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Background: Hypermucoviscous carbapenem-resistant Klebsiella pneumoniae (hmCRKp) is emerging globally and approaching the worst-case scenario in health care system. Aims: The main objective in this study was to determine the hypermucoviscous characteristics among the carbapenem-resistant K. pneumoniae (CRKp) isolated from a teaching hospital in Malaysia. The association of hypermucoviscous phenotype with the virulence traits and clinical presentations were also investigated. Methods: A retrospective study was conducted in University Malaya Medical Centre (UMMC). The presence of hypermucoviscous K. pneumoniae was identified among a collection of CRKp clinical isolates (first isolate per patient) from 2014 to 2015 using string test. Correlation between clinical and microbial characteristics of the hmCRKp was investigated. Results: A total of nine (7.5%) hmCRKp were detected among 120 CRKp isolates. Majority of the isolates were hospital acquired or health care-associated infections. None of the patients had typical pyogenic liver abscess. All of the hmCRKp isolates harbored carbapenemase genes and were multidrug resistant. K1/K serotype, peg-344, allS, and magA were not identified among hmCRKp isolates, whereas aerobactin siderophore receptor gene (iutA), iroB, rmpA, and rmpA2 were detected. Only three hmCRKp isolates were resistant to serum bactericidal. Conclusions: All the isolates presented inconclusive evidence for the interpretation of hypervirulence. Therefore, more study should be performed in the future to have a better understanding of the virulence mechanisms in correlation with the clinical and microbial determinants.

Lianjiang Huang,1 Li Fu,2 Xiaoyan Hu,3 Xiaoliang Liang,1 Guozhong Gong,4 Chunhong Xie,1 Feiyang Zhang,3 Ying Wang,3 Yingshun Zhou3 1Department of Clinical Laboratory, The Second Affiliated Hospital of Xiamen Medical College, Xiamen, 361021, People’s Republic of China; 2The Affiliated Hospital of Southwest Medical University, Luzhou, 646000, People’s Republic of China; 3Department of Pathogen Biology, School of Basic Medicine, Public Center of Experimental Technology of Pathogen Biology Technology Platform, Southwest Medical University, Luzhou, 646000, People’s Republic of China; 4Department of Clinical Laboratory, Suining First People’s Hospital, Suining, 629000, People’s Republic of ChinaCorrespondence: Yingshun Zhou; Ying WangDepartment of Pathogen Biology, School of Basic Medicine, Public Center of Experimental Technology of Pathogen Biology Technology Platform, Southwest Medical University, No. 1, Xianglin Road, Luzhou, 646000, People’s Republic of ChinaTel +86-830-3160073Email yingshunzhou@swmu.edu.cn; wyingnbgg@163.comObjective: The aim of this study was to use whole-genome sequencing to characterize Klebsiella pneumoniae SKp2F and Klebsiella variicola SKv2E, both carrying blaKPC, co-isolated from the same sputum specimen.Methods: Antimicrobial susceptibility testing was performed using microbroth dilution. Biofilm formation was determined by crystal violet staining and virulence was measured by a serum killing assay. Whole-genome sequencing of SKp2F and SKv2E was performed using an Illumina sequencer and the genetic characteristics were analyzed by computer.Results: SKp2F and SKv2E were sensitive only to tigecycline and polymyxin among the tested antibiotics. The biofilm-forming ability of SKv2E is stronger than that of SKp2F. The grades of serum resistance of SKp2F and SKv2E are 4 and 3. MLST analysis of the 6,115,610 bp and 5,403,687 bp of SKv2E and SKp2F showed associations with ST1615 and ST631, respectively. SKv2E carried 13 resistance genes (blaKPC-2, blaTEM-1A, blaLEN17, aadA16, arr-3, qnrB4, oqxA/B, dfrA27, sul1, tetD, fosA, qacEΔ 1) and SKp2F carried 23 (blaKPC-2, blaCTX-M-3, blaTEM-1B, blaCTX-M-65, blaSHV-27, aac(6ʹ)-IIa, rmtB, arr-3, aph(3ʹ)-Ia, aadA16, qnrS1, aac(6ʹ)-Ib-cr, qnrB91, oqxA/B, mph(A), tet(A), fosA, dfrA27, and two copies of qacEΔ 1-sul1). Most of them were carried by various mobile genetic elements, such as IncFIB(K)/IncFII(K)/IncFII(Yp), IncFII(K) plasmid, Tn6338, and In469. Both SKv2E and SKp2F carried a large number of virulence factors, including type 1 and 3 fimbriae, capsule, aerobactin (iutA), ent siderophore (entABCDEFS, fepABCDGfes), and salmochelin (iroE/iroEN). SKv2E also carried type IV pili (pilW), fimbrial adherence (steB, stfD), and capsule biosynthesis gene (glf).Conclusion: blaKPC-2-carrying K. variicola and K. pneumoniae, which carried multiple resistance genes, virulence factors, and highly similar mobile genetic elements, were identified from the same specimen, indicating that clinical samples may carry multiple bacteria. We should avoid misidentification, and bear in mind that resistance genes carrying mobile genetic elements can be transmitted or integrated between bacteria in the same host.Keywords: Klebsiella variicola, Klebsiella pneumoniae, carbapenem-resistant Enterobacteriaceae, CRE, blaKPC

Objective Comparative phenotypic and genetic assessment of the pathogenic potential of E. coli strains isolated from patients with calculous pyelonephritis. Materials and methods 78 strains of E. coli isolated from urine of patients with calculous pyelonephritis in the acute phase (n=58) and in the remission phase (n=20). Escherichia were investigated for the presence of virulence genes papA, pap EF, papGII; afa, bma E, iutA, fyuA, feoB, kspMTII, usp multiplex PCR using selected primers. Phenotypically determined the ability to biofilm formation, antilysozyme, antihemoglobin, anticytokine, adhesive and sIgA-protease activity E. coli. Results The virulent potential of Escherichia coli at the pheno- and genotype levels was characterized. In strains of E. coli isolated from the urine of patients in the remission phase, the ability to form biofilms was more often and with high values of the trait; and in strains isolated in relapse - adhesive activity, the ability to inactivate pro- and anti-inflammatory cytokines, antihemoglobin activity, and genes encoding aphimbrial adhesin (afa), responsible for the synthesis of siderophore aerobactin (iutA), transporting bivalent iron (feoB). Conclusion The revealed differences in the pheno- and genotypic profiles between the cultures of Escherichia coli isolated from patients with calculous pyelonephritis in the phases of exacerbation and remission make it possible to differentiate the isolated strain and predict the course of the infectious-inflammatory process.

Cong Zhou,1 Qiang Wu,1 Leqi He,1 Hui Zhang,1 Maosuo Xu,1 Baoyu Yuan,2 Zhi Jin,3 Fang Shen1 1Department of Clinical Laboratory Medicine, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People’s Republic of China; 2Department of Clinical Laboratory, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 3Department of Neurology, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People’s Republic of ChinaCorrespondence: Fang ShenDepartment of Clinical Laboratory Medicine, Shanghai Fifth People’s Hospital, Fudan University, No. 128, Ruili Road, Minhang District, Shanghai, 200240, People’s Republic of ChinaTel +86 18021073261Email shenfang5th@aliyun.comBackground: The convergence of carbapenem-resistance and hypervirulence in Klebsiella pneumoniae has led to a significant public health challenge. In recent years, there have been more and more reports on carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates.Materials and Methods: Clinical data of patients infected with CR-hvKP from January 2019 to December 2020 in a tertiary hospital were retrospectively evaluated. The number of isolates of Klebsiella pneumoniae, hypermucoviscous Klebsiella pneumoniae (hmKP), carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-hmKP) and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) collected during the period of 2 years was calculated. The antimicrobial resistance gene, virulence-associated gene, capsular serotype gene and multilocus sequence typing (MLST) of CR-hvKP isolates were detected by PCR.Results: During the study period, a total of 1081 isolates of non-repeat Klebsiella pneumoniae were isolated, including 392 isolates of hypermucoviscous Klebsiella pneumoniae (36.3%), 39 isolates of CR-hmKP (3.6%), and 16 isolates of CR-hvKP (1.5%). About 31.2% (5/16) of CR-hvKP were isolated from 2019, and 68.8% (11/16) of CR-hvKP were isolated from 2020. Among the 16 isolates of CR-hvKP, 13 isolates were ST11 and serotype K64, 1 isolate was ST11 and serotype K47, 1 isolate was ST23 and serotype K1, and 1 isolate was ST86 and serotype K2. The virulence-associated genes entB, fimH, rmpA2, iutA, iucA were present in all of 16 CR-hvKP isolates, followed by mrkD (n=14), rmpA (n=13), aerobactin (n=2), allS (n=1). Sixteen CR-hvKP isolates all carry carbapenemase gene blaKPC-2 and extended-spectrum β-lactamase gene blaSHV. ERIC-PCR DNA fingerprinting results showed that 16 CR-hvKP isolates were highly polymorphic, and there were significant differences in bands among the isolates, presenting a sporadic state.Conclusion: Although CR-hvKP was sporadically distributed, it showed an increasing trend year by year. Therefore, clinical attention should be paid, and necessary measures should be taken to avoid the cloning and transmission of superbacterium CR-hvKP.Keywords: Klebsiella pneumoniae, carbapenem-resistant, hypervirulent, hypermucoviscous, epidemiology

Background: K57 Klebsiella pneumoniae (K57-KP) is associated with hypervirulence, but the basis and systematic data of K57-KP are limited. Materials and Methods: A retrospective study was conducted in 156 patients between January 2013 and January 2016. The clinical and molecular data, including antimicrobial susceptibility testing, multilocus sequence typing, antimicrobial resistance genes, and virulence determinants were assessed. Results: Among the 39 K57-KP isolates, 14 isolates (35.9%) were associated with various types of invasive infections. Diabetes, drainage, use of carbapenems and quinolone antibiotics were dependent risk factors for K57-KP infections. Sequence type (ST)412 was the most prevalent among K57-KP isolates. K57-KP isolates were more resistant to clinically often used antimicrobial agents than hvKP (K1/K2) strains, and 12.8% (5/39) of the strains were resistant to carbapenems, which all harbored blaKPC-2. The prevalence of hypermucoviscosity phenotype, aerobactin, rmpA, rmpA2, and ybts revealed 66.7%, 100%, 89.7%, 89.7%, and 30.8%, whereas wcaG, allS, magA and kfu revealed 0%, 0%, 0%, and 5.1%, which were significantly lower than that of hvKP (K1/K2). The serum sensitivity, neutrophil phagocytic rate, and biofilm formation capacity of K57-KP strains were higher than that of K1/K2. Conclusion: There were no significant differences in the prevalence of hypermucoviscosity phenotype, carriage of rmpA and aerobactin genes between K57 and K1/K2 isolates, but the composition and production of capsule polysaccharide of K57-KP may be different from that of K1/K2 strains. K57-KP isolates exhibited distinctive virulence-associated traits, most of which belonged to ST412. Physicians should enhance the management of K57-KP infections because of the emergence of more and more carbapenem-resistant K57-KP isolates.

Coastal sands are important natural recreational facilities that have become hotspots for tourism and economic development. However, these sands harbour diverse microbial assemblages that play a critical role in the balance between public health and ecology. In this study, targeted high-throughput sequencing analysis was used to identify sand-borne bacterial populations at four public beaches in Durban. The effect of heavy metal in shaping the distribution of bacterial metacommunities was determined using canonical correspondence analysis (CCA), while the functional gene profiles were predicted using PICRUSt2 analysis. Sequences matching those of the bacterial phylum Proteobacteria were the most abundant in all samples, followed by those of the phyla Firmicutes, Actinobacteria, Bacteroidetes, and Gemmatimonadetes. Genus-level taxonomic analysis showed the presence of 1163 bacterial genera in all samples combined. The distribution of bacterial communities was shaped by heavy metal concentrations, with the distribution of Flavobacteria, Bacteroidia, and Deltaproteobacteria influenced by Pb and Zn, while B and Cr influenced the distribution of Clostridia and Gammaproteobacteria, respectively. Identified antibiotic resistance genes included the peptidoglycan biosynthesis gene II, III, IV, and V, as well as the polymyxin resistance gene, while the virulence genes included the sitA, fimB, aerobactin synthase, and pilL gene. Our findings demonstrate that beach sand-borne bacteria are reservoirs of virulence and antibiotic resistance genes. Contamination of beach sands with heavy metals selects for both heavy metal resistance and antibiotic resistance in beach sand bacterial communities. Children and immunocompromised people engaging in recreational activities on beaches may be exposed to higher risk of infection.

BackgroundAntibiotic-resistantKlebsiella pneumoniaeare a major cause of hospital- and community-acquired infections, including sepsis, liver abscess, and pneumonia, driven mainly by the emergence of successful high-risk clonal lineages. TheK. pneumoniaesequence type (ST) 307 lineage has appeared in several different parts of the world after first being described in Europe in 2008. From June to October 2019, we recorded an outbreak of an extensively drug-resistant ST307 lineage in four medical facilities in north-eastern Germany.MethodsHere, we investigated these isolates and those from subsequent cases in the same facilities. We performed whole-genome sequencing to study phylogenetics, microevolution, and plasmid transmission, as well as phenotypic experiments including growth curves, hypermucoviscosity, siderophore secretion, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance for an in-depth characterization of this outbreak clone.ResultsPhylogenetics suggest a homogenous phylogram with several sub-clades containing either isolates from only one patient or isolates originating from different patients, suggesting inter-patient transmission. We identified three large resistance plasmids, carrying either NDM-1, CTX-M-15, or OXA-48, whichK. pneumoniaeST307 likely donated to otherK. pneumoniaeisolates of different STs and even other bacterial species (e.g.,Enterobacter cloacae) within the clinical settings. Several chromosomally and plasmid-encoded, hypervirulence-associated virulence factors (e.g., yersiniabactin, metabolite transporter, aerobactin, and heavy metal resistance genes) were identified in addition. While growth, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance were comparable to several control strains, results from siderophore secretion and hypermucoviscosity experiments revealed superiority of the ST307 clone, similar to an archetypical, hypervirulentK. pneumoniaestrain (hvKP1).ConclusionsThe combination of extensive drug resistance and virulence, partly conferred through a “mosaic” plasmid carrying both antibiotic resistance and hypervirulence-associated features, demonstrates serious public health implications.

Purpose To characterize the clinical, resistance, and virulence features of carbapenem-resistant Klebsiella pneumonaie (CRKP) and hypervirulent Klebsiella pneumoniae (hvKP) and also provide an effective selection of drug in CRKP and hvKP treatment. Materials and methods Twelve strains were collected and investigated these isolates for their antimicrobial susceptibility and molecular features. Resistance mechanisms, virulence-associated genes, multilocus sequence typing (MLST), and serotypes were detected by PCR and sequencing. Next general sequencing (NGS) was carried out to determine the features of carbapenem resistance and virulence. The synergistic activity of tigecycline-imipenem (TGC+IPM), tigecycline-meropenem (TGC+MEM), and tigecycline-aztreonam (TGC+ATM) combinations were performed by microdilution checkerboard method. Results Eleven CRKP and one hvKP strains were collected. All strains showed highly sensitive rates to tigecycline (TGC) and amikacin (AMK). NDM (33.3%, 4/12) was the main resistance mechanism and MLST assigned 3 of them to ST11. CTX-M-producing (n = 1) and KPC-2-producing (n = 1) isolates belonged to ST147 and ST11, respectively. The MICs of ATM and quinolones in NDM-1 CRKP and NDM-5 CRKP strains were different. The serotype of the majority strains was KL22KL137 (58.3%, 7/12), hvKP stain belonged to K64. CRKP strains harbored plasmid-mediated quinolone resistance genes (oqxA, oqxB, qnrS, qnrB), β-lactams (bla CTX-M-3), aminoglycosides, type I and type III fimbriae genes, siderophore genes, and transporter and pumps. SIM-producing ST1764 K64 showed typical features of hvKP, showing hypermucoviscosity phenotype. The virulence genes, including rmpA2, alls and aerobactin genes, linked to hvKP, were found in ST1764 hvKP. hvKP was sensitive to quinolone; also, oqxA gene was detected. All TGC combinations showed highly synergistic effects and TGC+IPM was more effective treatment. Conclusion We first identified the NDM-5-producing ST690 CRKP and SIM-producing ST1764 hvKP strains in Shanxi province. Tigecycline-carbapenem combinations were available treatments for CRKP.

Food stuffs of animal origin may present hazards, due to bacterial contamination. This study was conducted to determine the prevalence, serotypes, and virulence genes of Escherichia coli in raw beef meat and some of meat products (raw, minced meat, beef burger and sausage, 25 sample each).Bacteriological examination of total 100 samples were collected from different localities in port-said City showed that 10/100 (10%) of samples were infected with E. coli. The prevalence of E.coli strains in fresh and minced meat, beef burger and sausage samples were4% (1/25), 16 % (4/25), 8 %( 2/25), and 12% (3/25), respectively.Serological typing were conducted on 10 isolates and results obtained revealed that two (20%) O114, 2 (20%) O157, three (30%) O125, and one (10%) for each of O127; O18, and O158. Ten isolates were submitted to molecular studies for detection of the traT (transfer protein), iutA (encoding aerobactin) and iss (increased serum survival protein) genes, by using PCR technique. PCR assays for different virulence genes showed that sex (60%), four (40%), and sex (60%) strains carry iss, iutA, and Trat genes, respectively.

The molecular genetic characteristic of the Klebsiella pneumoniae KP254 clinical strain belonging to clonal group 23, are given based on the genome-wide sequencing results. It is known that these clonal group representatives can have highly virulent properties and be causative agents of community-acquired infections. Multidrug resistance, including carbapenems, characterizes the K. pneumoniae KP254 strain phenotypically. The determinants of antibiotic resistance (blaSHV-1, oqxAB, fosA) and pathogenicity encoding fimbriae 1, 3 types and the synthesis of siderophore yersineobactin were found in the chromosome structure. The absence of the conjugative element ICEKp1, the pathogenicity island KPHPI208, and the allantoin regulon genes, which are often possessed by highly virulent strains, have been established. The replicons of plasmids of incompatibility groups FII, FIA HI1 /FII K , Col440I, Col pVC , FIB K , FII pCRY were revealed by analysis of nucleotide sequences in silico . The presence of presumably two antibiotic resistance plasmids IncFII and IncFII pCRY and one virulence plasmid IncFIBK were determined as a result of combining contigs with respect to reference sequences using the BLASTN service. The determinants of the aerobactin siderophore, the RmpA2 mucoid phenotype regulator, as well as heavy metal resistance genes are includes to the virulence plasmid structure. The virulence plasmid nucleotide sequence coverage was 93% relative to the virulence plasmid pK2044 with 99.38% identity level; the genomic regions responsible for the salmochelin and RmpA protein synthesis were deleted. The set of antibiotic resistance determinants identified in the mobilome structure includes the genes for beta-lactamase LAP-2 (IncFII pCRY plasmid) - a TEM-1 analogue, as well as extended-spectrum beta-lactamase CTX-M-55 (IncFII plasmid). Both determinants are rarely registered in the Russian Federation. Additionally, widespread genes bla OXA-1 , aac(3')-IIa , ΔcatB4 , aac (6') - Ib-cr , tet(A) , qnrS1 , sul 2 , catA2 were found in the plasmid DNA. The carbapenemases genes are absent in the resistome structure, while the studied strain is carbapenem resistant. The analysis of the ompK35 and ompK36 porins genes translated sequences revealed mutational changes that led to the stop codon formation in the ompK35 gene, and the amino acid sequence OmpK36 contains a large number of substitutions, insertions, and deletions. The presence of such changes is one of the factors determining the resistance to carbapenems. The synergistic effect by the activity of the efflux pumps present in the structure of the K. pneumoniae KP254 genome, in particular, AcrAB-TolC and KpnEF, may be exerted. Thus, the studied strain retains the most significant signs characteristic of the highly virulent Klebsiella strains evolutionary branch representatives, and at the same time, acquires the multidrug resistance genetic determinants.

Background Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. Methods Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. Results The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antibiotic resistance plasmids and a virulent plasmid. The antibiotic resistance plasmid expressing blaKPC-2, blaCTX-M-15, aph(3'')-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of capsular polysaccharide genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. We also identified an isolate that expressed the alls gene. Conclusion Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

Hypervirulent Klebsiella pneumoniae (hvKP) strains are associated with vigorous clinical presentation and relapses. Initially reported from Asia, these variants have spread globally and become an emerging agent of significant health threat. This study was carried out to identify hvKP strains in a previously uninvestigated region and to evaluate the impact of commonly-employed phenotypic and genotypic markers as diagnostic assays. A total of 111 blood culture isolates, collected at a tertiary care center was investigated. The hvKP strains were sought by a string test and the amplification of partial magA, rmpA, iucA and peg344. All products were characterized via sequencing. Evidence for hvKP was observed in 10.8% via iucA amplification (7.2%), string test (2.7%) and magA amplification (0.9%). Specific products were not produced by assays targeting rmpA and peg344 genes. Antibiotic susceptibility patterns compatible with possible extensive or pan-antimicrobial resistance was noted in 66.7% of the hvKP candidate strains. Capsule type in the magA positive strain was characterized as K5. We have detected hvKP in low prevalence at a region with no prior documentation. Targetting the aerobactin gene via iucA amplification provided the most accurate detection in this setting. The epidemiology of hvKP in Anatolia requires elucidation for effective control and management.

A growing number of evidence shows that human-associated microbiota is an important contributor in health and disease. However, much of the complexity of host-microbiota interaction remains to be elucidated both at cellular and molecular levels. Siderophores are chemically diverse, ferric-specific chelators synthesized and secreted by microbes to secure their iron acquisition. The host defense peptide LL-37 is ubiquitously produced at epithelial surfaces modulating microbial communities and suppressing pathogenic strains. The present work demonstrates that LL-37 binds tightly siderocalin-resistant stealth siderophores which are important contributors to the virulence of several pathogens. As indicated by circular dichroism spectroscopic experiments, addition of aerobactin and rhizoferrin increases the membrane active α-helical conformation of the partially folded peptide. The cationic nature of LL-37 (+6 net charge at pH 7.4) and the multiple carboxylate groups present in siderophores refer to the dominant contribution of electrostatic interactions in the stabilization of peptide-chelator adducts. It is proposed that aside siderocalin proteins, LL-37 may be a complementary, less specific component of the siderophore scavenging repertoire of the innate immune system.

Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N(6)-acetyl-N(6)-hydroxylysine onto the primary carboxylates of citrate. IucA and IucC are members of a family of nonribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family have been solved, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed terreactant, steady state kinetic analysis, and herein provide evidence for an ordered mechanism in which chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with site-directed mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. Here, we report the genome of a non-clinical E. bugandensis strain. We used this and other several publicly available E. bugandensis genomes to obtain the species pangenome, investigate the conservation of important genes, and elucidate general population structure features of the species. Core- and whole-genome multilocus sequence typing (cgMLST and wgMLST, respectively) allowed the detection of five E. bugandensis phylogroups (PG-A to E). We found important antimicrobial resistance and virulence determinants associated with specific PGs, notably PG-A and PG-E. IncFII was the most prevalent plasmid replicon type in this species. We uncovered several extended-spectrum β-lactamases, including blaCTX-M-55 and blaNDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of blaNDM-5 revealed the resemblance of this plasmid with other IncX plasmids isolated from other bacteria from the same country. Further, three distinctive siderophore producing operons were found in the E. bugandensis pangenome: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). The latter operon is conserved in all PG-E isolates. Collectively, our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.

Unusual community-acquired invasive Klebsiella pneumoniae infection has been reported worldwide, particularly in Asia. Recently, several virulence-associated genes of the isolates have been investigated. We report a case of multifocal intramuscular and musculoskeletal abscesses caused by K. pneumoniae in a 61-year-old male diabetes patient. A string test of the K. pneumoniae isolate, which was recovered from abscesses obtained by surgical debridement and drainage, was positive. We used whole-genome sequencing to analyze the virulence-associated gene profile of the isolate. The isolate belonged to the K2 genotype with sequence type 375. The isolate harbored rmpA and rmpA2, which induce serum resistance (hypermucoviscosity). The isolate also carried siderophores, i.e., aerobactin and salmochelin, which are associated with enhanced bacterial growth. The isolate did not harbor K1-unique virulence factors, such as colibactin, microcin, and yersiniabactin. Our K2 strain harbored a combination of virulence plasmid-associated genes—rmpA/A2 and siderophores (aerobactin and salmochelin). Hence, we advocate that essential molecular virulence factors of isolates that cannot be identified by a string test and capsular serotyping alone may exist.

Salmonella enterica strains often harbor plasmids representing several incompatibility groups (Inc) including IncFIB, which have been previously associated with carrying antimicrobial resistance and virulence associated genes. To better understand the distribution of virulence genes on IncFIB plasmids, we analyzed 37 complete whole genome and plasmid sequences of different S. enterica isolates from multiple serovars. Many of the sequences analyzed carried multiple virulence-associated genes, including those associated with iron acquisition systems; thus we aimed to determine how iron-rich (IR) and various iron-depleted (ID) conditions affected the transcription of iron acquisition and virulence genes including sitA, iutA, iucA, and enolase at different time intervals. sitA, iutA, and enolase from S. enterica that were grown in Luria-Bertani broth (LB) ID (LBID) conditions were substantially upregulated when compared to LBIR conditions. For both S. enterica strains that were grown at various LBID conditions, addition of 200 μM bipyridyl in the growth medium yielded the highest transcription for all four genes, followed by the 100 μM concentration. An antibody using a peptide targeting aerobactin receptor gene iutA encoded by IncFIB was generated and used to examine the protein expression in the wild-type, recipient, and transconjugant strain in LB, LBID, and LBIR growth conditions using Western blot analyses. A 70 KDa protein band was detected in the wild-type and transconjugant that carried the IncFIB plasmid, while this band was not detected in the recipient strain that lacked this plasmid.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains have been increasingly reported, and it is important to understand the evolutionary mechanisms of these highly pathogenic and resistant bacterial pathogens. In this study, we characterized a ST11 carbapenem-resistant K. pneumoniae strain which harbored an IncFIB/IncHI1B type virulence plasmid and an IncFII/IncR type blaKPC–2-bearing plasmid. The virulence plasmid was found to be conjugative and harbored a 35-kbp fragment including aerobactin encoding cluster from virulence plasmid pLVPK and multiple resistance genes, resulting in a mosaic multi-drug resistance and virulence plasmid. This virulence plasmid could be transferred via conjugation to Escherichia coli and K. pneumoniae strains alone as well as together with the blaKPC–2-bearing plasmid. Co-transmission of virulence and blaKPC–2-bearing plasmids would directly convert a classic K. pneumoniae strain into CR-HvKP strain, leading to a sharp increase in the prevalence of CR-HvKP in clinical settings, which poses a great threat to human health.

The presence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) often cause life-threatening infections worldwide, but the therapeutic option is limited. In this study, whole-genome sequencing (WGS) was applied to assess the epidemiological characteristics and transmission dynamics of CRKP isolates recovered from two fetal outbreaks of nosocomial infections. Between April 2016 and March 2018, a total of 70 isolates of K. pneumoniae were collected from sterile samples in a tertiary hospital in Hangzhou, China. The minimal inhibitory concentrations (MICs) of 21 antimicrobial agents were determined using the broth microdilution methods. Pulsed-field gel electrophoresis (PFGE) was performed on 47 CRKP isolates, and 16 clonally related isolates were further characterized by Illumina sequencing. In addition, the complete genome sequences of three representative isolates (KP12, KP36, and KP37) were determined by Oxford Nanopore sequencing. The K. pneumoniae isolates were recovered from patients diagnosed with pulmonary infection, cancer, or encephalopathy. For all CRKP isolates, PFGE separated three clusters among all strains. The most predominant PFGE cluster contained 16 isolates collected from patients who shared close hospital units and represented a potential outbreak. All 16 isolates showed an extremely high resistance level (≥87.5%) to 18 antimicrobials tested but remain susceptible to colistin (CST). Multiple antimicrobial resistance and virulence determinants, such as the carbapenem resistance gene blaKPC-2, and genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA and rmpA2), were observed in the 16 CRKP isolates. These isolates belonged to sequence type 11 (ST11) and capsular serotype KL64. A core genome single nucleotide polymorphism (cgSNP)-based phylogenetic analysis indicated that the 16 CRKP isolates could be partitioned into two separate clades (≤15 SNPs), suggesting the two independent transmission scenarios co-occurred. Moreover, a high prevalence of IncFIB/IncHI1B type virulence plasmid with the iroBCDN locus deleted, and an IncFII/IncR type blaKPC-2-bearing plasmid was co-harbored in ST11-KL64 CRKP isolates. In conclusion, our data indicated that the nosocomial dissemination of ST11-KL64 CRKP clone is a potential threat to anti-infective therapy. The development of novel strategies for surveillance, diagnosis, and treatment of this high-risk CRKP clone is urgently needed.

IntroductionKlebsiella pneumoniae is recognized as an urgent threat to human health because of the emergence of multidrug-resistant (MDR) and hypervirulent strains. Development of novel highly effective and safe treatment options is the need of the hour. One of the ways of achieving this goal is by conducting molecular characterization studies of antibiotic resistant bacteria.Hypothesis/Gap StatementThe government of India is committed to generate validated AMR data from different parts of the country. Efforts are being made to perform molecular characterization of MDR and potentially virulent bacteria by whole-genome sequencing (WGS). However, the data that we have at present is skewed as many parts of the country remain underrepresented. Uttarakhand is one such state located in the Himalay an belt of India, with relatively poor access to healthcare and a paucity of research.AimThis study was performed to generate WGS based preliminary data about the population structure, multi-locus sequence types (MLST), and virulence factors of CRKp isolates recovered from patients in a tertiary care teaching hospital and institute of national importance, located in Rishikesh, Uttarakhand, India.MethodologyA cross-sectional study was conducted at a tertiary care teaching hospital. It included twenty-nine randomly selected and archived carbapenem resistant Klebsiella pneumoniae (CR-Kp) isolates obtained from various clinical samples submitted in the Bacteriology laboratory for culture and sensitivity testing, from July 2018 to August 2019. After preliminary identification (ID) and antibiotic susceptibility testing (AST), as per standard guidelines, these isolates were sent to Central Research Laboratory (CRL), Bengaluru, India, for further characterization & WGS.ResultsTwenty-seven out of twenty-nine test isolates were CRKp. Among the 27 CRKp isolates, ST14 was the most common sequence type (8, 29.6%), followed by ST231 (5, 18.5%) and ST147(3, 11.1%) respectively. KL2 (9/27, 33.3%) and KL51 (8/27, 29.6%) were dominant K loci types in this study. Out of 5 O antigens identified, O1 and O2 together accounted for 88.9% (n=27) CRKp isolates. Yersiniabactin and Aerobactin were identified in 88.9% (24/27) & 29.6% (8/27) of the CRKp isolates of the isolate. Regulatory genes rmpA2 and rmpADC were found in 14.8% (4/27) and 3.7% (1/27) isolates respectively. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%), IncFIB(pQil) (44.4%), IncFIB(K) (37%), IncR (33.3%) and Col44 0I (18.5%) respectively. A perfect agreement (100%) was observed between phenotypic and genotypic resistance profiles in the case of fluoroquinolones, penicillins, Beta Lactam/Beta Lactam Inhibitor combinations (BL/BLI), and cephalosporins respectively. As compared to phenotypic resistance, higher genotypic resistance for aminoglycosides (96.3%) and folate pathway inhibitors (92.6%) respectively, was observed.ConclusionThis study emphasizes the need for continued genomic surveillance of emerging CRKp and other MDR bacteria in Uttarakhand and neighbouring states of India. This in turn would help in generating critical information that can be used to assess the emergence, dissemination, and potential impact of important variants.

Comparative genomics, in particular, pan-genome analysis, provides an in-depth understanding of the genetic variability and dynamics of a bacterial species. Coupled with whole-genome-based taxonomic analysis, these approaches can help to provide comprehensive, detailed insights into a bacterial species. Here, we report whole-genome-based taxonomic classification and comparative genomic analysis of potential human pathogenic Enterobacter hormaechei subsp. hoffmannii isolated from chlorinated wastewater. Genome Blast Distance Phylogeny (GBDP), digital DNA-DNA hybridization (dDDH), and average nucleotide identity (ANI) confirmed the identity of the isolates. The algorithm PathogenFinder predicted the isolates to be human pathogens with a probability of greater than 0.78. The potential pathogenic nature of the isolates was supported by the presence of biosynthetic gene clusters (BGCs), aerobactin, and aryl polyenes (APEs), which are known to be associated with pathogenic/virulent strains. Moreover, analysis of the genome sequences of the isolates reflected the presence of an arsenal of virulence factors and antibiotic resistance genes that augment the predictions of the algorithm PathogenFinder. The study comprehensively elucidated the genomic features of pathogenic Enterobacter isolates from wastewaters, highlighting the role of wastewaters in the dissemination of pathogenic microbes, and the need for monitoring the effectiveness of the wastewater treatment process.

BackgroundThe incidence of hypervirulent (hv) carbapenem-resistant (CR)Klebsiella pneumoniae(Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity.MethodsTwenty-seven CRKp isolates were identified to possessrmpA2by whole-genome sequencing; and resistance and virulence determinants were characterized. Byin silicoprotein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp.ResultsThe CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncatedrmpAandrmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones.In silicoanalysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors.ConclusionIncreasing incidence of convergence of AMR and virulence is observed amongK. pneumoniaeglobally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

Klebsiella pneumoniae is a well-studied human pathogen for which antimicrobial resistant and hypervirulent clones have emerged globally. K. pneumoniae is also present in a variety of environmental niches, but currently there is a lack of knowledge on the occurrence and characteristics of K. pneumoniae from non-human sources. Certain environmental niches, e.g., animals, may be associated with high K. pneumoniae abundance, and these can constitute a reservoir for further transmission of strains and genetic elements. The aim of this study was to explore and characterize K. pneumoniae from healthy broilers and turkeys. A total of 511 cecal samples (broiler n = 356, turkey n = 155), included in the Norwegian monitoring program for antimicrobial resistance (AMR) in the veterinary sector (NORM-VET) in 2018, were screened for K. pneumoniae by culturing on SCAI agar. K. pneumoniae was detected in 207 (40.5%) samples. Among the broiler samples, 25.8% were positive for K. pneumoniae, in contrast to turkey with 74.2% positive samples (p < 0.01). Antibiotic susceptibility testing was performed, in addition to investigating biofilm production. Whole genome sequencing was performed on 203 K. pneumoniae isolates, and analysis was performed utilizing comparative genomics tools. The genomes grouped into 66 sequence types (STs), with ST35, ST4710 and ST37 being the most prevalent at 13.8%, 7.4%, and 5.4%, respectively. The overall AMR occurrence was low, with only 11.3% of the isolates showing both pheno- and genotypic resistance. Genes encoding aerobactin, salmochelin or yersiniabactin were detected in 47 (23.2%) genomes. Fifteen hypervirulent genomes belonging to ST4710 and isolated from turkey were identified. These all encoded the siderophore virulence loci iuc5 and iro5 on an IncF plasmid. Isolates from both poultry species displayed good biofilm-forming abilities with an average of OD595 0.69 and 0.64. To conclude, the occurrence of K. pneumoniae in turkey was significantly higher than in broiler, indicating that turkey might be an important zoonotic reservoir for K. pneumoniae compared to broilers. Furthermore, our results show a highly diverse K. pneumoniae population in poultry, low levels of antimicrobial resistance, good biofilm-forming abilities and a novel hypervirulent ST4710 clone circulating in the turkey population.

The aim of this study was to examine antibiotic resistance profiles and diversity of β-lactamases in Escherichia coli present within the population and the potential spread of resistant E. coli into the receiving environment using city-scale sewage surveillance. In E. coli isolates from ECC plates without antibiotics from ten influent samples (n = 300), highest resistance was observed against ampicillin (16.6%), sulfamethoxazole (9.7%) and trimethoprim (9.0%), while in effluent samples (n = 262) it was against sulfamethoxazole (11.8%), ampicillin (11.5%) and tetracycline (8.8%). All isolates (n = 123) obtained on cefotaxime-containing plates were multidrug-resistant. Several clinically important antibiotic resistance genes (ARGs) were detected in 46 E. coli isolates subjected to whole-genome sequencing, including carbapenemases like NDM-6, VIM-1 and OXA-48-variant, as well as tigecycline resistance gene tet(X4). CTX-M-15 was the most prevalent (42.9%) extended-spectrum β-lactamase among cefotaxime-resistant isolates, followed by CTX-M-27 (31.4%) and CTX-M-14 (17.1%), resembling clinical prevalence in Norway. Most of the sequenced isolates carried other clinically relevant ARGs, such as dfrA17, sul1, sul2, tet(A), aph(6)-Id, aph(3’’)-Ib and aadA5. Sixteen different sequence types (STs) were identified, including ST131 (39.1%), ST38 (10.9%) and ST69 (8.7%). One E. coli isolate belonging to novel ST (ST11874) carried multiple virulence factors including genotoxin, salmochelin, aerobactin and yersiniabactin, suggesting that this isolate has potential to cause health concerns in future. Our study reveals presence of clinically relevant ARGs like blaNDM-6 and tet(X4) in pathogenic strains, which have so far not been reported from the clinics in Norway. Our study may thus, provide a framework for population-based surveillance of antibiotic resistance.

Background Emergence of bla KPC and bla NDM co-producing Klebsiella pneumoniae strains have led to the limited therapeutic options for clinical treatment. Understanding the diversity and frequency of resistance and virulence genes of these isolates is of great significance. Purpose The aim of this study is to research the diversity and frequency of resistance and virulence genes in the bla KPC and bla NDM co-producing Klebsiella pneumoniae strains. Methods and results In this study, 117 K. pneumonia strains were isolated from China, and among of which, 24 were found to be bla KPC and bla NDM co-producing with significant resistance against almost all the commonly used antibiotics. Additionally, 4 strains were hypermucoviscous and 8 showed high serum resistance. Overall, bla SHV, bla CTX-M, tetA and sul1 resistance genes found in 100% of the isolates, followed by bla TEM (95.8%), oqxA/B (91.7%), qnrB (87.5%), aac(6')Ib-cr (83.3%), bla DHA (79.2%), rmtB (66.7%), qnrS (54.2%), cat(54.2%), floR (50.0%), sul2 (45.8%) cmlA (20.8%)andbla CMY (8.33%), respectively. What' more, seven bla CTX-M subtypes [bla CTX-M-14 (n=18), bla CTX-M-3(n=11), bla CTX-M-65 (n=4), bla CTX-M-15 (n=3), bla CTX-M-28 (n=2), bla CTX-M-55 (n=2), bla CTX-M-22 (n=1)] and six bla SHV subtypes [bla SHV-12(n=16), bla SHV-11 (n=4), bla SHV-2a(n=1), bla SHV-1(n=1), bla SHV-38(n=1) and bla SHV-28(n=1)] were detected. The frequency of virulence genes was as follows: 100% for entB, ybtS and irp, 95.8% for mrkD, 91.66% for fimH, 79.2% for iutA, 62.5% for iroBCDE, aerobactin and kfu, 66.7% for allS, 45.8% for wcaG, 37.5% for rmpA, 20.8% for pagO and 16.7% for magA. Conclusion From this study, we concluded that the bla KPC and bla NDM co-producing Klebsiella pneumoniae strains have a high diversity and frequency of resistance and virulence genes. This study may offer hospitals important information about the control of infections caused by bla KPC and bla NDM co-producing Klebsiella pneumoniae.

We researched 26 K. pneumoniae ssp. pneumoniae strains, isolated from patients and objects of the environment during the epidemic ill-being period in the pediatric hospital. Identification of strains and their clustering, study of antibiotic sensitivity, detection of antibiotic resistance determinants, whole genome sequencing were carried out. It is established that one strain characterized by identical RAPD patterns and sensitivity to antibiotics from the groups of cephalosporins, monobactams, aminoglycosides, carbapenems, fluoroquinolones, sulfonamides and glycylcyclides circulates in the department.The strain is resistant to ampicillin (MIC > 16) and nitrofurantoin (MIC > 64). Whole genome sequencing made it possible to characterize the strain as K. pneumoniae ssp. pneumoniae ST 3181K-54, the analysis of the whole genome sequence showed the presence of a fosomycin resistance gene (fosA) and virulence determinants namely a complex of siderophores (aerobactin and enterobactin), fimbria of the third type, iron utilization system kfu and cluster of genes responsible for allantoin utilization that allowed us to consider the strain as a microorganism with a high pathogenic potential capable to rapid prevalence and dissemination in tissues.

Objectives To investigate the pathogenicity of Klebsiella pneumoniae (KPN) possessing rmpA or the aerobactin gene in infected mice. Methods BALB/c mice were divided into four groups (n = 10 per group) and infected with: string test-positive and rmpA-positive or aerobactin-positive KPN (group 1), string test-negative but rmpA-positive KPN (group 2), string test-negative but aerobactin-positive KPN (group 3), or string test- and rmpA/aerobactin-negative KPN (group 4). Mouse survival time was compared among groups, and the infection of livers, spleens, lungs, and kidneys and KPN growth were assessed in infected mice. Results Compared with the negative group (group 4), the survival rates of mice infected with rmpA- or aerobactin-positive KPN (groups 1–3) were significantly lower, their multi-organ injuries were significantly more severe, and bacterial enumeration was significantly higher. Conclusions Despite being string test-negative, aerobactin- or rmpA-positive KPN still exhibit high virulence and anti-immune effect activity. Therefore, the combination of the string test and gene detection of aerobactin and rmpA will be helpful in screening hypervirulent KPN.

Thirty-five serotype K1 hypervirulent Klebsiella pneumoniae (K1-hvKP) isolates collected from a Chinese hospital during the whole year of 2017 were evaluated to characterize the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes. In total, 18 (51.4%) isolates were detected to carry PMQR genes, and the most frequently detected gene was qnrS1 (37.5%), followed by aac(6')-Ib-cr (15%) and qnrB4 (2.5%). Remarkably, some qnr-carrying strains had only a single or more quinolone resistance-determining region mutations in GyrA or ParC, and most exhibited high-level ciprofloxacin resistance. However, only 11.4% (4/35) isolates were resistant to quinolones. Furthermore, 34.3% (12/35) carried extended-spectrum β-lactamase (ESBL) genes, but only 14.3% (5/35) exhibited ESBL phenotype. The most prevalent virulence genes were mrkD (100%, 21/21), followed by magA (97.1%, 34/35), wabG (97.1%, 34/35), rmpA (97.1%, 34/35), aerobactin (94.3%, 33/35), kfuB (94.3%, 33/35), ycf (91.4%, 32/35), iutA (91.4%, 32/35), rmpA2 (62.9%, 22/35), wcaG (62.9%, 22/35), ybtS (51.4%, 18/35), allS (45.7%, 16/35), and iroN (22.9%, 8/35). Multilocus sequence typing (MLST) analysis assigned the 35 K1-hvKP isolates into 5 sequence types (STs), with ST23 encompassing 77.1% of the strains. Pulsed field gel electrophoresis (PFGE) typing showed that strains closely related by MLST clustered in major PFGE clusters, of which cluster A accounts for 16 ST23 isolates and cluster B includes 11 ST23 isolates. The analysis of the transconjugants showed decreased susceptibility to quinolones and revealed a cotransfer of blaCTX-M-15 with the qnrS1 alleles. Cumulatively, our findings showed that the PMQR-producing K1-hvKP strain is covertly spreading even when K1-hvKP is rarely resistant to fluoroquinolones (FQs) according to the Clinical and Laboratory Standards Institute criteria. It is therefore critical to continuously monitor the PMQR-producing K1-hvKP epidemiology and minimize potential risks from FQ-resistant K1-hvKP.

In the era of emerging antibiotic resistance, Salmonella enterica subsp. enterica serovar Typhi the causative agent of typhoid, is a threat for healthcare systems in developing countries especially India, where the disease is highly endemic. Genetic diversity among different strains may be the cause of variable severity of disease in different regions of the world. To explore this genetic diversity, genome annotation by rapid annotation using subsystem technology (RAST) was carried out for genomes of four Salmonella Typhi strains from two distinct areas available in the public domain. Two clinical strains were from India (P-stx-12 and E02-1180) and the other two strains considered as reference strains were from the endemic regions of Papua New Guinea (UJ308A and UJ816A). We report that Indian clinical strains possess several similar genes responsible for virulence and pathogenicity as those present in the reference strains. Interestingly, Indian clinical strains also possess 34 additional potential virulence genes that are absent in the reference strains, suggesting the more dreadful nature of Indian clinical strains as compared to those causing endemic typhoid. Indian strains contained genes coding for; Colicin V and bacteriocin production; multidrug resistance efflux pumps; ABC transporters; Type III and Type VI secretion systems, siderophore aerobactin, pathogenicity islands and Vi polysaccharide biosynthesis and transport. These unique genes are also reported in the genomes of other six clinical strains of India analyzed through RAST and IslandViewer 4 for validation purpose. This study highlights the presence of potential genes as molecular targets to overcome the future endemic outbreaks in India.

Purpose: This study was undertaken to characterise the virulence factors in clinical strains of Klebsiella pneumoniae and analyse their association with various infections caused and also to determine the association between virulence factors and antimicrobial resistance profile. Materials and Methods: A total number of 370 clinically significant, non-duplicate isolates of K. pneumoniae isolated from both hospitalised patients and patients attending clinics were included in this study. Polymerase chain reaction (PCR) was carried out for the detection of various virulence genes such as mucoviscosity-associated gene A (magA), gene associated with allantoin metabolism (allS), Klebsiella ferric iron uptake(Kfu), capsule-associated gene A (K2A), regulator of mucoid phenotype A (rmpA), enterobactin (entB), yersiniabactin (YbtS), aerobactin, Fimbrial adhesin (FimH) and uridine-diphosphate galacturonate 4-epimerase (uge). Antimicrobial susceptibility testing and PCR-based detection of beta-lactamase-encoding genes such as extended-spectrum beta-lactamases, AmpCs and carbapenemases were performed. Univariate analysis was done to find the association between virulence genes and mortality. Results: The siderophore, entB, was present in most (90.5%) of the isolates. Of the 370 isolates, 345 carried multiple virulence genes; 15 harboured single virulence genes and 10 did not harbour any of the studied virulence genes. The most common combination of occurrence was entB and FimH. A mortality rate of 12.75% (38/298) was observed among hospitalised patients. None of the virulence genes had any significant association with mortality. Conclusion: Pathogenic K. pneumoniae can harbour single to multiple virulence genes. Invasive infection with even a single virulence gene-harbouring K. pneumoniae can lead to poor outcomes. Both multidrug-resistant (MDR) and non-MDR K. pneumoniae can harbour a variety of virulence genes. None of the virulence genes have a significant association with mortality.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid. IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

BackgroundHypervirulent Klebsiella pneumoniae (hvKp) infections have mainly been described in Asia. Two patients in the age group of 30 to 50 years presented within a two month period to a tertiary referral hospital in Texas with septic shock, hepatic abscess and septic thrombophlebitis. Blood cultures were positive for Klebsiella pneumoniae (isolates 2020CK-00441 and 2021CK-00720 respectively).MethodsWhole genome sequencing was performed using paired-end Illumina MiSeq reads for both isolates. Nanopore sequencing to obtain a closed genome was performed for 2020CK-00441.Results2020CK-00441 belonged to ST23 type while 2021CK-00720 was a ST65 type isolate. Kleborate analyses predicted with high confidence that both the isolates were hvKp. Phylogenetic analyses showed that the two strains are not closely related to each other or to any other known hvKp isolates. Both the isolates had yersiniabactin, colibactin, aerobactin and salmochelin producing loci which likely confer these isolates hvKp phenotype. 2020CK-00441 had a unique pK2044 like plasmid.ConclusionsHvKp strains capable of causing devastating metastatic septic infections have emerged in Texas. These isolates are unique when compared to other hvKp strains of the world. Country wide surveillance and whole genome sequencing of these strains is essential to prevent a major public health emergency in USA.

Background Two different types of hypervirulent K. pneumoniae (HvKp), the MLST-11 and serotype K1/K2 strains, have been frequently described in recent studies. Although these two types of strains were described to be HvKp, their virulence was not compared. In this study, in vitro and in vivo approaches were used to assess differences in virulence. Materials and methods A total of twenty-nine isolates, including 6 strains of each of serotype K1 and K2 isolates and 17 strains of ST11 isolates, were selected for this study. Phenotypic tests of virulence were performed by the string test and analysis of the virulent associated genes was detected by PCR. In vitro models of serum resistance and phagocytosis were used as the parameters to assess the virulence. In-frame deletion of virulence-associated genes was performed to study their contributions to virulence. The median lethal dose, i.e., the LD50, in mice was determined following IP injection. Results Although serotype K1 and K2 strains and ST11 isolates had similar virulence gene profiles, the ST11 isolates showed less serum and phagocytic resistance than the serotype K1/K2 isolates. The mouse lethality test revealed that all ST11 isolates were unable to cause lethality, even at > 107 CFU, while serotypes K1 and K2 showed an LD50 at ≤ 103 CFU. Aerobactin or capsule knockout mutants exhibited a lower LD50 than the parental strain, while capsule mutants showed a more significant decrease in LD50. Conclusion Since there was a significant difference in virulence levels between the two types of HvKp when assessed in in vitro and in vivo models, it may be better to use the designation "HvKp" for some strains based on animal studies to avoid confusion. Virulence and non-virulence could be analysed in a relative manner, especially in comparison studies.

Jiaying Tan,1,* Mi Tian,1,* Feng Zhao,1 Shuixiang Deng,1 Peng Jin,1 Yao Wang,1 Huimei Wen,1 Xiaohua Qin,2,3 Ye Gong1,4 1Department of Critical Care Medicine, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 2Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 3Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, 200040, People’s Republic of China; 4Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaohua QinInstitute of Antibiotics, Huashan Hospital, Fudan University, No. 12 Middle Urumqi Road, Shanghai, 200040, People’s Republic of ChinaEmail 0456240@fudan.edu.cnYe GongDepartment of Critical Care Medicine, Huashan Hospital, Fudan University, No. 12 Middle Urumqi Road, Shanghai, 200040, People’s Republic of ChinaEmail gong_ye@fudan.edu.cnBackground: Male genitourinary abscess is one of the serious complications of urinary tract infections (UTIs). There were few researches on the clinical and pathogenic characteristics of male genitourinary abscess.Patients and Methods: A retrospective observational study was conducted between January 2004 and April 2019. Male patients with genitourinary abscess originated from urinary tract, including sites of scrotum, testis, epididymis, spermatic cord, and prostate, were enrolled. Clinical and microbial records were collected and analyzed, and antimicrobial susceptibility testings wereperformed according to CLSI standard. Whole-genome sequencing was applied to detect the β-lactamase genes and virulence genes, as well as to determine the multilocus-sequence typing (MLST) of the collected Klebsiella pneumoniae (K. pneumoniae) isolates.Results: A total of 22 male patients were included. The main clinical symptoms were fever (86.4%), scrotal swelling (68.2%), local skin warmth (59.1%) and ulceration (45.5%). Urinary irritation was often presented in prostate involved abscess. Ultrasound features had a 94.7% positive rate. Surgical treatment, including abscess drainage, was helpful to the prognosis. No matter where the specimens obtained from, including blood, urine or pus, multidrug-resistant K. pneumoniae was the dominant (11 cases, 50.0%) microorganism in positive cultures. Nine of eleven K. pneumoniae isolates had been preserved and recovered. As for MLST typing, all the nine available isolates of K. pneumoniae belonged to the ST11 type and characterized with blaKPC-2 carbapenemase gene. Virulence genes rmpA2, ybtS, kfuC, wzi, aerobactin genes (iucABCD and iutA) and type 3 fimbriae genes (mrkAD) were identified in all the K. pneumoniae isolates.Conclusion: It seemed that more patients under 35 years old were vulnerable to genitourinary abscess. There was an increasing trend that multidrug-resistant K. pneumoniae isolates with multiple virulence genes were involved in male genitourinary abscess. Prompt and proper antibiotic use, combined with adequate drainage of the abscess, was important to prognosis.Keywords: male genitourinary abscess, carbapenem-resistant Klebsiella pneumoniae, virulence genes, MLST type

The increase in infections with multidrug-resistant and virulent Klebsiella pneumoniae (K. pneumoniae) strains poses a serious threat to public health. However, environmental reservoirs and routes of transmission for Klebsiella spp. that cause infections in humans and in livestock animals are not well understood. In this study, we aimed to analyze the distribution of antibiotic resistance genes and important virulence determinants (ybt, clb, iro, iuc, rmpA/A2) among 94 Klebsiella spp. isolates from different animal and food sources isolated between 2013 and 2017 in Germany. Antibiotic susceptibility testing was performed, and the genomes were sequenced by Illumina and Nanopore technology. Genetic relationships were assessed by conducting core genome multilocus sequence typing (cgMLST). Kleborate was used to predict resistance and virulence genes, Kaptive was used to derive the capsule types. The results revealed that 72 isolates (76.6%) belonged to the K. pneumoniae sensu lato complex. Within this complex, 44 known sequence types (STs), 18 new STs, and 38 capsule types were identified. Extended-spectrum beta-lactamase (ESBL) genes were detected in 16 isolates (17.0%) and colistin resistance in one (1.1%) K. pneumoniae isolate. Virulence genes were found in 22 K. pneumoniae isolates. Overall, nine (9.6%) and 18 (19.1%) isolates possessed the genes ybt and iuc, respectively. Notably, aerobactin (iuc lineage 3) was only detected in K. pneumoniae isolates from domestic pigs and wild boars. This study provides a snapshot of the genetic diversity of Klebsiella spp. in animals and food products in Germany. The siderophore aerobactin was found to be more prevalent in K. pneumoniae strains isolated from pigs than other sources. Further investigations are needed to evaluate if pigs constitute a reservoir for iuc lineage 3.

We describe a case of Lemierre's syndrome (LS) caused by a hypervirulent strain of Klebsiella pneumoniae in a 63-year-old female with hypertension, hyperlipidemia, and diabetes mellitus, who presented with right neck pain and fevers. Computerized tomography of the neck and chest revealed an occluded right internal jugular vein secondary to thrombosis and septic emboli in lungs. Blood cultures grew K. pneumoniae. The patient was treated with ampicillin-sulbactam and then transitioned to amoxicillin-clavulanate to complete a 6-week course of antibiotics, and a 3-month course of rivaroxaban. String test of the K. pneumoniae isolate was positive at 2 cm. Whole genome sequencing identified several genes associated with the hypervirulent strain, notably the genes encoding for aerobactin (iucA and iucB) and salmochelin (iroB) iron acquisition systems. LS can rarely be caused by K. pneumoniae. Clinicians should monitor for known complications, such as septic emboli in patients with LS.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and the analysis of the plasmids associated with CR-hypervirulent-KP (CR-HvKP) in Egypt are not presented. Therefore, we attempt to decipher the plasmids sequences, which are responsible for transferring the determinants of carbapenem-resistance, particularly the blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383, and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed using Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as CR-HvKP strain, it harboured four plasmids, namely; pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes, blaNDM-1 and blaKPC-2, were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD, iutA). Thus, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We reported for the first time a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient from the ICU in a tertiary-care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid.IMPORTANCECRKP had been registered in the critical priority tier by the World Health Organization and became a significant menace to public health. Therefore, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. Herein, we reported for the first time (to the best of our knowledge) a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient in a tertiary-care hospital from the ICU in Egypt, which showed the cohabitation of a novel hybrid plasmid co-harbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid. Herein, the high rate of CRKP might be due to the continuous usage of carbapenems as empirical therapy, besides the failure to implement an antibiotic stewardship program in Egyptian hospitals. Thus, this study serves to alert the contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe3+. Siderophores chelate Fe3+for uptake into the cell, where it is reduced to soluble Fe2+. As iron is essential for bacterial survival, siderophores are key molecules in low soluble iron conditions. Bacteria have devised many strategies to synthesize proprietary siderophores to avoid siderophore piracy by competing organisms, e.g., by incorporating different polyamine backbones into siderophores, while maintaining the catechol moieties. We report thatSerratia plymuthicaV4 produces a variety of siderophores, which we term thesiderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin with diaminopropane,S. plymuthicautilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore described forPhotorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described inEnterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact serratiochelin production. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness. This study illuminates the interplay between siderophore biosynthetic pathways and polyamine production superpathways, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 μmol/g DW) and putrescine (30.83 μmol/g DW),S. plymuthicacan be exploited for the industrial production of these compounds.Significance StatementSiderophores are molecules crucial for bacterial survival in low iron environments. Bacteria have evolved the capacity to pirate siderophores made by other bacterial strains and to diversify the structure of their own siderophores, to prevent piracy. We found thatSerratia plymuthicaV4 produces five different siderophores using three gene clusters and a polyamine production superpathway. The most well studied siderophore, enterobactin, rather than the strain’s proprietary and by far most abundant siderophore, serratiochelin, displayed a crucial role in the fitness ofS. plymuthica. Our results also indicate that this strain is a good candidate for engineering the large-scale production of diaminopropane (DAP), as without any optimization it produced the highest amounts of DAP reported for wild-type strains.

Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). Enterobacteriaceae corresponded to 78.6% (n = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were Escherichia spp. (57.1%, n = 117), followed by Aeromonas spp. (16.1%, n = 33) and Klebsiella spp. (12.7%, n = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum β-lactamases (ESBL), of which blaCTX-M-15 was predominant (37 isolates, 68.5%). A KPC-3 carbapenemase-producing K. oxytoca was identified (n = 1), with blaKPC-3 included in a transposon Tn4401 isoform b. A higher number of virulence genes (VG) (19 genes) was found in the E. coli 5301 (O25b-ST131-B2) isolate compared with a commensal E. coli 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin, Aerobactin, CFA/1 (clade α), Type1 (clade γ1), Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing K. oxytoca, and, for the first time, a CTX-M-15-producing Ochromobactrum intermedium, a human opportunistic pathogen, and a SED-1-producing Citrobacter farmeri, an uncommon CTX-M-type extended-spectrum beta-lactamase.

Avian pathogenic Escherichia coli (APEC) can cause localized or systemic infection in poultry herds, i.e., colibacillosis, which is an economically devastating bacterial disease of the poultry industry worldwide. Additionally, some APEC may have zoonotic potential. In this study, we sequenced 125 APEC isolates from chickens and ducks with obvious clinical symptoms in poultry farms in China and performed genomic epidemiological analysis along with 16 APEC reference genomes downloaded from NCBI. The phylogenetic analysis indicated a great diversity of APEC isolates, and a total of 35 different O types, 22 H types, and 29 ST types were identified. Several virulence-associated genes (VAGs), such as ompT (96.45 %), iss (97.87 %), and hlyF (90.78 %), as well as four complete siderophore gene clusters, including the Sit transport system (86.52 %), aerobactin (89.36 %), salmochelin (79.43 %), and yersiniabactin (54.61 %), were detected in APEC isolates with high prevalence, which could serve as virulence markers of APEC. Several virulence-associated gene clusters, including the two T6SS systems and the K1 capsule biosynthesis gene clusters, were significantly associated with APEC of phylogroups B2, D, and F but very rarely encoded by the APEC from phylogroups C and E. In addition, several virulence-associated genes, which have been reported in other E. coli pathotypes but have not been reported in APEC, were identified in this study. Our findings in this study have implications for a better understanding of APEC evolution and pathogenesis and may lead to the development of new diagnostic tools for APEC.

We recently reported on the diversity of Klebsiella pneumoniae isolated from dairy herds in China. In our previous work, isolates from subclinical mastitis (SCM) had lower indices of diversity when compared with bacteria from other sources, possibly due to a contagious-like spread of udder adapted strains. Here we explored the virulence profile and capsular types of K. pneumoniae isolated from different sources on 2 dairy farms in China. Our overarching goal was to gain insights on the role of virulence genes toward the severity of mastitis caused by K. pneumoniae. A total of 1,484 samples were collected from clinical mastitis (CM; n = 355), SCM (n = 561), bulk tank milk (BTM; n = 130), and environmental and extramammary (EE) sites (n = 438). From those, 431 K. pneumoniae isolates were obtained, including 129, 77, 66, and 159 isolates from CM, SCM, BTM, and EE samples, respectively. Polymerase chain reactions were used to determine the capsular types and to detect potential virulence genes in all isolates. No significant farm effects were observed when comparing the distribution of most virulence genes in K. pneumoniae isolated from each source. K57 was the most prevalent capsular type in K. pneumoniae from all sources, but with increased detection rate in isolates from CM. entB, kfu, fimH1, mrkD, and β-d-lacZ were frequently detected in K. pneumoniae from all sources. β-d-lacZ, entB, and ituA were more prevalent in isolates from CM, whereas kfu, allS, and nif were more frequently detected in isolates from SCM. ybtS, aerobactin, and rpmA had increased prevalence in K. pneumoniae from BTM when compared with bacteria from other sources. No association was detected between virulence genes and the severity of CM. K57 and the nif gene had the highest discriminatory power to classify isolates from CM and SCM, respectively. Based on our findings, it is likely that K57 is the dominant capsular type in K. pneumoniae causing CM in large Chinese dairy herds. Likewise, we demonstrated that β-d-lacZ is disseminated in K. pneumoniae isolated from large Chinese dairy farms, irrespectively of the source of bacteria. Our results also suggest a low contribution of the virulence profile of K. pneumoniae toward CM severity. Finally, the role of nif in increasing the adaptability to the udder and promoting a contagious-like spread of K. pneumoniae warrants further investigation.

BackgroundKlebsiella pneumoniae (K. pneumoniae) strains have been divided into two major categories: classical K. pneumonia, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe disseminated community-acquired infections in immunologically normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood.MethodsWe examined consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois April 2015 – April 2017. Bloodstream isolates underwent whole genome sequencing, and clinically relevant data (sequence type [ST], capsule loci, virulence factors, and antimicrobial resistance genes) were inferred from the genome using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as phenotypic antimicrobial resistance of the isolate were extracted from the electronic medical record. Candidate hypervirulent isolates were tested in a murine model of pneumonia. Complete genome sequences of these candidate hypervirulent isolates were obtained and plasmid content was evaluated.ResultsK. pneumoniae bloodstream isolates (n=104) from NMH were highly diverse consisting of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n=58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone, 17 harbored extended-spectrum beta-lactamases, and 9 (8.7%) were resistant to meropenem. Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. Of particular concern, several of these plasmids contained tra conjugation loci suggesting the potential for transmission. Two of these hypervirulent strains belonged to the well characterized ST23 lineage and one to the emerging ST66 lineage.ConclusionsWhile NMH K. pneumoniae bloodstream infections are caused by highly diverse strains, the most prevalent STs were those of multidrug-resistant high-risk clones. A small number of hypervirulent K. pneumoniae were observed in patients with no recent travel history, suggesting that these isolates are undergoing community spread in the United States.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer “average days alive” for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

BackgroundIn recent years, the emergence of multidrug resistant hypervirulent K. pneumoniae (MDR hvKp) isolates poses severe therapeutic challenge to global public health. The present study used the complete genome sequence of two MDR hvKp isolates belonging to ST23 to characterize the phylogenetic background and plasmid diversity.MethodsTwo hvKp isolates from patients with bacteremia were sequenced using Ion Torrent PGM and Oxford Nanopore MinION platforms and assembled by hybrid genome assembly approach. Comparative genomics approaches were used to investigate the population structure, evolution, virulence, and antimicrobial resistance of MDR hvKp strains.ResultsThe study isolates exhibited typical features of hvKp phenotypes associated with ST23. The convergence of multidrug resistance and hypervirulence were attributed by the presence of multiple plasmids including a 216 kb virulence plasmid and MDR plasmids belonging to IncA/C2, IncFIB, IncX3, and ColKP3 groups. The insertion of catA1 gene into virulence plasmid was observed along with genetic factors such as aerobactin, salmochelin, and rmpA2 that confer hvKp’s hypervirulent phenotype. The core genome single nucleotide polymorphism (SNP) phylogenetic analyses of the isolates showed the evolution of ST23 hvKp was predominantly driven by ICEKp acquisitions.ConclusionTo the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes. ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp.

Escherichia coli is one of the most commonly isolated pathogens from surgical site infections (SSIs) that accounts for significant morbidity and mortality, especially with high virulent strains. The purpose of this study was to evaluate the virulence determinants of E. coli strains isolated from surgical sections. 51 E. coli isolates were assessed for biofilm formation, mannose-sensitive or -resistant hemagglutination, capsule, hemolysin and siderophores production. Antibiotic susceptibility against 14 common antibiotics was performed. As a result, 25 strains displayed 5 virulence factors and multiple drug resistance towards more than 11 antibiotics. DNA and plasmids were extracted from the 25 virulent isolates. PCR was used to investigate virulence genes on DNA encoding adhesins (fimH-1, mrkD), hemolysin (hlyA), siderophores (entB: enterobactin, iutA: aerobactin, irp-1: yersiniabactin- which was encoded on (HPI) high-pathogenicity island). MrkD, iutA and hlyA genes were also screened on extracted plasmids. The most common virulence genes were iutA (25/100%), mrkD (24/96%), entB (23/92%) and fimH-1 (21/84%). Irp1 was found at moderate rates (15 /60%) and at lower prevalence, was gene hlyA (2 / 8%). Plasmids were found in 16/25 strains. MrkD and iutA were present in 10 /16 plasmids, whereas none of them harbored hlyA. In conclusion, most E. coli isolates harbored high frequencies of (fimH1, mrkD, entB and iutA) which seem to be at the basis of pathogenicity. However, some strains, which carry HPI and have virulence plasmids can account for even more real threat if they spread among other Enterobacteriaceae members in surgical sections.

Klebsiella pneumoniae (Kp) reference strain Kp52.145 is widely used in experimental Klebsiella pathophysiology. Since 1935, only one other strain of the same sublineage (sequence type ST66, capsular serotype K2) was isolated (AJ210, Australia). Here, we describe a community-acquired invasive infection caused by a ST66-K2 Kp strain in France. Four hypermucoviscous Kp isolates responsible for acute otitis media, meningitis, bacteraemia and bacteriuria, respectively, were obtained from a patient with a history of chronic alcoholism and diabetes mellitus, and infected with HIV. The isolates were characterized by phenotypic and genomic methods. The four genetically identical ST66-K2 isolates presented a full antimicrobial susceptibility profile, including to ampicillin, corresponding to a single strain (SB5881), which was more closely related to AJ210 (135 SNPs) than to Kp52.145 (388 SNPs). Colibactin and yersiniabactin gene clusters were present on the integrative and conjugative element ICEKp10 in the chromosome. The two plasmids from Kp52.145 were detected in SB5881. In addition to carrying genes for virulence factors RmpA, aerobactin and salmochelin, plasmid II has acquired in SB5881, the conjugation machinery gene cluster from plasmid I. We report the first case of community-acquired infection caused by a hypervirulent ST66-K2 Kp strain in Europe. This demonstrates the long-term persistence of the high-virulence and laboratory model ST66-K2 sublineage. The combination of a conjugative apparatus and major virulence genes on a single plasmid may contribute to the co-occurrence of hypervirulence and multidrug resistance in single Kp strains.

Although the biochemistry of bacterial and fungal siderophores has been intensively studied in laboratory cultures, their distribution and impacts on nutrient cycling and microbial communities in soils remain poorly understood. The detection of siderophores in soil is an analytical challenge because of the complexity of the soil matrix and their structural diversity. Liquid chromatography-mass spectrometry (LC-MS) is a suitable method for the sensitive analysis of siderophores in complex samples; however, siderophore extraction into liquid phases for analysis by LC-MS is problematic because of their adsorption to soil particles and organic matter. To determine extraction efficiencies of structurally diverse siderophores, spike-recovery experiments were set up with standards representing the three main siderophore classes: the hydroxamate desferrioxamine B (DFOB), the α-hydroxycarboxylate rhizoferrin, and the catecholate protochelin. Previously used solvent extractions with water or methanol recovered only a small fraction (< 35%) of siderophores, including < 5% for rhizoferrin and protochelin. We designed combinatorial chemical extractions (22 total solutions) to target siderophores associated with different soil components. A combination of calcium chloride and ascorbate achieved high and, for some soils, quantitative extraction of DFOB and rhizoferrin. Protochelin analysis was complicated by potential fast oxidation and interactions with colloidal soil components. Using the optimized extraction method, we detected α-hydroxycarboxylate type siderophores (viz. rhizoferrin, vibrioferrin, and aerobactin) in soil for the first time. Concentrations reached 461 pmol g–1, exceeding previously reported concentrations of siderophores in soil and suggesting a yet unrecognized importance of α-hydroxycarboxylate siderophores for biological interactions and biogeochemical processes in soil.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant (CR) K. pneumoniae are on the rise in India. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in the present study.K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were tested for antimicrobial susceptibility as recommended by CLSI 2019 and subjected to 96-well microtitre plate biofilm formation assay. Based on optical density at 570 nm isolates were graded as strong, moderate and weak biofilm formers. Subset of strong biofilm formers were subjected to whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Biofilm formation was compared for an association with the carbapenem susceptibility and with patient outcome. Statistical significance, correlations and graphical representation were performed using SPSS v23.0.Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure. The biofilm forming ability had a significant association with the morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (PwcaG, magA, rmpA, rmpA2 and wzc with a lack of mutation for hyper-capsulation might be poor biofilm formers. Interestingly, ST15, ST16, ST307 and ST258 – reported global high-risk clones were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, mrkD) and pili (pilQ, ecpA).This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and antimicrobial resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

Objective The emergence of multidrug-resistant (MDR) and hypervirulent Klebsiella pneumoniae (hvKP) facilitates simultaneous dissemination of virulence and resistance in a single event, which poses serious threat to public health. Methods This study characterized the multidrug-resistant and moderately virulent ST11 K64 K. pneumoniae strain HB25-1 from a clinical case with microbiological and genomic approaches. Plasmids from strain HB25-1 were subjected to whole plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms. Klebsiella pneumoniae HB25-1 was subjected to a conjugation experiment and Galleria mellonella infection model to evaluate the transmission and virulence potential. Results We report the emergence of an ST11, serotype K64 K. pneumoniae isolate, which is resistant to third-generation cephalosporin and exhibited a moderate level of virulence. WGS revealed that this strain harboured a plasmid, pHB25-1, which carried multidrug resistance genes (blaDHA-1, qnrB4, dfrA12, aadA2, sul1, aac(3)-lld, blaTEM-1, mph(E)) and virulence-encoding genes (the regulator of mucoid phenotype A gene rmpA2 and the aerobactin gene cluster iutAiucABCD). Genomic analysis indicated that pHB25-1 was formed through co-integration of structural regions located in two different plasmids, enabling it to encode both resistance and virulent phenotypes. Conclusion Findings in this study provide evidence of active plasmid evolution in K. pneumoniae and suggest that surveillance of multidrug-resistant and hypervirulent K. pneumoniae is urgently needed.

When two or more bacterial species inhabit a shared niche, often, they must compete for limited nutrients. Iron is an essential nutrient that is especially scarce in the marine environment. Bacteria can use the production, release, and re-uptake of siderophores, small molecule iron chelators, to scavenge iron. Siderophores provide fitness advantages to species that employ them by enhancing iron acquisition, and moreover, by denying iron to competitors incapable of using the siderophore-iron complex. Here, we show that cell-free culture fluids from the marine bacterium Vibrio fischeri ES114 prevent the growth of other vibrio species. Mutagenesis reveals the aerobactin siderophore as the inhibitor. Our analysis reveals a gene, that we name aerE, encodes the aerobactin exporter, and LuxT is a transcriptional activator of aerobactin production. In co-culture, under iron-limiting conditions, aerobactin production allows V. fischeri ES114 to competitively exclude Vibrio harveyi, which does not possess aerobactin production and uptake genes. In contrast, V. fischeri ES114 mutants incapable of aerobactin production lose in competition with V. harveyi. Introduction of iutA, encoding the aerobactin receptor, together with fhuCDB, encoding the aerobactin importer are sufficient to convert V. harveyi into an "aerobactin cheater."