Complete genome sequence of Australian soil bacterium Rouxiella badensis DAR84756 resolved with Oxford Nanopore long-read and Illumina sequences.

The complete genome sequence of the bacterium Rouxiella badensis DAR84756, isolated from soil in Orange, NSW, Australia, was resolved using a combination of Nanopore long-read and Illumina short-read sequencing. The genome consists of a single, circular chromosome of 5,004,491 bp and a plasmid of 40,722 bp.

Dominance of Escherichia coli sequence types ST73, ST95, ST127 and ST131 in Australian urine isolates: a genomic analysis of antimicrobial resistance and virulence linked to F plasmids.

Extraintestinal pathogenic Escherichia coli (ExPEC) are the most frequent cause of urinary tract infections (UTIs) globally. Most studies of clinical E. coli isolates are selected based on their antimicrobial resistance (AMR) phenotypes; however, this selection bias may not provide an accurate portrayal of which sequence types (STs) cause the most disease. Here, whole genome sequencing (WGS) was performed on 320 E. coli isolates from urine samples sourced from a regional hospital in Australia in 2006. Most isolates (91%) were sourced from patients with UTIs and were not selected based on any AMR phenotypes. No significant differences were observed in AMR and virulence genes profiles across age sex, and uro-clinical syndromes. While 88 STs were identified, ST73, ST95, ST127 and ST131 dominated. F virulence plasmids carrying senB-cjrABC (126/231; 55%) virulence genes were a feature of this collection. These senB-cjrABC+ plasmids were split into two categories: pUTI89-like (F29:A-:B10 and/or >95 % identity to pUTI89) (n=73) and non-pUTI89-like (n=53). Compared to all other plasmid replicons, isolates with pUTI89-like plasmids carried fewer antibiotic resistance genes (ARGs), whilst isolates with senB-cjrABC+/non-pUTI89 plasmids had a significantly higher load of ARGs and class 1 integrons. F plasmids were not detected in 89 genomes, predominantly ST73. Our phylogenomic analyses identified closely related isolates from the same patient associated with different pathologies and evidence of strain-sharing events involving isolates sourced from companion and wild animals.

Complete Genome Sequences of the Type Strains Rouxiella badensis DSM 100043 and R. chamberiensis DSM 28324, Resolved Using Nanopore Long-Read Sequencing.

The complete genome sequences of Rouxiella badensis DSM 100043T and Rouxiella chamberiensis DSM 28324T were determined using Oxford Nanopore long-read sequencing and the Flye assembler. The former contains a circular chromosome of 4,964,479 bp and a circular plasmid of 116,582 bp; the latter contains a circular chromosome of 4,639,296 bp.

A pseudo-outbreak of Cyberlindnera fabianii funguria: Implication from whole genome sequencing assay.

Background: Although the yeast Cyberlindnera fabianii (C. fabianii) has been rarely reported in human infections, nosocomial outbreaks caused by this organism have been documented. Here we report a pseudo-outbreak of C. fabianii in a urology department of a Chinese hospital over a two-week period. Methods: Three patients were admitted to the urology department of a tertiary teaching hospital in Beijing, China, from Nov to Dec 2018, for different medical intervention demands. During the period Nov 28 to Dec 5, funguria occurred in these three patients, and two of them had positive urine cultures multiple times. Sequencing of rDNA internal transcribed spacer (ITS) region and MALDI-TOF MS were applied for strain identification. Further, sequencing of rDNA non-transcribed spacer (NTS) region and whole genome sequencing approaches were used for outbreak investigation purpose. Results: All the cultured yeast strains were identified as C. fabianii by sequencing of ITS region, and were 100% identical to the C. fabianii type strain CBS 5640T. However, the MALDI-TOF MS system failed to correctly identify this yeast pathogen. Moreover, isolates from these three clustered cases shared 99.91%-100% identical NTS region sequences, which could not rule out the possibility of an outbreak. However, whole genome sequencing results revealed that only two of the C. fabianii cases were genetically-related with a pairwise SNP of 192 nt, whilst the third case had over 26,000 SNPs on its genome, suggesting a different origin. Furthermore, the genomes of the first three case strains were phylogenetically even more diverged when compared to a C. fabianii strain identified from another patient, who was admitted to a general surgical department of the same hospital 7 months later. One of the first three patients eventually passed away due to poor general conditions, one was asymptomatic, and other clinically improved. Conclusion: In conclusion, nosocomial outbreaks caused by emerging and uncommon fungal species are increasingly being reported, hence awareness must be raised. Genotyping with commonly used universal gene targets may have limited discriminatory power in tracing the sources of infection for these organisms, requiring use of whole genome sequencing to confirm outbreak events.

Possible step-up in prevalence for Escherichia coli ST131 from fecal to clinical isolates: inferred virulence potential comparative studies within phylogenetic group B2.

BACKGROUND: Escherichia coli sequence type (ST)131 is an important urinary tract pathogen, and is responsible for considerable healthcare-associated problems and costs worldwide. A better understanding of the factors that contribute to its rapid worldwide spread may help in arresting its continual spread. We studied a large collection of fecal and urinary E. coli ST131 and E. coli non-ST131 phylogenetic group B2 isolates, from women, men and children, in regional NSW, Australia. RESULTS: We found out that there was a step up in ST131 prevalence (and possibly in virulence) from fecal to clinical (urinary) isolates in general, and specifically among ciprofloxacin resistant isolates, in the 3 host groups. Furthermore, our results revealed that the inferred virulence potential of the ST131 isolates (as measured by VF gene scores) was much higher than that of non-ST131 phylogenetic group B2 isolates, and this was much more pronounced amongst the urinary isolates. This finding suggests presence of possible E. coli phylogenetic B2 subgroups with varying levels of virulence, with ST131 being much more virulent compared to others. A strong association between ST131 and fluoroquinolone (FQ) resistance was also demonstrated, suggesting that FQ use is related to ST131 emergence and spread. Specifically, about 77% of ST131 isolates from women and men, and 47% from children, were extended spectrum ß- lactamase (ESBL) producers. Moreover, FQ resistant ST131 ESBL isolates on average harbored more VF genes than all other isolates. CONCLUSIONS: The strong association between ST131 prevalence and FQ resistance amongst the studied isolates suggests that FQ use is related to ST131 emergence and spread. Furthermore, our results demonstrate that FQ resistance and a plurality of VF genes can exist together in ST131, something that has traditionally been regarded as being inversely related. This may partly contribute to the emergence and worldwide spread of ST131.

Distribution of papG alleles among uropathogenic Escherichia coli from reproductive age women.

BACKGROUND: Extraintestinal Escherichia coli (E. coli) causing urinary tract infections (UTIs), and often referred to as uropathogenic E. coli (UPEC), are a major contributor to the morbidity of UTIs and associated healthcare costs. UPEC possess several virulence factors (VFs) for infecting and injuring the host. We studied the papG allele distribution, and its association with other VF genes and phylogenetic groups, amongst 836 UPEC and fecal isolates from reproductive age women. RESULTS: The papGII gene was highly prevalent amongst pyelonephritis isolates (68%), whilst the majority, albeit smaller proportion, of cystitis isolates (31%) harboured the papGIII gene. Among the pyelonephritis and cystitis isolates, papG positive isolates on average had higher VF gene scores, and were more likely to belong to phylogenetic group B2, than their negative counterparts. This was mostly due to the contribution of papGII isolates, which on average contained more VF genes than their papGIII counterparts, irrespective of the uro-clinical syndrome. However, the papGII isolates from the pyelonephritis cohort had higher VF gene scores than the cystitis ones, suggesting presence of possible papGII clones with differing inferred virulence potential. Furthermore, papGII isolates were more likely to possess an intact pap gene operon than their papGIII counterparts. Also of note was the high proportion of isolates with the papGI allele which was not associated with other pap operon genes; and this finding has not been described before. CONCLUSIONS: The association of the papGII gene with several VF genes compared to the papGIII gene, appears to explain the abundance of these genes in pyelonephritis and cystitis isolates, respectively.

Molecular Characterization of Penicillin-Binding Protein2x, 2b and 1a of Streptococcus pneumoniae Causing Invasive Pneumococcal Diseases in China: A Multicenter Study.

Streptococcus pneumoniae is a common human pathogen that can cause severe invasive pneumococcal diseases (IPDs). Penicillin-binding proteins (PBPs) are the targets for ß-lactam antibiotics (BLAs), which are the common empirical drugs for treatment of pneumococcal infection. This study investigated the serotype distribution and antibiotic resistance patterns of S. pneumoniae strains causing IPD in China, including exploring the association between penicillin (PEN) susceptibility and PBPs variations. A total of 300 invasive S. pneumoniae isolates were collected from 27 teaching hospitals in China (2010-2015). Serotypes were determined by Quellung reaction. Serotypes 23F and 19F were the commonest serotypes in isolates from cerebrospinal fluid (CSF), whilst serotypes 19A and 23F were most commonly seen in non-CSF specimens. Among the 300 invasive S. pneumoniae strains, only one strain (serotype 6A, MIC = 0.25 µg/ml) with PEN MIC value ≤ 0.25 µg/ml did not have any substitutions in the PBPs active sites. All the strains with PEN MIC value ≥ 0.5 µg/ml had different substitutions within PBPs active sites. Substitutions in PBP2b and PBP2x active sites were common in low-level penicillin-resistant S. pneumoniae (PRSP) strains (MIC = 0.5 µg/ml), with or without PBP1a substitution, while all strains with PEN MIC ≥ 1 µg/ml had substitutions in PBP1a active sites, accompanied by PBP2b and PBP2x active site substitutions. Based on the three PBPs substitution combinations, a high degree of diversity was observed amongst the isolates. This study provides some new insights for understanding the serology and antibiotic resistance dynamics of S. pneumoniae causing IPD in China. However, further genomic studies are needed to facilitate a comprehensive understanding of antibiotic resistance mechanisms of S. pneumoniae.

Rapid identification of Streptococcus pneumoniae serotypes by cpsB gene-based sequetyping combined with multiplex PCR.

BACKGROUND/PURPOSE: Streptococcus pneumoniae is an important human pathogen that causes invasive infections in adults and children. Accurate serotyping is important to study its epidemiological distribution and to assess vaccine efficacy. METHODS: Invasive S. pneumoniae isolates (n = 300) from 27 teaching hospitals in China were studied. The Quellung reaction was used as the gold standard to identify the S. pneumoniae serotypes. Subsequently, multiplex PCR and cpsB gene-based sequetyping methods were used to identify the serotypes. RESULTS: Based on the Quellung reaction, 299 S. pneumoniae isolates were accurately identified to the serotype level and 40 different serotypes were detected. Only one strain was non-typeable, and five most common serotypes were identified: 23F (43, 14.3%), 19A (41, 13.7%), 19F (41, 13.7%), 3 (31, 10.3%), and 14 (27, 9.0%). Overall, the multiplex PCR method identified 73.3 and 20.7% of the isolates to the serotype and cluster levels, respectively, with 1.7% of the isolates misidentified. In contrast, the cpsB sequetyping method identified 59.0 and 30.3% of the isolates to the serotype and cluster levels, respectively, and 7% were misidentified. CONCLUSIONS: The cpsB gene sequetyping method combined with multiplex PCR, can greatly improve the accuracy and efficiency of serotyping, besides reducing the associated costs.

Genomic comparisons of Escherichia coli ST131 from Australia.

Escherichia coli ST131 is a globally dispersed extraintestinal pathogenic E. coli lineage contributing significantly to hospital and community acquired urinary tract and bloodstream infections. Here we describe a detailed phylogenetic analysis of the whole genome sequences of 284 Australian ST131 E. coli isolates from diverse sources, including clinical, food and companion animals, wildlife and the environment. Our phylogeny and the results of single nucleotide polymorphism (SNP) analysis show the typical ST131 clade distribution with clades A, B and C clearly displayed, but no niche associations were observed. Indeed, interspecies relatedness was a feature of this study. Thirty-five isolates (29 of human and six of wild bird origin) from clade A (32 fimH41, 2 fimH89, 1 fimH141) were observed to differ by an average of 76 SNPs. Forty-five isolates from clade C1 from four sources formed a cluster with an average of 46 SNPs. Within this cluster, human sourced isolates differed by approximately 37 SNPs from isolates sourced from canines, approximately 50 SNPs from isolates from wild birds, and approximately 52 SNPs from isolates from wastewater. Many ST131 carried resistance genes to multiple antibiotic classes and while 41 (14 %) contained the complete class one integron-integrase intI1, 128 (45 %) isolates harboured a truncated intI1 (462-1014 bp), highlighting the ongoing evolution of this element. The module intI1-dfrA17-aadA5-qacEΔ1-sul1-ORF-chrA-padR-IS1600-mphR-mrx-mphA, conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, sulphonamides, chromate and macrolides, was the most common structure. Most (73 %) Australian ST131 isolates carry at least one extended spectrum ß-lactamase gene, typically blaCTX-M-15 and blaCTX-M-27. Notably, dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a unique feature of clade C ST131 isolates, were identified in some clade A isolates. The results of this study indicate that the the ST131 population in Australia carries diverse antimicrobial resistance genes and plasmid replicons and indicate cross-species movement of ST131 strains across diverse reservoirs.

Kodamaea ohmeri as an Emerging Human Pathogen: A Review and Update.

Background: Kodamaea ohmeri, previously known as Pichia ohmeri or Yamadazyma ohmeri, has been regarded as an emerging human pathogen in recent decades, and has caused various types of infections with high mortality. This study systematically reviewed all the published cases of K. ohmeri infection, aiming to have a better understanding of the clinical and epidemiological characteristics of the organism. Methods: All the published literature (as of March 31, 2021) on K. ohmeri, in four databases: PubMed, Embase, Web of Science, and CNKI, were systematically reviewed to select appropriate studies for summarizing the demographic information, clinical and microbiological characteristics of relevant infections. Results: A total of 51 studies involving 67 patients were included for final analysis, including 49 sporadic cases and two clusters of outbreaks. Neonates and the elderly constituted the majority of patients, and fungemia was the dominant infection type. Comorbidities (like malignancy, diabetes, and rheumatism), invasive operations, previous antibiotic use and prematurity, were commonly described in patients. Gene sequencing and broth microdilution method, were the most reliable way for the identification and antifungal susceptibility testing of K. ohmeri, respectively. Amphotericin B and fluconazole were the commonest antifungal therapies administered. The calculated mortality rates for K. ohmeri infection was higher than that of common candidemia. Conclusion: In this study, we systematically reviewed the epidemiology, clinical characteristics, microbiological features, treatment, and outcomes, of all the published cases on K. ohmeri. Early recognition and increased awareness of K. ohmeri as an emerging human pathogen by clinicians and microbiologists is important for effective management of this organism.

High Prevalence of Extended-Spectrum Beta-Lactamases in Escherichia coli Strains Collected From Strictly Defined Community-Acquired Urinary Tract Infections in Adults in China: A Multicenter Prospective Clinical Microbiological and Molecular Study.

OBJECTIVE: The objective of the study was to investigate the antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) positive rates of Escherichia coli from community-acquired urinary tract infections (CA-UTIs) in Chinese hospitals. MATERIALS AND METHODS: A total of 809 E. coli isolates from CA-UTIs in 10 hospitals (5 tertiary and 5 secondary hospitals) from different regions in China were collected during the period 2016-2017 according to the strict inclusion criteria. Antimicrobial susceptibility testing was carried out by standard broth microdilution method. Isolates were categorized as ESBL-positive, ESBL-negative, and ESBL-uncertain groups according to the CLSI recommended phenotypic screening method. ESBL and AmpC genes were amplified and sequenced on ESBL-positive and ESBL-uncertain isolates. RESULTS: The antimicrobial agents with susceptibility rates of greater than 95% included imipenem (99.9%), colistin (99.6%), ertapenem (98.9%), amikacin (98.3%), cefmetazole (97.9%), nitrofurantoin (96%), and fosfomycin (95.4%). However, susceptibilities to cephalosporins (varying from 58.6% to 74.9%) and levofloxacin (48.8%) were relatively low. In the phenotypic detection of ESBLs, ESBL-positive isolates made up 38.07% of E. coli strains isolated from CA-UTIs, while 2.97% were ESBL-uncertain. Antimicrobial susceptibilities of imipenem, cefmetazole, colistin, ertapenem, amikacin, and nitrofurantoin against ESBL-producing E. coli strains were greater than 90%. The percentage of ESBL-producing strains was higher in male (53.6%) than in female patients (35.2%) (p < 0.001). CTX-M-14 (31.8%) was the major CTX-M variant in the ESBL-producing E. coli, followed by CTX-M-55 (23.4%), CTX-M-15 (17.5%), and CTX-M-27 (13.3%). The prevalence of carbapenem-resistant E. coli among CA-UTI isolates was 0.25% (2/809). CONCLUSION: Our study indicated high prevalence of ESBL in E. coli strains from strictly defined community-acquired urinary tract infections in adults in China. Imipenem, colistin, ertapenem, amikacin, and nitrofurantoin were the most active antimicrobials against ESBL-positive E. coli isolates. bla CTX-M- 14 is the predominant esbl gene in ESBL-producing and ESBL-uncertain strains. Our study indicated that the use of cephalosporins and fluoroquinolone needs to be restricted for empirical treatment of CA-UTIs in China.

Continual Decline in Azole Susceptibility Rates in Candida tropicalis Over a 9-Year Period in China.

BACKGROUND: There have been reports of increasing azole resistance in Candida tropicalis, especially in the Asia-Pacific region. Here we report on the epidemiology and antifungal susceptibility of C. tropicalis causing invasive candidiasis in China, from a 9-year surveillance study. METHODS: From August 2009 to July 2018, C. tropicalis isolates (n = 3702) were collected from 87 hospitals across China. Species identification was carried out by mass spectrometry or rDNA sequencing. Antifungal susceptibility was determined by Clinical and Laboratory Standards Institute disk diffusion (CHIF-NET10-14, n = 1510) or Sensititre YeastOne (CHIF-NET15-18, n = 2192) methods. RESULTS: Overall, 22.2% (823/3702) of the isolates were resistant to fluconazole, with 90.4% (744/823) being cross-resistant to voriconazole. In addition, 16.9 (370/2192) and 71.7% (1572/2192) of the isolates were of non-wild-type phenotype to itraconazole and posaconazole, respectively. Over the 9 years of surveillance, the fluconazole resistance rate continued to increase, rising from 5.7 (7/122) to 31.8% (236/741), while that for voriconazole was almost the same, rising from 5.7 (7/122) to 29.1% (216/741), with no significant statistical differences across the geographic regions. However, significant difference in fluconazole resistance rate was noted between isolates cultured from blood (27.2%, 489/1799) and those from non-blood (17.6%, 334/1903) specimens (P-value < 0.05), and amongst isolates collected from medical wards (28.1%, 312/1110) versus intensive care units (19.6%, 214/1092) and surgical wards (17.9%, 194/1086) (Bonferroni adjusted P-value < 0.05). Although echinocandin resistance remained low (0.8%, 18/2192) during the surveillance period, it was observed in most administrative regions, and one-third (6/18) of these isolates were simultaneously resistant to fluconazole. CONCLUSION: The continual decrease in the rate of azole susceptibility among C. tropicalis strains has become a nationwide challenge in China, and the emergence of multi-drug resistance could pose further threats. These phenomena call for effective efforts in future interventions.

Comprehensive Pathogen Identification, Antibiotic Resistance, and Virulence Genes Prediction Directly From Simulated Blood Samples and Positive Blood Cultures by Nanopore Metagenomic Sequencing.

Bloodstream infection is a major cause of morbidity and mortality worldwide. We explored whether MinION nanopore sequencing could accelerate diagnosis, resistance, and virulence profiling prediction in simulated blood samples and blood cultures. One milliliter of healthy blood samples each from direct spike (sample 1), anaerobic (sample 2), and aerobic (sample 3) blood cultures with initial inoculation of ∼30 CFU/ml of a clinically isolated Klebsiella pneumoniae strain was subjected to DNA extraction and nanopore sequencing. Hybrid assembly of Illumina and nanopore reads from pure colonies of the isolate (sample 4) was used as a reference for comparison. Hybrid assembly of the reference genome identified a total of 39 antibiotic resistance genes and 77 virulence genes through alignment with the CARD and VFDB databases. Nanopore correctly detected K. pneumoniae in all three blood samples. The fastest identification was achieved within 8 h from specimen to result in sample 1 without blood culture. However, direct sequencing in sample 1 only identified seven resistance genes (20.6%) but 28 genes in samples 2-4 (82.4%) compared to the reference within 2 h of sequencing time. Similarly, 11 (14.3%) and 74 (96.1%) of the virulence genes were detected in samples 1 and 2-4 within 2 h of sequencing time, respectively. Direct nanopore sequencing from positive blood cultures allowed comprehensive pathogen identification, resistance, and virulence genes prediction within 2 h, which shows its promising use in point-of-care clinical settings.

Evaluation of Autof MS 1000 and Vitek MS MALDI-TOF MS System in Identification of Closely-Related Yeasts Causing Invasive Fungal Diseases.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been accepted as a rapid, accurate, and less labor-intensive method in the identification of microorganisms in clinical laboratories. However, there is limited data on systematic evaluation of its effectiveness in the identification of phylogenetically closely-related yeast species. In this study, we evaluated two commercially available MALDI-TOF systems, Autof MS 1000 and Vitek MS, for the identification of yeasts within closely-related species complexes. A total of 1,228 yeast isolates, representing 14 different species of five species complexes, including 479 of Candida parapsilosis complex, 323 of Candida albicans complex, 95 of Candida glabrata complex, 16 of Candida haemulonii complex (including two Candida auris), and 315 of Cryptococcus neoformans complex, collected under the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program, were studied. Autof MS 1000 and Vitek MS systems correctly identified 99.2% and 89.2% of the isolates, with major error rate of 0.4% versus 1.6%, and minor error rate of 0.1% versus 3.5%, respectively. The proportion of isolates accurately identified by Autof MS 1000 and Vitek MS per each yeast complex, respectively, was as follows; C. albicans complex, 99.4% vs 96.3%; C. parapsilosis complex, 99.0% vs 79.1%; C glabrata complex, 98.9% vs 94.7%; C. haemulonii complex, 100% vs 93.8%; and C. neoformans, 99.4% vs 95.2%. Overall, Autof MS 1000 exhibited good capacity in yeast identification while Vitek MS had lower identification accuracy, especially in the identification of less common species within phylogenetically closely-related species complexes.

In vitro and in vivo Effect of Antimicrobial Agent Combinations Against Carbapenem-Resistant Klebsiella pneumoniae with Different Resistance Mechanisms in China.

OBJECTIVE: This study aimed to evaluate the in vitro and in vivo effects of different combinations of antimicrobial agents against carbapenemase-producing and non-producing Klebsiella pneumoniae from China. METHODS: A checkerboard assay of meropenem (MEM), amikacin (AK), tigecycline (TGC), colistin (COL) and their combinations was carried out against 58 clinical carbapenem-resistant K. pneumoniae (CRKp) isolates, including 11 carbapenemase-non-producing K. pneumoniae isolates and 21 isolates producing KPC-2 enzyme, 11 NDM-1, 13 IMP, one VIM-1 and one OXA-48. The checkerboard assay was analyzed by the fractional inhibitory concentration index (FICI). A time-kill assay and Galleria mellonella infection model were conducted to evaluate the in vitro and in vivo effects of the four drugs alone and in combination. RESULTS: In the checkerboard assay, TGC+AK and MEM+AK combinations showed the highest synergistic effect against KPC-2 and NDM-1 carbapenemase-producing isolates, with synergy+partial synergy (defined as FICI <1) rates of 76.2% and 71.4% against KPC-2 producers, and 54.5% and 81.8% against NDM-1 producers. TGC+AK and MEM+COL combinations showed the highest rate of synergistic effect against IMP-producing isolates. Against carbapenemase-non-producing isolates, TGC+COL and TGC+AK combinations showed the highest rate of synergy effect (63.6% and 54.5%). MEM+AK showed a synergistic effect against one VIM-1 producer (FICI=0.31) and an additivite effect (FICI=1) against one OXA-48 producer. In the time-kill assay, COL+AK, COL+TGC, COL+MEM and AK+TGC showed good synergistic effects against the KPC-2-producing isolate D16. COL+MEM and COL+TGC combinations showed good effects against the NDM-1-producing isolate L13 and IMP-4-producing isolate L34. Against the carbapenemase-non-producing isolate Y105, MEM+TGC and COL+AK showed high synergistic effects, with log10CFU/mL decreases of 6.2 and 5.5 compared to the most active single drug. In the G. mellonella survival assay, MEM-based combinations had relatively high survival rates, especially when combined with colistin, against KPC-2 producers (90% survival rate) and with amikacin against metallo-beta-lactamase producers (95-100% survival rate). CONCLUSION: Our study suggests that different antimicrobial agent combinations should be considered against CRKp infections with different resistance mechanisms.

Serotype Distribution, Antimicrobial Susceptibility, Multilocus Sequencing Type and Virulence of Invasive Streptococcus pneumoniae in China: A Six-Year Multicenter Study.

Background: Streptococcus pneumoniae is an important human pathogen that can cause severe invasive pneumococcal diseases (IPDs). The aim of this multicenter study was to investigate the serotype and sequence type (ST) distribution, antimicrobial susceptibility, and virulence of S. pneumoniae strains causing IPD in China. Methods: A total of 300 invasive S. pneumoniae isolates were included in this study. The serotype, ST, and antimicrobial susceptibility of the strains, were determined by the Quellung reaction, multi-locus sequence typing (MLST) and broth microdilution method, respectively. The virulence level of the strains in the most prevalent serotypes was evaluated by a mouse sepsis model, and the expression level of well-known virulence genes was measured by RT-PCR. Results: The most common serotypes in this study were 23F, 19A, 19F, 3, and 14. The serotype coverages of PCV7, PCV10, PCV13, and PPV23 vaccines on the strain collection were 42.3, 45.3, 73.3 and 79.3%, respectively. The most common STs were ST320, ST81, ST271, ST876, and ST3173. All strains were susceptible to ertapenem, levofloxacin, moxifloxacin, linezolid, and vancomycin, but a very high proportion (>95%) was resistant to macrolides and clindamycin. Based on the oral, meningitis and non-meningitis breakpoints, penicillin non-susceptible Streptococcus pneumoniae (PNSP) accounted for 67.7, 67.7 and 4.3% of the isolates, respectively. Serotype 3 strains were characterized by high virulence levels and low antimicrobial-resistance rates, while strains of serotypes 23F, 19F, 19A, and 14, exhibited low virulence and high resistance rates to antibiotics. Capsular polysaccharide and non-capsular virulence factors were collectively responsible for the virulence diversity of S. pneumoniae strains. Conclusion: Our study provides a comprehensive insight into the epidemiology and virulence diversity of S. pneumoniae strains causing IPD in China.

Laboratory diagnosis of COVID-19 in China: A review of challenging cases and analysis.

Since the initial emergence of coronavirus disease 2019 (COVID-19) in Wuhan, Hubei province, China, a rapid spread of the disease occurred around the world, rising to become an international global health concern at pandemic level. In the face of this medical challenge threatening humans, the development of rapid and accurate methods for early screening and diagnosis of COVID-19 became crucial to containing the emerging public health threat, and prevent further spread within the population. Despite the large number of COVID-19 confirmed cases in China, some problematic cases with inconsistent laboratory testing results, were reported. Specifically, a high false-negative rate of 41% on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays was observed in China. Although serological testing has been applied worldwide as a complementary method to help identify SARS-CoV-2, several limitations on its use have been reported in China. Therefore, the use of both qRT-PCR and serological testing in the diagnosis of COVID-19 in China and elsewhere, presented considerable challenges, but when used in combination, can be valuable tools in the fight against COVID-19. In this review, we give an overview of the advantages and disadvantages of different molecular techniques for SARS-CoV-2 detection that are currently used in several labs, including qRT-PCR, gene sequencing, loop-mediated isothermal amplification (LAMP), nucleic acid mass spectrometry (MS), and gene editing technique based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas13) system. Then we mainly review and analyze some causes of false-negative qRT-PCR results, and how to resolve some of the diagnostic dilemma.

Genomic analysis of trimethoprim-resistant extraintestinal pathogenic Escherichia coli and recurrent urinary tract infections.

Urinary tract infections (UTIs) are the most common bacterial infections requiring medical attention and a leading justification for antibiotic prescription. Trimethoprim is prescribed empirically for uncomplicated cases. UTIs are primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) and ExPEC strains play a central role in disseminating antimicrobial-resistance genes worldwide. Here, we describe the whole-genome sequences of trimethoprim-resistant ExPEC and/or ExPEC from recurrent UTIs (67 in total) from patients attending a regional Australian hospital from 2006 to 2008. Twenty-three sequence types (STs) were observed, with ST131 predominating (28 %), then ST69 and ST73 (both 7 %). Co-occurrence of trimethoprim-resistance genes with genes conferring resistance to extended-spectrum ß-lactams, heavy metals and quaternary ammonium ions was a feature of the ExPEC described here. Seven trimethoprim-resistance genes were identified, most commonly dfrA17 (38 %) and dfrA12 (18 %). An uncommon dfrB4 variant was also observed. Two blaCTX-M variants were identified - blaCTX-M-15 (16 %) and blaCTX-M-14 (10 %). The former was always associated with dfrA12, the latter with dfrA17, and all blaCTX-M genes co-occurred with chromate-resistance gene chrA. Eighteen class 1 integron structures were characterized, and chrA featured in eight structures; dfrA genes featured in seventeen. ST131 H30Rx isolates possessed distinct antimicrobial gene profiles comprising aac(3)-IIa, aac(6)-Ib-cr, aph(3')-Ia, aadA2, blaCTX-M-15, blaOXA-1 and dfrA12. The most common virulence-associated genes (VAGs) were fimH, fyuA, irp2 and sitA (all 91 %). Virulence profile clustering showed ST131 H30 isolates carried similar VAGs to ST73, ST405, ST550 and ST1193 isolates. The sole ST131 H27 isolate carried molecular predictors of enteroaggregative E. coli/ExPEC hybrid strains (aatA, aggR, fyuA). Seven isolates (10 %) carried VAGs suggesting ColV plasmid carriage. Finally, SNP analysis of serial UTI patients experiencing worsening sequelae demonstrated a high proportion of point mutations in virulence factors.

Antimicrobial activity of omadacycline in vitro against bacteria isolated from 2014 to 2017 in China, a multi-center study.

BACKGROUND: Omadacycline (ZL-2401) is a semi-synthetic derivative of minocycline. It has a broadspectrum activity against Gram-positive and Gram-negative bacteria, and atypical pathogens. The objective of this study was to evaluate the antibacterial activity of omadacycline against recently collected bacterial isolates from Chinese patients. RESULTS: Omadacycline showed potent activity against all Gram-positive pathogens: S. aureus MICs were low regardless of susceptibility to methicillin (methicillin-resistant Staphylococcus aureus, MRSA: N = 97, MIC50/90 0.12/0.25 mg/L, 98.5% susceptible; methicillin-sensitive Staphylococcus aureus, MSSA: N = 100, MIC50/90 0.12/0.12 mg/L, 100.0% susceptible). Omadacycline was also very effective against ß-haemolytic streptococci (MIC50/90, 0.06/0.12 mg/L), viridans group streptococci (MIC50/90,<0.03/0. 06 mg/L), and enterococci (MIC50/90, 0.03/0.12 mg/L). Against S. pneumoniae, omadacycline was highly active regardless of penicillin-resistance (MIC90 0.06 mg/L) and despite the fact that less than 10.0% of these strains were susceptible to tetracycline. Omadacycline exhibited good in vitro activity against Enterobacterales isolates (MIC50/90, 2/8 mg/L), inhibiting 81.7% of the isolates at ≤4 mg/L. M. catarrhalis isolates (MIC50/90, 0.12/0.25 mg/L) were fully susceptible to omadacycline at ≤0.5 mg/L. CONCLUSIONS: Omadacycline showed potent in vitro activity against most common bacterial pathogens, and even against highly resistant problem pathogens, such as MRSA, penicillin-R and tetracycline-R S. pneumoniae and enterococci. The susceptibility rate of Chinese isolates was similar to those reported in other countries, but the decreased activity against K. pneumoniae isolates in the present study should be noted.

Antimicrobial Susceptibility and Virulence of mcr-1-Positive Enterobacteriaceae in China, a Multicenter Longitudinal Epidemiological Study.

This study was to investigate the prevalence of mcr-1-positive Enterobacteriaceae (MPE) in intra-abdominal infections (IAIs), urinary tract infections (UTIs), and lower respiratory tract infections (LRTIs) in China. A total of 6,401 Enterobacteriaceae isolates were collected consecutively from IAI, UTI, and LRTI patients in 19 hospitals across mainland China during 2014-2016. MPE isolates were screened by PCR detection for the mcr gene. The resistance profiles were tested by antimicrobial susceptibility test. All MPE isolates were characterized by pulsed-field gel electrophoresis (PFGE), multi-locus-sequence typing, O and H serotyping, and whole-genome sequencing. Among the 6,401 Enterobacteriaceae isolates, 17 Escherichia coli strains (0.27%) were positive for the mcr-1 gene. The MPE prevalence rates in IAI, UTI, and LRTI patients were 0.34% (12/3502), 0.23% (5/2154), and 0% (0/745), respectively. The minimum inhibition concentrations (MICs) of colistin against 3 isolates were of 0.5-2 mg/L, and 4-8 mg/L against other 14 isolates. All the 17 isolates were susceptible to meropenem, imipenem, tigecycline, and ceftazidime/avibactam. The 17 MPE isolates belonged to 14 different ST types, and those that belonged to the same STs were not clonal by PFGE. The mcr-1-harboring plasmid of ten MPE isolates could transfer to the recipients by conjugation and the colistin MICs of the transconjugants ranged from 0.5 to 8 mg/L. Mcr-1-carrying plasmids from the 17 MPE isolates could be grouped into four clusters, including 8 IncX4 type, 4 IncI2 type, 4 IncHI2A type, and 1 p0111 type. Multiple-drug resistance genes and virulence genes were detected. In conclusion, the prevalence of MPE in IAI, UTI, and LRTI were low in China, and no clonal transmission was identified in our study. Most MPE isolates exhibited low-level colistin resistance. However, our study indicated that MPE isolates always carried a variety of drug resistance and virulence genes, which should be paid more attention.