Multilocus Sequence Typing Analysis Research Articles

Epidemiological relationship of Clostridium perfringens isolated from feces and soil of horse farm using multi-locus sequence typing analysis

Epidemiological relationship of Clostridium perfringens isolated from feces and soil of horse farm using multi-locus sequence typing analysis

Atypical diarrheagenic Escherichia coli in milk related to a large foodborne outbreak.

A foodborne outbreak related to milk cartons served in school lunches occurred in June 2021, which involved more than 1,800 cases from 25 schools. The major symptoms were abdominal pain, diarrhoea, vomiting, and fever. Although major foodborne toxins and pathogens were not detected, a specific Escherichia coli strain, serotype OUT (OgGp9):H18, was predominantly isolated from milk samples related to the outbreak and most patients tested. The strains from milk and patient stool samples were identified as the same clone by core genome multilocus sequence typing and single-nucleotide polymorphism analysis. The strain was detected in milk samples served for two days related to the foodborne outbreak at a rate of 69.6% and levels of less than ten most probable number/100mL but not on days unrelated to the outbreak. The acid tolerance of the strain for survival in the stomach was similar to that of enterohaemorrhagic E. coli O157:H7, and the same inserts in the chu gene cluster in the acid fitness island were genetically revealed. The pathogenicity of the strain was not clear; however, it was indicated that the causative pathogen was atypical diarrhoeagenic E. coli OUT (OgGp9):H18.

Genomic Analysis and Antimicrobial Resistance of Campylobacter jejuni Isolated from Diarrheal Patients - Beijing Municipality, China, 2019-2021.

Campylobacter jejuni (C. jejuni) is the leading cause of human bacterial gastroenteritis worldwide and has a major impact on global public health. The objective of the present study was to conduct whole genome sequencing (WGS) to determine the genetic diversity, virulence factors, and determinants of antimicrobial resistance of C. jejuni during a 3-year surveillance period in Beijing, China. A total of 184 clinical isolates were obtained from sentinel hospital surveillance between 2019 and 2021. Antimicrobial susceptibility testing was conducted using the agar dilution method. WGS was employed to characterize the 184 C. jejuni strains. Multilocus sequence typing analysis revealed high genetic diversity among the 184 C. jejuni strains, identifying 71 sequence types (STs) and 19 clonal complexes (CCs). The most prevalent ST was ST760 (6.5%), and the most common CC was CC21 (24.5%), consisting of 11 STs. High resistance rates were observed for ciprofloxacin (76.6%), nalidixic acid (76.1%), and tetracycline (71.2%). A total of 77 C. jejuni isolates (41.8%) exhibited multidrug resistance with 43 resistance patterns. Virulome analysis disclosed the differential distribution of virulence factors related to adherence, colonization, chemotaxis, as well as lipo-oligosaccharide and capsular polysaccharide biosynthesis. Resistome analysis demonstrated widespread resistance to quinolones and tetracycline, but low rates of macrolides resistance. The phylogeny, based on whole genome single nucleotide polymorphisms, indicated a high degree of clonality and grouped the C. jejuni strains into six clades. Closely related isolates that were part of a genetic cluster mostly shared a homogenous clonal complex. The present study emphasizes the rising resistance to quinolones and tetracycline, as well as the virulence potential and diverse genotypes identified among C. jejuni strains isolated from diarrheal patients in Beijing.

Molecular characterization of fungal endophthalmitis and keratitis caused by yeasts.

Candida species are the most common causes of sight-threatening fungal ocular infections in temperate regions of the world. Despite their relevance, little is known about the emergence of novel species and the molecular epidemiology of these infections. Here, we molecularly characterized 38 yeast isolates collected from patients diagnosed with endophthalmitis or keratitis at Massachusetts Eye and Ear from 2014 to 2021. Sequencing of the ITS1-5.8S-/ITS2 regions demonstrated that this population of yeasts was dominated by Candida spp. (37 out of 38; 97%), with 58% of the cases caused by C. albicans (n=22) and the remaining by emerging non-albicans species, predominantly by C. parapsilosis (n=8) and C. dubliniensis (n=6). One isolate each was identified as C. tropicalis and Clavispora lusitaniae. Interestingly, all C. dubliniensis were isolated from endophthalmitis and most C. parapsilosis from keratitis. Multilocus sequence typing analysis of C. albicans showed a prevalence of CC-1 isolates that has DST69 as the putative founder, with 64% of them belonging to this clonal complex (CC). Isolates grouped within this cluster were more predominant in endophthalmitis (10 out of 14; 71%). One C. albicans CC-1 isolate was multi-azole resistant. In conclusion, we observed that nearly half of the ocular infections caused by yeasts are associated with C. albicans, with evidence for the emergence of non-albicans species that are differentially enriched in distinct ocular niches. Candida albicans isolates clustered within the predominant CC-1 group were particularly more common in endophthalmitis, demonstrating a potential pattern of ocular disease enrichment within this clade.

Description and Genome Characterization of Three Novel Fungal Strains Isolated from Mars 2020 Mission-Associated Spacecraft Assembly Facility Surfaces-Recommendations for Two New Genera and One Species.

National Aeronautics and Space Administration's (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains.

1190. Effect of Pulsed Xenon Ultraviolet Light (PX-UV) on Clonal Recovery of Escherichia coli in a Prospective, Sham-controlled, Double-blinded, Interventional, Crossover Trial Conducted in Two Detroit Hospitals

Abstract Background Healthcare-associated infections (HAIs) can be caused by some multidrug-resistant Escherichia coli, most commonly due to production of an extended spectrum beta-lactamase (ESBL), and lead to increased morbidity and mortality. Pulsed Xenon Ultraviolet light (PX-UV), in combination with terminal cleaning, has been shown to improve disinfection and has the potential to lower HAIs by reducing the horizontal spread of infections in hospitals. Here, we assess the effect of PX-UV on the clonal recovery pattern of several E. coli sequence types (STs) using Whole Genome Sequencing (WGS). Methods A prospective, sham-controlled, double-blinded, interventional, crossover trial was conducted to compare standard terminal cleaning with PX-UV (intervention, Group Q) and standard terminal cleaning with sham UV (control, Group W) in 2 Detroit hospitals from 2017 to 2020. Treatments lasted 12 months before crossover, with a 6-month washout period in between (Group R) during which UV was not used. A total of 67 E. coli samples were collected. WGS of the isolates was performed using the Nextseq 550 (Illumina). After de novo assembly, BioNumerics calculation engine (v7.6) was used to complete Whole Genome Multilocus Sequence Type (wgMLST) analysis, assembly free and assembly-based call, and construction of minimum spanning tree (MST). Results The total number of different STs found for the intervention UV device group (Q) and the sham UV device group (W) was 6 while the washout group (R) was 5. Out of the 9 total STs obtained, the most common was ST131 (Table 1). In Group Q, 11 of ST131 were found; 15 were found in Group W. During the washout period (R) 17 of ST131 were found. All other STs had 3 or less circulating clusters. After UV treatment ST1193, ST399, and ST7394 were not recovered. Fig. 1:Minimum spanning tree (MST) for E. coliTable 1:Total number of each sequence type (ST) per treatment group Conclusion During UV intervention 3 different STs (ST1193, ST399, ST7394) were not recovered, but there were negligible changes to the frequency of recovery of the other 6 STs as compared to sham UV treatment. ST131 was the dominant E. coli ST found in Detroit, which is consistent with previously published data stating ST131 as the predominant strain. While PX-UV has previously demonstrated effectiveness on decreasing bioburden, our data does not indicate any remarkable change in clonality and prevalence of E. coli STs after PX-UV use. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

1189. Clonal Recovery Pattern of Staphylococcus aureus during Sham-controlled, Interventional, Crossover Trial on the Effectiveness of Pulsed Xenon Ultraviolet Light (PX-UV) in Reducing Healthcare-associated Infections

Abstract Background PX-UV has shown great effectiveness at reducing Staphylococcus aureus (S. aureus) on healthcare surfaces. S. aureus is one of the most prevalent causes of healthcare-associated infections (HAIs). Here we assess the clonal recovery pattern and the disruption caused by PX-UV on S. aureus collected from patients, using Whole Genome Multilocus Sequence Type (wgMLST) analysis. Methods During a 3-year period, 2017 to 2020, a prospective sham-controlled double-blinded interventional crossover trial designed to assess the effectiveness of post-discharge PX-UV room disinfection on HAIs in 2 Detroit hospitals. The interventional group (Q) and the control group (W) was treated with PX-UV and sham UV light after terminal cleaning respectively, both treatments lasting 12 months. Crossover occurred after a 6-month washout period (R) during which no UV light was used. Whole Genome Sequencing (WGS) of the 291 S. aureus samples collected was performed using the Nextseq 550 (Illumina). Following de novo assembly, construction of minimum spanning tree (MST) and wgMLST analysis was completed using BioNumerics calculation engine (v7.6). Results A total of 20 sequence types (STs) were found throughout the trial, the most common being ST5 and ST8 with roughly 20 to 50 each found in all 3 treatment groups (Table 1. The other 18 STs were only found 7 times or less in each treatment group. ). During intervention (Q) 9 STs were found, however 11 were not recovered after PX-UV (ST1, ST1508, ST22, ST231, ST3287, ST3462, ST398, ST59, ST6790, ST840, ST97). During the sham treatment (W) 15 STs were found while 7 were found during the washout period (R). The minimum spanning tree (MST) shows clusters of related ST groups color coded by treatment type (Figure 1). Fig. 1:Minimum spanning tree (MST) for S. aureusTable 1:Total number of each sequence type (ST) per treatment group Conclusion The difference between the STs recovered during intervention and during sham UV treatment was inconsequential because many factors influence the amount of STs recovered (treatment, number of patients, sampling bias, and terminal cleaning methods). There was no clonality noted among any of the intervention group (Fig. 1). Both dominant strains (ST5, ST8) have been characterized as hypervirulent and predominant previously, consistent with our data. Our data does not demonstrate any notable change in the clonal prevalence of S. aureus after PX-UV use. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

1187. Analysis of the Clonal Recovery Pattern of Enterococcus faecium to Evaluate the Efficacy of Pulsed Xenon Ultraviolet Light (PX-UV) in Bioburden Reduction

Abstract Background Enterococcus faecium (E. faecium) is a common hospital-associated infection (HAI) that can lead to increased costs, morbidity, and mortality. Pulsed Xenon Ultraviolet light (PX-UV) has been shown to reduce bacterial bioburden levels on surfaces. This study aims to assess the effect of the addition of PX-UV to terminal cleaning on the clonal recovery of E. faecium sequence types (STs) using Whole Genome Sequencing (WGS) on patient isolates. Methods During 2017 to 2020 a prospective, randomized, double-blinded, sham-controlled, interventional, crossover trial in 2 separate Detroit hospitals (H1 and H2) compared HAI counts after the addition of either PX-UV or a non-UV sham device to terminal cleaning methods. The trial consisted of a total of 16 units randomized to have either the treatment of PX-UV (Group Q) or the sham control device with no UV (Group W) for 12 months. A washout period (Group R) of 6 months followed and the trial concluded with a 12-month crossover of treatments. A total of 60 E. faecium samples were collected, then WGS was performed by the Illumina Nextseq 550 instrument. de novo assembly was preformed using the SPAdes program. Whole Genome Multilocus Sequence Type (wgMLST) analysis was performed by BioNumerics (v7.6) to construct minimum spanning tree (MST). Results A total of 7 STs were obtained across the 2 hospitals (H1 and H2). ST117, ST17, and ST80 all had more than 10 total recovered isolates, ST117 being the most frequent with 24 isolates. Less than 3 isolates were recovered for all other STs. For all STs, Group Q (PX-UV) had 14, Group R (washout period) had 22, and Group W (non-UV sham) had 24. ST18 was only found in Group Q. ST412, ST584, and ST736 were not found in Group W. The data shows that the intervention PX-UV group had a reduction of clonal recovery by 10 STs as compared to the sham UV group. Table 1:Total number of each sequence type (ST) in H1 and H2 Conclusion The overall reductions in the number of isolates in the real UV units was driven by reductions in the ST117 a predominant strain in a hospital environment reported previously in Detroit and ST80. There were negligible differences in recovery of other ST between the groups. The reduction in clonal recovery of E. faecium isolates in Group Q as compared to Group R might be related to use of PX-UV following terminal room cleaning. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

2221. Use of PCR and MLST analysis to establish Treponema pallidum as the cause of a painful oral lesion

Abstract Background A syphilitic chancre is described classically as a single, indurated, and painless ulcer at the site of Treponema pallidum inoculation. Methods We present a 39-year-old heterosexual, monogamous, HIV-negative woman with a painful tongue lesion of one month’s duration. To exclude a malignancy, a biopsy was performed. Results Histopathology showed intense inflammatory reaction, and T. pallidum immunocytochemistry demonstrated a high density of spirochetes in the subepithelial connective tissue. Subsequent testing revealed a reactive anti-T. pallidum IgG EIA and an RPR titer of 1:8. PCR amplification and multi-locus sequence typing (MLST) confirmed that the visualized spirochetes were T. pallidum. This particular MLST strain pattern has been identified twice before and named ST-109; however, no clinical information is available. Diagnosis and appropriate treatment were delayed since syphilis was not suspected initially. When the results became available, the patient was treated with 2.4 million units of benzathine penicillin given IM with complete resolution of the lesion. The male partner, who reported no symptoms, had a positive T. pallidum EIA with an RPR titer of 1:64 and was also treated with penicillin. Within four months, a woman from an adjacent town presented to urgent care with three perineal severely painful lesions, testing negative for HIV, Herpes Simplex Virus (HSV) by PCR of an ulcer swab, and chlamydia and gonorrhea on a urine sample by a NAAT. The anti-T. pallidum EIA was reactive, and the RPR titer was 1:4. The lesions resolved with IM penicillin treatment. Conclusion The observations reported here indicate that a subset of early syphilis cases may present with painful lesions. Despite the commonly held belief that syphilitic chancres are painless, several case series have reported tender lesions, single or multiple, without evidence of concurrent HSV infection to explain the pain. This is the first PCR and MLST sequencing analysis of a T. pallidum strain within a painful chancre. Given the rise in the number of cases of syphilis in the USA, it is essential that clinicians be aware of this atypical presentation. It remains to be determined whether such painful lesions are only associated with certain T. pallidum genotypes. Disclosures All Authors: No reported disclosures.

1230. Identification of Subclinical Healthcare-Associated Clusters of Staphylococcus epidermidis in an Orthopedic Patient Population.

Abstract Background Prosthetic joint infections (PJIs) cause increased morbidity and mortality for patients. Staphylococcus epidermidis (S. epidermidis) can readily form biofilm on implanted medical devices, making this typically commensal species a common cause of PJIs. This study investigates genetic, epidemiologic, and environmental factors contributing to positive S. epidermidis joint cultures. Methods We identified S. epidermidis isolates from hip or knee cultures obtained between 2017-20 in patients with one or more prior corresponding intraarticular procedure at our hospital. Whole genome sequencing and single nucleotide polymorphism (SNP) based clonality analysis was performed using the epiXact® service at Day Zero Diagnostics, including species identification, in silico multi-locus sequence typing (MLST), phylogenomic analysis, along with genotypic assessment of the prevalence of specific antibiotic resistance and virulence genes. Additional epidemiologic review was performed to compare cluster and noncluster cases. Results Sixty phenotypically distinct S. epidermidis joint isolates were identified from 44 patients. After removal of duplicates and impure samples, 46 samples were used for genomic sequencing, and 45 were used for MLST. Four S. epidermidis strains showed phenotypic susceptibility to oxacillin yet harbored mecA, and two showed phenotypic resistance despite not having mecA (Figure 1). Notably, smr was found in all isolates. MupA positivity was not observed. We also identified six clonal clusters, all with SNP distances < 10 (Figure 2). Each cluster consisted of between 2-4 patients. Cluster isolates accounted for 14/45 (31.1%) of S. epidermidis isolates sequenced. Further epidemiologic investigation showed ties to common aspirations or operative procedures although no specific common source was identified. These subclinical clusters appear to be self-limited, suggesting benefit to common infection control practices. Phylogenetic tree of S. epidermidis population and heatmap showing the presence/absence of specific genomic features. Circle color on node tip corresponds to sequence type (MLST). Color strips (right panel) showing genomic feature (gene or marker) presence/absence inferred from WGS de novo assemblies. Green cells correspond to a full match found, and gray cells to no match found. Empty rows correspond to external reference genomes used as controls. *AMR: antimicrobial resistance; ST: sequence type; SNP distance values for all pairs of samples of matching multi-locus sequence types. Only samples belonging to matching multi-locus sequence types (MLSTs) with more than one sample are shown. Blue cells correspond to pairwise distance of < 25 SNPs indicating clonality. 13 isolates with unique MLSTs are not shown. 10 isolates were not included due to having multi-strain or multi-species contamination. *ST: sequence type; SNP: single-nucleotide polymorphism; Univariate analysis of clonal isolates versus non-clonal isolates. Samples not included in the multi-locus sequence type analysis due to contamination were excluded from the univariate analysis. *PJI: prosthetic joint infection; MSIS: Musculoskeletal Infection Society; Conclusion The majority of S. epidermidis isolated from clinical joint samples are diverse in origin, but we identified an important subset of 31% that belonged to subclinical healthcare-associated clusters. Clusters appeared to resolve spontaneously over time, suggesting benefit to routine infection control practices. Diagram of clonal isolate clusters Potential relationships and modes of transmission between clonal isolates in each cluster. *PJI: prosthetic joint infection; MSIS: Musculoskeletal Infection Society; SNP: single-nucleotide polymorphism; Disclosures Mohamad Sater, PhD, Day Zero Diagnostics: Employee|Day Zero Diagnostics: Stocks/Bonds Ian C. Herriott, Bachelor's of Science, Day Zero Diagnostics: Employee|Day Zero Diagnostics: Stocks/Bonds Miriam H. Huntley, PhD, Day Zero Diagnostics: Employee|Day Zero Diagnostics: Ownership Interest.

Optimized multilocus sequence analysis for laboratory identification of pathogens of ixodid tick-borne borreliosis

Introduction. The most common etiological agents of ixodid tick-borne borreliosis (ITBB) in Russia are Borrelia garinii, B. afzelii, B. bavariensis. Multilocus sequence typing and multilocus sequence analysis (MLSA) have been used in recent studies for Borrelia species identification. The results of using the MLSA scheme for identification of pathogens causing erythemic forms of ITBB have been presented earlier.
 The purpose of the study was to explore the possibility of MLSA optimization for laboratory identification of ITBB pathogens. Objectives: comparative analysis of nucleotide sequences of 6 conserved genes (rrs, hbb, fla, groEL, recA, ospA) and the rrfA-rrlB intergenic spacer, which are recommended by the MLSA protocol; identification of the minimum set of genes, the concatenated sequences of which are essential for species identification of Borrelia isolates.
 Materials and methods. The sequences of the above loci of 23 reference isolates collected from patients with ITBB and assigned, using MLSA, to B. bavariensis were compared with the sequences of similar genes of other Borrelia species available in international databases. The UPGMA method was used to build and analyze dendrograms based on the obtained data.
 Results. The sequences of ospA gene loci of reference species demonstrated the greatest difference (not less than 8.5%) from the sequences of the above gene in other analyzed species of Borrelia; approximately similar species-related differences (not less than 6.7%) were demonstrated by the comparison of recA gene sequences. The sequences of the identified variants of these two genes in B. bavariensis differed from the sequences of the similar genes in the most closely related species B. garinii. The dendrogram of the concatenated nucleotide sequences of recA and ospA genes demonstrated that it was totally consistent with the results of identification of the isolates based on the MLSA protocol.
 Conclusion. The optimized approach to MLSA of the B. burgdorferi sensu lato group suggests that species identification should be based on the concatenated analysis of loci of only two genes (recA and ospA) out of 7 loci recommended by the MLSA protocol.

Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae in a Tertiary Hospital in Northern China.

In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged rapidly in China with the abuse and overuse of antibiotics, and infections caused by CRKP pose a serious threat to global public health safety. The present study aimed to explore the epidemiological characteristics of CRKP isolates in Northern China and to elucidate their drug resistance mechanisms. 45 CRKP strains were consecutively collected at a teaching hospital from March 1st, 2018 to June 30th, 2018. Antimicrobial susceptibility was determined by the VITEK2 compact system and microbroth dilution method. Polymerase chain reaction (PCR) and sequencing were used to analyze multilocus sequence typing (MLST), drug resistance determinants, and plasmid types. The transfer of resistance genes was determined by conjugation. All statistical analysis was performed using SPSS 22.0 software. All 45 isolates showed multidrug resistance (MDR). MLST analysis showed ST11 (48.9%, 22/45) was the most frequent type. All of the 45 CRKP isolates contained carbapenemase genes, extended-spectrum β-lactamase (ESBL) genes, and plasmid-mediated quinolone resistance (PMQR) genes. For carbapenemase genes, KPC-2 (93.3%, 42/45) was the main genotype, and followed by GES (37.8%, 17/45) and NDM-1 (11.1%, 5/45). Plasmid typing analysis showed that IncFII and IncFIB were the most prevalent plasmids. The carbapenem resistance rate of K.pneumoniae was 11.4% and ICU was the main CRKP infection source. ST11 is the most frequent sequence type and KPC-2 is the predominant carbapenemase of CRKP strains in Northern China. KPC-2-ST11 are representative clonal lineages.

Low-level colonization of methicillin-resistant Staphylococcus aureus in pigs is maintained by slowly evolving, closely related strains in Finnish pig farms

BackgroundOver the past two decades, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has become widely prevalent in pig production in Europe. The carriage status of LA-MRSA is known to vary among individual pigs, but bacterial load in pigs has rarely been studied. We assessed the quantity of LA-MRSA in nasal and skin samples of pigs and investigated the genetic diversity of the strains together with sequenced strains from national surveillance and pathology samples from the Finnish Food Authority. On two farms with assumed MRSA-positive status, farm 1 and farm 2, 10 healthy pigs were sampled three times during 2 weeks from the nares and skin (study A). On farm 1, 54 additional pigs were sampled and from confirmed MRSA-positive animals, 10 were randomly selected and transported to a clean, controlled environment for further sampling (study B). From the samples taken on farms 1 and 2 and in the controlled environment, MRSA was isolated both by direct plating and enrichment on selective media. spa types, multilocus sequence types, staphylococcal cassette chromosome mec types, resistance and virulence genes were determined. Core genome multilocus sequence typing (cgMLST) analysis was performed, including the sequences deriving from the surveillance/pathology samples from the Finnish Food Authority.ResultsAll pigs on farm 1 carried LA-MRSA in the nares at all three time points and five pigs on farm 2 at one time point. Nasal quantity varied between 10 and 103 CFU/swab and quantity on the skin between 10 and 102 CFU/swab. In the controlled environment, MRSA was detected in at least one of the nasal samples from each animal. spa type t034 was predominant. cgMLST showed one cluster with minimum allele differences between 0 and 11.ConclusionsThe study shows predominantly low-level carriage (< 103 CFU/swab) of LA-MRSA on farms. In the controlled environment we observed a decline in nasal carriage but constant skin carriage. cgMLST showed that strains of spa type t034 are closely related at the national level.

Evidence of Sharing of Carbapenem-Resistant Klebsiella pneumoniae Strains Between Intensive Care Unit Patients and the Environment.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) has emerged as an important public health threat. Intestinal colonization with CR-KP increases the risk of infection and death, especially in intensive care unit patients. To clarify the source of colonizing bacteria is very important to prevent the spread of CR-KP, so the purpose of this study was to explore the relationship between the ward environment and intestinal colonization of CR-KP. In this study, 353 environmental swabs from ICU (Intensive Care Unit) wards and 241 anal swab samples from ICU patients were collected and screened on MacConkey plates containing 2 μg/mL ertapenem, and the origin and genotype of CR-KP were analyzed by PCR and sequencing. The sequence type of the strains was also obtained by multi-locus sequence type (MLST) analysis, and plasmid conjugation test was used to clarify whether CR-KP can promote the transmission of drug resistance genes through plasmid integration and rearrangement. A total of 20 CR-KP environmental strains and 7 intestinal strains were obtained, most of which were bla OXA-48 resistant genotypes. Four different STs were identified by multi-locus sequence type (MLST) analysis, among which the large logarithm was ST485 type, and PFGE clustering showed that the similarity between them was >85%. In the plasmid transcoupling assay, we report that one of the Klebsiella pneumoniae drug-resistant plasmids was successfully transferred to E. coli, indicating that it may promote the spread of drug-resistant genes through plasmid integration and rearrangement. Our research suggests that the environment may be a potential source of CR-KP and that there is a need for us to adopt more effective disinfection measures.

Genetic and Phenotypic Characteristics of Carbapenem-Resistant Klebsiella pneumoniae Isolates from a Tertiary Hospital in Beijing.

Klebsiella pneumoniae is a common multidrug-resistant pathogen that jeopardizes the health of hospitalized patients. We aimed to study the phenotypic and genotypic characteristics of carbapenem-resistant K. pneumoniae (CRKP) isolates from a hospital in Beijing. Twenty-four CRKP clinical isolates were collected within a half-year to investigate antimicrobial resistance and genomic characteristics. Illumina and Nanopore sequencing were performed to assemble and annotate genomes. All strains were multi-drug resistant. Twenty-two strains carried the bla KPC-2 gene and two harbored bla NDM-5. Multilocus sequence type(MLST) analysis identified five sequence types; most isolates belonged to ST11. Three strains were isolated from the same patient; each carried a different plasmid replicon, either IncFII (pHN7A8), IncX, or IncFIB (K). This study furthers the understanding of CRKP antimicrobial resistance genotypes, and may facilitate the control of nosocomial infections caused by antimicrobial-resistant pathogens.

Tandem Repeat of bla NDM-1 and Clonal Dissemination of a fosA3 and bla KPC-2 Co-Carrying IncR-F33: A-: B- Plasmid in Klebsiella pneumoniae Isolates Collected in a Southwest Hospital in China, 2010-2013.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been widespread in coastal cities of eastern China since 2009. However, how CRKP spreads and evolves in southwest China is unclear. We investigated the genetic characteristics and dissemination mechanisms of carbapenemase genes in forty-one non-repetitive CRKP isolates collected from a southwest hospital, Kunming, Yunnan, during 2010-2013. Drug susceptibilities were analyzed by using VITEK 2 compact system. Genetic relationships were ascertained based on multilocus sequence typing (MLST) and Pulsed-field gel electrophoresis (PFGE) analysis. Genetic backgrounds of bla KPC-2 and bla NDM-1 were revealed by DNA walking and high-throughput sequencing. All isolates were highly resistant to common antibiotics except for tigecycline. In total, 34 bla KPC-2, 3 bla NDM-1, 1 bla IMP-4 and 3 bla IMP-26 genes were identified and KP67 plasmid 1 co-harbored bla NDM-1 and bla IMP-26. Five sequence types, namely ST11, ST290, ST340, ST395 and ST437, were recognized by MLST. Surprisingly, bla KPC-2 was only detected in ST11 strains. We described a clonal dissemination of fosA3-positive IncR-IncF33:A-:B- multireplicon plasmid carrying the gene cassettes IS26-ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-korC-klcA-ΔrepB-Tn1721 in all ST11 isolates. Three bla NDM-1 positive isolates belonged to three different ST types and their bla NDM-1 genetic backgrounds were also distinct. Interestingly, the flanking regions of bla NDM-1 in KP67 and KP72 were duplicated into one to five copies in a form of tandem repeat by the transposition of IS91 like element. The bla NDM-1 of KP82 was carried on a common IncX3 plasmid. This study described the early epidemiological characteristics of bla KPC-2/bla NDM-1-carrying CRKP, and reported a new tandem repeat pattern of bla NDM-1 cluster in Yunnan. These findings extend our knowledge on the carbapenemase gene evolutions.

Relationship between Phage Lytic Spectra and Sequence Types in Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated in Japan.

The lytic spectrum of phages is usually limited to only a few strains of the same bacterial species that can lyse. In clinical molecular epidemiology, bacterial strains are commonly classified into sequence types (STs) using the multilocus sequence typing (MLST) approach. The aim of this study was to determine the association between the phage lytic spectrum and STs. MLST analysis of 11 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli clinical isolates revealed that most belonged to ST73 or ST131, with four isolates each. Phages were isolated from sewage samples using various E. coli strains as hosts. The relationship between phage lytic spectra of ESBL-producing E. coli isolates ST73 and ST131 and STs was evaluated using Fisher's exact test. The lytic spectra of phages were significantly dependent on the ST classification of ST73 or ST131, suggesting that a phage lysing an isolate belonging to a particular ST could lyse other isolates of the same ST. We successfully isolated wide-host-range phages lysing all clinical isolates belonging to two clinically important ST types (ST73 and ST131).

Molecular typing and drug resistance analysis of carbapenem-resistant Klebsiella pneumoniae from paediatric patients in China.

There are few studies on paediatric carbapenem-resistant Klebsiella pneumoniae (CRKP) in China. The present study investigated the molecular epidemiological and drug resistance characteristics of CRKP from paediatric patients in China to provide a reference for the prevention and control of CRKP infection. In total, 20 nonrepetitive clinical CRKP isolates were collected between February 2019 and February 2020 in a tertiary hospital in China. Strain identification and drug susceptibility testing were carried out using the VITEK® 2 Compact Bacterial Identification and Monitoring System. Sequence typing, phylogenetic relationships, and antibiotic resistance-associated genes were analysed by whole genome sequencing (WGS). sequence typing (MLST) and Core genome multilocus sequence typing (cgMLST) analysis revealed the most frequently represented were ST2407-CT3536 (30%), ST76-CT5893 (25%), and ST309-CT7864 (25%). All 20 CRKP isolates were divided into three clusters. All isolates were highly resistant to a variety of β-lactams and were highly susceptible to quinolones, aminoglycosides, and sulphonamides. All isolates mainly carried the carbapenem resistance genes blaNDM-1 and blaKPC-2, among which 10 isolates carried both blaNDM-1 and blaKPC-2 simultaneously. Sequence typing, phylogenetic relationships, and antibiotic resistance genes can be determined using WGS technology. This can guide CRKP infection control and clinical treatment for paediatric patients.

Successful Control of an Outbreak by Phenotypically Identified Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae in a Neonatal Intensive Care Unit.

Premature newborns represent a vulnerable population, at high risk of acquiring nosocomial infections during neonatal intensive care unit (NICU) admission. Multidrug-resistant organisms represent the greatest concern due to their intrinsic virulence and the limited therapeutic options. Resistant Enterobacterales are a growing threat for critically ill neonates, with increasing numbers of NICU outbreaks caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales being described. This study reports the early detection and successful control of an outbreak caused by ESBL-producing Klebsiella pneumoniae (ESBL-KP) in an Italian NICU in February 2021. A total of 13 newborns tested positive for ESBL-KP between 2-9 February 2021, of whom four (31%) had a bloodstream infection. Two were critically ill, extremely premature newborns who died because of multiple comorbidities, and two were cured after treatment with meropenem. All other patients survived and were either discharged home or moved to other hospitals/wards in good clinical condition. ESBL-KP ST45 was found in all isolates by multilocus sequence typing (MLST) analysis. An outbreak control plan was set, including surveillance cultures for all neonates, NICU environments, and medical devices, along with the extended use of contact precautions and cohorting. In addition, the infection control plan was carried out through reinforcement and enhancement measures to guarantee maximal compliance. The outbreak was successfully controlled in seven days, given that no further cases were identified after 9 February. The source of the ESBL-KP outbreak was not identified through environmental sampling. Thanks to multidisciplinary management, a threatening outbreak of ESBL-KP in a NICU was controlled in few days. The prompt recognition of the event onset and the adoption of infection control interventions helped contain the bacteria spread on the ward.

Serotype Distribution, Virulence Determinants and Antimicrobial Susceptibility of Streptococcus agalactiae Isolated from Young Infants

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common cause of serious infections in the first 3 months of life worldwide. The pathogenicity of GBS is closely related to serotypes, surface proteins and virulence factors, and the distribution of them may vary temporally and geographically. However, data related to GBS surface proteins and virulence determinants in China are very few. The aim of this study is to investigate the genetic characteristics of clinical GBS isolates from infected infants. Methods: We recovered GBS isolates from infected infants younger than 3 months during 2017−2021 at Maternal and Child Health Hospital of Hubei Province in China. We assessed the GBS serotypes, surface proteins, virulence determinants and antibiotic resistance genes distribution, by Multilocus sequence typing (MLST) and whole-genome sequencing analysis. Results: Among 97 isolates (81 EOD and 16 LOD), 5 serotypes were detected. Serotype III was the most represented (49.5%), followed by type Ib (20.6%). The isolates belonged to 17 different sequence types (STs) that grouped into the 8 clonal complexes (CCs). The most frequently identified ST was ST17 (23.7%). The most predominant surface protein of alpha-protein-like (alp) family (one of the protein components of the GBS surface antigen, resistant to trypsin) present was Rib (41.2%), which was mainly detected in serotype III. The srr1, which encodes Srr1 protein, was identified in 54.6% of isolates. The hvgA encoding for hypervirulent GBS adhesin can be detected in all 24 serotype III GBS. Among the pilus islands genes, 50% and 58.8% of the isolates were positive for pi-1 and pi-2a genes, respectively. The presence of pi-2b was mainly associated with serotype III/CC17 strains; 56.7% of isolates carried tetM, tetO/tetL, ermB antibiotic resistant genes. Among all the virulence genes detected, the cfb-cylE-lmb-pavA pattern was the main virulence gene profile (81.4%), mainly in serotype III/CC17. Conclusions: The whole genomic sequencing data revealed the high variation in surface proteins, determining virulence and antibiotic resistance in clinical isolates from 97 GBS infected infants. These data provide insightful characteristics of genetic features of GBS. Constant epidemiological surveillance is warranted to provide information on the GBS pathogenic dynamics and antibiotic resistance profiles in the surveyed areas for improving therapeutic outcomes.