Dynamics of Staphylococcus aureus in patients and the hospital environment in a tertiary care hospital in the Netherlands.

BACKGROUND: The dynamics of Staphylococcus aureus in patients and the hospital environment are relatively unknown. We studied these dynamics in a tertiary care hospital in the Netherlands. METHODS: Nasal samples were taken from adult patients at admission and discharge. Isolates cultured from clinical samples taken before and during hospitalization from these patients were included. Environmental samples of patient rooms were taken over a three-year period. Finally, isolates from clinical samples from patients with an epidemiological link to S. aureus positive rooms were included. Staphylococcal protein A (spa) typing was performed. RESULTS: Nasal samples were taken from 673 patients. One hundred eighteen (17.5%) were positive at admission and discharge, 15 (2.2%) patients acquired S. aureus during hospitalization. Nineteen patients had a positive clinical sample during hospitalization, 15.9% of the S. aureus were considered as from an exogenous source. One hundred and forty (2.8%) environmental samples were S. aureus positive. No persistent contamination of surfaces was observed. Isolates were highly diverse: spa typing was performed for 893 isolates, identifying 278 different spa types, 161 of these spa types were observed only once. CONCLUSION: Limited transmission could be identified between patients and the hospital environment, and from patient-to-patient. Exogenous acquisition was assumed to occur in 15% of clinical samples. Environmental contamination was infrequent, temporarily, and coincided with the strain from the patient admitted to the room at that time. MRSA was rare and not found in the environment.

Universal screening or a universal risk assessment combined with risk-based screening for multidrug-resistant microorganisms upon admission: Comparing strategies.

OBJECTIVE: Timely identification of patients who carry multidrug-resistant microorganisms (MDRO) is needed to prevent nosocomial spread to other patients and to the hospital environment. We aimed to compare the yield of a universal screening strategy upon admission to the currently installed universal risk assessment combined with risk-based screening upon admission. METHODS: This observational study was conducted within a prospective cohort study. From January 1, 2018, until September 1, 2019, patients admitted to our hospital were asked to participate. Nasal and perianal samples were taken upon admission and checked for the presence of MDRO. The results of the universal risk assessment and risk-based screening were collected retrospectively from electronic health records. RESULTS: In total, 1017 patients with 1069 separate hospital admissions participated in the study. Universal screening identified 38 (3.6%) unknown MDRO carriers upon admission (37 individual patients), all carrying extended-spectrum beta-lactamase-producing Enterobacterales. For 946 of 1069 (88.5%) patients, both the universal risk assessment and universal screening were performed. For 19 (2.0%) admissions, ≥1 risk factor was identified. The universal risk assessment identified one (0.1%) unknown carrier, compared to 37 out of 946 carriers for the universal screening (P<0.001). Of the 37 carriers identified through the universal screening, 35 (94.6%) reported no risk factors. CONCLUSIONS: Our results show that in our low endemic setting, a universal screening strategy identified significantly more MDRO carriers than the currently implemented universal risk-assessment. When implementing a universal risk-assessment, risk factors should be carefully selected to be able to identify ESBL-E carriers. While the universal screening identified more MDRO carriers, further research is needed to determine the cost-effectiveness of this strategy.

Contact tracing for vancomycin-resistant Enterococcus faecium (VRE): evaluation of the Dutch policy of quintuple screening cultures.

Detection of vancomycin-resistant Enterococcus faecium (VRE) is hampered by low sensitivity of rectal swab cultures. This study aimed to define the number of screening cultures needed to increase sensitivity to detect VRE transmission, and to determine time from presumed exposure to detectable colonization. In a tertiary care setting, we retrospectively analyzed data from 9 VRE outbreaks. As a proxy or estimation for time to detectable colonization, the time between first positive culture of the presumed index patient and that of their contacts was determined. Only 64% of secondary cases were positive in the first out of five cultures. By using the first three out of five rectal swabs, 89% (95%CI: 78-95%) of all secondary cases would have been identified. The median number of days between the positive culture of the index patient and the first positive culture of secondary cases was 9 days. Eleven percent of secondary cases would have been missed if only three rectal samples would have been obtained. Furthermore, our results show that one or more rectal swabs taken around day 9 after presumed exposure should at least be included in the screening approach. In our setting, obtaining a fourth and a fifth rectal swab showed a relevant additional value compared to only one to three swabs. Our findings are useful for determining the most effective VRE contact tracing approach to prevent transmission.

Clinical Impact of Polymerase Chain Reaction-Based Aspergillus and Azole Resistance Detection in Invasive Aspergillosis: A Prospective Multicenter Study.

BACKGROUND: Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. METHODS: In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. RESULTS: Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). CONCLUSIONS: Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).

Limiting spread of VIM-positive Pseudomonas aeruginosa from colonized sink drains in a tertiary care hospital: A before-and-after study.

BACKGROUND: In healthcare environments, sinks are being increasingly recognized as reservoirs for multidrug-resistant Gram-negative bacteria. In our hospital, carbapenemase-producing, Verona Integron-encoded Metallo-beta-lactamase (VIM)-positive Pseudomonas aeruginosa (VIM-PA) was detected at low endemicity in patients, and environmental culturing revealed that sink drains were primary reservoirs. Therefore, an intervention was initiated in several wards to install sink drain plugs as physical barriers against splashing to prevent transmission of VIM-PA from drain reservoirs to the surrounding sink environment. AIM: To assess the efficacy of the intervention on limiting spread of VIM-PA. METHODS: Swabs were taken from inner sink environments (i.e. drains), and outer sink environments (i.e. wash basins, faucet aerators, and countertops) twice before and three times after the intervention. Siphon water and drain wells were also sampled before and at the moment of the intervention, respectively. All samples were screened for VIM-PA, and isolates were typed with multiple-locus variable-number tandem repeat analysis (MLVA). RESULTS: There was a significant reduction in VIM-PA positivity in both inner (P-value <0.001) and outer (P-value 0.001) sink environments after the intervention. However, VIM-PA recolonization was observed in the inner sink environments of patient rooms, and also in rooms exclusive to healthcare personnel, over time. Surfaces in the outer sink environment were rarely positive for VIM-PA after the intervention. MLVA revealed three genetic clusters, with one found in all wards and room types during the study period. CONCLUSIONS: Drain plugs are a simple and effective infection prevention and control measure to contain spread of VIM-PA from drain reservoirs.

Environmental contamination with highly resistant microorganisms after relocating to a new hospital building with 100% single-occupancy rooms: A prospective observational before-and-after study with a three-year follow-up.

INTRODUCTION: Inanimate surfaces within hospitals can be a source of transmission for highly resistant microorganisms (HRMO). While many hospitals are transitioning to single-occupancy rooms, the effect of single-occupancy rooms on environmental contamination is still unknown. We aimed to determine differences in environmental contamination with HRMO between an old hospital building with mainly multiple-occupancy rooms and a new hospital building with 100% single-occupancy rooms, and the environmental contamination in the new hospital building during three years after relocating. METHODS: Environmental samples were taken twice in the old hospital, and fifteen times over a three-year period in the new hospital. Replicate Organism Direct Agar Contact-plates (RODACs) were used to determine colony forming units (CFU). Cotton swabs premoistened with PBS were used to determine presence of methicillin-resistant Staphylococcus aureus, carbapenemase-producing Pseudomonas aeruginosa, highly resistant Enterobacterales, carbapenem-resistant Acinetobacter baumannii, and vancomycin-resistant Enterococcus faecium. All identified isolates were subjected to whole genome sequencing (WGS) using Illumina technology. RESULTS: In total, 4993 hospital sites were sampled, 724 in the old and 4269 in the new hospital. CFU counts fluctuated during the follow-up period in the new hospital building, with lower CFU counts observed two- and three years after relocating, which was during the COVID-19 pandemic. The CFU counts in the new building were equal to or surpassed the CFU counts in the old hospital building. In the old hospital building, 24 (3.3%) sample sites were positive for 49 HRMO isolates, compared to five (0.1%) sample sites for seven HRMO isolates in the new building (P < 0.001). In the old hospital, 89.8% of HRMO were identified from the sink plug. In the new hospital, 71.4% of HRMO were identified from the shower drain, and no HRMO were found in sinks. DISCUSSION: Our results indicate that relocating to a new hospital building with 100% single-occupancy rooms significantly decreases HRMO in the environment. Given that environmental contamination is an important source for healthcare associated infections, this finding should be taken into account when considering hospital designs for renovations or the construction of hospitals.

Whole Genome Multi-Locus Sequence Typing and Genomic Single Nucleotide Polymorphism Analysis for Epidemiological Typing of Pseudomonas aeruginosa From Indonesian Intensive Care Units.

We have previously studied carbapenem non-susceptible Pseudomonas aeruginosa (CNPA) strains from intensive care units (ICUs) in a referral hospital in Jakarta, Indonesia (Pelegrin et al., 2019). We documented that CNPA transmissions and acquisitions among patients were variable over time and that these were not significantly reduced by a set of infection control measures. Three high risk international CNPA clones (sequence type (ST)235, ST823, ST357) dominated, and carbapenem resistance was due to carbapenemase-encoding genes and mutations in the porin OprD. Pelegrin et al. (2019) reported core genome analysis of these strains. We present a more refined and detailed whole genome-based analysis of major clones represented in the same dataset. As per our knowledge, this is the first study reporting Single Nucleotide Polymorphisms (wgSNP) analysis of Pseudomonas strains. With whole genome-based Multi Locus Sequence Typing (wgMLST) of the 3 CNPA clones (ST235, ST357 and ST823), three to eleven subgroups with up to 200 allelic variants were observed for each of the CNPA clones. Furthermore, we analyzed these CNPA clone clusters for the presence of wgSNP to redefine CNPA transmission events during hospitalization. A maximum number 35350 SNPs (including non-informative wgSNPs) and 398 SNPs (ST-specific_informative-wgSNPs) were found in ST235, 34,570 SNPs (including non-informative wgSNPs) and 111 SNPs (ST-specific_informative-wgSNPs) in ST357 and 26,443 SNPs (including non-informative SNPs) and 61 SNPs (ST-specific_informative-wgSNPs) in ST823. ST-specific_Informative-wgSNPs were commonly noticed in sensor-response regulator genes. However, the majority of non-informative wgSNPs was found in conserved hypothetical proteins or in uncharacterized proteins. Of note, antibiotic resistance and virulence genes segregated according to the wgSNP analyses. A total of 8 transmission chains for ST235 strains followed by 9 and 4 possible transmission chains for ST357 and ST823 were traceable on the basis of pairwise distances of informative-wgSNPs (0 to 4 SNPs) among the strains. The present study demonstrates the value of detailed whole genome sequence analysis for highly refined epidemiological analysis of P. aeruginosa.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect ß-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of ß-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the ß-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, ANT(2 ' ' )-I, and APH(3')-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6')-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.

Madurella mycetomatis grains within a eumycetoma lesion are clonal.

Eumycetoma is a neglected tropical infection of the subcutaneous tissue, characterized by tumor-like lesions and most commonly caused by the fungus Madurella mycetomatis. In the tissue, M. mycetomatis organizes itself in grains, and within a single lesion, thousands of grains can be present. The current hypothesis is that all these grains originate from a single causative agent, however, this hypothesis was never proven. Here, we used our recently developed MmySTR assay, a highly discriminative typing method, to determine the genotypes of multiple grains within a single lesion. Multiple grains from surgical lesions obtained from 11 patients were isolated and genotyped using the MmySTR panel. Within a single lesion, all tested grains shared the same genotype. Only in one single grain from one patient, a difference of one repeat unit in one MmySTR marker was noted relative to the other grains from that patient. We conclude that within these lesions the grains originate from a single clone and that the inherent unstable nature of the microsatellite markers may lead to small genotypic differences. LAY ABSTRACT: In lesions of the implantation mycosis mycetoma many Madurella mycetomatis grains are noted. It was unknown if grains arose after implantation of a single isolate or a mixture of genetically diverse isolates. By typing the mycetoma grains we showed that all grains within a single lesion were clonal and originated from a single isolate.

Pre-COVID-19 international travel and admission to hospital when back home: travel behavior, carriage of highly resistant microorganisms, and risk perception of patients admitted to a large tertiary care hospital.

BACKGROUND: When people who recently travelled abroad are admitted to a hospital back home, there is a risk of introducing highly resistant microorganisms (HRMO) into the hospital. To minimize this risk, a feasible infection prevention strategy should be developed. In this study, we investigated patients' travel history and behavior during travel and analyzed whether this was correlated to HRMO carriage at admission. METHODS: From May 2018 until August 2019, adult patients admitted to a large tertiary care center in the Netherlands were asked upon hospital admission to participate in the study. Included patients received a questionnaire about risk perception, travel history in the last year, and behavior during travel, and were screened for HRMO carriage at admission using a perianal swab. RESULTS: Six hundred and eight questionnaires were handed out, of which 247 were returned (40.6%). One hundred and thirty (52.6%) patients did not travel abroad in the last year, of whom eight (6.2%) were HRMO carrier at admission. One hundred seventeen (47.4%) patients travelled in the preceding year, of whom seven patients (6.0%) were HRMO carrier at admission. Thirty patients (12%) travelled outside of Europe; in this group HRMO prevalence was 13.3% (4 out of 30). The majority of patients (71.3%) were aware that international travel could lead to carriage of HRMO, and an even larger majority (89.5%) would support a screening strategy upon hospital admission in case of a travel history, to minimize the risk of introducing HRMO. CONCLUSIONS: We identified that half of admitted patients to a large tertiary care hospital travelled abroad in the last year, with only a small percentage outside Europe. We discuss several screening strategies and propose a strategy of screening and preemptive isolation of patients who travelled to Asia or Africa in the 2 months before their hospital admission; a strategy that patients would support.

The effect of 100% single-occupancy rooms on acquisition of extended-spectrum beta-lactamase-producing Enterobacterales and intra-hospital patient transfers: a prospective before-and-after study.

BACKGROUND: Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) are a well-known cause of healthcare-associated infections. The implementation of single-occupancy rooms is believed to decrease the spread of ESBL-E. Additionally, implementation of single-occupancy rooms is expected to reduce the need for intra-hospital patient transfers. We studied the impact of a new hospital with 100% single-occupancy rooms on the acquisition of ESBL-E and on intra-hospital patient transfers. METHODS: In 2018, the Erasmus MC University Medical Center moved from an old, 1200-bed hospital with mainly multiple-occupancy rooms, to a newly constructed 522-bed hospital with 100% single-occupancy rooms. Adult patients admitted between January 2018 and September 2019 with an expected hospitalization of ≥ 48 h were asked to participate in this study. Perianal samples were taken at admission and discharge. Patient characteristics and clinical information, including number of intra-hospital patient transfers, were collected from the patients' electronic health records. RESULTS: Five hundred and ninety-seven patients were included, 225 in the old and 372 in the new hospital building. Fifty-one (8.5%) ESBL-E carriers were identified. Thirty-four (66.7%) patients were already positive at admission, of which 23 without recent hospitalization. Twenty patients acquired an ESBL-E, seven (3.1%) in the old and 13 (3.5%) in the new hospital building (P = 0.801). Forty-one (80.4%) carriers were only detected by the active screening performed during this study. Only 10 (19.6%) patients, six before and four during hospitalization, showed ESBL-E in a clinical sample taken on medical indication. Fifty-six (24.9%) patients were transferred to other rooms in the old hospital, compared to 53 (14.2%) in the new hospital building (P = 0.001). Intra-hospital patient transfers were associated with ESBL-E acquisition (OR 3.18, 95%CI 1.27-7.98), with increasing odds when transferred twice or more. CONCLUSION: Transitioning to 100% single-occupancy rooms did not decrease ESBL-E acquisition, but did significantly decrease the number of intra-hospital patient transfers. The latter was associated with lower odds on ESBL-E acquisition. ESBL-E carriers remained largely unidentified through clinical samples. TRIAL REGISTRATION: This study was retrospectively registered in the Dutch National Trial Register on 24-02-2020, with registration number NL8406.

High Prevalence of ST502 Carrying an OXA-24 Carbapenemase gene in Carbapenem-Nonsusceptible Acinetobacter baumannii-calcoaceticus Isolates in Romania.

Background: Acinetobacter baumannii can cause difficult-to-treat infections because it can acquire extensive antimicrobial resistance mechanisms. We aim to describe the antimicrobial resistance pattern and the genetic basis of carbapenem-nonsusceptible A. baumannii isolates in a University Hospital in Romania, a country where multidrug-resistant A. baumannii is widespread. Methods: We collected 104 consecutive meropenem-nonsusceptible A. baumannii isolates from 104 patients (36% female, mean age [SD] of 63 [16] years) between May 2015 and August 2017 from a large tertiary center in Romania. Whole-genome sequencing of representative isolates from amplified fragment length polymorphism clusters was used to determine clonality and resistance patterns. Results: All isolates were resistant to piperacillin/tazobactam, ceftazidime, and ciprofloxacin; 88.5% to gentamicin; and 90.4% to trimethoprim/sulfamethoxazole. In contrast, 79.8% and 99.0% were susceptible to tobramycin and colistin, respectively. The only isolate resistant to colistin had an minimum inhibitory concentration (MIC) of ≥16 mg/L. The blaOXA-24 gene was detected in 79.1% and blaOXA-23 in 20.9% of the isolates. In one isolate, blaOXA-23 was copresent with blaOXA-24. ST502 (Oxford scheme) was the most prevalent sequence type and was exclusively associated with blaOXA-24. Conclusions: ST502 associated with blaOXA-24 was frequently observed in the region where carbapenem-nonsusceptible A. baumannii was found to be endemic. In these isolates, tobramycin and colistin might be the remaining therapeutic options. Due to differences in gentamicin and tobramycin resistance in these isolates, surveillance data should not group gentamicin, tobramycin, and amikacin together as aminoglycosides.

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 ß-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem.

While Extended-Spectrum ß-Lactamases (ESBL) and AmpC ß-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC ß-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC ß-lactamase, and the plasmid-mediated CMY-2 ß-lactamase. Bla CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like ß-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli.

Confirmatory testing of Neisseria gonorrhoeae in a sexual health clinic: implications for epidemiology and treatment policy.

OBJECTIVES: European guidelines advise the use of dual nucleic acid amplification tests (NAAT) in order to minimise the inappropriate diagnosis of Neisseria gonorrhoeae (Ng) in urogenital samples from low prevalence areas and in extragenital specimens. In this cross-sectional study, we investigated the effect of confirmatory testing and confirmation policy on the Ng-positivity in a population visiting the sexual health clinic in Rotterdam, the Netherlands. METHODS: Apart from urogenital testing, extragenital (oropharyngeal/anorectal) testing was performed for men who have sex with men (MSM) and according to sexual exposure for women and heterosexual men. Ng detection using NAAT was performed using BD Viper and for confirmatory testing BD MAX. Sexual transmitted infection consultation data were merged with diagnostic data from August 2015 through May 2016. RESULTS: In women (n=4175), oral testing was performed in 84% and 22% were tested anally. In MSM (n=1828), these percentages were 97% and 96%, respectively. Heterosexual men (n=3089) were tested urogenitally. After confirmatory testing, oropharyngeal positivity rates decreased from 7.3% (95% CI 6.5 to 8.2) to 1.5% (95% CI 1.1 to 1.8) in women and from 13.9% (95% CI 12.3 to 15.5) to 5.4% (95% CI 4.3 to 6.4) in MSM. Anorectal positivity rates decreased from 2.6% (95% CI 1.6 to 3.7) to 1.8% (95% CI 0.9 to 2.6) in women and from 9.3% (95% CI 7.9 to 10.7) to 7.2% (95% CI 6.0 to 8.5) in MSM. Urogenital Ng-positivity rate ranged between 3.0% and 4.4% and after confirmation between 2.3% and 3.9%. When confirming oropharyngeal samples, Ng-positivity was 3.8% in women, 3.0% in heterosexual men and 12.5% in MSM. Additional confirmation of urogenital and anorectal samples led to 3.0% Ng positivity in women, 2.7% in heterosexual men and 11.4% in MSM. CONCLUSIONS: Confirmation of urogenital and anorectal samples reduced the Ng-positivity rates, especially for women. However, as there is no gold standard for the confirmation of Ng infection, the dilemma within public health settings is to choose between two evils: missing diagnoses or overtreatment. In view of the large decrease in oropharyngeal positivity, confirmation Ng-positivity in oropharyngeal samples remains essential to avoid unnecessary treatment.

National surveillance pilot study unveils a multicenter, clonal outbreak of VIM-2-producing Pseudomonas aeruginosa ST111 in the Netherlands between 2015 and 2017.

Verona Integron-encoded Metallo-beta-lactamase (VIM) is the most frequently-encountered carbapenemase in the healthcare-related pathogen Pseudomonas aeruginosa. In the Netherlands, a low-endemic country for antibiotic-resistant bacteria, no national surveillance data on the prevalence of carbapenemase-producing P. aeruginosa (CPPA) was available. Therefore, in 2016, a national surveillance pilot study was initiated to investigate the occurrence, molecular epidemiology, genetic characterization, and resistomes of CPPA among P. aeruginosa isolates submitted by medical microbiology laboratories (MMLs) throughout the country. From 1221 isolates included in the study, 124 (10%) produced carbapenemase (CIM-positive); of these, the majority (95, 77%) were positive for the blaVIM gene using PCR. Sequencing was performed on 112 CIM-positive and 56 CIM-negative isolates (n = 168), and genetic clustering revealed that 75/168 (45%) isolates were highly similar. This genetic cluster, designated Group 1, comprised isolates that belonged to high-risk sequence type ST111/serotype O12, had similar resistomes, and all but two carried the blaVIM-2 allele on an identical class 1 integron. Additionally, Group 1 isolates originated from around the country (i.e. seven provinces) and from multiple MMLs. In conclusion, the Netherlands had experienced a nationwide, inter-institutional, clonal outbreak of VIM-2-producing P. aeruginosa for at least three years, which this pilot study was crucial in identifying. A structured, national surveillance program is strongly advised to monitor the spread of Group 1 CPPA, to identify emerging clones/carbapenemase genes, and to detect transmission in and especially between hospitals in order to control current and future outbreaks.

Characterization of Staphylococcus roterodami sp. nov., a new species within the Staphylococcus aureus complex isolated from a human foot infection.

This article introduces a new Staphylococcus species cultivated from a human foot wound infection in a Dutch traveller returning from the island of Bali, Indonesia: Staphylococcus roterodami sp. nov. Based on the genomic sequence, there is strong molecular evidence for assigning the strain to a novel species within the S. aureus complex. Differences in cellular fatty acid spectrum and biochemical tests underline these findings. Its ecological niche and pathogenicity require further study. The type strain is DSM111914T (JCM34415T).

Genetic Analysis of mcr-1-Carrying Plasmids From Gram-Negative Bacteria in a Dutch Tertiary Care Hospital: Evidence for Intrapatient and Interspecies Transmission Events.

The role of plasmids in the complex pandemic of antimicrobial resistance is increasingly being recognized. In this respect, multiple mobile colistin resistance (mcr) gene-carrying plasmids have been described. However, the characteristics and epidemiology of these plasmids within local healthcare settings are largely unknown. We retrospectively characterized the genetic composition and epidemiology of plasmids from mcr-1-positive bacterial isolates identified from patients from a large academic hospital in the Netherlands. Clinical Gram-negative bacteria with an MIC > 2 µg/mL for colistin, obtained from patients hospitalized at the Erasmus MC University Medical Center Rotterdam during the years 2010-2018, were screened for presence of the mcr-1 gene. Extracted plasmids from mcr-1-positive isolates were sequenced using a combination of short- and long-read sequencing platforms, characterized by incompatibility type and genetic composition and compared to publicly available mcr-1-carrying plasmid sequences. In 21 isolates from 14 patients, mcr-1 was located on a plasmid. These plasmids were of diverse genetic background involving Inc types IncX4, IncI2(delta), IncHI2, as well as double Inc types IncHI2/IncN and IncHI2/IncQ. mcr-1-carrying plasmids were found in Escherichia coli, Klebsiella pneumoniae, and Kluyvera georgiana, and within the chromosome of an ST147 K. pneumoniae isolate. In depth analysis indicated intrapatient, interpatient, and interspecies transmission events of mcr-1-carrying plasmids. In addition, our results show that the mcr-1 gene resides in a rich environment full of other (mcr-1 negative) plasmids and of many different Inc types, enabling interplasmidal transfer events and facilitating widespread dissemination of the mcr-1 gene. Multiple mcr-1-carrying plasmid transmission events had likely occurred among isolates from hospitalized patients. Recognition and identification of plasmid transmission events within hospitals is necessary in order to design and implement effective infection control measures.

Investigation of possible transmission of a susceptible microorganism through a contaminated duodenoscope; a case report.

BACKGROUND: Despite compliance to extensive reprocessing protocols, duodenoscopes have been linked to outbreaks of susceptible and multi-drug resistant organisms (MDRO) due to persistent duodenoscope contamination. Duodenoscope-associated infections (DAIs) based on transmission of susceptible microorganisms are likely to be underreported due to detection bias. CASE PRESENTATION: We describe the retrospective detection of a DAI case caused by a susceptible microorganism which at the time of clinical infection was not recognized as such. During 2017 and 2018, duodenoscopes were cultured on a daily basis due to research activities. While analyzing this data, it was found that a duodenoscope had been contaminated with Enterobacter cloacae complex over a period of 3 months. We checked whether patients treated with this duodenoscope had developed infections and found one patient with an E. cloacae cholangitis 3 months after the ERCP (Endoscopic retrograde cholangiopancreaticography) procedure. The isolates on the duodenoscope and in the patients' blood culture were indistinguishable by amplified fragment length polymorphism (AFLP). By classical multi-locus sequence typing (MLST), both strains were of the same (but novel) sequence type. Application of whole genome MLST showed 93 (out of 3757) allelic differences. CONCLUSION: This case report describes a plausible link between a contaminated duodenoscope and a patient infection with E. cloacae. Transmission of susceptible E. cloacae was highly suspected from AFLP and MLST results; by WGS, 93 allelic differences were found which proves closely related strains. This report shows that DAIs by susceptible microorganisms can be easily missed and therefore its true prevalence remains underscored.

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics.

Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.