Clonal Spread Research Articles

Emergence of a blaNDM-5-carrying extensively drug-resistant Enterobacter cloacae ST1718 in Saudi Arabia: Insights from comprehensive genome analysis.

Patients with severe COVID-19 may require intensive care unit (ICU) admission to manage life-threatening complications. However, ICU admission is associated with an increased risk of acquiring nosocomial infections caused by multidrug-resistant (MDR) bacteria, particularly carbapenem-resistant Enterobacterale (CRE). Enterobacter cloacae complex (ECC), a group of closely related species including Enterobacter cloacae, is a common cause of healthcare-associated infections (HAIs). The study conducted a comprehensive genomic analysis of an extensively drug-resistant (XDR) E. cloacae strain (ECloa-JZ71) isolated from the blood of a critically ill COVID-19 patient in Jazan, Saudi Arabia. ECloa-JZ71 exhibited resistance to multiple antimicrobial agents, except for amikacin, gentamycin, and fosfomycin. Whole-genome sequencing revealed that ECloa-JZ71 had a rarely reported sequence type, ST1718. Resistance to β-lactam antibiotics was primarily mediated by the genes blaCMH-3, blaTEM-1B, blaLAP-2, and blaNDM-5. The strain was found to harbor IncFIB(pECLA) and IncX3 plasmid replicons, with the latter encoding the blaNDM-5 gene. The IncX3 plasmid was identified as a significant contributor to the dissemination of the blaNDM-5 gene among Enterobacterale species The coexistence of blaNDM-5 and other carbapenem-hydrolyzing enzymes explains the reduced efficacy of β-lactam drugs in ECloa-JZ71. The coexistence of blaNDM-5 and other carbapenem-hydrolyzing enzymes explains the reduced efficacy of β-lactam drugs in ECloa-JZ71. The presence of specific virulence factors along with carbapenem resistance in ECloa-JZ71 may enhance its pathogenesis, complicating treatment and control efforts. The findings highlight the need for monitoring the spread of multidrug-resistant clones, conducting molecular epidemiological studies, and implementing effective infection control measures to prevent the dissemination of antimicrobial resistance in healthcare settings.

Antimicrobial resistance and epidemiological patterns of Streptococcus pyogenes in Türkiye.

Drug-resistant Group A beta-hemolytic streptococci remain significant infectious agents globally. This study investigated the major S. pyogenes strains responsible for infections in Türkiye and their susceptibility to beta-lactam and macrolide antibiotics. We determined the minimum inhibitory concentration using the penicillin gradient test and performed emmtyping and DNA fingerprinting via pulsed-field gel electrophoresis (PFGE) to analyze the clonal spread of 92 S. pyogenes strains isolated from two hospitals in Türkiye between 2020 and 2022. Our findings revealed the predominant S. pyogenes strains causing infections in the population and provided insights into the epidemiological relatedness of these drug-resistant strains. This study also evaluated the correlation between emm typing and PFGE in tracking S. pyogenes epidemiology. In this study, the current resistance patterns of S. pyogenes strains in Türkiye identified erythromycin resistance in a few strains, but no resistance to penicillin was detected. This study revealed that emm types 1, 12 and 89 as S. pyogenes strain genotypes were responsible for epidemic infections in Türkiye. PFGE genotyping and emm typing were found to provide better phylogenetic classification in the investigation of S. pyogenes epidemiology.

Molecular Characterization of Colistin- and Carbapenem-Resistant Klebsiella pneumoniae: mgrB Mutations and Clonal Diversity in Pediatric Intensive Care Isolates.

Colistin- and carbapenem-resistant Klebsiella pneumoniae (ColR CrKp) cause important health problems in pediatric intensive care units (PICUs) due to its ability to harbor multiple resistance genes and spread of high-risk clones. In this study, molecular epidemiological characteristics, transferable resistance genes, and mgrB alterations of ColR CrKp isolated from PICU were investigated. Isolates were identified by MALDI-TOF MS, and antimicrobial susceptibility tests were performed using disk diffusion method, gradient strip test, and broth microdilution method. Extended spectrum beta-lactamase, AmpC beta-lactamase, carbapenemase, 16S rRNA methyltransferase, plasmid-mediated quinolone resistance, and mcr-1 to -5 genes were investigated by polymerase chain reaction. Sanger sequencing was performed to obtain blaOXA-48-like and mgrB sequences. Pulsed-field gel electrophoresis and multilocus sequence typing were used to determine the clonal spread of the isolates. Ten ColR CrKp harboring blaOXA-48 (70%), blaOXA-232 (20%), blaCTX-M (90%), armA (20%), qnrB (20%), and qnrS (50%) were identified. No mcr genes were found, whereas mgrB mutations through modifications (A7T, C88T, and A121G) and insertion of an IS-1-like insertion sequence were determined. Isolates belonged to ST 14, ST 37, ST 101, ST 147, ST 661, ST 985, and ST 2096. It is crucial to determine the antimicrobial resistance properties and the clonal spread of the isolates to guide the treatment decisions, implement effective infection control measures, and develop novel antimicrobial strategies.

Risk of Colonization with Multidrug-Resistant Gram-Negative Bacteria Among Travellers and Migrants: A Narrative Review.

Globalization in the 21st century has posed several challenges. In particular, the spread of multidrug-resistant bacterial strains, especially Gram-negative bacteria, which are prevalent in certain regions of the world, is one of the most critical issues. This raises concerns about the risks associated with the booming tourism industry and migratory flows. In fact, even transient colonization with multidrug-resistant strains can present significant challenges to individual, family, and public health. Understanding the epidemiology and mechanisms of resistance, associated risk factors and prevention policies is therefore essential to ensure that strategies are in place to limit the global spread of high-risk bacterial clones and thereby protect public health.

Mechanisms of blaIMP-4 dissemination across diverse carbapenem resistant clinical isolates.

The IMP-4 carbapenemase is an endemic cause of carbapenem resistance in the Asia-Pacific region. Our aim was to determine the dissemination mechanism of the blaIMP-4 gene. Twelve representative Australian IMP-4 clinical isolates from The Alfred Hospital, were characterised using antimicrobial susceptibility testing and genome and plasmid assemblies analysed. Conjugation efficiencies of different plasmids were investigated using filter mating with four recipient strains across two species. Selected IMP-4 isolates included six species and four genera (Enterobacter, Klebsiella, Serratia and Acinetobacter), whereby isolates of the same species belonged to the same sequence type and were closely related. Four IMP-4 plasmid types were noted: IncHI2A type 1 and 2 (Klebsiella spp. and Enterobacter hormaechei, respectively), IncC (Serratia marcescens and Klebsiella pneumoniae), and a novel type in Acinetobacter pittii. Sequence homology was observed across all plasmids at the blaIMP-4 location, termed Region I, with IS26- on IncHI2A and IS5075-and Tn3 resistance gene cassettes present IncC plasmids, respectively. Genomic rearrangements mediated by IS26 or Tn3 and IS5075 were identified in Region I of plasmids from the same Inc type. Plasmids of each Inc type were capable of conjugative transfer with varying efficiency. IncH12A plasmids and K. pneumoniae IncC displayed higher transfer efficiencies than other plasmids examined in this study when using the recipient E. coli strain J53 (with conjugation efficiencies of 1.17×10-2 - 5.02×10-5, p<0.001). Clonal spread, Inc type, homologous region and insertion sequences are important mobility factors in the dissemination and evolution of blaIMP-4 plasmids.

Clonal Spread and Genetic Mechanisms Underpinning Ciprofloxacin Resistance in Salmonella enteritidis.

Salmonella enteritidis is a major cause of foodborne illness worldwide, and the emergence of ciprofloxacin-resistant strains poses a significant threat to food safety and public health. This study aimed to investigate the prevalence, spread, and mechanisms of ciprofloxacin resistance in S. enteritidis isolates from food and patient samples in Shanghai, China. A total of 1625 S. enteritidis isolates were screened, and 34 (2.1%) exhibited resistance to ciprofloxacin. Pulsed-field gel electrophoresis (PFGE) results suggested that clonal spread might have persisted among these 34 isolates in the local area for several years. Multiple plasmid-mediated quinolone resistance (PMQR) genes, GyrA mutations in the quinolone resistance-determining region (QRDR), and overexpression of RND efflux pumps were identified as potential contributors to ciprofloxacin resistance. PMQR genes oqxAB, qnrA, qnrB, and aac(6')-Ib-cr as well as GyrA mutations S83Y, S83R, D87Y, D87G, D87N, and S83Y-D87Y were identified. The co-transfer of the PMQR gene oqxAB with the ESBL gene blaCTX-M-14/55 on an IncHI2 plasmid with a size of ~245 kbp was observed through conjugation, highlighting the role of horizontal gene transfer in the dissemination of antibiotic resistance. Sequencing of the oqxAB-bearing plasmid p12519A revealed a 248,746 bp sequence with a typical IncHI2 backbone. A 53,104 bp multidrug resistance region (MRR) was identified, containing two key antibiotic resistance determinants: IS26-oqxR-oqxAB-IS26 and IS26-ΔISEcp1-blaCTX-M-14-IS903B. The findings of this study indicate that ciprofloxacin-resistant S. Enteritidis poses a significant threat to food safety and public health. The persistence of clonal spread and the horizontal transfer of resistance genes highlight the need for enhanced surveillance and control measures to prevent the further spread of antibiotic resistance.

Clinical and molecular characteristics of KPC-producing Klebsiella pneumoniae bloodstream infections: results of a multicentre study.

KPC-producing Klebsiella pneumoniae (KPC-Kp) is a great cause of concern and it is often associated with bloodstream infections (BSIs) and a high mortality rate. We identified the risk factors of KPC-Kp BSIs observed in three Italian hospitals and studied the epidemiology of KPC-Kp strains. We performed a retrospective analysis of KPC-Kp BSIs from 2014 to 2019 in three hospitals in Central Italy (Ancona, Pesaro-Fano, and Perugia). Uni- and multi-variable analyses were performed to evaluate the clinical variables associated with mortality. PFGE and WGS analysis of KPC-Kp isolates was carried out to identify the antibiotic resistance genes and epidemiological relationships among the strains. A total of 219 patients were considered. Mortality on day 30 was 32% being older age, Apache score II ≥11, Charlson Comorbidity Index ≥4, and solid tumors more frequent in patients with a negative outcome. Positive outcomes were related to combination therapy with at least two active drugs that emerged also in multivariate analysis. Most KPC-Kp strains belonged to three major sequence types (ST512, ST307, and ST101) while the most common carbapenem resistance gene variant was blaKPC-3. KPC-Kp BSIs remain a challenging infection with a high crude mortality rate. Patients' conditions and comorbidities correlate with negative outcomes, while active drugs are correlated with better outcomes. Although collected from different hospitals, KPC-Kp strains were epidemiologically related, suggesting an inter-hospital diffusion. Timely and effective therapy, together with epidemiological surveillance are crucial to reduce mortality and prevent the spread of nosocomial clones.

Inter-Institutional Dynamics and Impact of Fluconazole-Resistant Candida parapsilosis.

Infections with fluconazole-resistant Candida parapsilosis have been increasing in Israeli hospitals with unclear implications for patient outcomes. To determine the frequency, mechanisms, molecular epidemiology, and outcomes of azole-resistant C. parapsilosis bloodstream infections in four hospitals in Israel. C. parapsilosis bloodstream isolates were collected at four hospitals in central Israel during varying periods from 2005 to 2022. Antifungal susceptibility testing was done using CLSI broth microdilution. Risk factors for fluconazole resistance were investigated using logistic regression. ERG11 gene sequencing was performed on all isolates. Genetic relatedness was determined using multilocus microsatellite genotyping. Clinical cure, microbiological eradication, and mortality rates were compared between fluconazole-susceptible and resistant isolates. A total of 192 patient-specific C. parapsilosis isolates were analysed. Resistance to fluconazole and voriconazole was detected in 80 (41%) and 14 (7.2%) isolates, respectively. The ERG11 Y132F substitution was found in 91% of fluconazole-resistant and 1% of fluconazole-susceptible isolates. Increasing age, intensive care hospitalisation, haemodialysis, and recent exposure to antibiotics were risk factors for fluconazole-resistant C. parapsilosis. Distinct but related genotypes predominated at each centre, indicating extensive dissemination within hospitals and limited transmission among them. Fluconazole resistance was associated with increased likelihood of microbiological failure but no significant difference in clinical cure and mortality. We found high rates of fluconazole resistance in C. parapsilosis, attributable to nosocomial spread of hospital-specific clones bearing the Y132F substitution. Fluconazole resistance was associated with a higher risk of microbiological but not clinical failure. Strategies to limit nosocomial transmission of C. parapsilosis are needed.

Molecular Characterization and Risk Factors of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae Isolated from Chinese Tertiary Hospital.

Therefore, the objectives of this study were to investigate the prevalence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) in Fujian Medical University Union Hospital, identify their genetic characters, characterize their resistance profiles, and identify risk factors for their infection to improve prevention and treatment strategies for CR-hvKp in the area. Between January 2021 and January 2022, clinically identified carbapenem-resistant Klebsiella pneumoniae (CRKp) isolates were collected. A PCR assay was used to detect the K capsule type, virulence genes, carbapenemase genes, and membrane pore protein. ERIC-PCR was carried out for homology analysis. Antimicrobial susceptibility test was used to determine drug resistance. Logistic multivariate regression analysis was conducted to confirm the risk factors for CR-hvKp infection. In total, 239 CRKp isolates were obtained. The virulence genes with the highest detection rates were mrkD, iucA, and rmpA2. Of these isolates, 54 (22.59%) carried both iucA and rmpA2, thus classifying them as CR-hvKp. All CR-hvKp isolates carried bla KPC. Furthermore, capsular serotypes K64 (94.44%) and K47 (3.70%) were detected. Resistance was observed against most common antibiotics, with the exception of complete sensitivity to ceftazidime-avibactam. ERIC-PCR indicated a potential clonal spread among CR-hvKp. Multivariate analysis found that changing beds was a risk factor for CR-hvKp infection. Currently, the hospital predominantly carries K64 CR-hvKp that harbors the bla KPC. Our study found that changing patient beds was an independent risk factor for CR-hvKp infection.

Multiple regional outbreaks caused by global and local VIM-producing Klebsiella pneumoniae clones in Poland, 2006-2019.

This study was aimed at comprehensive genomic analysis of VIM-type carbapenemase-producing Klebsiella pneumoniae species complex (KpSC) in Poland. All non-duplicate 214 VIM-producing KpSC isolates reported in Poland in 2006-2019 were short-read sequenced and re-identified by the average nucleotide identity scoring. Their clonality/phylogeny was assessed by cgMLST and SNP in comparison with genomes from international databases. Serotypes, VIM-encoding integrons, resistomes, virulomes and plasmid replicons were identified by various bioinformatic tools. Structures of plasmids and genomic islands with VIM integrons were analysed for representative long-read sequenced isolates. The KpSC isolates were the second most prevalent VIM-positive Enterobacterales (23.1%) in Poland in 2006-2019, following Enterobacter spp. (40.1%). Their significance emerged in 2014 and then grew consequently, owing to eight regional outbreaks of K. pneumoniae sequence types (STs) ST437, ST147, ST15, ST277 and ST392. These carried different VIM integrons, mainly In238 and In916 types, located on IncFIB + IncHI2 (pNDM-MAR)-, IncA- or IncM-like plasmids, or clc-type integrative and conjugative elements. Despite relatedness of the outbreak clusters to isolates from other countries, e.g. Greece, Spain, Slovakia or Germany, most of them have apparently emerged on site by horizontal acquisition of resistance determinants from other species, including Enterobacter spp. and Pseudomonas spp. This work shows dynamic epidemiology of VIM-producing organisms, driven by a mix of circulation of different VIM-encoding elements, and parallel clonal spread of multiple organisms.

An outbreak of influenza A(H1N1)pdm09 antigenic variants exhibiting cross-resistance to oseltamivir and peramivir in an elementary school in Japan, September 2024.

An outbreak of influenza A(H1N1)pdm09 viruses exhibiting cross-resistance to oseltamivir and peramivir occurred in Yokohama, Japan, in September 2024. Among 24 students in a class, 11 were diagnosed with influenza or influenza-like illness, and viruses harbouring the NA H275Y and HA Q210H substitutions were isolated from four. Deep sequencing analysis confirmed the clonal spread of these mutants. Antigenic analysis revealed differences from the vaccine strain. Continued monitoring is crucial to assess the potential for further spread of these mutant viruses.

Molecular epidemiology and clinical characterization of carbapenemase-producing Enterobacter spp. from an international cohort.

Despite the global public health threat posed by carbapenem-resistant Enterobacter spp., clinical and molecular epidemiological studies on international isolates remain scarce. Historically, the taxonomy of Enterobacter has been challenging, limiting our understanding of the clinical characteristics and outcomes of carbapenemase-producing Enterobacter spp. infections. Hospitalized patients enrolled in the CRACKLE-2 study (ClinicalTrials.gov, NCT03646227) from 2016-2018 with cultures positive for carbapenemase-producing Enterobacter spp. were included. Clinical and microbiologic data were collected from health records. Whole genome sequencing was performed, and the population structures of selected predominant clones were analyzed. We enrolled 136 hospitalized patients with carbapenemase-producing Enterobacter spp. from 30 hospitals in 7 countries. Among the 136 isolates, eleven Enterobacter species were identified, with most isolates belonging to E. xiangfangensis (n=81, 60%) and E. hoffmannii (n=17, 13%), and carrying blaKPC (n=106, 78%) and blaNDM (n=12, 9%). Clinical characteristics and outcomes were similar among patients with E. xiangfangensis, E. hoffmannii or the other Enterobacter spp. 30-day mortality was 20% and older age at enrollment (adjusted odds ratio 1.42, 95% confidence interval 1.08-1.87) was associated with increased mortality. Sequence type (ST)171 E. xiangfangensis, ST78 E. hoffmannii, and ST93 E. xiangfangensis were the predominant clones, and the acquisition of fluoroquinolone resistance-associated mutations and carbapenemase-encoding plasmids contributed to their formation and global dissemination. Our findings demonstrated that E. xiangfangensis and E. hoffmannii are common species among international carbapenemase-producing Enterobacter spp., potentially linked to the clonal spread of a few predominant clones that have acquired fluoroquinolone resistance and carbapenemase-encoding plasmids.

Isolation and Characterization of Lytic Phages Infecting Clinical Klebsiella pneumoniae from Tunisia.

Background: Klebsiella pneumoniae is an opportunistic pathogen that causes a wide range of infections worldwide. The emergence and spread of multidrug-resistant clones requires the implementation of novel therapeutics, and phages are a promising approach. Results: In this study, two Klebsiella phages, KpTDp1 and KpTDp2, were isolated from wastewater samples in Tunisia. These phages had a narrow host range and specifically targeted the hypervirulent K2 and K28 capsular types of K. pneumoniae. Both phages have double-stranded linear DNA genomes of 49,311 and 49,084 bp, respectively. Comparative genomic and phylogenetic analyses placed phage KpTDp2 in the genus Webervirus, while phage KpTDp1 showed some homology with members of the genus Jedunavirus, although its placement in a new undescribed genus may be reconsidered. The replication efficiency and lytic ability of these phages, combined with their high stability at temperatures up to 70 °C and pH values ranging from 3.5 to 8.2, highlight the potential of these phages as good candidates for the control of hypervirulent multidrug-resistant K. pneumoniae. Methods: Phage isolation, titration and multiplicity of infection were performed. The stability of KpTDp1 and KpTDp2 was tested at different pH and temperatures. Genomic characterization was done by genome sequencing, annotation and phylogenetic analysis. Conclusions: The ability of KpTDp1 and KpTDp2 to lyse one of the most virulent serotypes of K. pneumoniae, as well as the stability of their lytic activities to pH and temperature variations, make these phages promising candidates for antibacterial control.

Two outbreak cases involving ST65-KL2 and ST11-KL64 hypervirulent carbapenem-resistant Klebsiella pneumoniae: similarity and diversity analysis

The rise of the convergence of hypervirulence and carbapenem resistance in Klebsiella pneumoniae has been increasingly reported in recent years, however, there are few outbreak cases for these producing NDM carbapenemase. In this study, ST65-KL2 and ST11-KL64 hypervirulent and carbapenem-resistant K. pneumoniae (hvCRKP) were identified from two different outbreak cases: (1) clonal spreading of ST65-KL2 in five patients within transplantation wards spanning three months; and (2) clonal transmission of ST11-KL64 in ten patients across 10 months. The representative strains of ST65-KL2 and ST11-KL64 hvCRKP, K22877 and K56649, produced carbapenemase NDM-5 and dual carbapenemases KPC-2 and NDM-13, respectively, and both exhibited high-level carbapenem resistance. Moreover, virulent analysis showed that K22877 and K56649 were hypervirulent and the former possessed stronger virulence. Evolutionary pathways suggested ST65-KL2 and ST11-KL64 hvCRKP could be classified as CR-hvKP (hvKP acquiring carbapenem resistance) and hv-CRKP (CRKP acquiring hypervirulence), respectively. Unexpectedly, ST65-KL2 CR-hvKP showed resistance to ciprofloxacin mediated by plasmid acquisition as its spread, and ST11-KL64 hv-CRKP developed into enhanced virulence and macrophage resistance. Furthermore, compared to the ST65-KL2 CR-hvKP, the ST11-KL64 hv-CRKP tends to cause occult and persistent infection. Global genome analysis revealed ST11-KL64 hv-CRKP and ST65-KL2 CR-hvKP mainly carried blaKPC-2 and had significant differences in Ompk35/36, ybt, resistance and virulence. Effective surveillance should be implemented and novel therapeutic strategies are urgently needed to deal with refractory infections.

The spread of molecular markers of artemisinin partial resistance and diagnostic evasion in Eritrea: a retrospective molecular epidemiology study

Eritrea was the first African country to discontinue the use of histidine rich protein 2 (HRP2)-detecting rapid diagnostic tests (RDTs) for malaria diagnosis following reports of a high prevalence of pfhrp2/3-deleted Plasmodium falciparum parasites causing false-negative results in the country. Eritrea was also the first African country to report partial artemisinin resistance due to the Pfalciparum kelch13 (pfk13) Arg622Ile mutation. We aimed tocharacterise the spatial distribution of pfk13 mutants and their interactions with pfhrp2/3 deletions in Eritrea andto assess the role of the use of HRP2-detecting RDTs and antimalarial (artesunate-amodiaquine) therapy in the spread of the two variants. We conducted a retrospective molecular epidemiological analysis of pfk13 mutations and pfhrp2/3 deletions in existing Pfalciparum-infected blood samples collected as part of previous pfhrp2/3 deletion and severe malaria studies. Samples were collected in March, 2016and between September, 2018, and January, 2020, from symptomatic patients seeking care at 15health centres in four administration zones (Semenawi Keyih Bahri, Gash Barka, Anseba, and Debub) in Eritrea. Afragment spanning the propeller region of pfk13 was amplified from samples and sequenced using Sanger sequencing or targeted amplicon sequencing to identify genetic mutations. Deletions of pfhrp2/3 genes in samples were determined using multiplex quantitative PCR. Parasite haplotypes and genetic relatedness of parasite haplotypes were determined previously using microsatellite marker typing. The primary objective was to determine the prevalence of pfk13 mutations at health centres and administrative zones. The secondary objective was to investigate whether pfk13 mutants and pfhrp2/3 deleted parasites converge. We sequenced 50samples collected in March, 2016from the Semenawi Keyih Bahri zone and identified no pfk13 mutations. By contrast, in 587samples included in this study that were collected from health centres in GashBarka, Anseba, and Debub in 2018-20, we detected five different single non-synonymous mutations: Glu605Lys, Arg622Ile, Asn657Lys, Lys658Glu, and Ser679Leu. The most prevalent mutation was pfk13 Arg622Ile, which was detected in samples collected from all nine health centres where more than five samples were available across all three administration zones, with an overall prevalence of 11·9% (70 of 587samples; range 5·9-28·0%). Weidentified 22unique pfk13 Arg622Ile mutant haplotypes among 26samples tested, of which 13 (59·1%) were genetically related, whereas the remaining nine (40·9%) were not. The prevalence of pfk13 Arg622Ile was significantly higher in parasites with a single pfhrp3 deletion (46 [18·0%] of 255samples) than in parasites withoutpfhrp2/3 deletions (ten [6·2%] of 161samples; odds ratio 3·89 [95% CI 1·59-7·61]; p=0·0006) and with dual pfhrp2/3-deleted parasites (13 [9·0%] of 145; 2·23 [1·13-4·68]; p=0·018). The geographical spread of the pfk13 Arg622Ile mutation might have initially resulted from the clonal expansion and spread of pfhrp2/3 deletions under the test-and-treat policy using HRP2-detecting RDTs. Subsequently, selective pressure from artemisinin combination therapy could have further facilitated the spread of both pfk13 Arg622Ile and pfhrp2/3 deletions. Continuous monitoring of trends in pfk13 and pfhrp2/3 variants is needed to inform effective malaria control and elimination strategies in Eritrea and other African countries. US Department of Defense Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance Branch (AFHSD/GEIS), and Wellcome Trust.

Analysis of the virulence of a lethal, carbapenem-resistant hypervirulent KPC-33-producing Klebsiella pneumoniae: Emergence of ST11-KL64 hv-CRKP in ICU

ObjectiveHypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a serious threat to public health. Here, we analyse a case of systemic infection caused by a hv-CRKP, which ultimately led to the patient's death from sepsis. And a total of 30 CRKPs were analyzed to elucidate the molecular epidemiological features of CRKPs in the hospital, and to provide a basis for clinical anti-infective therapy. MethodsIn this case, a total of 7 K. pneumoniae strains were isolated from the blood, sputum, urine, and feces of the patient. The Vitek-2 compact system was used to identify the strains and perform antimicrobial susceptibility testing. Biofilm formation, siderophore production assays and Galleria mellonella infection model were used to verify the virulence phenotypes of the strains in the case. Whole-genome sequencing was conducted on the four hv-CRKP isolated from different samples in the case and 26 other CRKP collected in our hospital from September to November in 2022, using the Illumina Hiseq 6000 high-throughput sequencing platform to analyse the resistance and virulence genes. ResultsIn the case, after 7 days of treatment with ceftazidime-avibactam (CZA), the resistance profile of the strains changed. The strain that was initially sensitive to CZA developed to resistant, resistant to imipenem (IPM) developed to sensitive, and resistant to meropenem (MEM) developed to intermediate. Whole-genome sequencing revealed that the four strains in the case were all ST11-KL64 K. pneumoniae, and the change in resistance phenotype was due to the mutation from blaKPC-2 to blaKPC-33. KPN7 had a total of six plasmids, with siderophore-related genes iucABCD and iutA, and mucoid phenotype-related gene rmpA2 located on plasmid p4-KPN7; resistance genes blaKPC-33, blaTEM-1B, and blaCTX-M-65 located on plasmid p5-KPN7; and virulence genes fim, irp, iutA, and ybt located on the chromosome. Biofilm formation and siderophore production assays confirmed that the seven K. pneumoniae strains isolated in this case had strong biofilm formation and siderophore production capabilities. Galleria mellonella Infection Model showed that KPN4 and KPN7 was phenotypically highly virulent and KPN7 performed lower virulence compared to KPN4. Apart from the 4 hv-CRKP strains, other 26 CRKP strains all carried blaKPC-2, and 69.2% (18/26) were ST-11 and 30.8%(8/26) were ST-15. And 83.3% (15/18) were ST11-KL64 strains, followed by ST11-KL25 strains 11.1%(2/18) and ST11-KL47 strain 5.6%(1/18). All the eight ST-15 strains were KL-19. ConclusionThe ST11-KL64 hv-CRKP clone spread widely in ICU carried numerous resistance and virulence genes, and under antibiotic pressure, they easily underwent mutations resulting in changes in resistance phenotypes, especially in mutations of blaKPC-2 gene in acquiring resistance to CZA. Therefore, clinical attention should be paid to such strains, and the use of antibiotics should be adjusted promptly based on the susceptibility of the strains to antimicrobial agents.

Whole genome sequencing revealed high proportions of ST152 MRSA among clinical Staphylococcus aureus isolates from ten hospitals in Ghana.

Previous studies in Ghana indicated low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and predominance of ST152 methicillin-susceptible S. aureus (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 S. aureus isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. mecA (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the mecA gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with blaZ (97%) being the most prevalent. Other predominant resistance genes included tetK (46%), cat (42%), and dfrG (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins, and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of S. aureus from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of S. aureus in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating S. aureus strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.

Limited Evidence of Spillover of Antimicrobial-Resistant Klebsiella pneumoniae from Animal/Environmental Reservoirs to Humans in Vellore, India.

Klebsiella pneumoniae is a common opportunistic pathogen in humans, often associated with both virulence and antimicrobial resistance (AMR) phenotypes. K. pneumoniae have a highly plastic genome and can act as a vehicle for disseminating genetic information. Aiming to assess the impact of the human-animal-environment interface on AMR dissemination in K. pneumoniae we sampled and genome sequenced organisms from a range of environments and compared their genetic composition. Representative K. pneumoniae isolated from clinical specimens (n = 59), livestock samples (n = 71), and hospital sewage samples (n = 16) during a two-year surveillance study were subjected to whole genome sequencing. We compared the taxonomic and genomic distribution of K. pneumoniae, AMR gene abundance, virulence gene composition, and mobile genetic elements between the three sources. The K. pneumoniae isolates originating from livestock were clonally distinct from those derived from clinical/hospital effluent samples. Notably, the clinical and hospital sewage isolates typically possessed a greater number of resistance/virulence genes than those from animals. Overall, we observed a limited overlap of K. pneumoniae clones, AMR genes, virulence determinants, and plasmids between the different settings. In this setting, the spread of XDR and hypervirulent clones of K. pneumoniae appears to be restricted to humans with no obvious association with non-clinical sources. Emergent clones of K. pneumoniae carrying both resistance and virulence determinants are likely to have emerged in hospital settings rather than in animal or natural environments. These data challenge the current view of AMR transmission in K. pneumoniae in a One-Health context.

Molecular Characterization of Multidrug-Resistant and Hypervirulent New Delhi Metallo-Beta-Lactamase Klebsiella pneumoniae in Lazio, Italy: A Five-Year Retrospective Study.

Antimicrobial resistance represents a challenge to public health systems because of the array of resistance and virulence mechanisms that lead to treatment failure and increased mortality rates. Although for years the main driver of carbapenem resistance in Italy has been the Klebsiella pneumoniae KPC carbapenemase, recent years have seen an increase in VIM and NDM metallo-beta-lactamases (MBLs). We conducted a five-year survey of New Delhi Metallo-beta-Lactamase (NDM)-producing Klebsiella pneumoniae (NDM-Kpn) clinical isolates from the Lazio region, Italy; the study aimed to elucidate the molecular mechanisms underpinning their resistant and virulent phenotype. Antimicrobial susceptibility was evaluated by automated systems and broth microdilution. In silico analysis of acquired resistance and virulence genes was performed using whole-genome sequencing (WGS), molecular typing through MLST, and core genome multi-locus sequence typing (cgMLST). A total of 126 clinical NDM-Kpn isolates were collected from 19 distinct hospitals in the Lazio region. Molecular analysis highlighted the existence of NDM-1 (108/126) and NDM-5 (18/126) variants, 18 Sequence Types (STs), and 15 Cluster Types (CTs). Notably, 31/126 isolates displayed a virulence score of 4, carrying ybt, ICEKp, iuc, and rmp genes. This study identified a variety of NDM-Kpn STs, mainly carrying the blaNDM-1 gene, with a significant number linked to high-risk clones. Of these isolates, 24.6% showed high-level resistance and virulence, emphasizing the risk of the spread of strains that combine multi-drug-resistance (MDR) and virulence. Proactive surveillance and international collaborations are needed to prevent the spread of high-risk clones, as well as further research into new antimicrobial agents to fight antibiotic resistance.

Steady existence of Escherichia coli co-resistant to carbapenem and colistin in an animal breeding area even after the colistin forbidden

Carbapenem- and colistin-resistant Escherichia coli (CCREC) cause high mortality rates and health costs, and have become serious health concerns. Total 1764 samples were collected from 60 animal farms in 2019 and 2021, including worker and animal faeces, wastewater, well water, air, vegetables, human hands, object surfaces, throat swabs, soil, and flies to investigate the prevalence and potential transmission pathways of CCREC. Eleven CCREC were detected: 9 (5 in 2019 and 4 in 2021) from 5 worker faeces, 3 animal faeces, 1 wastewater, and 2 from 1 flies sample. Chicken farms had the highest number of CCREC (n = 9). The detection rate was low (<1.1%) overall, and there was no significant difference in both years, indicating that CCREC existed stably after 4 years of colistin ban. The combinations of chromosomes and plasmids harbouring blaNDM and mcr-1.1 were divided into 4 patterns: IncX3 plasmid-blaNDM & chromosome-mcr.1.1 (n = 5); IncX3 plasmid-blaNDM & IncHI2 plasmid-mcr.1.1 (n = 3); IncFII plasmid-blaNDM & IncI2 plasmid-mcr.1.1 (n = 2); both chromosome (n = 1). The blaNDM located on plasmids was surrounded by similar genetic structures: Tn3-IS-blaNDM-bleMBL-TrpF-DsbD-IS. The genetic contexts of mcr-1.1 were highly similar, with ‘ISApl1-mcr-1.1-PAP2’ and ‘mcr-1.1-PAP2’. All plasmids can be successfully transferred into E. coli J53, except for the IncHI2 plasmids with the transfer rate of 33.3%. The IncFII and IncI2 plasmids from same strain of flies could be co-transferred. The clonal spread of CCREC from humans to humans occurred on the same pig farm (P4) or different chicken farms (BC9 and LH7). This study suggested that flies, chromosomes, and plasmids jointly contribute to the steady existence of CCREC.