Outbreak of Pseudomonas aeruginosa producing VIM carbapenemase in an intensive care unit and its termination by implementation of waterless patient care.

BACKGROUND: Long-term outbreaks of multidrug-resistant Gram-negative bacilli related to hospital-building water systems have been described. However, successful mitigation strategies have rarely been reported. In particular, environmental disinfection or replacement of contaminated equipment usually failed to eradicate environmental sources of Pseudomonas aeruginosa. METHODS: We report the investigation and termination of an outbreak of P. aeruginosa producing VIM carbapenemase (PA-VIM) in the adult intensive care unit (ICU) of a Swiss tertiary care hospital with active case finding, environmental sampling and whole genome sequencing (WGS) of patient and environmental strains. We also describe the implemented control strategies and their effectiveness on eradication of the environmental reservoir. RESULTS: Between April 2018 and September 2020, 21 patients became either infected or colonized with a PA-VIM strain. For 16 of them, an acquisition in the ICU was suspected. Among 131 environmental samples collected in the ICU, 13 grew PA-VIM in sink traps and drains. WGS confirmed the epidemiological link between clinical and environmental strains and the monoclonal pattern of the outbreak. After removing sinks from patient rooms and implementation of waterless patient care, no new acquisition was detected in the ICU within 8 months after the intervention. DISCUSSION: Implementation of waterless patient care with removal of the sinks in patient rooms was successful for termination of a PA-VIM ICU outbreak linked to multiple environmental water sources. WGS provides highly discriminatory accuracy to investigate environment-related outbreaks.

Evaluation of the performance of rapid tests for screening carriers of acquired ESBL-producing Enterobacterales and their impact on turnaround time.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales constitute a global burden for hospital infection, and the identification of carriers by screening patients at risk is recommended by several guidelines. AIM: To evaluate the impact of rapid ESBL tests on the turnaround time (TAT) of screening. METHODS: Rectal swabs were analysed by culture and synergism tests for identification of non-Esherichia coli Enterobacterales that produce ESBLs (NEcESBL-producing Enterobacterales). The Rapid ESBL NP and NG CTX-M MULTI tests were performed on colonies grown on chromogenic media. The results of polymerase chain reaction and sequencing of ESBL genes were used as the gold standard. RESULTS: Among 473 analysed swabs, 75 (15.9%) grew NEcESBL-producing Enterobacterales, leading to 89 isolates. Sensitivities of the synergism, Rapid ESBL NP and NG CTX-M MULTI tests were 0.97 [95% confidence interval (CI) 0.88-0.99], 0.81 (95% CI 0.69-0.89) and 0.90 (95% CI 0.80-0.96), respectively. Specificities were 0.92 (95% CI 0.73-0.99), 0.85 (95% CI 0.64-0.95) and 0.96 (95% CI 0.78-1.00), respectively. Considering the 473 rectal swabs, ESBL screening using the synergism, Rapid ESBL NP and NG CTX-M MULTI tests was calculated. Sensitivities were 0.96 (95% CI 0.86-0.99), 0.81 (95% CI 0.68-0.90) and 0.91 (95% CI 0.79-0.97); specificities were 1.00 (95% CI 0.98-1.00), 0.99 (95% CI 0.98-1.00) and 1.00 (95% CI 0.99-1.00); positive predictive values were 0.96 (95% CI 0.86-0.99), 0.94 (95% CI 0.81-0.98) and 1.00 (95% CI 0.91-1.00); and negative predictive values were 1.00 (95% CI 0.98-1.00), 0.98 (95% CI 0.96-0.99) and 0.99 (95% CI 0.97-1.00), respectively. When no NEcESBL-producing Enterobacterales were observed, the mean TAT was 30 h. When NEcESBL-producing Enterobacterales were identified, the mean TATs were 74.7, 38.0 and 36.7 h for the synergism, Rapid ESBL NP and NG CTX-M MULTI tests, respectively. CONCLUSION: The two rapid ESBL tests showed good performance and allowed a reduction in TAT for screening protocols to identify patients carrying ESBL-producing Enterobacterales.

Evaluation of three consecutive versions of a commercial rapid PCR test to screen for methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Screening for methicillin-resistant Staphylococcus aureus (MRSA) is part of many recommendations to control MRSA. Several rapid PCR tests are available commercially and updated versions are constantly released. We aimed to evaluate the performance of three consecutive versions (G3, Gen3 and NxG) of the XpertMRSA test. METHODS: Routine samples for MRSA screening were simultaneously tested by culture and rapid PCR. The three versions of XpertMRSA were used successively and compared with culture. RESULTS: A total of 3512, 2794 and 3288 samples were analysed by culture and by the G3, Gen3 and NxG XpertMRSA versions, respectively. The rates of positive-by-culture in the three groups were 5.0%, 4.7% and 4.3%, respectively. The sensitivity improved over time (71.4, 95% CI 64.0-77.9; 82.3, 95% CI 74.4-88.2; and 84.3%, 95% CI 77.0-89.7, respectively), but not significantly. The specificity (98.4, 95% CI 97.9-98.8; 96.8, 95% CI 96.0-97.4; and 99.1, 95% CI 98.7-99.4, respectively) and the positive likelihood ratios (45.7, 95% CI 34.4-60.8; 25.6, 95% CI 20.5-32.0; and 97.1, 95% CI 66.3-142.4) were significantly lower in the Gen3 version (p < 0.00001). CONCLUSIONS: These significant differences in performance show the importance of evaluating each new version of a commercial test.

Global emergence of the widespread Pseudomonas aeruginosa ST235 clone.

OBJECTIVES: Despite the non-clonal epidemic population structure of Pseudomonas aeruginosa, several multi-locus sequence types are distributed worldwide and are frequently associated with epidemics where multidrug resistance confounds treatment. ST235 is the most prevalent of these widespread clones. In this study we aimed to understand the origin of ST235 and the molecular basis for its success. METHODS: The genomes of 79 P. aeruginosa ST235 isolates collected worldwide over a 27-year period were examined. A phylogenetic network was built, using a Bayesian approach to find the Most Recent Common Ancestor, and we identified antibiotic resistance determinants and ST235-specific genes. RESULTS: Our data suggested that the ST235 sublineage emerged in Europe around 1984, coinciding with the introduction of fluoroquinolones as an antipseudomonal treatment. The ST235 sublineage seemingly spread from Europe via two independent clones. ST235 isolates then appeared to acquire resistance determinants to aminoglycosides, ß-lactams and carbapenems locally. Additionally, we found that all the ST235 genomes contained the exoU-encoded exotoxin and identified 22 ST235-specific genes clustering in blocks and implicated in transmembrane efflux, DNA processing and bacterial transformation. These unique combinations of genes may have contributed to the poor outcome associated with P. aeruginosa ST235 infections and increased the ability of this international clone to acquire mobile resistance elements. CONCLUSION: Our data suggest that P. aeruginosa ST235 (a) has become prevalent across the globe potentially due to the selective pressure of fluoroquinolones and (b) readily became resistant to aminoglycosides, ß-lactams and carbapenems through mutation and acquisition of resistance elements among local populations.

Whole-Genome Sequencing Reveals the Contribution of Long-Term Carriers in Staphylococcus aureus Outbreak Investigation.

Whole-genome sequencing (WGS) makes it possible to determine the relatedness of bacterial isolates at a high resolution, thereby helping to characterize outbreaks. However, for Staphylococcus aureus, the accumulation of within-host diversity during carriage might limit the interpretation of sequencing data. In this study, we hypothesized the converse, namely, that within-host diversity can in fact be exploited to reveal the involvement of long-term carriers (LTCs) in outbreaks. We analyzed WGS data from 20 historical outbreaks and applied phylogenetic methods to assess genetic relatedness and to estimate the time to most recent common ancestor (TMRCA). The findings were compared with the routine investigation results and epidemiological evidence. Outbreaks with epidemiological evidence for an LTC source had a mean estimated TMRCA (adjusted for outbreak duration) of 243 days (95% highest posterior density interval [HPD], 143 to 343 days) compared with 55 days (95% HPD, 28 to 81 days) for outbreaks lacking epidemiological evidence for an LTC (P = 0.004). A threshold of 156 days predicted LTC involvement with a sensitivity of 0.875 and a specificity of 1. We also found 6/20 outbreaks included isolates with differing antimicrobial susceptibility profiles; however, these had only modestly increased pairwise diversity (mean 17.5 single nucleotide variants [SNVs] [95% confidence interval {CI}, 17.3 to 17.8]) compared with isolates with identical antibiograms (12.7 SNVs [95% CI, 12.5 to 12.8]) (P < 0.0001). Additionally, for 2 outbreaks, WGS identified 1 or more isolates that were genetically distinct despite having the outbreak pulsed-field gel electrophoresis (PFGE) pulsotype. The duration-adjusted TMRCA allowed the involvement of LTCs in outbreaks to be identified and could be used to decide whether screening for long-term carriage (e.g., in health care workers) is warranted. Requiring identical antibiograms to trigger investigation could miss important contributors to outbreaks.

Universal screening and decolonization for control of MRSA in nursing homes: follow-up of a cluster randomized controlled trial.

In 2010-11, a trial conducted in nursing homes showed no benefit of meticillin-resistant Staphylococcus aureus (MRSA) universal screening and decolonization over standard precautions to reduce the prevalence of MRSA carriage. Accordingly, no routine screening was performed from 2012. A five-year follow-up shows no new evidence supporting the intervention. Recommendations issued after trial (no screening and decolonization of MRSA residents) were retained.

New genotyping method discovers sustained nosocomial Pseudomonas aeruginosa outbreak in an intensive care burn unit.

BACKGROUND: Pseudomonas aeruginosa is a leading cause of healthcare-associated infections in the intensive care unit (ICU). AIM: To investigate an unexplained increase in the incidence of P. aeruginosa recovered from clinical samples in the ICU over a two-year period. METHODS: After unsuccessful epidemiological investigation by conventional tools, P. aeruginosa clinical isolates of all patients hospitalized between January 2010 and July 2012 were typed by a novel double-locus sequence typing (DLST) method and compared to environmental isolates recovered during the investigation period. FINDINGS: In total, 509 clinical isolates from 218 patients and 91 environmental isolates were typed. Thirty-five different genotypic clusters were found in 154 out of 218 patients (71%). The largest cluster, DLST 1-18, included 23 patients who were mostly hospitalized during overlapping periods in the burn unit. Genotype DLST 1-18 was also recovered from floor traps, shower trolleys and the shower mattress in the hydrotherapy rooms, suggesting environmental contamination of the burn unit as the source of the outbreak. After implementation of appropriate infection control measures, this genotype was recovered only once in a clinical sample from a burned patient and twice in the environment, but never thereafter during a 12-month follow-up period. CONCLUSION: The use of a novel DLST method allowed the genotyping of a large number of clinical and environmental isolates, leading to the identification of the environmental source of a large unrecognized outbreak in the burn unit. Eradication of the outbreak was confirmed after implementation of a continuous epidemiological surveillance of P. aeruginosa clones in the ICU.

Common skin infection due to Panton-Valentine leucocidin-producing Staphylococcus aureus strains in asylum seekers from Eritrea: a genome-based investigation of a suspected outbreak.

Since late 2014, multiple cases of abscesses and boils due to methicillin-susceptible Staphylococcus aureus (MSSA) expressing the Panton-Valentine leucocidin (PVL) were observed in Eritrean asylum seekers in Lausanne, Switzerland. Strains isolated from infected Eritrean and non-Eritrean patients were compared by whole genome sequencing to determine whether these numerous cases result from an outbreak. The genome of S. aureus PVL-producing strains were sequenced and compared. Clinical and epidemiological characteristics of patients infected by PVL-producing strains were investigated. This work reports 15 cases of infections due to PVL-producing strains affecting mostly asylum seekers (n = 10), people working with refugees and/or exposed to Africans (n = 3). Most infections were due to closely related strains of CC152 (n = 8) and CC15 (n = 3), two distantly related (>34 000 core single nucleotide polymorphisms) clonal complexes. An epidemiological link between the 15 cases could be ruled out by whole genome sequencing (33 to 172 core single nucleotide polymorphisms between the different strains of a given complex). Altogether, these results reflect the probable high incidence of CC15 and CC152 PVL-producing strains in eastern Africa. Clinicians facing unusual skin infections in African refugees (or in any person returning from this region of high endemicity) should consider S. aureus PVL-producer before suspecting rare infections such as leishmaniasis or rickettsiosis. Clinicians should also remember that PVL are frequently expressed by MSSA in some regions of the world and that antibiotics that are efficient on toxin expression, such as clindamycin, represent the best therapeutic option.

Hand soap contamination by Pseudomonas aeruginosa in a tertiary care hospital: no evidence of impact on patients.

BACKGROUND: During an environmental investigation of Pseudomonas aeruginosa in intensive care units, the liquid hand soap was found to be highly contaminated (up to 8 × 10(5)cfu/g) with this pathogen. It had been used over the previous five months and was probably contaminated during manufacturing. AIM: To evaluate the burden of this contamination on patients by conducting an epidemiological investigation using molecular typing combined with whole genome sequencing (WGS). METHODS: P. aeruginosa isolates from clinical specimens were analysed by double locus sequence typing (DLST) and compared with isolates recovered from the soap. Medical charts of patients infected with a genotype identical to those found in the soap were reviewed. WGS was performed on soap and patient isolates sharing the same genotype. FINDINGS: P. aeruginosa isolates (N = 776) were available in 358/382 patients (93.7%). Only three patients (0.8%) were infected with a genotype found in the soap. Epidemiological investigations showed that the first patient was not exposed to the soap, the second could have been exposed, and the third was indeed exposed. WGS showed a high number of core single nucleotide polymorphism differences between patients and soap isolates. No close genetic association was observed between soap and patient isolates, ruling out the hypothesis of transmission. CONCLUSION: Despite a highly contaminated soap, the combined investigation with DLST and WGS ruled out any impact on patients. Hand hygiene performed with alcohol-based solution for >15 years was probably the main reason. However, such contamination represents a putative reservoir of pathogens that should be avoided in the hospital setting.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Characterization of the staphylococcal cassette chromosome mec insertion site in 108 isolates lacking the mecA gene and identified as methicillin-resistant Staphylococcus aureus by the Xpert MRSA assay.

During a 3-year period, 848 patients were detected as carriers of methicillin-resistant Staphylococcus aureus (MRSA) by the Xpert MRSA assay (Cepheid). Among them, 108 patients (12.7 %) were colonized with strains showing methicillin-susceptible phenotypes and absence of the mecA gene, despite being positive with the rapid polymerase chain reaction (PCR) assay. DNA sequences of the staphylococcal cassette chromosome mec (SCCmec) insertion site of these "false-positive" strains was determined by direct sequencing of the genomic DNA. More than half (53.7 %) of the strains had DNA sequences unrelated to either SCC or SCCmec and one-third had DNA sequences related to non-mec SCC. Only 10.2 % of the strains carried sequences related to SCCmec, suggesting that a sequence containing the mecA gene was lost from an SCCmec. These findings differ from the general idea that all methicillin-susceptible S. aureus having positive Xpert MRSA assay results are essentially MRSA that lost the mecA gene.

Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method.

Although the molecular typing of Pseudomonas aeruginosa is important to understand the local epidemiology of this opportunistic pathogen, it remains challenging. Our aim was to develop a simple typing method based on the sequencing of two highly variable loci. Single-strand sequencing of three highly variable loci (ms172, ms217, and oprD) was performed on a collection of 282 isolates recovered between 1994 and 2007 (from patients and the environment). As expected, the resolution of each locus alone [number of types (NT) = 35-64; index of discrimination (ID) = 0.816-0.964] was lower than the combination of two loci (NT = 78-97; ID = 0.966-0.971). As each pairwise combination of loci gave similar results, we selected the most robust combination with ms172 [reverse; R] and ms217 [R] to constitute the double-locus sequence typing (DLST) scheme for P. aeruginosa. This combination gave: (i) a complete genotype for 276/282 isolates (typability of 98%), (ii) 86 different types, and (iii) an ID of 0.968. Analysis of multiple isolates from the same patients or taps showed that DLST genotypes are generally stable over a period of several months. The high typability, discriminatory power, and ease of use of the proposed DLST scheme makes it a method of choice for local epidemiological analyses of P. aeruginosa. Moreover, the possibility to give unambiguous definition of types allowed to develop an Internet database ( http://www.dlst.org ) accessible by all.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Switzerland: sampling only invasive isolates does not allow a representative description of the local diversity of clones.

We conducted a molecular study of MRSA isolated in Swiss hospitals, including the first five consecutive isolates recovered from blood cultures and the first ten isolates recovered from other sites in newly identified carriers. Among 73 MRSA isolates, 44 different double locus sequence typing (DLST) types and 32 spa types were observed. Most isolates belonged to the NewYork/Japan, the UK-EMRSA-15, the South German and the Berlin clones. In a country with a low to moderate MRSA incidence, inclusion of non-invasive isolates allowed a more accurate description of the diversity.

Very low prevalence of meticillin-resistant Staphylococcus aureus carrying the mecC gene in western Switzerland.

We report the first case of meticillin-resistant Staphylococcus aureus (MRSA) with the mecC gene in a patient in western Switzerland. After this first identification, a polymerase chain reaction protocol was established to investigate the occurrence of this new mecC gene in the population of this region. Enrichment broths were investigated from 1062 patients screened for MRSA, meticillin-susceptible Staphylococcus aureus isolates from clinical specimens from 475 patients, and 80 MRSA isolates (from 2005 to 2011) showing discrepancies between genotypic and phenotypic meticillin resistance. None was positive for mecC, suggesting that it is rare in the patient population of this region.

MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs.

The performance of the Xpert MRSA polymerase chain reaction (PCR) assay on pooled nose, groin, and throat swabs (three nylon flocked eSwabs into one tube) was compared to culture by analyzing 5,546 samples. The sensitivity [0.78, 95 % confidence interval (CI) 0.73-0.82] and specificity (0.99, 95 % CI 0.98-0.99) were similar to the results from published studies on separated nose or other specimens. Thus, the performance of the Xpert MRSA assay was not affected by pooling the three specimens into one assay, allowing a higher detection rate without increasing laboratory costs, as compared to nose samples alone.

Evaluation of adding a second marker to overcome Staphylococcus aureus spa typing homoplasies.

The utility of sequencing a second highly variable locus in addition to the spa gene (e.g., double-locus sequence typing [DLST]) was investigated to overcome limitations of a Staphylococcus aureus single-locus typing method. Although adding a second locus seemed to increase discriminatory power, it was not sufficient to definitively infer evolutionary relationships within a single multilocus sequence type (ST-5).

Which anatomical sites should be sampled for screening of methicillin-resistant Staphylococcus aureus carriage by culture or by rapid PCR test?

The nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat.

Investigation of classical epidemiological links between patients harbouring identical, non-predominant meticillin-resistant Staphylococcus aureus genotypes and lessons for epidemiological tracking.

According to molecular epidemiology theory, two isolates belong to the same chain of transmission if they are similar according to a highly discriminatory molecular typing method. This has been demonstrated in outbreaks, but is rarely studied in endemic situations. Person-to-person transmission cannot be established when isolates of meticillin-resistant Staphylococcus aureus (MRSA) belong to endemically predominant genotypes. By contrast, isolates of infrequent genotypes might be more suitable for epidemiological tracking. The objective of the present study was to determine, in newly identified patients harbouring non-predominant MRSA genotypes, whether putative epidemiological links inferred from molecular typing could replace classical epidemiology in the context of a regional surveillance programme. MRSA genotypes were defined using double-locus sequence typing (DLST) combining clfB and spa genes. A total of 1,268 non-repetitive MRSA isolates recovered between 2005 and 2006 in Western Switzerland were typed: 897 isolates (71%) belonged to four predominant genotypes, 231 (18%) to 55 non-predominant genotypes, and 140 (11%) were unique. Obvious epidemiological links were found in only 106/231 (46%) patients carrying isolates with non-predominant genotypes suggesting that molecular surveillance identified twice as many clusters as those that may have been suspected with classical epidemiological links. However, not all of these molecular clusters represented person-to-person transmission. Thus, molecular typing cannot replace classical epidemiology but is complementary. A prospective surveillance of MRSA genotypes could help to target epidemiological tracking in order to recognise new risk factors in hospital and community settings, or emergence of new epidemic clones.

Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007).

Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeruginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in categories A and B were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category B varied from 1.9 to 20 cases per 1000 admissions. This number exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases considered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.