Viral genome sequencing to decipher in-hospital SARS-CoV-2 transmission events.

The SARS-CoV-2 pandemic has highlighted the need to better define in-hospital transmissions, a need that extends to all other common infectious diseases encountered in clinical settings. To evaluate how whole viral genome sequencing can contribute to deciphering nosocomial SARS-CoV-2 transmission 926 SARS-CoV-2 viral genomes from 622 staff members and patients were collected between February 2020 and January 2021 at a university hospital in Munich, Germany, and analysed along with the place of work, duration of hospital stay, and ward transfers. Bioinformatically defined transmission clusters inferred from viral genome sequencing were compared to those inferred from interview-based contact tracing. An additional dataset collected at the same time at another university hospital in the same city was used to account for multiple independent introductions. Clustering analysis of 619 viral genomes generated 19 clusters ranging from 3 to 31 individuals. Sequencing-based transmission clusters showed little overlap with those based on contact tracing data. The viral genomes were significantly more closely related to each other than comparable genomes collected simultaneously at other hospitals in the same city (n = 829), suggesting nosocomial transmission. Longitudinal sampling from individual patients suggested possible cross-infection events during the hospital stay in 19.2% of individuals (14 of 73 individuals). Clustering analysis of SARS-CoV-2 whole genome sequences can reveal cryptic transmission events missed by classical, interview-based contact tracing, helping to decipher in-hospital transmissions. These results, in line with other studies, advocate for viral genome sequencing as a pathogen transmission surveillance tool in hospitals.

Extensive Expression of the Virulome Related to Antibiotic Genotyping in Nosocomial Strains of Klebsiella pneumoniae.

The emergence of hyper-virulent and multidrug-resistant (MDR) strains of Klebsiella pneumoniae isolated from patients with hospital- and community-acquired infections is a serious health problem that increases mortality. The molecular analysis of virulome expression related to antimicrobial-resistant genotype and infection type in K. pneumoniae strains isolated from patients with hospital- and community-acquired infections has been poorly studied. In this study, we analyzed the overall expression of the virulence genotype associated with the antimicrobial resistance genotype and pulse field gel electrophoresis (PFGE) type (PFtype) in K. pneumoniae. We studied 25 strains of K. pneumoniae isolated from patients who developed bacteremia and pneumonia during their hospital stay and 125 strains from outpatients who acquired community-acquired infections. Susceptibility to 12 antimicrobials was determined by Kirby-Bauer. The identification of K. pneumoniae and antibiotic-resistance genes was performed using polymerase chain reaction (PCR). To promote the expression of the virulence genes of K. pneumoniae, an in vitro infection model was used in human epithelial cell lines A549 and A431. Bacterial RNA was extracted with the QIAcube robotic workstation, and reverse transcription to cDNA was performed with the Reverse Transcription QuantiTect kit (Qiagen). The determination of the expression of the virulence genes was performed by real-time PCR. In addition, 57.3% (n = 86) of the strains isolated from patients with hospital- and community-acquired infections were multidrug-resistant (MDR), mainly to beta-lactam antibiotics (CB, AM, CFX, and CF), aminoglycosides (GE), quinolones (CPF and NOF), nitrofurantoin (NF), and sulfamethoxazole/trimethoprim (SXT). The most frequently expressed genes among strains isolated from hospital- and community-acquired infections were adhesion-type, ycfm (80%), mrkD (51.3%), and fimH (30.7%); iron uptake, irp2 (84%), fyuA (68.7%), entB (64.7%), and irp1 (56.7%); and protectins, rpmA (26%), which were related to antibiotic-resistance genes, blaTEM (96%), blaSHV (64%), blaCITM (52.6%), blaCTXM-1 (44.7%), tetA (74%), sul1 (57.3%), aac(3)-IV (40.7%), and aadA1 (36%). The results showed the existence of different patterns of expression of virulome related to the genotype of resistance to antimicrobials and to the PFtypes in the strains of K. pneumoniae that cause hospital- and community-acquired infections. These findings are important and may contribute to improving medical treatment strategies against infections caused by K. pneumoniae.

Isolation and characterization of a novel lytic bacteriophage vB_Efm_LG62 infecting Enterococcus faecium.

Enterococcus faecium has been classified as a "high priority" pathogen by the World Health Organization. Enterococcus faecium has rapidly evolved as a global nosocomial pathogen with adaptation to the nosocomial environment and the accumulation of resistance to multiple antibiotics. Phage therapy is considered a promising strategy against difficult-to-treat infections and antimicrobial resistance. In this study, we isolated and characterized a novel virulent bacteriophage, vB_Efm_LG62, that specifically infects multidrug-resistant E. faecium. Morphological observations suggested that the phage has siphovirus morphology, with an optimal multiplicity of infection of 0.001. One-step growth tests revealed that its latent growth was at 20 min, with a burst size of 101 PFU/cell. Phage vB_Efm_LG62 was verified to have a double-stranded genome of 42,236 bp (35.21% GC content), containing 66 predicted coding sequences as determined by whole genomic sequencing. No genes were predicted to have functions associated with virulence factors or antibiotic resistance, indicating that the phage vB_Efm_LG62 has good therapeutic potential. Our isolation and characterization of this highly efficient phage aids in expanding our knowledge of E. faecium-targeting phages, and provides additional options for phage cocktail therapy.

Detailed analysis of in-hospital transmission of SARS-CoV-2 using whole genome sequencing.

BACKGROUND: Hospital transmission of SARS-CoV-2 has proved difficult to control, with healthcare-associated infections troublesome throughout. AIM: To understand factors contributing to hospital transmission of infections, which is necessary for containing spread. METHODS: An outbreak of 56 staff and patient cases of COVID-19 over a 31-day period in a tertiary referral unit is presented, with at least a further 29 cases identified outside of the unit and the hospital by whole genome sequencing (WGS). FINDINGS: Transmission is documented from staff to staff, staff to patients, and patients to staff, showing disruption of a tertiary referral service, despite implementation of nationally recommended control measures, superior ventilation, and use of personal protective equipment. There was extensive spread from the index case, despite this patient spending only 10 h bed bound on the ward in strict cubicle isolation and with an initial single target low level (CT = 32) polymerase chain reaction test. CONCLUSION: This investigation highlights how effectively and rapidly SARS-CoV-2 can spread in certain circumstances. It raises questions about infection control measures in place at the time and calls into question the premise that transmissibility can be reliably detected by using lower sensitivity rapid antigen lateral flow tests. We also highlight the value of early intervention in reducing impact as well as the value of WGS in understanding outbreaks.

Comparison of fast Fourier transform infrared spectroscopy biotyping with whole genome sequencing-based genotyping in common nosocomial pathogens.

Early detection of bacterial transmission and outbreaks in hospitals is important because nosocomial infections can result in health complications and longer hospitalization. Current practice to detect outbreaks uses genotyping methods amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS), which are not suitable methods for real-time transmission screening of both susceptible and resistant bacteria. The aim was to assess the typing technique Fourier transform infrared (FTIR) spectroscopy as real-time screening method to discriminate large amounts of susceptible and resistant bacteria at strain level when there is no evident outbreak in comparison with the WGS reference. Isolates of past hospital outbreak strains of Acinetobacter baumannii/calcoaceticus complex (n = 25), Escherichia coli (n = 31), Enterococcus faecium (n = 22), Staphylococcus aureus (n = 37) and Pseudomonas aeruginosa (n = 30) were used for validation of FTIR. Subsequently, Enterococcus faecalis (n = 106) and Enterococcus faecium (n = 104) isolates from weekly routine screening samples when no potential outbreak was present were analysed. FTIR showed reproducibility and congruence of cluster composition with WGS for A. baumannii/calcoaceticus complex and E. faecium outbreak isolates. The FTIR results of E. faecalis and E. faecium isolates from routine samples showed reproducibility, but the congruence of cluster composition with WGS was low. For A. baumannii/calcoaceticus complex and E. faecium outbreak isolates, FTIR appears to be a discriminatory typing tool. However, our study shows the discriminatory power is too low to screen real-time for transmission of E. faecium and E. faecalis at patient wards based on isolates acquired in routine surveillance cultures when there is no clear suspicion of an ongoing outbreak.

Whole genome sequence of pan drug-resistant clinical isolate of Acinetobacter baumannii ST1890.

Acinetobacter baumannii is an opportunistic gram-negative bacteria typically attributed to hospital-associated infection. It could also become multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) during a short period. Although A. baumannii has been documented extensively, complete knowledge on the antibiotic-resistant mechanisms and virulence factors responsible for pathogenesis has not been entirely elucidated. This study investigated the drug resistance pattern and characterized the genomic sequence by de novo assembly of PDR A. baumannii strain VJR422, which was isolated from a catheter-sputum specimen. The results showed that the VJR422 strain was resistant to any existing antibiotics. Based on de novo assembly, whole-genome sequences showed a total genome size of 3,924,675-bp. In silico and conventional MLST analysis of sequence type (ST) of this strain was new ST by Oxford MLST scheme and designated as ST1890. Moreover, we found 10,915 genes that could be classified into 45 categories by Gene Ontology (GO) analysis. There were 1,687 genes mapped to 34 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The statistics from Clusters of Orthologous Genes (COG) annotation identified 3,189 genes of the VJR422 strain. Regarding the existence of virulence factors, a total of 59 virulence factors were identified in the genome of the VJR422 strain by virulence factors of pathogenic bacteria databases (VFDB). The drug-resistant genes were investigated by searching in the Comprehensive Antibiotic Resistance Database (CARD). The strain harbored antibiotic-resistant genes responsible for aminoglycoside, ß-lactam-ring-containing drugs, erythromycin, and streptogramin resistance. We also identified resistance-nodulation-cell division (RND) and the major facilitator superfamily (MFS) associated with the antibiotic efflux pump. Overall, this study focused on A. baumannii strain VJR422 at the genomic level data, i.e., GO, COG, and KEGG. The antibiotic-resistant genotype and phenotype as well as the presence of potential virulence associated factors were investigated.

Salmonella Typhi from Northwest Pakistan: Molecular Strain Typing and Drug Resistance Signature.

Objective: To determine the molecular strain typing and drug resistance pattern of Salmonella enterica serovar Typhi prevalent in Northwest Pakistan. Methodology: A total of 2,138 blood samples of suspected typhoid patients from Northwest Pakistan were collected followed by identification of Salmonella Typhi through biochemical, serological, and species-specific fliC-d gene amplification. These isolates were typed by variable-number tandem repeat (VNTR) profiling and investigated for drug resistance. Results: The overall prevalence of Salmonella Typhi was found to be 8.8% (n = 189). Thirty different VNTR strain types of Salmonella Typhi were detected and the most prevalent strain types were T1 and T4, whereas T27 was less prevalent strain. Among the 189 isolates 175 (92.5%) isolates were multidrug resistant, whereas 12 (5.8%) isolates were extensively drug resistant. Resistance to imipenem in Salmonella Typhi was not observed. Most of the isolates have genes encoding for resistance to fluoroquinolones, including gyrA (n = 164), gyrB (n = 160), parC (n = 164), parE (n = 160), ac(6')-ib-cr (n = 163), qnrS (n = 15), and qnrB (n = 3). Similarly, chloramphinicol (cat; n = 147), azithromycin (msrA; n = 3), and co-trimoxazole (dfrA7; n = 145) resistance genes were detected among Salmonella Typhi isolates. Conclusion: In this study, T1 and T4 type Salmonella Typhi strains were predominantly prevalent in Northwest Pakistan. Antibiotic resistance among Salmonella Typhi isolates were observed. Findings of the study would be helpful to devise an appropriate antibiotic policy to control the emergence of drug-resistant Salmonella Typhi in Pakistan.

Ward-specific clustering of methicillin-resistant Staphylococcus aureus spa-type t037 and t045 in two hospitals in South Africa: 2013 to 2017.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a highly clonal pathogen causing infections in various settings. The aim of this study was to determine if healthcare-associated (HA) MRSA isolates with the same spa-type originating from two geographically distinct hospitals in South Africa were genetically related based on PFGE. Furthermore, a small subset of MRSA isolates were characterised with WGS and then compared to PFGE to determine if PFGE is still a reliable method to define outbreaks and/or transmission chains. METHODS: Staphylococcus aureus isolated from blood cultures (BC) were submitted to the Centre for Healthcare-Associated Infections, Antimicrobial Resistance and Mycoses (CHARM) as part of a laboratory-based surveillance programme (GERMS-SA). The identified HA-MRSA isolates underwent molecular characterisation [Staphylococcal Chromosome Cassette (SCC) mec and spa-typing]. Pulsed-field gel electrophoresis (PFGE) was performed on selected isolates with the same spa-type. Twenty-one MRSA isolates were selected for whole-genome sequencing (WGS) based on spa-type, PFGE clustering, time and place of isolation. RESULTS: Eighteen percent (n = 95/529) and 33% (n = 234/710) of isolates collected, from two public tertiary academic hospitals in the Gauteng (GAU) and the Western Cape (WC) provinces, were identified as MRSA, respectively. The most dominant clone in the GAU hospital was t037-III-MRSA (43.2%; n = 41/95). The most dominant clones in the WC hospital was t037-III-MRSA (23.9%, n = 56/234) and t045-I-MRSA (23.5%, n = 55/234). The GAU-t037-III-MRSA cases and WC-t045-I-MRSA cases occurred in the paediatric patient population, whereas the WC-t037-III-MRSA cases occurred in the adult patient population. A novel spa-type (t19935) was detected in the GAU hospital. PFGE showed that the GAU- and WC-t037-III-MRSA isolates were genetically indistinguishable, as well as most of the WC-t045-I-MRSA isolates. The Vienna/Hungarian/Brazilian clone and British EMRSA-3 clone were in circulation and a low frequency of single nucleotide polymorphisms (SNP) (≤20) differences was observed among isolates with the same spa-type. CONCLUSION: The low number of SNP differences is suggestive of uninterrupted strain transmission and the persistence of t037-III-MRSA and t045-I-MRSA from 2013 to 2017 in the two studied hospitals. Alternative infection prevention and control strategies should be considered to supplement control efforts.

Multidrug-resistant Klebsiella pneumoniae harboring extended spectrum ß-lactamase encoding genes isolated from human septicemias.

Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of ß-lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZR) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, blaVIM, blaOXA-48, blaCMY-2, blaDHA-1, blaFOX, blaMOX, and blaACC) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6')-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZR K. pneumoniae isolates and 11 of them carried the blaCTX-M-15 gene (together with blaTEM), and the remaining isolate carried the blaSHV-106 gene. All the blaCTX-M-15 harboring isolates also contained a blaSHV gene (blaSHV-1, blaSHV-11 or blaSHV-27 variants). Both blaSHV-27 and blaSHV-106 genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (blaKPC2/3 and blaOXA-48) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.

Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations.

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.

Prevalence of Enterobacteriaceae spp. and its multidrug-resistant rates in clinical isolates: A two-center cross-sectional study.

Enterobacteriaceae spp., owing to their high durability and antibiotic-resistant mechanisms, are described as an eminent part of health treatments in hospital-acquired infections. This study aimed to estimate the prevalence of clinical isolated Enterobacteriaceae spp., and their multidrug-resistant rate in the north of Iran. In this cross-sectional study, over two years (2017-2019), clinical isolates were collected and Enterobacteriaceae spp. were identified using the standard media culture and Analytical Profile Index (API 20E) kit from two centers in the north of Iran. Isolates were confirmed by targeting the rpoB gene. Moreover, the susceptibility patterns of isolates were assessed using disc diffusion methods according to the Clinical Laboratory Standard Institute (CLSI) guidelines. Out of 2645 clinical specimens, 297 (11.2%) were confirmed as Enterobacteriaceae spp. containing Eshershia. coli 93 (31%), Citrobacter freundii 65 (21.9%), Klebsiella pneumoniae 48 (16.2%), Enterobacter spp. 43 (14.5%), and Proteus spp. 23 (7.7%). As much as 8.7% of other spp. Ampicillin (81.1%) and cephalexin (80.9%) have been shown to have the greatest resistant, and nalidixic acid (65%) and amikacin (59.2%) were the most sensitive drugs. Multidrug-resistance (MDR) strains are more isolated in the Burn and Burn intensive care unit (BICU) than other wards. The MDR frequency in Bouali and Zareh hospitals were 65 (49.61%) and 130 (78.31%), respectively. Considering the high isolation rates of MDR Enterobacteriaceae spp., preventive measures need to be taken to remove the mentioned bacteria from hospital wards.

Evaluation of nosocomial infections through contact patterns in a small animal hospital using social network analysis and genotyping techniques.

Nosocomial infections or hospital-acquired infections (HAIs) are common health problems affecting patients in human and animal hospitals. Herein, we hypothesised that HAIs could be spread through human and animal movement, contact with veterinary medical supplies, equipment, or instruments. We used a combination of social network analysis and genotyping techniques to find key players (or key nodes) and spread patterns using Escherichia coli as a marker. This study was implemented in the critical care unit, outpatient department, operation room, and ward of a small animal hospital. We conducted an observational study used for key player determination (or key node identification), then observed the selected key nodes twice with a one-month interval. Next, surface swabs of key nodes and their connecting nodes were analysed using bacterial identification, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, and pulsed-field gel electrophoresis. Altogether, our results showed that veterinarians were key players in this contact network in all departments. We found two predominant similarity clusters; dendrogram results suggested E. coli isolates from different time points and places to be closely related, providing evidence of HAI circulation within and across hospital departments. This study could aid in limiting the spread of HAIs in veterinary and human hospitals.

Microbiological and Clinical Characteristics of Klebsiella pneumoniae Isolates of K57 Capsular Serotype in China.

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

Novel ST1465/CC216 Nosocomial Lineage of Carbapenem-Resistant Acinetobacter baumannii Harboring an Unusual Plasmid Carrying blaNDM-1 Gene.

This study used whole-genome sequencing to analyze the first case of NDM-1-producing Acinetobacter baumannii belonging to the novel sequence type 1465/CC216 recovered in Brazil. The study identified an unusual plasmid carrying blaNDM-1 gene, in which some genes of the Tn125 transposon were lost. Besides, on the chromosome, the strain reported here presented blaOXA-106 gene, a variant of blaOXA-51 gene, and blaADC-25 with ISAba1 upstream. The isolation of new STs of A. baumannii carrying blaNDM-1 genes elicits our concerns about the possible spread of these genes among clinically relevant bacteria.

Outbreak of Multidrug-Resistant Acinetobacter baumannii ST208 Producing OXA-23-Like Carbapenemase in a Children's Hospital in Shanghai, China.

Aims: Acinetobacter baumannii is notorious for acquiring antibiotic resistance and causing nosocomial infections worldwide. This study aimed to investigate the prevalence and molecular characteristics of A. baumannii isolates obtained from inpatients and the intensive care unit (ICU) environment of a pediatric hospital in Shanghai, China. Methods: Between July 2017 and January 2018, a total of 88 A. baumannii isolates, including three obtained from ICU environmental specimens, were characterized by antibiotic susceptibility, multilocus sequence typing, and resistance genes. Results: Carbapenem-resistant A. baumannii (CRAB) isolates, which were resistant to all the antibiotics tested except colistin, accounted for 69.3% (61/88) of all isolates. Three sequence types (STs) were identified among the CRAB isolates, and the predominant clone was ST208 (93.4%, 57/61), which included three environmental isolates and 54 clinical isolates collected from ICU patients. Carbapenem-susceptible isolates, none of which was multidrug resistant (MDR), showed a more diverse genetic background with three known STs and 21 novel STs identified. Intrinsic blaOXA-51-like and blaAmpC were detected in all isolates, while blaOXA-23-like was only detected in all CRAB isolates. ISAba1-blaOXA-23-like, ISAba1-blaOXA-51-like, and ISAba1-blaAmpC were identified in 69.3% (all CRAB isolates), 0%, and 65.9% (58 CRAB isolates) of all isolates, respectively. Conclusions: A nosocomial outbreak of MDR A. baumannii ST208 producing OXA-23-like carbapenemase occurred, highlighting the necessity for strict infection control interventions in the ICU.

Candida parapsilosis Infection: A Multilocus Microsatellite Genotyping-Based Survey Demonstrating an Outbreak in Hospitalized Patients.

Microsatellite analysis identifies specific genotypes and the genetic relationship between strains. Our objective was to analyze the genotypes of C. parapsilosis strains isolated on different wards of a Tertiary-Referral University Center. We evaluated 70 C. parapsilosis strains in total, isolated from samples of patients admitted to five different wards over two years (January 2015-December 2016). Eight microsatellite markers were selected, and two multiplex PCR assays were set up for microsatellite analysis. The 70 strains, examined at eight microsatellite loci, showed 46 different multilocus genotypes profiles. A total of 74 alleles were detected, with an average of 9.25 alleles per locus. The most variable loci were CP6 and CP4, with 20 and 15 alleles, respectively. Four clusters were detected in four out of five wards. A significant cluster that involved 16 patients in the General Surgery department was also found in two patients who had been transferred to the General Medicine ward. Two multiplex PCRs allowed us to minimize costs, define genotypes and study the isolates' genetic diversity with extreme accuracy, demonstrating the high discriminative power of the microsatellite markers. Molecular epidemiology constitutes an appropriate tool for evaluating horizontal transmission of C. parapsilosis in different clinical settings. Microsatellite genotyping and the utilization of Bruvo's genetic distance are suitable for detecting and appraising nosocomial fungal infections.

Emergence of high-risk multidrug-resistant Enterococcus faecalis CC2 (ST181) and CC87 (ST28) causing healthcare-associated infections in India.

High-risk hospital-associated multidrug-resistant (MDR) Enterococcus faecalis clonal complexes (CCs) such as CC2 and CC87 are enriched with virulence determinants that help to accumulate, colonize, and cause serious nosocomial infections. The aim of this study was to establish the epidemiology and clonal composition of 134 clinical E. faecalis isolates and to link molecular typing data with antimicrobial resistance and virulence determinants. All isolates were identified by conventional methods and confirmed by polymerase chain reaction (PCR) (16srRNA gene and ddl genes of E. faecalis/ E. faecium) in 5-years. Disc diffusion test was performed on all strains. We screened all E. faecalis for aac(6')-aph(2″), vanA, and vanB resistance genes, and aggregation substance-asa1, cytolysin-cylA, collagen-binding protein-ace, enterococcal surface protein-esp, gelatinase-gelE, and hyaluronidase-hyl virulence genes by PCR. Representative isolates of E. faecalis were characterized by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Out of 539 patients with enterococcal infections, 134 (24.9%) had E. faecalis infections, 366 (67.9%) had E. faecium infections, and 39 (7.2%) had infections due to other enterococcal species. Of the 134 isolates, 79.1% and 61.9% isolates were high-level gentamicin resistant (HLGR) and MDR. In multivariate analysis, independent predictor for infection due to MDR E. faecalis strains was a surgical intervention (OR 2.41, 95% CI 1.17-4.96, P = 0·017). Overall, the observed rate of in-hospital mortality was 11.9%. The gelE, asa1, ace, cylA, esp and hyl genes were detected in 87.3%, 78.4%, 54.5%, 53.7%, 36.6% and 3.0%, respectively in E. faecalis isolates. The asaI, cylA, and gelE genes were significantly correlated with MDR E. faecalis. The PFGE analysis showed 28 clones with four major clones. MLST analysis revealed two sequence types-ST28 (CC87) and ST181 (CC2). This is the first Indian report on the emergence of the high-risk hospital-associated worldwide-disseminated ST28 (CC87) and ST181 (CC2), which have enriched with multiple virulence determinants and resistance to antibiotics, paticularly ampicillin. This report indicates serious health concern and calls for on-going surveillance, close monitoring, and improved infection control procedures to stop further spread of these isolates.

Genomic analysis of cardiac surgery-associated Mycobacterium chimaera infections in Italy.

One hundred and twenty-two Mycobacterium chimaera strains isolated in Italy from cardiac surgery-related patients, cardiac surgery-unrelated patients and from heater-cooler units, were submitted to whole-genome sequencing and to subsequent SNP analysis. All but one strains isolated from cardiac surgery-related patients belonged to Subgroup 1.1 (19/23) or Subgroup 1.8 (3/23). Only 28 out of 79 strains isolated from heater-cooler units belonged to groupings other than 1.1 and 1.8. The strains isolated from cardiac surgery-unrelated patients were instead distributed across the phylogenetic tree. Our data, the first on isolates from Italy, are in agreement with a recent large genomic study suggesting a common source, represented by strains belonging to Subgroups 1.1 and 1.8, of cardiac surgery-related Mycobacterium chimaera infections. The strains belonging to groupings other than 1.1 and 1.8 isolated from heather-cooler units evidently resulted from contaminations at hospital level and had no share in the Mycobacterium chimaera outbreak. One Mycobacterium chimaera strain investigated in this study proved distant from every previously known Mycobacterium chimaera Groups (1, 2, 3 and 4) and we propose to assign to a novel group, named "Group 5".

Systematic detection of horizontal gene transfer across genera among multidrug-resistant bacteria in a single hospital.

Multidrug-resistant bacteria pose a serious health threat, especially in hospitals. Horizontal gene transfer (HGT) of mobile genetic elements (MGEs) facilitates the spread of antibiotic resistance, virulence, and environmental persistence genes between nosocomial pathogens. We screened the genomes of 2173 bacterial isolates from healthcare-associated infections from a single hospital over 18 months, and identified identical nucleotide regions in bacteria belonging to distinct genera. To further resolve these shared sequences, we performed long-read sequencing on a subset of isolates and generated highly contiguous genomes. We then tracked the appearance of ten different plasmids in all 2173 genomes, and found evidence of plasmid transfer independent from bacterial transmission. Finally, we identified two instances of likely plasmid transfer within individual patients, including one plasmid that likely transferred to a second patient. This work expands our understanding of HGT in healthcare settings, and can inform efforts to limit the spread of drug-resistant pathogens in hospitals.

Bacteria are able to pass each other genes that make them invulnerable to antibiotics. This exchange of genetic material, also called horizontal gene transfer, can turn otherwise harmless bacteria into drug-resistant 'superbugs'. This is particularly problematic in hospitals, where bacteria use horizontal gene transfer to become resistant to several antibiotics and disinfectants at once, leading to serious infections that are difficult to treat. How can scientists stop bacteria from sharing genes with one another? To answer this question, first it is important to understand how horizontal gene transfer happens in the bacteria that cause infections in hospitals. To this end, Evans et al. examined the genomes of over 2000 different bacteria, collected from a hospital over 18 months, for signs of horizontal transfer. First the experiments identified the genetic material that had potentially been transferred between bacteria, also known as 'mobile genetic elements'. Next, Evans et al. examined the data of patients who had been infected with the bacteria carrying these mobile genetic elements to see whether horizontal transfer might have happened in the hospital. By combining genomics with patient data, it was determined that many of the mobile genetic elements identified were likely being shared among hospital bacteria. One of the mobile genetic elements identified was able to provide resistance to several drugs, and appeared to have been horizontally transferred between bacteria infecting two separate patients. The findings of Evans et al. show that the horizontal transfer of mobile genetic elements in hospital settings is likely frequent, but complex and difficult to study with current methods. The results of this study show how these events can now be tracked and analyzed, which may lead to new strategies for controlling the spread of antibiotic resistance.