Healthcare worker protection against mpox contamination: position paper of the French Society for Hospital Hygiene.

In the context of the recent re-emergence of mpox worldwide, the French Society for Hospital Hygiene (SF2H) performed a literature review of the transmission paths and proposed specific recommendations for healthcare workers (HCWs) caring for patients with suspected or confirmed MPXV. In developed countries, the risk of contamination among HCWs in healthcare facilities seemed to be very low, limited to contamination through needle stick injuries. Two additional contamination cases were reported and not fully explained. Beyond healthcare settings, the analysis of the literature highlighted (i) a main contamination route during sexual intercourse, mainly among men who have sex with men, and (ii) a very low secondary attack rate in other contexts, such as schools or jails. Numerous studies have reported molecular or virus identification on surfaces or in the air surrounding patients, without any association with the low secondary case incidence; moreover, the minimum infectious dose through air or mucosal exposure is still unknown. Owing to the lack of evidence of MPXV respiratory transmission in the healthcare setting, the SF2H recommends the implementation of standard and contact precautions combined with medical/surgical mask use. Owing to the lack of evidence of transcutaneous contamination, the SF2H recommends the use of gloves only if contact with cutaneous lesions or mucous membranes occurs. Regarding the risk of contamination from the environment in healthcare facilities, additional studies must be conducted to investigate this.

Using a stacker crane for sterile storage in the operating theatre: initial environmental microbiological qualification.

BACKGROUND: Adequate storage of sterile surgical devices must prevent contamination and the introduction of microbial contaminants inside the operating room. For functional and economic purposes, stacker cranes (STCs) could replace the traditional sterile storage room (TSSR). STCs are large, multi-stage, computer-assisted systems used to automatically store and retrieve loads from defined locations. However, their microbiological performance has not been evaluated. AIM: As part of the opening of a new building that included an operating theatre, we qualified a new STC and compared its microbiological control performance to that of the previous TSSR. METHODS: From December 2020 to March 2021, 590 environmental specimens (air, N = 56; surfaces, N = 534) were collected and interpreted according to the NF S90-351 French Association for Standardization standards. FINDINGS: Thorough surface disinfection was not sufficient for controlling microbial contamination in the STC. Thus, the initial qualification testing was conducted following an aggressive aerial chemical decontamination of the STC. Despite the lack of a HEPA filtered air system, the overall non-conformity rates were lower in the STC than in the TSSR (8.3% vs 21.4%, P=0.33 for air, respectively, and 9.7% vs 41.7% P<0.001 for surfaces). The air-controlled barrier in front of the loading zone appeared to be sufficient to prevent bacterial contamination. The presence of fungi must be carefully monitored. CONCLUSION: This is the first study supporting the contribution of STCs in saving space and improving the maintenance of sterile surgical device storage and availability under acceptable environmental conditions. Further studies are needed to assess the long-term microbiological contamination inside the STC.

Mortality in Escherichia coli bloodstream infections: antibiotic resistance still does not make it.

BACKGROUND: Escherichia coli bloodstream infections (BSIs) account for high mortality rates (5%-30%). Determinants of death are unclear, especially since the emergence of ESBL producers. OBJECTIVES: To determine the relative weight of host characteristics, bacterial virulence and antibiotic resistance in the outcome of patients suffering from E. coli BSI. METHODS: All consecutive patients suffering from E. coli BSI in seven teaching hospitals around Paris were prospectively included for 10 months. E. coli isolates were sequenced using Illumina NextSeq technology to determine the phylogroup, ST/ST complex (STc), virulence and antimicrobial resistance gene content. Risk factors associated with death at discharge or Day 28 were determined. RESULTS: Overall, 545 patients (mean ± SD age 68.5 ±âŸ16.5 years; 52.5% male) were included. Mean Charlson comorbidity index (CCI) was 5.6 (± 3.1); 19.6% and 12.8% presented with sepsis and septic shock, respectively. Portals of entry were mainly urinary (51.9%), digestive (41.9%) and pulmonary (3.5%); 98/545 isolates (18%) were third-generation cephalosporin resistant (3GC-R), including 86 ESBL producers. In-hospital death (or at Day 28) was 52/545 (9.5%). Factors independently associated with death were a pulmonary portal of entry [adjusted OR (aOR) 6.54, 95% CI 2.23-19.2, P = 0.0006], the iha_17 virulence gene (aOR 4.41, 95% CI 1.23-15.74, P = 0.022), the STc88 (aOR 3.62, 95% CI 1.30-10.09, P = 0.014), healthcare-associated infections (aOR 1.98, 95% CI 1.04-3.76, P = 0.036) and high CCI (aOR 1.14, 95% CI 1.04-1.26, P = 0.006), but not ESBL/3GC-R. CONCLUSIONS: Host factors, portal of entry and bacterial characteristics remain major determinants associated with mortality in E. coli BSIs. Despite a high prevalence of ESBL producers, antibiotic resistance did not impact mortality. (ClinicalTrials.gov identifier: NCT02890901.).

Local outbreak of extended-spectrum ß-lactamase SHV2a-producing Pseudomonas aeruginosa reveals the emergence of a new specific sub-lineage of the international ST235 high-risk clone.

BACKGROUND: Pseudomonas aeruginosa is a major bacterial pathogen responsible for hospital-acquired infections. Although its epidemiology is considered as non-clonal, certain international high-risk multidrug-resistant clones have been recognized. AIM: From the first report of an intra-hospital outbreak due to an SHV2a-producing P. aeruginosa strain, to describe the emergence of a new ST235-specific lineage harbouring this rare extended-spectrum ß-lactamase (ESBL). METHODS: Between May and October 2018, four patients hospitalized in the cardiovascular intensive care unit of a French teaching hospital were infected by a multidrug-resistant P. aeruginosa isolate. Serotype and antimicrobial susceptibility were tested; multi-locus sequence type (MLST), core genome MLST, and resistome were determined through whole genome sequencing. A phylogenetic analysis based on single nucleotide polymorphism was performed using available ST235 genomes. FINDINGS: The four strains were susceptible to colistin, ciprofloxacin, ceftazidime-avibactam, and ceftolozane-tazobactam. blaSHV2a was identified in each genome of this ST235-O11 serotype cluster that showed an identical cgMLST profile (0-2 out of 4162 different alleles). The phylogenic analysis of 162 ST235 genomes showed that only four other strains harboured a blaSHV2a, originating from France and USA, clustering together although being different from the outbreak strains. CONCLUSIONS: Among the ST235 P. aeruginosa strains, a sub-lineage sharing a common genetic background and harbouring the blaSHV2a ESBL seems to emerge from different locations, yielding secondary local outbreaks.

Molecular testing for respiratory pathogens in sickle cell disease adult patients presenting with febrile acute chest syndrome.

BACKGROUND: Differentiating acute chest syndrome (ACS) from community-acquired pneumonia (CAP) is challenging in adults presenting with major sickle cell disease (SCD) (semiological similarity, rare microbiological documentation). We aimed to assess the usefulness of nucleic acid amplification test (NAAT) for respiratory pathogens, in combination with standard bacteriological investigations, in febrile ACS adult patients presenting with major SCD. METHODS: We performed a prospective, monocentric, observational study of 61 SCD adults presenting with febrile ACS from February 2015 to April 2016. Systematic blood, urine, and respiratory specimens were collected, before antibiotic initiation, for culture, urinary antigen tests, serology, and NAAT for respiratory pathogens. RESULTS: A pathogen was detected in 12 febrile ACS (19.7%): four viruses (6.6%) (Rhinovirus; Influenza A/B), seven bacteria (11.4%) (S. aureus, S. pneumoniae, K. pneumoniae, L. pneumophila, M. pneumoniae), one mixed infection (1.6%) (S. aureus and Influenza B). NAAT only detected L. pneumophila in one case (serogroup 2). Apart from a significantly shorter antibiotic therapy duration (6.1 vs. 7.8 days, P=0.045), no difference was observed between undocumented and microbiologically-documented febrile ACS. CONCLUSION: Using NAAT for the detection of respiratory pathogens in adults presenting with SCD slightly improved the microbiological diagnostic of febrile ACS, although respiratory infections are not the main etiological factor.

Pathogen identification by shotgun metagenomics of patients with necrotizing soft-tissue infections.

BACKGROUND: Necrotizing soft-tissue infections (NSTIs) are life threatening, requiring broad-spectrum antibiotics. Their aetiological diagnosis can be limited by poor performance of cultures and administration of antibiotics before surgery. OBJECTIVES: We aimed (i) to compare 16S-targeted metagenomics (TM) and unbiased semiquantitative panmicroorganism DNA- and RNA-based shotgun metagenomics (SM) with cultures, (ii) to identify patients who would best benefit from metagenomics approaches and (iii) to detect the microbial pathogens in surrounding non-necrotic 'healthy' tissues by SM-based methods. METHODS: A prospective observational study was performed to assess the analytical performance of standard cultures, TM and SM on tissues from 34 patients with NSTIs. Pathogen identification obtained with these three methods was compared. RESULTS: Thirty-four necrotic and 10 healthy tissues were collected from 34 patients. The performance of TM was inferior to that of the other methods (P < 0·05), whereas SM performed better than standard culture, although the result was not statistically significant (P = 0·08). SM was significantly more sensitive than TM for the detection of all bacteria (P = 0·02) and more sensitive than standard culture for the detection of anaerobic bacteria (P < 0·01). There was a strong correlation (r = 0·71, Spearman correlation coefficient) between the semiquantitative abundance of bacteria in the culture and the bacteria-to-human sequence ratio in SM. Low amounts of bacterial DNA were found in healthy tissues, suggesting a bacterial continuum between macroscopically 'healthy' and necrotic tissue. CONCLUSIONS: SM showed a significantly better ability to detect a broader range of pathogens than TM and identify strict anaerobes than standard culture. Patients with diabetes with NSTIs appeared to benefit most from SM. Finally, our results suggest a bacterial continuum between macroscopically 'healthy' non-necrotic areas and necrotic tissues. What's already known about this topic? Necrotizing soft-tissue infections (NSTIs) are characterized by rapidly progressive necrosis of subcutaneous tissues and high mortality, despite surgical debridement combined with broad-spectrum antibiotics. The spectrum of potentially involved pathogens is very large, and identification is often limited by the poor performance of standard cultures, which may be impaired by previous antibiotic intake. Metagenomics-based approaches show promise for better identification of the pathogens that cause these infections, but they have not been evaluated in this medical context. What does this study add? Shotgun metagenomics (SM) showed higher sensitivity than 16S rRNA gene sequencing and a better ability than culture to detect anaerobic bacteria. As a result, a significant proportion of infections with bacteria, such as Pasteurella multocida or Clostridium perfringens, were detected only by SM. SM bacterial quantification enabled better detection of low amounts of bacterial DNA from macroscopically 'healthy' tissue, suggesting a subclinical infectious extension. What is the translational message? The high analytical performance of SM shown in this study should allow its future implementation for the diagnosis of necrotizing fasciitis, complementing or replacing routine methods. The large amount of data, including additional information on antimicrobial resistance, virulence profiles and metabolic adaptation of the pathogens, will improve microbiological documentation. Our results will improve our understanding of infectious pathophysiology in the future, leading to potentially better medical care.

Asymptomatic carriage of extensively drug-resistant bacteria (eXDR), a simple way to assess spontaneous clearance.

The duration of eXDR carriage depends on several factors that might be difficult to recover. We aim to assess the duration of eXDR carriage by using a simple to recover parameter: the number of consecutive negative screening. 131 eXDR carriers (51 VRE and 80 CPE) were included. The number of consecutive negative screenings was strongly associated with eXDR clearance. All patients displaying at least three negative screenings over a seven-month period were never screened positive thereafter. Taking into account the number of negative screenings as a part of a case-by-case risk assessment would be helpful for the decision to maintain or lift eXDR-focused precautions.

Are bacterial culture quantifications reliable? Comparative performance of the WASP automated inoculation instrument in the era of ISO 15189 accreditation.

PURPOSE: Isolating colonies and obtaining accurate colony counts from bacterial cultures are critical steps for the optimal management of infected patients. The uncertainties in the colony count results from the bacterial cultures were evaluated by verifying the performance of the WASP inoculation system according to the International Organization for Standardization (ISO) 15189 standard. METHODOLOGY: We first (i) evaluated the cross-contamination and precision of the WASP instrument (Copan Diagnostics, Italy) and (ii) established enumeration reading grids for urine, swab, bronchopulmonary specimens (BPSs) and catheter tip cultures. Subsequently, 72 clinical samples were tested to compare the results of the WASP, PREVI Isola (bioMérieux, France) and manual inoculation methods. RESULTS: The WASP method did not show cross-contamination. The coefficient of variation for the colony counts in the repeatability experiment was evaluated for 10 µl and 30 µl loop protocols and determined to be 29 and 14 %, respectively. The agreement between the automated and manual methods and between the automated methods for the colony counts was high (94.4 and 100 %, respectively). The WASP method yielded better isolation quality compared to the manual method (P=0.020) and to the PREVI Isola only when polymicrobial specimens were considered (P=0.014). For quantification evaluation, the measurement uncertainty was evaluated to 1.8×103 c.f.u. ml-1 for a suspension of Escherichia coli at 104 c.f.u. ml-1. CONCLUSION: We report the verification of the performance of the WASP instrument and describe a rapid procedure for achieving semi-quantitative cultures from BPSs and catheter tips. Quantitative interpretation of the bacterial cultures should be performed with caution.

PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level.

Plasmid prediction may be of great interest when studying bacteria of medical importance such as Enterobacteriaceae as well as Staphylococcus aureus or Enterococcus. Indeed, many resistance and virulence genes are located on such replicons with major impact in terms of pathogenicity and spreading capacities. Beyond strain outbreak, plasmid outbreaks have been reported in particular for some extended-spectrum beta-lactamase- or carbapenemase-producing Enterobacteriaceae. Several tools are now available to explore the 'plasmidome' from whole-genome sequences with various approaches, but none of them are able to combine high sensitivity and specificity. With this in mind, we developed PlaScope, a targeted approach to recover plasmidic sequences in genome assemblies at the species or genus level. Based on Centrifuge, a metagenomic classifier, and a custom database containing complete sequences of chromosomes and plasmids from various curated databases, PlaScope classifies contigs from an assembly according to their predicted location. Compared to other plasmid classifiers, PlasFlow and cBar, it achieves better recall (0.87), specificity (0.99), precision (0.96) and accuracy (0.98) on a dataset of 70 genomes of Escherichia coli containing plasmids. In a second part, we identified 20 of the 21 chromosomal integrations of the extended-spectrum beta-lactamase coding gene in a clinical dataset of E. coli strains. In addition, we predicted virulence gene and operon locations in agreement with the literature. We also built a database for Klebsiella and correctly assigned the location for the majority of resistance genes from a collection of 12 Klebsiella pneumoniae strains. Similar approaches could also be developed for other well-characterized bacteria.

The Odyssey of the Ancestral Escherich Strain through Culture Collections: an Example of Allopatric Diversification.

More than a century ago, Theodor Escherich isolated the bacterium that was to become Escherichia coli, one of the most studied organisms. Not long after, the strain began an odyssey and landed in many laboratories across the world. As laboratory culture conditions could be responsible for major changes in bacterial strains, we conducted a genome analysis of isolates of this emblematic strain from different culture collections (England, France, the United States, Germany). Strikingly, many discrepancies between the isolates were observed, as revealed by multilocus sequence typing (MLST), the presence of virulence-associated genes, core genome MLST, and single nucleotide polymorphism/indel analyses. These differences are correlated with the phylogeographic history of the strain and were due to an unprecedented number of mutations in coding DNA repair functions such as mismatch repair (MutL) and oxidized guanine nucleotide pool cleaning (MutT), conferring a specific mutational spectrum and leading to a mutator phenotype. The mutator phenotype was probably acquired during subculturing and corresponded to second-order selection. Furthermore, all of the isolates exhibited hypersusceptibility to antibiotics due to mutations in efflux pump- and porin-encoding genes, as well as a specific mutation in the sigma factor-encoding gene rpoS. These defects reflect a self-preservation and nutritional competence tradeoff allowing survival under the starvation conditions imposed by storage. From a clinical point of view, dealing with such mutator strains can lead microbiologists to draw false conclusions about isolate relatedness and may impact therapeutic effectiveness. IMPORTANCE Mutator phenotypes have been described in laboratory-evolved bacteria, as well as in natural isolates. Several genes can be impacted, each of them being associated with a typical mutational spectrum. By studying one of the oldest strains available, the ancestral Escherich strain, we were able to identify its mutator status leading to tremendous genetic diversity among the isolates from various collections and allowing us to reconstruct the phylogeographic history of the strain. This mutator phenotype was probably acquired during the storage of the strain, promoting adaptation to a specific environment. Other mutations in rpoS and efflux pump- and porin-encoding genes highlight the acclimatization of the strain through self-preservation and nutritional competence regulation. This strain history can be viewed as unintentional experimental evolution in culture collections all over the word since 1885, mimicking the long-term experimental evolution of E. coli of Lenski et al. (O. Tenaillon, J. E. Barrick, N. Ribeck, D. E. Deatherage, J. L. Blanchard, A. Dasgupta, G. C. Wu, S. Wielgoss, S. Cruveiller, C. Médigue, D. Schneider, and R. E. Lenski, Nature 536:165-170, 2016, https://doi.org/10.1038/nature18959) that shares numerous molecular features.

Contribution and limits of clinical specimens for the screening of intestinal multi-drug-resistant bacteria in view of laboratory automation.

The detection of multi-drug-resistant bacteria carriers constitutes a race against time for infection preventionists. Alongside standard analysis for diagnostic purposes and a rectal screening strategy, the authors tested a heavy-loaded selective method against 562 clinical specimens from 439 patients to detect extended-spectrum beta-lactamase-producing (ESBL) or carbapenemase-producing Enterobacteriaceae (CPE) and vancomycin-resistant enterococci (VRE). The approach identified five more specimens positive for ESBL-producing Enterobacteriaceae than standard analysis, and six out of nine known VRE/CPE carriers (three new CPE/VRE strains were also identified in this cohort). In view of the ongoing automation of laboratories, this approach focusing on urine and stool specimens may be an alternative or complementary approach to dedicated rectal screening.

Mortality of necrotizing fasciitis: relative influence of individual and hospital-level factors, a nationwide multilevel study, France, 2007-12.

BACKGROUND: Necrotizing soft-tissue infections (NSTI) are rare, life-threatening conditions. OBJECTIVES: To assess whether admitting hospital characteristics were associated with NSTI mortality. METHODS: We studied the French nationwide hospital discharge database (retrospective national cohort). All patients admitted in the period 2007-12 with an International Classification of Diseases 10 code of necrotizing fasciitis were eligible. We extracted data on the patients (age, sex, intensive care unit admission, comorbidities) and hospitals (public vs. private proprietary; for public hospitals, teaching, yes/no; and number of NSTI admissions, ≥ 3 NSTI cases/year, yes/no). Multivariable analyses were performed to identify independent predictors of 28-day mortality and in-hospital mortality using mixed logistic regression and Cox proportional hazards models, respectively. RESULTS: We identified 1537 patients (915 males) with a median age of 60 years (interquartile range 48-75), admitted to 326 hospitals, public (82%) and admitting < 3 NSTI cases/year (93%). Overall, 364 patients died [23·7%; 95% confidence interval (CI) 21·6-25·9]. Patients treated in public teaching centres with ≥ 3 NSTI cases annually had lower 28-day mortality (adjusted odds ratio 0·68; 95% CI 0·46-0·99; P = 0·045) and in-hospital mortality rates than patients treated in local hospitals, even after adjusting for potentially relevant individual risk factors. No significant association was found between mortality and interhospital transfer. CONCLUSIONS: Our findings highlight an increased survival in teaching centres with high-volume NSTI procedures. If confirmed in other settings, these findings reinforce the importance of expertise in early diagnosis and management of this condition.

Stenotrophomonas maltophilia healthcare-associated infections: identification of two main pathogenic genetic backgrounds.

BACKGROUND: Stenotrophomonas maltophilia is an opportunistic multi-drug-resistant bacterium responsible for healthcare-associated infections. Strategies for in-hospital infection control and management of carriers and environmental reservoirs remain controversial. AIM: To determine the population structure of S. maltophilia strains in hospitalized infected patients and to identify putative highly pathogenic subpopulations that require upgraded infection control measures. METHODS: Eighty-three diverse human strains of various clinical origins from 18 geographically distant hospitals were characterized phenotypically and genotypically using a multi-locus sequence typing (MLST) approach. FINDINGS: Neither a predominant nor emerging sequence type (ST) was identified. Among the 80 typeable strains, only 29% corresponded to described STs, especially ST5 (N=6) and ST4/26/31 (N=2). The ST distribution and the phylogenic tree based on the concatenated MLST genes did not account for geographical, clinical origin or antimicrobial susceptibility clustering. A phylogenic tree that included 173 ST profiles from the MLST database and the 80 typeable strains confirmed the high genetic diversity of S. maltophilia, the previously reported genogroup organization and the predominance of genogroup 6, as it represented 41% (33/80) of the strains. Unexpectedly, genogroup 2 was the second most prevalent genogroup and included 16% (13/80) of the strains. These genogroups represented 57% (20/35) of the strains in respiratory patients and 75% (9/12) of the strains in patients with cystic fibrosis. CONCLUSION: Beyond MLST, the over-representation of some genogroups among strains responsible for healthcare-associated infections was confirmed. Genogrouping affiliation is recommended to implement infection control measures selectively for the most pathogenic strains isolated from patient or environmental reservoirs.