Protective effect of SARS-CoV-2 preventive measures against ESKAPE and Escherichia coli infections.

BACKGROUND/OBJECTIVES: We investigated whether behavioral precautions adopted during Coronavirus disease (COVID-19) pandemic also influenced the spreading and multidrug resistance (MDR) of ESKAPEEc (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii [AB], Pseudomonas aeruginosa, Enterobacter spp and Escherichia Coli, [EC]) among Intensive Care Unit (ICU) patients. SUBJECTS/METHODS: We performed a single-center retrospective study in adult patients admitted to our COVID-19-free surgical ICU. Only patients staying in ICU for more than 48 hours were included. The ESKAPEEc infections recorded during the COVID-19 period (June 1, 2020 - February 28, 2021) and in the corresponding pre-pandemic period (June 1, 2019 - February 28, 2020) were compared. An interrupted time series analysis was performed to rule out possible confounders. RESULTS: Overall, 173 patients in the COVID-19 period and 132 in the pre-COVID-19 period were investigated. The ESKAPEEc infections were documented in 23 (13.3%) and 35 (26.5%) patients in the pandemic and the pre-pandemic periods, respectively (p = 0.005). Demographics, diagnosis, comorbidities, type of surgery, Simplified Acute Physiology Score II, length of mechanical ventilation, hospital and ICU length of stay, ICU death rate, and 28-day hospital mortality were similar in the two groups. In comparison with the pre-pandemic period, no AB was recorded during COVID-19 period, (p = 0.017), while extended-spectrum beta-lactamase-producing EC infections significantly decreased (p = 0.017). Overall, the ESKAPEEc isolates during pandemic less frequently exhibited multidrug-resistant (p = 0.014). CONCLUSIONS: These findings suggest that a robust adherence to hygiene measures together with human contact restrictions in a COVID-19 free ICU might also restrain the transmission of ESKAPEEc pathogens.

Pneumocystis jirovecii among patients with cystic fibrosis and their household members.

We conducted a pilot study of patients with cystic fibrosis (CF) to assess intra-family transmission of P. jirovecii and compare it with data on other prevalent pathogens such as P. aeruginosa and S. pneumoniae, in which respiratory transmission has already been documented. Oral swab samples from 10 patients with CF and 15 household members were collected at baseline and 2 weeks later. P. aeruginosa and S. pneumoniae were assessed using standardized culture methods and PCR, and P. jirovecii was assessed using real and nested PCR, genotyping the positive samples by direct sequencing. P. aeruginosa cultures were positive for 7/10 (70%) of patients with CF at baseline and was identified by PCR in 8/10 (80%) of cases at baseline and 2 weeks later. S. pneumoniae cultures were negative for all patients, but the microorganism was identified by PCR in two cases. P. jirovecii was detected by real time and nested PCR in 5/10 (50%) of the patients at the two time points. In the household members, P. aeruginosa and P. jirovecii were identified in 7/15 (46.7%), and S. pneumoniae was identified in 8/15 (53,3%). The concordance of positive or negative pairs of patients with CF and their household members was 33.3% (5/15) for P. aeruginosa, 46.7% (7/15) for S. pneumonia and 93.3% (14/15) for P. jirovecii. The concordance for P. jirovecii genotypes among five pairs with available genotype was 100%. This study suggests for the first time the possible transmission of Pneumocystis in the home of patients with CF, indicating that patients and their household members are reservoirs and possible sources of infection. LAY SUMMARY: This study suggests for the first time the possible transmission of Pneumocystis in the family environment of patients with cystic fibrosis, indicating that patients and their household members are reservoirs and possible sources of this infection.

Transmission, adaptation and geographical spread of the Pseudomonas aeruginosa Liverpool epidemic strain.

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.

Pseudomonas aeruginosa infection in augmented care: the molecular ecology and transmission dynamics in four large UK hospitals.

BACKGROUND: Pseudomonas aeruginosa is a common opportunistic pathogen and molecular typing in outbreaks has linked patient acquisition to contaminated hospital water systems. AIM: To elucidate the role of P. aeruginosa transmission rates in non-outbreak augmented care settings in the UK. METHODS: Over a 16-week period, all water outlets in augmented care units of four hospitals were sampled for P. aeruginosa and clinical isolates were collected. Outlet and clinical P. aeruginosa isolates underwent whole-genome sequencing (WGS), which with epidemiological data identified acquisition from water as definite (level 1), probable (level 2), possible (level 3), and no evidence (level 4). FINDINGS: Outlets were positive in each hospital on all three occasions: W (16%), X (2.5%), Y (0.9%) and Z (2%); and there were 51 persistently positive outlets in total. WGS identified likely transmission (at levels 1, 2 and 3) from outlets to patients in three hospitals for P. aeruginosa positive patients: W (63%), X (54.5%) and Z (26%). According to the criteria (intimate epidemiological link and no phylogenetic distance), approximately 5% of patients in the study 'definitely' acquired their P. aeruginosa from their water outlets in the intensive care unit. This study found extensive evidence of transmission from the outlet to the patients particularly in the newest hospital (W), which had the highest rate of positive outlets. CONCLUSIONS: The overall findings suggest that water outlets are the most likely source of P. aeruginosa nosocomial infections in some settings, and that widespread introduction of control measures would have a substantial impact on infections.

Novel use of culturomics to identify the microbiota in hospital sink drains with and without persistent VIM-positive Pseudomonas aeruginosa.

In hospitals, Verona Integron-encoded Metallo-beta-lactamase (VIM)-positive Pseudomonas aeruginosa may colonize sink drains, and from there, be transmitted to patients. These hidden reservoirs are difficult to eradicate since P. aeruginosa forms biofilms that resist disinfection. However, little is known on the composition of these biofilms. Therefore, culturomics was used for the first time to investigate the viable microbiota in four hospital sink drain samples with longstanding VIM-positive P. aeruginosa drain reservoirs (inhabited by high-risk clone, sequence type ST111), and four drain samples where VIM-positive P. aeruginosa was not present. Microbial load and composition varied between samples, yielding between 471-18,904 distinct colonies and 8-20 genera. In two VIM-positive drain samples, P. aeruginosa was the most abundantly-isolated microorganism, and found in combination with other Gram-negative bacteria, Citrobacter, Enterobacter, or Stenotrophomonas. P. aeruginosa was in low abundance in the other two VIM-positive samples, and found with Gram-positive cocci (Enterococcus and Staphylococcus) or Sphingomonas. In VIM-negative drain samples, high abundances of Gram-negative non-fermenting bacteria, including Acinetobacter, non-aeruginosa Pseudomonas spp., Acidovorax, Chryseobacterium, Flavobacterium, and Sphingobium, as well as Candida, were cultured. Although additional experiments are needed to draw more firm conclusions on which microorganisms enable or inhibit VIM-positive P. aeruginosa persistence, our data provide unique insights into the microbial compositions of sink drain inlets.

Molecular and Functional Analysis of Pore-Forming Toxin Monalysin From Entomopathogenic Bacterium Pseudomonas entomophila.

Pseudomonas entomophila is a highly pathogenic bacterium that infects insects. It is also used as a suitable model pathogen to analyze Drosophila's innate immunity. P. entomophila's virulence is largely derived from Monalysin, a ß-barrel pore-forming toxin that damages Drosophila tissues, inducing necrotic cell death. Here we report the first and efficient purification of endogenous Monalysin and its characterization. Monalysin is successfully purified as a pro-form, and trypsin treatment results in a cleaved mature form of purified Monalysin which kills Drosophila cell lines and adult flies. Electrophysiological measurement of Monalysin in a lipid membrane with an on-chip device confirms that Monalysin forms a pore, in a cleavage-dependent manner. This analysis also provides a pore-size estimate of Monalysin using current amplitude for a single pore and suggests lipid preferences for the insertion. Atomic Force Microscope (AFM) analysis displays its structure in a solution and shows that active-Monalysin is stable and composed of an 8-mer complex; this observation is consistent with mass spectrometry data. AFM analysis also shows the 8-mer structure of active-Monalysin in a lipid bilayer, and real-time imaging demonstrates the moment at which Monalysin is inserted into the lipid membrane. These results collectively suggest that endogenous Monalysin is indeed a pore-forming toxin composed of a rigid structure before pore formation in the lipid membrane. The endogenous Monalysin characterized in this study could be a desirable tool for analyzing host defense mechanisms against entomopathogenic bacteria producing damage-inducing toxins.

Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections.

Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.

In-Depth Study of a Nosocomial Outbreak Caused by Extensively Drug-Resistant Pseudomonas aeruginosa Using Whole Genome Sequencing Coupled With a Polymerase Chain Reaction Targeting Strain-Specific Single Nucleotide Polymorphisms.

In 2013-2014, an outbreak involving 14 patients infected by an extensively drug-resistant strain of Pseudomonas aeruginosa was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy for investigating the outbreak in depth by means of molecular and genomic approaches. Pulsed-field gel electrophoresis (PFGE) was applied as a first-line approach, followed by a more refined whole genome sequencing analysis. Single nucleotide polymorphisms identified by whole genome sequencing were used to design a specific polymerase chain reaction (PCR) for screening unsuspected cases infected by the outbreak strain. Whole genome sequencing alerted us to the existence of greater genetic diversity than was initially assumed, splitting the PFGE-associated outbreak isolates into 4 groups, 2 of which represented coincidental transmission unrelated to the outbreak. A multiplex allele-specific PCR targeting outbreak-specific single nucleotide polymorphisms was applied to 290 isolates, which allowed us to identify 25 additional cases related to the outbreak during 2011-2017. Whole genome sequencing coupled with an outbreak-strain-specific PCR enabled us to markedly redefine the initial picture of the outbreak by 1) ruling out initially suspected cases, 2) defining likely independent coincidental transmission events, 3) predating the starting point of the outbreak, 4) capturing new unsuspected cases, and 5) revealing that the outbreak was still active.

Systemic infection facilitates transmission of Pseudomonas aeruginosa in mice.

Health care-associated infections such as Pseudomonas aeruginosa bacteremia pose a major clinical risk for hospitalized patients. However, these systemic infections are presumed to be a "dead-end" for P. aeruginosa and to have no impact on transmission. Here, we use a mouse infection model to show that P. aeruginosa can spread from the bloodstream to the gallbladder, where it replicates to extremely high numbers. Bacteria in the gallbladder can then seed the intestines and feces, leading to transmission to uninfected cage-mate mice. Our work shows that the gallbladder is crucial for spread of P. aeruginosa from the bloodstream to the feces during bacteremia, a process that promotes transmission in this experimental system. Further research is needed to test to what extent these findings are relevant to infections in patients.

Tracking Pseudomonas aeruginosa transmissions due to environmental contamination after discharge in ICUs using mathematical models.

Pseudomonas aeruginosa (P. aeruginosa) is an important cause of healthcare-associated infections, particularly in immunocompromised patients. Understanding how this multi-drug resistant pathogen is transmitted within intensive care units (ICUs) is crucial for devising and evaluating successful control strategies. While it is known that moist environments serve as natural reservoirs for P. aeruginosa, there is little quantitative evidence regarding the contribution of environmental contamination to its transmission within ICUs. Previous studies on other nosocomial pathogens rely on deploying specific values for environmental parameters derived from costly and laborious genotyping. Using solely longitudinal surveillance data, we estimated the relative importance of P. aeruginosa transmission routes by exploiting the fact that different routes cause different pattern of fluctuations in the prevalence. We developed a mathematical model including background transmission, cross-transmission and environmental contamination. Patients contribute to a pool of pathogens by shedding bacteria to the environment. Natural decay and cleaning of the environment lead to a reduction of that pool. By assigning the bacterial load shed during an ICU stay to cross-transmission, we were able to disentangle environmental contamination during and after a patient's stay. Based on a data-augmented Markov Chain Monte Carlo method the relative importance of the considered acquisition routes is determined for two ICUs of the University hospital in Besançon (France). We used information about the admission and discharge days, screening days and screening results of the ICU patients. Both background and cross-transmission play a significant role in the transmission process in both ICUs. In contrast, only about 1% of the total transmissions were due to environmental contamination after discharge. Based on longitudinal surveillance data, we conclude that cleaning improvement of the environment after discharge might have only a limited impact regarding the prevention of P.A. infections in the two considered ICUs of the University hospital in Besançon. Our model was developed for P. aeruginosa but can be easily applied to other pathogens as well.

An outbreak of Pseudomonas aeruginosa urinary tract infections following outpatient flexible cystoscopy.

The investigation of an outbreak of Pseudomonas aeruginosa urinary tract infections after ambulatory cystoscopies identified a damaged cystoscope contaminated by P aeruginosa and acting as a relay object. This outbreak urges us not to trivialize urinary tract infections occurring after an elective cystoscopy. Patients should be advised to signal the occurrence of urologic symptoms after urologic exploration.

Pseudomonas poae-Associated Fatal Septic Transfusion Reaction, Peoria, Illinois, USA, 2017.

In the United States, fatal transfusion-transmitted infections from red blood cell units are rare. Although this pattern mostly reflects how inhospitable refrigerated red blood cell units are to contaminant growth, fatalities caused by microorganisms that can grow at storage temperature (4°C), but not in standard clinical blood cultures at 37°C, are probably underestimated. We analyzed a fatal red blood cell transfusion in Peoria, Illinois, USA, that occurred in 2017. Samples from the patient's whole blood and the red blood cell unit remained culture-negative during the investigation, despite direct visualization of gram-negative bacilli within the unit immediately after transfusion. We identified the bacteria as Pseudomonas poae, a nonpathogenic pseudomonad carrying multiple cold-shock domain protein genes, and confirmed its cold tolerance and inability to grow at 37°C. Our work indicates transfusion reaction workups need to include testing for psychrophilic organisms, which could explain the cause of other apparently culture-negative transfusion reactions.

Challenge of drug resistance in Pseudomonas aeruginosa: clonal spread of NDM-1-positive ST-308 within a tertiary hospital.

OBJECTIVES: MDR Pseudomonas aeruginosa is a serious global threat to healthcare institutions. The mechanism by which drug resistance can be acquired is variable, but acquired carbapenemase production has been reported in P. aeruginosa. An investigation was performed to determine the rate and genomic epidemiology of New Delhi MBL (NDM) in ß-lactam-non-susceptible isolates. METHODS: P. aeruginosa isolates from a tertiary hospital in Singapore between January 2015 and February 2018 were investigated for the presence of NDM genes. RESULTS: Out of 298 pan-ß-lactam-non-susceptible isolates, 31 were found to be NDM positive (10.4%). WGS demonstrated that all 31 NDM-positive isolates were clonal, belonging to ST-308. blaNDM was chromosomally inserted within an integrative and conjugative element (ICE), ICETn43716385. The NDM-P. aeruginosa isolates possessed an extensive repertoire of both cell-associated [flagella, pili, alginate/biofilm, LPS, type III secretion system (T3SS) and type VI secretion system (T6SS)] and secreted virulence factors. Antibiograms revealed higher rates of drug resistance in NDM-positive isolates compared with their non-NDM counterparts. The NDM isolates remained 100% susceptible only to colistin. CONCLUSIONS: The combination of chromosomal mutations, acquired resistance genes and virulence factors likely facilitated the persistent and ongoing spread of the ST-308 clade of P. aeruginosa within the hospital. Our study illustrates the particular threat of NDM-positive P. aeruginosa in a tertiary hospital setting in the era of antimicrobial resistance.

Transmission of multi-drug resistant Pseudomonas aeruginosa between two flexible ureteroscopes and an outbreak of urinary tract infection: the fragility of endoscope decontamination.

OBJECTIVES: Flexible endoscopes are difficult to decontaminate, and endoscope-associated infections are increasing. This report describes an outbreak of multi-drug resistant Pseudomonas aeruginosa identified following an increase in incidence of clinical infections associated with flexible ureteroscopy at a tertiary care centre in the UK. METHODS: Clinical, laboratory and central decontamination unit (CDU) records were reviewed to determine the extent of the problem, and links to the used endoscopes. Audits of the ureteroscopy procedure, endoscopy unit and CDU were performed. Endoscopes were sampled, cultured and examined for structural integrity. All available isolates were typed. RESULTS: Thirteen patients developed clinical infections linked to two flexible ureteroscopes. The first ureteroscope was likely colonized from a known infected patient and the second ureteroscope after use on another patient infected by the first. Risk factors identified include surface cuts, stretching and puckering of the outer cover in both ureteroscopes, absence of bedside cleaning, overnight delay between the ureteroscopy and decontamination, inadequate drying after decontamination and non-traceability of connector valves. CONCLUSIONS: The adequacy of flexible endoscope decontamination depends on numerous steps. With the increasing global incidence of multi-drug resistant organisms, stringent monitoring of the flexible endoscopy process by users and decontamination units is essential.

Risk Assessment After a Severe Hospital-Acquired Infection Associated With Carbapenemase-Producing Pseudomonas aeruginosa.

Importance: Resistance of gram-negative bacilli to carbapenems is rapidly emerging worldwide. In 2016, the World Health Organization defined the hospital-built environment as a core component of infection prevention and control programs. The hospital-built environment has recently been reported as a source for outbreaks and sporadic transmission events of carbapenemase-producing gram-negative bacilli from the environment to patients. Objective: To assess risk after the identification of an unexpected, severe, and lethal hospital-acquired infection caused by carbapenemase-producing Pseudomonas aeruginosa in a carbapenemase-low endemic setting. Design, Settings, and Participants: A case series study in which a risk assessment was performed on all 11 patients admitted to the combined cardiothoracic surgery and pulmonary diseases ward and the hospital-built environment in the Radboud University Medical Center, the Netherlands, in February 2018. Exposures: Water and aerosols containing carbapenemase-producing (Verona integron-mediated metallo-ß-lactamase [VIM]) P aeruginosa. Main Outcomes and Measures: Colonization and/or infection of patients and/or contamination of the environment after the detection of 1 patient infected with carbapenemase-producing (VIM) P aeruginosa. Results: A total of 5 men (age range, 60-84 years) and 6 women (age range, 55-74 years) were admitted to the combined cardiothoracic surgery and pulmonary diseases ward. The risk assessment was performed after carbapenemase-producing (VIM) P aeruginosa was unexpectedly detected in a man in his early 60s, who had undergone a left-sided pneumonectomy and adjuvant radiotherapy. No additional cases (colonization or infection) of carbapenemase-producing (VIM) P aeruginosa were detected. Plausible transmission of carbapenemase-producing P aeruginosa from the hospital environment to the patient via the air was confirmed by whole-genome sequencing, which proved the relation of Pseudomonas strains from the patient, the shower drains in 8 patient rooms, 1 sink, and an air sample. Conclusions and Relevance: This study suggests that rethinking the hospital-built environment, including shower drains and the sewage system, will be crucial for the prevention of severe and potential lethal hospital-acquired infections.

Are our stethoscopes contaminated ?

BACKGROUND: Health care-associated infections are a real public health problem. Contaminated medical equipments such as stethoscopes are often an overlooked vector. In our study, we were interested in proving our doctors stethoscopes contamination and in studying the microbiological profile of isolated germs. METHODS: This was about a cross-sectional study that lasted 2 months (May and June 2014) including 39 personal stethoscopes of all grade doctors working in 8 different departments in Habib Thameur Hospital. The swabs were taken from the membranes of the stethoscopes and sent quickly to our bacteriology service. Then, the bacteriological samples were subcultivated on blood agar. The reading tooks place 24 hours later. Were considered positive the cultures that contained more than 104 colony forming units/ml. Then we proceeded to the identification of the germ. RESULTS: Fifteen samples from 39 were positive that was 38 %. The coagulase- negative Staphylococcus was the predominant germ. The pathogenic germs were found in 5 positive samples that was 12 % from all the samples. They were distributed as follows: 2 Methicillin-susceptible Staphylococci Aureus, 2 methicillin-resistant coagulase- negative Staphylococci and one Pseudomonas aerogenusa. The pathogenic germs were found in stethoscopes taken from cardiology and intensive care unit (ICU). In cardiology, 7/9 stethoscopes were positive (5 coagulase- negative Staphylococcus among them 2 resistant methicillin and 2 Staphylococcus aureus.) In ICU, 6/13 stethoscopes were positive among them one Pseudomonas aerogenusa. CONCLUSION: The stethoscopes constitute bacterial reservoirs. It is necessary to perform a procedure for the good practices of disinfection.

Cystic fibrosis pathogens survive for extended periods within cough-generated droplet nuclei.

The airborne route is a potential pathway in the person-to-person transmission of bacterial strains among cystic fibrosis (CF) populations. In this cross-sectional study, we investigate the physical properties and survival of common non-Pseudomonas aeruginosa CF pathogens generated during coughing. We conclude that Gram-negative bacteria and Staphylococcus aureus are aerosolised during coughing, can travel up to 4 m and remain viable within droplet nuclei for up to 45 min. These results suggest that airborne person-to-person transmission is plausible for the CF pathogens we measured.

Tap out: reducing waterborne Pseudomonas aeruginosa transmission in an intensive care unit.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous and important opportunistic pathogen in immunocompromised or critically ill patients. Nosocomial P. aeruginosa outbreaks have been associated with hospital water sources. AIM: To describe engineering interventions to minimize contamination of water outlets and the subsequent clinical impact. METHODS: New tap outlets were fitted at selected outlets across the intensive care unit (ICU). Laboratory testing demonstrated that, following artificial contamination with P. aeruginosa, these taps could be effectively decontaminated using a thermal washer-disinfector. Water samples were collected weekly from new outlets on the ICU over an eight-month period and tested for the enumeration of P. aeruginosa via membrane filtration. Surveillance of P. aeruginosa from clinical specimens was routinely undertaken. FINDINGS: Prior to the interventions, water sampling on ICU indicated that 30% of the outlets were positive for P. aeruginosa at any one time, and whole genome sequencing data suggested at least 30% transmission from water to patient. Since their installation, weekly sampling of the new tap outlets has been negative for P. aeruginosa, and the number of P. aeruginosa clinical isolates has fallen by 50%. CONCLUSION: Installation and maintenance of tap outlets free of P. aeruginosa can substantially reduce the number of P. aeruginosa clinical isolates in an ICU.