An XDR Pseudomonas aeruginosa ST463 Strain with an IncP-2 Plasmid Containing a Novel Transposon Tn6485f Encoding blaIMP-45 and blaAFM-1 and a Second Plasmid with Two Copies of blaKPC-2.

The increased carbapenem resistance among Pseudomonas aeruginosa has become a serious health issue worldwide. We reported an extensively drug-resistant (XDR) P. aeruginosa PA30 isolate which belonged to sequence type ST463 and contained an IncP-2 plasmid (pPA30_1) carrying two genes, namely, blaIMP-45 and blaAFM-1, which encoded the metallo-ß-lactamases AFM-1 and IMP-45, respectively. Additionally, the strain had a plasmid (pPA30_2) with two copies of the blaKPC-2 genes embedded. The plasmid pPA30_1 was highly similar to the previously reported plasmid pHS17-127, which has the same genetic architecture. This plasmid contained blaIMP-45, located in a second gene cassette of the integron In786, carried by a Tn1403-derivative transposon acquiring an ISCR27n3-blaAFM-1 structure. Interestingly, the transposon in pPA30_1 acquired an extra ISCR1-qnrVC6 module and formed a novel transposon, which was subsequently annotated as Tn6485f. The blaKPC-2 genes in pPA30_2 underwent duplication due to the inversion of the IS26-blaKPC-2-IS26 element, which resulted in two copies of blaKPC-2. IMPORTANCE The ST463 clone is an emerging high-risk sequence type that is spreading with blaKPC-2-containing plasmids. The core blaKPC-2 genetic platform is ISKpn27-blaKPC-2-ISKpn6 in almost all samples, and the adjacent region beyond the core platform varies by IS26-mediated inversion or duplication events, amplifying the blaKPC-2 gene copies. The ST463 P. aeruginosa strain PA30 in our study contains another two metallo-ß-lactamase genes, namely, blaIMP-45 and blaAFM-1, in a novel transposon Tn6485f that is harbored by the IncP-2 megaplasmid. The pPA30_1 carrying blaIMP-45 and blaAFM-1 is highly related to pHS17-127 from the ST369 P. aeruginosa strain, indicating the putative dissemination of the megaplasmid between different clones.

Distribution and spread of the mobilised RND efflux pump gene cluster tmexCD-toprJ in clinical Gram-negative bacteria: a molecular epidemiological study.

BACKGROUND: TMexCD1-TOprJ1, which is associated with phenotypic resistance to multiple classes of antibiotics, is a transmissible resistance-nodulation-division (RND) family efflux pump. However, the prevalence and genomic and phenotypic characteristics of clinical isolates with this important resistance determinant are poorly understood. In this study, we aimed to survey tmexCD-toprJ among clinical Gram-negative isolates collected from hospitals in China between 1991 and 2020 and characterise tmexCD-toprJ-positive clinical isolates. METHODS: We conducted online data retrieval and active nationwide surveillance in China to screen tmexCD-toprJ-positive strains. We characterised tmexCD-toprJ-positive clinical strains for their antimicrobial susceptibility, genetic and functional characteristics, and the potential inter-species transmission route of tmexCD-toprJ with whole genome sequencing and bioinformatics analyses. The function of tmexCD-toprJ in Pseudomonas sp and Proteus sp was investigated by tmexD gene knockdown using an isopropylthio-ß-galactoside-inducible CRISPR interference system. FINDINGS: Data retrieval obtained 53 strains carrying tmexCD-toprJ, comprising 32 Pseudomonas spp, 11 Klebsiella pneumoniae, one Aeromonas spp, one Citrobacter freundii, and one uncultured bacterium from diverse niches. 48 (0·64%) of 7517 clinical isolates from China, including seven Klebsiella spp, one Proteus mirabilis, and 40 Pseudomonas spp, carried tmexCD-toprJ. These isolates exhibited multidrug resistance phenotypes and co-harboured resistance genes, such as mcr and carbapenemases genes. tmexCD-toprJ was encoded on both plasmids and chromosomes in all Klebsiella spp that carried plasmid-borne tmexCD-toprJ (n=7), P mirabilis carried chromosome-borne tmexCD-toprJ, and Pseudomonas spp carried either plasmid-borne (n=19) or chromosome-borne (n=21) ones. tmexCD-toprJ had undergone clonal and horizontal transmission among clinical pathogens. Eight different types of genetic context of tmexCD-toprJ were identified, each of which was associated with different mobile elements, including IntI, IS6100, TnAs1-like, ISRor5, ISVsa3, ISCfr-like, Tn5393, and IS222-like, which might facilitate its transmission. Knockdown of tmexD led to a four times decrease in tigecycline minimum inhibitory concentrations in both Pseudomonas spp and Proteus spp. INTERPRETATION: Our study provides evidence to suggest that tmexCD-toprJ contributes to the antimicrobial resistance phenotypes in different bacterial species. tmexCD-toprJ has disseminated among diverse species of clinical pathogens, which warrants timely monitoring in clinical pathogens. FUNDING: National Natural Science Foundation of China, Guangdong Major Project of Basic and Applied Basic Research, Natural Science Foundation of Jiangsu Province.

Epidemiological and genetic characteristics of clinical carbapenem-resistant Acinetobacter baumannii strains collected countrywide from hospital intensive care units (ICUs) in China.

Acinetobacter baumannii is one of the key Gram-negative pathogens that can cause serious nosocomial infections. In China, a large proportion of clinical A. baumannii strains are multidrug resistant, among which strains resistant to carbapenems are particularly worrisome, as infections caused by such strains may limit the choice of existing antibiotics. We conducted a nationwide and genome-based surveillance on the prevalence and antibiotic susceptibility profile of carbapenem-resistant A. baumannii (CRAB) strains collected from intensive care units (ICUs) in hospitals in different provinces and investigated the routes of transmission and mechanism of resistance by whole-genome sequencing and phylogenetic analysis. We found that CRAB strains were prevalent in 71.4% (55/77) of the ICUs surveyed. Clonal spread of CRAB was found in 37.6% (29/77) of ICUs and a total of 22 different clones were identified. Most clones were transmissible within one ICU, but up to six clones could be detected in at least three hospitals. In addition, carbapenem-hydrolysing class D ß-lactamases (CHDL) were found to be mainly responsible for carbapenem-resistance in A. baumannii and the ST2 global-clone is the predominant type of CRAB in China. Importantly, we found that CRAB isolates currently exhibited an extremely low rate of resistance to colistin (0.4%) and tigecycline (2.5%), but a high rate of resistance to ceftazidime-avibactam (70.2%). Findings in this work shall facilitate development of appropriate antimicrobial regimens for treatment of CRAB infections. Further surveillance and research on the evolutionary and epidemiological features of clinical CRAB strains are necessary.

Emergence of an ST1326 (CG258) Multi-Drug Resistant Klebsiella pneumoniae Co-harboring mcr-8.2, ESBL Genes, and the Resistance-Nodulation-Division Efflux Pump Gene Cluster tmexCD1-toprJ1 in China.

CG258 is the dominant carbapenemase-producing Klebsiella pneumoniae clone worldwide and treatment of infections caused by this clone relies largely on the last-line antibiotics, colistin, and tigecycline. However, the emergence and global dissemination of mcr and tmexCD1-toprJ1 genes have significantly compromised their clinical applications. CG258 K. pneumoniae carrying both mcr and tmexCD1-toprJ1 have not been reported. A colistin-resistant strain T698-1 belonging to ST1326, a member of CG258, was isolated from the intestinal sample of a patient and characterized by the antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis. It was resistant to colistin, tetracycline, aminoglycoside, fluoroqinolone, phenicols, sulfonamide, and some ß-lactams, and positive for mcr-8.2, tmexCD1-toprJ1, and ESBL genes (bla DHA-1 and bla CTX-M-15). The tmexCD1-toprJ1 gene cluster was located in an multi-drug resistant (MDR) region flanked by TnAs1 elements on an IncHI1B/FIB plasmid. The genetic context of tmexCD1-toprJ1 was slightly distinct from previously reported Tn5393-like structures, with an IS26 element disrupting the upstream Tn5393 and its adjacent genetic elements. The mcr-8.2 gene was inserted into the backbone of an IncFII/FIA plasmid with the genetic context of ISEcl1-mcr-8.2-orf-ISKpn26. To our knowledge, this is the first report of co-occurrence of mcr-8.2 and tmexCD1-toprJ1 in a CG258 K. pneumoniae strain. Though this strain is tigecycline sensitive, the acquisition of colistin and tigecycline resistance determinants by the endemic CG258 K. pneumoniae clone still poses a serious public health concern. CG258, which became resistant to multiple last resort antibiotics, would be the next emerging superbug.

Development of an In-House Rapid Antimicrobial Susceptibility Testing Protocol for Positive Blood Culture and Its Implementation in Routine Microbiology Laboratories.

Bloodstream infections (BSI) are associated with high morbidity and mortality and remain a leading cause of death. Blood culture (BC) including the identification and the antimicrobial susceptibility testing of the causative microorganisms should be performed as soon as possible. In this study, we developed an in-house rapid antimicrobial susceptibility testing (rAST) protocol for positive BC. First, the rAST was performed in the simulated positive BC of standard strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853) at three different times to assess the reproducibility and operability by dispensing four drops of BC broth onto a Mueller-Hinton agar plate after a positive signal. Furthermore, the rAST was performed in clinical positive BCs. The results of rAST at 4, 6, 8, and 18 h of incubation were compared with results of the standard 16- to 20-h disk diffusion method, and the preliminary breakpoints of the rAST method were established according to the inhibition diameter of sensitive strains and resistant strains. Finally, the rAST was performed in the simulated positive BC of clinical strains to evaluate the availability of the preliminary breakpoints. The rAST results of standard strains were distributed evenly at three different times. Among the 202 clinical strains used to establish the preliminary breakpoints, the number of zone diameters that could be read and interpreted (60, 87, 98, and 100%) increased with incubation time (4, 6, 8, and 18 h), and the categorical agreement was acceptable, with total error rates of 3.0, 2.3, 2.1, and 1.3% at 4, 6, 8, and 18 h of incubation, respectively. In conclusion, the in-house rAST protocol for positive BC can be implemented in routine laboratories. It provides reliable antimicrobial susceptibility testing results for BSI pathogens after 4-6 h of incubation.

A rare case of meningitis and septicemia caused by Streptococcus suis in a woman without a history of live pig contact or eating raw pork.

Streptococcus suis (S. suis) is a zoonotic pathogen that primarily inhabits the upper respiratory tract of pigs. Therefore, pigs that carry these pathogens are the major source of infection. Most patients are infected through contact with live pigs or unprocessed pork products and eating uncooked pork. S. Suis mainly causes sepsis and meningitis. The disease has an insidious onset and rapid progress. The patient becomes critically ill and the mortality is high. In this case report, we described a rare case of S. suis isolated from a middle-aged woman in Jinhua City, Zhejiang Province, China, who did not have any contact with live pigs and had not eaten uncooked pork. S. Suis was isolated from both the patient's blood and cerebrospinal fluid samples.

Correction to: Vogesella perlucida-induced bacteremia in an advanced-age patient: first case report.

An amendment to this paper has been published and can be accessed via the original article.

Vogesella perlucida-induced bacteremia in an advanced-age patient: first case report.

BACKGROUND: Vogesella species are common aquatic, Gram-negative rod-shaped bacteria, originally described in 1997. Vogesella perlucida was first isolated from spring water in 2008. Furthermore, bacterial pathogenicity of Vogesella perlucida has never been reported. Here, we report the first case of rare Vogesella perlucida-induced bacteremia in an advanced-age patient with many basic diseases and history of dexamethasone abuse. CASE PRESENTATION: A 71-year-old female was admitted with inflamed upper and lower limbs, rubefaction, pain and fever (about 40 °C). She had been injured in a fall at a vegetable market and then touched river snails with her injury hands. A few days later, soft tissue infection of the patient developed and worsened. Non-pigmented colonies were isolated from blood cultures of the patient. Initially, Vogesella perlucida was wrongly identified as Sphingomonas paucimobilis by Vitek-2 system with GN card. Besides, we failed to obtain an acceptable identification by the MALDI-TOF analysis. Finally, the isolated strain was identified as Vogesella perlucida by 16S rRNA gene sequences. In addition, the patient recovered well after a continuous treatment of levofloxacin for 12 days. CONCLUSION: Traditional microbiological testing system may be inadequate in the diagnosis of rare pathogenic bacteria. Applications of molecular diagnostics techniques have great advantages in clinical microbiology laboratory. By using 16S rRNA gene sequence analysis, we report the the first case of rare Vogesella perlucida-induced bacteremia.

First Report of OXA-181-Producing Klebsiella pneumoniae in China.

We present here the first report of an OXA-181-producing Klebsiella pneumoniae isolated from the fecal specimen of a patient in China. The OXA-181-encoding gene bla OXA-181 was located on a 51 kb IncX3-type plasmid. Conjugation assay and whole-genome sequencing analysis revealed that this transferrable plasmid in the K. pneumoniae isolate might have originated from Escherichia coli and have the potential to mediate the spread of bla OXA-181.