ST131 Strains Research Articles

Genomic characteristics of ST6115 carbapenem-resistant Klebsiella pneumoniae coharboring blaNDM-1 and blaIMP-4

ObjectivesThe aim of this study is to characterize the genomic features of ST6115 carbapenem-resistant Klebsiella pneumoniae (CRKP) co-harboring blaNDM-1 and blaIMP-4.MethodsThe genome sequences of one ST6115 Klebsiella pneumoniae (KP) strain and 12 ST17 KP strain were obtained through whole genome sequencing (WGS). Subsequently, a phylogenetic analysis was employed to ascertain the clonal relationship of these strains. The antimicrobial susceptibility testing (AST) was evaluated through the application of the minimum inhibitory concentration (MIC) methodology by the broth microdilution method. Annotation and analysis of the genome enabled the identification of the plasmid structure and the comparative analysis of the genetic background. Finally, the conjugation transfer experiment was conducted to assess the transferability of the plasmid pHD8428-NDM-IMP.ResultsA comparable phylogenetic analysis revealed that ST6115 HD8428 and the majority of ST17 strains (9/12) were clustered on the same clade, which suggests a close relationship between two ST types. Additionally, HD8428 showed particularly close genetic similarity to HD11490, with a single-nucleotide polymorphism (SNP) difference of only 273. The analysis of the antibiotic resistance genes carried by the 13 strains revealed that all strains carry extended-spectrum β-lactamase (ESBL) genes. AST revealed that HD8428 exhibited resistance to a diverse range of antibiotics. The blaNDM-1 and blaIMP-4 genes were identified as being located on the plasmid pHD8428-NDM-IMP. Further analysis demonstrated that the blaNDM-1 gene was present on ΔTn125, while the blaIMP-4 gene was located on In1377-2. The results of the conjugation experiment indicated that plasmid pHD8428-NDM-IMP may pose a risk for the transmission of antibiotic resistance in healthcare settings.ConclusionWe report a clinical ST6115 strain HD8428 and identify the coexistence of blaNDM-1 and blaIMP-4 in the IncHI5 type plasmid pHD8428-NDM-IMP. HD8428 was resistant to a wide range of antibiotics and harbored the transmissible plasmid pHD8428-NDM-IMP, which made it a potential threat to public health. Our study indicates that the healthcare system and services should remain vigilant regarding the spread and prevalence of ST6115.

Molecular analysis of AmpC-producing Escherichia coli isolated from pediatric patients.

AmpC-mediated cephalosporin resistance occurs in 1.0% to 3.3% of Escherichia coli isolates due to production of either plasmid-mediated AmpC (p-AmpC) or chromosomal AmpC (c-AmpC). Data on the prevalence and molecular characteristics of AmpC-producing E. coli in pediatric patients are limited. We analyzed E. coli clinical strains with resistance phenotype consistent with AmpC production isolated from patients at a pediatric hospital in Japan between 2015 and 2022. Sequence types, resistance genes, and relevant mutations were identified through whole genome sequencing. Promoter and attenuator regions of the chromosomal ampC gene were examined and the presence of plasmid-mediated ampC genes was determined. Among 2,081 E. coli strains, 80 (3.8%) from 27 patients demonstrated the AmpC phenotype. The median patient age was 55 months, with 92.6% having underlying diseases, mainly renal and urinary tract abnormalities. Of the 27 strains, p-AmpC was found in 9 strains including 6 strains belonging to ST131, while c-AmpC was identified in 18 strains including 9 ST73 strains and 4 ST12 strains. ST131 and ST73 were the major AmpC-E. coli lineages isolated from children with underlying diseases. Most ST131 strains harbored p-ampC, while all ST73 strains acquired cephalosporin resistance by c-AmpC production through promoter and attenuator mutations, suggesting the presence of both AmpC mechanisms in a lineage-specific manner in E. coli identified among hospitalized children.

Clostridioides difficile hypervirulent strain ST1 isolated from clinical stool specimens obtained from three Provinces in South Africa

Clostridioides difficile infection is a serious healthcare-associated infection linked to antimicrobial use. The severity of the disease can be associated with hypervirulent ribotypes such as RT027. The study aimed to investigate the molecular epidemiology and genomic characteristics of C. difficile isolates from private and public healthcare settings in South Africa. One hundred clinical stool specimens were cultured on cycloserine-cefoxitin-fructose agar. Conventional multiplex polymerase chain reaction (M-PCR) assays were conducted for isolate identification and detection of toxin genes. Genomic characteristics of the isolates were determined using whole genome sequencing (WGS) and data was analysed using pubMLST, EnteroBase, Pathogenwatch and CARD. One hundred clinically presumptive C. difficile positive stool specimens were collected, of which 62% (62/100) were confirmed as C. difficile by M-PCR assay. Among the 62 identified C. difficile isolates, 97% (60/62) were toxigenic, with the most dominant toxin profile being A+B+CDT+according to the M-PCR assay. The results showed that 93% (40/43) of the WGS analysed C. difficile strains clustered into clades 1 to 5. These 40 strains were categorized into 16 sequence types (STs), with ST1 (clade 2) being the most prevalent, representing 45% (18/40), this strain is an RT027-associated strain previously epidemic hypervirulent strain. One major cluster (n=18) comprising ST1 strains was identified in Gauteng Province and all the isolates associated with this cluster showed the same resistome (antimicrobial resistance genes and mutations: CDD-1, aac (6')-Ie-aph (2″)-Ia, PnimBG and Thr82Ile). The study also identified one strain as ST11, this strain is well known for its zoonotic potential, and two strains were identified as ST37 known as an epidemic strain. Strains from public healthcare settings exhibited genetic similarity, while those from private settings showed greater genetic diversity. The study reported, for the first time, hypervirulent strains ST1 in Africa and ST11 in South Africa, with a minimum spanning tree indicating an ongoing ST1 outbreak.

Comparative Analysis of In Vivo and In Vitro Virulence Among Foodborne and Clinical Listeria monocytogenes Strains.

Listeria monocytogenes is one of the most important foodborne pathogens that can cause invasive listeriosis. In this study, the virulence levels of 26 strains of L. monocytogenes isolated from food and clinical samples in Shanghai, China, between 2020 and 2022 were analyzed. There were significant differences among isolates in terms of their mortality rate in Galleria mellonella, cytotoxicity to JEG-3 cells, hemolytic activity, and expression of important virulence genes. Compared with other STs, both the ST121 (food source) and ST1930 (clinic source) strains exhibited higher G. mellonella mortality. The 48 h mortality in G. mellonella of lineage II strains was significantly higher than that in lineage I. Compared with other STs, ST1930, ST3, ST5, and ST1032 exhibited higher cytotoxicity to JEG-3 cells. Based on the classification of sources (food and clinical strains) and serogroups (II a, II b, and II c), there were no significant differences observed in terms of G. mellonella mortality, cytotoxicity, and hemolytic activity. In addition, ST121 exhibited significantly higher hly, inlA, inlB, prfA, plcA, and plcB gene expression compared with other STs. A gray relation analysis showed a high correlation between the toxicity of G. mellonella and the expression of the hly and inlB genes; in addition, L. monocytogenes may have a consistent virulence mechanism involving hemolysis activity and cytotoxicity. Through the integration of in vivo and in vitro infection models with information on the expression of virulence factor genes, the differences in virulence between strains or subtypes can be better understood.

From Traditional Typing to Genomic Precision: Whole-Genome Sequencing of Listeria monocytogenes Isolated from Refrigerated Foods in Chile.

Ready-to-eat (RTE) foods are the most common sources of Listeria monocytogenes transmission. Whole-genome sequencing (WGS) enhances the investigation of foodborne outbreaks by enabling the tracking of pathogen sources and the prediction of genetic traits related to virulence, stress, and antimicrobial resistance, which benefit food safety management. The aim of this study was to evaluate the efficacy of WGS in the typing of 16 L. monocytogenes strains isolated from refrigerated foods in Chile, highlighting its advantages in pathogen identification and the improvement of epidemiological surveillance and food safety. Using cgMLST, a cluster was identified comprising 2 strains with zero allele differences among the 16 strains evaluated. Ninety-four percent of the isolates (15/16) were serotype 1/2b, and 88% of them (14/16) were ST5. All strains shared identical virulence genes related to adhesion (ami, iap, lapB), stress resistance (clpCEP), invasion (aut, iapcwhA, inlAB, lpeA), toxin production (hly), and intracellular regulation (prfA), with only 13 strains exhibiting the bcrBC and qacJ gene, which confer resistance to quaternary ammonium. The pCFSAN010068_01 plasmids were prevalent, and insertion sequences (ISLs) and composite transposons (cns) were detected in 87.5% of the strains. The presence of various antibiotic resistance genes, along with resistance to thermal shocks and disinfectants, may provide L. monocytogenes ST5 strains with enhanced environmental resistance to the hygiene treatments used in the studied food production plant.

Evaluation of the Antibacterial Potential of Ethanolic Cannabis sativa L. (Hang Kra Rog Phu Phan ST1) Extracts Against Human Pathogenic Bacteria.

<b>Background and Objective:</b> Amid the escalating challenge of antibiotic resistance, the exploration of new sources has become essential, with plants serving as a promising reservoir of bioactive compounds. <i>Cannabis sativa</i> has attracted significant research interest for its antimicrobial properties and broad applications in medicine, industry and nutrition. This study aimed to investigate the antibacterial activity of ethanolic extracts from the stems and leaves of the Hang Kra Rog Phu Phan ST1 strain against twelve human pathogenic bacteria. <b>Materials and Methods:</b> Stems and leaves from the Hang Kra Rog Phu Phan ST1 strain were subjected to ethanol extraction. The primary antibacterial activity of ethanolic extracts from Tanao Si Kan Dang RD1 was assessed using the disc diffusion method, while the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined via the broth microdilution method. The inhibition zone diameter (mm) was analyzed using Duncan's Multiple Range Test (DMRT) with the SAS software. <b>Results:</b> The findings revealed that the ethanolic extract from the leaves of Hang Kra Rog Phu Phan ST1 produced the largest inhibition zone diameter of 10.00 mm against <i>Bacillus subtilis</i> TISTR 008. The MIC and MBC of the leaf extract showed the lowest values of 0.09 and 0.19 mg/mL, respectively, recorded against <i>Staphylococcus aureus</i> TISTR 1466. <b>Conclusion:</b> This is the first report on the antibacterial activity of the ethanolic extracts from the leaves and stems of Hang Kra Rog Phu Phan ST1, which offers potential benefits for developing natural antibiotic drugs to combat infections caused by the tested pathogenic bacteria.

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure.

It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to carbapenem resistance. Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two experimental evolutionary platforms to measure fast vs. slow adaptation. All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Increased transcript levels of genes present within the PCTn was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

Molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae in Gauteng South Africa

Klebsiella pneumoniae multidrug-resistant (MDR) high-risk clones drive the spread of antimicrobial resistance (AMR) associated infections, resulting in limited therapeutic options. This study described the genomic characteristics of K. pneumoniae MDR high-risk clones in Gauteng, South Africa. Representative carbapenem-resistant [K. pneumoniae carbapenemase (KPC)-2, New-Delhi metallo-beta (β)-lactamase (NDM)-1, oxacillinase (OXA)-181, OXA-232, OXA-48, Verona integron-encoded metallo-β-lactamase (VIM)-1] K. pneumoniae isolates (n = 22) obtained from inpatient and outpatient’s urine (n = 9) and inpatients rectal carriage (n = 13) were selected for short-read whole genome sequencing. Klebsiella pneumoniae population include sequence type (ST)-307 (n = 3), ST2497 (n = 5) and ST17 (n = 4). The ST17 strains were exclusively obtained from rectal screening. Ten isolates co-harboured carbapenemase genes including β-lactamase gene encoding KPC-2 + OXA-181, NDM-1 + OXA-48 and NDM-1 + OXA-181. One ST307 isolate (UP-KT-73CKP) co-harboured three carbapenemase genes (blaNDM-1 + blaOXA-48 + blaOXA-181), while all the ST2497 strains co-harboured (blaNDM-1 + blaOXA-232). Phenotypically, hypermucoviscosity was observed in a single ST307 isolate. The ST307 isolate UP-KT-151UKP harboured colibactin genotoxins. The following mobile genetic elements were detected: plasmids [incompatibility group (Inc)-FIB(K), IncX3], and bacteriophages [e.g. Klebsi_ST16_OXA48phi5.4_NC_049450, Klebsi_3LV2017_NC_047817(36)]. The study highlights the importance of local genomic surveillance systems to characterise K. pneumoniae MDR high-risk clones. This data will aid in designing infection and prevention measures for limiting the spread of carbapenemase-producing K. pneumoniae in Gauteng, South Africa.

Longitudinal genomic surveillance of a UK intensive care unit shows a lack of patient colonisation by multi-drug-resistant Gram-negative bacterial pathogens.

Vulnerable patients in an intensive care unit (ICU) setting are at high risk of infection from bacteria including gut-colonising Escherichia coli and Klebsiella species. Complex ICU procedures often depend on successful antimicrobial treatment, underscoring the importance of understanding the extent of patient colonisation by multi-drug-resistant organisms (MDROs) in large UK ICUs. Previous work on ICUs globally uncovered high rates of colonisation by transmission of MDROs, but the situation in UK ICUs is less understood. Here, we investigated the diversity and antibiotic resistance gene (ARG) carriage of bacteria present in one of the largest UK ICUs at the Queen Elizabeth Hospital Birmingham (QEHB), focusing primarily on E. coli as both a widespread commensal and a globally disseminated multi-drug-resistant pathogen. Samples were taken during highly restrictive coronavirus disease 2019 (COVID-19) control measures from May to December 2021. Whole-genome and metagenomic sequencing were used to detect and report strain-level colonisation of patients, focusing on E. coli sequence types (STs), their colonisation dynamics and antimicrobial resistance gene carriage. We found a lack of multi-drug resistance (MDR) in the QEHB. Only one carbapenemase-producing organism was isolated, a Citrobacter carrying bla KPC-2. There was no evidence supporting the spread of this strain, and there was little evidence overall of nosocomial acquisition or circulation of colonising E. coli. Whilst 22 different E. coli STs were identified, only 1 strain of the pandemic ST131 lineage was isolated. This ST131 strain was non-MDR and was found to be a clade A strain, associated with low levels of antibiotic resistance. Overall, the QEHB ICU had very low levels of pandemic or MDR strains, a result that may be influenced in part by the strict COVID-19 control measures in place at the time. Employing some of these infection prevention and control measures where reasonable in all ICUs might therefore assist in maintaining low levels of nosocomial MDR.

New Insights Into the Molecular Characteristics of Binary Toxin-Producing Clostridioides difficile Strains in China: A Comparative Genomics Study

Abstract Binary toxin (CDT)-producing Clostridioides difficile is associated with severe clinical symptoms and high mortality rates. However, the fundamental genomic factors that underpin the differentiated prevalence of CDT-producing C. difficile strains in China remain to be elucidated. To provide a comprehensive view of the genetic characteristics, this study comprehensively analyzed whole-genome sequences of CDT-producing C. difficile strains from China. The number of unique accessory genes in sequence type (ST)-5 strains, which are predominant in China, increased steadily with the increasing number of genomes sampled, while the accumulation curve for ST-1 strains was shallower. This suggests that ST-5 may have an open pangenome, whereas ST-1 may have a relatively conservative and stable pangenome. The pathogenicity locus (PaLoc) variants of Clades 1, 2 and 5 were found to share a common genetic organization, with the CDT locus (CdtLoc) showing lower variability than PaLoc. PaLoc in Clade 3 exhibits an insertion of transposon Tn6218, which is clade-specific. The flexibility of the C. difficile accessory genome has facilitated the development of divergent lineages characterized by the presence of evolutionarily advantageous genetic traits. This genetic diversification has led to enhanced antimicrobial resistance, increased virulence and improved metabolic capacities, which collectively augment the ability of C. difficile to survive and proliferate.

Whole-genome sequencing of toxigenic Clostridioides difficile reveals multidrug resistance and virulence genes in strains of environmental and animal origin

BackgroundClostridioides difficile has been recognized as an emerging pathogen in both humans and animals. In this context, antimicrobial resistance plays a major role in driving the spread of this disease, often leading to therapeutic failure. Moreover, recent increases in community-acquired C. difficile infections have led to greater numbers of investigations into the animal origin of the disease. The aim of this study was to evaluate the genetic similarities between 23 environmental and animal isolates by using whole-genome sequencing and to determine antimicrobial resistance and virulence factor genes in toxigenic C. difficile strains to provide important data for the development of diagnostic methods or treatment guidelines.ResultsThe most common sequence type was ST11 (87%), followed by ST2 (9%) and ST19 (4%). In addition, 86.95% of the strains exhibited multidrug resistance, with antimicrobial resistance to mainly aminoglycosides, fluoroquinolones, tetracycline and B-lactams; nevertheless, one strain also carried other resistance genes that conferred resistance to lincosamide, macrolides, streptogramin a, streptogramin b, pleuromutilin, oxazolidinone and amphenicol. In addition, a wide range of virulence factor genes, such as those encoding adherence factors, exoenzymes and toxins, were found. However, we observed variations between toxinotypes, ribotypes and sequence types.ConclusionsThe results of this study demonstrated significant genetic similarity between ST11 strains isolated from environmental sampling and from animal origin; these strains may represent a reservoir for community-acquired C. difficile infection, which is becoming a growing public health threat due to the development of multridug resistant (MDR) bacteria and the number of virulence factors detected.

The Comparison of Antibacterial Efficacy, Cell Proliferation, and Wound Healing Properties in Extracts Derived from Leaves and Inflorescences of Hang Kra Rog Phu Phan ST1 (Cannabis sativa L.)

The therapeutic potential of Cannabis sativa extracts in wound healing applications has garnered significant interest due to their diverse pharmacological properties. This study aimed to comparatively evaluate the antibacterial properties and wound-healing activity of ethanolic extracts derived from C. sativa leaves and inflorescences of the Hang Kra Rog Phu Phan ST1 cultivar. The extracts were prepared using ethanol, and their antibacterial properties, cytotoxicity and ability to promote cell proliferation and migration were investigated. The antibacterial activity was assessed against Staphylococcus epidermidis TISTR 518, Klebsiella pneumoniae TISTR 1383, Pseudomonas aeruginosa TISTR 2370, Staphylococcus aureus TISTR 1466 and Bacillus cereus TISTR 2373 using the disk diffusion method and broth microdilution assay. Cell viability and proliferation were evaluated using the MTT assay on human fibroblasts in serum-supplemented and serum-depleted media. The scratch wound healing assay was employed to quantify cell migration and wound closure. The results demonstrated that both leaf and inflorescence extracts exhibited antibacterial activity, with the inflorescence extract showing higher efficacy against S. aureus. Furthermore, the extracts enhanced fibroblast proliferation and migration in a concentration-dependent manner. Notably, the leaf extract outperformed the inflorescence extract in promoting fibroblast proliferation and migration, particularly at earlier time points. These findings suggest that C. sativa extracts, particularly from the leaves, possess wound-healing properties and could be developed as novel therapeutic agents for promoting efficient wound repair and managing chronic non-healing wounds. HIGHLIGHTS This research is the inaugural exploration into the antibacterial properties and wound healing capabilities of the Cannabis sativa L. Hang Kra Rog Phu Phan ST1 cultivar, a unique variant of Cannabis sativa. This strain has received official recognition and registration from the Rajamangala University of Technology Isan, Thailand. Our findings reveal that extracts derived from the leaves and inflorescences of the Hang Kra Rog Phu Phan ST1 strain display potent antibacterial activity against five tested pathogenic bacteria, namely Staphylococcus epidermidis TISTR 518, Klebsiella pneumoniae TISTR 1383, Pseudomonas aeruginosa TISTR 2370, Staphylococcus aureus TISTR 1466, and Bacillus cereus TISTR 2373. Moreover, these extracts stimulate the growth and movement of human fibroblasts. These compelling results underscore the potential for harnessing this plant as an efficacious agent for wound healing. GRAPHICAL ABSTRACT

A novel sequence type of carbapenem-resistant hypervirulent Klebsiella pneumoniae strains from a county-level tertiary hospital in Southeastern China.

The whole-genome sequencing of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains is required for investigating the molecular epidemiology because of their diverse molecular types across geographical regions. CRKP strains were collected from a tertiary hospital in Southeastern China from January 2017 to December 2020. Following species identification, drug susceptibility phenotypes were determined based on minimum inhibitory concentrations using the VITEK 2 Compact system. In addition, whole-genome sequencing was performed to identify the resistance genes and high virulence genes (rmpA, rmpA2, iucA, iroB, and peg-344). Finally, a phylogenetic tree was constructed based on the core genes. Forty CRKP strains were identified, and 25% of the involved patients (n = 10) died during hospitalization. The dominant sequence type (ST) was ST11 (65%), followed by ST290 (n = 4, 10%) and a novel ST (n = 4, assigned as ST6242, 10%). CRKP strains with this new ST were resistant to amikacin but susceptible to sulfamethoxazole-trimethoprim, and the phylogenetic tree indicated that they were derived from ST11 strains. All ST6242 strains were classified as the hypervirulent type (positive for rmpA, rmpA2, iucA, and peg-344). CRKP strains with this novel ST harbored highly virulent genes and a unique resistance phenotype. Thus, they should be epidemiologically monitored.

Serotype independent protection induced by a vaccine based on the IgM protease of Streptococcus suis and proposal for a new immunity-based classification system

The IgM protease (IdeSsuis gene; Gene ID 8153996) of Streptococcus suis is a putative virulence factor that has been shown to be a protective vaccine antigen for pigs (Seele et al. Vaccine 33:2207–12, 2015). To assess its potential as a cross-protective antigen, the amino acid variability among prevalent clinical isolates in various regions and among various serotypes was investigated. Multi-sequence alignment of full-length amino acid sequences of S. suis IgM protease, available in the public domain (status Jan-2022) supplemented with in-house sequences, i.e. a total of 1999 sequences, revealed that the IgM protease of S. suis clusters into three main evolutionary distinct branches: groups A, B and C. Group A, 82% of the sequences in the database, was associated with clinical isolates of various serotypes. Group B, 6% of the strains in the database, was associated with clinical isolates mainly in the EU and mainly belonging to serotype (st) 9. Group C, 12% of the strains in the database, was largely associated with healthy carrier isolates, i.e. nose or tonsil isolates of various serotypes but in particular with st9 and un-typable strains. Within the groups A, B and C, high levels of amino acid identity were observed (> 75%), whereas between groups A and B, the percentage amino acid identity was approximately 30% and between groups A and C approximately 55%. Experimental Escherichia coli expressed recombinant subunit vaccines based on the IgM protease group A sequence of st1 strain B10-99, st2 strain 10 or st7 strain 14009-1, induced serotype independent protection in pigs against challenge with all group A strains tested, i.e. strains of different parts of the phylogenetic tree and of different serotypes including st1, 2, 9 and 14. Protection was observed after vaccination of piglets at 3 and 5 weeks of age and subsequent challenge at 7 weeks but also after vaccination of gilts at 6 and 2 weeks before anticipated parturition and challenge of the offspring up to at least 8 weeks of age. No protection was observed against challenge with st9 strain SZ2000-6264 having group B IgM protease. A recombinant subunit vaccine based on the group B IgM protease sequence, also did not protect against challenge with the homologous group B st9 challenge strain. The results indicate that a vaccine based on a group A IgM protease induces protection against all S. suis strains that express the group A IgM protease. Depending on the geographical region such a vaccine is expected to protect against 60–100% of the virulent S. suis strains. Since the novel proposed IgM protease classification is highly relevant, a PCR was developed and validated, to be able to classify clinical isolates into IgM protease groups A, B and C and predict the cross-protection that can be expected from a group A based IgM protease vaccine.

Association of antibiotic resistance and biofilm formation in Escherichia coli ST131/O25b.

Urinary tract infections are becoming difficult to treat every year due to antibiotic resistance. Uropathogenic Escherichia coli (UPEC) isolates pose a threat with a combined expression of multidrug-resistance and biofilm formation. ST131 clone is a high-risk pandemic clone due to its strong association with antimicrobial resistance, which has been reported frequently in recent years. This study aims to define risk factors, clinical outcomes, and bacterial genetics associated with ST131/O25b UPEC. In this study, antibiotic susceptibility and species-level identification of 61 clinical E. coli strains were determined by automated systems. Detection of extended-spectrum beta-lactamases was assessed by double-disk synergy test. Biofilm formation was quantified by spectrophotometric method. Virulence genes (iutA, sfa cnf-1, iroN, afa, papA, fimA), antibiotic resistance genes (blaCTX-M, blaTEM, blaSHV, blaOXA, qnrA, qnrB, qnrS, ant(2')-Ia, ant(3)-Ia, aac(3)-IIa, mcr-1, mcr-2, mcr-3, mcr-4) were investigated by PCR. The following beta-lactamase genes were identified, blaTEM (n = 53, 86.8%), blaCTX-M (n = 59, 96.7%), blaSHV (n = 47, 77.0%), and blaOXA-1 (n = 27, 44.2%). Our data revealed that 93.4% of (57/61) E. coli isolates were biofilm-producers. O25pabBspe and trpA2 were investigated for the presence of ST131/O25b clone. Among multidrug resistant isolates, co-existence of O25pabBspe and trpA2 was detected in 29 isolates (47.5%). The fimH30 and H30Rx subclones were detected in four isolates that are strong biofilm-producers. These results suggest that clinical E. coli strains may become reservoirs of virulence and antibiotic resistance genes. This study demonstrates a significant difference in biofilm formation between E. coli ST131 and non-ST131 isolates. Moreover, 86.21% (n = 25) of ST131 isolates produced strong to moderate biofilms, while only 43.75% (n = 14) of non-ST131 isolates showed the ability to form strong biofilms. Presence of iutA and fimA genes in the majority of ST131 strains showed an important role in biofilm formation. These findings suggest application of iutA and fimA gene suppressors in treatment of infections caused by biofilm-producing drug-resistant ST131 strains.

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure.

It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/ H 30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P -value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing bla CTX-M-15 copy numbers via modular, insertion sequence 26 (IS 26 ) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/ H 30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

Clinical Course and Molecular Characterization of Human Bocavirus Associated with Acute Lower Respiratory Tract Infections in a Tertiary Care Hospital in Northern India

Respiratory samples from 139 hospitalized children were screened for the human bocavirus (HBoV) genome. Positive samples were sequenced for the partial VP1/VP2 gene followed by molecular and phylogenetic analyses. HBoV positivity was noted in 7.2% (10/139) of patients. All HBoV-positive children presented with fever, cough, and respiratory distress (90%, 9/10). Three children developed multisystemic viral illness, with one fatality. Eight children required intensive care management and five required mechanical ventilation. The nucleotide percent identity of the partial VP1/VP2 gene in the HBoV study strains ranged from 97.52% to 99.67%. Non-synonymous mutations in the VP1 protein were T591S (n = 8) and Y517S (n = 1) in the HBoV St1 strain and N475S (n = 8) and S591T (n = 2) in the HBoV St2 strain. One strain showed A556P, H556P, I561S, and M562R non-synonymous mutations. All the study strains belonged to the HBoV1 type. Seven HBoV strains belonged to the same lineage, and three belonged to another lineage. For evolutionary dynamics, GTR+I substitution model with uncorrelated relaxed lognormal clock and Bayesian Skyline tree prior showed 9.0 × 10-4 (95% highest probability density interval: 3.1 × 10-6, 2.1 × 10-3) nucleotide substitutions per site per year. Clinical suspicion and virological screening are necessary to identify HBoV infections in children.

Filtered handheld far-ultraviolet disinfection device in reducing environmental pathogens from high-touch clinical surfaces

Background: Healthcare-acquired infections (HAIs) continue to be a major challenge. In fact, an increased risk of HAIs has been linked to high-touch surfaces contaminated with multidrug-resistant organisms (MDROs), and enhanced environmental disinfection is linked to reduced HAI rates. Recently, more focus has been placed on emerging disinfection technologies, such as UV light-producing portable device that emits light at a wavelength of 222 nm, which has previously demonstrated germicidal capabilities at short contact times. In this study, we aim i) to evaluate the efficacy of a filtered far-UV-C handheld device (FFUHH) to reduce bacterial loads on high-touch surfaces in clinical workrooms in a cancer center, and ii) to isolate, identify and establish a genetic relationship between these environmental clinically significant pathogens and the ones recovered from patients. Methods: Samples were collected weekly on a rotating schedule over a 24-week period from five high-touch items (dictation device, mouse, armchair, desk, and keyboard) in multiple clinical work rooms on hematologic malignancy and stem cell transplant units. Contact plates for colony count and swabs were collected pre- and post-intervention with the FFUHH on standardized adjacent areas respectively for each surface. The swabs were enriched and cultured on selective media to isolate clinically significant pathogens. Whole genome sequencing (WGS) was then performed on environmental pathogens validated by MALDI-TOF as well as clinical samples collected from patients in the same unit around the time of environmental sample collection. Results: A total of 440 plates, 220 pre- and 220 post-interventions, were collected and analyzed. The highest mean colony count pre-treatment was detected from the armchairs and the lowest for the keyboards. The mean reduction of colony forming units (CFUs) ranged between 53% for the keyboard and 83% for the mouse. The reduction was statistically significant across all surfaces with P values &lt; 0.05, except for the keyboard (Figure 1). We isolated many pathogens of the human microbiota identified by MALDI-TOF such as Micrococcus luteus, S. capitis as well as methicillin-resistant S. epidermidis, S. haemolyticus and S. hominis. We also identified several Candida parapsilosis, Pseudomonas stutzeri, one Listeria grayi and one Acinetobacter baumanni. Finally, WSG allowed us to further characterize an environmental multi-drug resistant S. epidermidis ST5 strain associated with patient bacteremia, and ST16 strains detected on surfaces both pre- and post-FFUHH treatment. Conclusion: The FFUHH effectively reduced the microbial burden on high-touch surfaces in clinical workrooms on hematologic malignancy and stem cell units.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin

BackgroundGroup B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells.ResultsIn this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain.ConclusionBy combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

Whole genome sequencing analysis of seven unknown resistance mechanisms of carbapenem-resistant Klebsiella pneumoniae strains resistance to ceftazidime-avibactam.

To investigate alternative resistance mechanisms among seven ceftazidime-avibactam (CZA)-resistant carbapenem-resistant Klebsiella pneumoniae (CRKP) strains lacking common antimicrobial resistance genes (ARGs) using whole genome sequencing. ARG and virulence factors (VFs) were screened using the ARG database CARD and the VF database, respectively, and identified using genomic annotation data with BLAST+. Six strains were ST11 sequence types (STs), and one was ST2123. ST11 strains harbored more ARGs than the ST2123 strains. All seven strains carried multiple ARGs with efflux-mediated antibiotic resistance, including oqxA, oqxB, tet (A), qacEdltal, CRP, H-NS, Kpn-E, F, G, H, acrA, LptD, acrB, acrD, cpxA, mdtB, and mdtC. These efflux-mediated ARGs were identified in most strains and even all strains. Whole genome sequencing revealed that the ST11 strain carried multiple potential prophages, genomic islands, and integrative and conjugative elements, while the ST2123 strain carried an independent potential prophages and a genomic island. Whole genome sequencing analysis revealed that these seven CZA-resistant CRKP strains lacking common ARGs exhibited efflux-mediated antibiotic resistance-associated ARGs. The main mechanism by which CRKP resists CZA is antibiotic inactivation. Except for tet (A), no ARGs and validation experiments related to efflux were found. This study's results provide a new possibility for the resistance mechanism of CRKP to CZA, and we will verify this conclusion through experiments in the future.