Sequence Type 131 Research Articles

Gut microbiome predictors of Escherichia coli sequence type 131 colonization and loss

Escherichia coli sequence type 131 (ST131), specifically its fluoroquinolone-resistant H30R clade (ST131-H30R), is a global multidrug-resistant pathogen. The gut microbiome's role in ST131-H30R intestinal carriage is undefined. Veterans and their household members underwent longitudinal fecal swab surveillance for ST131 in 2014-2018. The fecal microbiome was characterized by 16S rRNA qPCR and sequencing. We evaluated associations between ST131-H30R carriage and gut microbiome at baseline by random forest models to identify the most informative gut bacterial phyla and genera attributes for ST131 and ST131-H30R carriage status. Next, we assessed longitudinal associations between fecal microbiome and ST131-H30R carriage using a mixed-effects logistic regression with longitudinal measures. Of the 519 participants, 78 were carriers of ST131, among whom 49 had ST131-H30R. At the baseline timepoint, H30R-positive participants had higher proportional abundances of Actinobacteria phylum (mean: 4.9% vs. 3.1%) than ST131-negative participants. H30R-positive participants also had higher abundances of Collinsella (mean: 2.3% vs. 1.1%) and lower abundances of Alistipes (mean: 2.1% vs. 2.6%) than ST131-negative participants. In the longitudinal analysis, Collinsella abundance correlated positively with ST131-H30R carriage status and negatively with the loss of ST131-H30R. Conversely, Alistipes corresponded with the loss and persistent absence of ST131-H30R even in the presence of a household exposure. Abundances of specific fecal bacteria correlated with ST131-H30R carriage, persistence, and loss, suggesting their potential as targets for microbiome-based strategies to reduce carriage of ST131-H30R, a significant risk factor for invasive infections. This work was supported in part by National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R21AI117654 and UM1AI104681 and the Office of Research and Development, Department of Veterans Affairs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs.

Whole-genome sequencing of extensively drug-resistant Salmonella enterica serovar Typhi clinical isolates from the Peshawar region of Pakistan

BackgroundTyphoid fever, caused by Salmonella enterica serovar Typhi, is a significant public health concern due to the escalating of antimicrobial resistance (AMR), with limited treatment options for extensively drug-resistant (XDR) S. Typhi strains pose a serious threat to disease management and control. This study aimed to investigate the genomic characteristics, epidemiology and AMR genes of XDR S. Typhi strains from typhoid fever patients in Pakistan. MethodologyWe assessed 200 patients with enteric fever symptoms, confirming 65 S. Typhi cases through culturing and biochemical tests. Subsequent antimicrobial susceptibility testing revealed 40 cases of extensively drug-resistant (XDR) and 25 cases of multi-drug resistance (MDR). Thirteen XDR strains were selected for whole-genome sequencing, to analyze their sequence type, phylogenetics, resistance genes, pathogenicity islands, and plasmid sequences using variety of data analysis resources. Pangenome analysis was conducted for 140 XDR strains, including thirteen in-house and 127 strains reported from other regions of Pakistan, to assess their genetic diversity and functional annotation. ResultsMLST analysis classified all isolates as sequence type 1 (ST-1) with 4.3.1.1. P1 genotype characterization. Prophage and Salmonella Pathogenicity Island (SPI) analysis identified intact prophages and eight SPIs involved in Salmonella's invasion and replication within host cells. Genome data analysis revealed numerous AMR genes including dfrA7, sul1, qnrS1, TEM-1, Cat1, and CTX-M-15, and SNPs associated with antibiotics resistance. IncY, IncQ1, pMAC, and pAbTS2 plasmids, conferring antimicrobial resistance, were detected in a few XDR S. Typhi strains. Phylogenetic analysis inferred a close epidemiological linkage among XDR strains from different regions of Pakistan. Pangenome was noted closed among these strains and functional annotation highlighted genes related to metabolism and pathogenesis. ConclusionThis study revealed a uniform genotypic background among XDR S. Typhi strains in Pakistan, signifying a persistence transmission of a single, highly antibiotic-resistant clone. The closed pan-genome observed underscores limited genetic diversity and highlights the importance of genomic surveillance for combating drug-resistant typhoid infections.

Aptitude of Uropathogenic Escherichia coli in Renal Transplant Recipients: A Comprehensive Review on Characteristic Features, and Production of Extended Spectrum β-Lactamase.

Urinary tract infection is the most common infection in almost half of the renal transplant patients. The development of UTI in these patients may progress to bacteremia, acute T cell-mediated rejection, impaired allograft function, or allograft loss, along with the increased risk of hospitalization and death. Among various pathogens implicated, Uropathogenic E. coli (UPEC), especially sequence type 131 (ST131), is the most virulent and multidrug-resistant pathogen. High antimicrobial resistance to most β-lactam antibiotics, mediated by extended spectrum β-lactamases (ESBLs) produced by UPEC, is a challenge in the clinical management of UTIs in kidney transplant recipients. Indeed, multidrug resistance to β-lactam antibiotics is a direct consequence of ESBL production. Resistance to other antibiotics such as aminoglycosides, fluoroquinolones, and trimethoprim-sulphamethoxazole has also been reported in ESBLs-producing UPEC, which reduces the therapeutic options, rising healthcare-associated costs and subsequently leads to renal failure or even graft loss. In this review, we aimed to discuss the post-transplant risk factors of UTI, UPEC virulence factors (VF), and the related factors including quorum sensing, and stress resistance genes. Furthermore, we searched for the current treatment strategies and some of the alternate approaches proposed as therapeutic options that may affirm the treatment of ESBL-producing UPEC.

The first genomic characterization of a stable, hemin-dependent small colony variant strain of Staphylococcus epidermidis isolated from a prosthetic-joint infection

Phenotype switching from a wild type (WT) to a slow-growing subpopulation, referred to as small colony variants (SCVs), supports an infectious lifestyle of Staphylococcus epidermidis, the leading cause of medical device-related infections. Specific mechanisms underlying formation of SCVs and involved in the shaping of their pathogenic potential are of particular interest for stable strains as they have been only rarely cultured from clinical specimens. As the SCV phenotype stability implies the existence of genetic changes, the whole genome sequence of a stable, hemin-dependent S. epidermidis SCV strain (named 49SCV) involved in a late prosthetic joint infection was analyzed. The strain was isolated in a monoculture without a corresponding WT clone, therefore, its genome was compared against five reference S. epidermidis strains (ATCC12228, ATCC14990, NBRC113846, O47, and RP62A), both at the level of the genome structure and coding sequences. According to the Multilocus Sequence Typing analysis, the 49SCV strain represented the sequence type 2 (ST2) regarded as the most prominent infection-causing lineage with a worldwide dissemination. Genomic features unique to 49SCV included the absence of the Staphylococcal Cassette Chromosome (SCC), ~12 kb deletion with the loss of genes involved in the arginine deiminase pathway, and frameshift-generating mutations within the poly(A) and poly(T) homopolymeric tracts. Indels were identified in loci associated with adherence, metabolism, stress response, virulence, and cell wall synthesis. Of note, deletion in the poly(A) of the hemA gene has been considered a possible trigger factor for the phenotype transition and hemin auxotrophy in the strain. To our knowledge, the study represents the first genomic characterization of a clinical, stable and hemin-dependent S. epidermidis SCV strain. We propose that previously unreported indels in the homopolymeric tracts can constitute a background of the SCV phenotype due to a resulting truncation of the corresponding proteins and their possible biological dysfunction. Streamline of genetic content evidenced by the loss of the SCC and a large genomic deletion can represent a possible strategy associated both with the SCV phenotype and its adaptation to chronicity.

Bla KPC-2 overexpression and bla GES-5 carriage as major imipenem/relebactam resistance mechanisms in Pseudomonas aeruginosa high-risk clones ST463 and ST235, respectively, in China.

Pseudomonas aeruginosa high-risk clones pose severe threats to public health. Here, we characterize the imipenem/relebactam (IR) resistance mechanisms in P. aeruginosa high-risk clones sequence type 235 (ST235) and ST463 in China. Minimum inhibitory concentrations (MICs) were determined, and Illumina short-read sequencing was performed for 1,168 clinical carbapenem-resistant P. aeruginosa (CRPA) isolates. The gene copy number and expression level were analyzed by Illumina sequencing depth and reverse transcription-quantitative PCR, respectively. Resistance conferred by bla GES-5 was evaluated by cloning experiments. ST463 and ST235 accounted for 9.8% (115/1,168) and 4.5% (53/1,168) of total isolates, respectively, and showed high frequencies of extensively drug-resistant and difficult-to-treat resistant phenotypes. The overall IR-resistant rate in CRPA was 21.0% (245/1,168). However, the IR resistance rate was 81.7% (94/115) in ST463-PA and 52.8% (28/53) in ST235-PA. Of the ST463 isolates, 92.2% (106/115) were Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-PA), and all 94 IR-resistant ST463-PA produced KPC-2. Compared to IR-susceptible ST463 KPC-2-PA, IR-resistant ST463 KPC-2-PA exhibited significantly higher bla KPC-2 copy numbers and expression levels. In ST463 KPC-2-PA, 16 mg/L relebactam resulted in additional fourfold reductions in imipenem MIC50/90 values compared to 4 mg/L relebactam. In ST235, 1.9% (1/53) carried bla IMP carbapenemase and 54.7% (29/53) carried bla GES carbapenemase. Other than the IMP producer, all 27 IR-resistant ST235-PA produced GES-5. Cloning experiments revealed that imipenem resistance in bla GES-5-carrying PAO1 transformants was generally unaffected by relebactam. In conclusion, IR-resistant CRPA isolates in China were mainly distributed in P. aeruginosa high-risk clones ST463 and ST235. The major underlying IR resistance mechanisms were bla KPC-2 overexpression and bla GES-5 carriage.

Clinical Characteristics, Antimicrobial Resistance, Virulence Genes and Multi-Locus Sequence Typing of Non-Typhoidal Salmonella Serovar Typhimurium and Enteritidis Strains Isolated from Patients in Chiang Mai, Thailand.

Non-typhoidal salmonellosis (NTS) caused by ingesting Salmonella enterica contaminated food or drink remains a major bacterial foodborne disease. Clinical outcomes of NTS range from self-limited gastroenteritis to life-threatening invasive NTS (iNTS). In this study, we isolated Salmonella spp. from the stool and blood of patients hospitalized at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, between 2016-2021 (a total of 395 cases). Then, serovar Typhimurium and Enteritidis were identified and further characterized by multiplex PCR, and multi-locus sequence typing. Our data show that multidrug resistance (MDR) sequence type 34 (ST34) and ST11 are the predominant sequence types for serovars Typhimurium and Enteritidis, respectively. Most S. Typhimurium ST34 lacks spvB, and most S. Enteritidis ST11 harbor sseI, sodCI, rpoS and spvB genes. NTS can be found in a wide range of ages, and anemia could be a significant factor for S. Typhimurium infection (86.3%). Both S. Typhimurium (6.7%) and S. Enteritidis (25.0%) can cause iNTS in immunocompromised patients. S. Typhimurium conferred MDR phenotype higher than S. Enteritidis with multiple antibiotic resistance indexes of 0.22 and 0.04, respectively. Here, we characterized the important S. Typhimurium, S. Enteritidis, and human clinical factors of NTS within the region.

Molecular and clinical epidemiology of carbapenem resistant Acinetobacter baumannii ST2 in Oceania: a multicountry cohort study

Carbapenem resistant Acinetobacter baumannii (CRAb) is categorised by the World Health Organization (WHO) as a pathogen of critical concern. However, little is known about CRAb transmission within the Oceania region. This study addresses this knowledge gap by using molecular epidemiology to characterise the phylogenetic relationships of CRAb isolated in hospitals in Fiji, Samoa, and other countries within the Oceania region including Australia and New Zealand, and India from South Asia. In this multicountry cohort study, we analysed clinical isolates of CRAb collected from the Colonial War Memorial Hospital (CWMH) in Fiji from January through December 2019 (n=64) and Tupua Tamasese Mea'ole Hospital (TTMH) in Samoa from November 2017 through June 2021 (n=32). All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole-genome sequencing. For CWMH, data were collected on clinical and demographic characteristics of patients with CRAb, duration of hospital stay, mortality and assessing the appropriateness of meropenem use from the treated patients who had CRAb infections. To provide a broader geographical context, CRAb strains from Fiji and Samoa were compared with CRAb sequences from Australia collected in 2016-2018 (n=22), New Zealand in 2018-2021 (n=13), and India in 2019 (n=58), a country which has close medical links with Fiji. Phylogenetic relationships of all these CRAb isolates were determined using differences in core genome SNPs. Of CRAb isolates, 49 (77%) of 64 from Fiji and all 32 (100%) from Samoa belonged to CRAb sequence type 2 (ST2). All ST2 isolates from both countries harboured blaOXA-23, blaOXA-66 and ampC-2 genes, mediating resistance to β-lactam antimicrobials, including cephalosporins and carbapenems. The blaOXA-23 gene was associated with two copies of ISAba1 insertion element, forming the composite transposon Tn2006, on the chromosome. Two distinct clusters (group 1 and group 2) of CRAb ST2 were detected in Fiji. The first group shared common ancestral linkage to all CRAb ST2 collected from Fiji's historic outbreak in 2016/2017, Samoa, Australia and 54% of total New Zealand isolates; they formed a single cluster with a median (range) SNP difference of 13 (0-102). The second group shared common ancestral linkage to 3% of the total CRAb ST2 isolated from India. Fifty eight of the 64 patients with CRAb infections at the CWMH had their first positive CRAb sample collected 72h or more following admission. Meropenem use was deemed inappropriate in 15 (48%) of the 31 patients that received treatment with meropenem in Fiji. Other strains of CRAb ST1, ST25, ST107, and ST1112 were also detected in Fiji. We identified unrecognised outbreaks of CRAb ST2 in Fiji and Samoa that linked to strains in other parts of Oceania and South Asia. The existence of Tn2006, containing the blaOXA-23 and ISAba1 insertion element, within CRAb ST2 from Fiji and Samoa indicates the potential for high mobility and dissemination. This raises concerns about unmitigated prolonged outbreaks of CRAb ST2 in the two major hospitals in Fiji and Samoa. Given the magnitude of this problem, there is a need to re-evaluate the current strategies used for infection prevention and control, antimicrobial stewardship, and public health measures locally and internationally. Moreover, a collaborative approach to AMR surveillance within the Oceania region with technical, management and budgetary support systems is required to prevent introduction and control transmission of these highly problematic strains within the island nation health systems. This project was funded by an Otago Global Health Institute seed grant and Maurice Wilkins Centre of Research Excellence (CoREs) grant (SC0000169653, RO0000002300).

Novel multidrug-resistant sublineages of Staphylococcus aureus clonal complex 22 discovered in India.

Staphylococcus aureus is a major pathogen in India causing community and nosocomial infections, but little is known about its molecular epidemiology and mechanisms of resistance in hospital settings. Here, we use whole-genome sequencing (WGS) to characterize 478 S. aureus clinical isolates (393 methicillin-resistant Staphylococcus aureus (MRSA) and 85 methicilin-sensitive Staphylococcus aureus (MSSA) collected from 17 sentinel sites across India between 2014 and 2019. Sequencing results confirmed that sequence type 22 (ST22) (142 isolates, 29.7%), ST239 (74 isolates, 15.48%), and ST772 (67 isolates, 14%) were the most common clones. An in-depth analysis of 175 clonal complex (CC) 22 Indian isolates identified two novel ST22 MRSA lineages, both Panton-Valentine leukocidin+, both resistant to fluoroquinolones and aminoglycosides, and one harboring the the gene for toxic shock syndrome toxin 1 (tst). A temporal analysis of 1797 CC22 global isolates from 14 different studies showed that the two Indian ST22 lineages shared a common ancestor in 1984 (95% highest posterior density [HPD]: 1982-1986), as well as evidence of transmission to other parts of the world. Moreover, the study also gives a comprehensive view of ST2371, a sublineage of CC22, as a new emerging lineage in India and describes it in relationship with the other Indian ST22 isolates. In addition, the retrospective identification of a putative outbreak of multidrug-resistant (MDR) ST239 from a single hospital in Bangalore that persisted over a period of 3 years highlights the need for the implementation of routine surveillance and simple infection prevention and control measures to reduce these outbreaks. To our knowledge, this is the first WGS study that characterized CC22 in India and showed that the Indian clones are distinct from the EMRSA-15 clone. Thus, with the improved resolution afforded by WGS, this study substantially contributed to our understanding of the global population of MRSA. IMPORTANCE The study conducted in India between 2014 and 2019 presents novel insights into the prevalence of MRSA in the region. Previous studies have characterized two dominant clones of MRSA in India, ST772 and ST239, using whole-genome sequencing. However, this study is the first to describe the third dominant clone, ST22, using the same approach. The ST22 Indian isolates were analyzed in-depth, leading to the discovery of two new sublineages of hospital-acquired Staphylococcus aureus in India, both carrying antimicrobial resistance genes and mutations, which limit treatment options for patients. One of the newly characterized sublineages, second Indian cluster, carries the tsst-1 virulence gene, increasing the risk of severe infections. The geographic spread of the two novel lineages, both within India and internationally, could pose a global public health threat. The study also sheds light on ST2371 in India, a single-locus variant of ST22. The identification of a putative outbreak of MDR ST239 in a single hospital in Bangalore emphasizes the need for routine surveillance and simple infection prevention and control measures to reduce these outbreaks. Overall, this study significantly contributes to our understanding of the global population of MRSA, thanks to the improved resolution afforded by WGS.

Occurrence of Acinetobacter baumannii genomic resistance islands (AbGRIs) in Acinetobacter baumannii strains belonging to global clone 2 obtained from COVID-19 patients.

The Acinetobacter baumannii genomic resistance islands (AbGRIs), which were characterized in the genome of the global clone 2 (GC2) A. baumannii contain resistance genes. Here, we aimed to determine the occurrence of AbGRIs in GC2 A. baumannii obtained from COVID-19 patients in a referral hospital in Tehran, Iran. A total of 19 carbapenem-resistant A. baumannii (CRAB) isolates belonging to GC2 and sequence type 2 (ST2), including 17 from COVID-19 patients and two from the devices used in the ICU that the COVID-19 patients were admitted, were examined in this study. Antibiotic susceptibility testing was performed by the disk diffusion method. PCR and PCR mapping, followed by sequencing, were performed to characterize the structure of AbGRI resistance islands in the isolates tested. The AbGRI3 was the most frequent resistance island (RI) detected, present in all the 19 isolates, followed by AbGRI1 (15 isolates; 78.9%) and AbGRI2 (three isolates; 15.8%). Notably, AbGRIs were identified in one of the A. baumannii strains, which was isolated from a medical device used in the ICU where COVID-19 patients were admitted. Furthermore, new structures of AbGRI1 and AbGRI3 resistance islands were found in this study, which was the first report of these structures. The present study provided evidence for the circulation of the GC2 A. baumannii strains harboring AbGRI resistance islands in a referral hospital in Tehran, Iran. It was found that resistance to several classes of antibiotics in the isolates collected from COVID-19 patients is associated with the resistance genes located within AbGRIs.

Ceftriaxone-resistant Salmonella Typhi isolated from paediatric patients in north India: Insights into genetic profiles and antibiotic resistance mechanisms

PurposeTo investigate the antibiotic resistance and genetic profile of ceftriaxone-resistant Salmonella Typhi isolated from the blood culture of two paediatric cases of typhoid fever and one from the stool culture of their household contact, in North India. MethodsIn this study, whole-genome sequencing was carried out with paired-end 2 ​× ​150 bp reads on Illumina MiSeq (Illumina, USA) employing v2 and v3 chemistry. To check data quality, adapters and low-quality sequences were removed through Trimmomatic-v0.36. High quality reads were then assembled de novo using A5-miseq pipeline. For further refinement, reference-guided contig ordering and orienting were performed on the scaffold assemblies using ABACAS (http://abacas.sourceforge.net/). The assembled genome was annotated using Prokka v1.12 to identify and annotate the gene content. Plasmid replicons in bacterial isolates were identified by PlasmidFinder, whereas mobile genetic elements were predicted using Mobile Element Finder. Referenced-based SNP tree with maximum likelihood method was built with CSI phylogeny v1.4. ResultsAll three isolates exhibited resistance to ceftriaxone, cefixime, ciprofloxacin, ampicillin, and co-trimoxazole, while demonstrating sensitivity to azithromycin and chloramphenicol. The whole-genome sequencing of these strains revealed the presence of blaCTX-M-15 gene for cephalosporin resistance in addition to gyrA, qnr and IncY plasmid replicon. A 5 ​kb IS91 Sbo1 gene cassette (IncY plasmid) was identified which carried extended spectrum β-lactamase blaCTX-M-15, blaTEM-1D (resistance to ampicillin and cephalosporin), sul2, dfrA14 (resistant to trimethoprim-sulfamethoxazole) and qnrS (resistant to ciprofloxacin). These isolates belong to H58 lineage and grouped as sequence type 1 (ST1) on multilocus sequence typing (MLST) analysis. ConclusionIn the present study we report the isolation of blaCTX-M-15 positive S. Typhi from two paediatric patients presenting with fever and one from stool culture of their contact from North India and highlight the need for further investigations to understand the different factors contributing to ceftriaxone resistance in Salmonella Typhi.

Cluster of Carbapenemase-Producing Carbapenem-Resistant Pseudomonas aeruginosa Among Patients in an Adult Intensive Care Unit - Idaho, 2021-2022.

Treatment of carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) infections is challenging because of antibiotic resistance. CP-CRPA infections are highly transmissible in health care settings because they can spread from person to person and from environmental sources such as sink drains and toilets. During September 2021-January 2022, an Idaho hospital (hospital A) isolated CP-CRPA from sputum of two patients who stayed in the same intensive care unit (ICU) room (room X), 4 months apart. Both isolates had active-on-imipenem metallo-beta-lactamase (IMP) carbapenemase gene type 84 (blaIMP-84) and were characterized as multilocus sequence type 235 (ST235). A health care-associated infections team from the Idaho Division of Public Health visited hospital A during March 21-22, 2022, to discuss the cluster investigation with hospital A staff members and to collect environmental samples. CP-CRPA ST235 with blaIMP-84 was isolated from swab samples of one sink in room X, suggesting it was the likely environmental source of transmission. Recommended prevention and control measures included application of drain biofilm disinfectant, screening of future patients who stay in room X (e.g., the next 10 occupants) upon reopening, and continuing submission of carbapenem-resistant P. aeruginosa (CRPA) isolates to public health laboratories. Repeat environmental sampling did not detect any CRPA. As of December 2022, no additional CP-CRPA isolates had been reported by hospital A. Collaboration between health care facilities and public health agencies, including testing of CRPA isolates for carbapenemase genes and implementation of sink hygiene interventions, was critical in the identification of and response to this CP-CRPA cluster in a health care setting.

Molecular characteristics and virulence factors of carbapenem-resistant Klebsiella pneumoniae among pediatric patients in Shanghai, China.

We retrospectively investigated CRKP isolates among 92 pediatric patients (32 neonates and 60 non‑neonates) in 2019 and 2020 (59 and 33 isolates, respectively) to investigate the molecular characteristics and virulence factors of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from pediatric patients,. All the CRKP isolates were subjected to antimicrobial susceptibility testing, string testing, molecular typing of virulence and carbapenemase genes, and multilocus sequence typing. Hypervirulent K. pneumoniae (Hvkp) was defined based on the detection of the regulator of mucoid phenotype A (rmpA).Sequence type 11 (ST11) accounted for the majority of infections in both neonates (37.5%) and non‑neonates (43.3%) (P>0.05), whereas it increased from 30.5% (18/59) in 2019 to 60.6% (20/33) in 2020 (P<0.05). Carbapenemase gene KPC-2 was predominant in both neonates and non‑neonates (46.9% vs. 51.7%, respectively), followed by New Delhi metallo-beta-lactamase 1 (NDM-1) (34.4% vs. 28.3%, respectively) (all P>0.05). Compared to 2019, the proportion of blaNDM-1 decreased (44.1% vs. 6.1%) (P<0.001), while that of blaKPC-2 increased (40.7% vs. 66.7%) (P=0.017) in 2020. ybtS and iutA had a higher positivity rate in KPC-2 and ST11 producers (all P<0.05); the KPC-2-, ybtS-, and iutA-positive isolates showed relatively higher resistance to fluoroquinolones and aminoglycosides, nitrofurantoin, and piperacillin/tazobactam, respectively. Furthermore, the combined expression (95.7%, 88/92) of carbapenemase and virulence-associated genes was detected, with the carbapenemase genes blaKPC-2 and blaTEM-1 combined with virulence-associated genes entB, mrkD, and ybtS accounting for the highest percentage (20.7%).Carbapenemase gene mutations in the CRKP strain from 2019 to 2020 highlight the importance of dynamic monitoring. The spread of hypervirulence-associated genes in CRKP strains and the high positivity rates of ybtS and iutA in KPC-2- and ST11-producing ones signify their high virulence potential in pediatric patients.

High Emergence of Multidrug-Resistant Sequence Type 131 Subclade C2 among Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Isolated from the University Hospital Bratislava, Slovakia.

The expansion of sequence type 131 (ST131) extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) represents major worldwide challenges. E. coli strains originating from healthcare facilities (labeled No. 1 and No. 2) of the University Hospital Bratislava (UHB) were analyzed for ST131 emergence, including its (sub)lineages and clonal relatedness. Antimicrobial resistance was determined in most strains. Of a total of 354 E. coli strains, 263 (74.3%) belonged to ST131; of these, 177 (67.3%) were from No. 1. Generally, among 260 ST131 E. coli, clades A/B were confirmed in 20 (7.7%), while clade C was noted in 240 (92.3%) strains; within them, subclades were detected as follows: C0 (17; 7.1%), C1 (3; 1.2%), and C2 (220; 91.7%). Among fifteen randomly selected E. coli strains that were investigated for ST and clonal relatedness, seven STs were identified: eight (53.3%) ST131, two (13.3%) ST73, and one each (6.7%) of ST10, ST12, ST14, ST1193, and ST1196. From No. 1, two ST131 in the first internal clinic and one ST131 from No. 2 in the aftercare department were highly clonally related, suggesting possible epidemiological association. Antimicrobial resistance was as follows: ciprofloxacin 93.8%, ceftazidime 78.4%, meropenem 0%, fosfomycin 2.9% and nitrofurantoin 1.4%. Prevention of ESBL-producing E. coli dissemination, especially for ST131 clade C2, is inevitably necessary for reducing drug resistance and decreasing healthcare-associated infections.

Short- and Long-Read Sequencing Reveals the Presence and Evolution of an IncF Plasmid Harboring blaCTX-M-15 and blaCTX-M-27 Genes in Escherichia coli ST131.

Escherichia coli sequence type 131 (ST131) has contributed to the spread of extended-spectrum beta-lactamase (ESBL) and has emerged as the dominant cause of hospital- and community-acquired urinary tract infections. Here, we report for the first time an in-depth analysis of whole-genome sequencing (WGS) of 4 ESBL-producing E. coli ST131 isolates recovered from patients in two hospitals in Armenia using Illumina short-read sequencing for accurate base calling to determine their genotype and to infer their phylogeny and using Oxford Nanopore Technologies long-read sequencing to resolve plasmid and chromosomal genetic elements. Genotypically, the four Armenian isolates were identified as part of the H30Rx/clade C2 (n = 2) and H41/clade A (n = 2) lineages and were phylogenetically closely related to isolates from the European Nucleotide Archive (ENA) database previously recovered from patients in the United States, Australia, and New Zealand. The Armenian isolates recovered in this study had chromosomal integration of the blaCTX-M-15 gene in the H30Rx isolates and a high number of virulence genes found in the H41 isolates associated with the carriage of a rare genomic island (in the context of E. coli ST131) containing the S fimbrial, salmochelin siderophore, and microcin H47 virulence genes. Furthermore, our data show the evolution of the IncF[2:A2:B20] plasmid harboring both blaCTX-M-15 and blaCTX-M-27 genes, derived from the recombination of genes from an IncF[F2:A-:B-] blaCTX-M-15-associated plasmid into the IncF[F1:A2:B20] blaCTX-M-27-associated plasmid backbone seen in two genetically closely related H41 Armenian isolates. IMPORTANCE Combining short and long reads from whole-genome sequencing analysis provided a genetic context for uncommon genes of clinical importance to better understand transmission and evolutionary features of ESBL-producing uropathogenic E. coli (UPEC) ST131 isolates recovered in Armenia. Using hybrid genome assembly in countries lacking genomic surveillance studies can inform us about new lineages not seen in other countries with genes encoding high virulence and antibiotic resistance harbored on mobile genetic elements.

Characterizing the molecular epidemiology of anaesthesia work area transmission of Staphylococcus aureus sequence type 5

Staphylococcus aureus sequence type 5 (ST5) is an emerging global threat. To characterize the epidemiology of ST5 transmission in the anaesthesia work area. The retrospective cohort study analysed transmitted, prophylactic antibiotic-resistant Staphylococcus aureus isolates involving anaesthesia work area reservoirs. Using whole-genome analysis, the epidemiology of ST5 transmission was characterized by reservoir(s) of origin, transmission location(s), portal of entry, and mode(s) of transmission. All patients were followed for at least 30 days for surgical site infection (SSI) development. Forty-one percent (18/44; 95% confidence interval: 28-56%) of isolates were ST5. Provider hands were the reservoir of origin for 28% (5/18) of transmitted ST5 vs 4% (1/26) for other STs. Provider hands were the transmission location for 28% (5/18) of ST5 vs 7% (2/26) of other STs. Stopcock contamination occurred for 8% (1/13) of ST5 isolates vs 12% (3/25) of other STs. Sixty-three percent of transmission events occurring between cases on separate operative dates involved ST5. ST5 was more likely to harbour resistance traits (ST5 median (interquartile range) 3 (2-3) vs 2 (1-2) other STs; P < 0.001) and had greater resistance to cefazolin, piperacillin-tazobactam, and/or ciprofloxacin (ST5: 3 (2-3) vs 2 (1-3) other STs; P = 0.02). ST5 was associated with three of six SSIs. ST5 is prevalent among transmitted, prophylactic antibiotic-resistant isolates in the anaesthesia work area. Transmission involves provider hands and one patient to another on future date(s). ST5 is associated with a greater number of resistance traits and reduced in-vitro susceptibility vs other intraoperative meticillin-resistant S.aureus.

Multiplicity of blaKPC Genes and pKpQIL Plasmid Plasticity in the Development of Ceftazidime-Avibactam and Meropenem Coresistance in Klebsiella pneumoniae Sequence Type 307.

In 2021, Klebsiella pneumoniae sequence type 307 (ST307) strains causing pulmonary and bloodstream infections identified in a hospital in Rome, Italy, reached high levels of resistance to ceftazidime-avibactam (CZA). One of these strains reached high levels of resistance to both CZA and carbapenems and carried two copies of blaKPC-3 and one copy of blaKPC-31 located on plasmid pKpQIL. The genomes and plasmids of CZA-resistant ST307 strains were analyzed to identify the molecular mechanisms leading to the evolution of resistance and compared with ST307 genomes at local and global levels. A complex pattern of multiple plasmids in rearranged configurations, coresident within the CZA-carbapenem-resistant K. pneumoniae strain, was observed. Characterization of these plasmids revealed recombination and segregation events explaining why K. pneumoniae isolates from the same patient had different antibiotic resistance profiles. This study illustrates the intense genetic plasticity occurring in ST307, one of the most worldwide-diffused K. pneumoniae high-risk clones.

Multicenter Study of Colistin Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae Strains in China.

Colistin has been considered a last-line option for the treatment of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). Heterogeneous resistance to colistin leads to unexplained clinical colistin treatment failure for CRKP. Our study aimed to investigate the extent of colistin heteroresistance among CRKP strains in China. A total of 455 colistin-susceptible strains, collected from six tertiary care hospitals in China, were characterized. The overall rate of colistin heteroresistance was 6.2%, as determined by the population analysis profiles (PAPs). Genomic analysis revealed that 60.7% of the colistin-heteroresistant isolates belonged to the epidemic sequence type 11 (ST11) clone. Single-nucleotide polymorphisms (SNPs) suggested that 6 ST5216 strains shared the same origin. Each of the subpopulations had a ≥8-fold decrease in colistin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which indicated that heteroresistance could be suppressed by an efflux pump inhibitor. In addition, our results suggested that the PhoPQ pathway plays an important role in the mechanisms of heteroresistance. IMPORTANCE The problem of CRKP has raised alarms concerning global health. Our study enriches the epidemiological study of colistin heteroresistance among CRKP strains in China, where the prevalence of this phenomenon was previously unknown. Importantly, colistin-heteroresistant strains may cause the failure of clinical treatment with colistin, even if the clinical laboratory reports that the strains are sensitive. The commonly used broth microdilution method is unable to detect this special phenomenon. Additionally, our results indicate that efflux pumps play a major role in colistin heteroresistance, and inhibitors can effectively reverse it. Our study is the first to provide a detailed analysis of the prevalence of colistin heteroresistance in China, as well as an analysis of the genetic mechanisms of this phenomenon.

Genomic epidemiology of mcr carrying multidrug-resistant ST34 Salmonella enterica serovar Typhimurium in a one health context: The evolution of a global menace

The rapid global dissemination of Salmonella enterica sequence type 34 (ST34) has sparked significant concern due to its resistance to critical antimicrobials and its ability to spread across various sectors. In order to investigate the evolution and transmission dynamics of this epidemic clonal lineage, as well as the horizontal transfer of mcr-carrying plasmids within the One Health framework, we conducted a comprehensive genomic epidemiological study. This study focused on the 11 mcr-carrying S. enterica isolates obtained from clinical settings in China, while also considering 2337 publicly available genomes of mcr-carrying S. enterica collected from 20 countries and diverse sources spanning over a 22-year period. Among the mcr-positive Salmonella isolates, ST34 was found to be the predominant lineage, comprising 30.12 % (704/2337) of the total collection. These isolates were identified as either serovar Typhimurium or its monophasic variant, which were obtained from both clinical and non-clinical sources. Phylogeographic analyses traced the global spread of the mcr-carrying ST34 lineage, which was divided into three distinct clusters, with 83.10 % of them carrying mcr-1 or/and mcr-9 genes. Notably, the mcr-1 positive ST34 isolates were primarily found in China (190/298, 63.76 %), with only four from the United States. Conversely, mcr-9 positive ST34 isolates were predominantly identified in the United States (261/293, 89.08 %), while none were observed in China. The mcr-1 positive ST34 isolates was predicted to have originated from clinical sources in United Kingdom, whereas mcr-9 positive ST34 isolates was likely derived from environmental sources in Germany. The most recent common ancestor for mcr-1 and mcr-9 carrying ST34 S. enterica was estimated to have emerged around 1983 and 1951. These findings provided thorough and intuitive insights into the intercontinental spread of mcr-carrying S. enterica ST34 lineage in a One Health context. Ongoing surveillance is crucial for effectively monitoring the worldwide dissemination of this multidrug-resistant high-risk clone.

Overexpression of blaGES-1 due to a strong promoter in the class 1 integron contributes to decreased ceftazidime-avibactam susceptibility in carbapenem-resistant Pseudomonas aeruginosa ST235.

Sequence type 235 (ST235) Pseudomonas aeruginosa, harboring so-called international, high-risk, or widespread clones, is associated with relatively high morbidity and mortality, partly due to multiantibiotic and high-level antibiotic resistance. Treatment of infections caused by such strains with ceftazidime-avibactam (CZA) is often successful. However, CZA resistance in carbapenem-resistant P. aeruginosa (CRPA) strains has been consistently reported with the increasing use of this drug. Likewise, we identified thirty-seven CZA-resistant ST235 P. aeruginosa strains from among 872 CRPA isolates. A total of 10.8% of the ST235 CRPA strains were resistant to CZA. Site-directed mutagenesis, cloning, expression, and whole-genome sequencing analysis revealed that overexpression of blaGES-1, which was carried in a class 1 integron of the complex transposon Tn6584, occurred due to a strong promoter, contributing to CZA resistance. Moreover, such overexpression of blaGES-1 combined with an efflux pump resulted in high-level resistance to CZA, considerably reducing the therapeutic options available for treating infections caused by ST235 CRPA. Considering the widespread presence of ST235 P. aeruginosa strains, clinicians should be aware of the risk of CZA resistance development in high-risk ST235 P. aeruginosa. Surveillance initiatives for preventing further dissemination of high-risk ST235 CRPA isolates with CZA resistance are essential.

Clinical and Molecular Characteristics and Antibacterial Strategies of Klebsiella pneumoniae in Pyogenic Infection.

Treatment of Klebsiella pneumoniae causing pyogenic infections is challenging. The clinical and molecular characteristics of Klebsiella pneumoniae causing pyogenic infections are poorly understood, and antibacterial treatment strategies are limited. We analyzed the clinical and molecular characteristics of K. pneumoniae from patients with pyogenic infections and used time-kill assays to reveal the bactericidal kinetics of antimicrobial agents against hypervirulent K. pneumoniae (hvKp). A total of 54 K. pneumoniae isolates were included, comprising 33 hvKp and 21 classic K. pneumoniae (cKp) isolates, and the hvKp and cKp isolates were identified using five genes (iroB, iucA, rmpA, rmpA2, and peg-344) that have been applied as hvKp strain markers. The median age of all cases was 54 years (25th and 75th percentiles, 50.5 to 70), 62.96% of individuals had diabetes, and 22.22% of isolates were sourced from individuals without underlying disease. The ratios of white blood cells/procalcitonin and C-reactive protein/procalcitonin were potential clinical markers for the identification of suppurative infection caused by hvKp and cKp. The 54 K. pneumoniae isolates were classified into 8 sequence type 11 (ST11) and 46 non-ST11 strains. ST11 strains carrying multiple drug resistance genes have a multidrug resistance phenotype, while non-ST11 strains carrying only intrinsic resistance genes are generally susceptible to antibiotics. Bactericidal kinetics revealed that hvKp isolates were not easily killed by antimicrobials at susceptible breakpoint concentrations compared with cKp. Given the varied clinical and molecular features and the catastrophic pathogenicity of K. pneumoniae, it is critical to determine the characteristics of such isolates for optimal management and effective treatment of K. pneumoniae causing pyogenic infections. IMPORTANCE Klebsiella pneumoniae may cause pyogenic infections, which are potentially life-threatening and bring great challenges for clinical management. However, the clinical and molecular characteristics of K. pneumoniae are poorly understood, and effective antibacterial treatment strategies are limited. We analyzed the clinical and molecular features of 54 isolates from patients with various pyogenic infections. We found that most patients with pyogenic infections had underlying diseases, such as diabetes. The ratio of white blood cells to procalcitonin and the ratio of C-reactive protein to procalcitonin were potential clinical markers for differentiating hypervirulent K. pneumoniae strains from classical K. pneumoniae strains that cause pyogenic infections. K. pneumoniae isolates of ST11 were generally more resistant to antibiotics than non-ST11 isolates. Most importantly, hypervirulent K. pneumoniae strains were more tolerant to antibiotics than classic K. pneumoniae isolates.