Within-patient plasmid dynamics in Klebsiella pneumoniae during an outbreak of a carbapenemase-producing Klebsiella pneumoniae

INTRODUCTION: Knowledge of within-patient dynamics of resistance plasmids during outbreaks is important for understanding the persistence and transmission of plasmid-mediated antimicrobial resistance. During an outbreak of a Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae, the plasmid and chromosomal dynamics of K. pneumoniae within-patients were investigated. METHODS: During the outbreak, all K. pneumoniae isolates of colonized or infected patients were collected, regardless of their susceptibility pattern. A selection of isolates was short-read and long-read sequenced. A hybrid assembly of the short-and long-read sequence data was performed. Plasmid contigs were extracted from the hybrid assembly, annotated, and within patient plasmid comparisons were performed. RESULTS: Fifteen K. pneumoniae isolates of six patients were short-read whole-genome sequenced. Whole-genome multi-locus sequence typing revealed a maximum of 4 allele differences between the sequenced isolates. Within patients 1 and 2 the resistance gene- and plasmid replicon-content did differ between the isolates sequenced. Long-read sequencing and hybrid assembly of 4 isolates revealed loss of the entire KPC-gene containing plasmid in the isolates of patient 2 and a recombination event between the plasmids in the isolates of patient 1. This resulted in two different KPC-gene containing plasmids being simultaneously present during the outbreak. CONCLUSION: During a hospital outbreak of a KPC-producing K. pneumoniae isolate, plasmid loss of the KPC-gene carrying plasmid and plasmid recombination was detected within the isolates from two patients. When investigating outbreaks, one should be aware that plasmid transmission can occur and the possibility of within- and between-patient plasmid variation needs to be considered

Prevalence of Livestock-Associated MRSA in Communities with High Pig-Densities in The Netherlands

Contains fulltext : 124345.pdf (publisher's version ) (Open Access)BACKGROUND: Recently, livestock-associated methicillin-resistant Staphylococcus aureus CC398 has been discovered in animals, livestock farmers and retail meat. This cross-sectional study aimed to determine the spread to persons not in direct contact with livestock in areas with a high density of pig farms. METHODOLOGY/PRINCIPAL FINDINGS: With a random mailing in 3 selected municipalities in The Netherlands, adult persons were asked to fill in a questionnaire and to take a nose swab. In total, complete information was obtained on 583 persons. Of the 534 persons without livestock-contact, one was positive for MRSA (0.2%; 95% confidence interval, <0.01-1.2). Of the 49 persons who did indicate to be working at or living on a livestock farm, 13 were positive for MRSA (26.5%; 95% confidence interval, 16.1-40.4). All spa-types belonged to CC398. CONCLUSIONS/SIGNIFICANCE: Livestock-associated MRSA has a high prevalence in people with direct contact with animals. At this moment it has not spread from the farms into the community

Viral and bacterial aetiology of community-acquired pneumonia in adults

BACKGROUND: Modern molecular techniques reveal new information on the role of respiratory viruses in community-acquired pneumonia. In this study, we tried to determine the prevalence of respiratory viruses and bacteria in patients with community-acquired pneumonia who were admitted to the hospital. METHODS: Between April 2008 and April 2009, 408 adult patients (aged between 20 and 94 years) with community-acquired pneumonia were tested for the presence of respiratory pathogens using bacterial cultures, real-time PCR for viruses and bacteria, urinary antigen testing for Legionella and Pneumococci and serology for the presence of viral and bacterial pathogens. RESULTS: Pathogens were identified in 263 (64·5%) of the 408 patients. The most common single organisms in these 263 patients were Streptococcus pneumoniae (22·8%), Coxiella burnetii (6·8%) and influenza A virus (3·8%). Of the 263 patients detected with pathogens, 117 (44·5%) patients were positive for one or more viral pathogens. Of these 117 patients, 52 (44·4%) had no bacterial pathogen. Multiple virus infections (≥2) were found in 16 patients. CONCLUSION: In conclusion, respiratory viruses are frequently found in patients with CAP and may therefore play an important role in the aetiology of this disease

Recent Emergence of Staphylococcus aureus Clonal Complex 398 in Human Blood Cultures

Background: Recently, a clone of MRSA with clonal complex 398 (CC398) has emerged that is related to an extensive reservoir in animals, especially pigs and veal calves. It has been reported previously that methicillin-susceptible variants of CC398 circulate among humans at low frequency, and these have been isolated in a few cases of bloodstream infections (BSI). The purpose of this study was to determine the prevalence of S. aureus CC398 in blood cultures taken from patients in a geographic area with a high density of pigs. Methodology/Principal Findings: In total, 612 consecutive episodes of S. aureus BSI diagnosed before and during the emergence of CC398 were included. Three strains (2 MSSA and 1 MRSA) that were isolated from bacteremic patients between 2010-2011 were positive in a CC398 specific PCR. There was a marked increase in prevalence of S. aureus CC398 BSI isolated between 2010-2011 compared to the combined collections that were isolated between 1996-1998 and 2002-2005 (3/157, 1.9% vs. 0/455, 0.0%; p = 0.017). Conclusions/Significance: In conclusion, in an area with a relative high density of pigs, S. aureus CC398 was found as a cause of BSI in humans only recently. This indicates that S. aureus CC398 is able to cause invasive infections in humans and that the preva

Prevalence and Clinical Presentation of Health Care Workers With Symptoms of Coronavirus Disease 2019 in 2 Dutch Hospitals During an Early Phase of the Pandemic

Importance: On February 27, 2020, the first patient with coronavirus disease 2019 (COVID-19) was reported in the Netherlands. During the following weeks, at 2 Dutch teaching hospitals, 9 health care workers (HCWs) received a diagnosis of COVID-19, 8 of whom had no history of travel to China or northern Italy, raising the question of whether undetected community circulation was occurring. Objective: To determine the prevalence and clinical presentation of COVID-19 among HCWs with self-reported fever or respiratory symptoms. Design, Setting, and Participants: This cross-sectional study was performed in 2 teaching hospitals in the southern part of the Netherlands in March 2020, during the early phase of the COVID-19 pandemic. Health care workers employed in the participating hospitals who experienced fever or respiratory symptoms were asked to voluntarily participate in a screening for infection with the severe acute respiratory syndrome coronavirus 2. Data analysis was performed in March 2020. Main Outcomes and Measures: The prevalence of severe acute respiratory syndrome coronavirus 2 infection was determined by semiquantitative real-time reverse transcriptase-polymerase chain reaction on oropharyngeal samples. Structured interviews were conducted to document symptoms for all HCWs with confirmed COVID-19. Results: Of 9705 HCWs employed (1722 male [18%]), 1353 (14%) reported fever or respiratory symptoms and were tested. Of those, 86 HCWs (6%) were infected with severe acute respiratory syndrome coronavirus 2 (median age, 49 years [range, 22-66 years]; 15 [17%] male), representing 1% of all HCWs employed. Most HCWs experienced mild disease, and only 46 (53%) reported fever. Eighty HCWs (93%) met a case definition of fever and/or coughing and/or shortness of breath. Only 3 (3%) of the HCWs identified through the screening had a history of travel to China or northern Italy, and 3 (3%) reported having been exposed to an inpatient with a known diagnosis of COVID-19 before the onset of symptoms. Conclusions and Relevance: Within 2 weeks after the first Dutch case was detected, a substantial proportion of HCWs with self-reported fever or respiratory symptoms were infected with severe acute respiratory syndrome coronavirus 2, likely as a result of acquisition of the virus in the community during the early phase of local spread. The high prevalence of mild clinical presentations, frequently not including fever, suggests that the currently recommended case definition for suspected COVID-19 should be used less stringently

The Population Genetics of Pseudomonas aeruginosa Isolates from Different Patient Populations Exhibits High-Level Host Specificity

Objective To determine whether highly prevalent P. aeruginosa sequence types (ST) in Dutch cystic fibrosis (CF) patients are specifically linked to CF patients we investigated the population structure of P. aeruginosa from different clinical backgrounds. We first selected the optimal genotyping method by comparing pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and multilocus variable number tandem-repeat analysis (MLVA). Methods Selected P. aeruginosa isolates (n = 60) were genotyped with PFGE, MLST and MLVA to determine the diversity index (DI) and congruence (adjusted Rand and Wallace coefficients). Subsequently, isolates from patients admitted to two different ICUs (n = 205), from CF patients (n = 100) and from non-ICU, non-CF patients (n = 58, of which 19 were community acquired) were genotyped with MLVA to determine distribution of genotypes and genetic diversity. Results Congruence between the typing methods was >79% and DIs were similar and all >0.963. Based on costs, ease, speed and possibilities to compare results between labs an adapted MLVA scheme called MLVA9-Utrecht was selected as the preferred typing method. In 363 clinical isolates 252 different MLVA types (MTs) were identified, indicating a highly diverse population (DI = 0.995; CI = 0.993–0.997). DI levels were similarly high in the diverse clinical sources (all >0.981) and only eight genotypes were shared. MTs were highly specific (>80%) for the different patient populations, even for similar patient groups (ICU patients) in two distinct geographic regions, with only three of 142 ICU genotypes detected in both ICUs. The two major CF clones were unique to CF patients. Conclusion The population structure of P. aeruginosa isolates is highly diverse and population specific without evidence for a core lineage in which major CF, hospital or community clones co-cluster. The two genotypes highly prevalent among Dutch CF patients appeared unique to CF patients, suggesting specific adaptation of these clones to the CF lung

COVID-19 in health-care workers in three hospitals in the south of the Netherlands:A cross-sectional study

Background: 10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers. Methods: We did a cross-sectional study at three of the nine hospitals located in the south of the Netherlands. We screened health-care workers at the participating hospitals for SARS-CoV-2 infection, based on clinical symptoms (fever or mild respiratory symptoms) in the 10 days before screening. We obtained epidemiological data through structured interviews with health-care workers and combined this information with data from whole-genome sequencing of SARS-CoV-2 in clinical samples taken from health-care workers and patients. We did an in-depth analysis of sources and modes of transmission of SARS-CoV-2 in health-care workers and patients. Findings: Between March 2 and March 12, 2020, 1796 (15%) of 12 022 health-care workers were screened, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete and near-complete genome sequences from 50 health-care workers and ten patients. Most sequences were grouped in three clusters, with two clusters showing local circulation within the region. The noted patterns were consistent with multiple introductions into the hospitals through community-acquired infections and local amplification in the community. Interpretation: Although direct transmission in the hospitals cannot be ruled out, our data do not support widespread nosocomial transmission as the source of infection in patients or health-care workers. Funding: EU Horizon 2020 (RECoVer, VEO, and the European Joint Programme One Health METASTAVA), and the National Institute of Allergy and Infectious Diseases, National Institutes of Health

National laboratory-based surveillance system for antimicrobial resistance: a successful tool to support the control of antimicrobial resistance in the Netherlands

An important cornerstone in the control of antimicrobial resistance (AMR) is a well-designed quantitative system for the surveillance of spread and temporal trends in AMR. Since 2008, the Dutch national AMR surveillance system, based on routine data from medical microbiological laboratories (MMLs), has developed into a successful tool to support the control of AMR in the Netherlands. It provides background information for policy making in public health and healthcare services, supports development of empirical antibiotic therapy guidelines and facilitates in-depth research. In addition, participation of the MMLs in the national AMR surveillance network has contributed to sharing of knowledge and quality improvement. A future improvement will be the implementation of a new semantic standard together with standardised data transfer, which will reduce errors in data handling and enable a more real-time surveillance. Furthermore, the

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

Objectives: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. Methods: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). Results: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. Conclusions: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem

Outbreak of Infection with a Multiresistant Klebsiella pneumoniae Strain Associated with Contaminated Roll Boards in Operating Rooms

An outbreak with a multiresistant Klebsiella pneumoniae (MRKP) strain among seven patients admitted to the adult intensive care unit (ICU) of a regional teaching hospital in The Netherlands was investigated. Epidemiologic investigations revealed a short delay between an operation and the acquisition of the MRKP strain. A case-control study comprising 7 cases and 14 controls was conducted to identify the risk factors associated with the acquisition of the MRKP strain. An operation at each of two operation rooms was strongly associated with the acquisition of the MRKP strain: odds ratio of 36 (95% confidence interval, 2.7 to 481.2; P = 0.003, Fisher exact two-tailed test). Cultures of environmental specimens of the operation rooms revealed contamination of the roll boards used to transport patients from the bed to the operation table with the MRKP strains. Molecular genotyping of the isolates revealed clonal similarity between the isolates of the seven cases, isolates from environmental specimen cultures, and in addition, an MRKP isolate from a repatriated ICU patient from earlier that year. The outbreak ended after cleaning and replacement of the roll boards in the operation rooms and implementation of additional barrier precautions for colonized or infected patients. It was concluded that two operation rooms played a significant role in the transmission of an MRKP strain between ICU patients during the presented outbreak