P. aeruginosa to the four antibiotics. Additionally, although downregulation of oprD and upregulation of efflux-associated genes was evident in heteroresistant subpopulations, their expression was not regulated by LasI and RhlI. Moreover, fitness cost measurements disclosed higher growth rates of PAO1ΔlasI and PAO1ΔrhlI in the presence of sub-MIC antibiotic as compared with that of wild-type PAO1. Our data suggest that under temporary antibiotic pressure, downregulation of QS might result in less fitness cost and promote HR of P. aeruginosa.

In response to the spread of colistin resistance gene mcr-1, China banned the use of colistin in livestock fodders. We used a time-series analysis of inpatient colonization data from 2011–2019 to accurately reveal the associated fluctuations of mcr-1 that occurred in inpatients in response to the ban.

ABSTRACT Pseudomonas aeruginosa is an opportunistic and nosocomial pathogen of humans with hundreds of its virulence factors regulated by quorum sensing (QS) system. Small noncoding RNAs (sRNAs) are also key regulators of bacterial virulence. However, the QS regulatory sRNAs (Qrrs) that have been characterized in P. aeruginosa are still largely unknown. Here, sRNA AmiL (PA3366.1) in the amiEBCRS operon of PAO1 was identified as a novel Qrr by transcriptome sequencing (RNA-Seq). The expression of AmiL was negatively regulated by the las or rhl system, of which RhlR probably inhibited its transcription. AmiL deletion mutant and overexpressing strains were constructed in PAO1. Broad phenotypic changes were found, including reduced pyocyanin synthesis, elastase activity, biofilm formation, hemolytic activity, and cytotoxicity, as well as increased rhamnolipid production and swarming motility. AmiL appears to be a new regulator that influences diverse QS-mediated virulence. Furthermore, AmiL directly targeted PhzC, a key member of pyocyanin synthesis. AmiL also negatively regulated lasI expression in the early growth of PAO1, but predominantly increased rhlI expression and C4-HSL production in the middle and late stages. Therefore, a novel QS-sRNA signaling cascade of las/rhl (RhlR)-AmiL-PhzC/las/rhl was demonstrated, and it will help to shed new light on the virulence regulatory network of P. aeruginosa PAO1. IMPORTANCE P. aeruginosa is a common nosocomial pathogen that causes diverse opportunistic infections in humans. The virulence crucial for infection is mainly regulated by QS. Small noncoding RNAs (sRNAs) involved in virulence regulation have also been identified in many bacteria. Recently, there is a growing interest in the new sRNA species, QS regulatory sRNAs (Qrrs). Understanding Qrrs-mediated regulation in P. aeruginosa virulence is therefore important to combat infection. In this study, a previously uncharacterized sRNA AmiL in PAO1 has been identified as a novel Qrr. It has been found to influence diverse QS-mediated virulence factors including pyocyanin, elastase, rhamnolipid, and hemolysin, as well as biofilm formation, swarming motility, and cytotoxicity. Furthermore, PhzC essential for pyocyanin synthesis was a direct target of AmiL. QS gene expression and C4-HSL production were also regulated by AmiL. This study provides insights into the roles of Qrr AmiL in modulating P. aeruginosa virulence.

ABSTRACT Corynebacterium, particularly Corynebacterium kroppenstedtii, has been increasingly recognized as an important pathogen causing mastitis. However, no clear taxonomic, microbiological, or clinical identification for C. kroppenstedtii-related Corynebacterium species is recognized. During the investigation of isolates cultured from female patients with mastitis, 27 lipophilic C. kroppenstedtii-like isolates were obtained from clinical breast specimens from 2017 to 2019 in Guangzhou, China. These isolates were identified by phenotypic characterization, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), partial sequencing of the 16S rRNA, rpoB, and fusA genes, and whole-genome sequencing methods. By phylogenetic analyses, two major clusters were identified that were closely related to C. kroppenstedtii DSM 44385T. Comparative genome analyses suggested that these isolates formed two distinct genospecies within the genus Corynebacterium. The digital DNA–DNA hybridization (dDDH) values for the two genospecies were 45.5 to 47.8% between them and 47.4 to 47.7% and 49.9% to C. kroppenstedtii DSM 44385T, respectively. Based on these results, it can be concluded that these isolates need to be recognized as two new species of the genus Corynebacterium, for which we proposed the names Corynebacterium parakroppenstedtii sp. nov. and Corynebacterium pseudokroppenstedtii sp. nov. The type strain for the novel species Corynebacterium parakroppenstedtii is MC-26T (NBRC 115146T; CCTCC AB 2020210T), and that for Corynebacterium pseudokroppenstedtii is MC-17XT (NBRC 115143T; CCTCC AB 2020199T). IMPORTANCE In this study, we characterized two novel species that were closely related to but hard to distinguish from C. kroppenstedtii by routine identification methods used in clinical laboratories. Since all 27 C. kroppenstedtii-like isolates were obtained from breast specimens of female patients with mastitis, they may be potential pathogens causing mastitis. We hope to perform further epidemiological investigation of these strains and explore their role in mastitis.

ABSTRACT Clostridioides difficile, which causes life-threatening diarrheal disease, presents an urgent threat to health care systems. In this study, we present a retrospective genomic and epidemiological analysis of C. difficile in a large teaching hospital. First, we collected 894 nonduplicate fecal samples from patients during a whole year to elucidate the C. difficile molecular epidemiology. We then presented a detailed description of the population structure of C. difficile based on 270 isolates separated between 2015 and 2020 and clarified the genetic and phenotypic features by MIC and whole-genome sequencing. We observed a high carriage rate (19.4%, 173/894) of C. difficile among patients in this hospital. The population structure of C. difficile was diverse with a total of 36 distinct STs assigned. In total, 64.8% (175/270) of the isolates were toxigenic, including four CDT-positive (C. difficile transferase) isolates, and 50.4% (135/268) of the isolates were multidrug-resistant. Statistically, the rates of resistance to erythromycin, moxifloxacin, and rifaximin were higher for nontoxigenic isolates. Although no vancomycin-resistant isolates were detected, the MIC for vancomycin was higher for toxigenic isolates (P < 0.01). The in-hospital transmission was observed, with 43.8% (110/251) of isolates being genetically linked to a prior case. However, no strong correlation was detected between the genetic linkage and epidemiological linkage. Asymptomatic colonized patients play the same role in nosocomial transmission as infected patients, raising the issue of routine screening of C. difficile on admission. This work provides an in-depth description of C. difficile in a hospital setting and paves the way for better surveillance and effective prevention of related diseases in China. IMPORTANCE Clostridioides difficile infections (CDI) are the leading cause of healthcare-associated diarrhea and are known to be resistant to multiple antibiotics. In the past decade, C. difficile has emerged rapidly and has spread globally, causing great concern among American and European countries. However, research on CDI remains limited in China. Here, we characterized the comprehensive spectrum of C. difficile by whole-genome sequencing (WGS) in a Chinese hospital, showing a high detection rate among patients, diverse genome characteristics, a high level of antibiotic resistance, and an unknown nosocomial transmission risk of C. difficile. During the study period, two C. difficile transferase (CDT)-positive isolates belonging to a new multilocus sequence type (ST820) were detected, which have caused serious clinical symptoms. This work describes C. difficile integrally and provides new insight into C. difficile surveillance based on WGS in China.

Objectives: Ongoing acquisition of antimicrobial resistance genes has made Morganella morganii a new clinical treatment challenge. Understanding the molecular epidemiology of M. morganii will contribute to clinical treatment and prevention.Methods: We undertook a 6-year clinical molecular epidemiological investigation of M. morganii from three tertiary hospitals in China since 2014. Antimicrobial susceptibility testing was performed using a VITEK-2 system. All isolates were screened for β-lactam and plasmid-mediated quinolone resistance genes by PCR. Isolates carrying carbapenem-resistant genes were subjected to whole-genome sequencing (WGS). The variation and evolution of these mobile genetic elements (MGEs) were then systematically analyzed.Results: Among all M. morganii isolates (n = 335), forty (11.9%) were recognized as multidrug resistant strains. qnrD1, aac(6′)-Ib-cr, blaTEM–104, and blaCTX–M–162 were the top four most prevalent resistance genes. Notably, phylogenomic and population structure analysis suggested clade 1 (rhierBAPS SC3 and SC5) associated with multiple resistance genes seemed to be widely spread. WGS showed a blaOXA–181-carrying IncX3 plasmid and a Proteus genomic island 2 variant carrying blaCTX–M–3, aac(6′)-Ib-cr coexisted in the same multidrug resistant strain zy_m28. Additionally, a blaIMP–1-carrying IncP-1β type plasmid was found in the strain nx_m63.Conclusion: This study indicates a clade of M. morganii is prone to acquire resistance genes, and multidrug resistant M. morganii are increasing by harboring a variety of MGEs including two newly discovered ones in the species. We should be vigilant that M. morganii may bring more extensive and challenging antimicrobial resistance issue.

ABSTRACTSmall regulatory RNAs (sRNAs) act as key regulators in many bacterial signalling cascades. However, in P. aeruginosa, the sRNAs involved in quorum sensing (QS) regulation and their function are still largely unknown. Here, we explored how the prrH locus sRNA influences P. aeruginosa virulence in the context of the QS regulatory network. First, gain- and loss-of-function studies showed that PrrH affects pyocyanin, elastase and rhamnolipid production; biofilm formation; and swimming and swarming motility and impaired the viability of P. aeruginosa in human whole blood. Next, our investigation disclosed that LasI and PhzC/D were directly repressed by PrrH. In addition, RhlI, the key member of the rhl QS system, diminished the expression of PrrH and enhanced the expression of downstream genes. Bioinformatics analysis found two binding sites of RhlR, the transcription factor of the rhl system, on the promoter region of prrH. Further β-galactosidase reporter and qPCR assays confirmed that PrrH was transcriptionally repressed by RhlR. Collectively, our data identified a novel RhlI/R-PrrH-LasI/PhzC/PhzD regulatory circuitry that may contribute to P. aeruginosa pathogenesis. Our findings indicate that PrrH is a quorum regulatory RNA (Qrr) in P. aeruginosa and provide new insight into PrrH’s function.

Indole works as an interspecies signal molecule to regulate multiple physiological activities, like antibiotic resistance, acid resistance, and virulence. However, the effect of indole on conjugation is unknown. Here, with Escherichia coli SM10λπ as a donor strain that carries a chromosomally integrated conjugative RP4 plasmid, we explored the effect of indole on conjugation of a mobilizable pUCP24T plasmid imparting gentamycin resistance. The results showed that exogenous indole treatment inhibited conjugative transfer of pUCP24T from SM10λπ to recipient strains, Pseudomonas aeruginosa PAO1 and E. coli EC600. Furthermore, raising endogenous indole production through overexpression of TnaA, a tryptophanase, in SM10λπ significantly inhibited both SM10λπ-PAO1 and SM10λπ-EC600 conjugation, whereas deficiency of tnaA reversed the phenotype. Subsequent mechanistic studies revealed that exogenous indole significantly inhibited the expression of mating pair formation gene (trbB) and the DNA transfer and replication gene (trfA), mainly due to the promotion of regulatory genes (korA and korB), and the result was confirmed in tnaA knockout and overexpression strains. Additionally, we found that both extracellular indole production and tnaA expression of SM10λπ were downregulated by ciprofloxacin (CIP). Intriguingly, one-eighth minimum inhibitory concentration of CIP treatment clearly facilitated both SM10λπ-PAO1 and SM10λπ-EC600 conjugation, and indole inhibited CIP-induced conjugation frequency. These data suggest that indole may play a negative role in the process of CIP-induced conjugation. This is the first study to reveal the biological function of indole-inhibiting conjugation and its role in CIP-induced conjugation, which may be developed into a new way of controlling the spread of antibiotic resistance.

BackgroundThis study aimed to examine the impact of an intervention carried out in 2011 to combat multi-drug resistance and outbreaks of imipenem-resistant Acinetobacter baumannii (IRAB), and to explore its resistance mechanism.MethodsA total of 2572 isolates of A. baumannii, including 1673 IRAB isolates, were collected between 2007 and 2014. An intervention was implemented to control A. baumannii resistance and outbreaks. Antimicrobial susceptibility was tested by calculating minimal inhibitory concentrations (MICs), and outbreaks were typed using pulsed-field gel electrophoresis (PFGE). Resistance mechanisms were explored by polymerase chain reaction (PCR) and whole genome sequencing (WGS).ResultsFollowing the intervention in 2011, the resistance rates of A. baumannii to almost all tested antibiotics decreased, from 85.3 to 72.6% for imipenem, 100 to 80.8% for ceftriaxone, and 45.0 to 6.9% for tigecycline. The intervention resulted in a decrease in the number (seven to five), duration (8–3 months), and departments (five to three) affected by outbreaks; no outbreaks occurred in 2011. After the intervention, only blaAMPC (76.47 to 100%) and blaTEM–1 (75.74 to 96.92%) increased (P < 0.0001); whereas blaGES–1 (32.35 to 3.07%), blaPER–1 (21.32 to 1.54%), blaOXA–58 (60.29 to 1.54%), carO (37.50 to 7.69%), and adeB (9.56 to 3.08%) decreased (P < 0.0001). Interestingly, the frequency of class B β-lactamase genes decreased from 91.18% (blaSPM–1) and 61.03% (blaIMP–1) to 0%, while that of class D blaOXA–23 increased to 96.92% (P < 0.0001). WGS showed that the major PFGE types causing outbreaks each year (type 01, 11, 18, 23, 26, and 31) carried the same resistance genes (blaKPC–1, blaADC–25, blaOXA–66, and adeABC), AdeR-S mutations (G186V and A136V), and a partially blocked porin channel CarO. Meanwhile, plasmids harboring blaOXA–23 were found after the intervention.ConclusionThe intervention was highly effective in reducing multi-drug resistance of A. baumannii and IRAB outbreaks in the long term. The resistance mechanisms of IRAB may involve genes encoding β-lactamases, efflux pump overexpression, outer membrane porin blockade, and plasmids; in particular, clonal spread of blaOXA–23 was the major cause of outbreaks. Similar interventions may also help reduce bacterial resistance rates and outbreaks in other hospitals.

The acid tolerance mechanism is important for Escherichia coli to resist acidic conditions encountered in mammalian host digestive tract environment. Here, we explored how the LuxR protein SdiA influenced E. coli acid tolerance ability in the context of the glutamate- and glutamine-dependent acid resistance system (AR2). First, using a growth and acid shock assay under different acid stresses, we demonstrated that the deletion of sdiA in SM10λpir or BW25113 led to impaired growth under the acidic environment of pH 3–6, which was restored by complementary expression of SdiA. Next, transcriptome sequencing and qPCR disclosed that the expression of glutamate decarboxylase W (GadW) and GadY, the key members of the AR2 system, were regulated by SdiA. Further, β-galactosidase reporter assays showed that the promoter activity of gadW and gadY was positively regulated by SdiA. Moreover, qPCR and β-galactosidase reporter assays confirmed that the regulation of SdiA on GadW, but not GadY, could be enhanced by quorum sensing (QS) signal molecules AHLs. Collectively, these data suggest that SdiA plays a crucial role in acid tolerance regulation of E. coli. Our findings provide new insights into the important contribution of quorum sensing system AHLs–SdiA to the networks that regulate acid tolerance.

Due to the increasing multidrug resistance and limited antibiotics, polymyxin B revived as the last resort for the treatment of carbapenemase-producing Klebsiella pneumoniae (CRKP). Unfortunately, the heteroresistance hampers polymyxin B monotherapy treatment via the amplification of resistant subpopulation. Reliable polymyxin B based combinations are demanded. Ceftazidime/avibactam has been regarded as a new salvage therapy against CRKP. The occurrence of heteroresistance was confirmed by population analysis profiling (PAP). Our study demonstrated that polymyxin B and ceftazidime/avibactam combinations improved the in vitro antimicrobial activity of polymyxin B and delayed or suppressed the regrowth of resistant subpopulation by time-kill studies. Ceftazidime/avibactam at around MIC values (0.5–1 × MIC) plus clinically achievable concentrations of polymyxin B (0.5–2 mg/L) resulted in sustained killing against polymyxin B-heteroresistant isolates. Active PmrAB and PhoPQ systems and a pmrA mutation (G53R) in resistant subpopulation might associate with heteroresistance, but further investigation was required. Our findings suggested that the heteroresistance represented barriers to polymyxin B efficacy, and the combination of polymyxin B with ceftazidime/avibactam could be potentially valuable for the treatment of heteroresistant CRKP. Further, in vivo studies need to be performed to evaluate the efficacy of this combination against heteroresistant strains.

Enterobacter cloacae has recently emerged as one of the most common carbapenem-resistant Enterobacteriaceae. The emergence and spread of metallo-β-lactamase-producing E. cloacae have posed an immediate threat globally. Here, we investigated the molecular characteristics of 84 carbapenem-resistant Enterobacter cloacae (CREL) collected from three tertiary hospitals in China between 2012 and 2016. Species identification and antimicrobial susceptibility testing were performed using a VITEK-2 system. Carbapenems, polymyxins B, and tigecycline were tested by broth microdilution method. The carbapenem in activation method (CIM) and cefoxitin three-dimensional test were used to detect carbapenemase and AmpC β-lactamase, respectively. Isolates were screened for β-lactam resistance genes by PCR, and expression of ompC and ompF was determined by qRT-PCR. Genetic relatedness was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), while selected isolates were subjected to whole-genome sequencing. Among the 84 CREL isolates, 50 (59.5%) were detected as carbapenemase producers. NDM-1 was the dominant carbapenemase (80.0%), followed by IMP-26 (8.0%) and IMP-4 (6.0%). Notably, we identified the first NDM-1 and IMP-1 co-producing E. cloacae, carrying plasmids of several incompatibility (Inc) groups, including IncHI2, IncHI2A, and IncN. Most isolates showed decreased expression of ompC and/or ompF, and contained a broad distribution of ESBLs and AmpC β-lactamases. These findings suggested that different molecular mechanisms, including carbapenemase, ESBL and/or AmpC plus loss of porins, have contributed to carbapenem resistance. The blaNDM−1-harboring plasmids contained highly conserved gene environment around blaNDM−1 (blaNDM−1-bleMBL-trpF-dsbD-cutA1-groES-groEL), which could be associated with the potential dissemination of blaNDM−1. IMP-type MBL was located within a variety of integrons and usually contained various gene cassettes encoding multidrug resistance. These isolates produced 54 different pulsotypes, and were classified into 42 STs by MLST. Nineteen blaNDM−1-positive E. cloacae isolates obtained from Ningxia had the same pulsotype (PFGE type 1), belonging to ST78 within clonal complex 74 (CC74). The plasmid-based replicon typing indicated that IncX3 plasmids mediated the dissemination of blaNDM−1 among these homologous strains. This is the first report on the outbreak of NDM-1-producing E. cloacae ST78 with contribution of IncX3 plasmids in Northwestern China. There's an immediate need to intensify surveillance attentively to prevent and control the further spread of NDM-1 in China.

To assess the effectiveness and safety of linezolid in comparison with glycopeptides (vancomycin and teicoplanin) for the treatment of Staphylococcus aureus infections, we conducted a meta-analysis of relevant randomized controlled trials. A thorough search of Pubmed and other databases was performed. Thirteen trials on 3863 clinically assessed patients were included. Linezolid was slightly more effective than glycopeptides in the intent-to-treat population (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.01-1.10), was more effective in clinically assessed patients (OR 95% CI: 1.38, 1.17-1.64) and in all microbiologically assessed patients (OR 95% CI: 1.38, 1.15-1.65). Linezolid was associated with better treatment in skin and soft-tissue infections (SSTIs) patients (OR 95% CI: 1.61, 1.22-2.12), but not in bacteraemia (OR 95% CI: 1.24, 0.78-1.97) or pneumonia (OR 95% CI: 1.25, 0.97-1.60) patients. No difference of mortality between linezolid and glycopeptides was seen in the pooled trials (OR 95% CI: 0.98, 0.83-1.15). While linezolid was associated with more haematological (OR 95% CI: 2.23, 1.07-4.65) and gastrointestinal events (OR 95% CI: 2.34, 1.53-3.59), a significantly fewer events of skin adverse effects (OR 95% CI: 0.27, 0.16-0.46) and nephrotoxicity (OR 95% CI: 0.45, 0.28-0.72) were recorded in linezolid. Based on the analysis of the pooled data of randomized control trials, linezolid should be a better choice for treatment of patients with S. aureus infections, especially in SSTIs patients than glycopeptides. However, when physicians choose to use linezolid, risk of haematological and gastrointestinal events should be taken into account according to the characteristics of the specific patient populations.

The etiologies of infectious diseases (IDs) of classic fever of unknown origin (FUO) are multitudinous. Different etiologies affect medication decisions. Here, we have made an attempt to predict the types of etiology on the basis of a machine learning (ML) model for IDs of classic FUO for adults. Ten years clinical data of 408 classic FUO were retrospectively collected from August 2012 to August 2022 in Huzhou Central Hospital. A total of 256 adult patients with ID of classic FUO were divided into four subgroups for clinical characteristic analysis. Random forest (RF), light gradients boosting (Light GBM), and extreme gradient boosting (XGBoost) were used to construct prediction models of 10-fold crossvalidation. The micro average and weighted average of F1 score were calculated to evaluate the performance of the models. SHapley Additive exPlanations (SHAP) was used to explain the relationship between features and the predicted results. Clinical characteristic analysis showed that 25 indices were statistically different (P <0.05). RF, LightGBM and XGBoost models, were constructed on the basis of these indices. Among them, the XGBoost model showed the best performance (micro-F1=0.7129, weighted-F1=0.6618). The areas under the ROC curve of the four subgroups were 0.7477, 0.7162, 0.9200 and 0.7500, respectively. C-reactive protein (CRP), N%, C3, and C4 with high SHAP values were positively correlated with the bacterial ID model output, which was used to distinguished other causes. Bacterial infections were the main cause of IDs. The XGBoost model could be regarded as an auxiliary tool to predict the etiological types of IDs of classic FUO, improve the etiological diagnostic rate, and provide evidence for clinical drug application. [ABSTRACT FROM AUTHOR]