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Abstract Identification, sorting, and sequencing of individual cells directly from in situ samples have great potential for in‐depth analysis of the structure and function of microbiomes. In this work, based on an artificial intelligence (AI)‐assisted object detection model for cell phenotype screening and a cross‐interface contact method for single‐cell exporting, we developed an automatic and index‐based system called EasySort AUTO, where individual microbial cells are sorted and then packaged in a microdroplet and automatically exported in a precisely indexed, “One‐Cell‐One‐Tube” manner. The target cell is automatically identified based on an AI‐assisted object detection model and then mobilized via an optical tweezer for sorting. Then, a cross‐interface contact microfluidic printing method that we developed enables the automated transfer of cells from the chip to the tube, which leads to coupling with subsequent single‐cell culture or sequencing. The efficiency of the system for single‐cell printing is >93%. The throughput of the system for single‐cell printing is ~120 cells/h. Moreover, >80% of single cells of both yeast and Escherichia coli are culturable, suggesting the superior preservation of cell viability during sorting. Finally, AI‐assisted object detection supports automated sorting of target cells with high accuracy from mixed yeast samples, which was validated by downstream single‐cell proliferation assays. The automation, index maintenance, and vitality preservation of EasySort AUTO suggest its excellent application potential for single‐cell sorting.

Background: Rapid and reliable diagnostic methods for Aspergillus fumigatus infection are urgently needed. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 13a (Cas13a) has high sensitivity and specificity in the diagnosis of viral infection. However, its potential use in detecting A. fumigatus remains unexplored. A highly sensitive and specific method using the CRISPR/Cas13a system was developed for the reliable and rapid detection of A. fumigatus. Methods: The conserved internal transcribed spacer (ITS) region of A. fumigatus was used to design CRISPR-derived RNA (crRNA) and the corresponding recombinase polymerase amplification (RPA) primer sequence with the T7 promoter for the CRISPR assay. Twenty-five clinical isolates and 43 bronchoalveolar lavage fluid (BALF) remaining from routine examinations of patients with confirmed pulmonary aspergillosis were collected to further validate the CRISPR assay. Results: No amplification signal was observed when genomic DNA from closely clinically related Aspergillus species, such as Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, as well as Monascus purpureus Went and Escherichia coli, was tested by this assay, and the detection limit for A. fumigatus was 3 copies in a single reaction system. Validation experiments using the 25 clinical isolates demonstrated 91.7% specificity for the A. fumigatus section, and the sensitivity was 100% when first-generation sequencing was used as the standard. There was no significant difference between the PCR and CRISPR methods (P = 1.0), and the diagnosis results of the two methods were consistent (Kappa = 0.459, P = 0.003). Conclusion: The study offers a new validated CRISPR/Cas13a technique for A. fumigatus detection, providing a simple, rapid and affordable test that is ready for application in the diagnosis of A. fumigatus infection.

BackgroundThe emergence of highly drug-resistant K. pneumoniae, has become a major public health challenge. In this work, we aim to investigate the diversity of species and sequence types (STs) of clinical Klebsiella isolates and to characterize the prevalence and structure of class 1 integrons.MethodsBased on the whole genome sequencing, species identification was performed by 16S rRNA gene homology and average nucleotide identity (ANI) analysis. STs were determined in accordance with the international MLST schemes for K. pneumoniae and K. variicola. Integron characterization and comparative genomic analysis were performed using various bioinformatic tools.ResultsSpecies identification showed that the 167 isolates belonged to four species: K. pneumoniae, K. variicola subsp. variicola, K. quasipneumoniae and K. aerogenes. Thirty-six known and 5 novel STs were identified in K. pneumoniae, and 10 novel STs were identified in K. variicola subsp. variicola. Class 1 integrons were found in 57.49% (96/167) of the isolates, and a total of 169 resistance gene cassettes encoding 19 types of resistance genes, including carbapenem resistance gene (blaIPM-4) and class D β-lactamases gene (blaOXA-1 and blaOXA-10), were identified. Among the 17 complete genomes, 29 class 1 integrons from 12 groups were found, only 1 group was encoded on chromosomes. Interestingly, one plasmid (pKP167-261) carrying two copies of approximately 19-kb IS26-Int1 complex resistance region that contains an integron and a multidrug resistance gene fragment.ConclusionThe results of this work demonstrated that the species and STs of the clinical Klebsiella isolates were more complex by the whole genome sequence analysis than by the traditional laboratory methods. Finding of the new structure of MGEs related to the resistance genes indicates the great importance of deeply exploring the molecular mechanisms of bacterial multidrug resistance.

In this study, we identified and characterized a novel chromosomally-encoded class B metallo-β-lactamase (MBL) gene designated blaWUS-1 in a carbapenem-resistant isolate Myroides albus P34 isolated from sewage discharged from an animal farm. Comparative analysis of the deduced amino acid sequence revealed that WUS-1 shares the highest amino acid similarities with the function-characterized MBLs MUS-1 (AAN63647.1; 70.73%) and TUS-1 (AAN63648.1; 70.32%). The recombinant carrying blaWUS-1 exhibited increased MICs levels against a number of β-lactam antimicrobials such as carbenicillin, ampicillin and imipenem, and β-lactamase inhibitors (clavulanic acid and tazobactam). The metallo-β-lactamase WUS-1 could also hydrolyze these antimicrobials and the hydrolytic activities could be inhibited by EDTA. Genetic context analysis of blaWUS-1 revealed that no mobile genetic element was found in its surrounding region. The plasmid pMA84474 of Myroides albus P34 harbored 6 resistance genes (blaOXA-347, aadS, blaMYO-1, ereD, sul2 and ermF) within an approximately 17 kb multidrug resistance (MDR) region. These genes, however, were all related to mobile genetic elements.

Florfenicol is widely used for the treatment of bacterial infections in domestic animals. The aim of this study was to analyze the molecular mechanisms of florfenicol and oxazolidinone resistance in Enterococcus isolates from anal feces of domestic animals. The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method. Polymerase chain reaction (PCR) was performed to analyze the distribution of the resistance genes. Whole-genome sequencing and comparative plasmid analysis was conducted to analyze the resistance gene environment. A total of 351 non-duplicated enteric strains were obtained. Among these isolates, 22 Enterococcus isolates, including 19 Enterococcus. faecium and 3 Enterococcus. faecalis, were further studied. 31 florfenicol resistance genes (13 fexA, 3 fexB, 12 optrA, and 3 poxtA genes) were identified in 15 of the 19 E. faecium isolates, and no florfenicol or oxazolidinone resistance genes were identified in 3 E. faecalis isolates. Whole-genome sequencing of E. faecium P47, which had all four florfenicol and oxazolidinone resistance genes and high MIC levels for both florfenicol (256 mg/L) and linezolid (8 mg/L), revealed that it contained a chromosome and 3 plasmids (pP47-27, pP47-61, and pP47-180). The four florfenicol and oxazolidinone resistance genes were all related to the insertion sequences IS1216 and located on two smaller plasmids. The genes fexB and poxtA encoded in pP47-27, while fexA and optrA encoded in the conjugative plasmid pP47-61. Comparative analysis of homologous plasmids revealed that the sequences with high identities were plasmid sequences from various Enterococcus species except for the Tn6349 sequence from a Staphylococcus aureus chromosome (MH746818.1). The current study revealed that florfenicol and oxazolidinone resistance genes (fexA, fexB, poxtA, and optrA) were widely distributed in Enterococcus isolates from animal in China. The mobile genetic elements, including the insertion sequences and conjugative plasmid, played an important role in the horizontal transfer of florfenicol and oxazolidinone resistance.

Research on resistance against polymyxins induced by the mcr-1 gene is gaining interest. In this study, using agar dilution method, polymerase chain reaction, and comparative genomic analysis, we investigated the colistin resistance mechanism of clinical E. coli isolates. The minimum inhibitory concentration (MIC) analysis results revealed that of the 515 isolates tested, bacteria with significantly increased MIC levels against colistin were isolated in 2019. Approximately one-fifth (17.14% to 19.65%) of the isolates showed MIC values ≥1 mg/L against colistin in 2015, 2016, and 2017. However, in 2019, up to three-quarters (74.11%, 146/197) of the isolates showed MIC values ≥1 mg/L against colistin indicating an increase in colistin resistance. Six isolates (EC7518, EC4968, EC3769, EC16, EC117, EC195, 1.13%, 6/515) were found to carry the mcr-1 gene and a novel mcr-1 variant with Met2Ile mutation was identified in EC3769. All six strains showed higher MIC levels (MIC=4 mg/L) than any mcr-1-negative strains (MIC ≤ 2 mg/L). Whole-genome sequencing of the six mcr-1-positive isolates revealed that EC195 carried the highest number of resistance genes (n = 28), nearly a half more than those of the following EC117 (n = 19). Thus, EC195 showed a wider resistance spectrum and higher MIC levels against the antimicrobials tested than the other five isolates. Multi-locus sequence typing demonstrated that these mcr-1-positive strains belonged to six different sequence types. The six mcr-1 genes were located in three different incompatibility group plasmids (IncI2, IncHI2 and IncX4). The genetic context of mcr-1 was related to a sequence derived from Tn6330 (ISApl1-mcr-1-pap2-ISApl1). Investigations into the colistin resistance mechanism and characterization of the molecular background of the mcr genes may help trace the development and spread of colistin resistance in clinical settings.

Pseudomonas aeruginosa can cause infections in the blood, lungs (pneumonia), or other parts of the body after surgery. To investigate the molecular characteristics of β-lactam antibiotic resistance of P. aeruginosa isolated from a hospital population between 2015 and 2017, in this study, the antimicrobial susceptibility and the resistance gene profile of the bacteria were determined. The Pulsed-field gel electrophoresis (PFGE) was used to characterize the clonal relatedness and sequencing and comparative genomic analysis were performed to analyze the structure of the resistance gene-related sequences. As a result, of the 260 P. aeruginosa strains analyzed, the resistance rates for 6 β-lactam antibiotics ranged from 4.6 to 9.6%. A total of 7 genotypes of 44 β-lactamase genes were identified in 23 isolates (8.9%, 23/260). Four transconjugants from different donors carrying blaCARB-3 exhibited a phenotype of reduced susceptibility to piperacillin–tazobactam, ceftazidime, and cefepime, and 2 transconjugants harboring blaIMP-45 exhibited a phenotype of reduced susceptibility to carbapenems. blaCARB positive isolates (n = 12) presented six PFGE patterns, designated groups A to F. Two bla genes (blaIMP-45 and blaOXA-1) in PA1609 related to a class 1 integron (intI1-blaIMP-45-blaOXA-1-aac(6′)-Ib7-catB3-qacE∆1-sul1) were encoded on a plasmid (pPA1609-475), while the blaCARB-3 gene of PA1616 also related to a class 1 integron was located on the chromosome. The results suggest that β-lactam antibiotic resistance and clonal dissemination exist in this hospital population. It indicates the necessity for molecular surveillance in tracking β-lactamase-producing strains and emphasizes the need for epidemiological monitoring.

ABSTRACTUnexplained pneumonia (UP) caused by a novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) emerged in China in late December 2019 and has infected more than 9000 cases by 31 January 2020. Shanghai reported the first imported case of COVID-19 (Coronavirus Disease 2019) in 20 January 2020. A combinative approach of real-time RT–PCR, CRISPR-based assay and metagenomic next-generation sequencing (mNGS) were used to diagnose this unexplained pneumonia patient. Real-time RT–PCR and CRISPR-based assay both reported positive. This sample belonged to Betacoronavirus and shared a more than 99% nucleotide (nt) identity with the Wuhan SARS-CoV-2 isolates. We further compared pros and cons of common molecular diagnostics in UP. In this study, we illustrated the importance of combining molecular diagnostics to rule out common pathogens and performed mNGS to obtain unbiased potential pathogen result for the diagnosis of UP.

Clinical value of metagenomic next-generation sequencing (mNGS) in pneumonia management is still controversial. A prospective study was conducted to evaluate the clinical impact of PneumoSeq in 57 immunocompetent (ICO) and 75 immunocompromised (ICH) pneumonia patients. The value of PneumoSeq for both etiological and clinical impact investigation in pneumonia was assessed. Among the 276 potential pathogens detected with PneumoSeq in our cohort, 251 (90.9%) were cross-validated. Clinical diagnoses of the causative pathogens were obtained for 97 patients, 90.7% of which were supported by PneumoSeq. Compared to conventional testing, PneumoSeq suggested potentially missed diagnoses in 16.7% of cases (22/132), involving 48 additional pathogenic microorganisms. In 58 (43.9%) cases, PneumoSeq data led to antimicrobial treatment de-escalation (n = 12 in ICO, n = 18 in ICH) and targeted treatment initiation (n = 7 in ICO, n = 21 in ICH). The PneumoSeq assay benefited the diagnosis and clinical management of both ICH and ICO pneumonia patients in real-world settings.

A novel plasmid-encoded aminoglycoside 3''-nucleotidyltransferase ANT(3")-IId, was discovered in Acinetobacter lwoffi strain H7 isolated from a chick on an animal farm in Wenzhou, China. The whole-genome of A. lwoffii H7 consisted of one chromosome and five plasmids (pH7-250, pH7-108, pH7-68, pH7-48, and pH7-11). ant(3")-IId was identified as being encoded on pH7-250, sharing the highest amino acid identity of 50.64% with a function-known resistance gene, ant(3")-IIb (KB849358.1). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 3"-nucleotidyltransferase. The ant(3")-IId gene conferred resistance to spectinomycin and streptomycin [the minimum inhibitory concentration (MIC) levels of both increased 16-fold compared with the control strain]. Consistent with the MIC data, kinetic analysis revealed a narrow substrate profile including spectinomycin and streptomycin, with Kcat/Km ratios of 4.99 and 4.45×103M−1 S−1, respectively. Sequencing analysis revealed that the ant(3")-IId gene was associated with insertion sequences (IS) element [ΔISAba14-ΔISAba14-hp-orf-orf-orf1-ant(3")-IId], and ant(3")-IId were identified in plasmids from various Acinetobacter species. This study of the novel aminoglycoside 3"-nucleotidyltranferase ANT(3")-IId helps us further understand the functional and sequence characteristics of aminoglycoside 3"-nucleotidyltranferases, highlights the risk of resistance gene transfer among Acinetobacter species and suggests that attention should be given to the emergence of new aminoglycoside 3"-nucleotidyltranferase genes.

Coagulase-negative Staphylococcus warneri is an opportunistic pathogen that is capable of causing several infections, especially in patients with indwelling medical devices. Here, we determined the complete genome sequence of a clinical S. warneri strain isolated from the blood culture of a 1-year-old nursling patient with acute upper respiratory infection. Genome-wide phylogenetic analysis confirmed the phylogenetic relationships between S. warneri and other Staphylococcus species. Using comparative genomics, we identified three cell wall-anchored (CWA) proteins at the same locus (sdr), named SdrJ, SdrK, and SdrL, on the chromosome sequences of different S. warneri strains. Structural predictions showed that SdrJ/K/L have structural features characteristic of Sdr proteins but exceptionally contained an unusual N-terminal repeat region. However, the C-terminal repetitive (R) region of SdrJ contains a significantly larger proportion of alanine (142/338, 42.01%) than the previously reported SdrI (37.00%). Investigation of the genetic organization revealed that the sdrJ/K/L genes were always followed by one or two glycosyltransferase genes, gtfA and gtfB and were present in an ∼56 kb region bordered by a pair of 8 bp identical direct repeats, named Sw-Sdr. This region was further found to be located on a 160-kb region subtended by a pair of 160-bp direct repeats along with other virulence genes and resistance genes. Sw-Sdr contained a putative integrase that was probably a remnant of a functional integrase. Evidence suggests that Sw-Sdr is improbably an efficient pathogenicity island. A large-scale investigation of Staphylococcus genomes showed that sdr loci were a potential hotspot of insertion sequences (ISs), which could lead to intraspecific diversity at these loci. Our work expanded the repository of Staphylococcus Sdr proteins, and for the first time, we established the connection between sdr loci and phylogenetic relationships and compared the sdr loci in different Staphylococcus species, which provided large insights into the genetic environment of CWA genes in Staphylococcus.

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 β-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.

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.

Soil is home to an enormous and complex microbiome that features arguably the highest genomic diversity and metabolic heterogeneity of cells on Earth. Their in situ

ABSTRACTRapid and simple-to-use diagnostic methods for tuberculosis are urgently needed. Recent development has unveiled the diagnostic power of the CRISPR system in the detection of viral infections. However, its potential use in detecting the Mycobacterium tuberculosis complex (MTB) remained unexplored. We developed a rapid CRISPR-based assay for TB detection and conducted a retrospective cohort study of 179 patients to evaluate the CRISPR-MTB test for identifying MTB in various forms of direct clinical samples. Its diagnostic performance was compared, in parallel with culture and the GeneXpert MTB/RIF assay (Xpert). The CRISPR-MTB test is highly sensitive with a near single-copy sensitivity, demands less sample input and offers shorter turnaround time than Xpert. When evaluated in the clinical cohort of both pulmonary and extra-pulmonary tuberculosis, the CRISPR-MTB test exhibited an overall improved sensitivity over both culture (79% vs 33%) and Xpert (79% vs 66%), without comprise in specificity (62/63, 98%). The CRISPR-MTB test exhibits an improved overall diagnostic performance over culture and Xpert across a variety of sample types, and offers great potential as a new diagnostic technique for both pulmonary and extra-pulmonary tuberculosis.

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.

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 β-lactamase gene with the ability to confer resistance to β-lactam antibiotics, designated blaYOC–1, was identified in the genome of Y. regensburgei W13. Kinetic analysis revealed that the β-lactamase YOC-1 has a broad spectrum of substrates, including penicillins, cefazolin, cefoxitin and cefotaxime. The two functionally characterized β-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 blaYOC–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 (blaOXA–10, blaLAP–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.

Bacterial resistance to antibiotics has become an important concern for public health. This study was aimed to investigate the characteristics and the distribution of the florfenicol-related resistance genes in bacteria isolated from four farms. A total of 106 florfenicol-resistant Gram-negative bacilli were examined for florfenicol-related resistance genes, and the positive isolates were further characterized. The antimicrobial sensitivity results showed that most of them (100, 94.33%) belonged to multidrug resistance Enterobacteriaceae. About 91.51% of the strains carried floR gene, while 4.72% carried cfr gene. According to the pulsed-field gel electrophoresis results, 34 Escherichia coli were subdivided into 22 profiles, the genetic similarity coefficient of which ranged from 80.3 to 98.0%. The multilocus sequence typing (MLST) results revealed 17 sequence types (STs), with ST10 being the most prevalent. The genome sequencing result showed that the Proteus vulgaris G32 genome consists of a 4.06-Mb chromosome, a 177,911-bp plasmid (pG32-177), and a 51,686-bp plasmid (pG32-51). A floR located in a drug-resistant region on the chromosome of P. vulgaris G32 was with IS91 family transposase, and the other floR gene on the plasmid pG32-177 was with an ISCR2 insertion sequence. The cfr gene was located on the pG32-51 flanked by IS26 element and TnpA26. This study suggested that the mobile genetic elements played an important role in the replication of resistance genes and the horizontal resistance gene transfer.

Staphylococcus caprae, Staphylococcus capitis, and Staphylococcus epidermidis belong to the “Epidermidis Cluster Group” (ECG) and are generally opportunistic pathogens. In this work, whole genome sequencing, molecular cloning and pan-genome analysis were performed to investigate the genetic characteristics of the resistance, virulence and genome structures of 69 ECG strains, including a clinical isolate (S. caprae SY333) obtained in this work. Two resistance genes (blaZ and aadD2) encoded on the plasmids pSY333-41 and pSY333-45 of S. caprae SY333 were confirmed to be functional. The bla region in ECG exhibited three distinct structures, and these chromosome- and plasmid-encoded bla operons seemed to follow two different evolutionary paths. Pan-genome analysis revealed their pan-genomes tend to be “open.” For the virulence-related factors, the genes involved in primary attachment were observed almost exclusively in S. epidermidis, while the genes associated with intercellular aggregation were observed more frequently in S. caprae and S. capitis. The type VII secretion system was present in all strains of S. caprae and some of S. epidermidis but not in S. capitis. Moreover, the isd locus (iron regulated surface determinant) was first found to be encoded on the genomes of S. caprae and S. capitis. These findings suggested that the plasmid and chromosome encoded bla operons of ECG species underwent different evolution paths, as well as they differed in the abundance of virulence genes associated with adherence, invasion, secretion system and immune evasion. Identification of isd loci in S. caprae and S. capitis indicated their ability to acquire heme as nutrient iron during infection.

The diversity of class D β-lactamases mediating resistance to β-lactams has been increasingly reported recently. In this study, a novel class D oxacillinase named OXA-830 was identified in a fully sequenced Aeromonas simiae strain, which was isolated from sewage discharged from a farm in southern China. OXA-830 shares the highest amino acid identity of 79.3% with an OXA-12-like variant named OXA-725. When expressed in E. coli DH5α, OXA-830 conferred resistance to penicillins and selected β-lactamase inhibitors but not to cephalosporins and carbapenems. Kinetic analysis of OXA-830 revealed a broad substrate profile including penicillins, cefazolin, cefoxitin, and ceftazidime but not carbapenems. The hydrolytic activity was significantly inhibited by sulbactam, followed by tazobactam, but was less effectively inhibited by clavulanic acid. The blaOXA–830 gene was located on the A. simiae A6 chromosome and the blaOXA–830-related region was bracketed by a pair of perfect inverted repeats.

Staphylococcus aureus is a zoonotic pathogen that causes various life-threatening diseases. The mechanisms of action of amoxicillin against S. aureus are unclear. Here, we established a rabbit tissue cage infection model to evaluate the relationship between the pharmacokinetic/pharmacodynamic (PK/PD) parameters of amoxicillin and selective enrichment of resistant strains of S. aureus and to elucidate the evolution of its resistance to amoxicillin. S. aureus was injected into the tissue cages at 1010 colony forming units (CFU)/mL. We injected different intramuscular concentrations of amoxicillin at doses of 5, 10, 20, and 30 mg/kg body weight once a day for 5 days and 5, 10, 20, and 30 mg/kg body weight twice a day for 2.5 days. Differences in gene expression between two differentially resistant strains and a sensitive strain were evaluated using Illumina sequencing followed by COG and KEGG analysis. RT-qPCR was carried out to validate the difference in protein translation levels. Our results demonstrated that the emergence of resistant bacteria was dose dependent within a given time interval. In the same dosage group, the appearance of resistant bacteria increased with time. The resistant bacteria showed cumulative growth, and the level of resistance increased over time. The resistant bacteria were completely inhibited when the cumulative percentage of time over a 24-h period that the drug concentration exceeded the mutant prevention concentration (MPC) (%T > MPC) was ≥52%. We also found that mecA and femX in S. aureus played a leading role in the development of resistance to amoxicillin. In conclusion, it provide references for optimizing amoxicillin regimens to treat infections caused by S. aureus.

Similar to other CTX-M family enzymes, KLUC is a recently identified and emerging determinant of cefotaxime resistance that has been recovered from at least three Enterobacteriaceae species, including Kluyvera cryocrescens, Escherichia coli, and Enterobacter cloacae. Whether this extended-spectrum β-lactamase (ESBL) has been disseminated among commonly isolated Enterobacteriaceae is worthy of further investigation. In this study, we screened 739 nosocomial Enterobacteriaceae isolates (240 Klebsiella pneumoniae and 499 E. coli strains) and found that one K. pneumoniae and four E. coli isolates harbored the blaKLUC gene. Three blaKLUC determinants isolated from E. coli were entirely identical to a blaKLUC-3 gene previously recovered in the same hospital. PFGE of four blaKLUC-harboring E. coli strains showed that prevalence of these determinants was most likely mediated by horizontal gene transfer but not clonal dissemination. However, the variant isolated from K. pneumoniae belonged to a novel member of the KLUC enzyme group. This newly identified enzyme (KLUC-5) has an amino acid substitution compared with previously identified KLUC-1 (G18S) and KLUC-3 (G240D). Antimicrobial susceptibility tests showed that KLUC-5 significantly reduced resistance activity to almost all the selected antimicrobials compared to previously identified KLUC-3. Site-directed mutagenesis showed that blaKLUC-5-D240G and blaKLUC-5-S18G significantly enhanced the MIC against its best substrate. Conjugation and S1-PFGE indicated that blaKLUC-5 was located on a transferable plasmid, which was further decoded by single-molecule, real-time sequencing. Comparative genome analysis showed that its backbone exhibited genetic homology to the IncA/C incompatibility group plasmids. A transposable element, ISEcp1, was detected 256-bp upstream of the blaKLUC-5 gene; this location was inconsistent with the previously identified blaKLUC-1 but congruent with the variants recovered from E. coli in the same hospital. These data provide evidence of the increasingly emerging KLUC group of ESBLs in China.

Many Pseudomonas aeruginosa virulence traits that contribute to human infections are accepted as being associated with its environmental lifestyle. Therefore, identifying the molecular mechanisms that govern the lifestyle choice is of high significance. We previously reported that a mutation in suhB results in a decrease in swimming motility and increased biofilm formation compared to the wild-type strain. Yet, little is known about how this occurs. In this study, we demonstrated that SuhB inversely regulates motility and biofilm formation through the GacA-RsmY/Z-RsmA cascade. Mutations in gacA or the two small RNAs rsmY/rsmZ, or overproduction of the RsmA protein essentially rescued the motility defect of the suhB mutant. Additionally, we identified a c-di-GMP mediated mechanism for SuhB regulation of motility and biofilm formation. We showed that the ΔsuhB mutant displayed elevated levels of c-di-GMP, and the ΔsuhB motility and biofilm phenotypes could be switched by artificially decreasing c-di-GMP levels. Further experiments led to the identification of the diguanylate cyclase GcbA responsible for regulating the c-di-GMP concentration in ΔsuhB and hence the switch between planktonic and surface-associated growth. Together, our results demonstrate a novel mechanism for SuhB regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks in P. aeruginosa.