Expansion and transmission dynamics of high risk carbapenem-resistant Klebsiella pneumoniae subclones in China: An epidemiological, spatial, genomic analysis.

AIMS: Carbapenem-resistant Klebsiella pneumonia (CRKP) is a global threat that varies by region. The global distribution, evolution, and clinical implications of the ST11 CRKP clone remain obscure. METHODS: We conducted a multicenter molecular epidemiological survey using isolates obtained from 28 provinces and municipalities across China between 2011 and 2021. We integrated sequences from public databases and performed genetic epidemiology analysis of ST11 CRKP. RESULTS: Among ST11 CRKP, KL64 serotypes exhibited considerable expansion, increasing from 1.54% to 46.08% between 2011 and 2021. Combining our data with public databases, the phylogenetic and phylogeography analyses indicated that ST11 CRKP appeared in the Americas in 1996 and spread worldwide, with key clones progressing from China's southeastern coast to the inland by 2010. Global phylogenetic analysis showed that ST11 KL64 CRKP has evolved to a virulent, resistant clade with notable regional spread. Single-nucleotide polymorphism (SNP) analysis identified BMPPS (bmr3, mltC, pyrB, ppsC, and sdaC) as a key marker for this clade. The BMPPS SNP clade is associated with high mortality and has strong anti-phagocytic and competitive traits in vitro. CONCLUSIONS: The high-risk ST11 KL64 CRKP subclone showed strong expansion potential and survival advantages, probably owing to genetic factors.

Clonal transmission of polymyxin B-resistant hypervirulent Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaKPC-2 in a tertiary hospital in China.

BACKGROUND: The prevalence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP) has gradually increased. It poses a severe threat to human health. However, polymyxin-resistant hvKP is rare. Here, we collected eight polymyxin B-resistant K. pneumoniae isolates from a Chinese teaching hospital as a suspected outbreak. RESULTS: The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. HvKP was identified by detecting virulence-related genes and using a Galleria mellonella infection model. Their resistance to serum, growth, biofilm formation, and plasmid conjugation were analyzed in this study. Molecular characteristics were analyzed using whole-genome sequencing (WGS) and mutations of chromosome-mediated two-component systems pmrAB and phoPQ, and the negative phoPQ regulator mgrB to cause polymyxin B (PB) resistance were screened. All isolates were resistant to polymyxin B and sensitive to tigecycline; four were resistant to ceftazidime/avibactam. Except for KP16 (a newly discovered ST5254), all were of the K64 capsular serotype and belonged to ST11. Four strains co-harbored blaKPC-2, blaNDM-1, and the virulence-related genes prmpA, prmpA2, iucA, and peg344, and were confirmed to be hypervirulent by the G. mellonella infection model. According to WGS analysis, three hvKP strains showed evidence of clonal transmission (8-20 single nucleotide polymorphisms) and had a highly transferable pKOX_NDM1-like plasmid. KP25 had multiple plasmids carrying blaKPC-2, blaNDM-1, blaSHV-12, blaLAP-2, tet(A), fosA5, and a pLVPK-like virulence plasmid. Tn1722 and multiple additional insert sequence-mediated transpositions were observed. Mutations in chromosomal genes phoQ and pmrB, and insertion mutations in mgrB were major causes of PB resistance. CONCLUSIONS: Polymyxin-resistant hvKP has become an essential new superbug prevalent in China, posing a serious challenge to public health. Its epidemic transmission characteristics and mechanisms of resistance and virulence deserve attention.

Transmission of ST45 and ST2407 extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in neonatal intensive care units, associated with contaminated environments.

OBJECTIVES: Given the increasing frequency of infections due to extended-spectrum ß-lactamase (EBSL)-producing Klebsiella pneumoniae in humans over recent decades, infection control against this pathogen is of high importance. METHODS: In this study, the transmission mode of ESBL-producing K. pneumoniae in neonatal intensive care units (NICU) was investigated. We collected K. pneumoniae isolates from patients admitted to the NICU and performed environmental screening of the NICU and nearby obstetrics department. All isolates were analysed using antimicrobial susceptibility testing, whole-genome sequencing, molecular typing, and antimicrobial and virulence determinant screening. The phylogenetic relationships of all the isolates were analysed using core-genome multi-locus sequence type and single-nucleotide polymorphism-based analysis, and their plasmids harbouring antimicrobial resistance genes in ST2407 were compared. RESULTS: Eighteen K. pneumoniae isolates were collected, of which 10 isolates from patients belonged to ST45 and ST2407, and eight isolates from the environment belonged to various other clones. Although 80% and 100% of isolates from patients were ESBL-positive (blaCTX-M-14 and blaCTX-M-55) and possessed siderophores, respectively; fewer environmental isolates harboured antimicrobial resistance and virulence genes. For both ST45 and ST2407 isolates, the phylogenetic assessment revealed a close relationship between clinical and environmental isolates, indicating that bloodstream infections were associated with the contaminated environments. CONCLUSIONS: Based on these results, the environmental prevalence of K. pneumoniae should be considered given its pathogenicity in humans. Early and active infection control measures could decrease the spread of multidrug-resistant K. pneumoniae.

regentrans: a framework and R package for using genomics to study regional pathogen transmission.

Increasing evidence of regional pathogen transmission networks highlights the importance of investigating the dissemination of multidrug-resistant organisms (MDROs) across a region to identify where transmission is occurring and how pathogens move across regions. We developed a framework for investigating MDRO regional transmission dynamics using whole-genome sequencing data and created regentrans, an easy-to-use, open source R package that implements these methods (https://github.com/Snitkin-Lab-Umich/regentrans). Using a dataset of over 400 carbapenem-resistant isolates of Klebsiella pneumoniae collected from patients in 21 long-term acute care hospitals over a one-year period, we demonstrate how to use our framework to gain insights into differences in inter- and intra-facility transmission across different facilities and over time. This framework and corresponding R package will allow investigators to better understand the origins and transmission patterns of MDROs, which is the first step in understanding how to stop transmission at the regional level.

Clonal spread of carbapenem-resistant Klebsiella pneumoniae among patients at admission and discharge at a Vietnamese neonatal intensive care unit.

BACKGROUND: The increasing prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is a growing problem globally, particularly in low- to middle-income countries (LMICs). Previous studies have shown high rates of CRE colonisation among patients at hospitals in LMICs, with increased risk of hospital-acquired infections. METHODS: We isolated carbapenem-resistant Klebsiella pneumoniae (CRKP) from faecal samples collected in 2017 from patients at admission and discharge at a Vietnamese neonatal intensive care unit (NICU). 126 CRKP were whole-genome sequenced. The phylogenetic relationship between the isolates and between clinical CRKP isolates collected in 2012-2018 at the same hospital were investigated. RESULTS: NDM-type carbapenemase-(61%) and KPC-2-encoding genes (41%) were the most common carbapenem resistance genes observed among the admission and discharge isolates. Most isolates (56%) belonged to three distinct clonal clusters of ST15, carrying blaKPC-2, blaNDM-1 and blaNDM-4, respectively. Each cluster also comprised clinical isolates from blood collected at the study hospital. The most dominant ST15 clone was shown to be related to isolates collected from the same hospital as far back as in 2012. CONCLUSIONS: Highly resistant CRKP were found colonising admission and discharge patients at a Vietnamese NICU, emphasising the importance of continued monitoring. Whole-genome sequencing revealed a population of CRKP consisting mostly of ST15 isolates in three clonally related clusters, each related to blood isolates collected from the same hospital. Furthermore, clinical isolates collected from previous years (dating back to 2012) were shown to likely be clonally descended from ST15 isolates in the largest cluster, suggesting a successful hospital strain which can colonise inpatients.

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.

Droplets generated from toilets during urination as a possible vehicle of carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: In the health care setting, infection control actions are fundamental for containing the dissemination of multidrug-resistant bacteria (MDR). Carbapenemase-producing Enterobacterales (CPE), especially Klebsiella pneumoniae (CR-KP), can spread among patients, although the dynamics of transmission are not fully known. Since CR-KP is present in wastewater and microorganisms are not completely removed from the toilet bowl by flushing, the risk of transmission in settings where toilets are shared should be addressed. We investigated whether urinating generates droplets that can be a vehicle for bacteria and explored the use of an innovative foam to control and eliminate this phenomenon. METHODS: To study droplet formation during urination, we set up an experiment in which different geometrical configurations of toilets could be reproduced and customized. To demonstrate that droplets can mobilize bacteria from the toilet bowl, a standard ceramic toilet was contaminated with a KPC-producing Klebsiella pneumoniae ST101 isolate. Then, we reproduced urination and attached culture dishes to the bottom of the toilet lid for bacterial colony recovery with and without foam. RESULTS: Rebound droplets invariably formed, irrespective of the geometrical configuration of the toilet. In microbiological experiments, we demonstrated that bacteria are always mobilized from the toilet bowl (mean value: 0.11 ± 0.05 CFU/cm2) and showed that a specific foam layer can completely suppress mobilization. CONCLUSIONS: Our study demonstrated that droplets generated from toilets during urination can be a hidden source of CR-KP transmission in settings where toilets are shared among colonized and noncolonized patients.

ESBL-producing Klebsiella pneumoniae in a University hospital: Molecular features, diffusion of epidemic clones and evaluation of cross-transmission.

The worldwide spread of Klebsiella pneumoniae producing extended-spectrum ß-lactamase (ESBL-Kp) is a significant threat. Specifically, various pandemic clones of ESBL-Kp are involved in hospital outbreaks and caused serious infections. In that context, we assessed the phenotypic and molecular features of a collection of ESBL-Kp isolates in a French university hospital and evaluated the occurrence of potential cross-transmissions. Over a 2-year period (2017-2018), 204 non-duplicate isolates of ESBL-Kp were isolated from clinical (n = 118, 57.8%) or screening (n = 86, 42.2%) sample cultures. These isolates were predominantly resistant to cotrimoxazole (88.8%) and ofloxacin (82.8%) but remained susceptible to imipenem (99.3%) and amikacin (93.8%). CTX-M-15 was the most frequent ESBL identified (83.6%). Multilocus sequence typing and pulse-field gel electrophoresis analysis showed an important genetic variability with 41 sequence types (ST) and 50 pulsotypes identified, and the over representation of the international epidemic clones ST307 and ST405. An epidemiological link attesting probable cross-transmission has been identified for 16 patients clustered in 4 groups during the study period. In conclusion, we showed here the dissemination of pandemic clones of ESBL-Kp in our hospital on a background of clonal diversity.

OXA-181-Like Carbapenemases in Klebsiella pneumoniae ST14, ST15, ST23, ST48, and ST231 from Septicemic Neonates: Coexistence with NDM-5, Resistome, Transmissibility, and Genome Diversity.

Studies on the epidemiology and genomes of isolates harboring OXA-48-like genes in septicemic neonates are rare. Here, isolates producing these carbapenemases which emerged and persisted in an Indian neonatal unit were characterized in terms of their resistome, transmissibility, and genome diversity. Antibiotic susceptibility and whole-genome sequencing were carried out. The sequence types, resistome, virulome, mobile genetic elements, and transmissibility of carbapenem-resistant plasmids were evaluated. Core genome analysis of isolates was shown in a global context with other OXA-48-like carbapenemase-harboring genomes, including those from neonatal studies. Eleven OXA-48-like carbapenemase-producing Klebsiella pneumoniae (blaOXA-181, n = 7 and blaOXA-232, n = 4) isolates belonging to diverse sequence types (ST14, ST15, ST23, ST48, and ST231) were identified. blaOXA-181/OXA-232 and blaNDM-5 were found in a high-risk clone, ST14 (n = 4). blaOXA-181/OXA-232 were in small, nonconjugative ColKP3 plasmids located on truncated Tn2013, whereas blaNDM-5 was in self-transmissible, conjugative IncFII plasmids, within truncated Tn125 Conjugal transfer of blaOXA-181/OXA-232 was observed in the presence of blaNDM-5 The study strains were diverse among themselves and showed various levels of relatedness with non-neonatal strains from different parts of the world and similarity with neonatal strains from Tanzania and Ghana when compared with a representative collection of carbapenemase-positive K. pneumoniae strains. We found that blaOXA-181/OXA-232-harboring isolates from a single neonatal unit had remarkably diverse genomes, ruling out clonal spread and emphasizing the extent of plasmid spreading across different STs. This study is probably the first to report the coexistence of blaOXA-181/232 and blaNDM-5 in neonatal isolates.IMPORTANCE Neonatal sepsis is a leading cause of neonatal mortality in low- and middle-income countries (LMICs). Treatment of sepsis in this vulnerable population is dependent on antimicrobials, and resistance to these life-saving antimicrobials is worrisome. Carbapenemases, enzymes produced by bacteria, can make these antimicrobials useless. Our study describes how OXA-48-like carbapenemases in neonatal septicemic Klebsiella pneumoniae shows remarkable diversity in the genomes of the strains and relatedness with strains from other parts of world and also to some neonatal outbreak strains. It is also the first to describe such resistance due to coproduction of dual carbapenemases, (OXA)-48 and New Delhi metallo-ß-lactamase-5, in Klebsiella pneumoniae from neonatal settings. Carbapenemase genes situated on plasmids within high-risk international clones, as seen here, increase the ease and transfer of resistant genetic material. With the WHO treatment protocols not adequately poised to handle such infections, prompt attention to neonatal health care is required.

Identifying the drivers of multidrug-resistant Klebsiella pneumoniae at a European level.

Beta-lactam- and in particular carbapenem-resistant Enterobacteriaceae represent a major public health threat. Despite strong variation of resistance across geographical settings, there is limited understanding of the underlying drivers. To assess these drivers, we developed a transmission model of cephalosporin- and carbapenem-resistant Klebsiella pneumoniae. The model is parameterized using antibiotic consumption and demographic data from eleven European countries and fitted to the resistance rates for Klebsiella pneumoniae for these settings. The impact of potential drivers of resistance is then assessed in counterfactual analyses. Based on reported consumption data, the model could simultaneously fit the prevalence of extended-spectrum beta-lactamase-producing and carbapenem-resistant Klebsiella pneumoniae (ESBL and CRK) across eleven European countries over eleven years. The fit could explain the large between-country variability of resistance in terms of consumption patterns and fitted differences in hospital transmission rates. Based on this fit, a counterfactual analysis found that reducing nosocomial transmission and antibiotic consumption in the hospital had the strongest impact on ESBL and CRK prevalence. Antibiotic consumption in the community also affected ESBL prevalence but its relative impact was weaker than inpatient consumption. Finally, we used the model to estimate a moderate fitness cost of CRK and ESBL at the population level. This work highlights the disproportionate role of antibiotic consumption in the hospital and of nosocomial transmission for resistance in gram-negative bacteria at a European level. This indicates that infection control and antibiotic stewardship measures should play a major role in limiting resistance even at the national or regional level.

Convergence of carbapenem resistance and hypervirulence in a highly-transmissible ST11 clone of K. pneumoniae: An epidemiological, genomic and functional study.

Co-occurrence of hypervirulence and KPC-2 carbapenem resistant phenotypes in a highly-transmissible ST11 clone ofKlebsiella pneumoniae has elicited deep concerns from public health stand point. To address this puzzle, we conducted a large-scale epidemiological, clinical and genomic study of K. pneumonia ST11 clones with both hypervirulence and carbapenem resistance in two tertiary hospitals in Zhejiang province. Most of the patients (15/23) were diagnosed with exclusively carbapenem-resistant K. pneumoniae (CRKP) infections. Ten death cases were reported, some of which are due to the failure of antibiotic therapies. As a result, we identified one new rare sequence types (ST449) to KPC-2-producing CRKP, in addition to the dominant ST11. These clinical isolates of K. pneumoniae are multi-drug resistant and possess a number of virulence factors. Experimental infections of wax moth larvae revealed the presence of hypervirulence at varied level, suggesting the complexity in bacterial virulence factors. However, plasmid curing assays further suggested that the rmpA2-virulence plasmid is associated with, but not sufficient for neither phenotypic hypermucoviscosity nor virulence of K. pneumoniae. Intriguingly, all the rmpA2 genes were found to be inactive due to genetic deletion. In total, we reported 21 complete plasmid sequences comprising 13 rmpA2-positive virulence plasmids and 8 blaKPC-2-harboring resistance plasmids. In addition to the prevalent pLVKP-like virulence plasmid variants (~178kb), we found an unexpected diversity among KPC-2-producing plasmids whose dominant form is IncFII-IncR type (~120kb), rather than the previously anticipated version of ~170kb. These findings provide an updated snapshot of convergence of hypervirulence and carbapenem resistance in ST11 K. pneumoniae.

Forensic genomics of a novel Klebsiella quasipneumoniae type from a neonatal intensive care unit in China reveals patterns of colonization, evolution and epidemiology.

During March 2017, a neonatal patient with severe diarrhoea subsequently developed septicaemia and died, with Klebsiella isolated as the causative microorganism. In keeping with infection control protocols, the coincident illness of an attending staff member and three other neonates with Klebsiella infection triggered an outbreak response, leading to microbiological assessment of isolates collected from the staff member and all 21 co-housed neonates. Multilocus sequence typing and genomic sequencing identified that the isolates from the 21 neonates were of a new Klebsiella sequence type, ST2727, and taxonomically belonged to K. quasipneumoniae subsp. similipneumoniae (formerly referred to as KpIIB). Genomic characterization showed that the isolated ST2727 strains had diverged from other K. quasipneumoniae subsp. similipneumoniae strains at least 90 years ago, whereas the neonatal samples were highly similar with a genomic divergence of 3.6 months. There was no relationship to the Klebsiella isolate from the staff member. This demonstrates that no transmission occurred from staff to patient or between patients. Rather, the data suggest that ST2727 colonized each neonate from a common hospital source. Sequence-based analysis of the genomes revealed several genes for antimicrobial resistance and some virulence features, but suggest that ST2727 is neither extremely-drug resistant nor hypervirulent. Our results highlight the clinical significance and genomic properties of ST2727 and urge genome-based measures be implemented for diagnostics and surveillance within hospital environments. Additionally, the present study demonstrates the need to scale the power of genomic analysis in retrospective studies where relatively few samples are available.

Animal Model To Study Klebsiella pneumoniae Gastrointestinal Colonization and Host-to-Host Transmission.

An important yet poorly understood facet of the life cycle of a successful pathogen is host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae, a Gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat, as K. pneumoniae has become multidrug resistant. Epidemiological studies suggest that K. pneumoniae host-to-host transmission requires close contact and generally occurs through the fecal-oral route. Here, we describe a murine model that can be utilized to study mucosal (oropharynx and gastrointestinal [GI]) colonization, shedding within feces, and transmission of K. pneumoniae through the fecal-oral route. Using an oral route of inoculation, and fecal shedding as a marker for GI colonization, we showed that K. pneumoniae can asymptomatically colonize the GI tract in immunocompetent mice and modifies the host GI microbiota. Colonization density within the GI tract and levels of shedding in the feces differed among the clinical isolates tested. A hypervirulent K. pneumoniae isolate was able to translocate from the GI tract and cause hepatic infection that mimicked the route of human infection. Expression of the capsule was required for colonization and, in turn, robust shedding. Furthermore, K. pneumoniae carrier mice were able to transmit to uninfected cohabitating mice. Lastly, treatment with antibiotics led to changes in the host microbiota and development of a transient supershedder phenotype, which enhanced transmission efficiency. Thus, this model can be used to determine the contribution of host and bacterial factors toward K. pneumoniae dissemination.

Toilet drain water as a potential source of hospital room-to-room transmission of carbapenemase-producing Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) have rapidly emerged in Europe, being responsible for nosocomial outbreaks. AIM: Following an outbreak in the burn unit of Ghent University Hospital, we investigated whether CPE can spread between toilets through drain water and therefrom be transmitted to patients. METHODS: In 2017, the burn centre of our hospital experienced an outbreak of OXA-48-producing Klebsiella pneumoniae that affected five patients staying in three different rooms. Environmental samples were collected from the sink, shower, shower stretcher, hand rail of the bed, nursing carts, toilets, and drain water to explore a common source. Whole-genome sequencing and phylogenetic analysis was performed on K. pneumoniae outbreak isolates and two random K. pneumoniae isolates. FINDINGS: OXA-48-producing K. pneumoniae was detected in toilet water in four out of six rooms and drain water between two rooms. The strain persisted in two out of six rooms after two months of daily disinfection with bleach. All outbreak isolates belonged to sequence type (ST) 15 and showed isogenicity (<15 allele differences). This suggests that the strain may have spread between rooms by drain water. Unexpectedly, one random isolate obtained from a patient who became colonized while residing at the geriatric ward clustered with the outbreak isolates, suggesting the outbreak to be larger than expected. Daily application of bleach tended to be superior to acetic acid to disinfect toilet water; however, disinfection did not completely prevent the presence of carbapenemase-producing K. pneumoniae in toilet water. CONCLUSION: Toilet drain water may be a potential source of hospital room-to-room transmission of carbapenemase-producing K. pneumoniae.

Transmission of novel Klebsiella pneumoniae carbapenemase-producing Escherichia coli sequence type 1193 among residents and caregivers in a community-based, residential care setting-Nevada, 2018.

We describe transmission of Klebsiella pneumoniae carbapenemase-producing Escherichia coli sequence type (ST) 1193 in a group home. E. coli ST1193 is an emerging multidrug-resistant clone not previously shown to carry carbapenemases in the United States. Our investigation illustrates the potential of residential group homes to amplify rare combinations of pathogens and resistance mechanisms.

Comparison of core-genome MLST, coreSNP and PFGE methods for Klebsiella pneumoniae cluster analysis.

In this work we compared the most frequently used Klebsiella pneumoniae typing methods: PFGE, cgMLST and coreSNP. We evaluated the discriminatory power of the three methods to confirm or exclude nosocomial transmission on K. pneumoniae strains isolated from January to December 2017, in the framework of the routine surveillance for multidrug-resistant organisms at the San Raffaele Hospital, in Milan. We compared the results of the different methods to the results of epidemiological investigation. Our results showed that cgMLST and coreSNP are more discriminant than PFGE, and that both approaches are suitable for transmission analyses. cgMLST appeared to be inferior to coreSNP in the K. pneumoniae CG258 phylogenetic reconstruction. Indeed, we found that the phylogenetic reconstruction based on cgMLST genes wrongly clustered ST258 clade1 and clade2 strains, conversely properly assigned by coreSNP approach. In conclusion, this study provides evidences supporting the reliability of both cgMLST and coreSNP for hospital surveillance programs and highlights the limits of cgMLST scheme genes for phylogenetic reconstructions.

Klebsiella variicola causing nosocomial transmission among neonates - an emerging pathogen?

Introduction. Transmission of Enterobacterales in neonatal intensive care units (NICU) can cause outbreaks of colonization and invasive infections among neonates. Two clusters of nosocomial transmission of Klebsiella pneumoniae identified by MALDI-ToF mass-spectrometry were suspected at two NICUs in July and August 2016.Aim. To assess the potential transmission of K. pneumoniae among neonates.Methodology. Whole-genome sequencing (WGS) was performed of K. pneumoniae isolates obtained through targeted surveillance of patients and environmental sampling.Results. WGS data revealed that patient and environmental isolates represented two species, K. pneumoniae and K. variicola. Core-genome multi-locus sequence typing (cgMLST) of the isolates identified three separate transmission clusters, in Hospital A a cluster of K. pneumoniae isolates in 12 children and two environmental samples and a second cluster of K. variicola isolates in five children. In Hospital B a cluster of K. pneumoniae isolates from three children and five unrelated isolates of K. pneumoniae and two unrelated isolates of K. variicola were found.Conclusion. K. variicola can cause hospital outbreaks of colonization and infection similar to other Klebsiella spp.Preliminary results from this study were presented at the 27th European Congress of Clinical Microbiology and Infectious Diseases, April 22-25, 2018, Vienna, Austria.

Investigation of possible clonal transmission of carbapenemase-producing Klebsiella pneumoniae complex member isolates in Denmark using core genome MLST and National Patient Registry Data.

OBJECTIVES: The aim of this study was to identify clonally-related carbapenemase-producing Klebsiella pneumoniae complex members that could be involved in outbreaks among hospitalized patients in Denmark, and to identify possible epidemiological links. METHODS: From January 2014 to June 2018, 103 isolates belonging to the K. pneumoniae complex were collected from 102 patients. From the whole-genome sequencing (WGS) data, presence of genes encoding carbapenemase and multilocal sequence typing (MLST) data were extracted. Core genome MLST (cgMLST) cluster analysis was performed. Using data from the Danish National Patient Registry (DNPR) and reported travel history, presumptive outbreaks were investigated for possible epidemiological links. RESULTS: The most common detected carbapenemase gene was blaOXA-48, followed by blaNDM-1. The 103 K. pneumoniae complex isolates belonged to 47 sequence types (STs) and cgMLST subdivided the isolates into 80 different complex types. cgMLST identified 13 clusters with 2-4 isolates per cluster. For five of the 13 clusters, a direct link (the patients stayed at the same ward on the same day) could be detected between at least some of the patients. In two clusters, the patients resided simultaneously at the same hospital, but not the same ward. A possible link (same ward within 1-13 days) was detected for the patients in one cluster. For five clusters detected by cgMLST, no epidemiological link could be detected using data from DNPR. CONCLUSION: In this study, cgMLST combined with patient hospital admission data and travel information was found to be a reliable and detailed approach to detect possible clonal transmission of carbapenemase-producing K. pneumoniae complex members.

Successful control of an extended-spectrum beta-lactamase-producing Klebsiella pneumoniae ST307 outbreak in a neonatal intensive care unit.

BACKGROUND: In this study, we evaluated the genetic relatedness of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KPN) isolates from an outbreak in a neonatal intensive care unit (NICU) in August 2017, We implemented an active countermeasure to control this outbreak successfully. METHODS: The incidence of healthcare-associated ESBL-KPN bacteremia was evaluated before and after initiating enhanced infection control (IC) practices in January 2018. Surveillance cultures were set up and monitored for neonates, medical personnel, and NICU environments. Molecular analyses, including pulse-field gel electrophoresis (PFGE), sequence typing, and ESBL genotyping, were performed for the isolated KPN strains. RESULTS: After implementing the enhanced IC procedures, the healthcare-associated bacteremia rate decreased from 6.0 to 0.0 per 1000 patient-days. Samples from neonates (n = 11/15, 73.3%), medical personnel (n = 1/41, 2.4%), and medical devices and the environments (6/181, 3.3%) tested positive for ESBL-KPN in the surveillance cultures in December 2017. Active surveillance cultures revealed that 23 of 72 neonates who were screened (31.9%) were colonized with ESBL-KPN between January and March 2018. All the isolates demonstrated closely related PFGE patterns and were identified as ST307 strain carrying the CTX-M-15 gene. CONCLUSIONS: Contaminated NICU environments and medical devices, as well as transmission by medical personnel, appeared to be the source of the outbreak of ESBL-KPN infection. We employed an enhanced IC strategy during January-March 2018 and successfully controlled the clonal outbreak of CTX-M-15-positive KPN. ST307 has emerged as an important bacteremia-causing pathogen in the NICU and should be carefully monitored.

Intra-hospital acquisition of colonization and infection by Klebsiella pneumoniae strains producing carbapenemases and carriage evolution: A longitudinal analysis in an Italian teaching hospital from January 2017 to August 2019.

OBJECTIVES: We present an updated picture (1/1/2017-31/08/2019) of the frequency of carbapenemase producing Klebsiella pneumoniae (CPKP) in surveillance rectal swabs (SRS) and in clinical samples (CS) of patients admitted to a tertiary level hospital, focusing on longitudinal evolution of CPKP detected in SRS and on colistin resistant strains. METHODS: Retrospective longitudinal analysis. Only the first positive CPKP strain isolated from each patient was included. RESULTS: 638 CPKP strains were identified (471 in SRS and 167 in CS). SRS frequency increased over time in the medical department, remained high in the surgical department (SD) and decreased in the intensive care department. Most SRS-71.3%-and 49.1% of CS had nosocomial origin; about half of the SRS were identified in the SD. Regarding SRS evolution, carriage was confirmed in 39.5% of patients, no more testing in 25.5%, clinical involvement in 24.8 %, and negative result in 10.2%. Rates of colistin resistance were 20.1% in 2017, 31.2% in 2018 and 26.9% in 2019. CONCLUSIONS: CPKP diffusion is still an important issue despite the surveillance program. It is vital to enhance medical staff's awareness on this because most CPKP first detections in SRS occurred during hospital stay due to a nosocomial acquisition with a comparable picture over time. Colistin resistance is increasing.